Novel molecular mechanisms and signaling pathways of microglial responses in amyotrophic lateral sclerosis (ALS)

! ! 1! ! Universidad de Sevilla - Facultad de Medicina Tesis Doctoral Novel molecular mechanisms and signaling pathways of microglial responses in amyotrophic lateral sclerosis (ALS) María Magdalena Leal Lasarte Octubre, 2018 ! ! 3! ! El Prof. Dr. David Pozo Pérez, Profesor Titular e Investigador Principal de la Universidad de Sevilla y miembro del Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), junto a la Profa. Dra. Cintia Roodveldt, Personal Docente Investigador (PDI) de la Universidad de Sevilla CERTIFICAN: Que el trabajo titulado “Novel molecular mechanisms and signaling pathways of microglial responses in amyotrophic lateral sclerosis (ALS)” presentado por María Magdalena Leal Lasarte, para optar al Grado de Doctor por la Universidad de Sevilla, ha sido realizado bajo su supervisión en el laboratorio de Terapias Avanzadas en Neuroprotección e Inmunoregulación del Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), y reúne, a su juicio, originalidad y contenidos suficientes para que sea defendido ante el tribunal correspondiente. Y, para que conste a los efectos oportunos, expedimos y firmamos el presente certificado en Sevilla a 15 de Octubre de 2018. Dra. Cintia Roodveldt Directora de tesis Prof. Dr. David Pozo Pérez Codirector de tesis ! 4! ! ! ! ! 5! TABLE!OF!CONTENTS! ! INDEX! 7! ABBREVIATIONS! 13! FIGURES!AND!TABLES!LIST!17! SUMMARY! 21! INTRODUCTION! 25! OBJECTIVES!51! METHODS!AND!MATERIALS! 55! RESULTS! 75! DISCUSSION!108! CONCLUSIONS!117! REFERENCES! 123! ! ! ! 7! ! ! ! ! ! ! ! ! ! Index! ! ! 9! 1!ABBREVIATIONS! 15! 2!FIGURES!AND!TABLES!LIST!19! 3!SUMMARY! 23! 4!INTRODUCTION! 27! 4.1!Amyotrophic!Lateral!Sclerosis!(ALS)! 27! 4.1.1! Etiology!and!clinical!features!of!ALS! 27! 4.1.2! Pathological!mechanism!in!ALS! 29! 4.1.3! Molecular!chaperones!and!ALS! 38! 4.2!Neuroinflammation!and!ALS:!the!central!role!of!microglia! 39! 4.3!TAR!DNA!Binding!protein!(TDP-43)!misfolding!and!aggregation!43! 4.3.1! Characterization! 43! 4.3.2! Subcellular!localization,!aggregation!and!redistribution! 45! 4.3.3!Post-translational!modifications!in!ALS! 46! 4.3.4!TDP-43!and!microglia!in!ALS! 48! 5!OBJECTIVES!53! 6! METHODS!AND!MATERIALS! 57! 6.1!Cell!cultures!57! 6.2! Maintenance!of!the!hSOD1G93A!mouse!colony!58! 6.3!Organotypic!slice!cultures!59! 6.4!Preparation!of!TDP-43!and!mock!aggregates! 60! 6.5!Hsp27!and!Hsp70!protein!overexpression!and!purification!61! 6.6! Microglial!cells!viability!assay!62! ! 16! LPS:!lipopolysaccharide! MALDI:!Matrix!Assisted!Laser!Desorption/Ionization! MAPK:!mitogen-activated!protein!kinase! MCT1:!monocarboxylate!transporter!1! MOK:!MAPK/MAK/MRK!overlapping!kinase! mRNA:!messenger!ribonucleic!acid! MS:!mass!spectrometry! mSOD1:!mutant!superoxide!dismutase!1! MTT:!3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium!bromide! NC:!non-carrier! NEAA:!non-essential!aminoacids! NES:!nuclear!export!signal! NF-κB:!Nuclear!factor!kappa!B! NLRP3:!NLR!family!pyrin!domain!containing!3! NLS:!nuclear!localization!signal! NTD:!N-terminal!domain! PAMP:!pathogen-associated!molecular!pattern! PD:!Parkinson’s!disease! PRR:!pattern-recognition!receptor! RNA:!ribonucleic!acid! ROS:!reactive!oxygen!species! RRM:!RNA!recognition!motifs! sALS:!sporadic!amyotrophic!lateral!sclerosis! SOD1:!superoxide!dismutase!1! TDP-43:!transactive!response!DNA-binding!protein-43! TGF-β:!transforming!growth!factor-β! TNF-α:!tumor!necrosis!factor-α! WT:!wild!type! α-syn:!α-synuclein! ! ! ! ! ! 17! ! ! ! ! ! ! ! ! Figures(and(tables( list! ! ! ! ! 19! 2 Figures!and!tables!list! ! Figure!4-1!A!model!of!Ithe!inflammatory!mediators!associated!to!ALS!.....................!41! figure!4-2!Microglia!phenotypes!in!neurodegenerative!diseases.!.............................!42! Figure!4-3!TDP-43!protein!under!healthy!and!pathological!conditions.!....................!47! Figure!7-1!TDP-43!aggregates!are!internalized!and!modified!by!microglial!cells,! mobilizing!endogenous!TDP-43!protein.!...........................................................!78! Figure!7-2!TDP-43!aggregates!are!internalized!and!modified!by!microglial!cells,! mobilizing!endogenous!TDP-43!protein.!...........................................................!80! Figure!7-3!TDP-43!aggregates!activate!NLRP3-dependent!and!independent! neuroinflammatory!responses.!.......................................................................!83! Figure!7-4!Alternative!processing!of!IL-18!into!ca.!16!kDa!species!in!primary!microglial! culture.!............................................................................................................!85! Figure!7-5!TDP-43!aggregates!induce!a!neuroinflammatory!response!involving! caspase-3!(part!I).!............................................................................................!86! Figure!7-6!TDP-43!aggregates!induce!a!neuroinflammatory!response!involving! caspase-3!(part!II).!...........................................................................................!88! Figure!7-7!Microglial!response!to!TDP-43!aggregates!and!cotreatment!with!Hsp70! chaperone.!.......................................................................................................!89! Figure!7-8!Intracellular!co-localization!between!exogenously!added!Hsp27!with!TDP- 43.!...................................................................................................................!90! Figure!7-9!Alternative!processing!of!IL-18!into!ca.!16!kDa!species!in!primary!microglial! culture.!............................................................................................................!91! Figure!7-10!Caspase-3!in!TDP-43!activated!microglia!modifications!by!co-stimulating! Hsp27!and!Hsp70!chaperones.!.........................................................................!93! Figure!7-11!Effect!of!co-stimulating!Hsp27!and!Hsp70!chaperones!on!TDP-43-elicited! microglial!response.!.........................................................................................!95! Figure!7-12!Extracellular!TDP-43!aggregates!target!MOK!signaling!pathway!in! microglia!upon!internalization.!........................................................................!97! Figure!7-13!MOK!and!TDP-43!aggregates!co-localization!inside!microglial!cells!of! organotypic!cultures.!.......................................................................................!98! ! 20! Figure!7-14!Modification!of!MOK!phosphorylated!status!by!extracellular!TDP-43! aggregates.!....................................................................................................!100! Figure!7-15!MOK!is!involved!in!microglia!neuroinflammatory!response!and!possibly! regulates!activated!caspase-3.!.......................................................................!102! Figure!7-16!MOK!protein!is!altered!in!the!mutant!hSOD1G93A!mouse!model.!..........!105! Figure!7-17!MOK!protein!is!altered!in!ALS!patients.!...............................................!107! Table!1!The!genetics!of!ALS:!genes!associated!with!ALS!identified!between!1993!and! 2016!.................................................................................................................!31! ! ! 21! ! ! ! ! ! ! ! ! ! Summary! ! ! ! 23! 3 Summary! Amyotrophic! Lateral! Sclerosis!(ALS)! is! an! incurable! and! fatal! neurodegenerative! disease,!with! survival! expectancy! between!3-5! years! from! the! clinical! diagnose.! This! disease!is!characterized!for!the!progressive!death!of!motor!neurons.!A!typical!hallmark! of! ALS! is! neuroinflammation,! with! extensive! microgliosis! and! astrogliosis! within! the! affected! areas! of! the! central! nervous! system! (CNS).! It! is! believed! that! neuroinflammation,! especially! microglia! activation,! is! involved! in! the! onset! and! progression!of!the!disease.!This!is! known! as! the! non-cell! autonomous! theory! where! motor! neurons! die! not! only! due! to! intrinsic! factors! but! also! as! a! consequence! of! a! chronic!and!exacerbated!inflammation!state.! Another! feature! of! ALS,! which!is! considered! as! a! protein! misfolding! disease! together! with! other! neurodegenerative! diseases! such! as! frontotemporal! lobar! dementia! (FTLD)! or! Parkinson´s! disease! (PD),! is! the! accumulation! of! intracellular! protein!inclusions.!TAR!DNA!binding!protein!43!(TDP-43)!is!the!principal!component!of! almost! all! intracellular! aggregates! found! in! ALS! cases! and! in! ca.! 50%! of! those! with! Frontotemporal!dementia!(FTD).!TDP-43!is!a!nuclear!multifunctional!protein!known!for! its! RNA/DNA! binding! activities,! which! is! believed! to! play! a! central! role! in! these! disorders.! TDP-43!aberrantly!aggregates!in!the!cytoplasm!of!motor!neurons!in!pathological! scenarios.! Under! these! conditions,! TDP-43! aggregates! are! formed! in! the! cytoplasm,! containing!truncated,!ubiquitylated!and/or!hyperphosphorylated!forms!of!the!protein,! and,!additionally,!retro-translocation!of!the!protein!from!the!nucleus!to!the!cytoplasm! of!degenerating!motor!neurons!occurs.!Until!this!date!there!is!not!much!data!on!how! TDP-43!proteinopathy! affects! protein! homeostasis! of! microglial! cells.! More! recently,! increased!levels!of!TDP-43!have!been!found!in!biological!fluids,!including!cerebrospinal! fluid!(CSF)!and!plasma,!of!ALS!and!FTD!patients.!The!extracellular!content!of!TDP-43! may! be! a! result! of! cell! death! caused! by! cytotoxic! intracellular! TDP-43! inclusions!or,! additionally,! be! released! from! cells! to! CSF! via! exosomes! in! ALS! patients.! Moreover,! extracellular!TDP-43!could!play!a!critical!role!in!sustained!neuroinflammation!in!these! disorders.!! ! 24! It!is!known!that! microglial!cells!can! be!activated!by!extracellular!aggregates!of! mutant!superoxide!dismutase!1!(mSOD1)!via!activation!of!Toll!like!receptor!2!(TLR2)! and!CD14!receptors! but! until!now! no! data! involving! extracellular! TDP-43!aggregates! exist.! In! order! to! gain! further! insight! into! the! physiopathological! roles! of! TDP-43! aggregates!in!ALS,!we!studied!the!impact!of!exogenously!added!TDP-43!aggregates!on! the! neuroinflammatory! response! of! microglial! cells! in! culture,! and! explored! the! molecular! mechanisms! operating! upon! exposure! to! such! ALS-associated! species.! Furthermore,!we!investigated!whether!certain!molecular!chaperones!that!have!been! found!at!abnormal!levels!in!extracellular!fluids!of!ALS!animal!models!and!patients,!are! able! to! modify! TDP-43-elicited! microglial! responses.! Heat-shock! proteins! (HSPs)! and! other! molecular! chaperones! are! known! for! their! traditional! role! linked! to! the! maintenance! of! cellular! proteostasis! both! under! physiological! and! pathological! scenarios!such!as!in!neurodegenerative!proteinopathies.!Importantly,!several!HSPs,!in! addition! to! binding! and! remodeling! protein! aggregates,! have! been! found! to! display! immunological! activities,! including! the! alteration! of! α-synuclein! (α-syn)!misfolding! protein-elicited!responses!in#vitro!and!in!immunized!mice.! In! attempt! to! elucidate! new! molecular! elements! involved! in! the! signaling! pathways! of! the! neuroimmune! response,! we! searched! for! TDP-43-interacting! molecules!in!microglial!cells.!Remarkably,!we!uncovered!MAPK/MAK/MRK!overlapping! kinase!(MOK)!as!a!new!TDP-43!interactor!and!cell!signalling!mediator!engaged!not!only! in!TDP-43-exposed!microglia!but!also!in!a!general!inflammation!model.!Moreover,!we! were! able! to! demonstrate! that! MOK! protein! kinase! is! indeed! involved! in! the! neuroinflammatory!response!and!that!it!might!be!a!central!player!in!ALS!disease!and! other!neurodegenerative!diseases.!! !!! ! ! ! ! ! 25! ! ! ! ! ! ! ! ! Introduction! ! ! 32! ! 4.1.2.2 Protein!aggregation!and!prion-like!spread!! Another!hallmark!of!ALS!is!the!intracellular!accumulation!of!protein!inclusions! containing!misfolded!TDP-43,!and!in!certain!cases,!FUS!or!SOD1!proteins23.!Since!the! first! report! in! 2006,! TDP-43! —a! multifunctional! protein! with! RNA/DNA! binding! activities! and! typically! located! in! the! cell! nucleus—! has! been! identified! as! a! major! component!of!intraneuronal!and!glial!inclusions!in!virtually!all!patients!with!ALS!and!in! ca.! 50%! of! those! with! FTD8,24,! thus! indicating! that! TDP-43! has! a! key! participation! in! these!disorders25.! Aberrant! protein! aggregation! is! a! common! feature! in! neurodegenerative! diseases,! including! ALS,! and! it! seems! to! play! a! central! role! in! these! disorders.! The! aggregation!process!is!thought!to!initiate!with!abnormal!structural!conformation!of!the! protein!monomers.!These!misfolded!monomers!could!self-associate!and!reorganize!to! form!relatively!stable!intermediate!species!referred!to!as!‘oligomers’,!or!form!amyloidlike!fibrils!by!linear!addition26,27.!Oligomers!or!protofibrils!could!also!self-assemble!to! form!amyloid!fibrils!or!amorphous!aggregates!that!do!not!contain!fibrils.!Amorphous! aggregates! grow! without! any! major! structural! conversion! retaining! the! oligomers! characteristic!while!amyloid!fibrils!often!suffer!dramatic!reorganizations!conferring!a! high!level!of!structural!order26,27.!!Whether!this!process!is!a!mechanism!used!by!cells!to! protect!themselves!from!misfolded!proteins!is!still!under!debate!as!well!as!which!are! the!toxic!species!along!the!aggregation!pathway.!! Misfolded! proteins! accumulation! can! cause! aggregation! of! their! native! counterparts!through!a!mechanism!similar!to!the!infectious!prion!protein’s!induction! of!a!pathogenic!conformation!of!other!molecules!of!that!protein.!The!term!‘prion-like’! is!used!to!describe!molecular!events!that!share!similarities!with!the!infectious!cycle!of! the! mammalian! prion! protein’s! self-perpetuating! seeded! aggregation! and! spreading.! Because!the!pattern!of!clinical!progression!in!ALS!suggests!that!the!disorder!spreads! centrifugally! from! an! affected! area! to! adjacent! regions,! it! is! proposed! that! normal! SOD1,! TDP-43! and! possibly! also! FUS/TLS,! can! undergo! seeded! aggregation! that! can! spread!from!cell!to!cell!through!a!prion-like!mechanism!after!an!initiating!event15,28.! Indeed,! prion-like! propagation! has! been! seen! for! both! TDP-43! and! SOD1! where! ! ! 33! aggregated! forms! of! either! proteins! can! seed! the! misfolding! and! aggregation! of! the! endogenous!wild-type!protein!in#vitro!28,29.! The!mechanisms!of!aggregation!and!how!they!contribute!to!neurodegeneration! are!still!poorly!understood!and!whether!those!aggregates!are!toxic!per#se!(gain-of-toxic! fuction)!or!it!is!rather!protein!sequestration!that!causes!a!loss-of-physiological!function! of!the!protein,!or!a!combination!of!both!mechanisms,!is!currently!under!strong!debate.! Gain-of-toxic-function!mechanisms:!! Accumulated! studies! suggest! that! protein! inclusions! or! their! precursor! oligomers! contribute! to! ALS! pathogenesis26.! It! has! been! observed! that! protein! aggregates! induce! mitochondrial! dysfunction,! produce! endoplasmic! reticulum! (ER)! stress,!Golgi!fragmentation!and!the!formation!of!pores!on!cellular!membranes.!Protein! aggregates! may! also! overwhelm! the! protein! degradation! pathways! that! end! in! the! impairment!of!the!proteasomal!and!lysosomal!protein!degradation!pathways30–32.! Previous! studies! have! demonstrated! that! disease-linked! protein! variants! present! in! the! extracellular! medium! and! extracellular! protein! aggregates! linked! to! various! neurodegenerative! diseases! such! as! α-syn! (PD),! amyloid! β!(AD),! and! SOD1! (ALS)! are! able! to! activate! microglia! and! trigger! strong! immune! responses33,34.! These! aggregates!have!been!shown!to!induce!the!activation!of!different!pathways!such!as!the! nuclear!factor!κB!(NF-κB),!the!NRLP3!imflammasome!that!promotes!proinflammatory! cytokine!production,!reactive!oxygen!species!(ROS)!and!other!neurotoxic!factors35.! The! interaction! of! ALS-associated! misfolding! proteins! such! as! SOD1,! TDP-43,! FUS,!C9orf72,!with!the!mitochondria!appears!to!induce!mitochondrial!damage.!Some! of!these!proteins!accumulate!and/or!aggregate!in!mitochondria,!interfering!with!their! normal! function36.! Abnormal! release! of! ATP! and! ROS,! impairment! of! energy! homeostasis,! activation! of! apoptosis! and! altered! calcium! buffering! capacity! is! observed.! Moreover,! disrupted! axonal! transport,! as! seen! in! ALS,! has! been! found! to! produce!metabolic!alterations!in!neurons36.!!!! Loss-of-function!mechanisms:! There!is!also!evidence!that!the!inclusions!found!in!the!motor!neurons!or!glial! cells!not!only!contribute!to!the!disease!by!a!gain!of!toxic!function,!but!also!through!the! loss!of!the!protein’s!native!function!due!to!its!abnormal!aggregation!and!mislocation.! For! example,! it! is! believed! that! many! of! the! TDP-43! normal! functions! that! are! ! 34! performed! in! the! nucleus! are! disabled! because! of! its! translocation! to! the! cytoplasm30,32.! Mutations! in! ALS-related! genes! such! as! C9orf72,! serine/threonine! kinase!1!and!ubiquilin-2,!among!others,!that!are!related!to!the!ubiquitin-proteasome! system! (UPS)! or! are! autophagy! regulator! genes! might,! by! their! loss! of! function,! contribute!to!the!toxic!accumulation!of!TDP-43,!SOD1!or!other!related!proteins!4,31.!On! the! other! hand,! a! decrease/loss! in! the! enzymatic! function! of! SOD1! has! also! been! described! for! mSOD1! in! cellular! and! animal! models,! thus! increasing! vulnerability! of! cells!to!oxidative!stress37.! ! 4.1.2.3 Axonal!disorganization!! Motor!neurons!are!highly!polarized!cells!with!long!axons!that!can!reach!more! than!1!meter!of!length.!Axonal!transport!is!of!high!importance!because!components! that! are! synthesized! in! the! cell! bodies! must! be! delivered! to! axons! and! synapses.! Disorganization!of!axonal!cytoskeleton!is!a!key!feature!of!both!fALS!and!sALS37.!Direct! evidence!for!axonal!transport!defects!in!ALS!was!obtained!following!the!development! of!mutant!SOD1!transgenic!mouse.!ALS-linked!mutant!SOD1!has!been!demonstrated!to! slow! both! anterograde! and! retrograde! transport! routs! months! before! neurodegeneration38.!However,! defects!in!axonal!transport!are!not! limited! to! SOD1- related!ALS.!Overexpression!of!TDP-43,!the!ALS!mutant!vesicle-associated!membrane! protein-associated! protein! B! (VAPB),! FUS! and! transgenic! expression! of! C9orf72! GGGGCC!(G4C2)!repeat!expansion!constructs!also!have!detrimental!effects!on!axonal! transport38.!The!mechanisms!underlying!defective!axonal!transport!are!unknown,!but! are!likely!to!involve!several!pathways.! ! 4.1.2.4 Oxidative!stress!and!mitochondrial!dysfuction!! Oxygen!free!radicals,!or!ROS,!are!natural!products!of!oxygen!metabolism.!When! the! production! of! ROS! is! higher! than! the! capacity! of! the! cell! to! remove! them,! an! oxidative! stress! is! produced.! With! the! discovery! of! mutations! in! SOD1,! the! major! enzyme!to!prevent!oxidative!damage,!as!a!cause!of!fALS,!this!hypothesis!has!gained! strength.!Increased!levels!of!free!radicals!and!of!oxidative!damage!have!been!found!in! ! ! 35! CSF,!serum!and!urine!samples!from!ALS!patients39.!Oxidative!damage!in!proteins,!lipids! and!DNA!has!also!been!observed!in!post-mortem!samples!from!ALS!patients37.!! There!is!multiple!evidence!of!mitochondrial!involvement!in!ALS.!Mitochondria! are!implicated!in!the!production!of!intracellular!energy!and!are!important!organelles! for!calcium! homeostasis.! Moreover,! they!produce! high! levels! of!ROS! and! play! a! key! role! in! apoptosis! mediated! by! the! activation! of! caspase! cascades.! Disruption! of! mitochondrial! structure,! dynamics,! bioenergetics! and! calcium! buffering! has! been! extensively! reported! in! spinal! cord! and! skeletal! muscle! of! ALS! patients! and! model! systems40.!! 4.1.2.5 Non-cell!autonomous!toxicity! ! Many! pathological! conditions,! including! neurodegenerative! diseases,! have! traditionally! been! considered! mechanistically! cell! autonomous.! This! means! that! damage!in!a!selective!population!of!neurons!is!sufficient!to!produce!disease.!! In! the! sporadic! case! of! almost! all! neurodegenerative! diseases! stressors! that! are! thought! to! be! the! cause! of! disease! are! unknown,! whereas! the! expression! of! a! particular!mutant!gene!is!the!cause!of!the!familial!forms.!Those!mutant!genes!that!are! the! cause! of! the! inherited! forms! are! widely! or! ubiquitously! expressed! so! it! was! inevitable! to! ask! whether! neurodegenerative! diseases,! including! ALS,! are! fully! cell! autonomous! or! whether! other! cell! types! are! also! involved! in! the! disease! drive!or! progression.!In!the!last!few!years,!it!has!become!established!that!disease!is!a!result!of! a! combination! of! damage! in! both! motor! neurons! and! their! glial! partners,! such! as! microglia,!astrocytes!and!oligodendrocytes41.!! Studies!in!mutant!SOD1!(mSOD1)!mice!have!shown!that!cell-specific!expression! in!motor!neurons!of!either!high!or!low!levels!of!mSOD1!is!sufficient!to!modify!the!fate! of!disease.!However,!whereas!high!levels!of!mSOD1!in!motor!neurons!is!not!enough!to! cause!early!disease!onset42,!a!reduction!in!SOD1!protein!synthesis!does!not!slow!the! progression! of! symptoms! after! the! onset! of! disease43.! Therefore,! not! only! motor! neurons!are!involved!in!the!pathogenesis!of!ALS!but!also!are!associated!cells!of!several! types! where! glia! play! a! crucial! role15,41,44.! ! In! summary,! it! is! currently! thought! that! expression! of! mSOD1! in! motor! neurons! initiates! a! damaging! process! that! drives! ! 36! disease!onset,!and!that!activation!of!microglia!and!astrocytes!by!mSOD1!exacerbates! disease!progression44.! Another! pathological! hallmark! of! ALS! is! the! presence! of! activated! microglia! sorrounding! damaged! neurons! within! the! spinal! cord! and! certain! brain! regions,! indicative! of! a! sustained! neuroinflammatory! state,! since! the! early! stages! of! disease! and! along! disease! progression45,46! Many! studies! have! demonstrated! that! microglia,! which! are! the! innate! immune! cells! of! the! nervous! system,! become! activated! and! neurotoxic! in! ALS47,48.! In! samples! from! ALS! patients! tissue,! obtained#post-mortem,# there!is!extensive!microgliosis!in!many!regions!exhibiting!motor!neuron!loss,!including! the! ventral! horn! of! the! spinal! cord,! motor! cortex,! and! the! corticospinal! tract.! In! addition,! the! level! of! microglial! activation! correlates! with! the! end-stage! clinical! measures! of! executive! function41.! Furthermore,! genome-wide! association! studies! (GWASs)! have! shown! that! many! genes! associated! to! a! risk! to! develop! ALS,! such! as! C9ORF72,!hnRNPA1,!TANK!Binding!Kinase!1!(TBK1),!Valosin!Containing!Protein!(VCP),! are!highly!expressed!in!microglia41.!! Interestingly,!a!role!for!microglia!in!the!onset!of!ALS!has!also!been!described!in! the!human!SOD1G93A!(hSOD1G93A)!mouse!model,!the!most!widely!used!ALS!pre-clinical! model41.! Furthermore,! another! study! in! this! same! animal! model! revealed! microglia! activation!even!before!motor!neuron!death48.!Studies!have!also!shown!that!when!the! mSOD1!gene!is!silenced!in!microglial!cells!in!mouse!models,!the!progression!of!disease! slows!down49,50.!This!also!occurred!when!cell!grafts!replaced!mutant!SOD1-!expressing! microglia! with! healthy! microglia15.! In! another! study! supporting! these! findings,! the! inhibition! of! inflammation-related! transcription! factor! NF-κB! was! found! to! reduce! neurotoxicity!of!microglia!in!co-cultured!motor!neurons15.!!!! Another!mechanism!that!was!found!to!produce!damage!by!mSOD1-expressing! microglia!is! the! excessive!production!of!superoxide.! Misfolded! mSOD1! can!associate! with! the! small! GTPase! RAC1,! which! controls! the! activation! of! NADPH! oxidase,! a! complex! that! produces! superoxide! ion,! thereby! increasing! the! levels! of! extracellular! superoxide!produced!by!microglia15.!!! ! ! 37! It!is!believed!that!microglia!plays!a!key!role!in!the!progression!of!ALS!and!evidence!also! indicates! that! it! may! play! a! role! in! the! onset! of! ALS! as! well.! Recent! studies! have! reinforced!this!concept!and!increased!our!knowledge!of!microglial-mediated!non-cell! autonomous! pathways! that! contribute! to! the! disease45.! However,! how! microglia! contribute!to!ALS!is!not!fully!understood!and!much!research!is!still!needed.! Studies! have! shown! that! astrocytes! also! have! a! non! cell-autonomous! role! in! ALS.!The!astrocytes!provide!motor!neurons!with!nutrients,!ion!buffering!and!recycling! of! the! neurotransmitter! glutamate.! Several! research! teams! have! used! in# vitro# cocultures!of!motor!neurons!and!astrocytes!(or!astrocyte-conditioned!medium)!to!show! that! astrocytes! expressing! ALS-linked! mutations! produce! a! toxicity! that! diffuses! to! motor!neurons15,41,44,51.!Astrocytes-differentiated!induced!pluripotent!stem!(iPS)!cells! from! sALS! patients! were! found! to! reduce! viability! of! motor! neurons! in! co-culture! experiments,!and!the!reduction!of!mSOD1expression!in!mouse!models!were!seen!to! slow!down!the!onset!and!progression!of!the!disease!15,44.!!! Another! proposed! pathogenic! mechanism! in!ALS! is! glutamate-induced! excitotoxicity.!Increased!levels!of!glutamate!were!detected!in!CSF!from!ALS!patients52.! Moreover,! glutamate! transport! deficits! were! found! in! spinal! cord! and! motor! cortex! samples!from!ALS!patients!and!mSOD1!mouse!models.!A!reduction!in!the!levels!of!the! astrocytes!excitatory! amino!acid!transporter!2! (EAAT2)! was!also!reported,!indicating! that! astrocytes! are! in! part! responsible! for! excitotoxicity15,44,51.! Finally,! evidence! has! shown! that! mSOD1-expressing! astrocytes! lose! the! ability! to! control! expression! of! the!GluR2!subunit!of!AMPA-type!glutamate!receptors!in!motor!neurons,!leading!to!an! elevated!influx!of!calcium!and!the!degeneration!of!motor!neurons15,51.!! More! recently,! oligodendrocytes! have! also! ! been! implicated! in! the! pathophysiology!of!ALS.!Removal!of!mutant!SOD1!from!oligodendrocytes!early!in!the! maturation! process! was! found! to! produce! a! delay! in! the! disease! onset! in! the! hSOD1G93A!ALS!mouse!model15.!In#vitro,!mSOD1!mouse!oligodendrocytes!induce!wildtype! (WT)! motor! neuron! hyperexcitability! and! death53.! Moreover,! human! oligodendrocytes! from! patients! with! sALS! and! fALS! induced! motor! neuron! death! through! conditioned! medium! and! in! co-culture! studies53.! Oligodendrocytes! also! support!motor!neuron!function!by!directly!supplying!the!energy!metabolite!lactate!to! the!axon!through!the!action!of!monocarboxylate!transporter!1!(MCT!1)!53.!mSOD1!was! ! 38! found! to! impair! the! expression! of! MCT1! by! oligodendrocytes! in! a! mouse! model! of! ALS15.! Interestingly,! a! similar! reduction! in! the! accumulation! of! MCT1! was! found! in! sporadic! ALS15.! Furthermore,! a! recent! study! reported! that! early! mSOD1! knockdown! rescued! lactate! impairment! and! cell! toxicity! in! lines! tested,! with! the! exception! of! samples!carrying!C9orf72!repeat!expansions53.!! In!summary,!accumulating!gene!data!and!pre-clinical!and!clinical!investigations! have!lately!established!that!the!physiopathology!of!ALS!is!multifactorial!and!it!is!now! clear! that! other,! non-neuronal! cell! types! are! involved! in! the! disease! where! motor! neurons! die,! at! least! in! part,! through! non-cell-autonomous! toxicity! processes! and! where!different!mechanisms!of!action!lead!to!the!degeneration!of!motor!neurons.!! Although!our!understanding!of!non-cell!autonomous!toxicity!came!from!SOD1! mutant-mediated! ALS! models,! other! mutations! in! widely! or! ubiquitously! expressed! genes,! such! as! TDP-43! or! FUS,! have! been! reported! indicating! that! the! non-cell! autonomous!characteristic!of!the!disease!seems!likely!to!be!a!universal!feature!of!ALS! rather! than! a! specific! signature! of! mSOD1! cases.! Moreover,! and! as! said! before,! mislocation! and! accumulation! of! TDP-43! not! only! in! motor! neurons! but! also! in! glial! cells24,54!are!seen!in!almost!all!patients!with!sALS!or!fALS!reinforcing!this!concept.!! ! 4.1.3 Molecular!chaperones!and!ALS! Heat-shock!proteins!(HSPs)!and!other!molecular!chaperones!are!key!elements! of! cellular! proteostasis! both! under! physiological! and! certain! pathological! scenarios,! such! as! in! neurodegenerative! proteinopathies55,56.! HSPs,! normally! associated! to! intracellular! functions,! have! been! also! found! in! the! extracellular! medium! under! physiological! conditions! and! increased! under! pathological! states.! Higher! immunoreactivity!against!HSPB5!(αB-crystallin)!was!found!in!brains!from!ALS!patients! and! also! in! the! hSOD1G93A!mouse! model,! both! in! the! cytoplasm! of! neuronal! and! reactive!glial!cells56.!In!the!same!mouse!model,!HSPB1!(Hsp27)!levels!were!found!to!be! upregulated56,57.!Moreover,!missense!mutations!in!the!Hsp27! gene!are!linked! to! the! sensory!and!motor!neuropathies!in!Charcot-Marie-Tooth!disease!and!distal!hereditary! motor!neuropathy58.!Also,!co-expression!of!TDP-43!with!heat-shock!factor!1!(HSF1),!a! master!transcription!factor!that!upregulates!HSPs!during!the!stress!response,!led!to!a! ! ! 39! significant!reduction!in!the!level!of!aggregated!TDP-43!in!cultured!cells59.!Importantly,! HSF1!was!decreased!in!mutant!TDP-43!transgenic!mice,!also!showing!lower!levels!of! DnaJ! (Hsp40)! and! HSPA1! (Hsp70).! Even! though! HSF1! levels! were! not! significantly! altered,!lower!levels!of!Hsp40!and!Hsp70!were!detected!in!tissue!from!sALS!patients! compared!to!healthy!controls59.!! Certain! molecular! chaperones! have! been! found! at! abnormal! levels! in! extracellular! fluids! of! ALS! animal! models! and! patients60.! Serum! levels! of! Hsp90! and! Hsp70! were! found! to! be! significantly! higher! in! ALS! patients! than! in! healthy! individuals60.! Remarkably,! in! addition! to! binding! to! and! remodelling! protein! aggregates61,62,! chaperones! have! been! more! recently! reported! to! display! immunological! activities,! triggering! innate! and! adaptive! immune! responses! though! several!pathways63,64.!Indeed,!it!is!believed!that!extracellular!HSPs!act!as!stress!signals! by! priming! other! cells65,! and! that! their!immunomodulary! effect! could! involve! either! anti-!or!pro-inflammatory!responses64.!Moreover,!certain!chaperones!have!been!found! to! modify! the! immune! response! elicited! by! a! neurodegenerative-linked! misfolding! protein,!α-syn,!in#vitro!and!in#vivo!with!immunized!PD!model!mice66,67.! ! 4.2 Neuroinflammation!and!ALS:!the!central!role!of!microglia! Neuroinflammation! is! a! common! hallmark! of! neurodegenerative! diseases,! including! ALS,! PD! and! AD68,69.! It! is! principally! mediated! by! activated! microglia! and! astroglia,! and! is! usually! accompanied! by! lymphocyte! infiltration70.! Even! though! the! CNS!has!traditionally!been!considered!“immune!privileged”,!i.e.!isolated!from!the!rest! of!the!body!and!partially!dependent!on!the!blood-brain!barrier!(BBB)!and!the!bloodspinal! cord! barrier! (BSB),! both! innate! and! adaptive! inflammatory! responses! are! currently!known!to!occur.!Activation!of!the!innate!immune!system!is!the!first!line!of! defense,! and! microglia! are! the! main! immunocompetent! cells! in! the! CNS,! and! are! essential!to!clear!apoptotic!cells!and!misfolded!or!aggregated!proteins68.!Although!not! all! immune! responses! are! detrimental,! chronic! immune! response,! in! particular! exacerbated!microglia!response,!is!recognized!to!have!a!key!role!in!neurodegenerative! diseases.!Furthermore,!innate!immune!responses!recruit!cells!of!the!adaptive!immune! ! 40! system!by!secreting!various!cytokines!and!chemokines!that!induce!adhesion!molecules! on!the!BBB71.!! In! ALS,! sustained! inflammation! has! been! found! in! association! with! neuronal! loss! in! human!tissue!and!in!animal!models!even!before!the!onset!of!the!disease72.!Moreover,! activation!of!the!immune!system!in!peripheral!blood!and!in!CSF!has!been!observed!in! ALS! patients73.! A! meta-analysis! study! demonstrated! that! levels! of! pro-inflammatory! cytokines!such!as!tumor!necrosis!factor-α!(TNF-α),!TNF!receptor!1!(TNFR1),!IL-6,!IL-1β,! IL-8,! and! vascular! endothelial! growth! factor! (VEGF! —! a! protein! that! stimulates! the! growth!of!new!blood!vessels)!in!the!blood!are!significantly!elevated!in!patients!with! ALS,! compared! with! controls74.! It! is! believed! that! in! the! initial! phase! of! slow! progression! of! the! disease! the! immune! response! is! beneficial,! but! the! unresolved! exposure! to! a! pro-inflammatory! environment! exerts! a! deleterious! effect! on! motor! neurons,!and!having!a!key!role!in!disease!progression70.!However!the!mechanisms!that! are!involved!are!poorly!understood.! Microglia! are! the! first! line! of! defence! in! the! brain! and! spinal! cord.! They! are! constantly! surveying! the! environment! and! respond! to! danger! signals! from! damaged! tissues35,75.! Microglia! recognize! these! signals! through! conserved! pattern-recognition! receptors!(PRRs)!that!include!Toll-like!receptors!(TLRs),!retinoic!acid!inducible!gene!I- like! receptors! (RIGLs),! etc.! The! PRRs! trigger! the! immune! response! by! recognition! of! pathogen-associated! molecular! patterns! (PAMPS)! or! danger-associated! molecular! patterns!(DAMPS)35,70,75-!The!activation!of!downstream!signalling!cascades!ultimately! leads!to!the!expression!and!secretion!of!several!cytokines!and!chemokines45,76.! It! has! been! reported! that! proteins! found! in! the! extracellular! medium! under! pathological! conditions,! such! as! mSOD1,! activate! microglia! through! the! receptors! CD14,! TLR2,! TLR4,! and! scavenger! receptor-dependent! pathways46.! Activation! of! microglia!by!extracellular!proteins!has!been!described!not!only!for!ALS-linked!proteins! but!also!for!proteins!related!to!other!neurodegenerative!diseases33,!for!example,!PD- lined!α-syn34!and!AD-associated!amyloid!β77.!! ! ! 41! ! Figure 4-1 A model of Ithe inflammatory mediators associated to ALS ALS is characterized by the degeneration and loss of motor neurons. Mutations in SOD1 were the first to be described as responsible for fALS. Currently, it is accepted that the non-cell autonomous theory where motor neuron lost is not only a result of intrinsic mechanisms that occur in neurons but also of neighbor glial cells. Toxic aggregates somehow released by motor neurons induced the activation of microglia mediated by TLR2 and CD14 receptors. Microglial cells might induce astrocytes activation by the production cytokines. Activation of both, microglia and astrocytes, amplify the motor neurons injury by induction of AP-1 y NF-κβ pathways and the secretion of pro-inflammatory cytokines and apoptosis induced molecules such as TNF-α y FASL. TNF-α e IL-1β. ATP released by dying motor neurons axacerbate microglia activation through the activation of the P2X7 purinergic receptors expressed in microglia. Figure adapted from (Glass et al. 2010-Cell). Traditionally,! microglia! has! been! described! to! exist! in! two! general! states:! ‘resting’! or! activated.! The! concept! of! resting! has! been! lately! modified! to! a! more! descriptive!concept!and!now!it!is!referred!to!as!´surveilling´.!Surveilling!microglia!are! constantly! screening! the! environment! and! have! a! crucial! role! in! maintaining! CNS! homeostasis.! Microglia! can! rapidly! react! to! danger! signals,! adopting! an! amoeboid! shape!called!‘activated!microglia’.!Activated!microglia!may!adopt!at!least!two!different! ! 48! the nucleus to the cytoplasm where it aggregates into inclusions. These aggregates contain cleavede forms of TDP-43, which are found to be hyperphosphorylated and/or ubiquitylated.. Figure adapted from (Chen-Plotkin, A. et al. 2010 - Nat. Rev. Neurol.) ! 4.3.4 TDP-43!and!microglia!in!ALS!! Apart!from!their!role!in!motor!neurons,!abnormal!cytoplasmic!accumulation!of! TDP-43!was!observed!in!glial!cells!of!spinal!cord,!motor!cortex!and!brainstem!of!ALS! patients112,113.!However,!there!is!no!specific!data!in!microglial!cells.!Intriguingly,!it!was! recently!reported!that!LPS-triggered!inflammation!induced!the!clearing!of!nuclear!TDP- 43!and!the!cytoplasmic!aggregation!in!microglial!cells!in!culture114.!In#vivo,!the!same! work!showed!that!chronic!LPS!administration!in!a!transgenic!mouse!overexpressing!a! mutant!form!of!TDP-43!enhanced!cytoplasmic!retro-translocation!and!aggregation!of! TDP-43!in!the!spinal!cord,!suggesting!that!chronic!inflammation!may!contribute!to!the! formation!of!TDP-43!pathological!species114!.!!!!! It! was! also! reported! that! TDP-43! overexpression! in! BV-2! microglial! cell! line! exacerbates! the! production! of! pro-inflammatory! cytokines! and! cytotoxic! mediators! with!detrimental!effects!on!neurons115.!It!was!also!shown!that!TDP-43!interacts!and!colocalizes! with! the! p65! subunit! of! the! NF-κB! and,! additionally,! that! TDP-43! and! p65! mRNA!levels!are!elevated!in!patients!with!ALS115.!NF-κB!is!a!major!transcription!factor! that!plays!a!key!role!in!regulating!the!immune!response.!A!recent!study!addressed!the! effect! of! non-aggregated,! soluble! TDP-43! on! microglial! immune! response.! Interestingly,! it! was! observed! that! treatment! of! BV-2! microglial! cell! line! with! exogenously!added!TDP-43-GST!fusion!protein!induced!microglial!activation!via!NF-κB! and!NLRP3!inflammasome!pathways,!accompanied!by!enhanced!neurotoxicity116.!! On! the! other! hand,! pathologically! relevant! TPD-43! species! are! currently! recognized!to!exist!in!the!extracellular!medium,!as!higher!levels!of!TDP-43!have!been! measured!in!biological!fluids!in!the!context!of!ALS.!In!particular,!higher!levels!of!TDP-43! have! been! found! in! CSF! from! sALS! patients! compared! to! healthy! controls117,118! and,! interestingly,!pathological!TDP-43!immunoreactivity! in!brain!tissue!is!correlated!with! elevated!levels!of!TDP-43!in!the!CSF119.!Extracellular!TDP-43!species!may!be!a!result!of! cell!death!caused!by!cytotoxic!intracellular!TDP-43!inclusions.!Additionally,!it!has!been! recently!shown!that!pathogenic! TDP-43! species,! including! aggregation! seeds,! can! be! ! ! 49! released!from!cells!to!CSF!via!exosomes!in!ALS!patients120,121.!!This!raises!the!possibility! that! extracellular! TDP-43! species! could! play! a! principal! role! in! microglial-mediated! pathological! neuroinflammation! in! these! disorders.! However,! the! contribution! of! pathologically!relevant,!aggregated!TDP-43!species!to!microglial!activation,!underlying! molecular! mechanisms! and! elicited! immune! responses,! has! thus! far! not! been! addressed.! !!!!!!!!!!!!!!!!!!! ! ! ! 51! ! ! ! ! ! ! Objectives! ! ! ! 53! 5 Objectives!!! ! v To!characterize!TDP-43!species!in!microglial!cells!exposed!to!pre-formed!TPD- 43! aggregates,! including! subcellular! localization! and! the! presence! of! TDP-43! post-translational!modifications!and!inclusions!similar!to!those!linked!to!ALS.!! v To! describe! the! effects! of! extracellular! TDP-43! aggregates! on! microglial! immune!response!(no!‘neuroimmne!response’!en!general),!and!determination! of!the!pathways!involved.!! v To! investigate! the! effect! of! specific! molecular! chaperones! on! microglial! immune!responses!as!possible!physiopathological!modulators,!and!as!potential! molecular!tools!for!intervention.!! v To!explore!the!molecular!mechanisms!linking!the!exposure!of!microglial!cells!to! ALS-linked!TDP-43-aggregates!and!the!elicited!immune!responses.!! v To!validate!the!pathological!relevance!of!such!findings!in!spinal!cord!samples! from!an!ALS!pre-clinical!model!and!from!ALS!patients.!! ! ! ! ! ! ! ! ! ! ! ! ! ! 55! ! ! ! ! ! ! ! ! Methods(and( materials! ! ! ! 57! 6 Methods!and!materials! ! 6.1 Cell!cultures! Mixed!glial!cultures!were!prepared!from!cerebral!cortices!of!1-3!day-old!C57BL/6!male! mice! (University! of! Seville! Animal! Core! Facility,! Seville,! Spain),! and! microglia! were! isolated!by!mild!trypsinization!as!previously!described122,!with!minor!modifications!as! follows.! Sixteen! mouse! brains! were! used! per! culture! batch.! Each! cerebrum! with! removed! cerebellum! was! dissected! in! ice-cold! HBSS,! carefully! stripped! of! their! meninges,! and! digested! with! 0.25%! trypsin! for! 15! min! at! 37ºC.! Trypsinization! was! stopped!by!addition!of!an!equal!volume!of!DMEM-F12!culture!medium!(Sigma-Aldrich,! St!Louis,!USA)!supplemented!with!10%!FBS!(Sigma-Aldrich),!2!mM!L-glutamine!(Gibco,! Thermo!Fisher!Scientific,!Massachusetts,!USA),!non-essential!aminoacids!(NEAA,!Gibco)! 100! U/ml! penicillin! (Gibco),! 100! μg/ml! streptomycin! (Gibco),! with! 0.02%! deoxyribonuclease!I!(Sigma-Aldrich).!The!cells!were!dispersed!into!a!single!cell!level!by! repeated! pipetting,! then! pelleted! and! resuspended! in! culture! medium.! The! cell! suspension!was!passed!through!a!70!μm-pore!cell!strainer.!4-5!cerebral!cortices!were! plated!in!6-well!plates!and!cultured!at!37°C!in!humidified!5%!CO2,!95%!air.!The!medium! was!replaced!every!4–5!days.!These!cultures!(mixed!glia)!reached!confluence!after!7– 10! days! and! were! used! between! 15! and! 20! days! after! preparation.! Microglia! were! separated!from!the!rest!of!the!cells!by!mild!trypsinization!as!described!before123,!with! certain! modifications.! ! The! confluent! mixed! glial! cultures! were! treated! with! trypsin! (0.05%)!diluted!1/3!in!medium!without!FBS,!causing!an!intact!layer!of!cells!containing! almost! all! the! astrocytes! to! become! detached,! leaving! behind! a! highly! enriched! population! of! microglia! (>98%),!as! determined! by! flow! cytometric!analysis! using! PE- conjugated! CD11b! antibody! (Abcam,! Cambridge,! UK).! The! attached! microglia! were! allowed! to! recover! in! conditioned! medium! (half! new! culture! medium,! half! mixed! microglia-used! culture! medium)! for! 24! h! before! being! subjected! to! the! different! treatments.! BV-2!and!N13!cell!lines!were!kindly!provided!by!Prof.!Diego!Ruano!(Institute!of! Biomedicine!of!Seville!and!University!of!Seville,!Spain).!Both!cell!lines!were!maintained! ! 64! α! levels! were! assayed! using! Mouse! IL-1β/TNF-α! BD! OptEIA! ELISA! set! (BD! Biosciences,!Madrid,!Spain)!according!to!the!manufacturer's!protocol.!IL-18!levels!were! assay!using!an!ELISA!KIT!(MBL,!MA,!USA)!according!to!the!manufacturer´s!protocol.!For! detecting! activated! caspase-3! protein! levels,! the! Human/Mouse! Cleaved! Caspase-3! (Asp175)-DuoSet! ELISA! kit! (R&D! Systems,! Abingdom,! UK)! was! used.! For! this,! total! protein! was! extracted! from! treated! microglial! cells! in! culture! with! ‘lysis! buffer’! according!to!the!manufacturer’s!instructions,!and!quantified!with!DC®!protein!assay!kit! (Bio-Rad! Laboratories,! Hercules! California,! CA,! USA).! Cytokine! profiles! shown! were! performed! with! sample! duplicates! and! are! representative! of! more! than! three! independent!experiments.!! ! 6.10 Western!blot!assays!(except!for!organotypic!cultures!and!for!MOK! detection)! Cells! were! cultured! on! 24-well! plates! (Nunclon! Delta! Surface,! Thermo! Scientific,! Massachusetts,!USA)!at!a!seeding!concentration!of!9x104!cells/mL!and!treated!for!24!h! as!described!before.!After!incubation,!cells!were!washed!once!with!ice-cold!PBS,!and! lysed!in!ice-cold!lysis!buffer!(3%!sodium!dodecyl!sulfate,!10%!(v/v)!glycerol,!and!10!mM! disodium! hydrogen! phosphate)! containing! Complete! EDTA-free! Protease! inhibitor! cocktail! tablets! (Roche,! Basel,! Switzerland)! for! 30! min! at! 4! ºC.! The! lysates! were! centrifuged!at!21,000!g!at!4!ºC!for!20!min,!supernatants!were!collected!and!stored!at! −80!°C! until! use.! The! protein! concentrations! of! these! extracts! were! determined! by! using! DC®! protein! assay! kit! (Bio-Rad! Laboratories).! Equal! volumes! of! cell! lysate! with! comparable!protein!content!(30-40!μg)!were!loaded!onto!15%!or!12!%!SDS-PAGE!gel! (the!loading!buffer!containing!0.1!M!DTT)!and!subjected!to!electrophoresis!at!180!V.! All! samples! were! electrophoretically! transferred! to! a! nitrocellulose! membrane! (GE! Healthcare! Life! Sciences,! Germany).! As! a! control! for! IL-18! Western! blots,! 0.05! µg! murine!mature!IL-18!recombinant!protein!(GenWay!Biotech,!San!Diego,!CA,!USA)!was! used.!After!blocking!with!5%!non-fat!milk!in!Tris-buffered!saline!(TBS)!containing!0.05%! Tween!20!(TBST)!for!1!h!at!RT!and!washed!with!TBST,!the!membranes!were!incubated! with! the! corresponding! primary! antibody:! 1:1000! rabbit! polyclonal! anti-caspase-1! (ab189796,! Abcam,! Cambridge,! UK)! for! analyses! of! cell! cultures,! or! 1:1000! mouse! ! ! 65! monoclonal!anti-caspase-1!(clone!Casper-1,!AG-20B0042,!Adipogen!Life!Sciences,!CA,! USA)! for! organotypic! cultures,! 1:750! rabbit! polyclonal! anti-IL-18! (210-401-323,! Rockland!Inc.,!Limerick,!PA,!USA),! 1:750! rabbit! anti-TDP-43! (12892-1-AP,!Proteintech! Group,! Inc.! Rosemont,! USA),! or! 1:5000! anti-! glyceraldehyde-3-phosphate! dehydrogenase!(GAPDH)!antibody!from!mouse!(clone!6C5,!MAB374,!Merck!Millipore,! Darmstadt,!Germany).!In!all!cases!the!membrane!was!incubated!o/n!at!4!ºC!with!the! primary!antibody.!The!membrane!was!washed!with!TBST!and!incubated!with!an!antirabbit! IgG! HPR-conjugate! secondary! antibody! (P0448,! DAKO,! Madison,! WI,! USA)! or! anti-mouse!IgG!(H+L)!HRP!conjugate!(W4021,!Promega),!for!1!h!at!RT.!The!antibodybound! proteins! were! detected! with! the! ECL! chemiluminescence! reagent! (Merck! Millipore)!and!the!signal!revealed!with!ChemiDoc™!MP!System!(Bio-Rad!Laboratories)! Western!Blot!images!were!analyzed!and!quantified!with!Image!Lab™!software!(Bio-Rad! Laboratories).! The! relative! expression! of! proteins! was! normalized! to! that! of! loading! controls.! ! 6.11 IL-18! immunoprecipitation! (IP)!assay! and! IL-18! Western! blot! from! organotypic!supernatants!! Spinal!cord!organotypic!cultures!supernatants!were!incubated!o/n!at!4º!C!with!4!µg!of! anti-IL-18!polyclonal!antibody!from!rabbit!(MBS175182,!Mybiosource,!San!Diego,!CA,! USA).! Subsequently,! Protein! G-coated! Dynabeads! (Invitrogen)! were! used! to! immunoprecipitate! IL-18! from! supernatants,! according! to! the! manufacturer’s! instructions.!Elution!of!bound!proteins!was!performed!in!30!mL!of!Glycine!50!mM!pH! 2.8!(Sigma-Aldrich).!The!eluted!fractions!from!protein!G-coated!Dynabeads-antibody- protein! complex! were! resolved! by! 15%! SDS-PAGE.! Western! blot! analysis! was! conducted!as!described!before.! ! 6.12 Preparation! of! cell! lysates,! co-immunoprecipitation! (co-IP)! assays! and!MALDI-MS!analysis!of!bound!proteins! 2x106! primary! microglial! cells! were! lysed! in! NP-40! cell! lysis! buffer! (Invitrogen)! ! 66! according! to! the! manufacturer´s! instructions.! Half! of! the! volume! of! total! protein! extract!was!incubated!with!20!µg!of!TDP-43-containing!IBs!and!half!with!20!µg!of!mock! IBs,!for!30!min!at!RT.!Subsequently,!the!samples!were!incubated!o/n!at!4!ºC!with!4!µg! anti-TDP-43! polyclonal! antibody! from! rabbit! (12892-1-AP,! Proteintech! Group,! Inc.! Rosemont,!USA)!in!order!to!favor!binding!of!the!protein!to!the!antibody.!Subsequently,! Protein! G-coated! Dynabeads! (Invitrogen)! were! used! to! immunoprecipitate! TDP-43- bound! proteins,! according! to! the! manufacturer’s! instructions.! Elution! of! bound! proteins!was!performed!in!20!mL!of!Glycine!50!mM!pH!2.8!(Sigma-Aldrich,!St.!Louis,! USA).! For! identification! of! TDP-43-bound! proteins,! elution! fractions! and! the! second! and! third! washes! after! formation! of! protein! G-coated! Dynabeads-antibody-protein! complex!were!resolved!by!12%!SDS-PAGE!and!silver!staining!with!SilverQuest™!Silver! Staining!Kit!(Life!Technologies,!Carlsbad,!CA,!USA).!Selected!bands!were!excised!from! the!gel!and!subjected!to!MALDI-TOF!MS!analysis!or!to!Western!blot!for!bound!protein! confirmation.!! ! 6.13 Protein!preparation!for!mass!spectrometry!and!MALDI-TOF/MS! Protein! preparation! was! performed! at! the! Institute! of! Plant! Biochemistry! and! Photosynthesis,! cicCartuja! (CSIC-University! of! Seville,! Seville)! based! on! a! previously! described!protocol!(35)!with!the!following!modifications.!After!the!washing!step,!the! selected!proteins!were!incubated!for!10!min!with!100%!acetonitrile!and!air-dried.!In! the!digestion!step,!samples!were!incubated!for!4!h!with!13.1!ng/mL!sequencing!grade! trypsin!(Promega,!Madison,!WI,!USA)!in!25!mM!ammonium!bicarbonate!(pH!8.5)!at!30! °C.!! The! MALDI-TOF-MS! analysis! was! done! as! previously! described124,! except! that! proteins! ambiguously! identified! by! peptide! mass! fingerprinting! were! subjected! to! MS/MS! sequencing! analysis! using! the! 5800! system! MALDI-TOF/TOF! Analyzer! (AB! SCIEX,!Connecticut,!USA).!! For! protein! identification,! NCBI! non-redundant! database! restricted! to! mouse! sequences! was! searched! using! license-free! MASCOT! server! through! Global! Protein! Server!v.!3.6!from!Applied!Biosystems!(Thermo!Fisher!Scientific,!Waltham,!MA,!USA).! In!all!protein!identifications,!probability!scores!were!greater!than!the!score!established! ! ! 67! by!MASCOT!as!significant,!with!a!p!value!lower!than!0.05.! ! 6.14 Co-IP!and!Western!blot!for!MOK!detection! After!co-IP!(as!described!before),!the!elution!and!flow-through!fractions!were!resolved! by! 12! %! SDS-PAGE.! Western! blot! analysis! was! conducted! as! described! before! with! minor!modifications.!Briefly,!for!detection!of!MOK!protein,!rabbit!anti-MOK!antibody! (ab135623,!Abcam,! Cambridge,! UK)!diluted!1:500,!was!used.! After! incubation! of! the! membrane!o/n! at! 4! ºC!with! the!primary! antibody,! mouse! anti-rabbit! IgG! light! chain! (Cell!Signaling!Technology!Inc.,!Danvers,!USA)!was!used!in!order!to!avoid!masking!of! MOK’s! band! (ca.! 48! kDa)! with! rabbit! IgG! heavy! chain! band! from! the! Co-IP! capture! antibody!(ca.!50!kDa).!The!antibody!was!incubated!for!1!h!at!RT.!After!washing,!antimouse!IgG!HPR-conjugate!secondary!antibody!(Promega,!Madison,WI,!USA)!was!used.!! ! 6.15 Western!blot!for!assaying!phosphorylated!MOK! Treated! cells! were! lysed! in! NP40! cell! lysis! buffer! (Invitrogen)! containing! EDTA-free! protease! inhibitor! cocktail! (Roche,! Basel,! Switzerland)! and! PhoStop! (Roche,! Basel,! Switzerland)! according! to! the! manufacturer´s! protocol.! The! primary! antibodies! used! were! 1:1000! MOK! (phospho-Tyr161)! antibody! from! rabbit! (12518,! SignalWay! Antibody,! Baltimore,! USA)! and! anti-a-tubulin! antibody! from! mouse! (clone! DM1A,! T6199,!Sigma-Aldrich),!and!the!secondary!antibodies!were!peroxidase-conjugated!goat! anti-rabbit! immunoglobulin! (P0448,! DAKO)! and! anti-mouse! IgG! (H+L)! HRP! conjugate! (W4021,!Promega),!respectively.! ! 6.16 RNA!isolation,!cDNA!synthesis!and!real-time!qPCR! Expression! levels! of! cIAP1/2! and! hypoxanthine-guanine! phophoribosyl! transferase! (HPRT)!genes!were!determined!by!using!a!two-step!quantitative!real-time!PCR!(qRT- PCR)! method.! Total! RNA! from! treated! microglial! cells! was! extracted! by! using! the! Tripure!Isolation!Reagent!(Roche)!according!to!the!manufacturer's!protocol.!RNA!(1!µg)! was! reverse-transcribed! by! using! the! Quantitect! Reverse! Transcription! kit! (Qiagen! GmbH,! Hilden,! Germany)! according! to! the! manufacturer's! protocol.! qRT-PCR! was! ! 68! performed! with! SensiFAST™! SYBR! Lo-ROX! Kit! (Bioline,! London,! UK)! on! an! ABI! Prism! 7500! Real! Time! PCR! System.! Primer! pairs! (Sigma-Aldrich,! St.! Louis,! USA)! were! especially! designed! to! anneal! in! different! exons,! and! they! were:! HPRT_For:! 5’- GTAATGATCAGTCAACGGGGGAC-3’,! HPRT_Rev:!5’-CCAGCAAGCTTGCAACCTTAACCA-3’;! cIAP2_For:!5’-! ATCTTCCGAACTTTCTCCAGGG-3’;! cIAP2_Rev:!5’-! TGTCAGCCAAGTTCAAGCTG-3’.! Multiple! transcripts! were! analyzed! simultaneously! for! 40!cycles!by!using!an!optimized!qRT-PCR!thermal!profile.!Changes!in!gene!expression! were!determined!by!using!the!∆Ct!value!taking!HPRT!as!endogenous!control.!The!∆∆Ct! values!were!calculated!by!subtracting!the!average!∆Ct!values!obtained!for!non-treated! microglia!samples.! ! 6.17 Immunofluorescence!analyses!! For! immunofluorescence! (IF)! analyses,! highly! pure! microglial! cell! cultures! were! obtained! as! described! above! except! that! in! this! case! cultures! were! seeded! on! pretreated!poly-D-Lys! (Sigma-Aldrich,! St.!Louis,!USA)!glass!lamellas.!48!h!after! isolation,! microglial!cells!were!treated!as!described!before!for!4!h!or!24!h.! For! the! immunolabeling! step,! treated! microglial! cultures! were! fixed! in! cold! PBS! containing! 4%! p-formaldehyde! (PFA)! (Sigma-Aldrich)! for! 20! min! at! 4! °C! and! then! washed!with!PBS!prior!to!being!permeabilized!and!blocked!with!PBS!containing!0.5!%! (v/v)!triton!X-100!(Sigma-Aldrich)!and!3%!BSA!(Sigma-Aldrich)!for!1!h!at!4!°C.!Cells!were! incubated!o/n!at!4!°C!with!different!primary!antibodies:!(1:100)!rabbit!polyclonal!anti- TDP-43!(12892-1-AP,!Proteintech!Group,!Inc.!Rosemont,!USA)!(TDP-43),!(1:100)!rabbit! polyclonal!anti-IL-18!(MBS175182,!Mybiosource),!(1:1000)!mouse!monoclonal!anti-His- tag! (HIS.H8)! (MA1-21315,! Thermo! Scientific,! Rockland,! USA),! (1:500)! mouse! monoclonal! anti-ubiquitin! (MA1-10035,! Thermo! Scientific),! (1:500)! rabbit! polyclonal! anti-HSP27! (#ADI-SPA-803,! Enzo! Life! Sciences,! New! York,! USA),! mouse! monoclonal! (1:500)!anti-HSP70!(SPA-8132,!Enzo!Life!Sciences)!were!used.!Cells!were!incubated!for! 1!h!at!RT!with!their!corresponding!conjugated!secondary!antibodies!(Invitrogen).!After! washes! with! PBS,! the! nuclei! were! counterstained! by! incubating! cells! with! PBS! containing! 1! µg/mL! DAPI! (Sigma-Aldrich)! for! 15! min! at! 4! °C! and! examined! under! a! fluorescence! microscope.! Fluorescence! images! were! captured! with! an! automated! ! ! 69! upright!microscope!(DM6000B,!Leica!Microsystems,!Wetzlar,!Germany).!Fluorescence! images!were!taken!at!x63!or!x100!magnification!from!randomly!chosen!fields.! ! 6.18 Organotypic!culture!immunofluorescence!analyses! Spinal!cord!slices!were!fixed!with!4%!paraformaldehyde!in!phosphate-buffered!saline! for!3h!at!4ºC.!After!blocking!with!5%!normal!donkey!serum!(EMD!Millipore,!Darmstadt,! Germany)!and!0.5%!(v/v)!Triton-X-100!in!PBS!(PBS-TX),!sections!were!incubated!with! primary!antibodies!against!anti-mouse-CD11b!(ab8878,!Abcam,!Cambridge,!UK),!anti- TDP43,! anti-His-tag,! anti-MOK! and! anti-caspase-3! diluted! 1:200,! 1:100,! 1:1000,! 1:25! and!1:200,!respectively!in!blocking!buffer.!Slices!were!thoroughly!washed!in!PBS!with! 0.2%! Tween-20! (PBS-T)! and! incubated! them! with! appropriate! secondary! antibody! Alexa! Fluor®488donkey! anti-rabbit! IgG! (1:1000),! Alexa! Fluor®674!chicken! anti-rat! IgG! (1:1000),!Dylight®549!donkey!anti-mouse!IgG!(1:800)!(Invitrogen),!diluted!in!PBS-TX!with! 1! %! of! normal! donkey! serum! for! 2! h! at! RT.! Finally,! cell! nuclei! were! labelled! with! 1! mg/mL!DAPI!for!15!min!in!PBS!and!sections!were!mounted!with!Vectashield!medium! (Vector! Laboratories,! Burlingame,! CA,! USA).! We! analyzed! the! slides! under! confocal! microscope! (Confocal! Laser! Scanning! Microscope! TCS! SP5,! Leica! Microsystems,! Wetzlar,!Germany).! ! 6.19 Determination!of!cleaved!caspase-3!levels!by!immunofluorescence! (IF)! For!immunofluorescence!(IF)!analysis!of!caspase-3,!highly!pure!primary!microglial!cell! cultures! were! seeded! on! pre-treated! poly-D-Lys! (Sigma-Aldrich)! glass! lamellas! and! treated!as!described!before!for!4!h!or!24!h.!For!the!immunelabeling!step,!the!protocol! used!was!the!one!described!above!but!cells!were!incubated!o/n!at!4!°C!with!cleaved! caspase-3! (Asp175)! specific! antibody! (9661,! Cell! Signaling! Technology! Inc.,! Danvers,! USA)! diluted! 1:200! in! PBS! containing! 3%! BSA! and! 0.5%! (v/v)! Triton! X-100.! As! a! secondary!antibody,!donkey!anti-rabbit!IgG-AlexaFluor®594!(Invitrogen)!was!used!in!the! same! buffer.! Fluorescence! images! were! captured! with! a! confocal! laser! scanning! microscope!(TCS!SP5,!Leica!Microsystems,!Wetzlar,!Germany)!and!images!were!taken! ! 70! at!x63!magnification!from!randomly!chosen!fields.!For!quantitative!analysis!of!cleaved! caspase-3!levels!by!IF,!the!imaging!software!MetaMorph!Offline!(version!7.5.1.0,!MDS! Analytical!Technologies,!USA)!was!used.!Analysis!was!performed!by!using!the!intensity! values!of!AlexaFluor®594!(RF)!and!DAPI!(BF)!above!background!(areas!lacking!cells),!and! the! data! were! expressed! as! arbitrary! units! (AU)! and! exported! automatically! to! Microsoft!Excel!software!through!a!summary!log.!Quantification!of!cleaved!caspase-3! (total)!levels!was!performed!by!analyzing!the!average!intensity!levels!of!RF!per!cell.!For! quantifying!the!number!of!cleaved!caspase-3!granules!per!cell,!the!intensity!levels!of! RF! and! granularity! size! were! considered.! The! cut-off! size! selected! for! activated! caspase-3!granules!was!>6!pixels.!The!nuclear!or!cytoplasmic!localization!of!granules! were!analyzed!according!the!DAPI!nuclear!staining!of!cells.!For!quantitative!analyses!of! cleaved! caspase-3! levels! by! IF,! ten! images! from! random! fields! (each! containing! 6-8! cells)!were!used!for!each!sample!condition!in!each!experiment,!and!three!independent! experiments! (N=3)! were! used! to! calculate! the! mean! RF/BF! and! SEM! values! represented!in!the!figures.! ! 6.20 Co-localization!studies!of!internalized!TDP-43!and!MOK!! For!assaying!the!co-localization!of!exogenously!added!TDP-43!with!MOK!in!microglia,! the!treatment!protocol!was!the!same!as!the!one!described!before!with!an!incubation! period!of!4!h,!except!that!in!the!case!of!BV-2!cells!a!seeding!concentration!of!1.2x105! cell/mL! was! used.! The! immunolabeling! protocol! was! the! same! as! described! before! except!that!in!this!case!(1:25)!anti-MOK!rabbit!polyclonal!antibody!(Abcam,!Cambridge,! UK)! and! (1:1000)! anti-His-tag! mouse! monoclonal! antibody! (Thermo! Scientific,! Rockland,!USA)!for!detection!of!exogenous!TDP-43,!were!used!as!primary!antibodies.! Secondary! antibodies! used! were! 1:800! donkey! anti-rabbit! IgG-AlexaFluor®594! (Invitrogen)! (RF)! for! MOK! protein! detection,! and! 1:800! donkey! anti-mouse! IgG- AlexaFluor®488! (GF)! (Invitrogen)! for! His-tag! detection.! After! washes! with! PBS,! the! nuclei! were! counterstained! by! incubating! cells! with! PBS! containing! 1! µg/mL! DAPI! (Sigma-Aldrich)! for! 30! min! at! 4! °C! and! examined! under! a! fluorescence! microscope.! Fluorescence! images! were! captured! with! a! confocal! laser! scanning! microscope! (TCS! SP5,! Leica! Microsystems,! Wetzlar,! Germany)! and! images! were! taken! at! x63! ! ! 71! magnification! from! randomly! chosen! fields.! For! quantitative! co-localization! analysis,! the! imaging! software! MetaMorph! Offline! (version! 7.5.1.0,! MDS! Analytical! Technologies,!USA)!was!used.!Analysis!was!performed!by!using!the!area!parameters!of! AlexaFluor®594!(RF)!and!AlexaFluor®488!!(GF)!above!background!(areas!lacking!cells),! and! the! data! were! expressed! as! percentage! (%)! of! co-localization! and! exported! automatically!to!Microsoft!Excel!software!through!a!summary!log.!! ! 6.21 NSC-34!cells!neurotoxicity!assay! NSC-34!motor!neuron!cells!were!seeded!at!a!density!of!7.5x104!cells!per!well!in!400!μ L!of!NSC-34!culture!medium!on!a!24-well!tissue!culture!plate!provided!with!0.1!μm! (pore! size)! PET! membrane! inserts! (Guangzhou! JET! Bio-Filtration! Co.,! Guangzhou,! China).!After!24!h!in!culture,!conditioned!medium!of!primary!microglia!treated!for!4!h,! as!described!before,!was!added!in!to!the!inserts!in!order!to!avoid!contact! of!TDP43! aggregates! with! motor! neuron! cells.! After! 24! h! or! 48! h! in! culture,! lactate! dehydrogenase! (LDH)! cytotoxicity! detection! assay! (Roche,! Basel,! Switzerland)! was! performed!according!to!the!manufacturer's!protocol.!!! ! 6.22 MOK!inhibition!assay! Lyophilized! fluoromethylketone! or! compound! 13! (C13)! was! a! kindly! gift! from! Prof.! Kevan!Shokat´s!lab,!University!California,!San!Francisco!(UCFS,!CA,!USA).!Stock!solution! was!prepared!in!DMSO!at!a!concentration!of!10!mM.!Microglia!or!organotypic!cultures! were!pre-treated,!for!1!h!and!2!h!respectively,!with!C13!at!a!final!concentration!of!10! µM!diluted!in!culture!medium!before!performing!the!treatments!as!described!before! for!24!h!without!removing!the!compound.!Controls!were!added!with!the!same!volume! of!DMSO!as!the!one!added!to!the!C13!treatments!in!order!to!discriminate!any!effect! cause!by!the!DMSO.! ! ! 72! 6.23 Immunohistochemistry!(IHC)!of!cryopreserved!mouse!samples!! Mouse! spinal! cords! were! carefully! extracted! from! 4%! PFA! perfused! mice! previously! anesthetized!as!described!for!organotypic!spinal!cord!cultures.!Spinal!cords!were!cut! into! 30! µm! transverse! sections! with! a! vibrating! blade! microtome! (VT1000S,! Leica! Biosystems,! Nussloch,! Germany)! and! freeze! at! -20! ºC! in! a! cryopreservative! solution! (30%! sucrose! and! 10! %! glycerol! in! PBS).! Cryopreserved! spinal! cord! sections! were! washed! for! 10! min! with! PBS! before! endogenous! peroxidase! inactivation! with! 3%! of! H2O2!in!PBS!for!45!min!in!the!dark.!Tissue!permeabilization!and!blocking!of!non-specific! antibody!binding!were!performed!with!blocking!solution!containing!1%!BSA,!0.5%!FBS,! and!0.1%! Triton!TX-100!in!PBS!for! 1! h.!MOK! (phospho-Tyr161)!antibody!from!rabbit! (SignalWay! Antibody)! and! MOK! (phospho-Thr159+Tyr161)! antibody! from! rabbit! (SP1030,!StressMarq!Bioscience,!Victoria,!Canada)!primary!antibodies!(1:100!and!1:50,! respectively)! were! incubated! overnight! in! blocking! solution! at! 4ºC.! Non-specific! primary!antibody!binding!was!removed!with!3!washes!in!PBS.!Biotinylated!secondary! antibody! staining! was! performed! with! a! Vectastain! ABC! kit! (vector! laboratories,! Burlingame,! CA! 94010,! USA)! following! manufacturers! instructions.! To! remove! nonspecific! secondary! antibody,! 3! washes! in! PBS! were! done.! A! DAB! (3,3ʹ- Diaminobenzidine)!Peroxidase!(HRP)!Substrate!Kit!from!Vector!Laboratories!was!used! for! developing! the! staining! following! its! instructions.! Immunostained! images! were! captured! with! an! automated! upright! microscope! (DM6000B,! Leica! Microsystems,! Wetzlar,! Germany).! Images! were! taken! at! x40! or! x63! magnification! from! randomly! chosen!fields.! 6.24 Immunohistochemestry!of!paraffin-embedded!human!tissue! Spinal! cord! sections! from! ALS! patients! and! controls! were! supply! by! Dr! Salvador! Martinez! perez,! M.D.,! Instituto! de! Neurociencias! de! Alicante.! Tissue! sections! were! dewaxed! by! heating! at! 62°C! for! 1! h! before! washing! twice! with! xilol.! Afterwards,! sections!were!rehydrated!with!ethanol!steps:!first,!two!washes!were!performed!with! 100°!ethanol!followed!by!two!washes!with!96°!and!70°!ethanol,!!respectively.!Finally,! tissue!sections!were!washed!with!distilled!water.!After!dewaxing!and!rehydrating,!the! sections!were!incubated!in!0.1!M!sodium!citrate!for!30!min!at!sub-boiling!temperature! ! ! 73! for! epitope! retrieval.! Tissue! sections! were! then! washed! for! 10! min! with! PBS! and! subsequent!steps!were!performed!as!described!for!mouse!samples.!! ! 6.25 Statistical!analyses! All! values! of! results! derived! from! at! least! three! independent! experiments! (N≥3)! are! expressed! as! the! mean! ±! SEM,! whereas! results! from! two! independent! experiments! (N=2)!are!expressed!as!mean!±!SD.!For!all!determined!parameters,!statistical!analysis! was!performed!with!the!non-parametric!Mann-Whitney!U!(two-sided)!test!using!SPSS! Statistics!22.0!(IBM!Company,!Chicago,!USA).!Statistically!significant!differences!were! those!with!p<0.05.! ! ! 80! ! Figure 7-2 TDP-43 aggregates are internalized and modified by microglial cells, mobilizing endogenous TDP-43 protein. A) IF analysis by confocal microscopy of CD11b (white), His-tag (red) and TDP-43 (green) immunolabelling of spinal cord organotypic slice cultures after 24 h of treatment with 5 µg/mL of TDP43 (or mock control) showing the presence of His-tag/TDP-43-containing inclusions in the cytoplasm of microglial cells and the clearing of nuclear TDP-43, in the sample treated with exogenous TDP-43 aggregates. Cell nuclei were counterstained with DAPI (blue). Scale bars: 25 µm. Cultures without treatment and mock-treated cultures were used as controls in all experiments. Images and results shown are representative of at least three independent experiments in all cases. Finally,!to!test!whether!these!effects!of!extracellular!TDP-43!aggregates!can!be! extrapolated! to! a! more! physiopathological! context,! we! performed! IF! studies! with! spinal! cord! organotypic! cultures! from! adult-like! mice,! after! treatment! with! TDP-43! aggregates!(or!mock!control)!for!24!h!(Fig.!2A).!Organotypic!slice!cultures!offer!unique! advantages!compared!to!other!in#vitro!and!cell!culture!approaches!because!they!retain! the! tissue! architecture,! homeostasis! and! inter-cellular! communication,! and! preserve! neuronal! activity.! Specifically,! organotypic! spinal! cord! cultures! can! be! maintained! in! culture!for!several!weeks!and!are!a!useful!platform!for!assessment!of!a!wide!range!of! cellular!interactions126,127.!Remarkably,!confocal! microscopy! analysis! of! CD11b-,! TDP- 43-!and!His6-tag-immunolabeled!samples!clearly!demonstrated!that!the!extracellular,! exogenously! added! TDP-43! aggregates! are! indeed! internalized! by! microglial! cells,! among! other! cell! types,! which! could! be! observed! as! His-tag-! and! TDP-43-containing! ! ! 81! cytoplasmic!inclusions,!and!promote!the!clearing!of!nuclear!TDP-43.!Taken!together,! these!results!are!in!line!with!previous!observations!in!neuronal!cells!and!are!the!first!to! point!to!abnormal!mobilization!of!intracellular,!endogenous!TDP-43!protein!caused!by! exposure!to!extracellular!TDP-43!aggregates!in!microglia.!! 7.2 TDP-43! aggregates! activate! inflammasome-dependent! and! independent!neuroinflammatory!responses!involving!caspase-3!! Having!verified!that!microglial!cellular!viability!is!not!significantly!compromised! by!exposure!to!TDP-43!at!<5!μg/mL!for!up!to!48!h!as!measured!by!MTT!analyses!(Fig.! 3A),!we!decided!to!study!the!effect!of!such!TDP-43!aggregates!on!primary!microglial! activation!and!immune!response.!It!has!been!reported!that!protein!aggregates!found! in! the! extracellular! medium! have! the! ability! to! activate! microglia! and! trigger! a! proinflammatory! immune! response34,128.! .The! secretion! of! the! key! proinflammatory! cytokines!IL-1b!and!IL-18!is!typically!enabled!by!activation!of!the!NLRP3!inflammasome! system! and! finally! cleaved! into! their! mature! and! secretable! forms,! by! caspase-1! protease129.!The!NRLP3!inflammasome!has!been!involved!in!many!neurodegenerative! diseases!such!as!PD!and!AD,!and!many!studies!have!shown!that!mature!forms!of!IL-1β! and! IL-18! are! upregulated! in! ALS130.! The! NLRP3! inflammasome! activation! typically! occurs! in! two! steps.! The! first! step! involves! a! priming! or! initiating! signal,! leading! to! activation! of! NF-κB-mediated! signaling! which! up! regulates! imflammasome-related! components,! including! inactive! pro-IL-1b! and! pro! IL-18.! The! second! step! is! the! assembly! of! NRLP3,! Apoptosis-associated! speck! like! protein! containing! a! caspase! recruitment! domain! (ASC)! and! pro-caspase-1! into! a! multi-protein! complex.! This! complex!triggers!the!transformation!of!pro-caspase-1!into!activated!caspase-1,!as!well! as!the!production!and!secretion!of!active!IL-1β!and!IL-18131,132.!By!using!lysates!from! 24!h-treated!cells!in!a!Western!blot!against!caspase-1,!we!observed!a!strong!and!highly! specific!band!of!ca.!45!kDa!corresponding!to!pro-caspase-1!in!TDP-43-exposed!cells,!as! opposed!to!the!mock!control!(Fig.!3B).! ! 82! Furthermore,!by!quantifying!the!levels!of!key!cytokines!released!to!the!medium,! we!noticed!significant!increments!in!IL-1β!!and!TNF-α!secretion!as!a!result!of!exposure! to! TDP-43! aggregates! (Fig.! 3C).! As! expected,! pre-incubation! of! cells! with! the! NLRP3! inflammasome! inhibitor! Bay11-7082! resulted! in! the! complete! abrogation! of! IL-1β!! release,! demonstrating! that! IL-1β! ! secretion! occurs! via! activation! of! the! canonical! NLRP3! inflammasome! system! (Fig.! 3C).! These! results! are! consistent! with! those! previously!reported!for!microglial!cells!stimulated!with!non-aggregated,!soluble!TDP- 43116,! therefore! demonstrating! a! comparable! activation! of! the! NF-κB/NLRP3! inflammasome!pathway!as!a!result!of!microglial!exposure!to!exogenous!TDP-43!in!an! aggregated!form. ! ! 83! ! Figure 7-3 TDP-43 aggregates activate NLRP3-dependent and independent neuroinflammatory responses. A) Cytotoxicity determination assays with BV-2 cells microglial cells exposed for 24 h and 48 h to TDP43 or mock at 5 mg/mL or lower, were performed by the MTT assay. B) Pro-caspase-1 expression in treated cells analyzed by Western blot with cell lysates from primary microglial cells treated for 24 h with 2.5 µg/mL of TDP43 or mock. Blot is representative of 5 independent experiments (N=5). C) Quantitative determination by ELISA of IL-1b and TNF-a cytokine levels in supernatants from N13 (TNF-a) or primary microglial cells (IL-1b), 6 h or 24 h after treatment with 5, 2.5 or 1.25 μg/mL of TDP43 or mock, with or without pre-treatment with Bay11-7082. Bars are the mean of at least 3 independent experiments (N≥3), each one performed with duplicate samples. D) Quantitative determination by ELISA of secreted, activated IL-18 in culture supernatants of primary microglia 24 h after treatment with 2.5 or 5 µg/mL of TDP43 or mock. Absolute measured levels of IL-18 for TDP-43- treated cells ranged between 60 and 90 pg/mL between independent experiments. Bars are mean of six ! 84! independent experiments (N=6), each one performed with duplicate samples. E) Western blot analysis of cleaved IL-18 in supernatants from primary microglial cells collected 24 h after treatment with 5 µg/mL of TDP43 or mock. Recombinant murine IL-18 was used as a control in the same gel (right end). Bottom: band density quantification (relative values) from three independent experiments (N=3). F) IF analysis of intracellular IL-18 in N13 cells, 24 h after treatment with 5 µg/mL of TDP43 or mock. Scale bars: 20 µm. G) Western blot analysis of cleaved IL-18 in cell lysates from primary microglial cells treated with 5 µg/mL TDP43 or mock, for 24 h. Quantitative analysis of ca. 16 kDa IL-18 (relative values) from four independent experiments (N=4). Cells without treatment and mock-treated cells were used as controls in all experiments. Unless indicated otherwise, error bars correspond to ± SEM of at least 3 independent experiments. In all cases, *p<0.05, **p<0.01, ***p<0.001 denote significant differences for results from N≥3 independent experiments. Similarly!to!IL-1β,!IL-18!is!a!proinflammatory!cytokine!that!is!typically!activated! by!caspase-1-mediated!cleavage!via!the!NLRP3!inflammasome!to!generate!biologically! active!18!kDa!IL-18,!which!is!then!secreted!to!the!extracellular!medium133.!Unlike!IL-1 β!—which!is! involved! in! Th17!cell! responses—! IL-18! has! a! role! in! Th1! immunity134.! Importantly,!IL-18!cytokine!has!been!measured!at!higher!levels!in!serum,!spinal!cord! and!brain!of!sporadic!ALS!patients130,135,136.!Moreover,!in-situ!expression!of!IL-18!and! all! its! associated! mediators! have! been! recently! detected! in! brains! of! sporadic! ALS! patients136,! indicating! that! IL-18! may! have! an! important! role! in! neurodegenerative! progression.! Based! on! this,! we! decided! to! test! the! possible! activation! of! IL-18! as! a! result!of!exposure!of!microglia!to!extracellular!TDP-43!aggregates.!Indeed,!ELISA!assays! allowed! us! to! detect! a! low! level! of! activated! IL-18! cytokine! in! supernatants! from! primary!microglial!cells!that!had!been!exposed!to!TDP43,!which!was!suppressed!by!the! addition! of! the! NLRP3! inflammasome! inhibitor! Bay11-7082! (Fig.! 3D).! These! results! were! confirmed! by! the! presence! in! the! supernatant! from! TDP-43-treated! cells! of! a! differential!band!corresponding!to!a!18!kDa!species!as!analyzed!by!Western!blot!(Fig.! 3E).!Importantly,!secretion!of!IL-18!induced!by!TDP43!treatment!(and!not!by!the!mock! control)! was! reproduced! in! an! analogous! experiment! performed! with! organotypic! spinal!cord,!as!revealed!by!IL-18!immunoprecipitation!of!culture!supernatants!followed! by! Western! blot! analysis! (Fig.! 4C).! Moreover,! elevated! levels! of! pro-caspase-1! were! found!in!lysates!from!spinal!cord!organotypic!cultures!treated!with!TDP-43!aggregates,! suggesting!that!IL-18!had!been!processed!by!the!canonical!NLRP3!pathway!(Fig.!4B).!!In! addition! to! this,! our! results! from! IF! studies! of! treated! N13! microglial! cells! revealed! significant!levels!of!intracellular!IL-18!species!as!a!result!of!TDP-43!treatment!(Fig.!3F),! ! ! 85! which!led!us!to!examine!the!intracellular!IL-18!content!by!Western!blot!in!cell!lysates! from! treated! primary! microglial! cells.! Unexpectedly,! our! results! showed! a! strong! differential! band! corresponding! to! ca.! 16! kDa,! in! samples! from! TDP-43-treated! cells! (Fig.!3G).!Furthermore,!the!accumulation!of!such!IL-18!processing!product!showed!to! be!unaltered!by!the!addition!of!the!NLRP3!inflammasome!inhibitor!Bay11-7082!(Fig.! 4A),!suggesting!that!an!NLRP3-independent!mechanism!of!IL-18!proteolytic!processing! also!takes!place.! ! ! Figure 7-4 Alternative processing of IL-18 into ca. 16 kDa species in primary microglial culture. Caspase-1 expression and IL-18 secretion in spinal cord organotypic slice culture. A) Western blot of total lysate from primary microglia treated for 24 h with 2.5 μg/mL of TDP43 or mock, and co-treatment with NLRP3 inflammasome inhibitor Bay11-7082, using an anti-IL-18 antibody (left). Quantitative analysis by band densitometry of ca. 16 kDa IL-18 (right). Shown values (mean ± SEM) are relative to the untreated control (Ctrl) in each experiment, and derive from four independent experiments (N=4). B) Western blot of total lysate from organotypic slice cultures from 4-week old mouse spinal cord, after 24 h of treatment with 2.5 μg/mL of TDP43 (or mock control), using anti-caspase-1 antibody (left). Quantitative analysis shown (relative levels) derives from two independent experiments (N=2). Values are mean ± SD. C) Western blot with anti-IL-18 antibody of supernatants from treated organotypic slice cultures, after IP with anti-IL-18 antibody for purposes of concentration. Image shown is representative of two independent experiments. ! 86! Apart!from!its!well-known!function!as!executioner!of!apoptosis,!caspase-3!has! been! recently! demonstrated! to! play! a! central! role! in! regulating! microglia! proinflammatory!activation!associated!to!neurotoxicity,!as!observed!in!brain!tissue!from! Alzheimer’s! disease! (AD)! and! Parkinson’s! disease! (PD)! patients137.! It! has! also! been! shown! that! such! different! functions! of! caspase-3! in! microglia! are! related! to! its! intracellular!location!—either!cytoplasmic!(proinflammatory)!or!nuclear!(apoptotic)!—! of! the! cleaved! protein137,138.! Based! on! this,! we! sought! to! investigate! the! expression! profile!of!activated!caspase-3!in!primary!microglia!that!had!been!treated!with!TDP43! or!mock,!by!using!IF!and!confocal!microscopy!analysis.!As!can!be!observed,!microglial! cells!that!had!been!exposed!to!TDP-43!aggregates!display!a!higher!signal!for!activated! caspase-3!as!compared!to!the!mock!control!after!4!h!and!24!h!(Fig.!5).!Quantification! of!total!activated!caspase-3!content!from!the!IF!images!confirmed!such!increment!in! cells! exposed! to! TDP43! (Fig.! 5B).! In! addition,! we! observed! an! increase! in! cleaved! caspase-3!in!the!form!of!granules!—similar!to!those!previously!described!in!viable!mast! cells139—!as!a!result!of!TDP43!treatment!!(Fig.!5C).!! ! Figure 7-5 TDP-43 aggregates induce a neuroinflammatory response involving caspase-3 (part I). A) Analysis by IF and confocal microscopy of activated caspase-3 levels in primary microglial cells 4 h after treatment with 5 μg/mL TDP43 or mock. Treatment with 1 µM staurosporine (STS) for 4 h and Leal et al. Fig. S2 LPS$ Casp3* DAPI Merge 24h 4h Ctrl STS LPS TDP43 Mock Casp3* DAPI Merge A 0 Casp-3* fluor/cell (AU) Ctrl STS LPS TDP43 Mock B C Ctrl STS LPS TDP43 Mock Casp-3* granules (no./cell) Total Cytoplasm 2 4 6 0 5 10 15 20 * #" ! ! 87! with 1 µg/mL LPS for 24 h served as ‘apoptosis’ and ‘inflammation’ controls, respectively. Merged images include DAPI fluorescence. Scale bars: 25 μm. B, C) Quantitative assessment of activated caspase-3 levels from IF and confocal microscopy images (as shown in A), based on total activated caspase-3 (B) or activated caspase-3 granules in the cytoplasmic and nuclear compartments of primary microglial cells (C). Values shown were normalized by the untreated control (Ctrl) within each of the three experiments, and subsequently referred to the average of granules/cell of the three Ctrl samples. Moreover,! this! result! is! further! supported! by! analogous! experiments! carried! out! with! organotypic! spinal! cord! cultures! showing! a! higher! content! of! activated! caspase-3! dots! within! CD11b+!cells,! in! TDP-43-treated! samples! (Fig.! 6A).! These! dots! were!mostly!located!in!the!cytoplasmic!compartment!and!in!close!proximity!to!the!cell! membrane.!Overall,!our!results!showed!increased!activated!caspase-3!levels!caused!by! exposure!of!microglial!cells!to!TDP-43!aggregates,!especially!in!the!number!of!cleaved! caspase-3!granules,!which!were!mainly!located!in!the!cytoplasmic!compartment.! Finally,!we!assayed!the!neurotoxic!capacity!of!stimulated!microglia!by!exposing! NSC-34!motor!neuron!cells!for!24!h!or!48!h!to!aggregate-free!conditioned!media!from! primary!microglial!cultures!(see!Materials!and!Methods!section).!Our!results!from!LDH! assays! evidenced! a! cytotoxic! effect! of! TDP-43-exposed! microglia! on! NSC-34! cells,! as! compared! to! the! mock! control,! indicating! microglial-mediated! cytotoxicity! of! extracellular! TDP-43! aggregates! on! motor! neurons! (Fig.! 6B).! Therefore,! our! results! show! that! TDP-43! aggregates! trigger! different! responses! associated! to! neurodegeneration,! including! not! only! NLRP3! inflammasome-dependent! IL-1β! secretion,!but!also!canonical!and!non-canonical!IL-18!processing,!as!well!as!caspase-3!a ctivation.!! ! 88! ! Figure 7-6 TDP-43 aggregates induce a neuroinflammatory response involving caspase-3 (part II). A) IF analysis by confocal microscopy of CD11b (red) and caspase-3 (green) immunolabeled of spinal cord organotypic slice cultures after 24 h of treatment with 5 µg/mL of TDP43 (or mock control) showing the presence of caspase-3 granules in the cytoplasm of microglial cells of the sample treated with exogenous TDP-43 aggregates. Cell nuclei were counterstained with DAPI (blue). Scale bars: 25 µm. B) Microglial-mediated neurotoxicity study performed by LDH assaying of NSC-34 motor neuron cultured cells after incubation for 24 h or 48 h with aggregate-free conditioned media from primary microglial cultures treated with 5 μg/mL of TDP43 or mock. Diagram is representative of three independent experiments each one performed by duplicate samples. Cells without treatment and mock-treated cells were used as controls in all experiments. Images and results shown are representative of at least three independent experiments in all cases. Unless indicated otherwise, error bars correspond to ± SEM of at least 3 independent experiments (N≥3). In all cases, *p<0.05, **p<0.01, ***p<0.001 denote significant differences for results from N≥3 independent experiments. 7.3 Microglial!response!to!TDP-43!aggregates!is!modified!by!exogenous! co-treatment!with!Hsp27!and!Hsp70!chaperones! We! then! sought! to! explore! whether! certain! HSPs! that! may! be! present! in! the! extracellular!medium!in!an!ALS/FTLD!pathophysiological!context!or!added!exogenously! for! therapeutic! purposes,! could! affect! the! neuroinflammatory! response! elicited! by! TDP-43! aggregates! in! microglia.! We! decided! to! analyze! the! effect! of! two! HSPs! in! B LDH release (%) 0 10 20 30 40 24 h 48 h Ctrl STS LPS TDP43 Mock NSC-34 * #" CD11b Casp-3* Merge DAPI CD11b Casp-3* Merge DAPI TDP43 Mock Ctrl LPS A ! ! 89! particular,! Hsp27! and! Hsp70.! These! HSPs! are! major! chaperones! with! known! immunological! activities64,65,! and! both! have! been! found! in! the! extracellular! medium! and!dysregulated!in!ALS!patients!and!animal!models59,60,65.!Importantly,!as!they!both! display!immunological!activities,!they!could!potentially!be!used!as!intervention!tools! over!the!inflammatory!response63,140.!To!that!aim,!we!prepared!mixtures!of!TDP-43!(or! mock)!in!the!presence!or!absence!of!Hsp27!or!Hsp70!(see!Materials!and!Methods!for! further! details),! as! well! as! samples! containing! either! component! alone! as! controls,! which!were!used!to!treat!microglial!cells!in!culture!for!4!h!or!24!h.!First,!we!examined! whether! exogenously! added! Hsp27! and! Hsp70! chaperones! were! internalized! into! primary!microglial!cells!in!a!TDP-43-dependent!manner!by!co-IF!by!using!anti-TDP-43! antibodies! (recognizing! both! exogenous! and! endogenous! TDP-43! protein)! and! anti- Hsp27!(Fig.!8A)!or!anti-Hsp70!specific!antibodies!(Fig.!7A).!No!significant!co-localization! between!TDP-43!and!Hsp70!was!seen!in!such!cells.! ! Figure 7-7 Microglial response to TDP-43 aggregates and cotreatment with Hsp70 chaperone. A TDP-43 Hsp70 Merge Ctrl TDP43 Mock Hsp70 TDP43/Hsp70 Mock/Hsp70 ! 96! 7.4 Internalization! of! extracellular! TDP-43! aggregates! by! microglia! disrupts! the! MAPK/MAK/MRK! overlapping! kinase! (MOK)! signaling! pathway! To! shed! light! into! the! molecular! mechanisms! of! microglial! activation! and! response!elicited!by!exogenous!TDP-43!aggregates,!we!first!carried!out!a!Co-IP!assay! with!anti-TDP-43!capturing!antibody!after!incubation!of!TDP-43!aggregates!(or!mock! control)! with! primary! microglial! cell! lysates.! Next,! we! analyzed! the! protein! bands! resolved! after! SDS-PAGE! electrophoresis! and! silver! staining! from! samples! corresponding!to!the!fractions!obtained!upon!elution!of!bound!proteins!(Fig.!12A).!A! differential!band!close!to!the!46-50!kDa!gel!resolving!region!in!the!TDP-43!sample!and! the! corresponding! region! in! the! mock! lane,! were! subjected! to! MALDI/MS! analysis.! According!to!the!peptide!fingerprint!obtained,!the!TDP-43!interactor!was!identified!as! murine! MAPK/MAK/MRK! overlapping! kinase! (MOK)! protein! (gi7106391),! while! that! protein!was!not!detected!in!the!mock!sample.!! MOK!is!a!recently!identified!member!of!the!ICK/MAK/MOK!family,!and!known!to! be!expressed!in!the!brain!but!whose!functions!remain!to!be!elucidated141,142.!In!order! to! confirm! such! interaction,! we! repeated! the! co-IP! assay,! this! time! followed! by! a! Western!blot!with!a!specific!anti-MOK!antibody.!In!addition!to!a!faint!band!that!could! be!seen!for!the!mock!sample!at!a!similar!migration!distance!(which!could!have!been! caused!by!interaction!through!the!endogenous!TDP-43!protein!present!in!the!lysate),!a! strong!band!for!the!sample!treated!with!exogenous!TDP-43!aggregates,!corresponding! to!the!ca.!48!kDa!expected!mass!for!MOK,!was!observed!(Fig.!12B).!Furthermore,!by! applying!His-tag/MOK!co-IF!studies!with!whole!primary!microglial!and!BV-2!cells!after! incubation!with!exogenous!TDP-43!aggregates!for!4!h,!we!were!able!to!observer,!apart! from!a!general!increase!in!MOK!signal,!a!significant!degree!of!co-localization!between! the!His6-containing!internalized!TDP-43!aggregates!and!MOK!protein!in!TDP43-exposed! cells!(Fig.!12C-F).!! ! ! 97! ! Figure 7-12 Extracellular TDP-43 aggregates target MOK signaling pathway in microglia upon internalization. A) Silver-stained SDS-PAGE analysis of the eluted fraction in Co-IP assay performed after incubating TDP43 (or mock control) with primary microglial lysates, by using anti-TDP-43 capture antibody. White arrows indicates a differential band/s and the regions in the gel (for both lanes) that were excised and analyzed by MALDI/MS. B) Western blot from SDS-PAGE of eluted fraction after Co-IP assay, with ! 98! anti-MOK antibody, confirms the interaction between TDP-43 aggregates and microglial MOK. C) Co-IF studies with His-tag (red) and MOK (green) immunolabeling by confocal microscopy of BV-2 cells exposed to 5 μg/mL TDP43 or mock show co-localization between internalized TDP-43 species and MOK protein in cells exposed to exogenous TDP-43 aggregates. Merged images include DAPI fluorescence. Scale bars: 25 μm. D) Quantitative analysis of His6-tag/MOK co-localization (over total MOK) based on co-IF and confocal microscopy analysis. E) Co-IF studies by confocal microscopy with His-tag and MOK-immunolabeled proteins of primary microglial cells exposed to 5 μg/mL TDP43 or mock show co-localization between internalized TDP-43 species and MOK in cells exposed to exogenous TDP-43 aggregates. Merged images include DAPI fluorescence. Scale bars: 25 μm. F) Quantitative analysis of His6-tag/MOK co-localization based on co-IF and confocal microscopy analysis. ! Notably,! such! general! up-regulation! of! MOK! protein! and! His-tag/MOK! colocalization! were! also! observed! by! IF! confocal! microscopy! analysis! of! organotypic! spinal!cord!after!treatment!for!24!h!with!exogenously!added!TDP-43!aggregates!(Fig.! 13A).!These!results!strongly!indicate!that!internalized!TDP-43!aggregates!interact!in!a! stable!and!specific!manner!with!MOK!protein!kinase!within!microglial!cells.!! ! Figure 7-13 MOK and TDP-43 aggregates co-localization inside microglial cells of organotypic cultures. A) Co-IF analysis by confocal microscopy of CD11b (white), His-tag (green) and MOK (red) immunolabelling of spinal cord organotypic slice cultures from 4-week old mice, after 24 h of treatment with 2.5 µg/mL of TDP43 (or mock control) showing the co-localization (yellow) in the cytoplasm of A 70# His-tag Merge DAPI CD11b Mock TDP43 Ctrl MOK ! ! 99! MOK with internalized (His6-cotaining) TDP43 species, in the sample treated with exogenous TDP-43 aggregates. Cell nuclei were counterstained with DAPI (blue). Scale bars: 25 µm. ! Next,!we!sought!to!establish!whether!MOK’s!activity!and!downstream!signaling! pathways!could!be!potentially!affected!as!a!consequence!of!its!abnormal!interaction! with!internalized!TDP-43!aggregates.!Knowing!that!MOK’s!kinase!activity!is!regulated! by!phosphorylation!of!two!residues,!Thr159!and!Tyr161,!within!a!critical!TEY!motif142,!we! performed! a! Western! blot! assay! with! lysates! from! microglial! cells! (primary! cultures! and! BV-2! cells)! previously! treated! with! LPS,! TDP43! or! mock! for! 30! min,! by! using! a! phospho-Tyr161-MOK! (pMOK)! specific! antibody.! Blot! results! showed! a! faint! band! corresponding!to!MOK’s!expected!molecular!mass!(48.4!kDa)!for!all!samples!without! changes!in!response! to! stimulation! (Fig.! 14A).!Intriguingly,!this! was! accompanied! by! up-regulation! of! another! band! corresponding! to! a! molecular! mass! of! ca.! 46! kDa,! consistent! with! the! size! predicted! for! MOK! isoforms! 2! and! 3! (NCBI! non! redundant! database).! Therefore,! we! focused! on! that! particular! species! for! assaying! MOK’s! phosphorylation! status! as! a! result! of! cell! treatment! with! TDP-43! aggregates.! Remarkably,! we! detected! a! ca.! 30%! decrease! in! phosphorylation! levels! of! MOK’s! critical! Tyr161! residue! in! TDP43-treated! vs.! mock-treated! cells,! in! both! primary! microglia! (Fig.! 14B)! and! BV-2! cells! (Fig.! 14C).! Such! reduction! in! MOK’s! relative! phosphorylation!levels!following!exposure!to!exogenous!TDP-43!aggregates!are!in!line! with!our!finding!that!MOK!kinase!protein!is!affected!by!internalized!TDP-43!aggregates! and!strongly!support!the!idea!that!its!signaling!function!may!be!disrupted!in!TDP-43- exposed!microglia.! ! ! 100! ! Figure 7-14 Modification of MOK phosphorylated status by extracellular TDP-43 aggregates. A) Western blot with anti-MOK and anti-phospho-Tyr161 MOK-specific antibodies of total lysate from BV-2 and primary microglia, 30 min after treatment with 1 µg/mL LPS or 5 µg/mL TDP-43 (or mock control). Cells without treatment were used as a negative control. B) Western blot of total lysate from primary microglial cells, 30 min after treatment with 5 µg/mL of TDP-43 or mock, by using phospho- Tyr161 MOK-specific antibody (left). Quantitative analysis by band densitometry of phoshp-Tyr161 MOK levels in Western blot assay (right). Normalization was performed against GAPDH, and mock-treatment was considered as 100%. Values are mean ± SD and derive from two independent experiments (N=2), each one performed in triplicates. In all other cases, images and results shown are representative of three independent experiments (N=3). C) Western blot of total lysate from BV-2 cells, 30 min after treatment with 5 µg/mL of TDP-43 or mock, by using phospho-Tyr161 MOK-specific antibody (left). Quantitative analysis by band densitometry of phospho-Tyr161 MOK levels in Western blot assay (right). Normalization was performed against a-tubulin, and mock-treatment was considered as the reference (100%) 7.5 Inhibition!of!MOK!kinase!decreases!the!inflammatory!response! Induction! and! regulation! of! the! inflammatory! response! is! at! least! in! part! regulated!by!the!mitogen-activated!protein!kinases!(MAPKs)!that!are!a!well-studied,! important!family!of!signaling!molecules143,144.!MAPK!signaling!pathways!encompass! ! ! 101! cascades!of!kinases,!which!convert!extracellular!signals!into!cellular!responses!and,! importantly,! many! members! of! this! family! have!been! reported! to! be! involved! in! neurodegenerative! diseases! such! as! AD,! PD! and! ALS143,145.! Moreover,! several! MAPKs,!especially!the!p38!member,!have!been!involved!in!the!neuroinflammatory! response!mediated!by!microglia!145–147.!! MOK,! a! member! of! the! MAPK! superfamily,!is! a! virtually! uncharacterized! Ser/Thr!kinase!with! as! yet! unknown! substrates! and! whose! only!reported!function! thus!far!is!the!regulation!of!ciliary!growth!in!epithelial!cells148.!In!order!to!explore! whether!MOK!has!a!role!in!the!neuroinflammatory!response!associated!to!microglia,! we! performed! inhibition! assays! in# vitro# with! a! small-molecule! compound,! C13,! which! was! identified!as! a! specific! MOK! inhibitor149.! In! this! case,! we! used! LPS! stimulation!as!an!inflammatory!model!to!study!the!involvement!of!MOK!in!a!more! general! inflammatory!context!and! because! LPS! stimulation! is! a! well-known! and! established!model!that!allows!us!to!easily!perform!several!experiments!and!obtain! larger!amounts!of!data.!To!that!aim,!we!pre-treated!primary!microglial!cultures!with! 10! μM! of!C13! (or! DMSO)! for! 1! h.! Next,! we! treated! cells! with! 1! μg/mL! of! LPS! (or! medium)!for!24h!without!changing!the!medium!(Fig.!15).!Remarkably,!we!observed! a! significant!reduction! in! the!secretion! of!pro-inflammatory! cytokines.! IL1-β! and! TNF-α! showed!a! reduction!in! their! secretion!levels!of!ca.! 50%,! whereas!IL-6! was! reduced!by!ca.! 25%,! as! measured! by! ELISA!(Fig.! 15D-F).! On! the! other! hand,! no! significant!differences!were!observed!in!the!secretion!of!immunoregulatory!cytokine! IL-10!(Fig.!15C).!In!an!attempt!to!elucidate!possible!mechanisms!in!which!MOK!could! be! involved! we! measured! the! levels! of! intracellular! caspase-1! by! Western! blot! of! treated!microglia!lysates!!(Fig.!15B).!Indeed,!we!found!that!C13!reduced!the!levels!of! pro-caspase-1,! suggesting! an! involvement! of!MOK! in!the! NLRP3! inflammasomemediated!response.! Finally,! we! wanted! to! address! the! question! whether!MOK! is! involved! in! regulating!the!levels!of!cytoplasmic!activated!caspase-3,!which!has!been!found!to!be! involved!in!neurotoxic!inflammatory!responses!of!microglia137,138.!Interestingly,!even! though!no!significant!differences!were!observed!for!total!activated!caspase-3,!our! results! showed!that! the!number! of!activated! capase-3! granules! showed! a! trend! towards! decrease!when! cells! were!pre-treated! with! C13!in!LPS-treated! cells!(Fig.! ! 102! 15G,H).!This!suggests!that!MOK!could!have!a!role!in!regulating!caspase-3!activation! in!an!inflammatory!environment.!! ! ! Figure 7-15 MOK is involved in microglia neuroinflammatory response and possibly regulates activated caspase-3. A) Molecular structure of the MOK inhibitor C13 compound. B) Western blot of total lysate from primary microglial cells, 24 h after treatment with 1 µg/mL of LPS, pre-treated or not with C13 (for 1 h), by using an anti-caspase-1 antibody. C-F) Determination by ELISA of cytokine levels in cells incubated with 1 µg/mL LPS or medium alone, with or without pre-treatment for 1 h with C13, after 6 h (TNF-a) or 24 h (IL-1b, IL-6, IL-10). At least three independent experiments (N=3) were performed, each one with A B C13 -" -" +" +" ctrl ctrl LPS LPS IL-1β (rel. to ctrl) C13 -" -" +" +" ctrl ctrl LPS LPS IL-6 (rel. to ctrl) C13 -" -" +" +" ctrl ctrl LPS LPS TNF-α (rel. to ctrl) C13 -" -" +" +" ctrl ctrl LPS LPS IL-10 (rel. to ctrl) kDa 43 - 55 - α-Tubulina Ctrl LPS anti-casp-1 pro-casp-1 C13 -" -" +" +" Casp-3* DAPI Merge Ctrl C13/Ctrl LPS C13/LPS Casp-3* DAPI Merge C13 -" -" +" +" ctrl ctrl LPS LPS 4 0 1 2 3 Relative Casp-3* (cytoplasm granules) C13 -" -" +" +" ctrl ctrl LPS LPS 0 1 2 Casp-3* (Total average inten.) C D E F G H ! ! 103! duplicate samples. G) Representative images of activated caspase-3 IF studies levels in cells co-treated for 24 h with 1 µg/mL LPS or medium, with or without pre-treatment of 1 hour with C13 inhibitor. Scale bars: 25 µm. H) Quantification of activated caspase-3 cytoplasmic granules per cell and total levels based on two independent IF studies (N=2) and confocal microscopy analyses. Error bars correspond to ± SD 7.6 Expression!of!MAPK/MAK/MRK!overlapping!kinase!(MOK)!in!spinal! cord!from!hSOD1G93A!mice!and!ALS!patients! ! After!the!identification!of!MOK!and!its!involvement!in!the!neuroinflammatory! response!elicited!by!ALS-associated!TDP-43!aggregates!in#vitro,!we!wondered!whether! MOK!would!also!be!altered!in!cells!of!the!spinal!cord!from!hSOD1G93A!ALS!mice!and!ALS! patients.! Previously,! MOK! had! been! shown,! in! a! cell! line,! to! be! a! predominantly! cytoplasmic! protein141! but! another! group! observed! both! cytoplasmic! and! nuclear! localizations!in!human!cell!lines142.!Moreover,!they!suggested!that!MOK!might!shuttle! from! the! nucleus! to! the! cytoplasm.! Until! now,! no! evidence! of! MOK! subcellular! localization!in!CNS!cells!had!been!reported.!!! First!of!all,! we!characterized!the!levels!and!subcellular!localization!of!MOK!in! the! hSOD1G93A!mouse! model! vs.! non-carrier! (NC)! controls! by! immunohistochemistry! (IHC)! of! tissue! slices.! We! decided! to! use! the! standard! hSOD1G93A! transgenic! mouse! model! (from! a! colony! established! in! our! lab)! because! it! has! been! lately! shown! to! display!altered! TDP-43! and! TDP-43-containing!cytoplasmic! inclusions!in! spinal!motor! neurons!and!glial!cells,!similarly!to!ALS!patients150–154.!Furthermore,!and!as!opposed!to! the! hSOD1G93A!model,! the! available! TPD-43-transgenic! mouse! models! do! not! recapitulate!human!ALS!to!a!sufficient!level155.!We!focused!on!the!distal!section!of!the! spinal!cord! as! this! is! the! region!where!disease! shows! to! be! more! severe! in!this! ALS! model.!We!used!two!time!points!along!the!disease!course,!8!weeks-old!(asymptomatic! phase)!and!16!weeks-old!(end-stage),!to!monitor!MOK’s!profile!along!the!evolution!of! pathology.! We! began! by! analysing! the! Tyr161-phospho-MOK! (pMOK)! form! of! the! protein,! followed! by! analysis! of! the! Thr159/Tyr161!bi-phospho-MOK! protein! (bpMOK)! form.! The! importance! of! the! bi-phosphorylated! form! of! MOK! is! that! MOK’s! kinase! activity!is!thought!to!be!regulated!by!the!phosphorylation!of!these!two!residues142.!In!8! weeks-old!NC!mice,! pMOK! was! observed! at! both! nuclear! and! cytoplasmic! locations,! ! 104! with!higher!nuclear!intensity,!in!motor!neurons.!Another!cell!type,!possibly!neuron!cell! bodies,! showed! a! predominant! nuclear! staining! whereas! no! pMOK! staining! was! observed!in!microglial!cells!(Fig.!16A).!!! When!analyzing!the!hSOD1G93A!model,!we!observed!that!whereas!some!motor! neurons!had!similar!staining!than!the!NC!controls,!some!motor!neurons!showed!very! intense! pMOK! nuclear! staining! accompanied! by! complete! loss! of! pMOK! in! the! cytoplasm.!In!addition,!as!opposed!to!NC!controls,!we!observes!pMOK!signal!in!glial! cells.! We! then! wondered! whether! pMOK! features! might! change! along! the! disease! course.!Interestingly,!whereas!16-weeks!old!NC!mice!showed!nuclear!and!cytoplasmic! localization!for!pMOK!similar!at!what!was!seen!at!8!weeks,!the!staining!in!the!ALS!mice! was!exclusively!cytoplasmic,!with!little!staining!in!the!nucleus!(Fig.!16B).!Importantly,! we!observed!pMOK!staining!not!only!in!neurons!but!also!in!glial!cells.!Furthermore,!we! observed!many!pMOK-positive!processes!also!compared!to!what!was!seen!at!8!weeks,! possibly!due!to!the!extensive!gliosis!seen!at!the!end-stage!of!the!disease!(Fig.!16B).!!! Next,!we!wanted!to!evaluate!if!bpMOK!showed!a!similar!pattern!than!pMOK.!In! 8-weeks!old!animals,!we!identified!similar!localization!and!intensity!levels!for!bpMOK! than!for!pMOK!in!both!the!NC!and!transgenic!mice.!In!the!NC!controls,!bpMOK!showed! nuclear! and! cytoplasmic! localization! with! higher! intensity! in! the! nucleus.! For! transgenic! mice,! some! motor! neurons! displayed! a! very! intense! nuclear! staining! of! bpMOK! with! a! loss! of! cytoplasmic! protein.! Again,! staining! of! glial! cells! was! also! observed!only!in!mSOD1!animals!(Fig.!16A).!At!16!weeks!of!age,!towards!the!end-stage! of! the! disease,! we! observed! a! decrease! in! the! intensity! levels! of! bpMOK! in! motor! neurons! in! the! transgenic! animals! but,! as! with! the! pMOK! staining,! the! protein! was! found!in!the!nucleus!and!in!the!cytoplasm,!although!possibly!at!lower!levels!(Fig.!16B).! Remarkably,!as!for!pMOK,!bpMOK!expression!was!significantly!augmented!in!glial!cells! (Fig.! 16B).! The! intensity! of! bpMOK! in! these! cells! was! even! higher! than! for! pMOK,! suggesting!that!activated!MOK!could!have!an!important!function!in!pathology-linked! glial!responses.! ! ! 105! ! Figure 7-16 MOK protein is altered in the mutant hSOD1G93A mouse model. A-B) Immunohistochemical reactivity of distal transverse spinal cord sections showing pMOK and bpMOK in non-carrier mice and hSOD1G93A mouse model of 8 and 16 weeks old, respectively. Images are representative of at least two different mice. Images magnification are 10x and 40x. ! Importantly,!we!got!access!to!human!spinal!cord!samples!from!an!ALS!patient! and!a!control!subject!to!characterize!the!presence!of!MOK!by!IHC.!In!this!case,!we!also! analyzed! ‘total’! MOK! (MOKt),! pMOK! and! bpMOK.! First,! we! checked! how! MOKt! was! distributed!in!cells!of!the!lumbar!spinal!cord.!For!the!control!subject,!MOKt!in!motor! neurons!was!mostly!localized!in!the!cytoplasm!with!little!or!no!nuclear!staining!(Fig.! 17A).!The!ALS!patient!also!showed!a!predominantly!cytoplasmic!distribution!but!the! intensity! of! staining! was! lower! compared! to! the! control.! In! addition,! some! cells,! A pMOK Ctrl SOD1 bpMOK 8 weeks B pMOK Ctrl SOD1 bpMOK 16 weeks ! 112! brain!samples!from!ALS!patients.!This!could!be!highly!relevant!as!extracellular!HSPs!are! currently!considered!as!major!intercellular!signaling!molecules64!and!may!indeed!act!as! alarm! signals.! Furthermore,! it! has! become! established! that! certain! HSPs,! including! Hsp70!and!Hsp27,!display!immunological!capabilities,!and!therefore!these!chaperones! could!be!proposed!to!have!an!impact!on!TDP-43-exerted!responses!in!the!context!of! ALS.!! Indeed,!the!results!obtained!in!the!present!study!demonstrate!that!the!presence! of!certain!HSPs!in!the!extracellular!medium!could!radically!affect!the!aggregates!fate! and!intracellular!processing!in!TDP-43-exposed!microglia!(Fig.!7,8).!In!particular,!while! Hsp70!does!not!co-localize!with!TDP-43!aggregates!nor!does!it!visibly!alter!the!features! of!internalized!TDP-43!aggregates,!Hsp27!promotes!the!aggregates!entrance!into!the! cell! and! co-localizes! with! internalized! TDP-43! in! the! cytoplasm.! Interestingly,! extracellular!Hsp27!chaperone! was! not! seen! to! be! uptaken! by!microglial!cells! on! its! own,! but! only! in! association! with! TDP-43! aggregates.! On! the! other! hand,! Hsp70,! as! opposed!to!Hsp27,!is!readily!uptaken!by!microglial!cells!independently!of!the!presence! of!TDP-43!extracellular!aggregates.! We! then! focused! on! the! effect! of! extracellular! HSPs! on! the! immune! response! elicited!by!TDP-43!aggregates!in!microglia!(Fig.!9-11).!Previously,!exogenous!Hsp27!had! been!shown! to!promote!a! proinflammatory! response!in!astrocytes! isolated!from!AD! and! cerebral! amyloid! angiopathy! patient’s! brain! by! altering! IL-8,! CD40L! and! TGF-β,! levels168! and! to! activate! NF-κB! cell! signaling! accompanied! by! up-regulation! of! both! pro-! and! anti-inflammatory! cytokine! genes,! in! THP-1! macrophages169.! On! the! other! hand,! Hsp70s! have! been! reported! to! display! both! pro-inflammatory! and! antiinflammatory! properties! depending! on! the! target! cell64,170,171! and! several! receptors! have! been! proposed! to! mediate! such! functions! in! the! case! of! extracellular! Hsp7064,172,173.! In! this! study,! extracellular! Hsp27! —unlike! Hsp70—! was! found! to! exacerbate!the!pro-inflammatory!response!of!TDP-43-exposed!microglia,!including!the! release! of! IL-1β! and! IL-18! cytokines(Fig.! 9,10).! Furthermore,! Hsp27! was! found! to! dramatically!suppress!the!number!of!caspase-3!granules!of!TDP-43-exposed!cells!and! to!slightly!increase!the!activated!caspase-3!total!levels!(Fig.!10,11),!supporting!a!role!of! caspase-3! IL-18! alternative! processing! in! microglia.! Interestingly,! active! caspase-3! granule-like! compartments! have! been! previously! found! in! viable! mast! cells139.! ! ! 113! Therefore,!the!observed!Hsp27-induced!reduction!in!the!content!of!caspase-3!granules! with! a! modest! increase! in! activated! caspase-3! total! levels! may! suggest! an! actual! increase!in!caspase-3!intracellular!availability.!If!so,!the!incremented!neurotoxic!effects! of! TDP-43-activated! microglia! by! Hsp27! co-treatment! may! be! related! to! an! exacerbated!neuroinflammatory/neurotoxic!profile!linked!to!IL-1β/IL-18!and!activated! caspase-3137,174,!in!activated!microglia.!On!the!other!hand,!exposure!of!cells!to!TDP-43! aggregates!co-treated!with!Hsp70!caused!an!increase!in!expression!levels!of!the!cIAP2! anti-apoptotic! gene,! which! had! been! proposed! to! regulate! the! shifts! between! proinflammatory! and! apoptotic! profiles! in! microglia138,! without! clearly! altering! the! TDP43-excerted!neurotoxicity!(Fig.!11).! Overall,!these!results!provide!a!new!mechanistic!insight!linking!previous!clinical! observations!related!to!increased!levels!of!extracellular!TDP-43!aggregates!in!ALS/FTLD! patients!with!particular!microglial!responses.!Furthermore,!it!reveals!the!potential!of! certain! molecular! chaperones! to! modify! such! TDP-43-induced! microglial! activation! profile.! Importantly,! our! study! provides! novel! molecular! targets! that! could! be! modulated!for!therapeutic!purposes!in!ALS!and!other!related!diseases.!! ! To!shed!light!on!the!molecular!mechanisms!leading!to!TDP-43-caused!microglial! activation,! we! explored! the! protein-protein! interactions! established! between! the! exogenously!added!TDP-43!aggregates!and!microglial!proteins.!Our!results!from!Co-IP! and!Co-IF!studies!allowed!us!to!uncover!MOK!kinase,!a!recently!identified!member!of! the!unique!ICK/MAK/MOK!family!of!protein!kinases,!as!a!protein!interacting!and!colocalizing! in! the! cytoplasm! with! internalized! TDP-43,! either! in! a! direct! or! indirect! manner!(Fig.!12,13).!Intriguingly,!MOK’s!RNA!sequence!at!the!3’!region!is!identical!to! that! of! the! receptor!of! advanced! glycation-end!products! (RAGE)141.! RAGE! is! a! multiligand! protein! involved! in! a! number! of! neurodegenerative! diseases,! which! has! been! found! to! bind! to! a! myriad! of! molecules,! including! AD-linked! amyloid! β!and!other! misfolding! proteins175.! Even! though! no! substrates! for! MOK! have! thus! far! been! identified,! MOK! has! been! found! to! undergo! auto-phosphorylation! and! to! possess! protein!kinase!activity!toward!exogenous!substrates,!in#vitro!(47).!Indeed,!even!though! it!is!suspected!that!this!group!of!signaling!kinases!belonging!to!the!MAPK!superfamily! may!have!important!biological!functions!in!different!physiological!processes!as!well!as! in!disease142,!many!questions!are!still!open.!Recently,!high!gene!expression!levels!MOK! ! 114! have!been!in!blood!monocytes!from!patients!with!high!blood!pressure176.!Remarkably,! we!found!that!exposure!of!microglial!cells!to!extracellular!TDP-43!aggregates!leads!to! lower! levels! of! phospho-Tyr161-MOK! (Fig.! 14).! This! phenomenon! might! be! a! direct! consequence! of! abnormal! engagement! of! MOK! with! internalized! TDP-43! aggregates! ⎯which! was! observed! within! microglial! cells! both! in! cellular! and! organotypic! cultures⎯! and! therefore! disruption! of! MOK’s! activation! state! could! be! proposed! to! affect! cellular! responses! that! are! modulated! by! downstream! signalling! pathways.! Moreover,! we! found! that! MOK! is! indeed! involved! in! the! inflammatory! response! mediated!by!microglia!(Fig.!15).!MOK!kinase!inhibition!assays!showed!that!the!immune! response! is! strongly! affected! when! its! kinase! activity! is! blocked!by! using! C13! MOK- specific! inhibitor.! In! particular,! we! observed! a! down! regulation! of! pro-inflammatory! cytokines! and! cytoplasmic! granules! of! activated! caspase-3.! The! results! also! suggest! that! MOK! is! involved! in! the! activation! of! the! NLRP3! inflammasone! pathway! as! a! reduction! in! the! levels! of! pro-caspase-1! were! observed! accompanied! by! a! dramatic! reduction!in!IL-1β!cytokine.!MAPKs!have!been!described!to!participate!in!microglial! inflammatory! pathways,! being! p38! the! most! extensively! characterized! MAPK! implicated!in!this!function145,146.!! Importantly,!these!data!show!for!the!first!time!not!only!that!MOK!is!involved!in! the!inflammatory!response!but!they!also!demonstrate!that!MOK!protein!is!altered!in! ALS,! both! in! a! pre-clinical! animal! model! and! in! an! ALS! patient! ! Furthermore,! ,! we! described! that! pMOK! and! bpMOK! is! highly! expressed! in! glial! cells.! Moreover,! we! observed! that! MOK! is! also! expressed! in! neuronal! cells,! and! that! its! subcellular! localization! in! motor! neurons! differs! depending! on! its! phosphorylation! state,! suggesting!that!MOK!has!different!functions!depending!on!the!expressing!cell!and!on! its! subcellular! localization.! Strikingly,! during! the! course! of! the! disease! the! different! forms!of!the!phosphorylated!protein!modified!their!localization!and!expression!levels.! Importantly,!we!suggest!that!MOK!has!an!important!role!in!both!motor!neurons!but! also!a!key!role!in!the!immune!response!mediated!by!glial!cells.!! These! data! indicate! that! MOK! could! be! involved! in! the! disease! via!neuronautonomous! and! non-autonomous! mechanisms,! having! a! key! role! in! neuroinflammation!and,!therefore,!in!disease!progression.!Based!on!these!results,!we! propose! MOK! as! a! novel! therapeutic! target! in! ALS! and,! probably,! other! ! ! 115! neurodegenerative!diseases.!Protein!kinases!represent!a!large!group!of!enzymes!that! are!involved!in!numerous!cell!signaling!pathways.!In!the!last!decade,!protein!kinases! have!emerged!as!promising!drug!targets!for!the!treatment!of!several!disorders!ranging! from!cancer,!autoimmune!pathologies,!inflammation,!or!neurodegenerative!diseases.! Several! MAPKs! inhibitors! are! currently! being! tested! in! clinical! trials! in! certain! neurodegenerative! diseases,! particularly! in! AD! and! PD177,178.! Interestingly,! a! phase-3! clinical! trial! is! ongoing! for! a! tyrosine! kinase! inhibitor,! masitinib179,! the! only! protein! kinase!inhibitor!in!ALS!in!clinical!trials,!thus!far.!The!increasing!number!of!new!protein! kinases!inhibitors,!some!of!which!in!different!phases!of!clinical!trials!at!the!moment,! indicate! the! major! involvement! of! certain! signaling! kinases! in! the! pathogenic! mechanisms! in! neurodegenerative! diseases.! Importantly,! this! highlights! the! importance!of!discovering!key!signaling!mediators,!such!as!MOK,!that!could!serve!as! novel!and!effective!therapeutic!targets!against!ALS.!! These!results!show!that!MOK!indeed!plays!a!role!in!ALS!pathogenesis!and!they! suggest!that!it!could!possibly!be!involved!in!other!neurodegenerative!diseases!such!as! AD,!PD!or!FTD.!Overall,!this!work!sheds!light!on!a!new!mechanistic!element!within!the! complex! orchestra! involved! in! ALS,! and! we! propose! that! MOK! could! be! used! as! an! intervention! tool! to! modify! the! progression! of! ALS! and! other! neurodegenerative! diseases.! ! ! ! 117! ! ! ! ! ! ! ! ! ! Conclusions! ! ! ! 119! 9 Conclusions! v Pre-formed! TDP-43! aggregates! are! internalized! by! murine! primary! microglial! cells! and! suffer! ubiquitylation! and! proteolytic! proccesing! similar! as! what! is! observed!in!TDP-43!aggregates!found!under!pathological!conditions.!Moreover,! TDP-43! aggregates! sequester! and! induce! retr-translocation! of! endogenous! nuclear! TDP-43.! Importantly,! internalization! by! microglial! cells! was! also! observed!in!adult-like!mouse!spinal!cord!organotypic!cultures.! v !Extracellular! TDP-43! aggregates! induce! a! pro-inflammatory! response! in! primary!microglial!cells!and!microglia!within!organotypic!cultures!of!spinal!cord.! TDP-43!aggregates!increase!levels!of!pro-caspase-1!and!cytokines!processed!by! the!NLRP3!inflammasome!such!as!IL-1β!and!IL-18.!They!also!raise!the!levels!of! TNF-α,!a!key!pro-inflammatory!cytokine.!! v In! addition,! a! 16! kDa,! alternative! form! of! IL-18! is! found! intracellularly! when! microglia!is!exposed!to!TDP-43!aggregates.! v Total! levels! of! activated! caspase-3! are! increased! for! TDP-43-aggregates! treatment.!Importantly,!cytoplasmic!levels!of!activated!caspase-3,!associated!to! a! neurotoxic! pro-inflammatory! state! of! microglia,! are! also! increased! in! microglia! exsposed! to! TDP-43! aggregates.! These! cytoplasmic! caspase-3! granules! are! also! observed! in! spinal! cord! organotypic! cultures! as! a! result! of! TDP-43-aggregates!treatment.! v !Exposure!of!a!motor!neuron!cell!line!to!conditioned!medium!from!microglial! primary! cultures! previously! treated! with! TDP-43! aggregates,! reduces! cell! viability.! v Exogenously!added!HSBPB1!(Hsp27)!is!internalized!by!microglia!and!co-localizes! with!intracellular!TDP-43!aggregates!only!when!it!is!pre-incubated!with!TDP-43.!! v Pre-incubated! mixtures! of! Hsp27:TDP-43-aggregates! modify! the! immune! responses! of! microglia.! Hsp27! exacerbates! the! pro-inflammatory! profile! induced! by! TDP-43! aggregates,! increasing! the! levels! of! secreted! proinflammatory!cytokines!such!as!IL-1β!and!IL-18.!Moreover,!Hsp27!co-treatment! ! 120! dramatically! reduces! the! number! of! activated! caspase-3! granules! with! an! increase!of!total!levels!of!caspase-3.! v Hsp27:TDP-43! co-treatment! increased! the! neurotoxic! effect! as! compared! to! TDP-43-treated!microglia.! v Contrary! to! Hsp27,! Hsp70! does! not! co-localize! with! TDP-43! aggregates! in! microglial! cells! as! a! result! of! co-treatment! with! Hsp70:TDP-43-aggregates.! Furthermore,! uptaken! Hsp70! is! independent! of! the! presence! of! extracellular! TDP-43!aggregates.!! v Expression!of!the!anti-apoptotic!gene!cIAP2!is!increased!when!microglia!is!cotreated!with!Hsp70:TDP-43!mixture.!! v Hsp70!does!not!significantly!modify!the!pro-inflammatory!profile!of!microglia! nor! does! it! increase! the! neurotoxic! effect! caused! by! treatment! of! motor! neurons! with! conditioned! medium! from! Hsp70:TDP-43-aggregates-treated! microglia.!! v TDP-43! strongly! binds! to! the! protein! kinase! MOK! (MAPK/MAK/MRK! overlapping!kinase),!a!poorly!characterized!Ser/Thr!kinase!with!no!previous!link! to!the!CNS,!as!a!result!of!exposure!of!microglial!cells!to!TDP-43!aggregates.! v Exposure! of! microglia! to! extracellular! TDP-43! aggregates! leads! to! reduced! levels!of!Tyr161-phospho-MOK!levels!in!BV2!and!primary!microglial!cells.!An!upregulation!of!MOK!protein!levels!was!observed!in!organotypic!cultures!of!adultlike!mouse!spinal!cord.!! v Treatment! of! microglial! cells! with! MOK-specific! inhibitor! C13! leads! to! suppression!of!pro-caspase-1!and!of!secreted!pro-inflammatory!cytokines!TNF- α!and!IL-1β,!induced!by!LPS-stimulation.!! v The!mono-!(pMOK)!and!bi-!(bpMOK)!phosphorylated!forms!of!MOK!are!located! principally!in!the!nucleus!but!also!in!the!cytoplasm!of!motor!neurons!in!wildtype! mice! at! 8! and! 16! weeks.! Whereas! both! species! are! increased! in! the! nuclear! compartment! at! 8! weeks! in! the! hSOD1G93A! mouse! model,! especially! pMOK! for! which! depletion! of! the! cytoplasmic! form! of! the! protein! in! motor! neurons!was!observed,!pMOK!is!mostly!cytoplasmic!at!16!weeks!in!the!same! model.!As! opposed! to! wild-type! spinal! cord,!higher!levels!of! both! pMOK! and! ! ! 121! bpMOK!in!the!hSOD1G93A! spinal!cord!tissue!are!seen!at!16!weeks!in!glial!cells! with!the!most!striking!increase!for!bpMOK.! v In!lumbar!spinal!cord!human!tissue,!MOK!protein!is!mostly!cytoplasmic!in!both! the!ALS!patient!and!control!subject,!but!in!the!former!the!staining!intensity!was! lower! in! motor! neurons.! 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