Therapeutical approaches for prion diseases and characterization of recombinant prion strains from different perspectives

ESCOLA DE DOUTORAMENTO INTERNACIONAL DA USC Nuria López Lorenzo Tese de doutoramento Therapeutical approaches for prion diseases and characterization of recombinant prion strains from different perspectives Santiago de Compostela, 2023 Programa de Doutoramento en Medicina Molecular TESE DE DOUTORAMENTO THERAPEUTICAL APPROACHES FOR PRION DISEASES AND CHARACTERIZATION OF RECOMBINANT PRION STRAINS FROM DIFFERENT PERSPECTIVES Autor Nuria López Lorenzo Director: Jesús R. Requena PROGRAMA DE DOUTORAMENTO EN MEDICINA MOLECULAR SANTIAGO DE COMPOSTELA DECLARACIÓN DA AUTORA DA TESE Dna. Nuria López Lorenzo Título da tese: Therapeutical approaches for prion diseases and characterization of recombinant prion strains from different perspectives Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De ser o caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. 4) A tese é a versión definitiva presentada para a súa defensa e coincide a versión impresa coa presentada en formato electrónico E comprométome a presentar o Compromiso Documental de Supervisión no caso de que o orixinal non estea na Escola. En Santiago de Compostela, 18 de Agosto de 2023. Sinatura electrónica A doutoranda declara non ter ningún conflito de interese en relación coa tese de doutoramento. A doutoranda declara ser autora de todas as figuras recollidas nesta tese de doutoramento agás daquelas que o indiquen no pé de imaxe. A presente tese de doutoramento contou coas seguintes fontes de financiamiento: - Financiamiento do Ministerio de Economía, Industria y Competitividad (MINECO),Plan Nacional 2017 (BFU201786692-P) - Financiamento da Agencia Española de Investigación (PID2020-117465GB-I00), financiado parcialmente polo Fondo Europeo de Desenvolvemento Rexional. - Axuda de investigación “Sergio Rubio Muñóz” 2020-2021, da Fundación Española de Enfermedades Priónicas. - A doutoranda foi beneficiaria dunha axuda de apoio a etapa predoutoral do Sistema galego de I+D+i, cofinanciadas parcialmente no ámbito das universidades do Sistema universitario de Galicia polo programa operativo Fondo Social Europeo Galicia 2014-2020, na súa convocatoria no ano 2019, pola Consellería de Cultura, Educación e Ordenación Universitaria de la Xunta de Galicia. - xii de TFM, TFG y prácticas que habéis pasado por el laboratorio, gracias a vosotros también por recordarme a mí misma de dónde vengo y por qué estoy aquí. Gracias también a las chicas del laboratorio de al lado, las plaquetas. A Irene y María, cuyas tesis fueron mis primeras referencias y mi camino a seguir. Gracias especialmente a Lidia y Alberto (que te incluyo aquí aunque no seas de este grupo) por los cafés y las eternas conversaciones de pasillo. Por compartir conmigo vuestras experiencias, consejos y por escucharme, aún ahora, que ya terminasteis vuestra etapa en el CiMUS. Por último, solo me queda agradecerles a esas personas que están a mi lado cuando salgo del laboratorio. A mis amigas, que sin tener ni idea de priones me preguntan cómo me va la tesis y cómo se encuentran los ratones; que a día de hoy se ofrecen para que practique con ellas las presentaciones de congresos o la de la tesis, y que me animan diciendo que en unos meses seré “Dra. López”. A mis padres por apoyarme en todas mis decisiones y por enseñarme que los límites me los pongo yo. Por alegrarse conmigo de los pequeños logros y por intentar entender de qué va todo esto. A mi hermano, Iván, porque sí, porque me achucha cada vez que llego de Santiago y con eso le basta para demostrarme su apoyo. Y a Germán, porque lo que llevamos vivido en estos cuatro años de tesis solo los sabemos tú y yo. Porque una tesis no es un camino de rosas y las alegrías se celebran con los compañeros y los amigo, pero la frustración se lleva y se paga en casa. Gracias por soportarme en los malos momentos y no dejar que tire la toalla. Gracias también por aguantar que antepusiera la tesis a todo lo demás. Gracias por estar ahí y ser un pilar fundamental de esta tesis. Abstract xv Abstract Transmissible spongiform encephalopathies, also known as prion diseases, are neurodegenerative disorders caused by prions. These infectious proteins result from misfolding of the cellular prion protein, PrPC, into its pathogenic isoform, PrPSc. Transmissible spongiform encephalopathies affect humans and other mammals and can manifest as idiopathic, genetic, or acquired diseases. Although PrPC and PrPSc share the same amino acid sequence, they differ in their secondary, tertiary, and quaternary structures, leading to distinct biological and biochemical properties. Furthermore, PrPSc can exhibit conformational variants called strains, which display different phenotypes and even resistance to certain compounds with anti-prion properties. Prion diseases are fatal. Despite the evaluation of various candidates with therapeutic potential, there are currently no treatments or vaccines available for them. In this thesis, three therapeutic candidates are assessed as proof of concept, tackling prion diseases from different aspects of their biology in a rapid murine model of prion disease. The first candidate is a porphyrin termed Fe(III)-TMPyP, which acts as a molecular chaperone, stabilizing the native conformation of PrPC and hindering its conversion into PrPSc. The second therapeutic strategy proposed involves the use of a single-chain antibody, scFv-G1, designed to recognize PrPSc specifically. The third therapeutic approach employs a recombinant self-propagating PrP amyloid that aims to prevent the conversion of PrPC into PrPSc and compete with PrPSc for the substrate. Unfortunately, the results obtained from these therapeutic approaches were not as expected; similar to other studies in search of treatments for prion diseases, they were unable to demonstrate beneficial effects in animal models. NURIA LÓPEZ LORENZO xvi One reason for therapeutic failure in prion diseases is attributed to the ability of prions to evade treatment through adaptation to the presence of the pharmacological agent or the selection of prion strains that propagate more effectively in its presence. Both mechanisms result in the generation of drug-resistant strains and are mainly due to slight conformational arrangements in prion structure. Therefore, a thorough characterization of diverse prion strains can provide crucial information about the differences between them, allowing for the design of effective therapies and addressing the adaptation and selection mechanisms that lead to therapeutic failure. Furthermore, studying the diversity of prion strains and their differences involves significant biochemical and structural interest. For these reasons, the last chapter of this doctoral thesis aims to study various recombinant prion strains generated spontaneously by PMSA using different biological, biochemical, and biophysical approaches. Overall, this work addresses prion diseases through the use of three pharmacological candidates, each approaching prion disorders from different perspectives. Although the results have not met expectations in terms of efficacy, they have shed light on the limitations of each candidate. Additionally, it has been possible to characterize recombinant prion strains from different approaches, establishing differences among them and redefining the role of cofactors in the diversity of prion strains. Resumen xvii Resumen Las encefalopatías espongiformes transmisibles, también conocidas como enfermedades priónicas, son trastornos neurodegenerativos causados por priones, proteínas infecciosas que surgen del malplegamiento de la proteína priónica celular, PrPC, en su isoforma patogénica, PrPSc. Las encefalopatías espongiformes transmisibles afectan al ser humano y otros mamíferos y pueden presentarse como enfermedades idiopáticas, genéticas, o adquiridas. Aunque la PrPC y la PrPSc comparten la misma secuencia aminoacídica, difieren en sus estructuras secundaria, terciaria y cuaternaria, lo que se traduce en distintas propiedades biológicas y bioquímicas. Por otra parte, la PrPSc puede presentar variantes conformacionales, denominadas cepas, que se manifiestan con distintos fenotipos e incluso resistencia a ciertos compuestos con propiedades antipriónicas. Las enfermedades priónicas son mortales. Aunque se han estudiado diversos candidatos con potencial terapéutico, a día de hoy no existen tratamientos ni vacunas para ellas. En la presente tesis se evalúan tres candidatos terapéuticos como prueba de concepto, abordando las enfermedades priónicas desde distintos aspectos de su biología, en un modelo murino rápido de enfermedad priónica. El primer candidato es una porfirina, llamada Fe(III)-TMPyP, que actúa como una chaperona molecular estabilizando la conformación nativa de la PrPC e impidiendo su conversión en la PrPSc. La segunda estrategia terapéutica que se plantea es el uso del anticuerpo de cadena sencilla, scFv-G1, diseñado para reconocer específicamente la PrPSc. El tercer abordaje terapéutico consiste en el uso de un amiloide de PrP recombinante autopropagativo que tiene como objetivo bloquear la conversión de la PrPC en PrPSc y competir con la PrPSc por el sustrato. NURIA LÓPEZ LORENZO xviii Desafortunadamente, los resultados obtenidos con los abordajes terapéuticos no fueron los esperados; al igual que otros trabajos en la búsqueda de un tratamiento para las enfermedades priónicas, no pudieron mostrar efectos beneficiosos en modelos animales. Un motivo de fracaso terapéutico en las enfermedades priónicas se atribuye a la capacidad de los priones de evadir el tratamiento mediante la adaptación a la presencia del agente farmacológico o a la selección de las cepas de priones que mejor se propagan en su presencia. Ambos mecanismos tienen como consecuencia la generación de cepas resistentes al tratamiento y se deben principalmente a ligeros cambios conformacionales en la estructura de los priones. Por este motivo, una caracterización minuciosa de diversas cepas puede proporcionar información crucial sobre las diferencias entre ellas, lo que permitiría diseñar terapias efectivas y abordar los mecanismos de adaptación y selección que conducen a la ineficacia del tratamiento. Por otro lado, el estudio de la diversidad de cepas de priones y las diferencias existentes entre ellas es de gran interés bioquímico y estructural. Por todo ello, el último capítulo de la presente tesis de doctorado tiene como propósito el estudio mediante métodos biológicos, bioquímicos y biofísicos de diversas cepas de priones recombinantes, generadas espontáneamente mediante PMSA. En definitiva, ha sido posible estudiar el efecto terapéutico de tres candidatos que abordan las enfermedades priónicas desde distintas perspectivas y, aunque los resultados no han sido los esperados en términos de eficacia, han arrojado luz sobre las limitaciones que presenta cada una de ellas en la terapia de las enfermedades priónicas. Además, ha sido posible caracterizar desde distintas perspectivas cepas recombinantes de priones, estableciendo diferencias entre ellas y redefiniendo el role de los cofactores en la diversidad de cepas de priones. Resumo xix Resumo As encefalopatías esponxiformes transmisíbeis, tamén coñecidas como enfermidades priónicas, son trastornos neurodexenerativos causados por prións, proteínas infecciosas que xorden do mal-pregado da proteína priónica celular, PrPC, na súa iso-forma patóxena, PrPSc. As encefalopatías esponxiformes transmisíbeis son enfermidades raras; a súa incidencia é de 1-2 casos por cada millón de habitantes e ano, fronte aos 11000 casos por millón de habitantes e ano que se diagnostican de enfermidade de Alzheimer, a doenza neurodexenerativa máis común. As enfermidades priónicas non só afectan ao ser humano, tamén a outros mamíferos, e poden presentarse como enfermidades xenéticas, adquiridas ou idiopáticas, sendo estas últimas as máis comúns. As enfermidades priónicas xenéticas son aquelas que están vinculadas con mutacións no xene PRNP, que codifica a PrPC e para as que hai máis de 40 mutacións descritas. Son enfermidades dominantes e con elevada penetrancia, o que quere dicir que un portador dunha mutación relacionada cunha destas doenzas xenéticas, ten unha alta probabilidade de desenrolar a enfermidade ao longo da súa vida. Por outra banda, as enfermidades priónicas menos comúns son as adquiridas, que se relacionan co consumo de alimentos, co contacto con materiais cirúrxicos, ou coa recepción de órganos ou tecidos contaminados con prións. Aínda que as enfermidades priónicas adquiridas son as menos común, son probablemente as máis coñecidas debido á encefalopatía esponxiforme bovina, coñecida popularmente como a enfermidade das vacas tolas. Esta doenza xurdiu en Reino Unido como consecuencia da alimentación do gando vacún con fariñas cárnicas, que resultaron estar contaminadas con prións. O brote de encefalopatía esponxiforme bovina levou ao sacrificio de máis de 4 millóns de reses e incontábeis perdas económicas. Pero o máis desalentador foi que, como consecuencia do consumo de carne contaminada, esta enfermidade deu orixe a unha nova zoonose, coñecida como a variante da enfermidade NURIA LÓPEZ LORENZO xx de Creutzfeldt-Jakob. Este feito desencadeou unha crise de saúde pública en Europa a finais dos anos 90 e comezos dos 2000. A PrPC é una glicoproteína de membrana altamente conservada entre os mamíferos. A súa función non está ben definida, aínda que se coñece que participa en diversos procesos biolóxicos e patolóxicos coma o mantemento da mielina, a homeostase de certos metais, proporcionando protección contra o estrés oxidativo, preservando a plasticidade sináptica, regulando o ritmo circadiano, regulando o sistema inmune, tamén participa na sensibilidade á dor, na bioloxía do cancro e en enfermidades neurodexenerativas. Porén, o desenrolo de ratos transxénicos que carecen desta proteína demostrou que a súa función non é vital ou que na súa ausencia existen mecanismos compensatorios que permiten o desenrolo da vida. Malia que a PrPC e a PrPSc comparten a mesma secuencia aminoacídica, ambas iso-formas presentan distintas arquitecturas nas súas estruturas secundaria, terciaria e cuaternaria. Mentres que a PrPC é una proteína globular, cunha gran presencia de hélices α na súa estrutura secundaria e cun extremo N-terminal intrinsecamente desordenado, a PrPSc ten unha elevada presenza de follas β na súa estrutura secundaria e presenta unha arquitectura de follas β intermolecular en rexistro paralelo, PIRIBS (do inglés, Parallel In-register Intermolecular β-Sheet Architectures). Ademais, as súas diferencias estruturais vense reflectidas nas propiedades biolóxicas e bioquímicas de ámbalas dúas iso-formas. Mentres que a PrPC é unha proteína fisiolóxica, que se atopa como monómero ancorada ás membranas celulares e é solúbel e sensíbel á dixestión con proteasas; na PrPSc, os monómeros asócianse formando fibras amiloides que son insolúbeis e crean depósitos no cerebro, ademais a PrPSc é parcialmente resistentes á dixestión con proteasas. Por outra banda, a PrPSc pode presentar variantes conformacionais que difiren lixeiramente na súa arquitectura, denominadas cepas, e que presentan certas diferenzas biolóxicas e bioquímicas, como distinta resistencia á dixestión con proteasas, á desnaturalización con axentes caotrópicos, distinta afinidade polas Resumo xxi áreas cerebrais, distintos tempos de incubación, e incluso resistencia a certos compostos con propiedades antipriónicas. A arquitectura da PrPSc foi recentemente descuberta. Ata ese momento existían dous modelos conformacionais para a iso-forma patóxena da PrPC, o PIRIBS e o β-solenoide de 4 chanzos (4RβS, do inglés, 4-Runβ-Solenoid). Na conformación PIRIBS, cada monómero está pregado en follas β que se atopan completamente aliñadas, de tal xeito que cada aminoácido se atopa enriba ou debaixo do mesmo aminoácido do monómero consecutivo. Pola contra, o 4RβS implica que cada monómero se atopa pregado sobre sí mesmo nunha configuración helicoidal, na que cada monómero distribúese ao longo de 4 chanzos. A escasa resolución dos métodos disponíbeis ata o momento, as dificultades que incumben á purificación da PrPSc de cerebro e as limitacións das ferramentas informáticas non permitían determinar cal dos dous modelos era o correcto e as evidencias que se obtiñan eran compatíbeis cos dous. Non foi ata o ano 2021 cando Allison Kraus e os seus colegas obtiveron imaxes por criomicroscopía electrónica que permitiron a resolución da primeira cepa de prións illada de cerebro de hámsteres inoculados. En cuestión de meses, o mesmo grupo e outros foron quen de resolver a estrutura doutras cepas de prións, evidenciando que as diferencias entre elas residen en lixeiras variacións estruturais. A resolución da estrutura dos prións considérase un dos grandes fitos dos últimos anos no campo dos prións. Desafortunadamente, as enfermidades priónicas son mortais. Ata o momento diversas estratexias terapéuticas foron avaliadas intentando abordar as enfermidades priónicas dende distintos aspectos da súa bioloxía, pero con escasa ou nula efectividade. Dado que a presenza da PrPC é imprescindíbel para o desenrrolo das enfermidades priónicas, moitos esforzos centráronse en diminuir ou impedir a expresión da PrPC. Esta estratexia sería altamente vantaxosa para os portadores de mutacións relacionadas con enfermidades priónicas xenéticas, xa que se fora posíbel anular por completo a expresión da PrPC, eliminaríase o risco de padecer estas enfermidades. Esta estratexia terapéutica é a máis NURIA LÓPEZ LORENZO xxviii biolóxicos. As análises mediante espectrometría de masas, resonancia magnética de estado sólido e Western blot, determinaron que os confórmeros analizados presentaban distintas propiedades bioquímicas e biofísicas, indicando que eran estruturalmente diferentes. Ademais, os resultados obtidos por espectrometría de masas comparáronse con datos previos procedentes de mostras de pacientes, animais inoculados, o doutras cepas de prión recombiantes xeradas con outras técnicas, demostrando que os nosos datos eran consistentes co previamente descrito e concluíndo que os confórmeros xerados mediante PMSA compartían propiedades con prións bona fide. Por outra banda, para caracterizar as propiedades biolóxicas dos confórmeros, estes foron inoculados intracerebralmente no mesmo modelo de rato transxénico empregado nas abordaxes terapéuticas, o TgVole. Trala inoculación, todos os animais adoeceron, presentando signos compatíbeis con enfermidades priónicas. Cando os seus cerebros foron analizados, atopáronse en todos eles PrP resistente a proteasa, e a análise histopatolóxica confirmou que os ratos padecerán unha enfermidade priónica. Estes resultados demostraron que os confórmeros eran infecciosos, e polo tanto trátabanse de prións bona fide. Os prións xerados con PrPC recombinante a miúdo sofren un proceso de adaptación cando son inoculados en animais. Esta adaptación débese principalmente ás diferencias entre a PrPC recombinante e a nativa do cerebro; esta última presenta modificación postraducionais, en forma de glicosilacións e unha áncora de glicofosfatidilinositol. Ademais o ambiente cerebral é moito máis complexo que os reactivos que se empregan para xerar prións in vitro. Para comparar as características biolóxicas de cada un dos confórmeros realizouse un segundo pase, para asegurar que o material xerado nos cerebros, froito da inoculación dos confórmeros, estaba totalmente adaptado ao cerebro dos animais. Ao igual que no primeiro pase, todos os animais sucumbiron a unha enfermidade priónica. Neste segundo pase, estudáronse os tempos de incubación, os patróns electroforéticos, e os perfís de lesións cerebrais. Resumo xxix Aínda que moitos tempos de incubación se viron unificados, estas análises permitiron identificar cepas de prións que presentaban diferente afinidade polas áreas cerebrais e con patróns de resistencia a proteasa distintos. Estes resultados permitiron concluir que diferentes cepas de prións foron xeradas nas mesmas condición e en presenza do mesmo cofactor, pero por outra banda os resultados tamén indicaron que confórmeros xerados con cofactores distintos deron lugar a enfermidades con características moi similares, suxerindo que con cofactores distintos podería xerarse a mesma cepa. En resumo, neste traballo foi posíbel estudar o efecto terapéutico de tres candidatos con potencial antipriónico e, malia que os resultados non foron os esperados en termos de eficacia, arroxaron luz sobre as limitacións que presenta cada un deles na terapia das enfermidades priónicas. Ademais, foi posíbel caracterizar por medios biolóxicos, bioquímicos e biofísicos cepas recombinantes de prións, establecendo diferenzas entre elas e redefinindo o papel dos cofactores na diversidade de cepas de prións. List of Abbreviations xxxi List of Abbreviations °C Å % Celsius degree Angstroms Percentage µg Microgram µL Microliter µM Micromolar λex λem 12B2 22L 263K 3F10 4RβS 6C2 9A2 Wavelength of excitation Wavelength of emission Anti-PrP monoclonal antibody Mouse-adapted prion strain Hamster-adapted prion strain Anti-PrP monoclonal antibody 4-rung-β-solenoid Anti-PrP monoclonal antibody Anti-PrP monoclonal antibody A Aβ a22L AAV ADAM Amyloid-β GPI-anchorless 22L prion strain Adeno-associated viruses a disintegrin and metalloproteinase Adgrg6 ALS APP aRML ASA Adhesion G Protein-Coupled Receptor G6 Amyotrophic lateral sclerosis Amyloid precursor protein GPI-anchorless RML prion strain Amyloid Seed Assay ASO Antisense oligonucleotide ATM AU Atomic force microscopy Arbitrary units B BBB Blood-brain barrier BSE Bovine spongiform encephalopathy NURIA LÓPEZ LORENZO xxxii BV BVPrP Bank vole Bank vole prion protein C C1 C2 cAMP CB C-BSE CC1 CC2 CD CJD C-terminal fragment generated by α-cleavage C-terminal fragment generated by β-cleavage Cyclic adenosine monophosphate Conversion buffer Classical bovine spongiform encephalopathy Charged cluster 1 Charged cluster 2 Circular dichroism Creutzfeldt-Jakob disease CNS Central nervous system CoS CPD Chondroitin sulfate Camel prion disease Cryo-EM Cryo-electron microscopy CSF CTF12/13 Cerebrospinal fluid C-terminal fragments of 12 and 13kDa CWD Chronic wasting disease D Da DAPI DARR Dalton 4 ',6-diamidino-2-fenilindol Dipolar assisted rotational resonance Dex Dextran sulfate DNA Deoxyribonucleic acid DNase OD600 dpi Deoxyribonuclease Optical density at 600nm Days post-inoculation Dy Drowsy, hamster prion strain E E. coli Escherichia coli EEG ELISA Electroencephalogram Enzyme-linked immunosorbent assay List of Abbreviations xxxiii EM Electron microscopy ER ERAD Endoplasmic reticulum Endoplasmic-reticulum-associated protein degradation F FDA Food and drug administration Fe(III)-TMPyP Fe(III) tetrakis(4N-methylpyridinium)porphyrin FFI FTIR Fatal familial insomnia Fourier-transform infrared spectroscopy G g gCJD GFAP Centrifuge force Genetic Creutzfeldt-Jakob disease Glial fibrillary acidic protein Gdn Guanidine GPI Gpr126 Glycosylphosphatidylinositol G-protein coupled receptor Adgrg6 GSS Gerstmann-Sträussler-Scheinker syndrome H H H-BSE Hours H-type atypical bovine spongiform encephalopathy HC Hydrophobic core Hep HR HRP Heparin Hydrophobic region Horseradish peroxidase Hu Human Hy Hyper, hamster prion strain I i.c. Intracerebral iCJD Iatrogenic Creutzfeldt-Jakob disease i.p. Intraperitoneal IPTG Isopropyl β-d-1-thiogalactopyranoside K NURIA LÓPEZ LORENZO xxxiv kDa kHz Kilo Dalton Kilohertz L L Liter LB L-BSE LCP LOTSS LTP Luria Bertoni, bacteria growth medium L-type atypical bovine spongiform encephalopathy Luminescent conjugated polymers Low Toxicity Synthetic Strain Long-term potentiation M M M7 MBM Molar Mouse-adapted prion strain Meat and bone meal MeOH Methanol MES 2-(N-morpholino)ethanesulfonic acid mg Milligram min Minutes miRNA Micro ribonucleic acid mL Milliliter mM Millimolar Mo Mouse mQ MRI Milli-Q water Magnetic resonance imaging mRNA Messenger ribonucleic acid MS ms MSE Mass spectrometry Milliseconds Mink spongiform encephalopathy MW Molecular weight NBH Normal brain homogenate N N1 N2 N3 N-terminal fragment generated by α-cleavage N-terminal fragment generated by β-cleavage N-terminal fragment generated by γ-cleavage List of Abbreviations xxxv NaPTA Ni-NTA NMDA NMR Nor98 ns Sodium phosphotungstate Nickel-nitrilotriacetic acid N-methyl-D-aspartate Nuclear Magnetic Resonance Norway 98, atypical prion strain Nanoseconds O OD on Optical density Overnight OPR OR Open reading frame Octarepeats P PBS Phosphate-buffered saline PERK PKR-like endoplasmic reticulum kinase PIRIBS Parallel-in-register-intermolecular-β-sheets PK Proteinase K PMCA Protein misfolding cycling amplification PMSA ppm Protein misfolding shaking amplification Parts per million PPS PRND Pentosan polysulfate sodium Prion-like protein Doppel gene PRNP PRNT PrP27-30 Prion protein gene Putative testis-specific prion protein gene Scrapie isoform of the prion protein PK treated PrP Prion protein PrPC Cellular prion protein PrPres PrPSc PK-resistant PrP Scrapie prion protein PrPSs Sslow prion protein PVDF Polyvinylidene difluoride Q q.s. Quantum Satis NURIA LÓPEZ LORENZO xxxvi R recPrP Recombinant prion protein RML Rocky Mountain Laboratory prion strain RNAi Ribonucleic acid interference rpm RT Revolutions per minute Room temperature RT-QuIC Real-time quaking-induced conversion S s Seconds SAF SAF83 SAF84 SAXS Scrapie associated fibrils Anti-PrP monoclonal antibody Anti-PrP monoclonal antibody Small Angle X-ray Scattering scFv Single chain variable fragment sCJD SD SEM Sporadic Creutzfeldt-Jakob disease Standard deviation Standard error of the mean sFI siRNA Sporadic fatal insomnia Small interfering ribonucleic acid shRNA Short hairpin ribonucleic acid SMA SPRN SSLOW Spinal muscular atrophy Shadow of prion protein gene Synthetic Strain Leading to OverWeight ssNMR Solid-state nuclear magnetic resonance T TBS-T TEM Tg TgVole Tris buffer saline - tween Transmission electron microscopy Transgenic Transgenic mice bearing bank vole PrP sequence ThT TI TMB Thioflavin-T Thymus-independent antigens 3,3´,5,5´-tetramethylbenzidine TSE Transmissible spongiform encephalopathy List of Abbreviations xxxvii U UPR Unfolded protein response UPS Ubiquitin proteasome system V vCJD v/v Variant Creutzfeldt-Jakob disease Volume/volume VPSPr Variably protease-sensitive prionopathy W w/v Weight/Volume WT Wild-type Amino acid Three-letter abbreviation One-letter abbreviation Amino acid Three-letter abbreviation One-letter abbreviation Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamic acid Glu E Serine Ser S Glutamine Gln Q Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V NURIA LÓPEZ LORENZO xliv 1. GENERAL INTRODUCTION General Introduction 3 1.1. PRION DISEASES Prion diseases are neurodegenerative disorders that belong to the group of protein misfolding diseases or proteinopathies, a heterogeneous group of conditions that also includes cystic fibrosis, Alzheimer's disease, type II diabetes, and several others. All these diseases are characterized by the aggregation of structurally abnormal proteins that failed in acquiring their native three-dimensional conformation or lost it, leading to the accumulation of protein aggregates in different organs and tissues that disrupt their normal functioning 1. In the case of prion diseases, the central event in their pathogenesis is the conformational conversion of the host-encoded cellular prion protein (PrPC) into its pathogenic isoform (PrPSc). This conformational rearrangement involves important changes in the biochemical and biological properties of the protein, such as the formation of insoluble proteins and partial resistance to digestion with proteases. Prion diseases are fatal and incurable conditions that affect humans and other species of mammals consisting of an extended asymptomatic incubation period followed by a rapidly progressive disease that leads to the death of the patients in a matter of months or few years. While patients manifest various clinical signs depending on the prion disease, the predominant symptoms include motor dysfunctions, cognitive impairment, behavior abnormalities, and cerebellar ataxia. Unfortunately, these clinical presentations are not pathognomonic; quite the opposite, they are common to many other neurodegenerative disorders. This fact, added to the low incidence of these illnesses, the rapid progression of the disease, and the lack of specific biomarkers, difficult an early and accurate diagnosis. Although techniques such as electroencephalogram (EEG), magnetic resonance imaging (MRI), or cerebrospinal fluid (CSF) analysis can be helpful in the diagnosis, the NURIA LÓPEZ LORENZO 4 confirmation of prion disease is generally post-mortem after neuropathological brain examinations 2,3. In this regard, prion diseases are also known as transmissible spongiform encephalopathies (TSE) in reference to the appearance of the brain tissue that presents multiple rounded vacuoles in the grey matter that provide a sponge-like aspect. Apart from the spongiform degeneration, other histopathological features that also characterize prion diseases are the presence of reactive gliosis involving astrocytes and microglia, neuronal loss, and accumulation of prion protein aggregates (Fig 1.1). Figure 1.1 Neuropathological features of prion diseases in comparison with healthy brains. Abnormalities in the brain of affected individuals include vacuolation visible with hematoxylin-eosin staining, accumulation of prion protein deposits detectable with immunohistochemical staining with an anti-PrP antibody, and astrogliosis perceivable after staining with anti-glial fibrillary acidic protein antibody (GFAP). Adapted from Raymond et al 20194. 1.1.1 Human prion diseases Human prion diseases are rare neurodegenerative conditions that affect approximately 1-2 persons per million of population worldwide and annually 5. They comprise a heterogeneous group of diseases with different clinical presentations, molecular and histopathological General Introduction 5 features. According to their etiology, prion diseases can be classified into three subgroups: sporadic, genetic, and acquired disorders (Table 1.1). Sporadic prion diseases Sporadic prion diseases are the most common subgroup of prion diseases that encompasses 85% of the cases. Sporadic prion diseases have been documented globally, displaying a random distribution and lacking any association with environmental, social, or occupational risk factors. Although the incidence varies among countries, these divergences are most likely due to limitations in the detection and reporting of the cases rather than a different distribution. The event that triggers the development of sporadic prion disease is unknown. Epidemiological studies have ruled out the hypothesis that an external exposure to the infectious agent triggered sporadic cases, and there is no evidence of spatial or temporal clustering, or a connection with outbreaks of animal prion diseases. Instead, studies suggest that sporadic cases arise from a random conversion of the protein into its pathogenic form 6. The most common sporadic prion disease is sporadic Creutzfeldt-Jakob disease (sCJD). In Spain, the incidence of sCJD is 1,14 cases per million population and year 7, sharing a similar frequency with other European countries. The distribution of patients by gender and age reveals that the frequency in women is slightly higher than in men (1,4:1), and the mean age at the diagnosis was between 60 and 79 in both sexes 8. NURIA LÓPEZ LORENZO 6 Table 1.1 Classification of human prion diseases according to their etiology. General Introduction 7 sCJD exhibits a wide range of clinical manifestations: dementia, ataxia, myoclonic involuntary movements, language disorders, visual impairments, behavioral changes, or spasticity, for instance6. Moreover, it also displays various molecular and histological features, such as the electrophoretic pattern of PrP resistant to digestion with the protease proteinase K (PK), or the brain lesion profile. The clinical symptoms, life expectancy, and molecular properties strongly correlate with the polymorphic variations of the prion protein gene (PRNP) 9, to the point that sCJD has been categorized according to amino acid present at codon 129 and their molecular features. Therefore, six subtypes of sCJD exist based on the presence of methionine, valine or both, in case of heterozygous: MM1, MV1, VV2, MV2, MM2-C, VV1, and MM2-T, this last subtype is also known as sporadic fatal insomnia (sFI) due to the main clinical sign is the difficulty to fall asleep or stay asleep, resulting in severe sleep deprivation2,3. In 2008, a new prion disease was described, termed variably proteasesensitive prionopathy (VPSPr) 10,11, and due to its lack of association with mutations in the PRNP gene, it was classified as a sporadic prion disease. The progression of this disease is slightly slower than sCJD (˃2 years), and the most common clinical signs are cerebellar ataxia, movement disorders, cognitive impairments, or extrapyramidal symptoms. Neuropathological examinations show spongiform degeneration and PrP amyloid plaques. However, the most remarkable feature of this disorder, which gives this disease its name, is the high sensitivity of the misfolded form of the prion protein to digestion with PK, which is reflected as a characteristic multiple-band pattern upon immunoblotting 12. Genetic prion diseases While the majority of prion diseases are idiopathic approximately 1015% of the cases are linked to a mutation in the PRNP sequence and are referred to as genetic prion diseases. Genetic prion disorders are autosomal-dominant diseases with high penetrance, meaning that NURIA LÓPEZ LORENZO 14 have been reported to date, some studies carried out in humanized transgenic mice and non-human primates have demonstrated a zoonotic potential 43. Bovine spongiform encephalopathy In 1986 a new prion disease that affected cattle emerged, commonly known as mad cow disease 44. Bovine spongiform encephalopathy (BSE) spread to 28 countries and soon became an epidemic. In the United Kingdom, the most affected country, around 4,4 million animals were culled to contain the spread of the disease; of those, roughly 180.000 were infected. The economic impact resulted in losses of £4 billion 45, and, despite the efforts, it has been estimated that over 1 million BSE-infected animals could have entered the human food chain 43. The clinical signs of those animals affected with BSE were cachexia, ataxia, behavioral changes, lethargy, and hyperreactivity. The origin of BSE remains unknown; however, since the ban on the use of meat and bone meal (MBM) to feed livestock in 1988 resulted in a drastic decline in BSE cases, the most likely explanation is that the MBM used to feed livestock was contaminated with prions46 and the disease was transmitted orally to cattle. Moreover, three hypotheses about the provenance of the contaminated MBM were formulated. The first hypothesis proposed that the contamination may have come from sheep infected with atypical scrapie, as indicated by the biochemical similarities observed when Nor98 is transmitted to bovine transgenic mice 47,48. The second explanation suggests that MBM could be contaminated with remains of animals with subclinical sporadic or genetic bovine spongiform encephalopathy 49,50. Lastly, the third theory proposed that MBM could contain human remains infected with CJD 51. The outbreak of BSE was responsible for the emergence of vCJD (Fig 1.3); additionally, BSE was also transmitted to other animals fed with contaminated meat, such as domestic cats and some zoological ruminants, ungulates, felines, and non-human primates 52. Moreover, General Introduction 15 BSE has also been transmitted experimentally to hamsters, mice, minks, sheep, goats, and pigs, demonstrating that BSE has a high capacity to cross the species barrier 42. The active surveillance to detect BSE cases found out atypical BSE variants, namely H-BSE or L-BSE, according to their biochemical properties 53,54. Since atypical BSE variants have a low incidence and mostly affect old animals, it was suggested that they may consist of sporadic cases; however, the zoonotic risk and the potential transmission of these atypical variants have been demonstrated experimentally and were proposed as the origin of the BSE epidemic 55–58. Figure 1.3 Comparison between BSE and vCJD cases. The graph represents the reported cases of bovine spongiform encephalopathy and the subsequent emergence of variant Creutzfeldt-Jakob disease in the United Kingdom. Graph available in Watson et al 20215. Chronic wasting disease Chronic wasting disease (CWD) is a prion disease that affects freeranging and captive cervids. It was first reported in 1967, in Colorado, NURIA LÓPEZ LORENZO 16 and in a few years, it had rapidly spread in North America promoted by natural migrations of free-ranging populations and commercial exports. Furthermore, some cases have been reported in South Korea due to animal importations from Canada. Recently, in 2016, CWD was also identified in wild reindeer and moose in Norway, followed by the detection of more cases in a semi-isolated reindeer population in the region of Nordfjella. In an attempt to control the spread of cases in this area, the Norwegian authorities took the drastic decision to cull this reindeer population and test them for CWD, finding 18 positive cases out of 2400 samples. Currently, CWD cases have also been reported in Sweden and Finland (Fig 1.4) 59. CWD affects different species of cervids, such as deer, reindeer, elk, and moose, among others, and sick animals are popularly called zombie deers because of the clinical signs that they present, characterized by cachexia, excessive salivation, behavioral changes such as loss of fear to humans, dropping ears and lowered head. CWD is highly contagious among individuals60; prion infectivity was found in saliva61, urine62, feces63, and placenta, confirming vertical and horizontal transmission. Likewise, prions have been detected in grazing areas, soil64, and water65, raising concern about significant environmental contamination and the risk of transmission among individuals 66. Moreover, the high prevalence of CWD in some states of the U.S. is a major cause for concern, particularly in Wyoming, where the population of mule deer is facing significant declines as a result of this prion disease67 . While no natural transmission to other species or humans has been reported to date, experimental studies have successfully transmitted CWD to various animal species that cohabitate with cervids or act as scavengers, such as cattle, sheep, goats, ferrets, minks, raccoons, mice, and non-human primates as well 68, supporting the potential zoonotic risk and a possible cross-species transmission. General Introduction 17 Figure 1.4. Cases of CWD in North America and Northern Europe. Maps of CWD distribution in North America (left panel) and the Nordic countries (right panel). Maps of public domain obtained from the National Wildlife Health Center (available on https://www.usgs.gov/centers/nwhc/science/expanding-distribution-chronicwasting-disease) and the Norwegian Institute for Nature Research (available on https://www.hjortevilt.no/skrantesjuke-cwd/cwd-in-norway-english/), respectively. NURIA LÓPEZ LORENZO 18 Mink spongiform encephalopathy Mink spongiform encephalopathy (MSE) was first detected in 1947 in farmed minks for fur in Wisconsin and Minnesota. Subsequently, more cases were reported in Canada, Germany, or Finland, always in captive animals 69. Although the origin of MSE is unknown, it was most likely transmitted by scrapie-contaminated feed since simultaneous outbreaks occurred in different farms sharing the same feed supplier 70. Infected animals display behavioral changes like aggressiveness, compulsive biting, or depression, and also motor impairments such as ataxia, incoordination, and hindlimb weakness 52, and horizontal transmission is the most plausible spreading route among individuals. While MSE did not hold as much socioeconomic impact as BSE or CWD, it provided significant contributions to prion research. After transmission of MSE to hamsters, two prion variants were isolated, which triggered different clinical signs, biochemical properties, and neuropathological features. These two variants, called “hyper”(Hy) and “drowsy” (Dy), have been extensively studied and characterized, contributing to the understanding of the diversity and complexity of prion strains. Camel prion disease In 2018, camel prion disease (CPD) was identified in three individuals of dromedary camel in Algeria, and one year later, some cases were also reported in Tunisia, increasing concern about a possible epizootic and zoonotic risk that this disease could entail 71,72. It should be noted that in some countries in Africa and the Middle East, camels are an essential source of meat, milk, and leather. Likewise, they are also used for the transportation of both people and goods, and they are used for leisure, such as dromedary races. As a result, camels hold important economic value for the local population. CPD was detected in young and old animals, suggesting both horizontal and vertical transmission. Nonetheless, the origin remains unknown, and a cross-species transmission seems unlikely since neither scrapie nor BSE cases have been reported in these areas 5. General Introduction 19 1.2 HISTORICAL LANDMARKS IN PRION RESEARCH Prion diseases have over 300 years of history. Despite numerous progresses in deciphering the infectious agent, especially in recent decades, many details still need to be unveiled. For centuries, a neurological disorder was known to affect sheep. The earliest written documentation of scrapie date back to 1732 and consisted of a request to the United Kingdom Parliament urging the regulation of the entry of sheep into the country after some farmers reported outbreaks originating from imported livestock. At that time, various causes were attributed to it, from parasites to viruses, thunderstorms or hereditary diseases 24. It was in the 1930s when Cuille and Chelle demonstrated the infectious nature of the disease, transmitting scrapie experimentally to healthy sheep and goats and suggesting that the infectious agent should be a filterable virus73. Simultaneously and serendipitously, Gordon conducted an experiment that supported Cuille and Chelle’s evidence. Between 1931 and 1934, Gordon was developing a vaccine for the looping-ill disease; for this purpose, he inoculated some sheep intracranially with the looping-ill virus. To prepare the vaccines, he obtained the infected animals' brains, spinal cords, and spleens and mixed these tissues with 0.35% formalin to inactivate the virus and subsequently, he immunized sheep with these preparations. Years later, the vaccine was demonstrated to be effective against the looping-ill virus, but, unexpectedly, immunized animals developed scrapie. This fact evidenced that scrapie was transmissible between individuals and, most importantly, the infectious agent was resistant to formalin 74. On the other side of the world, in Papua New Guinea, another neurodegenerative disease was emerging 75. In 1950, Vincent Zigas, an Estonian doctor, moved to Papua New Guinea where he worked as a medical officer. Soon after arriving, he heard about a unique unreported disorder affecting people in the Fore tribe. Along with Carleton Gajdusek, an American physician, thoroughly studied the disease named kuru23. Despite the extensive clinical description and post- NURIA LÓPEZ LORENZO 20 mortem findings, Gajdusek and Zigas did not identify the etiological agent; hence, they proposed that the disease might be hereditary or caused by a toxic sustance. Later, Igor Klazto, a Russian neuropathologist, joined Gajdusek and Zigas in describing the neuropathological findings in the brains of kuru-affected patients76. During those examinations, Klazto noted some similarities with another neurological disorder reported a few years earlier, in 1920 and 1921, by two German neuropathologists, Alfons Maria Jakob and Hans Gerhardt Creutzfeldt 34, who independently reported cases of patients with motor impairments and progressive dementia associated with neuronal degeneration. In 1959, Klazto participated in a convention in London where he presented kuru-affected brain micrographs. It was in this meeting where William Hadlow, a veterinary pathologist, recognized histopathological analogies between kuru and scrapie77. Thus, the intricate puzzle linking kuru, scrapie, and CJD gradually came together. During the following years, significant advances were made in the experimental transmission of these disorders, demonstrating that they consisted of infectious diseases and evidencing the existence of different variants, termed strains, responsible for various clinical and neuropathological features. In this regard, Gajdusek and Gibbs proved the transmission of kuru and CJD to human and non-human primates 25,26,78, Pattison and Millson transmitted scrapie to goats 79, and Chandler transmitted scrapie to mice 80. The demonstration that kuru was an infectious disease led Gajdusek to win the Nobel Prize in 1976. At that point, the most accepted hypothesis was that slow viruses caused spongiform encephalopathies; however, unsuccessful attempts to isolate the infectious agent, its minute size81, and certain resemblances to the protein known to induce allergic encephalomyelitis 82 raised doubts about the viral origin of the scrapie agent. To determine the size of the scrapie agent, Alper and colleagues irradiated samples with ionizing radiation and ultraviolet light. According to their results, they proposed that the scrapie agent may lack nucleic acids given its high resistance to radiation and small size 83. Regarding these findings, a General Introduction 21 mathematician called Griffith proposed the breakthrough idea that proteins could self-replicate, and presented three models based on mathematical equations to explain how proteins could propagate themselves without the involvement of nucleic acids. One of the models explained that a distinct structural version of the same protein could self-template, propagating its conformation to native proteins in thermodynamically feasible ways84. This model laid the foundations of the “protein-only hypothesis”, a controversial theory that proposed that proteins could propagate their conformation. Although this hypothesis went unnoticed at the time, years later it would be the key to understanding the propagation of prions85. In 1980, Cho determined that a protein component was required for scrapie infectivity 86, and in 1982, Prusiner, Bolton, and McKinley extracted a protein from scrapie-infected hamster brains that was not present in healthy animals 87,88. Such protein was highly hydrophobic, presented an elevated resistance to proteolytic digestion with PK, was resistant to inactivation with non-denaturing detergents, and had a molecular weight of 27-30kDa. Based on these findings, Prusiner enunciated the prion concept, which postulated that the causative agent of prion disease lacked nucleic acids and consisted of proteinaceous infectious particles, termed prions89. The identification of the causal agent of prion diseases was the first support for the protein-only hypothesis and opened a new paradigm for comprehending not only prion diseases, but also other neurodegenerative disorders, and challenged the previous understandings of the protein biology. Prusiner would win the Nobel Prize in Physiology or Medicine in 1997 for his enunciation of the prion concept. Later on, the term prion would be used not only to designate the infectious agent of TSE, but also to refer to other self-propagating proteins, such as the fungal and yeast proteins HET-s, or URE3, among others 90. Prion purification led to the identification and cloning of the encoding gene for the prion protein, the PRNP gene. The PRNP gene encodes NURIA LÓPEZ LORENZO 22 both isoforms of the prion protein, the cellular prion protein, and the scrapie isoform 91,92. Additionally, the documentation of the prion protein gene also permitted the identification of prion diseases linked to mutations in this gene 93 or the development of transgenic mice. Almost in parallel with these discoveries, the outbreak of BSE emerged in the United Kingdom 44. Another remarkable finding that resulted from the identification of the PRNP gene was that the ablation of this gene prevented the acquisition of prion diseases. Büeler and Weissmann developed transgenic mice lacking PRNP and demonstrated that these animals did not succumb to prion disease, not even presented a subclinical prion replication; in addition, heterozygous animals with a lower expression of PrP showed longer periods of incubation 94–97. This crucial role of the prion protein in developing prion diseases was the last piece of evidence confirming the protein-only hypothesis. Moreover, the cellular prion protein was established as a potential therapeutic target for treating prion disorders. Once the causal agent and the involvement of the prion protein in prion disease were discovered, efforts shifted toward studying the disparities between the two isoforms and the triggers that led to their conversion. In this regard, Kocisko and colleagues developed the first method of prion propagation in vitro using cell extracts and partially purified prions, called cell-free conversion assay 98; this method was later substituted for more efficient techniques for prion detection or amplification, such as the protein misfolding cyclic amplification (PMCA) 99, real-time quaking-induced conversion (RT-QuIC)100, or protein misfolding shaking amplification (PMSA)101. These techniques have not only contributed greatly to the understanding of the propagation process, but have also been useful in studying species barrier phenomena, as diagnostic tools, and in structural studies. Moreover, assays for therapeutic candidates have also benefited from these methods, which will be described in detail below. General Introduction 23 Figure 1.5 Timeline summarizing the historical hallmarks in the prions field. Image created with BioRender. NURIA LÓPEZ LORENZO 30 tissues, such as iron, copper, and zinc, presenting signs of anemia that evidenced the relation between metal ions and the PrPC 139. Another phenotypic difference between wild-type and PrPC-devoid mice was demyelination of the peripheral nervous system, suggesting that PrPC would be involved in myelin maintenance. Bremer and colleagues confirmed this hypothesis demonstrating that the demyelination could be rescued by triggering neuron-specific PrPC expression 140. This function depends on the interaction between the region of the PrPC comprising amino acids 23-33 and the G-protein coupled receptor Adgrg6 (Gpr126) on the Schwann cell surface. In this regard, the N1 fragment that contains such sequence is released to the extracellular compartment after α-cleavage; subsequently, it binds to the Gpr126 expressed on Schwann cells activating Adgrg6, which via cyclic adenosine monophosphate (cAMP), triggers a downstream signaling cascade that leads to the myelination 141. PrPC is mostly localized in pre-and postsynaptic compartments along axons and in pre-synaptic terminals. This expression pattern suggests that PrPC could also be involved in preserving normal synaptic transmission and plasticity. Moreover, while knockout mice presented weakened inhibitory GABAergic synaptic transmission and reduced long-term potentiation (LTP) in hippocampal cells, animals overexpressing PrPC presented supra physiological synaptic transmission142. Additionally, LTP is related to synaptic plasticity, and, due to memory and learning processes lie in the hippocampus, deficits in synaptic plasticity in this brain area would be related to prionassociated cognitive impairment. The prion protein may also play a role in Alzheimer’s disease and other neurodegenerative diseases. It has been reported that PrPC inhibits βsecretase 1, an enzyme that participates in the synthesis of amyloid-β (Aβ) by cleaving the amyloid precursor protein (APP). According to this hypothesis, the expression of PrPC would be protective against Alzheimer’s disease, while the ablation of PrP expression would increase the formation of Aβ 143. However, studies exploring this theory General Introduction 31 found contradictory results, and while patients of Alzheimer’s disease non-associated to genetic mutations presented a lower expression of PrPC, familial cases of Alzheimer’s disease did not display altered levels of PrPC144. Likewise, PrPC-null mice did not display altered levels of Aβ 145. On the other hand, the direct interaction between the N-terminal domain of the PrPC and Aβ oligomers has also been described 146,147. It has been suggested that PrPC may act by transducing the toxic signaling pathway that triggers the pathological phenotype in neurons. It was suggested that the neurotoxicity would be mediated by the dysregulation of the glutamate receptor mGluR5, which causes dendritic spine loss and long-term depression. Moreover, PrPC, may also be involved in Aβ oligomer-induced tau hyperphosphorylation, which is associated with neurofibrillary tangle formation148. PrPC also binds other amyloids related to neurodegenerative disorders, such as α-synuclein and the scrapie isoform of the prion protein, reinforcing the idea that PrPC could act as receptor for these amyloids triggering a neurotoxic signaling cascade149,150. Since the most notable sign of FFI patients is sleep-wake cycle disruption, the role of PrPC in circadian rhythm was suspected 151. Studies with PrPC-null mice showed that animals exhibited sleep dysregulation; 152,153 this sleep-wake cycle alteration was related to melatonin serum levels 154, and this phenotype was rescued after inducing PrPC expression. This evidence suggested that the FFI phenotype could be related to a loss of function of the PrPC. Moreover, in rat forebrain and suprachiasmatic nucleus, PrP mRNA seemed to be subjected to circadian oscillations155. However, many studies disagree about the causes of the sleep-wake cycle dysregulation, and some authors suggested that it was related to the malfunction of cation channels and receptors such as calcium-dependent potassium channels, voltage-gated calcium channels or NMDA receptors that lead to hyperexcitability. On the other hand, other works suggested that the effect of PrPC on NMDA receptors containing the subunit GluN2D NURIA LÓPEZ LORENZO 32 confered neuroprotection facing synaptotoxicity156, due to the fact that it was shown that PrPC modulates NMDA-mediated excitotoxicity after acute cerebral ischemia in vivo 157. This interaction showed that, while PrPC-deficient mice presented more extended brain lesions after cerebral ischemia, in mice overexpressing PrPC the extent of the damage was minor 158. Figure 1.8 Schematic representation of the functions of the cellular prion protein. Image created with BioRender on biorender.com Apart from the nervous system, PrPC is highly expressed in cells of the immune system, such as mast cells, T-lymphocytes, natural killer cells, General Introduction 33 and macrophages. In vitro experiments showed that activation of mast cells is translated into the secretion of the inflammatory mediators, but also in the release of the N3 fragment, suggesting that PrPC may be involved in inflammatory responses 159. PrPC has also been associated with T lymphocyte differentiation, since PrPC knock-out mice present more T cells with pro-inflammatory phenotype. Another evidence of PrPC role in the immune system was provided by the aforementioned wild-type goats lacking PrPC. These animals presented higher levels of neutrophils in comparison with other animals; however, the implications of this alteration remain unknown 132,133. PrPC has also been related to pain sensibility, and some works demonstrated that the expression of PrPC protects against both inflammatory and neuropathic pain when comparing wild-type and PrPC-null mice. This effect is due to the inhibition of NMDA receptors related to the transmission of pain at the spinal cord level160. While the primary implications of PrPC are typically associated with the proper functioning of the nervous system or neurodegenerative disorders, this protein has also been linked to various biological aspects of cancer development, such as cell proliferation, migration, and chemoresistance161. PrPC has been identified as an upregulated protein in various cancers, indicating that its involvement is attributed to a gain of function. Overall, PrPC is associated with several signaling pathways that activate the transcription of genes involved in cell growth, metabolism (e.g., GLUT1 gene) 162, multidrug resistance proteins (e.g., MDR1)163,164, and cytoskeleton rearrangement, facilitating epithelialto-mesenchymal transition165. Moreover, it has been demonstrated that PrPC directly interacts with doxorubicin, a chemotherapeutic drug, sequestering the molecule through the utilization of its N-terminal tail166. NURIA LÓPEZ LORENZO 34 1.3.2 The scrapie prion protein (PrPSc) The scrapie isoform of the prion protein (PrPSc) is the pathogenic conformer associated with prion disorders. While PrPSc and PrPC have the same primary sequence and post-translational modification, they differ in their secondary, tertiary, and quaternary structures. Although PrPC has been solved by NMR and X-ray crystallography, the conformational change of the PrPSc confers it distinct biological and biochemical properties. Unlike PrPC, this isoform tends to form insoluble aggregates making it impossible to use those structural techniques that require the protein to remain in a soluble state; thus, the elucidation of the structure of PrPSc posed a great challenge for decades. However, after many efforts, the structure of PrPSc has been recently deciphered and it is expected that, in the near future, further research based on these findings will shed light on mechanisms that are still poorly understood, such as the conversion or the pathogenesis. Notwithstanding, it is important to note that this thesis was already in progress when the structure of PrPSc was published and, even though we now know that classical PrPSc consists of a parallel in-register βsheet (PIRIBS), it is crucial to comprehend the previous structural models as well as the analytical pieces of evidence that supported them, in order to fully understand some of the hypotheses and experiments conducted in the present work. 1.3.2.1 Understanding the structure of PrPSc While cryogenic electron microscopy was the technique used to solve the structure of PrPSc, various imaging, biochemical, and spectroscopic techniques were also employed to gather data from ex vivo or synthetic prions. Lowand high-resolution methods were equally crucial in providing valuable insights into the architecture of PrPSc. Electron microscopy was the first technique employed to visualize prion fibrils 104,167. Despite the high tendency to form aggregates, negative stain electron microscopy observations led to perceiving some similarities between PrPSc and the fibrils of Aβ present in the brains of Alzheimer’s disease patients. Some years after these first observations, General Introduction 35 negative stain electron microscopy was also used in independent works to measure the fibrils diameter, suggesting that 10-20nm fibrils may be constituted by two 4-6nm width intertwined protofilaments 168,169. Early structural studies based on FTIR170,171 and CD analyses determined that, unlike PrPC, the secondary structure of PrPSc contained more β-sheet and substantially less α-helices. Nonetheless, a later reinterpretation of this data, revealed that PrPSc is spared of α-helical structures. The misinterpretation was due to the presence of an absorption band in the spectrum at ∼1660 cm−1 attributed to α-helical structures. However, this band was also identified in amyloids lacking any α-helix in their structure. Such band overlaps with the assignment of other structural motifs, such as turn or coils, which are present in amyloid structures 106,107,172 X-ray diffraction experiments importantly contributed to shedding light on the PrPSc structure173. The application of this technique was possible thanks to the improvements in prion purification methods, using phosphostungstate anions to precipitate the protein, and advances in the alignment of fibril preparation. X-ray diffraction determined that the amyloid fibril was composed of the repetition of 1,92nm units along the fibril axis, and most importantly, the absence of equatorial diffraction at 1,0nm was indicative of stacked β-sheet amyloid structures169. Altogether, these findings suggested that the PrPSc structure may be a β-helical architecture, such as a β-solenoid174. Also based on X-ray technology, synchrotron-based small-angle X-ray scattering (SAXS) was applied to describe prion features. The results obtained by this method corroborated previous findings indicating that the fibril had a diameter of ~11nm. The sizes of the diameter and the units of repetition were compatible with two intertwined protofilaments consisting of four β-strands in a β-solenoid configuration. This hypothesis was supported by solid-state nuclear magnetic resonance (ssNMR) spectroscopic analyses of a fungal prion, HET-s, which structure was a two-rungs βsolenoid175,176. NURIA LÓPEZ LORENZO 36 Another imaging technique employed to visualize PrPSc was atomic force microscopy (ATM). This approach permits the visualization of different surfaces or biomolecules immobilized on different materials providing a 3D image. Using ATM, PrPSc was observed on the surface of neuroblastoma cells persistently infected with murine prions177. Moreover, it was also used to compare and estimate the height and helical periodicity of purified or synthetic prions178. One of the main biochemical differences between PrPC and PrPSc is the partial resistance of PrPSc to proteolytic degradation with PK. This proteinase cleaves unstructured and flexible regions of the PrPSc, whereas β-strand and inaccessible motifs would remain intact. PKresistant fragments of hamster-adapted prion strains, 263K, Dy179, and GPI-anchorless RML180, were analyzed by mass spectrometry to identify the PK cleavage sites. Overall, the analyses of these three strains agreed on the high resistance of the C-terminal region to proteolytic degradation 181,182. Moreover, minor differences in the cleavage sites were reported. Limited proteolysis analyses of samples from CJD patients agreed with these results reporting similar cleavage sites; further, N-terminal truncated PK-resistant fragments were also described 183. Altogether, these data allowed designing a preliminary map of the β-strands of the PrPSc 184. Hydrogen/deuterium exchange mass spectrometry provides structural information based on the accessibility of different structural motifs to the solvent; those flexible secondary structures present rapid hydrogen for deuterium exchange, whereas the exchange is moderately or largely slower when the structure consists of α-helices and β-sheets, respectively. In this regard, hydrogen/deuterium exchange analyses determined that the region comprising amino acids 90-224 presented low exchange rates, suggesting that this region has a high content of βstrands connected by turns or loops 185,186. ssNMR is a spectroscopic method employed to determine the chemical structure, three-dimensional arrangement, and dynamic properties of General Introduction 37 solids or semi-solids. It has been successfully employed to solve the architecture of amyloids such as the fungal prion, HET-s, which was a β-solenoid configuration 175, or Aβ, which unlike HET-s, presents a parallel in-register β-sheet conformation 187,188. Structural characterization of prions using ssNMR had been restricted due to the limited amount obtained from in vitro amplification techniques; nonetheless, the development of the protein misfolding shaking amplification (PMSA), a technique that allowed obtaining a large amount of recombinant prions isotopically labeled needed for ssNMR analysis 101,189. Although the prion structure could not be solved by this method, measurements of distances between amino acids typically located in β strand were compatible with a PIRIBS architecture. The application of cryo-EM technology has revolutionized structural studies in the prion field representing a major milestone. In recent years cryo-EM technology improved so much that the images obtained reached a near-atomic resolution 190. Moreover, the structural model supported by this technique also changed with the progressive improvement in the cryo-EM resolution. Vazquez-Fernández and colleagues employed Cryo-EM to examine GPI-anchorless RML prions directly, in the absence of any staining that could introduce possible artifacts and provided consistent measurements of the prion fibrils. The helical symmetry within the PrPSc fibrils made it possible to perform 3D reconstruction and analyses of the individual fibrils. These determinations indicated that each PrPSc particle had an average height of 1,8nm, which coincided with the expected height of a four-rung β-solenoid monomer, 1,92nm (4 rungs x 0,48nm) 191; these measurements were also consistent with single particle averaging, an alternative processing approach that classifies and averages hundreds of images of fibril sections. Moreover, volumetric analyses supported the idea that fibrils were composed of two intertwined protofilaments, which agreed with previous findings by X-ray diffraction 169. Unfortunately, this attempt to solve the prion NURIA LÓPEZ LORENZO 38 structure did not obtain a sufficient resolution to achieve an all-atom reconstruction. Figure 1.9. Cryo-EM data obtained from ex vivo GPI-anchorless RML prions. Improvements in purification, resolution, and cryo-EM data collection were translated into a different interpretation regarding the structure of the same prion strain. Comparison of the data collected by Vázquez-Fernández et al., (A-C) and Kraus et al., (D-F). A and D correspond to 2D class averages; B and E consist of the lateral view of the 3D reconstruction; and C and F correspond to the cross-section projection of the density map. Adapted from Vázquez-Fernández et al 2016 191 (open access) and Kraus et al 2021 192. General Introduction 39 The definitive confirmation that bona fide brain-derived prions have a PIRIBS architecture was the fruit of the work of Kraus and colleagues, who solved the hamster-adapted 263K prion strain by cryo-EM with a 3,1Å resolution 192. Soon after these findings, more ex vivo prion strains were solved, reinforcing Kraus’s results 190,193,194. Thus, ex vivo classical prions, such as 263K, RML, 22L, or M7, consist of left-handed helical twist single-protofilament. PrPSc monomers display a parallel-in register β-sheet architecture and present two lobes, an N-terminal and a C-terminal lobe which are connected by continuous electron density, refusing the idea that both halves belong to independent protofilaments. The fibril core of 263K comprises amino acids ~95-227 and is ~3,5nm in its narrowest dimension, whereas it is ~13nm in its longest. 1.3.2.2 PrPSc models Based on the pieces of evidence provided by all the aforementioned structural methods, at the beginning of the present thesis, two possible conformational models were competing: the 4-Rung β-Solenoid (4RβS) and the Parallel In-Register Intermolecular β-Sheet (PIRIBS). 4-Rung β-Solenoid (4RβS) Overall, the β-solenoid configuration consists of elongated fibrils comprising repetitive arrangements of β-strands connected by loops resulting in a helical shape. Coils are made up of linear β-strands joined with tight turns, termed β-arcs, or loops around the axis of the solenoid. This architecture creates a stable and rigid structure in which stacked coils are coordinated by hydrogen bonds contributing to strands of different coils forming parallel β‐sheets. The β-solenoid inter-coil distance is well established and consists of the distance between hydrogen-bonded β‐strands, 4.8±0.2Å. β-solenoids have the distinctive property of having a clear boundary between the interior side chains, that compose the hydrophobic core, and the side chains on the surfaces exposed to solvent. This boundary is formed by a tightly packed layer of polypeptide backbones linked by hydrogen bonds, which wraps around the hydrophobic core 195. NURIA LÓPEZ LORENZO 46 To summarize, the study of prions represented, and still represents an amazing challenge to the scientific community. The concept of prion has expanded the traditional concept of infectious agents by demonstrating that misfolded proteins can also be transmissible among individuals. In addition, although transmissible spongiform encephalopathies are rare diseases, their contemplation can also have a significant impact on advancing other neurodegenerative diseases, such as Alzheimer's and Parkinson’s, which share common features with prion diseases, such as protein misfolding and the formation of toxic aggregates in the brain. Finally, in view of the common features between prions and proteins involved in other neurodegenerative disorders, by better understanding prions, it is possible to gain valuable insights for the development of therapies and therapeutic approaches for these more common diseases that currently lack a cure. 2. OBJECTIVES Objectives 49 The present thesis is organized into three chapters, two of which are dedicated to the assessment of three therapeutic candidates for prion diseases. The third chapter tackles the study of the biochemical and biological properties of different recombinant prion strains. Thus, the objectives of this work were classified according to the chapter they belong. Prion diseases are rare and fatal neurodegenerative disorders for which there is no treatment. The global objective of the first chapter is to evaluate two therapeutic strategies in a fast murine model of prion disease; these pharmacological approaches tackle prion diseases from two different perspectives, targeting PrPC and PrPSc. The global goal of the first chapter has been broken up into two specific objectives according to the therapeutic compound employed in each approach: 1. Evaluation of the chronic administration of a tetrapyrrole, Fe(III)-TMPyP, as a treatment against prion diseases. 2. Evaluation of the single-chain fragment variable, scFv-G1, as a chronic treatment for prion diseases. Similarly, the global objective of the second chapter was to assess the therapeutic potential of a PrP amyloid in a fast murine model of prion disease. However, in view of the results that arose from the first experiments performed, new objectives were incorporated: 1. Evaluation of the prophylactic administration of a recombinant PrP amyloid as a treatment of prion diseases. 2. Serial transmission of the infectious PrP amyloid. 3. Biochemical and biological characterization of the infectious PrP amyloid. NURIA LÓPEZ LORENZO 50 Due to the involvement of diversity of prions strains in the capacity to cause diseases with different phenotypes and their implication in the development of resistances to pharmacological treatments, the characterization of different prion strains could shed light on the mechanisms associated to therapeutical failure. Hence, the main objective of the third chapter is to generate de novo different PrP conformers by PMSA in the presence of different polysaccharide cofactors. This global objective has been broken up into the following specific aims: 4. Determination of the biological, biochemical, and biophysical properties of the recombinant conformers. 5. Comparison of the properties of the generated conformers to determine if they exhibit different features. 6. Assessment of the infectious potential of the generated conformers to determine if they exhibit characteristics of bona fide prions. 7. Comparison of the properties of the conformers in vivo to determine if they consist of different prion strains. 8. Comparison of the effect of the different cofactors on the generation of the conformers. 3. CHAPTER I: THERAPEUTICAL POTENTIAL OF A PORPHYRIN AND A scFv ANTIBODY FOR PRION DISEASES Chapter I: Introduction 53 3.1 INTRODUCTION Prion diseases or transmissible spongiform encephalopathies are fatal neuropathological disorders for which there is no cure. Three decades ago, it was discovered that prions cause transmissible spongiform encephalopathies; however, the efforts to find a treatment that could extend survival or even ameliorate the symptoms of those patients suffering from these conditions failed. Many attempts have been made in order to find an effective treatment against prion diseases. Nonetheless, there are critical aspects still poorly understood, completely unknown, or which have been recently discovered that could be contemplated as possible therapeutic targets, such as the PrPC function, the pathogenesis, the mechanism of conversion of PrPC into PrPSc and the PrPSc structure110. A large variety of drugs have been tested in vitro, in cellula or in vivo 215–220: from small compounds commonly used as a treatment for other disorders to large and complex molecules, immunotherapie, or gene therapies specially designed for these diseases. However, only six treatments have been clinically tested: amantadine, flupirtine, pentosan polysulfate, quinacrine, doxycycline, and PRN100 221–228, unfortunately, all of them with little or no improvement over controls. The search for a prion treatment was tackled from different points of view regarding the target molecule (Fig 2.1). Most of the approaches were based on the PrPC because it is the best-known molecule involved in prion disorders and its essential role in prion pathogenesis as the substrate for PrPSc propagation; however, other key points of the prion biology have also been explored, tackling TSE from the expression of NURIA LÓPEZ LORENZO 54 the PrP, to the conversion of PrPC into PrPSc or the toxicity derived from the conversion, among others. Targeting the PrP expression PrPC is essential for the development of prion diseases. This fact was demonstrated in the early ’90s when the first PRNP knock-out mice revealed to be resistant to prion diseases96,207. Moreover, heterozygous and knock-down animals had an extended incubation time compared to wild-type animals, which means that even a partial suppression of PrP expression could have beneficial effects96,229. Hence, reducing the PrP expression is currently the most promising approach to finding a therapy for prion diseases. Although this approach would be beneficial for all patients, it would be especially advantageous for carriers of genetic prion diseases, since a complete the suppression of the expression of the PrP would permit them to evade the disease, even after the onset. In this regard, two strategies based on decreasing mRNA have been assessed: small interference RNA (siRNA) and antisense oligonucleotides (ASOs). RNA interference (RNAi) is a noncoding RNA naturally synthesized as a conserved response against double-stranded RNA, generally from a viral origin. Its function is to regulate gene expression in a sequencespecific manner230. This biological mechanism could be used from a therapeutic perspective by designing an RNAi targeting PrP mRNA. Two varieties of RNAi share the exact mechanism of action, although they differ in the way they are synthesized: small interfering RNA (siRNA) and microRNA (miRNA) (also called short hairpin RNA, shRNA, if its origin is synthetic). SiRNA was successfully tested in chronically infected cells showing PrPSc inhibition231. However, its inability to cross the blood-brain barrier was evidenced in the first attempt to use it in infected animals 232. This handicap was overcome by binding the siRNA to the rabies Chapter I: Introduction 55 virus glycoprotein (RVG), which uses the nicotinic acetylcholine receptor (AchR) to enter into the neurons overcoming the BBB233, or encapsulating the siRNA in liposomes-siRNA-peptide complexes. This last strategy showed an increase in the survival of mice of up to threefold in comparison with the control group234,235. Figure 2.1. Scheme of therapeutic approaches for prion diseases. 1. Reducing the expression level of PrP. 2. Targeting PrPC. 3. Targeting PrP trafficking. 4. Impeding PrPSc propagation 5. Increasing the PrPSc clearance. 6. Increasing the degradation of misfolded proteins. 7. Interrupting the cell signaling cascade that triggers the toxicity. Picture created with BioRender.com On the other hand, shRNA encoded in adeno-associated virus (AAV) vector type 2 was also tested in vivo by thalamic infusion. However, NURIA LÓPEZ LORENZO 62 generate antibodies against PrP, denoting that misfolded PrP can promote some sort of immune response294,295. To explore this concept, bacterially expressed C-terminal (211-230) or PrP peptides from the central region (131-150) were inoculated in wild-type mice and demonstrated to trigger an important immune response in these animals. Moreover, antibodies generated by the host did not recognize the native PrPC, which means that such epitopes could be hidden in the mature PrPC after post-translational modifications296–298. This immunoreactivity was also obtained by inoculating truncated, modified, crosslinked PrP peptides or even heterologous PrP. However, the effect on the survival of prion-infected animals was minimal299–303. The use of adjuvants such as alum or Freund’s adjuvant304–306, but specially CpG-oligodeoxynucleotides (CpG) also demonstrated to boost the immune response and break the self-tolerance307–309. The efforts mentioned above were attempts to overcome self-tolerance, enhance the immunogenicity of PrP peptides and promote an immune response against PrPC; nevertheless, PrPSc and its structural difference regarding PrPC would be the ideal target of active immunization. Some attempts were focused on identifying epitopes that only were available in PrPSc. However, the PrPSc structure was not available at that moment, and the selected epitopes had not a preventive effect or even accelerated prion disease310–313. On the other hand, passive immunization is based on the use of PrPC or PrPSc-specific antibodies. This promising approach would offer a complete abolition of PrP propagation by three mechanisms: stabilizing PrPC, sterically impeding the interaction between PrPC and PrPSc, and stabilizing PrPSc. In this regard, knock-out mice entailed a critical tool to develop a large variety of antibodies targeting PrP epitopes which were tested in prion-infected animals to assess their specificity and capability to delay the onset of prion diseases314,315. Although several antibodies demonstrated to decrease prion pathology, their efficacy essentially depends on the epitope, and targeting the Chapter I: Introduction 63 wrong epitope could trigger neurotoxicity, such as nerve cell loss, gliosis, and microglial activation. Thus, antibodies targeting the Nterminal flexible tail resulted in neuroprotective, while antibodies targeting the globular domain, especially helices α1 and α3, are neurotoxic. It was hypothesized that the toxicity of such antibodies was mediated by the PrP N-terminal tail and activated the same toxic pathways to PrPSc, including PERK, activating calpains and promoting the production of reactive oxygen species316. Apart from ASOs, currently, the most promising treatments against prion diseases are therapeutic antibodies. In vivo, the most encouraging results were obtained by intraperitoneal treatment with ICSM18 (IgG1 recognizing PrP 146–159) or ICSM35 (IgG2 targeting PrP 91-110) after inoculation. These mice accumulated much less PrPSc in the spleen and brain and lived more than 500 days with no clinical signs; thus, they were postulated for clinical trials317. Nonetheless, independent studies showed controversial results regarding the safety of both antibodies: one of them reported no-drug-related toxicity, whereas the other one demonstrated dose-dependent neurotoxic effects 215,317–319. Recently, in 2018 the first-in-human treatment with a therapeutic antibody for prion diseases was conducted. The procedure enrolled 6 patients diagnosed with CJD, 5 with sCJD, and 1 with iCJD, and untreated historical controls. This study was conducted under a UK Special Licence instead of a clinical trial; the UK Special Licence allows treating individual patients with an unlicensed drug if the clinical condition cannot be treated with a licensed product on the market, such as prion diseases. The antibody, PRN100, is a humanized monoclonal antibody derived from ICSM18 (IgG4κ) and was administered intravenously. The number of patients was too small to draw conclusions about the therapeutic properties of PRN100. However, it permitted monitoring of the concentrations of the antibody in serum and CSF and to assess the safety. This study allowed to conclude that the intravenous treatment with PRN100 can cross the BBB and reach the expected therapeutic concentrations in CSF. Moreover, the absence of NURIA LÓPEZ LORENZO 64 side effects and the results of the autopsy showed that the treatment did not induce neurotoxicity, demonstrating that the treatment is safe 228. Limitations in prion disease therapies Despite years of effort looking for a treatment for prion diseases, these disorders are still uncurable. Researchers found many obstacles to overcome, some of them physiological, others derived from the pathology, and others due to the wrong designs of the experiments that were fruitless in drawing conclusions. One of the main issues is that a large percentage of the chemical compound tested for prion diseases does not cross the BBB216,243,251,254,320. In animals, this handicap is overcome by injecting the treatment directly into the brain; however, this approach can hardly be translated to humans. An alternative is an intrathecal administration or the use of osmotic pumps, which connect an intraventricular catheter to allow a continuous administration of the treatment into the brain; these pumps have a homologous device for use in humans, called the Ommaya reservoir. All these approaches carry risks like infections, partial loss of brain function, or subdural hematoma, among others321,322. On the other hand, when PrPSc-targeting compounds were tested, it was discovered that some molecules were only effective against specific prion strains. This is the case of cpd-B or some tetracyclic antibiotics323,324. Although PrPSc is the same molecule, there are some architectural differences between prion strains that make them susceptible or not to be inactivated by different drugs, called strain specificity325. Also related to prion strains, another difficulty that limits the treatment with some promising drugs is the emergence of drug resistance326,327. This disappointing effect was seen after the treatment with quinacrine or IND24 and could be due to two reasons: 1) the presence of two different strains in the same host and the selection of the resistant one after the treatment or 2) the adaptation of the prion strain to the treatment325. The combination of different treatments was Chapter I: Introduction 65 proposed as a strategy to evade this obstacle; nevertheless, the combination of IND24 and Anle138b also concluded with the acquisition of drug resistance328. Finally, regarding clinical trials, many factors difficult obtaining statistically significant data. The low incidence, the rapid progression, the difficulties in obtaining an accurate diagnosis in the early stages, and the paperwork involving a clinical trial contribute to a small number of participants enrolled and patients in an advanced stage of the disease, who sometimes died before receiving the treatment. Moreover, the diversity of prion diseases regarding the origin, polymorphisms, mutations, and age at the onset entail a high heterogeneity. Finally, the difference in clinical trial design, sample collection, and the lack of standardized outcome measures make the comparison of different studies and meta-analyses a challenge215,218,329. Palliative cares The absence of therapies for curing prion diseases limits the treatments to palliative care to ameliorate the condition. Most patients suffer from psychological and psychiatric symptoms, such as depression, anxiety, psychosis, and hallucinations, but also motor disorders like myoclonic jerking, rigidity, or akinesia, which require pharmacological treatments such as anxiolytics, antipsychotics, or neuroleptics. However, nonpharmacological assistance is also essential to maintain motor capabilities as long as possible; for example, a physical or speech therapist to alleviate swallowing difficulties and avoid choking and suffocation risks330,331 Prion diseases are rare fatal disorders with rapid progression. The diagnosis supposes to be a severe blow, not only for patients but for relatives as well; for this reason, psychological support is fundamental to deal with the situation. NURIA LÓPEZ LORENZO 66 Given the null therapeutic alternatives for prion, we aimed to contribute to the search for candidates to treat these disorders. Thus, this chapter describes the evaluation of two therapeutic candidates, a molecular chaperone and a conformational scFv antibody. One of the possible therapeutical strategies against TSE is PrPC stabilization by molecular chaperones, which avoid the conversion of PrPC into PrPSc. Certain cyclic tetrapyrroles, such as porphyrins, were demonstrated to bind to PrPC, inhibiting prion propagation in cellula and animal models332. Fe(III)-TMPyP is a cationic tetrapyrrole that binds to the structured domain of huPrP, interacting with the C-terminal region of helix α3 and the first β-strand, (Fig 2.2) and acts as a pharmacological chaperone preserving its native conformation and preventing it from misfolding. Fe(III)-TMPyP was demonstrated to inhibit prion replication in vitro333 and in cellula, against different prion strains334–336. Based on the recognized activity of Fe(III)-TMPyP, we selected it as a potential candidate for the chronic treatment of prion disorders. Figure 2.2. Fe(III)-TMPYP and its interaction with PrPC. A) Chemical structure of Fe(III)-TMPyP. B) Cartoon image of huPrP (PDB 1QLX) and Fe(III)-TMPyP (PubChem 126456051). Fe(III)-TMPyP (purple) interacts with the C-terminal region of helix α3 and the first β-strand (light blue) of human PrP91-231(gray). C) Surface model of huPrP (gray) and Fe(III)-TMPyP (purple) interaction. Light blue areas of huPrP indicate the site of interaction. Chapter I: Introduction 67 The second candidate was based on a passive immunization with a scFv antibody. This scFv antibody was obtained from an IgG isolated from mice immunized with a vaccine based on the 4RβS structural model, but which recognized PrPSc from brain homogenates. Given the significant interest in passive immunization for diseases like Alzheimer's, exemplified by the approval of aducanumab and lecanemab, we considered this approach based on passive immunization could also be useful in treating prion diseases. To assess the potential anti-prion activity of these two candidates, we conducted experiments using a fast murine model of prion disease. This murine model involved transgenic mice bearing the bank vole (109I) prion protein (TgVole). Such model was selected based on the high susceptibility of bank voles to developing prion disease within a relatively short timeframe. Thus, we considered these TgVole mice as an appropriate model for evaluating the proof-of-concept of in vivo antiprion activity. Chapter I: Specific aims 69 3.2 SPECIFIC AIMS Prion diseases are rare and fatal neurodegenerative disorders for which there is no treatment. The global objective of this chapter is to evaluate two therapeutic strategies in a fast murine model of prion disease. These pharmacological approaches tackle prion diseases from two different perspectives, targeting PrPC and PrPSc. This global goal has been broken up into two specific objectives according to the therapeutic compound employed in each approach: 1. Evaluation of the chronic administration of a tetrapyrrole, Fe(III)-TMPyP, as a treatment against prion diseases. 2. Evaluation of the single-chain fragment variable, scFv-G1, as a chronic treatment for prion diseases. Chapter I: Methods 71 3.3 METHODS Reagents, materials, buffers composition, and the equipment used in this doctoral thesis are collected in Annexes I, II, III, IV, and V. 3.3.1 ETHICAL STATEMENT All described experiments were in compliance with the guidelines included in the current Spanish regulations “Real Decreto 118/2021 de 23 de Febrero” which modifies the “Real Decreto 53/2013 de 1 de Febrero” on the protection of animals used for experimental and other scientific purposes. Both are based on the European Directive 2010/63/EU on Laboratory Animal Protection. All experiments described were performed under animal experiment permit 15005/16/006, approved by the Ethical Committee of the University of Santiago de Compostela and the Consellería del Medio Rural of the Xunta de Galicia. The procedures were carried out at Centro de Biomedicina Experimental (CEBEGA) de la Universidad de Santiago de Compostela, registered in the Spanish Register of breeding, supplier and user centers with the number ES150780292901. 3.3.2 ANIMAL MODEL AND HOUSING CONDITIONS Bank voles (Myodes glareolus) are wild rodents used in prion research. Their experimental use in the prion field was first reported in the 70’s decade, in a test with different rodents; in this study, bank voles (BV) were the fastest animal model in developing prion disease337. Later experiments demonstrated that BVs were also prone to propagate different prion strains, even from other species, developing prion NURIA LÓPEZ LORENZO 78 350. Briefly, fourteen different brain areas were examined (Fig 2.4): piriform cortex (Pfc), hippocampus (H), occipital cortex (Oc), temporal cortex (Tc), parietal cortex (Pc), frontal cortex (Fc), striatum (S), thalamus (T), hypothalamus (HT), mesencephalon (M), medulla oblongata (Mobl), cerebellar nuclei (Cm), cerebellar vermis (Cv) and cerebellar cortex (Cc), and the spongiform lesion for each area was scored from (0) absence of spongiosis, (1) mild, (2) moderate, (3) intense and (4) maximum level of lesion. Each area was investigated globally as region for the scoring. Brain lesion profiles were plotted considering the anatomical regions and the mean of the semiquantitative score for each area. Error bars represent the standard error of the mean 350. Figure 2.4. Brain sections for histopathological analysis. Mice brains were cut in four sections (A, B, C and D) and the following brain areas were examined: A) frontal cortex (Fc), in light blue; striatum (S), in dark blue; B) parietal cortex (Pc), in light blue; temporal cortex (Tc), in dark blue; piriform cortex (Pfc), in dark purple; hippocampus (H),in turquois; thalamus (T) in light lilac; hypothalamus (HT), in pink; C) occipital cortex (Oc), in light blue; mesencephalon (M), in dark blue; D) cerebellar cortex (Cc), in light bue; cerebellar nuclei (Cm), in dark purple; cerebellar vermis (Cv), in dark blue and medulla oblongata (Mobl), in light lilac. Chapter I: Methods 79 3.3.9 KINETICS OF FE(III)-TMPYP DISTRIBUTION IN BRAIN A group of 16 mice was anesthetized with Avertin, as previously described. An incision was performed to expose the skull and Fe(III)- TMPyP was injected intracerebrally approximately 1mm lateral to the bregma and 3mm deep perpendicularly to the skull surface in order to reach the lateral ventricle. Mice were euthanized immediately after the injection or after 10min, 30min, 1h, 2h, 3h, or 24h. Two mice received saline buffer instead of Fe(III)-TMPyP as negative controls and were euthanized immediately after the administration. Mice were euthanized at different time points, and their brains were collected and immersed in 4% paraformaldehyde in PBS for 24h at 4°C. Subsequently, brains were cryopreserved and immersed in 30% sucrose at room temperature. Samples were frozen at -20°C, and caudal sections at 30µm thickness were obtained using a cryostat. The cryosections were stained with 4 ',6-diamidino-2-fenilindol (DAPI), and slides were visualized using a microscope of fluorescence Leica DM4 B, with the 10X objective. Images were then analyzed with LAS X software. 3.3.10 STATISTICAL ANALYSIS Statistical analyses were performed using Graph Pad Prism 8. Descriptive statistics such as mean, standard deviation, or the standard error of the mean were calculated for each group of mice. Survival time was expressed as mean±SEM. Kolmogorov–Smirnov test for individual datasets was tested for normality. Student’s t-test was applied to all datasets with two tails, according to the result of the Kolmogorov–Smirnov test. The graphical representation of survival time was plotted as the percentage of survival and was analyzed by the Kaplan-Meier test. The results were contrasted by the log-rank (Mantel-Cox) test, and a ρ value≤0.05 was considered statistically significant. NURIA LÓPEZ LORENZO 80 All groups of mice were tested for outliers by Grubbs’ test with α=0.05. Identified outliers were removed. A one-way ANOVA followed by post hoc tests was also performed to assess the difference between treated and control groups. A ρ value≤0.05 was considered statistically significant. Chapter I: Results 81 3.4 RESULTS 3.4.1 ASSESSMENT OF THE ANTI-PRION ACTIVITY OF FE(III)-TMPYP IN A FAST MURINE MODEL OF PRION DISEASE The broad knowledge about the mechanism of action of Fe(III)-TMPyP and its previous encouraging results led us to test a chronic administration in a fast murine model of prion disease. This study was a proof of concept with a view to assessing it in a human model of prion disease in case of promising results were achieved. A total of 28 mice were inoculated intracerebrally with 20μL of 1% brain homogenate from a terminally sick mouse inoculated with a voleadapted CWD strain. Two weeks after inoculation, mice were divided into two groups: one group of mice was treated intracerebrally with 20μL 0.5mM Fe(III)-TMPyP, and the other group, a saline control group, received 20μL of a saline buffer. After the treatment, mice were observed in order to identify possible side effects due to the intracranial administration of Fe(III)-TMPyP. Some animals displayed slight pruritus at the site of inoculation minutes after administration. This local irritation lasted a few hours after the treatment. The treatment was repeated weekly for 5 weeks. At the end of the treatment, 7 out of the 28 mice had died because of the anesthesia, the procedure, or intercurrent diseases; 3 animals belonged to the treated group, while 4 mice belonged to the saline group. The attack rate for both groups was 100%, and all the mice presented neurological signs such as ataxic gait, head bobbing, and hyperactivity. The mean survival time of mice treated with Fe(III)-TMPyP was 71,2 ± 1,2dpi, while the saline group had an average survival time of 71,0 ± 2,1dpi; the log-rank statistical analysis demonstrated that there were no statistically significant differences between both groups indicating that NURIA LÓPEZ LORENZO 82 Fe(III)-TMPyP did not delay the onset of clinical signs neither prolonged the survival time of mice inoculated with a vole-adapted CWD strain (Fig 2.5). Figure 2.5. Chronic treatment with Fe(III)-TMPyP did not prolong the survival of prion-infected mice. A) Two groups of mice were inoculated with 20μL of 1% brain homogenate from a terminally ill mouse inoculated with vole-adapted CWD. All the mice succumbed to prion disease, and the mean incubation time was calculated for both groups. Log-rank test showed no statistically significant differences between the group of mice that received Fe(III)-TMPyP and the saline group (p=0.4970). Asterisk (*) indicates that one or more mice died during the procedure or due to intercurrent diseases; these mice were not included in the statistical analysis or the Kaplan-Meier curve. B) Time course of the treatment. Two weeks after inoculation, mice were treated intracerebrally with Fe(III)-TMPyP or saline. The treatment was repeated weekly for 4 weeks. C) Survival curve of mice treated with Fe(III)-TMPyP or saline. The survival endpoint of animals treated with Fe(III)-TMPyP (light purple)(n=11) or saline (dark blue)(10) was plotted on a Kaplan-Meier survival curve. Chapter I: Results 83 Proteinase K digestion and Western blot analysis performed with frozen brains demonstrated that both groups accumulated PrPSc. Unexpectedly, densitometric and statistical analysis of Western blots showed that the amount of PrPSc in the brains of mice treated with Fe(III)-TMPyP was lower in comparison with non-treated animals (ρ=0,0246), displaying a reduction of the PrPSc accumulation of 49%, while there was no statistically significant difference in the amount of total PrP (ρ=0,6773) (Fig 2.6). Figure 2.6. Chronic treatment with Fe(III)-TMPyP did not modify the pathology compared to the saline group A) The accumulation of PK-resistant material was NURIA LÓPEZ LORENZO 84 assessed by Western blot using SAF83 antibody (1:400) (epitope 130-160). B) Western blot of total PrP and tubulin of treated and control mice. Undigested samples from brain homogenates were subjected to Western blot analysis to assess the total amount of PrP and the loading control, β-tubulin (1:1000). C) Densitometric analysis of total PrP (circle) and PrPSc (triangle) normalized by the loading control showed statistically significant less amount of PrPSc (ρ=0,0246) in brains of mice treated chronically with Fe(III)-TMPyP(light purple) in comparison with control mice (dark blue), whereas the total PrP of mice treated with Fe(III)-TMPyP (light purple) and control mice (dark blue) is essentially the same (ρ=0,6773). Error bars represent the SD and asterisk (*) indicates statistical significance. D) The brain lesion profile of mice traeated with Fe(III)-TMPyP (light purple line) (n=6) or saline buffer (dark blue line) (n=7) represents the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions against the following brain regions: Pfc: piriform cortex, H: hippocampus, Oc: occipital cortex, Tc: temporal cortex, Pc: parietal cortex, Fc: frontal cortex, S: striatum, T: thalamus, HT: hypothalamus, M: mesencephalon, Mob: medulla oblongata, Cm: cerebellar nuclei, Cv: cerebellar vermis, Cc: cerebellar cortex. The spongiosis score was essentially similar for both groups that presented moderate vacuolation in the thalamus, and slight affectation of the striatum, mesencephalon and medulla oblongata. Error bars represent the SEM and hash (#) indicates that the pointed-out brain area could not be examined in all the samples. To determine the effect of the treatment on the microscopic pathology, fixed brains were stained with hematoxylin and eosin. Histopathological examination of the brains of both groups showed a characteristic spongiform lesion profile of prion disease. This pattern was essentially the same for both groups demonstrating that the chronic treatment with Fe(III)-TMPyP did not modify the pathology in comparison with the saline group. The spongiosis score determined moderate vacuolation in the thalamus and mild affectation of the basal ganglia, concretely the corpus striatum, in addition to the mesencephalon and medulla oblongata. Immunohistochemical staining using the monoclonal antibody 6C2 did not successfully detect PKresistant PrP (PrPres) deposits in the brains of both animal groups. 3.4.2 SCFV-G1 ANTIBODY AS A THERAPEUTIC STRATEGY FOR PRION DISEASES Passive immunization is one of the few therapeutic strategies that has achieved successful enough results in vivo to reach clinical trials, not only for prion diseases228 but also for other neurodegenerative diseases Chapter I: Results 85 such as Alzheimer’s disease351, Parkinson’s disease292, Huntington disease293 or ALS352. Based on the structural differences among the PrPC and the 4RβS model proposed for the PrPSc structure353, Holger Wille and colleagues from the University of Alberta designed a structure-based vaccine for prion diseases. For this purpose, they used the fungal prion HET-s as the scaffold to harbor the epitope based on specific amino acids displayed on the surface of the 4RβS. HET-s has a two-rung β-solenoid architecture 354 ; thus, to simulate the 4RβS structure and bear the epitope, two molecules of HET-s were linked (HET-2s) and mutated strategically to replace certain amino acids for others present in the prion protein (353)355,356. This molecule was bacterially expressed and fibrillated, assessing that the fibrils morphology was similar to the native HET-s fibrils. The epitope of the vaccine was composed of two clusters of amino acids located in the intrinsically disordered region and the helix α1, respectively, Lys 105, Ans 107, Lys109, and His110, and Asp143, Glu145, and Asp146 (mouse sequence), in the PrPC conformation. These two clusters of amino acids are separated by a distance of 35Å in the native conformation, while in the model of the 4RβS, both groups of amino acids are located in the first and second rung, one on top of the other, specifically separated by a distance of 4,8 Å, which is the distance between hydrogen-bonded β-strands. In contrast, in the PIRIBS architecture, the distance that separates both clusters of amino acids is approximately 27Å (measured with ChimeraX). Against all odds, Wille and collaborators showed that mice immunized with this vaccine developed antibodies that specifically recognized PrPSc present in brain homogenates from mice inoculated with different prion strains357. Furthermore, TgP101L mice immunized with this vaccine, a mouse model that naturally develops a GSS syndrome, experimented an extension of the survival of 250% in comparison with NURIA LÓPEZ LORENZO 86 unimmunized animals or animals that received the HET-2s scaffold without the PrP epitope (Fig 2.7) 357. Figure 2.7 Epitope locations in different PrP architectures. The epitope, Lys105, Asn107, His110, and Lys109 are in light red, Asp143, Glu145, and Asp146 are in dark red. A) HET-s (PDB 2KJ3) dimer with the epitope in red. The HET-s sequence was mutated to link two monomers and display the epitope on its surface. HET-s βsolenoid architecture is similar to the 4RβS model and served as a scaffold to harbor the epitope354. B) 4RβS model with the epitope in red. The 4RβS was proposed as possible architecture of PrPSc; in this conformation, a monomer or PrP is organized in 4 rungs, and the epitope is comprised of amino acids located in the first and second rungs196. C) Structure of mouse PrPC obtained from AlphaFold (UniProt P04925) with the amino acids involved in the antibody epitope in red358; one part of the epitope is located in the intrinsically unstructured domain (light red), while the other amino acids are in the helix α 1 (dark red)359 D) PIRIBS architecture with the epitope in red 110. In contrast with the 4RβS model, in the PIRIBS conformation (PDB 7LNA) the amino acids that comprise the epitope are separated by a distance of 27Å approximately. Image created with ChimeraX(359) Chapter I: Results 87 A monoclonal IgG antibody (YEG mAb G1) was selected and engineered to be expressed in E.coli as humanized recombinant single chain variable region antibody (scFv-G1). In this thesis, the anti-prion activity of scFv-G1 was assessed in TgVole mice from the point of view of a chronic treatment. Several studies demonstrated that chronic passive immunization extended the survival of prion-infected mice when they were administered either via intraperitoneal or intraventricular infusion320,360–362. Based on these previous studies, we decided to try a therapeutic approach focused on chronic treatment via intracerebral. Thus, a group of 24 mice was inoculated intracerebrally with 20µL of 1% brain homogenate of a vole-adapted CWD strain. Two weeks after inoculation, animals were divided into 2 groups of 12 mice each and were treated weekly for 5 weeks with 20µL 0.5mg/mL of scFv-G1 or 20µL of a saline buffer. Mice were observed after administering the antibody in search of acute side effects, but fortunately, the antibody was well-tolerated, and none was observed. Subsequently, animals were monitored and euthanized when they were terminally sick. At the end of the treatment 4 mice, 2 of each group, had died during the procedure or because of intercurrent diseases. All mice developed clinical signs characterized by kyphosis, ataxia, hyperactivity, head bobbing, and body weight loss. The group of mice that receive the antibody had an average survival time of 63,3±0,7 dpi, whereas the incubation period for control mice was 61,9±1,0 dpi. The log-rank test showed no statistically significant differences between the group of mice that received the treatment and the saline group (ρ=0,3094). The Western blot performed with brain homogenates from animals of both groups revealed that the presence of PK-resistant material in the samples was essentially the same (Fig 2.8). Anatomopathological analysis of brains of mice belonging to both groups showed that, independently of the treatment, all the animals NURIA LÓPEZ LORENZO 94 Figure 2.10. Fluorescence images of brain cryosections. Cryosections of the lateral ventricle were visualized with the blue (DAPI) and red (Fe(III)-TMPyP) channels, and both images were merged. A group of 16 mice was anesthetized, and Fe(III)-TMPyP was injected into the brain, taking as reference the bregma to deposit the treatment in the lateral ventricle. Two mice received saline instead of Fe(III)-TMPyP. The mice that received saline and two mice injected with Fe(III)-TMPyP were euthanized immediately after the administration. The remaining mice were euthanized after 10min, 30min, 1h, 2h, 3h, and 24h. Their brains were fixed and cryopreserved before taking coronal slides with a cryostat. Nuclei were stained with DAPI while it was taken advantage of the natural autofluorescence of Fe(III)-TMPyP to visualize the distribution. It can be observed the red fluorescence of Fe(III)-TMPyP surrounding the site of inoculation at different time points. 3.4.3.3 A therapeutic administration of scFv-G1 does not delay the clinical course of prion disease with a diluted inoculum To assess if the treatment with scFv-G1 antibody was effective in more favorable conditions, a group of 26 TgVole mice receive an intracranial inoculation with 20µL of 0,1% brain homogenate of a vole-adapted CWD strain. After 2 weeks, mice were grouped into treatment and control groups, which received 20µL 0.5mg/mL of scFv-G1 antibody or 20µL of a saline buffer. The treatment was repeated weekly for 5 weeks via intracerebral. At the end of the treatment, mice were monitored twice a week before the onset of the prion disease or daily during the course of the illness until they reach the terminal stage. At this point, mice were euthanized. All the animals developed neurological signs characterized by kyphosis, ataxia, head bobbing, and body weight loss. The group of mice that receive the antibody had an average survival time of 72,8±1,6 dpi, whereas the incubation period for control mice was 75,1±1,7 dpi. The log-rank test showed no statistically significant differences between both groups of mice (ρ=0,2508). The Western blot analysis performed with brain homogenates from animals of both groups revealed that both groups accumulated PKresistant material in equal proportions. Chapter I: Results 95 Figure 2.11 Therapeutic treatment with scFv-G1 did not extend the survival time of prion-infected mice nor modified the pathology in comparison with the saline group even when the inoculum was diluted. A) Two groups of mice were inoculated with 20μL of 0,1% brain homogenate from a terminally ill mouse inoculated with voleadapted CWD. All the mice succumbed to prion disease and the mean incubation time was calculated for both groups. Log-rank test showed no statistically significant differences between the group of mice that received scFv-G1 and the saline group (p=0.2508). Hash (#) indicates that outliers were detected by Grubbs’ test; these outliers were removed before the statistical analysis (Annex VII Fig S1). B) Survival curve of mice treated with scFv-G1(n=13) or saline buffer (n=13). Mice were monitored twice a week to identify clinical signs, after the onset of the clinical phase, mice were observed daily and euthanized when they were terminally sick and the survival endpoint of animals treated with scFv-G1 (light green) or saline (dark blue) was plotted on a Kaplan-Meier survival curve. C) The accumulation of PKresistant material was assessed by Western blot using SAF83 antibody (1:400) (epitope 130-160). NURIA LÓPEZ LORENZO 96 3.4.4. A CHRONIC INTRACRANIAL TREATMENT DOES NOT REDUCE THE MICE LIFE EXPECTANCY The results presented above showed that neither a chronic treatment with Fe(III)-TMPyP nor with scFv-G1 were effective in TgVole mice inoculated with a vole-adapted CWD strain. These results contrasted with preliminary studies in vitro and in cell-based bioassays that suggested that both treatments would be active against prion diseases. We reasoned that one possible argument could be that the continuous intracerebral treatment could weaken the mice. This effect, plus the proper prion disease, could hide the beneficial effect of both treatments. In order to assess whether the repetition of intracranial injections affected the lifespan of infected mice, a control group was inoculated with 20µL of 0.1% brain homogenate from a terminally sick mouse inoculated with vole-adapted CWD and the average survival time was then compared with the saline group of mice that was previously used in the section 3.4.2.3 A therapeutic administration of scFv-G1 does not delay the clinical course of prion disease with a diluted inoculum. The mean survival time of mice just inoculated with a 0,1% brain homogenate (that did not receive repeated intracranial administrations of saline buffer) was 76,6±1,8 dpi, whereas the mean incubation period of mice inoculated and treated intracerebrally weekly for 5 weeks, was 75,1±1,7 dpi. The log-rank test showed that the difference was not statistically significant, with a ρ value=0,3953. Moreover, the histopathological analysis did not show brain damage as a consequence of repetitive brain injection derived from the chronic treatments, in comparison with those mice that were just inoculated. Chapter I: Results 97 Figure 2.12 The chronic intracranial treatment did not affect the mice lifespan. A) Two groups of mice inoculated with 20μL of 0,1% brain homogenate from a terminally ill mouse inoculated with vole-adapted CWD were compared. One group of mice was just inoculated (pink) while the other group was inoculated and treated weekly for 5 weeks with saline buffer (blue). All the mice succumbed to prion disease and the mean incubation time was calculated for both groups. Log-rank test showed no statistically significant differences between both groups of mice (p=0.3953). B) Kaplan-Meier survival curve. The survival curve of both groups was practically similar; the mean survival time of mice just inoculated (pink) was 76,6±1,8dpi (n=11), while the survival of mice that were also treated with saline (blue) was 75,1±1,7 dpi (n=13). C) The histopathological profile of mice just inoculated (pink line)(n=5) or inoculated and treated with saline buffer (blue line) (n=4) represents the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions against these brain regions: Pfc: piriform cortex, H: hippocampus, Oc: occipital cortex, Tc: temporal cortex, Pc: parietal cortex, Fc: frontal cortex, S: striatum, T: thalamus, HT: hypothalamus, M: mesencephalon, Mob: medulla oblongata, Cm: cerebellar nuclei, Cv: cerebellar vermis, Cc: cerebellar cortex. The spongiform change of both groups is similar, meaning that the chronic intracranial treatment did not cause a change in the lesion profile. Hash (#) indicates that the pointed-out brain area could not be examined in all the samples. Chapter I: Dicussion 99 3.5 DISCUSSION 3.5.1 ASSESSMENT OF THE ANTI-PRION ACTIVITY OF FE(III)-TMPYP IN A FAST MURINE MODEL OF PRION DISEASE Porphyrins are organic heterocyclic molecules with essential functions in animals, plants, algae, and also bacteria; in consequence, they are known as “pigments of life”. They play crucial roles in oxygen transport, photosynthesis, and enzymatic reactions. Moreover, porphyrins have intrinsic properties with industrial and medical interest; for instance, one of the main porphyrin features is their capability to absorb light in the visible region and transmission of energy through the coordinated metal, called light-harvesting system. This process is the basis of their function in photosynthesis; for this reason, porphyrin-based materials have been used in the fabrication of organic photovoltaic devices. Furthermore, their flat and symmetric architecture is useful for building units of supramolecular polymers364. Medical interest in porphyrins lies in the porphyrin-mediated photodynamic therapy. Their natural fluorescence and the capability to chelate superparamagnetic metal ions allow in vivo imaging and guiding of porphyrins to the place of interest by MRI. Moreover, porphyrins can encapsulate drugs and molecules of interest and release them by ultrasounds in concrete tissue252. These examples show the potential and interest harbored in porphyrin compounds. Anti-prion properties of porphyrins and phthalocyanines have been studied in vitro, in cellula for decades with encouraging results251,332– 335,346,365. Fe(III)-TMPyP is a cationic tetrapyrrole that acts as a molecular chaperone binding to huPrPC residues 160-180 in an equimolar ratio365. Previous studies demonstrated that such porphyrin NURIA LÓPEZ LORENZO 100 inhibits prion propagation by PMCA and in cell cultures infected with different prion strains in a micromolar range: 6,21μM in vitro and 1.61μM in cellula. Moreover, Fe(III)-TMPyP also demonstrated to reduce the synaptotoxicity of PrPC-mediated Aβ oligomers335. In addition, experiments in transgenic mice overexpressing hamster PrP (Tg7) showed 90 days of prolongation of survival over the untreated animals. These animals were inoculated and treated intraperitoneally with Fe(III)-TMPyP, 3 times a week for 4 weeks, starting the treatment on the day of the inoculation. Nonetheless, when the treatment started in an advanced stage of the disease (56dpi), it did not report an increase in the incubation time. Additionally, further experiments demonstrated that the effect was due to an inactivation of the infectious agent in the place of inoculation. As consequence, the authors of this work concluded that Fe(III)-TMPyP would be better used as a prophylactic agent in case of peripheral incidental contact with prions, rather than as a therapeutic agent346. In the present thesis, Fe(III)-TMPyP was assessed in a fast murine model of prion disease. For this purpose, a group of TgVole mice was inoculated intracranially with a vole-adapted CWD strain. This CWD strain causes a prion disease in a short incubation period of approximately 70dpi, which was considered to be helpful in getting fast results that could be useful as proof of concept. Furthermore, the i.c. route was selected over the i.p. via, as it better mimics the pathogenesis of sporadic prion diseases, which are the most common forms of prion diseases, while i.p. route would be more suitable for targeting acquired prion diseases. The treatment with Fe(III)-TMPyP started 2 weeks after inoculation in order to avoid the inactivation of the infectious agent at the site of inoculation, as was previously reported346. The treatment was repeated weekly for 4 weeks and finished approximately 2 weeks before the onset of clinical signs. Chapter I: Dicussion 101 Unfortunately, our attempt failed to prolong the survival or delay the onset of clinical signs. Based on the previous experiments, our first interpretation was that the prion strain might be extremely aggressive or that prion titters in the inoculum was too high to be neutralized by Fe(III)-TMPyP. For this reason, a second attempt was performed with a higher dilution of the inoculum, 0,1% instead of 1%, while the administration route or dosing regimen were preserved. This second approach did not provide the expected results either, dismissing the hypothesis that the prion concentration in the inoculum was too harmful. Although mice did not experience an extension on the survival, analysis of the PrPSc present in their brains revealed a reduced amount of protease-resistant PrP. Despite the fact that Western blot is not a quantitative technique and the statistical results derived from its densitometric analysis should be cautiously interpreted, this reduction in the PrPSc was consistent, since it was observed in both groups of animals treated with Fe(III)-TMPyP regardless the dilution of the inoculum. The reason why this reduction was not translated into a lifespan extension could be that Fe(III)-TMPyP cannot distribute throughout the brain parenchyma. The kinetics of Fe(III)-TPMyP diffusion revealed that after 24h, it remained in the site of administration. This should be enough to obtain a local reduction of PrPSc but not sufficient to obtain a global effect reflected in a delay in the onset of the disease. Moreover, since stereotaxic devices were not used during the treatment, the site of administration of Fe(III)-TMPyP slightly varied among the repetitive injections and most likely allowed to cover a wider treated area, reinforcing the hypothesis that Fe(III)-TMPyP had local effect reflected in the amount of PrPSc, though insufficient to increase the survival. Very recently, Chiesa and colleagues identified another cationic tetrapyrrole with anti-prion properties, Zn(II)-BnPyP. Although Fe(III)- TMPyP and Zn(II)-BnPyP share the same chemical nature, they differ NURIA LÓPEZ LORENZO 102 on the mechanism of action. While Fe(III)-TMPyP acts as a molecular chaperones stabilizing PrPC, Zn(II)-BnPyP binds to two distinct sites of PrPC and destabilizes its native conformation; as a result, Zn(II)-BnPyP shows a dual effect: on one hand, it hinders PrPC conversion into PrPSc and, on the other hand, it promotes the degradation of the complex Zn(II)-BnPyP - PrPC. Despite the encouraging results that Zn(II)- BnPyP showed in vitro and in cell-based assays, attempts to treat prion infected mice failed in showing an effect on the survival over the vehicle-treated mice, even when Zn(II)-BnPyP was administered either by direct intracerebral injections and chronic infusion with osmotic pumps to bypass the BBB. Consistently with our findings, Zn(II)- BnPyP presented poor diffusion into the brain parenchyma366; thus, despite the interesting anti-prion properties that porphyrins show in vitro these results suggest that their poor pharmacokinetics is responsible for their pharmacological failure in vivo. In addition, since Fe(III)-TMPyP had apparently a local effect, it would be expected that this effect would also have an impact on the histopathological profile of treated mice. Specifically, since the treatment was attempted to be deposited in the lateral ventricle, a reduction in vacuolization in the brain areas adjacent to the lateral ventricles could be expected. Considering that the inoculated CWD strain mainly affects the brainstem and basal ganglia, brain areas such as the striatum and thalamus might be expected to show less vacuolation. However, the anatomopathological examinations did not reveal any differences in spongiosis in any region. Moreover, the immunohistochemical labeling failed in exhibiting PrPres deposition in both, control and treated mice, even when two different primary antibodies were tested, 6C2 and SAF84 (data not shown). The absence of PrPres may be due to the incubation time of this strain, which may be too short to obtain an accumulation of PrPres deposits. On the other hand, it may also be due to the fact that this strain is affected by the unmasking process to which samples are subjected during their preparation for the immunohistochemical analysis. Chapter I: Dicussion 103 A reasonable argumentation for the discrepancy between the absence of activity of Fe(III)-TMPyP regarding the survival or the lesion profile, and the levels of PrPSc, could be that the amount of PrPSc might not be directly related to the neurotoxicity. This idea was proposed by Collinge and colleagues208,209,286 and hypothesizes that prion disease takes place in two distinct phases. During the silent phase 1, infectious prion titers exponentially rise until they reach a plateau (phase 2) In this second phase, clinical signs begin, and the duration is inversely related to the PrPC expression levels, being shorter in mice that overexpress PrPC and longer in heterozygous animals. Classical PrPSc just emerges at the end of the incubation period, implying that the neurotoxicity might be mediated by different PrP species such as PK-sensitive PrP species or PrP oligomers. Consistent with this data and based on the mechanism of action, we consider that Fe(III)-TMPyP might bind PrPC and decrease the conversion into the PrPSc; however, it does not affect the toxic PrP conformers or does not interrupt the toxic signaling cascade. In summary, the most reasonable argument for why Fe(III)-TMPyP did not ameliorate the prion diseases is because it cannot diffuse throughout the brain parenchyma or it does not avoid the formation of toxic species of PrP. 3.5.2 SCFV-G1 ANTIBODY AS A THERAPEUTIC STRATEGY FOR PRION DISEASES In 1986, muromonab, the first therapeutic antibody was approved by the FDA. Nowadays, there are more than 130 therapeutic antibodies authorized; most of them, 45%, are commonly used in oncology, while antibodies against neurological disorders represent approximately 7%. The reason why the number of antibodies targeting neuropathological diseases is too low is that most of them do not cross the BBB due to their large size, added to the t he tight junctions formed by endothelial cells in the BBB, for these reasons typically less than 0,05% of the initial dose reaches the targeted brain region. This fact implies the use NURIA LÓPEZ LORENZO 206 Figure 4.14 Mass spectrometry of Ust09. A) Deconvoluted spectrum of the PKresistant fragments of Ust09; tagged peaks are those that were identified. The chromatogram and mass/charge spectrum are included in Annex VIII. B) Table of the identified peptides mass and the corresponding sequence. C) Schematic representation of bands densitometry based on the electrophoretic pattern. Chapter III: Results 207 Of the seven conformers that were analyzed, none of them presented the same distribution of PK-resistant peptides: even though Hep01 and Ust02 shared an electrophoretic pattern with similar ~16, ~9, ~8, and ~6kDa bands, mass spectrometry revealed that the cleavage sites were not precisely the same; for example, the contribution to the ~6kDa band in the case of Hep01 was restricted to the PK-resistant peptides N97Y150 and Q98-Y150, while the same fragment in Ust02 was comprised by N97-Y150 and Q98-Y150, but H96-Y150, Q98-N153, and W99Y150. Since the digestion conditions were the same (regarding the concentration of proteinase, pH, temperature, or duration of the digestion), these differences, could be due to different structural arrangements, which provided better accessibility of the PK to the cleavage sites or promoted the generation of ragged ends (Fig 4.15). Figure 4.15 PK cleavage sites. Schematic representation of the nicksites of the PK and the resultant PK-resistant fragments. NURIA LÓPEZ LORENZO 208 Although the total protein staining showed a different PK-resistant fragments proportion, the mass spectrometry-based analysis is not a quantitative technique, and the intensity of the peaks is not directly related to the abundance of each peptide. 5.4.3 STUDY OF THE INFECTIVITY OF THE CONFORMERS GENERATED BY PMSA In order to assess if the new conformers generated by PMSA with heparin, chondroitin sulfate, and pentosan polysulfate were bona fide prions and different strains, the product of the PMSA was inoculated intracerebrally into TgVole mice. Mice were then monitored twice a week until the onset of clinical signs; thereafter, they were observed daily until they reached the terminal stage. All the inoculated mice developed clinical signs compatible with a transmissible spongiform encephalopathy. The prion disease was confirmed by detecting PK-resistant PrPSc in their brains by Western blot and verifying the presence of spongiosis characteristic of prion diseases. Interestingly, the average incubation time varied widely among the conformers: Hep03 presented the shorter period of incubation, 105±2 dpi, which was similar to the average incubation time described for one of the prion strains generated with dextran, Ust02, 101±4 dpi 533. On the other hand, the slowest conformer was Hep01, also generated using heparin as a cofactor, whose incubation period was 363±7dpi. Given that the amount of PK-resistant PrP was similar in all the conformers regarding the total protein staining, it would be reasonable to think that the difference in the incubation was not due to the titers of PrPSc; thus, the variability in the incubation would be due to intrinsic differences among the conformers, suggesting that they could be different prion strains. Moreover, one-way ANOVA and post hoc multiple comparisons test (Annex IX) demonstrated that conformers generated with the same cofactor presented statistically significant differences Chapter III: Results 209 regarding the mean incubation time. These differences, added to the variable electrophoretic mobility, supported the idea that different prion strains could be generated under the same conditions. On the contrary, the same test revealed that conformers, with different patterns, such as Hep02 and CoS01 or Hep03 and CoS03, could behave similarly from a biological point of view, and share the same average incubation time. To further characterize these prion strains, TgVole mice were inoculated intracranially with 1% brain homogenates of mice from the first passage. Like with the first passage, mice were monitored and euthanized when in the terminal stage of the prion disease. All the inoculated animals displayed clinical signs and the attack rate was 100%; moreover, the presence of PK-resistant PrP was detected in all the brains analyzed; hence it was confirmed that the different conformers were de novo generated bona fide recombinant prions (Fig 4.16). NURIA LÓPEZ LORENZO 210 Figure 4.16 The recombinant conformers generated with different cofactors were bona fide prions. A) Table summarizing the average incubation time of the first and second passages. The attack rate represents the animals that presented clinical signs compatible with prion disease when euthanized. The asterisk (*) indicates that animals from those groups died of intercurrent diseases; such animals were excluded from the analysis. Hash (#) represents groups with animals whose brains were not possible to extract (e.g., they were found dead), although they presented clinical signs, and for this reason, the attack rate and the brains with PrPSc do not match. B) Kaplan-Meier curve of mice from the first passage. C) Kaplan-Meier curve of mice from the second passage. It should be noted that the X-axis of both curves does not have the same graduation to better distinguish the incubation time. Chapter III: Results 211 In general terms, the average incubation time on the second passage was shorter in comparison with the first passage, as well as the dispersion. On the first passage, the average incubation time varied between 105 and 363dpi; on the contrary, on the second passage the periods of incubation of all the stains tended to unify between 74 and 105dpi. Moreover, some conformers presented a significant decrease in the incubation period between the first and the second passages, such as Hep01, whose average incubation time moved from 363 to 86dpi. In contrast, Hep03 displayed a low variation in the incubation period, from 105 to 95 dpi. On the other hand, conformers CoS01 and PPS01 displayed similar incubation times in both passages, showing a parallel adaptation to the host; however, it is not possible to confirm that they consist of the same strain just based on this feature, since different prion strains can share the same period of incubation 449. Overall, these data revealed that all the strains had to adapt to the brain PrP, although such adaptation was different depending on the strain: while some strains suffered from an important adaptation reflected in a great reduction of the incubation period, other strains most likely had a structure in which fully glycosylated brain PrP fitted efficiently. 5.4.4 BIOCHEMICAL FEATURES OF THE DIFFERENT RECOMBINANT PRION STRAINS Brains of mice belonging to both passages were subjected to Western blot analysis in order to assess the presence of PrPSc. A clear accumulation of classical PrPSc was detected in all groups of animals and on both passages, supporting the idea that these recombinant conformers are bona fide prions. the nature. Interestingly, after PK digestion, all the conformers present a predominance of the diglycosylated band, and they greatest variety was perceived in the nonglycosylated band. This band highly varied in intensity, and molecular weight. NURIA LÓPEZ LORENZO 212 Figure 4.17 Western blot of brain homogenates of mice inoculated with conformers generated with different cofactors. A) Western blot of brain homogenates of terminally ill mice inoculated with different conformers generated by PMSA using different cofactors. B) Western blot of brain homogenates of terminally ill mice from the second passage. These animals were inoculated intracranially with brain homogenates of mice from the first passage and succumbed Chapter III: Results 213 to prion disease. A non-digested normal brain homogenate (NBH) was included as a reference. Regarding the second passage, while the non-glycosylated band of PPS02, PPS03, Ust02 or Ust09 presented a weak intensity, or it was even absent, like in CoS03, such band showed a strong presence in Hep03, CoS01, CoS02 or PPS01. In terms of the electrophoretic mobility, the non-glycosylated band seemed to display a higher molecular weight in CoS02, PPS02 or Ust09. Also with respect to electrophoretic mobility in the second passage, CoS03 and PPS01 seemed to have a diglycosylated band of lower molecular weight than the other conformers (Fig 4.17). These differences would suggest structural differences among the conformers. To better characterize the material that is propagating in the brains of the mice inoculated with the different conformers and also with the aim of assessing that the conformers give place to different prion strains, fixed brains were examined in search of spongiform changes and PrPres deposits. Notwithstanding brains of animals from the first passage were inspected to confirm the presence of vacuolation and deposits of PrPres, a detailed histopathological characterization was performed with the brains of mice from the second passage. The reason why an in-depth analysis was only performed with the second passage was that on the first passage the recombinant prion strains are not completely adapted to the host, as was reflected in the reduction in the incubation period, and we most likely would find certain variability in the brains of mice from the same inoculum. Since all the mice were culled in the terminal stage of the disease, it was assumed that the differences in terms of severity of the spongiform changes or in the amount of PrPres depositions were characteristic properties of the strain. NURIA LÓPEZ LORENZO 214 Figure 4. 18. Histopathological features of TgVole mice inoculated with conformers generated with heparin. The histopathological profile represents the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions against these brain regions: Pfc: piriform cortex, H: hippocampus, Oc: occipital cortex, Tc: temporal cortex, Pc: parietal cortex, Fc: frontal cortex, S: striatum, T: thalamus, HT: hypothalamus, M: mesencephalon, Mob: medulla oblongata, Cm: cerebellar nuclei, Cv: cerebellar vermis, Cc: cerebellar cortex. Hash (#) indicates that the pointed-out brain area could not be examined in all the samples. Hematoxylin-eosin staining revealed vacuolation and PrP immunolabeling showed PK-resistant PrP deposits; the brain areas from which the images were obtained are indicated in parenthesis. A schematic representation of the different brain areas summarizes the histopathological features of each conformer. The intensity of the color is in accordance with the semiquantitative score of spongiform changes and the brown dots indicate the areas where PK-resistant PrP was found. Chapter III: Results 215 Figure 4. 19. Histopathological features of TgVole mice inoculated with conformers generated with chondroitin sulfate. The histopathological profile represents the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions against these brain regions: Pfc: piriform cortex, H: hippocampus, Oc: occipital cortex, Tc: temporal cortex, Pc: parietal cortex, Fc: frontal cortex, S: striatum, T: thalamus, HT: hypothalamus, M: mesencephalon, Mob: medulla oblongata, Cm: cerebellar nuclei, Cv: cerebellar vermis, Cc: cerebellar cortex. Hash (#) indicates that the pointed-out brain area could not be examined in all the samples. Hematoxylin-eosin staining revealed vacuolation and PrP immunolabeling showed PK-resistant PrP deposits; the brain areas from which the images were obtained are indicated in parenthesis. A schematic representation of the different brain areas summarizes the histopathological features of each conformer. The intensity of the color is in accordance with the semiquantitative score of spongiform changes and the brown dots indicate the areas where PK-resistant PrP were found. NURIA LÓPEZ LORENZO 222 to other intra or intermolecular 13C nuclei that are close in space539,540 (Fig 4.22). Since this experiment was performed with U-(13C, 15N)-Phe full-length BVPrP, it would provide information about the chemical shift of phenylalanines. In case of showing similar chemical shifts, the results would provide evidence that the three phenylalanine residues shared conformational environments. On the contrary, in case that they presented different chemical shifts, these results would suggest that the phenylalanine residues are in different conformational environments with different levels of rigidity. Depending on the chemical shift, the results would support that the chemical environment of phenylalanines would be compatible with a PIRIBS or 4RβS architectures. It should be noted that this experiment was performed before the structural resolution of the 263K strain by cryo-EM. Figure 4.22 Schematic representation of the basis of 13C-13C Dipolar Assisted Rotational Resonance (DARR). Hydrogen magnetization is transferred to 13C nuclei (yellow arrows). Subsequently, magnetized 13C nuclei transfer the magnetization to other nearby intra (light green arrows) or intermolecular (cyan arrows) 13C nuclei. Image created with bioRender.com adapted from https://protein-nmr.org.uk/541 Intriguingly, the Sst01 13C-13C-DARR spectrum presented two sets with three signals for each set, while only one signal for each Chapter III: Results 223 phenylalanine was expected. Moreover, the chemical shift of one set of signals was more characteristic of a coil, while the other one was compatible with a β-sheet secondary structure 542; although it was not possible to make a further interpretation of these data from the structural point of view, this experiment evidenced that phenylalanines would be in different chemical environments; therefore, more than one conformer would be present in Sst01. In order to characterize more recombinant prion strains by ssNMR and to compare their spectra, large amounts of U-(13C, 15N)-Phe Ust02 and Ust09 were obtained by PMSA. Like Sst01, these strains were propagated in the presence of dextran sulfate as a cofactor, with the aim to reduce variability among strains, since the interaction between glycosaminoglycans can vary amyloids DARR chemical shift543. After amplification, the recombinant prion strains were digested with PK, pelleted, dried, and loaded in a 1.3mm rotor. Interestingly, like Sst01, Ust02 and Ust09 spectra also presented two sets of three signals, indicating that phenylalanine residues are located in structures with different levels of rigidity: the chemical shift of one set would be compatible with a relatively rigid coil or a flexible β-sheet, and the other set would present a chemical shift compatible with a more rigid β-sheet (Fig 4.23)542. Comparing the three spectra it can be noticed that the signals were not completely overlapped, indicating that the chemical environment of the phenylalanines of the three strains was significatively different. While some of the coil signals of Sst01 and Ust02 could be superposed, Ust09 presented a chemical shift slightly displaced. On the other hand, β-sheet chemical shift signals of Sst01 and Ust09 were completely overlapped; whereas only one of the signals of the β-sheet chemical shifts set of Ust02 merged with the corresponding signals of Sst01 and Ust09. Definitely, these results demonstrated distinct structural arrangements among the recombinant prion strains. NURIA LÓPEZ LORENZO 224 Figure 4.23 13C-13C DARR spectra (τmix=100ms; MAS 20kHz). Spectra of expanded CO/CA regions of A) Sst01, B)Ust02, and C) Ust09. Diamonds indicate peaks corresponding to phenylalanine signals with chemical shifts characteristic of a coil (green) or β-sheet (blue). D) Merged spectra of the three recombinant prion strains: Sst01 (blue), Ust02 (yellow), and Ust09 (green). Chapter III: Discussion 225 5.5 DISCUSSION 5.5.1 GENERATION AND CHARACTERIZATION OF DIFFERENT RECOMBINANT PRIONS STRAINS DE NOVO GENERATED BY PMSA WITH DIFFERENT COFACTORS The wide variety of prion strains is of structural and biochemical interest. However, since prion strains cause specific pathological phenotypes, the characterization of these conformers is of vital importance from a pharmacological point of view, as well. Currently, there is no effective treatment for prion diseases, and one of the reasons is the strains capability to evade the treatment by mechanisms of adaptation and selection474,544. The presence of pharmacological compounds modifies the environment of prion propagation, which triggers the emergence of drug-resistant conformers415,472; therefore, a comprehensive characterization of diverse strains can provide crucial information about the underlying differences among them, enabling the design of effective therapies and to tackle the adaptation and selection mechanisms that lead to therapeutic failure. It has been suggested that cofactors are biomolecules involved in maintaining the biological and biochemical features of prion strains and closely related to prion infectivity545; additionally, cofactors have also been associated with prion strain selection. Since PrP is a membrane protein, it would be reasonable to consider as possible cofactors molecules that are present in the extracellular matrix and with which PrP could interact; thus, such molecules include membrane phospholipids, proteoglycans, glycosaminoglycans, and other proteins. Although prions isolated from infected animals had associated different cofactors, glycosaminoglycans are frequently related to amyloid NURIA LÓPEZ LORENZO 226 deposits, and in vitro assays demonstrated that they play a crucial role in amyloid formation and fibril stabilization546; additionally, in some amyloidoses, glycosaminoglycans have also been associated with cytotoxicity522. Moreover, heparan sulfate, which is the most common glycosaminoglycan present in the cell membrane, has been associated with diverse amyloidoses, such as Alzheimer’s disease, Parkinson’s disease, type II diabetes or light chain amyloidosis 524. In prion diseases, heparan sulfate affects the kinetics of propagation547 and the brain deposition in vivo, therefore, it changes the strain properties548. The aim of this work was to generate different synthetic conformers by PMSA using natural or synthetic glycosaminoglycans cofactors and characterize them by different biological, biochemical, and biophysical methods. This characterization would enable us to assess their infectivity and their properties; therefore, it would make it possible to confirm whether they are different bona fide prion strains. Nine different conformers were de novo generated by PSMA using recombinant full-length BVPrP (109I) and heparin, chondroitin sulfate, or pentosan polysulfate as cofactors. Concretely, three different conformers were generated with each cofactor. These conformers were stabilized by serial passages and compared with two recombinant prion strains generated by the same methodology but using dextran sulfate as a cofactor533. Attending to the electrophoretic pattern and independently of the cofactor, all the conformers shared three PK-resistant fragments detectable with total protein staining and Western blot of ~16, ~9, and ~6kDa; particularly, the ~16kDa PK-resistant fragment is present in recombinant infectious prions propagated by PMSA101 or de novo generated by PMCA using recombinant PrP 108, and corresponds to the PrP27-30 PK-resistant core of bona fide prions92. The characterization of the conformers by limited proteolysis coupled to mass spectrometry turned out to be an exceptionally revealing technique. It must be noted that the bases of limited proteolysis Chapter III: Discussion 227 establish that the tertiary structure, rather than the sequence of the protein, determines the site of proteolysis 534. Thus, loops, unstructured and flexible regions exposed to the solvent would be primarily cleaved since local deformations are required to reach the active site of the enzyme. Oppositely, β-strands are rigid structures, stabilized by inter and intrachain hydrogen bonds that involve energetic constraints and can hardly be deformed 535, for these reasons, limited proteolysis provided structural information. Moreover, the combination with mass spectrometry-based analysis permitted the identification of exact cleavage sites and the PK-resistant fragments that composed each conformer. In addition, although the intensity of the peaks was not directly related to the abundance of the peptide, a semiquantitative approach could be done by attending to the total protein staining, taking into consideration the intensity and the molecular weight of the bands. As well as the electrophoretic migration, mass spectrometry-based analysis showed that all the conformers shared three bands, identifying common cleavage sites at positions 98, 150, 153, and 154 which yielded the peptides 98-231, 153-231, 154-231, and 98-150, and corresponded to the aforementioned bands observed in the total protein staining. Apart from these common nicksites, specific cleavage sites were recognized for each conformer that agreed with the electrophoretic pattern. Moreover, all these conformers present a PK-sensitive 23-89 region, which is in complete agreement with previous works and with PrPSc solved structures 110,179,180,190,193,194,506,549. Nevertheless, we found the first particularity in the region 89-99, which corresponds with the frontier between the unstructured N-terminal domain and the C-terminal PIRIBS core. Different works have demonstrated that the PK-resistant core of some ex vivo wild-type prion strains from patients with the GSS P102L variant 549, sCJD 181, or from prion-infected hamsters,179 and mice 180 started at position ~74-81 with ragged termini fragments that could be extended until position 92. However, the PK-resistant core of mouse and BV prion strains propagated by PMCA506 or PMSA started at positions ~90-99. When NURIA LÓPEZ LORENZO 228 these cleavage sites were projected into the different structures available for mouse prion strains, aRML194, RML190, ME7193, and a22L205, most of them were placed in a region that corresponds to a βsheet which extend from position ~94 to ~99. Additionally, the size of the ~90-231 region determines the PK-resistant core of brain-derived prions, known as PrP27-30. In this regard, the cleavage site at the Nterminal extreme determines the electrophoretic mobility, differentiating between type 1 or type 2 patterns. Therefore, dominant nicksites at positions 78, 82, or 90 are preponderant in 263K or PrPSc derived from type 1 CJD patients, whereas Dy or PrPSc derived from type 2 CJD patients have preponderant cleavage sites at positions 92, 97, or 101179,550. Based on these data, BV recombinant strains propagated PMSA present common cleavage sites at positions 92, 97, or 98; therefore, it would be reasonable to expect a Dy-like electrophoretic mobility after inoculation (Annex VII Fig S2). Another cleavage site in the region ~150-154 was identified in all the recombinant prion strains studied in the present work, and two reciprocal fragments arise from this cleavage site ~90-150 and ~154231. These fragments, together with ~90-231, represent the most common PK-resistant fragments in recombinant prion strains 101,506. Additionally, cleavage sites at these positions have also been described in brain-derived classical179,180,182 and atypical181,183,549,551 prions. Notwithstanding, while in recombinant prion strains these cleavage sites are predominant, in brain-derived PrPSc these nicksites were detected in a very low proportion179,180. Although these cleavage sites were identified in both, classical and atypical PrPSc, the presence of the resultant N-terminal fragment, ~90-150, was widely associated with atypical PrPSc such as GSS P102L549, sCJD183, Nor98552, or VPSPr551 among others. On the contrary, the C-terminal fragment resultant of the cleave, ~154-231, involves the most resistant region of the PrPSc core to PK digestion or denaturalization 179,182. Many of the strains generated by PMSA presented a major nicksite at position ~163, which originated the complementary peptides ~97-163 Chapter III: Discussion 229 and ~163-231. While fragment ~97-163 was very unusual, the ~163231 peptide, together with the fragment ~153-231, were reflected as an intense doublet of ~9kDa in the electrophoretic pattern. Although this cleavage site was also described in ex vivo aRML180, the presence of this fragment in classical prion strains was minimal. However, this ~163-231 C-terminal truncated fragment, together with ~154-231 fragment, also known as CTF12/13, are much more common in brainderived samples from patients with different subtypes of CJD553, such as sCJD181,183 iCJD554, CJD E200K555, or M232R554, where they were found in coexistence with PrP27-30. Interestingly, recombinant PrP amyloids also present this ~163-231 PK-resistant core 413. When this cleavage site is projected into the available PrPSc structures, it could be perceived that it is placed in a β-sheet; thus, the cleavage at this residue should be unlikely. Zou et al suggested that this fragment would be generated following a different pathway than PrP27-30181. This idea suggests the existence of a mixture of distinct PrPSc conformers with slight differences in the tertiary structure; nonetheless, the role of the ~163-231 C-terminal fragments in human pathogenesis remains unclear556. Regarding the recombinant prion strains generated by PMSA, the simultaneous origin of a mixture of species with a slightly different architecture would be a reasonable explanation for the coexistence of both truncated forms: ~163-231 and ~153-231; and it is supported by the evidence that in vitro amplification techniques, like PMCA, can generate heterogeneous populations186,557,558. Overall, the data obtained by mass spectrometry-based analysis after digestion with PK indicate that the presence of different PK-resistant fragments in the different conformers is compatible with different structural arrangements, reinforcing the idea that they consist of different strains. Moreover, this information strongly suggests the existence of different conformational arrangements between wild-type and synthetic prion strains generated by PMSA, reflected in a higher sensitivity to PK digestion and the presence of different cleavage sites. Such structural differences could be a consequence of the lack of the bulky glycans that could interfere sterically in the adopted conformation. NURIA LÓPEZ LORENZO 230 Cleavage sites at positions 117/119 have been broadly described in both, ex vivo and recombinant prion strains; in fact, Kraus and colleagues proposed that these nicksites would be responsible for local destabilizations which would trigger partial unfolding and separation of residues ~90-118. This hypothesis would be supported by the fact that the hydrophobic clusters provide lateral and longitudinal intra and interchain polar interactions which stabilize the structure; the proteolytic digestion could disrupt these interactions causing local structural destabilizations559. Moreover, this phenomenon would allow the access of the solvent and PK to the hydrophobic region 120-140192 where minor cleavage sites have been also identified at positions 133, 135, or 139 (BV numbering)179,180,506. In contrast to this evidence, none of the recombinant conformers presented nick sites at positions 117/119, although cleavage sites at positions 133 and 136 were identified. Since in these samples nicksites at residues 133 and 136 generated the peptides 97-136 and 133-231, they are most likely independent cleavage sites rather than complementary; thus, there are some reasonable explanations for their existence that are not exclusive. One hypothesis for this event would be that cleavage sites at position 117 and 119 were present in the conformers, although they were not identified due to the low intensity of the peaks; moreover, reciprocal peptides, ~97-117 or ~97-119, would have such a low molecular weight that an accurate and precise identification would not be possible. This hypothesis would explain the origin of the peptides 133-231 recognized in CoS03 and Ust09 spectra. Another option would be that these strains generated by PMSA structurally differ from ex vivo or other recombinant prion strains. This idea would be supported by the fact that the cleavage site at position 136 exists in the absence of the nicksites at positions 117 and 119, since the peptide 97-136 was identified in Hep02, Hep03, and Ust09. This peptide would not exist if the cleavage site at 136 were a consequence of a primary cleavage at positions 117 or 119; on the other hand, based on Kraus’s hypothesis, this cleavage site would be secondary to an initial nick at residues 153-154, which are present in all the strains studied. Another interpretation is that the Chapter III: Discussion 231 structural arrangements of PMSA-generated recombinant BV strains expose some cleavage sites to the solvent and to the PK, while in wildtype strains these nicksites are buried in hydrophobic cores. Figure 4.24 Hypothetical structural differences between brain-derived and recombinant prion strains. A) Structure of RML with the representation of glycans as dark blue clouds. Gray arrows represent glycans demanding space that force the interaction of the N-terminal lobe. B) Hypothetical representation of a recombinant prion strain generated with recombinant PrP; therefore, lacking glycans. Dashed line indicates a flexible ~90-118 region, and gray icons illustrate how PK could access to different cleavage sites identified by mass spectrometry-based analysis. Image created with ChimeraX with the RML structure (PDB 7QIG)197 Since most of the differences regarding the PK cleavage sites between recombinant and brain-derived strains affect the N-terminal region of the PrPSc, it could be hypothesized that there exist structural differences in this area. Such structural divergences would affect the flexibility or the degree of compaction of the N-terminal lobe, leading to a higher accessibility of the PK to certain cleavage sites, which are unavailable in brain-derived prion strains. It is possible that in brain-derived strains the presence of bulky glycans at position 180 (mouse numbering) pushes region ~90-118, forcing it to interact with the region ~120-145. In case of prion strains generated in vitro from recombinant PrP, the 6. CONCLUSIONS Conclusions 241 1. The molecular chaperone, Fe(III)-TMPyP, neither had effect on the survival nor the pathology of TgVole mice inoculated with a vole adapted-CWD strain after a chronic treatment. 2. A chronic treatment with Fe(III)-TMPyP reduced the accumulation of PrPSc in the brain of mice inoculated with a vole adapted-CWD strain. 3. A preliminary kinetic evaluation of Fe(III)-TMPyP showed that the tetrapyrrole cannot diffuse throughout the brain parenchyma. 4. Given the poor penetrance of Fe(III)-TMPyP, the reduction in the accumulation of PrPSc might be due to a local effect in the site of administration. 5. The conformational antibody, scFv-G1, neither had effect on the survival nor the pathology of TgVole mice inoculated with a vole adapted-CWD strain after a chronic treatment. 6. The prophylactic administration of the recombinant PrP amyloid, termed theraloid, had no effect on the survival of prion infected mice. 7. Animals treated with the theraloid presented distinct histopathological features, indicating that the theraloid was propagating. 8. The theraloid resulted infectious in TgVole mice, triggering a prion disease in those animals that were solely inoculated with the amyloid. 9. Mice inoculated with the theraloid accumulated and propagated two different prion strains. NURIA LÓPEZ LORENZO 242 10. The amyloid was serially transmitted to TgVole mice, indicating that the recombinant PrP amyloid was a bona fide prion. 11. The biological and biochemical properties of the amyloid were maintained after serial transmission. 12. Multiple conformers have been spontaneously generated by PMSA in the presence of three different cofactors. 13. Biochemical and biophysical analyses determined that the conformers present different structural properties. 14. 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SUPPLEMENTARY INFORMATION ANNEX I: LISTING OF MATERIALS USED IN THIS THESIS Material Reference Brand 0.22µm filter JCAS 045025K Cytiva 0.45µm filter SLGP033RS Filter lab JA-10 rotor tubes 31410500 Thermo Scientific JA-20 rotor tubes 55.517 Sarstedt PMSA tubes 3468 Thermo Scientific Immobilon P PVDF membrane IPVH00010 Millipore Blotting paper RPN6101M Cytiva 3mL Slide-A-Lyzer dialysis cassettes G2 87718 Thermo Scalpel blade 21 0207 Swann-Morton Black-bottom 96-wells plate 353945 Falcon 96-wells plate M600 Deltalab 29-gauge needle (built-in syringe) 320926 BD Medical 25-gauge needle (built-in syringe) 4657853 Braun His Trap HP column 17524802 Cytiva 1mm Zirconia/Silica beads 11079110z Biospec products 1mm Glass beads 11079110 Biospec products 0.1mm Glass beads 11079101 Biospec products 2.3mm Zirconia/Silica beads 11079125z Biospec products Supplementary information 291 ChromXP C18 150×0.30 mm column 5022436 SCIEX Centrifugal filters ultracel 3K Amicon Ultra 15 UFC900324 Merck mPAGE 4-12% Bis-Tris gels MP41G10 Sigma 70Ti rotor tubes 355618 Beckman Coulter YMC-TRIART C18 trap column TA12S03-1503PTH Tecnokroma NURIA LÓPEZ LORENZO 292 ANNEX II: LISTING OF REAGENTS USED IN THE PRESENT THESIS Reagent Reference Brand 2,2,2-tribromoethanol T48402 Sigma 2-mercaptoethanol M3148 Sigma 2-methyl-2-butanol 152483 Sigma Ampicillin sodium salt A9518 Sigma Anti-β-tubulin antibody MA5-16308 Invitrogen APS (Ammonium persulfate) A6761 Sigma APS (ammonium persulfate) A3678 Sigma Benzonase Nuclease 70746 Millipore Biotin B4501 Sigma BlueSafe MB15201 NZYtech Bromophenol blue 114391 Sigma C0mplete (protease inhibition cocktail) 11697498001 Merck Ca2Cl (Calcium chloride) 2083 Merck Chondroitin sulfate from shark cartilage C4384 Sigma ClNH4 (Ammonium chloride) A9434 Sigma D+Glucose G7021 Sigma Dextran sodium sulfate 6500-10000 D4911 Sigma DNAse 04536282001 Merck E. coli Rossetta (DE3) Competent Cells 69450 Novagen EDTA (Ethylenediaminetetraacetic acid) E9884 Sigma Fe(III)-TMPyP [Fe(III) meso-Tetra (NMethyl-4-Pyridyl) porphine pentachloride] 1-800-364-9897 Cayman Chemical Company Supplementary information 293 Glycerol G9012 Sigma Goat anti-mouse IgG (HRP) Ab6789 Abcam Guanidine - HCl A13543.01 Alfa Aesar H2SO4 (Sulfuric acid) HCl (Chlorhydric acid) 30721 Sigma Heparin sodium from porcine mucosa SRE0027 Sigma Imidazole I2399 Sigma Immobilon Forte Western Substrate WBLUF0100 Millipore IPTG (Isopropil ß-D-1thiogalactopyranoside) IS758 Sigma KCl (Potassium chloride) 9541 Sigma KH2PO4 [Potassium dihydrogen phosphate] 1.04873.1000 Merck L-alanine AL00300100 Scharlau L-arginine AR12500100 Scharlau L-asparagine AS00150100 Scharlau L-aspartic acid AC05290100 Scharlau LB growth media L3022 Sigma L-cysteine CI03050025 Scharlau L-glutamic acid AC12250250 Scharlau L-glutamine GL01650100 Scharlau L-glycine G7126 Sigma L-histidine HI03950100 Scharlau L-isoleucine IS01400025 Scharlau L-leucine LE00550025 Scharlau L-lysine LI00351000 Scharlau NURIA LÓPEZ LORENZO 294 L-methionine 152882.1606 Panreac L-phenylalanine FE01800100 Scharlau L-proline PR00550100 Scharlau L-serine SE01050100 Scharlau L-threonine BP394-100 Fisher Bioreagents L-tryptophan TR04000025 Scharlau L-tyrosine TI03250100 Scharlau L-valine VA00550025 Scharlau Lysozyme from chicken egg white 62980 Sigma MES (morfolino etanesulfonic acid monohydrate) 1.06126.0250 Merck Methanol 20847.360 Normapur Reagents MgCl2 Magnesium chloride MA00360500 Scharlau MgSO4 Magnesium sulfate M7506 Sigma Molecular weight marker 161-0377 BioRad Na2HPO4 (Sodium hydrogen phosphate) 1.06586.1000 Merck NaAcO (Sodium acetate trihydrate) S7670 Sigma NaCl (Sodium chloride) 39781 Serva NaH2PO4 (Sodium hydrogen phosphate monohydrate) 1.06346.0500 Merck NaOH (Sodium hydroxide) 30620 Sigma NaPTA (sodium phosphotungstate dibasic hydrate) P6395 Sigma NiSO4 [Nickel (III) sulfate-6-hydrate] 131445.1209 Panreac NuPAGE LDS Sample Buffer 4x NP0008 Thermo Fisher Scientific NuPAGE MES SDS Running buffer 20x NP0002 Thermo Fisher Scientific Optiprep (Iodixanol density gradient medium) DI556 Sigma Supplementary information 295 Paraformaldehyde, 4% in PBS J61899 AlfaAesar Pefabloc 76307 Sigma Pierce BCA Protein Assay Kit 23227 Thermo Fisher Scientific Pierce ECL Western Blotting Substrate 32106 Thermo Fisher Scientific PMSF (phenylmethylsulfonyl fluoride) 78830 Sigma PNGase F P0704L New England Labs PPS (Pentosan polysulfate sodium) Pronase E from Stretomyces griseus 1.07433.0001 Merck Proteinase K recombinant PCR grade 03115801001 Roche SAF83 A03207 Bertin Bioreagents SAF84 A03208 Bertin Bioreagents Sarkosyl (N-laurosyl salkosine sodium salt) 8.14715.0500 Sigma SDS (sodium dodecyl sulfate) L4509 Sigma Skim milk powder 70166 Sigma SOC medium S1797 Sigma TEMED 1.10732.0100 Millipore TFA (trifluoroacetic acid) 363317 Panreac Thioflavin T (ThT) 8.40072.0005 Merck Tiamine hydrocloride T4625 Sigma Tris Base A1379.1000 Panreac Triton Tx-100 T8787 Sigma Tween-20 P9416 Sigma Uniformly labeled L-phenylalanine CC2000P1 Cortecnet NURIA LÓPEZ LORENZO 302 ANNEX VII: SUPPLEMENTARY IMAGES Figure S1. Survival curve of mice inoculated with 0,1% vole-adapted CWD inoculum and treated chronically with scFv-G1 including the outliers. Figure S2. Comparison of the electrophoretic mobility of five recombinant prion strains inoculated in TgVole mice and 263K. Supplementary information 303 ANNEX VIII: CHROMATOGRAM AND MASS/CHARGE SPECTRA Figure 1. Chromatogram of Hep01 Figure 2. Mass/charge spectrum of Hep01 NURIA LÓPEZ LORENZO 304 Figure 3. Chromatogram of Hep02 Figure 4. Mass/charge spectrum of Hep02 Supplementary information 305 Figure 5. Chromatogram of Hep03 Figure 6. Mass/charge spectrum of Hep03 NURIA LÓPEZ LORENZO 306 Figure 7. Chromatogram of CoS02 Figure 8. Mass/charge spectrum of CoS02 Supplementary information 307 Figure 9. Chromatogram of CoS03 Figure 10. Mass/charge spectrum of CoS03 NURIA LÓPEZ LORENZO 308 Figure 11. Chromatogram of Ust02 Figure 12. Mass/charge spectrum of Ust02 Supplementary information 309 Figure 13. Chromatogram of Ust09 Figure 14. Mass/charge spectrum of Ust09 NURIA LÓPEZ LORENZO 310 Supplementary information 311 ANNEX IX: GAMES-HOWELL’S MULTIPLE COMPARISON POST HOC TEST OF THE CONFORMERS. Table S1. Multiple comparison tests of the conformers from the first passage. Adjusted P Value Summary Significant? 95,00% CI of diff, Mean Diff, Mean 2 Mean 1 Games-Howell's multiple comparisons test 0,0004 *** Yes 79,00 to 207,9 143,4 220 363,4 Hep01 vs. Hep02 <0,0001 **** Yes 227,2 to 289,6 258,4 105 363,4 Hep01 vs. Hep03 0,3946 ns No -84,78 to 250,8 83,03 280,4 363,4 Hep01 vs. CoS01 <0,0001 **** Yes 187,6 to 250,4 219 144,4 363,4 Hep01 vs. CosS02 <0,0001 **** Yes 217,5 to 286,9 252,2 111,2 363,4 Hep01 vs. CoS03 0,2667 ns No -41,59 to 184,1 71,26 292,2 363,4 Hep01 vs. PPS01 <0,0001 **** Yes 190,9 to 253,4 222,1 141,3 363,4 Hep01 vs. PPS02 <0,0001 **** Yes 165,4 to 228,0 196,7 166,7 363,4 Hep01 vs. PPS03 0,0042 ** Yes 49,01 to 181,0 115 105 220 Hep02 vs. Hep03 NURIA LÓPEZ LORENZO 318 Table 3. Multiple comparison tests of the first and second passages of the same conformer. Adjusted P Value Summary Significant? 95,00% CI of diff, Mean Diff, Mean 2 Mean 1 Tamhane's T2 multiple comparisons test <0,0001 **** Yes 247,1 to 307,7 277,4 86 363,4 Hep01 vs. Hep01 0,0012 ** Yes 73,71 to 199,1 136,4 83,6 220 Hep02 vs. Hep02 0,477 ns No -8,441 to 28,77 10,17 94,83 105 Hep03 vs. Hep03 0,0429 * Yes 7,511 to 350,3 178,9 101,5 280,4 CoS01 vs. CoS01 0,0152 * Yes 9,992 to 74,20 42,1 102,3 144,4 CosS02 vs. CosS02 0,0112 * Yes 10,62 to 64,44 37,53 73,67 111,2 CoS03 vs. CoS03 0,0049 ** Yes 75,57 to 297,1 186,3 105,8 292,2 PPS01 vs. PPS01 <0,0001 **** Yes 52,66 to 72,58 62,62 78,67 141,3 PPS02 vs. PPS02 <0,0001 **** Yes 49,55 to 76,88 63,21 103,5 166,7 PPS03 vs. PPS03 Supplementary information 319 ANNEX X: ETHICS COMMITTEE REPORT / INFOME DEL COMITÉ DE ÉTICA 320 321 NURIA LÓPEZ LORENZO 322 ANNEX XI: COPYRIGHT PERMISSIONS Prion diseases are rare and fatal neurodegenerative disorders for which there is no cure or treatment. They are caused by infectious proteins that result from the misfolding of the cellular prion protein, PrPC, into its pathogenic form, PrPSc. Furthermore, PrPSc can appear as conformational variants called strains that exhibit distinct phenotypes and could present resistance to anti-prion compounds. This thesis aims to assess three different therapeutic candidates in a fast murine model of prion disease to tackle these disorders from different aspects of their biology. Additionally, due to the role of prion strains in drug resistance and their biochemical and structural interest, diverse recombinant prion strains were characterized in this thesis using biological, biochemical, and biophysical methods.