Neuronal survival and axonal regeneration after spinal cord injury: the role of neurotransmitters

TESE DE DOUTORAMENTO NEURONAL SURVIVAL AND AXONAL REGENERATION AFTER SPINAL CORD INJURY: THE ROLE OF NEUROTRANSMITTERS DANIEL SOBRIDO CAMEÁN ESCOLA DE DOUTORAMENTO INTERNACIONAL PROGRAMA DE DOUTORAMENTO EN NEUROCIENCIA E PSICOLOXÍA CLÍNICA SANTIAGO DE COMPOSTELA 2019 AGRADECEMENTOS DANIEL SOBRIDO CAMEÁN teu equipo, gracias por abrirme as portas do teu grupo para o futuro, gracias por facer que me plantease tantas preguntas sobre a ciencia e o meu futuro. Gracias a Matt por estar sempre disposto a axudarme en todo e por introducirme ao traballo nun laboratorio con moscas, gracias a Eva por axudarme sempre que o necesitei e por esa simpatía, gracias ás demais persoas do lab (Serene, Yi Xing and Hwei Minn) e ao demais do departamento de zooloxía, especialmente ao grupo de Jimena, todo o equipo é fantástico; e aos compañeiros da tea room. Ao persoal docente: Durante a carreira e máster tiven brillantes docentes pero por mencionar un, Francisco Martín Cora, quen me deu Neurobioloxía, unha forma de ensinar que fomenta moitísimo o traballo persoal dos alumnos e que conseguisches elevar en escala exponencial, o meu amor pola neurociencia. Outros docentes que tiven durante a miña vida e que quero mencionar, sen desmerecer aos demais, son Leonor, tutora en 5º e 6º de primaria, soubeches aumentar as ansias de saber e entender o mundo que xa habitaban dentro de min; a Anxela, profesora de filosofía no instituto, por ensinarme a pensar razoando; a Teresa, profesora de física; a Xulio César, profesor de bioloxía, que intensificastes o meu amor pola bioloxía; Margarita, profesora de historia, despois de toda a vida querendo ser biólogo entroume a dubida, xusto no momento de decidir carreira, gracias por axudarme a tomar unha boa decisión. Ás outras amizades: Moitas gracias a todas as persoas que me acompañaron e me acompañan polo camiño da vida. Gracias ás amizades todas que forman parte da miña vida, presente ou pasada. Aos “Monte Reinlles” e demais amizades que forman parte que gran grupo chegamos a formar. A Rocío (Pose) a primeira (ou das primeiras) persoas que coñecín ao empezar a carreira e dende entón a amizade sempre foi a máis. Entre as primeiras amizades da carreira están tamén María; Aldara, Roi, Miriam e Miguel (os mellores veciños que se pode ter); Paco, que camiñatas de praia e que comilonas nos temos dado; a Elisa, que momentazos de reirnos a dolor, “amantes de Camela” e concertos de rock...jajajajaja...que momentos, que tantas charlas, especialmente sobre a nosa afección (as plantas), gracias por esas cenas de “ensalada estilo Elisa” que gusto chegar de traballar e ter a cea lista, e gracias tamén polas natillas de leite de Paloma....manxar de deuses; e AGRADECEMENTOS Emilio, lémbrome do día que che coñecín, que personaxe único, non tes remedio pero non hai nada que remediar, quéreseche como es. Que ganas de unha reunión/festa con todos vos. Creo que nunca o dixen pero sodes parte importantísima de min. Que sorte coñecervos. Hai tanta xente que me gustaría mencionar...compañeiras de piso: Leti, Alba (Rodríguez), Lidia, Inés....gracias. Amistades da infancia: Mª José, Álvaro, Miguel, as supernenas (Elisa, Gema, Magdalena) que ben o temos pasado e pasa o tempo e os camiños que seguimos son distintos e aparece a distancia pero a amizade queda para sempre. Aos amigos de máster! Menudo grupazo somos, que sorte coincidir con todes vos, TODES, que ben o pasamos. Vanesa, es a mellor e sempre me acordarei de como me salvastes aquela semana, gracias; Uxía, que tan fácil se che colle cariño; ás miñas “zorritas”...jajaja...Mari e Clara, que maneira tan peculiar de empezar a levarnos ben. Clara, que tantos momentos compartidos, gracias por ser como es, quéroche moito! Á familia: Menos mal que a familia é a que nos toca e non a eliximos porque nin en mil vidas podería deseñar unha familia mellor. Que sorte teño e que orgullo ser parte desta familia. Tíos e tías, primeiros, segundos terceiros, etc, de sangue ou políticos, primos e primas...segundos, terceiros, etc, especialmente as/os curmáns: Que ben o temos pasado! Virginia, Rosina, Jorge, Vanesa, e imos aumentando o equipo...Alberto e Valeria. Bisavoas, nais ao cubo, Ramona, Manuela e Pepa, unha vida de loita, exemplos de fortaleza, sufrir a morte de seres queridos é unha dor brutal pero que sorte que cheguei a coñecervos. Avós e avoas, outros catro exemplos de modelos a seguir, persoas traballadoras, incansables e que pasaron o impensable para seguir para diante, quérovos moito. Gracias! Avoa María, tiven que aprender a vivir sen ti e, que difícil é, non hai día que non me acorde de ti, daría o que fose por falar un ratiño contigo outra vez e contarche como me vai. Bótoche de menos. Alba (Sayáns), non sei como definir o que es para min...¿amiga? sería quedarse corto, ¿unha parte de min con vida propia?, ¿es para min o que o aire para os demais humanos? chámalle “x”. Gracias a teus pais por facerche e a ti por existires. Papá e mamá, non lle quedan suficientes anos a este universo para ter tempo a devolvervos algo de todo o que facedes por min. Sempre estades aí para todo, infalibles, DANIEL SOBRIDO CAMEÁN capaces de dar a vida por nos (inclúo a Laura) se fixera falta. Gracias! E gracias tamén por faceres a Laura, isto supera todo o que ninguén pode facer por min nunca. Laura, última persoa que menciono pero a máis importante. ¿Como pode quedar sorte no mundo se a acabei eu toda téndoche como irmán? Es o mellor que me pasou na vida. Quéroche infinito! AGRADECEMENTOS Gracias tamén a: Rita Levi Montalcini Santiago Ramón y Cajal “Yo por qué voy a ser distinta a los setenta que el día siguiente que tengo setenta más un día. Yo considero que la jubilación es discriminatoria” Margarita Salas FINANCIACIÓN A investigación realizada en esta tese foi financiada por: • Study of the changes in neurotransmitter systems during spinal regeneration in lampreys. Spanish Ministry of Science and Innovation (BFU2010-17174/BFI). Principal investigator: María Celina Rodicio Rodicio. Duration: 4 years (1/1/201131/12/2014). Funding: 148,830 euros. • Research group consolidation grant of the Galician Government 2014. Xunta de Galicia (Grant number: GPC2014/030). Principal investigator: Victor Manuel Arce. Duration: 2.5 years (24/06/2014-31/12/2016). Funding: 70,000 euros. • Role of GABA in the survival and regeneration of spinal-projecting brain neurons after spinal cord injury in lampreys. Spanish Ministry of Economy and Competitiveness (BFU2014-56300P). Principal investigator: María Celina Rodicio. Duration: 3 years (01/01/2015-31/12/2017). Funding: 133,100 euros. • Role of neurotransmitters in axon regeneration following spinal cord injury: serotonin and a novel in vivo drug screen. Xunta de Galicia (Grant number: 2016-PG008). Principal investigator: Antón Barreiro-Iglesias. Duration: 2 years (01/08/201631/07/2018). Funding: 20,000 euros. • Searching for drugs to promote axonal regeneration following spinal cord injury. Crowdfunding campaign in the platform Precipita of the FECYT, Spanish Ministry of Economy and Competiveness, Spain (Grant number: 2017-CP0081). Principal Investigator: Antón Barreiro-Iglesias. Duration: 1 year (07/2017-07/2018). Funding: 11,620 euros. The project was the most visited and most supported project during its period in the public platform Precipita. • GABA promotes neuronal survival and axonal regeneration following spinal cord injury by inhibiting the Notch pathway. Spanish Ministry of Economy and Competitiveness (BFU2017-87079-P). Principal investigators: María Celina Rodicio and Antón Barreiro Iglesias. Duration: 3 years (01/01/2018-31/12/2020). Funding: 114,950 euros. A realización da estancia predoutoral no laboratorio do Dr. Mathias Langraf foi financiada pola European Molecular Biology Organization (EMBO): • EMBO Short-Term Fellowship. Duration 3 months (01/07/2019-31/09/2019). Funding: 8,990 euros. Daniel Sobrido Cameán ARTIGOS DERIVADOS DA TESE As publicacións realizadas durante a miña etapa predoutoral son: As 6 publicacións que forman da tese: Barreiro-Iglesias A, Sobrido-Cameán D, Shifman MI. (2017). Retrograde activation of the extrinsic apoptotic pathway in spinal-projecting neurons after a complete spinal cord injury in lampreys. BioMed Research International. 2017:5953674. doi: 10.1155/2017/5953674 Sobrido-Cameán D, Rodicio MC, Barreiro-Iglesias A. (2018). The role of serotonin in nervous system regeneration: lessons from regenerating animal models. Neural Regeneration Research. 13, 237-238. doi: 10.4103/1673-5374.226387. Sobrido-Cameán D, Barreiro-Iglesias A. (2018). Role of caspase-8 and Fas in cell death after spinal cord injury. Frontiers in Molecular Neuroscience. 3, 11:101. doi: 10.3389/fnmol.2018.00101. Sobrido-Cameán D, Rodicio MC, Barreiro-Iglesias A. (2018). Data on the effect of a muscimol treatment in caspase activation in descending neurons of lampreys after a complete spinal cord injury. Data in Brief. 21, 2037-2041. doi: 10.1016/j.dib.2018.11.003. Sobrido-Cameán D, Robledo D, Sánchez L, Rodicio MC, Barreiro-Iglesias A. (2019). Serotonin inhibits axonal regeneration of identifiable descending neurons after a complete spinal cord injury in lampreys. Disease Models and Mechanisms. 12(2). pii: dmm037085. doi: 10.1242/dmm.037085. Sobrido-Cameán D, Fernández-López B, Pereiro N, Lafuente A, Rodicio MC, BarreiroIglesias A. Taurine promotes axonal regeneration after a complete spinal cord injury in lampreys. Journal of Neurotrauma. doi: 10.1089/neu.2019.6604. Outras publicacións: Romaus-Sanjurjo, D, Fernández-López, B, Sobrido-Cameán, D, Barreiro-Iglesias, A, Rodicio, MC. (2017). Cloning of the gabaB Receptor Subunits B1 and B2 and their Expression in the Central Nervous System of the Adult Sea Lamprey. Frontiers in Neuroanatomy. 10:118. doi: 10.3389/fnana.2016.00118 Barreiro-Iglesias A, Fernández-López B, Sobrido-Cameán D, Anadón R. (2017). Organization of Alpha-Transducin Immunoreactive System in the Brain and Retina of Larval and Young Adult Sea Lamprey (Petromyzon marinus), and their Relationship with Other Neural Systems. Journal of Comparative Neurology. 525, 3683-3704. doi: 10.1002/cne.24296 Fernández-López B, Sobrido-Cameán D, Anadón R, Rodicio MC, Barreiro-Iglesias A. (2017). Restricted co-localization of glutamate and dopamine in neurons of the adult sea lamprey brain. Journal of Anatomy. 231, 776-784. doi: 10.1111/joa.12674 Sobrido-Cameán D, Yáñez-Guerra LA, Lamanna F, Conde-Fernández C, Kaessmann H, Elphick MR, Anadón R, Rodicio MC, Barreiro-Iglesias A. (2019). Galanin in an agnathan: precursor identification and localisation of expression in the brain of the sea lamprey Petromyzon marinus. Journal of Neuroanatomy. Daniel Sobrido Cameán Da Silva-Álvarez S, Guerra-Varela J, Sobrido-Cameán D, Quelle A, Barreiro-Iglesias A, Sánchez L, Collado M. (2019). Cell senescence contributes to tissue regeneration in Zebrafish. Aging Cell. CONTENTS DANIEL SOBRIDO CAMEÁN characteristic of lampreys as an animal model in neuroscience is that, in contrast to mammals, lampreys spontaneously recover locomotion after a complete SCI (see Rodicio and Barreiro-Iglesias, 2012). Lampreys have been used since the 1970's as a model system for studying the recovery of locomotor function after SCI (Parker, 2017). Brain descending neurons of lampreys include 36 identifiable giant reticulospinal neurons (Jacobs et al., 1997). These include the Mauthner neurons and several pairs of Müller cells. Interestingly, these identifiable descending neurons vary greatly in their survival and regenerative abilities (Jacobs et al., 1997; Shifman et al., 2008). Thus, in lampreys, there is an opportunity to study both enhancement and inhibition of death and/or regeneration in the same preparation. An additional advantage of the lamprey model of SCI is that the identifiable descending neurons and their descending axons can be visualised in vivo and in CNS whole-mounts due to the transparency of the lamprey brain. Thus, the lamprey is a convenient vertebrate model for the in vivo study of the mechanisms underlying the death/survival and/or regeneration of spinal-projecting neurons after SCI. The recovery process of lampreys following SCI involves the regeneration of descending spinal axons (Jacobs et al., 1997) and the formation of synaptic connections between the regenerated axons and neurons caudal to the lesion (Rovainen, 1976; Selzer, 1978; Wood and Cohen, 1979). However, regeneration of descending axons in lampreys is incomplete (Becker and Parker, 2015). So, changes are necessary in the connectome above and below the lesion site to compensate the reduced number of descending inputs (Parker, 2017). Different neurotransmitter systems are involved in plastic changes after a SCI and adjust in different ways to the post-injury situation: serotonergic (Cohen et al., 2005; Cornide-Petronio et al., 2014), GABAergic (Svensson et al., 2013; Fernández- GENERAL INTRODUCTION 5 López et al., 2014; Romaus-Sanjurjo et al., 2018a), glutamatergic (Fernández-López et al., 2016), dopaminergic (Fernández-López et al., 2015) and glycinergic (Valle-Maroto, 2017) systems. More recently, our group also showed that endogenous GABA promotes neuronal survival and axon re-growth after a complete SCI in lampreys (Romaus-Sanjurjo et al., 2018b). Thus, neurotransmitters seem to play important roles in recovery and regeneration after SCI. We aim to understand important aspects of the basic biology responsible for the amazing regenerative capacity of lampreys as compared to mammals. Understanding the molecular processes that are responsible for the recovery of function after SCI in lampreys will serve as a basis to open new research lines in mammalian pre-clinical models and to design new therapies for humans with spinal injuries. The general aims proposed to be developed in this Thesis project were: 1. To advance on our knowledge on the process of the cell death after SCI. It has been reported that a complete SCI induces delayed death of lamprey identifiable descending neurons (Shifman et al., 2008). Evidence for cell death included the disappearance of Nissl staining, the loss of neurofilament expression, the absence of labelling when using retrograde tracers (Shifman et al., 2008), and the earlier staining of these neurons with Fluoro-Jade C, a marker for degenerating neurons, (Busch and Morgan, 2012). The appearance of TUNEL staining (Shifman et al., 2008; Hu et al., 2013) and activated caspases (Barreiro-Iglesias and Shifman, 2012; Hu et al., 2013; Zhang et al., 2014) in the axotomized perikarya suggested that the death of these bad-regenerating neurons in lampreys was apoptotic. However, we do not yet know whether there is or not a correlation between caspase activation and the regeneration/death of descending DANIEL SOBRIDO CAMEÁN neurons in lampreys and which specific signalling pathway/s are involved in the death of these neurons. 2. To study the possible role of the GABAA receptors in caspase activation in descending neurons following SCI. As in mammals, there is a massive release of glutamate, GABA and glycine from most of the spinal neurons close to the lesion site in lampreys after SCI (Fernández-López et al., 2014). Interestingly, between 1 and 3 days after the injury there is an accumulation of GABA around some axotomized axons of descending neurons (Fernández-López et al., 2014). GABA accumulation correlated with a higher survival ability of the corresponding identifiable descending neurons (Fernández-López et al., 2014). Moreover, there is a correlation between the presence of increased GABA inhibition and a better recovery of function in spinal lesioned lampreys (Svensson et al., 2013). More recently, our group showed that endogenous GABA promotes axon re-growth in descending neurons, which could be caused by the inhibition of caspase activation in these neurons (Romaus-Sanjurjo et al., 2018b). The GABA effects appear to be mediated, at least in part, by the activation of GABAB receptors expressed in descending neurons. However, the role of the GABAA receptors in neuronal death after SCI in lampreys is not known. 3. To study the role of 5-HT in axon regeneration following SCI. The first report showing that 5-HT could be involved in neurite outgrowth came in 1984 (Haydon et al., 1984). Haydon and coworkers (1984) demonstrated that growth cones and elongating neurites of the snail Helisoma trivolvis neurons are inhibited by the application of 5-HT. The effect of 5-HT seems to be different at different developmental GENERAL INTRODUCTION 7 stages, because the comparison between the effect of application 5-HT on embryonic and adult H. trivolvis neurons shows the same inhibitory effects on actively growing neurites; however, 5-HT could reinitiate neurite elongation in non-growing neurites in embryos, but not in non-growing neurites from adults (Goldberg et al., 1991). More recently, a study showed that the application of a selective 5-HT reuptake inhibitor (fluoxetine) on snail neurons (serotonergic or non-serotonergic) inhibits neurite formation and induced growth cone collapse and neurite retraction (Xu et al., 2010). In mammals, a similar effect of 5-HT has been reported. The application of fluoxetine on rat cultured cortical neurons decreased neurite outgrowth (Xu et al., 2010). Moreover, the neurite length of cultured neurons from foetal rats decreases after the application of 5-HT (Sikich et al., 1990). In contrast, there are other reports in mammalian models whose results show the reverse effect of 5-HT. For example, the in vivo application of DL-P-chlorophenylalanine methyl ester hydrochloride (PCPA), an inhibitor of 5-HT synthesis, to embryonic rats reduces dendritic complexity in pyramidal neurons (Vitalis et al., 2007). The application of 5-HT to cultured rat neurons induced outgrowth of secondary neurites in embryonic neurons (Rojas et al., 2014) and in neurons from new-born rats (Whitaker-Azmitia and Azmitia, 1989). Interestingly, 5-HT application over ex vivo cerebellar slices from rats increases or decreases dendritic areas depending on the region and concentration (Kondoh et al., 2004). The heterogeneous responses to 5-HT supplementation or inhibition could be explained by the heterogeneity of 5-HT receptors depending on developmental stages and nervous system region. Despite the evident role of 5-HT in axon growth during development or in in vitro studies, much less is known about its possible effect on axon regeneration after CNS damage. DANIEL SOBRIDO CAMEÁN 4. To study the role of taurine on axonal regeneration following SCI. Taurine is one of the most abundant free amino acids in the brain. It is well known that taurine protects the brain from further damage after a traumatic event. Interestingly, methylprednisolone, which is the only pharmacological therapy approved for the treatment of traumatic SCI, enhances axon regeneration after SCI (Chen et al., 1996, Nash et al., 2002) and is known to cause an increase in the concentration of taurine in the spinal cord (Benton et al., 2001). In vertebrates, taurine can act as an agonist of a variety of neurotransmitter receptors, including GABA receptors (Albrecht and Schousboe, 2005). Recent work of our group has shown that endogenous GABA promotes axonal regeneration of identifiable reticulospinal neurons following SCI in lampreys (see above). These data led us to study the possible effect of taurine in axon regeneration after SCI in lampreys. To tackle these general aims, we defined the following specific aims: 1. Study if caspase activation in lamprey giant identifiable reticulospinal neurons correlates with their survival and regenerative abilities following SCI. 2. Propose a possible signalling pathway regulating the death of reticulospinal neurons following SCI using the results of the previous objective and the revision of existing literature. 3. Study the possible role of GABAA receptors in caspase activation in giant reticulospinal neurons of lampreys following SCI. 4. Compile the current knowledge on the role of 5-HT in neuronal regeneration as a basis for the study of the role of 5-HT in axonal regeneration following SCI in lampreys. 5. Study the role of 5-HT in axonal regeneration following SCI in lampreys GENERAL INTRODUCTION 9 6. Study the role of taurine in axonal regeneration following SCI in lampreys. The present Thesis has been organized in 6 chapters corresponding to the publications derived from the results obtained pursuing each specify aim. The first chapter corresponds to the specific aim 1. Here, a significant correlation between levels of activated caspases 2 weeks after SCI and the regenerative ability of giant identifiable neurons of lampreys is shown. The second chapter corresponds to the specific aim 2. Here, we hypothesize, based on the available literature, that Fas/caspase-8 signalling could be an important player in the degeneration of reticulospinal neurons after SCI in vertebrates. The third chapter corresponds to the specific aim 3. Here, the effect on caspase activation of a treatment with a specific GABAA receptor agonist (muscimol) is shown. A single dose of muscimol reduces the level of activated caspases 2 weeks after a complete SCI. The fourth chapter corresponds to the specific aim 4. A revision of the knowledge about the role of 5-HT in neuronal regeneration is presented. Here, we propose the possibility that 5-HT has a role in the regeneration of axons after SCI. The fifth chapter corresponds to specific aim 5. Here, we present data showing that endogenous 5-HT inhibits axonal regeneration of identifiable descending neurons after a complete spinal cord injury in lampreys. Moreover, we show that changes in the expression of genes that control axonal guidance process could be a key factor determining the 5-HT effects during regeneration. The sixth chapter corresponds to specific aim 6. Here, we present data showing that taurine promotes axon regeneration following a complete SCI in lampreys. DANIEL SOBRIDO CAMEÁN References Ahuja CS, Nori S, Tetreault L, Wilson J, Kwon B, Harrop J, Choi D, Fehlings MG. 2017. Traumatic Spinal Cord Injury-Repair and Regeneration. Neurosurgery. 80:S9S22. Albrecht J, Schousboe A. 2005. Taurine interaction with neurotransmitter receptors in the CNS: an update. Neurochem Res. 30:1615-1621. Barreiro-Iglesias A, Shifman MI. 2012. Use of fluorochrome-labeled inhibitors of caspases to detect neuronal apoptosis in the whole-mounted lamprey brain after spinal cord injury. Enzyme Res. 2012:835731. Becker MI, Parker D. 2015. Changes in functional properties and 5-HT modulation above and below a spinal transection in lamprey. Front Neural Circuits. 8:148. Benton RL, Ross CD, Miller KE. 2001. Spinal taurine levels are increased 7 and 30 days following methylprednisolone treatment of spinal cord injury in rats. Brain Res. 893:292-300. Busch DJ, Morgan JR. 2012. Synuclein accumulation is associated with cellspecific neuronal death after spinal cord injury. J Comp Neurol. 520:1751-1771. Chen A, Xu XM, Kleitman N, Bunge MB. 1996. Methylprednisolone administration improves axonal regeneration into Schwann cell grafts in transected adult rat thoracic spinal cord. Exp Neurol. 138:261-276. GENERAL INTRODUCTION 11 Cohen AH, Abdelnabi M, Guan L, Ottinger MA, Chakrabarti L. 2005. Changes in distribution of serotonin induced by spinal injury in larval lampreys: evidence from immunohistochemistry and HPLC. J Neurotrauma. 22:172-188. 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Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis. J Neurobiol. 22:182194. Haydon PG, McCobb DP, Kater SB. 1984. Serotonin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons. Science. 226:561-564. DANIEL SOBRIDO CAMEÁN Hu J, Zhang G, Selzer ME. 2013. Activated caspase detection in living tissuecombined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains. J Neurosci Methods. 220:92-98. Jacobs AJ, Swain GP, Snedeker JA, Pijak DS, Gladstone LJ, Selzer ME. 1997. Recovery of neurofilament expression selectively in regenerating reticulospinal neurons. J Neurosci. 17:5206-5220. Kondoh M, Shiga T, Okado N. 2004. Regulation of dendrite formation of Purkinje cells by serotonin through serotonin 1A and serotonin 2A receptors in culture. Neurosci Res. 48:101-109. Kuratani S, Kuraku S, Murakami Y. 2002. Lamprey as an evo-devo model: lessons from comparative embryology and molecular phylogenetics. Genesis. 34:175183. Langille RM, Hall BK. 1988. The organ culture and grafting of lamprey cartilage and teeth. In Vitro Cell Dev Biol. 24:1-8. Nash HH, Borke RC, Anders JJ. 2002. Ensheathing cells and methylprednisolone promote axonal regeneration and functional recovery in the lesioned adult rat spinal cord. J Neurosci. 22:7111-7120. Osório J, Rétaux S. 2008. The lamprey in evolutionary studies. Dev Genes Evol. 218:221-235. Parker D. 2017. The Lesioned Spinal Cord Is a "New" Spinal Cord: Evidence from Functional Changes after Spinal Injury in Lamprey. Front Neural Circuits. 11:84. GENERAL INTRODUCTION 13 Ramer LM, Ramer MS, Bradbury EJ. 2014. Restoring function after spinal cord injury: towards clinical translation of experimental strategies. Lancet Neurol. 13:12411256. Rodicio MC, Barreiro-Iglesias A. 2012. Lampreys as an animal model in regeneration studies after spinal cord injury. Rev Neurol. 55:157-166. Rojas PS, Neira D, Muñoz M, Lavandero S, Fiedler JL. 2014. Serotonin (5-HT) regulates neurite outgrowth through 5-HT1A and 5-HT7 receptors in cultured hippocampal neurons. J Neurosci Res. 92:1000-1009. Romaus-Sanjurjo D, Ledo-García R, Fernández-López B, Hanslik K, Morgan JR, Barreiro-Iglesias A, Rodicio MC. 2018b. GABA promotes survival and axonal regeneration in identifiable descending neurons after spinal cord injury in larval lampreys. Cell Death Dis. 9:663. Romaus-Sanjurjo D, Valle-Maroto SM, Barreiro-Iglesias A, Fernández-López B, Rodicio MC. 2018a. Anatomical recovery of the GABAergic system after a complete spinal cord injury in lampreys. Neuropharmacology. 131:389-402. Rovainen CM. 1976. Regeneration of Müller and Mauthner axons after spinal transection in larval lampreys. J Comp Neurol. 168:545-554. Selzer ME. 1978. Mechanisms of functional recovery and regeneration after spinalcord transection in larval sea lamprey. J Physiol. 277:395-408. Shifman MI, Zhang G, Selzer ME. 2008. Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys. J Comp Neurol. 510:269-282. DANIEL SOBRIDO CAMEÁN First, we demonstrated that in identifiable descending neurons of lampreys there is a significant correlation between the intensity of caspase activation 2 weeks following a complete SCI with their long-term survival and regenerative abilities (chapter 1). Thus, we validated that measurements of intensity of caspase activation 2 weeks after a complete SCI are a good approach to predict the survival/regenerative ability of neurons after axotomy. In all vertebrates, SCI causes the death of neurons and glial cells at the site of injury. This is due to the initial mechanical forces and through a cascade of secondary molecular events that exacerbate cell death (Ahuja et al., 2017). These secondary injury pathways include the ischemic cascade, inflammation and neurotransmitter imbalances. During the secondary injury, apoptotic processes are activated in neurons and glia. However, there is some controversy about retrograde neuronal of death of spinal projecting neurons of the brain following SCI in mammals (see chapter 2). Several studies have shown the death of brain neurons after SCI in mammals, including humans (see chapter 2). However, other studies in rodents showed that spinal projecting surviving neurons were significantly smaller in size after SCI than uninjured neurons but did not detect cell death (McBride et al., 1989; Kwon et al., 2002; Nielson et al., 2011). In lampreys, reticulospinal neurons clearly die after a complete SCI by a delayed caspasemediated apoptosis (Shifftman et al., 2008; Barreiro-Iglesias and Shifman, 2012, 2015; Hu et al., 2013; Chapter 1, 2 and 3). Our review article (chapter 2) shows that caspase-8 could play an important role in these processes (neuronal death or atrophy). In chapter 3 we showed that an acute treatment with muscimol, a GABAA receptor agonist, inhibits caspase activation 2 weeks after a complete SCI. Based on our knowledge of the correlation between caspase activation and axon regeneration, we are tempted to hypothesize that a treatment with muscimol might also result in an GENERAL DISCUSION 93 improvement in axon regeneration of descending neurons after SCI, although this should be experimentally tested in the future. Results previously reported by our group demonstrated that endogenous GABA promotes the regeneration of giant identifiable reticulospinal neurons after a complete SCI by acting through GABAB receptors (Romaus-Sanjurjo et al., 2018). Present results start to extend these analyses to GABAA receptors. The muscimol results suggest that the activation of GABAA receptors might also promote survival and axon regeneration in descending neurons. Interestingly, in chapter 6, we showed that taurine promotes axon regeneration following a complete SCI. Taurine can act as an agonist of GABAA receptors (Albretch et al., 2005), which further supports the idea that GABAA receptor activation could promote not only neuronal survival but also axon regeneration. Results of this thesis show that, in a regenerating vertebrate, endogenous 5-HT inhibits axon regeneration in descending neurons after SCI (chapter 5). It is known that neurotransmitters activate various signal transduction pathways. Cyclic adenosine 3′,5′- monophosphate (cAMP) is a second messenger molecule that plays fundamental roles in cellular responses to neurotransmitters (Greengard, 1976). 5-HT regulates the intracellular levels of cAMP by the activation of 5-HT receptors (Prasad et al., 2019; chapter 5). Different studies have revealed that cAMP modulates axon growth after SCI (Cai et al., 2001; Neumann et al., 2002; Qiu et al., 2002; Lu et al., 2004; Nikulina et al., 2004; Pearse et al., 2004 a, b; Whitaker et al., 2008; Jin et al., 2009; Costa et al., 2013; Lau et al., 2013; Pale et al., 2013; Qi et al., 2019; Chapter 4, 5). Interestingly, several studies have also shown that cAMP can play important roles in neuroprotection (Rydel and Greene, 1988; Hanson et al., 1998; Cui and So, 2004; see Silveira and Linden, 2006; Lau et al., 2013). So, in the future it would be of interest to analyse the 5-HT effects on DANIEL SOBRIDO CAMEÁN neuronal survival after SCI (see above). Recent studies show that 5-HT plays distinct roles in the process of neuronal regeneration after SCI (see chapter 4). For example, in zebrafish, endogenous 5-HT promotes motor neuron production in the spinal cord after a complete SCI by enhancing the proliferation of motor neuron progenitor cells (BarreiroIglesias et al., 2015); in turtles, 5-HT inhibits the emergence of serotonergic interneurons after SCI by inhibiting a change in neurotransmitter phenotype of non-serotonergic neurons (Fabbiani et al., 2018). For all of this, we suggest that 5-HT could be a target of interest in non-regenerating mammalian models of SCI, since it can modulate several aspects of the regenerative process (chapter 5). We studied molecular pathways involved in neuroprotection (chapter 1, 2, 3) and axon regeneration (chapter 4, 5, 6) following a complete SCI in lampreys. This work further suggests that a given signalling pathway can influence both neuroprotection and axon regeneration after axotomy. Recent studies from other groups have also revealed that molecular mechanisms that reduce neuronal apoptosis also promote neuron regeneration (Boczek et al., 2019; Lei et al., 2019). Our group recently reported that GABA and baclofen treatment inhibits caspase activation and promote axon regeneration following SCI in lampreys (Romaus-Sanjurjo et al., 2018). Here, we have obtained additional information about the role of neurotransmitters in spinal cord regeneration following complete SCI. This thesis corroborates that lampreys constitute a reliable model for the study of the molecular mechanisms that underlie spontaneous recovery after SCI. Moreover, these results establish a solid basis for the study of new therapies for the regeneration of the mammalian spinal cord after injury. Translation of all this knowledge to pre-clinical studies is of obvious and crucial importance. GENERAL DISCUSION 95 References Ahuja CS, Nori S, Tetreault L, Wilson J, Kwon B, Harrop J, Choi D, Fehlings MG. 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PLoS One 14: e0206069. CHAPTER 1 RETROGRADE ACTIVATION OF THE EXTRINSIC APOPTOTIC PATHWAY IN SPINAL-PROJECTING NEURONS AFTER A COMPLETE SPINAL CORD INJURY IN LAMPREYS https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733621/pdf/BMRI 2017-5953674.pdf CHAPTER 5 SEROTONIN INHIBITS AXONAL REGENERATION OF IDENTIFIABLE DESCENDING NEURONS AFTER A COMPLETE SPINAL CORD INJURY IN LAMPREYS https://dmm.biologists.org/content/12/2/dmm037085.long CHAPTER 6 TAURINE PROMOTES AXONAL REGENERATION AFTER A COMPLETE SPINAL CORD INJURY IN LAMPREYS https://www.liebertpub.com/doi/abs/10.1089/neu.2019.6604 Note: My contribution to this article is the experiments related to “taurine treatments” 101 CONCLUSIONS 102 CONCLUSIONS 103 1. Caspase activation in lamprey giant identifiable reticulospinal neurons correlates, negatively, with their survival ability following a complete SCI. 2. Caspase activation in lamprey giant identifiable reticulospinal neurons correlates, negatively, with their regenerative ability following a complete SCI. 3. Fas/Caspase-8 is an important signalling pathway regulating neuronal death following SCI. 4. Muscimol, a GABAA receptor agonist, reduces caspase activation in giant reticulospinal neurons of lampreys following a complete SCI. 5. 5-HT is an important modulator of neuronal regeneration in invertebrate and vertebrate species. 6. Endogenous 5-HT inhibits axonal regeneration following a complete SCI in lampreys. 7. Taurine promotes axonal regeneration following a complete SCI in lampreys. ´ DANIEL SOBRIDO CAMEÁN 105 RESUMO DANIEL SOBRIDO CAMEÁN axóns a partir de estudos in vitro, ex vivo e in vivo en diferentes especies (por exemplo, invertebrados e peixes). Esta revisión mostra que a 5-HT xoga un papel importante tanto na modulación do re-crecemento dos axóns como na xeración de novas neuronas despois da lesión do sistema nervioso en especies con capacidade para rexenerar. Estudos de rexeneración en modelos como invertebrados e peixes están a revelar un papel crucial da 5-HT na modulación de procesos rexeneradores tras lesións traumaticas do sistema nervioso. Non obstante, necesítase máis estudios para comprender plenamente o papel que xogan diferentes receptores de neurotransmisores neste proceso. É necesario o uso de ferramentas xenéticas ou fármacos máis específicos para descifrar a contribución de cada receptor de 5-HT na rexeneración axónica ou neuronal. Futuros estudios debería intentar trasladar os resultados en especies con capacidade rexeneradora a modelos “non rexeneradores”. Unha vez que teñamos unha imaxe clara do papel específico da 5-HT e de cada un dos seus receptores na rexeneración, a posible tradución deste coñecemento á clínica será facilitada pola existencia de medicamentos serotonérxicos que xa están en uso en pacientes humanos con outras problemas. No capítulo 5 utilizamos o modelo de lamprea en lesión medular para estudar o efecto da 5-HT na rexeneración dos axóns das neuronas descendentes identificables. As manipulacións farmacolóxicas e xenéticas tras unha SCI completa demostraron que a 5HT endóxena inhibe a rexeneración axonal en neuronas descendentes identificables mediante a activación dos receptores da serotonina 1A e unha posterior diminución dos niveis de cAMP. A secuenciación de ARN revelou que os cambios na expresión de xenes que controlan o crecemento dos axóns poderían ser un factor clave nos efectos da 5-HT durante a rexeneración. Este capítulo proporciona unha base para novos estudios en RESUMO 113 modelos de SCI de mamíferos non rexeneradores e amplía as funcións coñecidos da sinalización de 5-HT durante a rexeneración neuronal. Para revelar o efecto do 5-HT na rexeneración de neuronas descendentes identificables, as lampreas tratáronse con 5-HT durante un mes despois dunha SCI completa. 11 semanas despois da lesión vimos que, o tratamento con 5-HT, inhibiu significativamente a rexeneración das neuronas descendentes identificables das lampreas. É importante destacar que as análises de comportamento revelaron que o tratamento con 5-HT non causaron un efecto tóxico xeral xa que a recuperación locomotora non se viu afectada polo tratamento con 5-HT. Ademais, levamos a cabo un experimento de “rescate” no que os animais tratados con 5-HT como anteriormente tamén foron tratados con dibutiril-cAMP (db-cAMP). O tratamento db-cAMP bloqueou o efecto inhibidor do 5-HT. Os nosos experimentos de hibridación in situ para estudiar o transcrito do receptor de 5-HT1A revelaron que en neuronas malas rexeneradoras hai un aumento significativo na expresión do receptor 5-HT1A 4 semanas despois da SCI completa, mentres que nas neuronas boas rexeneradoras (neuronas M1 e I3) a expresión do receptor diminúe (non significativamente) nas primeiras semanas despois dunha SCI completa. O análise estatísticos revelou unha correlación significativa entre a porcentaxe de aumento/diminución da expresión do receptor e a capacidade rexenerativa a longo prazo de neuronas identificables. Estes datos suxiren que a presenza e actividade do receptor 5HT1A en neuronas descendentes pode inhibir a rexeneración axónica despois dunha SCI completa. Tamén tratamos a outro grupo de animais durante 4 semanas despois dunha SCI completa co antagonista do receptor 5-HT1A WAY-100,135. O tratamento con WAY- DANIEL SOBRIDO CAMEÁN 100.135 promoveu significativamente a rexeneración dos axóns en neuronas identificables en comparación cos animais tratados con vehículos control. Para confirmar que o efecto inhibidor da 5-HT endóxena débese á activación dos receptores 5-HT1A expresados en neuronas descendentes identificables, decidimos inhibir especificamente a expresión do receptor nestas neuronas empregando morfolinos dirixidos contra o sitio de iniciación da tradución. do receptor 5-HT1A. A aplicación deste morfolino promoveu significativamente a rexeneración dos axóns de neuronas identificables tras unha SCI completa. Como control da especificidade empregouse un segundo morfolino dirixido á rexión non traducida de 5 'do ARNm 5-HT1a (non solapada co primeiro morfolino). A aplicación deste segundo morfolino promoveu significativamente a rexeneración de axóns de neuronas identificables. Tanto os tratamentos co antagonista como con morfolinos indican que a 5-HT endóxena inhibe a rexeneración dos axóns en neuronas identificables despois da SCI mediante a activación de receptores 5-HT1A expresados nestas neuronas. Os experimentos de ganancia e perda de función revelaron que a 5-HT endóxena inhibe a rexeneración de axóns tras a SCI en lampreas a través da activación dos receptores 5-HT1A e que este efecto podería ser causado por unha diminución dos niveis de cAMP intracelulares. Para ver os xenes que poderían estar implicados no control intrínseco da rexeneración do axón e cuxa expresión está modulada pola actividade dos receptores 1A, decidimos repetir o tratamento WAY-100.135 e realizar un estudo de secuenciación de ARN no cerebro de lampreas 4 semans despois da SCI. A análise de vías mediante Reactoma revelou 29 vías enriquecidas de forma significativa. Entre estes, os máis interesantes son vías de "guía axónica", "Sinalización por receptores ROBO" e "Regulación de expresión de SLITs e ROBOs". RESUMO 115 No capítulo 6, demostramos que a taurina promove a rexeneración axonal despois dunha SCI completa. A taurina é un dos aminoácidos máis abundantes no cerebro e ten varias funcións fisiolóxicas, incluída a osmorregulación e a modulación dos niveis de calcio intracelular. Moitos estudos demostraron tamén que a taurina pode protexer o cerebro contra danos mecánicos. Non obstante, só algúns estudos suxeriron que a taurina tamén podería promover a rexeneración do axón despois de unha axotomía. A metilprednisolona, que é a única terapia farmacolóxica aprobada para o tratamento das SCI traumáticas, aumenta a rexeneración dos axóns despois da SCI e tamén provoca un aumento na concentración de taurina. En vertebrados, a taurina pode actuar como agonista de unha diversa variedade de receptores de neurotransmisores, incluídos os receptores de GABA. O GABA endóxeno promove a rexeneración axonal de neuronas reticulospinais identificables tras unha SCI en lampreas. Os efectos beneficiosos do GABA parecen estar mediados principalmente mediante a activación dos receptores GABAB. No capítulo 3 desta tese demostramos que a activación dos receptores de GABAA empregando o agonista muscimol tamén reduce a activación de caspasas en neuronas identificables tras SCI en lampreas. No capítulo 6, mostramos que os niveis de taurina aumentan 4 semanas despois dunha SCI completa na medula espiñal das lampreas e que un tratamento agudo con taurina promove aínda máis a rexeneración dos axóns das neuronas xigantes reticuloespiñais. 11 semanas despois da laesión, o tratamento con taurina mellorou significativamente a rexeneración de neuronas descendentes identificables das lampreas e aumentou significativamente o número de axóns rexenerados tras SCI. Estes resultados demostran que a subministración de taurina no momento da lesión mellora aínda máis a capacidade de rexeneración dos axóns da lamprea. Así, amosamos, por primeira vez, o efecto da taurina favorecendo a rexeneración DANIEL SOBRIDO CAMEÁN dos axóns tras unha SCI. En próximos estudios sería de interese descifrar os mecanismos subxacentes detrás do efecto proxenerativo da taurina. Os resultados desta tese demostran que, nun vertebrado con capacidade de rexenear, a 5-HT endóxena inhibe a rexeneración dos axóns das neuronas descendentes despois da SCI (capítulo 5). Sábese que os neurotransmisores activan varias vías de transducción de sinal. A adenosina cíclica 3 ', 5'-monofosfato (cAMP) é un segundo mensaxeiro que desempeña papeis fundamentais nas respostas celulares aos neurotransmisores. A 5-HT regula os niveis intracelulares de cAMP mediante a activación de receptores 5-HT (capítulo 5). Diferentes estudos revelaron que o cAMP modula o crecemento do axón tras SCI (capítulo 4, 5). Curiosamente, varios estudos tamén demostraron que a cAMP pode desempeñar importantes funcios neuroprotectoras. Así, no futuro sería de interese analizar os efectos da 5-HT sobre a supervivencia neuronal despois da SCI. Estudos recentes demostran que a 5-HT desempeña distintos papeis no proceso de rexeneración neuronal despois da SCI (ver capítulo 4). Por exemplo, no peixe cebra, a 5-HT endóxena promove a produción de neuronas motoras na medula espiñal despois dunha SCI completa aumentando a proliferación de células progenitoras de neuronas motoras; en tartarugas, a 5-HT inhibe a aparición de interneuronas serotonérxicas despois da SCI ao inhibir un cambio no fenotipo neurotransmisor de neuronas non serotonérxicas. Por todo isto, suxerimos que a 5-HT podería ser unha diana de interese para os modelos de mamíferos non rexeneradores , xa que pode modular varios aspectos do proceso de rexeneración (capítulo 5). Nesta tese estudamos rutas moleculares implicadas na neuroprotección (capítulo 1, 2, 3) e na rexeneración do axón (capítulo 4, 5, 6) despois de unha SCI completa en lampreas. Este traballo suxire ademais que unha vía de sinalización determinada pode RESUMO 117 influír tanto na neuroprotección como na rexeneración do axón despois da axotomía. Estudos recentes doutros grupos tamén revelaron que os mecanismos moleculares que reducen a apoptose neuronal tamén promoven a rexeneración de neuronas. O noso grupo descubriu recentemente que o tratamento con GABA e baclofen inhibe a activación das caspasas e promove a rexeneración do axón tras a SCI nas lampreas. Nesta tese obtivemos información adicional sobre o papel dos neurotransmisores na rexeneración da medula espiral tras unha SCI completa. Esta tese corrobora que as lampreas constitúen un modelo fiable para o estudo dos mecanismos moleculares subxacentes á recuperación espontánea despois da SCI. Ademais, estes resultados establecen unha base sólida para o estudo de novas terapias para a rexeneración da medula espiñal despois da lesión. A translación de todo este coñecemento a estudos preclínicos podería ter unha importancia relevante para a procura de un tratamento eficiente que promova a rexeneración da medula espiñal e que permita que as persoas que sufran este problema poidan ter unha solución. DANIEL SOBRIDO CAMEÁN