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Thepathophysiologyofamyotrophiclateral

sclerosis(ALS)andtheroleofC9orf72

Madeby:Marie-LouiseHoffman

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Content

1.Introduction ………………………………………………………………………… 32.Pathophysiology …………………………………………………………………………. 43.Etiology ………………………………………………………………………………………. 64.RoleofC9orf72 …………………………………………………………………………….. 85.Conclusionsandfuturereferences ……………………………………………. 116.References……………………………………………………………………………………….. 12

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1.Introduction Amyotrophic lateral sclerosis (ALS) is a disastrous neurodegenerativedisorder,wherepeoplediewithin3-5yearsafter theywerediagnosed,mostlydue to respiratory failure. ALS appears with an incidence of 2.6/100.000 peryear(Kumaretal.2016),andnormallydevelopsbetween50and75yearsofage,decreasingafterwards(Zufiriaetal.2016). In ALS,motor neurons in the spinal cord, brainstem, andmotor cortexdegrade (motor neuron dysfunction). The consequences are motor problems,muscle weakness, and paralysis (Boeynaems et al. 2016). As a result of theinvolvement of the upper and lowermotor neurons, neurological examinationconfirmsacombinationofuppermotorneuronsigns (spasticity,hyperreflexia,andextensorplantarresponse)andlowermotorneuronsigns(muscleatrophy,fasciculationsandcramps).Theclinicalphenotypesdiffer fromeachother.Theclassicalorspinalforms,withearlyinvolvementofthelimbs,aremosttypical(in65% of cases). In bulbar forms (impairment of the function of the cranialnerves), the disease starts with dysarthria (difficult or unclear articulation ofspeech), dysphagia (difficulty ordiscomfort in swallowing) orboth (in30%ofcases).5%ofcasesstartswithearlyrespiratoryfailure(Zufiriaetal.2016). ALS is considered to be a complex genetic disorder: there is acombination of multiple genes and environmental exposure needed for thepersontobesusceptible.Risksincludeage,malesex,cigarettesmoking,physicalstress,exposuretopesticides,andexcessivesporting(Gordon2011).Inaround10% of ALS patients, the disease runs in the family. These familial cases arecausedbyaheterogeneoussetofgenes,mostlyTARDBP,SOD1,FUS,andC9orf72.WiththegeneC9orf72,patientscarryahexanucleotide(GGGGCC)(Dafincaetal.2016) repeat expansions (HRE) in the first intronic region (of chromosome 9open reading frame 72 gene) (Boeynaems et al. 2016). While healthy peoplehaveanaverageof2repeats,with90%carrying8orless,therepeatsrangefrom500toseveralthousandsamongtheALSpatients(Kumaretal.2016).Thereisnoeffectofrepeatsizeonclinicalpresentation. Since Charcot has described the disease in 1874, the accumulatedknowledgehasnot been enough to find successful therapeutic strategies.Onlyriluzole, a glutamate agonist, has beendescribed to increase survival by a fewmonths(Zufiriaetal.2016). Even though there are these new insights, the exact pathophysiologicalmechanism in ALS is unknown (Boeynaems et al. 2016). In this paper, I willprovidefurtherinsightintothepathophysiologicalmechanismofALS,especiallythepathophysiologyrelatedtoC9orf72.

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2.Pathophysiology In this section, I describe the pathophysiology of mitochondria, axonaltransport, oxidative stress, excitotoxicity, inflammation, apoptosis and theirrelationtoALS. Morphologicalandultrastructuraldeformitiesofmitochondriahavebeenfound in autopsies of patientswithALS.Aggregates of abnormalmitochondriawereshowninskeletalmuscleandintramuscularnerves.Raisedmitochondrialvolume and elevated calcium levels in the mitochondria have been found inmusclebiopsies.Moreover,defectsintheactivitiesofmitochondrialrespiratorychaincomplexIandIVhavebeenrevealedinmuscleandspinalcord,indicatingthat a deterioration of the mitochondrial respiratory chain could be ofimportance in thepathogenesisofALS.However, therearealsocontradictions.Various lines of proof obtained in cellular models reveal that expression ofmutantSOD1(inmice)isnotonlycorrelatedwithmitochondrialmorphologicalchanges, but also with mitochondrial dysfunction (Brown et al. 2006). Thus,moreresearchhastobedonetofindoutwhattheexactcorrelationbetweenthemitochondria and ALS is. AxonaltransportalsohasarelationshipwithALS.Neurofilaments(NFs)are the most abundant intermediate filaments. NF concentrations incerebrospinal fluid and blood have been reported to be increased in patientswith both familial and sporadic forms of ALS (Brown et al. 2006). Therefore,neurofilament concentration is one of the most promising neurochemicaldiagnosticandprognosticbiomarkercandidates todate (Oeckletal.2016).TotesttherelationtautoALS,mousemodelshavebeencreatedbymakingchangesinthemicrotubule-associatedproteintau.Inoneofthestudies(Leeetal.2001),therewerespecifically tau45-230aggregatespresent in thespinalcordofpeoplewithALS.Different tau transgenicmicewere found tohaveprogressivemotorphenotypewithmuscle atrophy and paresis. Themotor axons had dilatationsthatconsistedofanaccumulationofNFs,mitochondria,andvesicles(Brownetal.2006). Inconclusion,alotisknownaboutthemitochondria:morphologicalandultrastructural deformities of mitochondria have been found in autopsies ofpatientswith ALS. Furthermore, therewas relationship found between axonaltransport and ALS. Increased neurofilament concentrations in cerebrospinalfluidandbloodhavebeenreported.However,ineachcase,theexactrelationtoALSisnotyetclear. Inthispart,ItalkaboutSOD1mutantmice,meaningmicewithamutationin the SOD1 gene. SOD1, superoxide dismutase (Cu-Zn), is an enzyme that inhumans is encrypted by theSOD1gene, located on chromosome 21. SOD1 is apowerfulantioxidantenzyme thatdefendscells from thedetrimentaleffectsofsuperoxide radicals. This enzyme binds both copper and zinc ions that arestraightlyinvolvedinthedeactivationoftoxicsuperoxideradicals(Brownetal.2006).MicewithamutationinSOD1haveprogressivemotordeficits,hindlimbparalysis,motorneurondegenerationandearlydeath(Joyceetal.2014).Thatis

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why it is the most used transgenic animal model to study ALS. ALShasalsobeenrelatedtodifferentprocessessuchasoxidativestress,excitotoxicity, inflammation, and apoptosis. Oxidative stressreflects animbalance between the systemic expression of reactive oxygen species and abiological system's ability to readily detoxify the reactive intermediates or torepairtheresultingdamage. SOD1-mediatedoxidativeabnormalitiesarenottheprimarycauseoftoxicity(Brownetal.2006). The next process is excitotoxicity. Glutamate-induced, AMPA receptor-mediatedexcitotoxicityaddstotheselectivemotorneurondegenerationinALS.Themost significant argument is that riluzole,which is theonly effectivedrugtreatmentforALSpatients, interfereswithglutamaterelease(Boeynaemsetal.2016).Furthermore,AMPAreceptorantagonistsextendsurvivalofmutantSOD1mice.Also,inhibitingglutamatecarboxypeptidaseIIincreasedthelifespaninthismousemodel (Brown et al. 2006). SoexcitotoxicitycanberelatedtoALS,butdoesinflammationalsoplayarole in ALS? This is not yet clear, however, evidence in human reveals thatmicroglial activation is an extensive phenomenon in patients who have ALS(Brownetal.2006).Theinvolvementofmicroglialactivationwasreportedinatransgenic mouse with general overexpression of the cytokine interleukine-3(IL-3)(Brown et al. 2006). In the figure below (figure 1) you see the differentprocessesI justmentioned.Youseethat foroxidativestressNrf2 isneededforsurvival and vitamin E/A is for disease prevention. In inflammation the mostimportantfactorsareinterleukine-1A,-1BandTNF.Andexcitotoxicityhastodowith glutamate.

Figure1Roleofinflammation,excitotoxicityandoxidativestressinmutantSOD1mice(Dennysetal.2014)

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Oneotherprocesssuspectedtohavearole inALS isapoptosis.There isgrowingevidencethatapoptosiscontributestoneuronallossinmanyacuteandchronicneurologicaldisease,includingALS.Apoptosisisahighlyregulatedformof cell death, inwhich cells diedue to activationof a pre-programmed suicidemechanism. It ismorphologically characterized by shrinking, collapsing of thecytoskeleton, disassembling of the nuclear envelope, and condensing of thenuclear chromatin. Furthermore, the cell also breaks up into fragments. It is amultistep, energy-dependent process that involves activation of positive andnegative regulatory components, for instance theBcl-2/Bax family of proteins,thep53tumorsuppressorgene,membersofthetumornecrosisfactorreceptors(TNFR) superfamily and cell cycle-related genes (Brown et al. 2006). TheargumentthatapoptosisisrelatedtoALSisthatoverexpressionofBcl-2protectsagainst motor neuron death from nerve transection in newborn mice(Sathasivametal.2001). However, there are some arguments against the role of apoptosis in ALSpathogenesis.Oneoftheseargumentsisthatthisformofcelldeathisveryrapidand for that reason it cannot be involved in chronic disease in which cellsdegenerate over a long period of time (Brown et al. 2006). 3.Etiology

Many studies have tried to establish genetic, environmental or lifestylefactorsintheetiologyofALS.DatafromtheUKandtheSwedishTwinregistrieshavefoundthatmonozygotictwinshaveahigherriskthandizygotictwins,butbothshowahigherriskthanthecontrolpopulation.TheriskofALSisincreasedin close relatives, but not in wives, supporting a significant role of geneticinfluencesoverunidentifiedenvironmentaldeterminants(Zufiriaetal.2016).Inthis section, the environmental and genetic factors related to ALS will beexplained. There is a wide list of studies of environmental factors such as heavymetals,pesticidecontaminationelectromagneticradiation,lifestyleandtobacco.Itiswellknownthatheavymetalsmightbethecauseofdisease,bothwhentheyare present in insufficient amounts and also when they are present in toxicconcentrations.Thepotentialroleofthesemetalshasbeenextensivelystudied.However,itisnotcompletelyunderstoodyet.Lead,mercury,andseleniumhavebeenthemoststudiedones.(Zufiriaetal.2016). High lead levels have been described in cerebrospinal fluid, blood andbone samples of ALS in US veterans (Zufiria et al. 2016). The association isespeciallymarked forblood lead.Veteransmaybeexposed to lead from firingpracticeandothermilitary-relatedsources,sotheobservedlead-ALSassociationmayat leastexplainthehigherriskofALSfoundformilitaryservicepersonnelcomparedwiththegeneralpopulation(Kameletal.2006). Anothermetal thathasbeenassociatedwithan increasedriskofALS ismercury.Mercuryhasbeenshowntobeneurotoxicininvitroorinvivostudiesand to have accumulated in neural tissues (Wang et al. 2016). Clinicalmanifestations in patients with long-term accidental exposure tomercury are

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verymuchaliketothosereportedinclassicalALS.ALSmiceexposedtomercuryshowa quicker andmore abrupt disease course, implying that thismetalmaypotentiate the development of the disease in patients who are geneticallypredisposed(Zufiriaetal.2016). Adifferentmetal,selenium,isametalloidelementthathasbeenbroadlystudiedsinceitwasdiscoveredtobebothatoxicandanessentialtraceelementformammals.ThefirstevidencesfortheassociationbetweenALSandseleniumcamefromtwoepidemiologicalinvestigationsthatdocumentedincreasedriskofALSinpopulationsinseleniferousregions.(Estevezetal.2012). The last metal associated with ALS is zinc. Zinc is co-released atglutamatergic synapses in the whole central nervous system and acts as aneuromodulator for glutamatergic neurotransmission (Zufiria et al. 2016).Currently,investigatorsfoundthatzincexposuremayhaveaneurotoxiceffectinmotorneurons.Someexperimentshaveconfirmedthatzincincreasesoxidativestressandraisesexcitotoxicity,asaresultofthispromotingmotorneurondeath(Nutinietal.2011). Anotherenvironmentalfactorispesticidecontamination.Thepesticides,whicharethemostused,areorganophosphates.Organophosphatesirreversiblyinhibit acetylcholinesterase, thereby inducing brain damage. Acute, extrastimulation of cholinergic receptors causes cholinergic neuronal excitotoxicityanddysfunction.Morahanetal.showedthatanimpairedabilityofsporadicALSpatients to detoxify pesticides could be associatedwith polymorphisms in themetallothioneinfamilyofgenes(Zufiriaetal.2016) Adistinctenvironmentalfactorislow-frequencies-electromagnetic-fields(EMF).Thereexistsonemeta-analysisshowingaslight,butsignificantincreasein the risk of developing ALS among intensely low-frequencies-EMF-relatedoccupations.Invitrostudiesconfirmthatprolongedexposuretoextremelylowfrequencies-EMF might induce oxidative stress, DNA damage, and apoptosis.However, theseresultshavenotbeenreplicatedinALSmurinemodels(Zufiriaetal.2016). The lifestyle is also considered as a risk factor for ALS. It has beensuggested that exercise and good physical conditions, which are usuallyconsidered to have advantageous effects on health, could be risk factors forneurodegeneration later in life (Zufiria et al. 2016). There are several case-control studies reporting that intense and powerful physical activity could beassociatedwith thedisease. Ina studyamongcross-country skiers, they foundthat longdistance cross-country skiing is associatedwith a higher risk ofALS,among the best skiers. However, recreational skiers appear to have a largelyreducedrisk(Fangetal.2015). Anotherenvironmental factorassociatedwiththeriskofALSistobacco.The correlation between smoking and ALS has been suggested, but it is notconfirmed. Even though several studies found a potential association, otherstudiesdidn’tfindthem(Zufiriaetal.2016)

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Amongall the factorssuspectedtocauseALS, thestrongestcorrelationshave been made to the different genetics factors, which play a role in ALS.Together, the 4 genes (SOD1, FUS, TARDBP, and C9orf72) account 60-80% offamilial ALS cases. C9orf72 expansions serve as about 50% of the mutationsfoundinfamilialform,butmutationsinFUSaccountfor35%ofcasesappearing.ThoughothergeneshavebeenrelatedtotheMendelianformsofthedisease,itsepidemiologicalimportanceisnearlyneglectable(Millecampsetal.2012).4.RoleofC9orf72 TheC9orf72geneisveryspecial,becauseitliesinanintronicregionandnormallyonlyexonsaretranscribed,notintrons. So what seems to be the normal function of C9orf72? BioinformaticstudieshavepredictedthatthemajorstructuralfeatureoftheC9orf72proteinisaDENNdomain.DENNdomainsarebestdescribedasregulatorsofintracellularmembranetrafficthroughtheiractionsasguanineexchangefactorsforspecificRab guanosine 5’-triphosphate (GTP)ases. Knockdown of C9orf72 leads toreducedendocytosisanddysregulatedautophagyinhumanneuroblastomacells(Amick et al. 2016;Mizielinska et al. 2014). This points out criticalmembranetrafficfunctionsforC9orf72(Amicketal.2016).Amicketal.alsofoundoutthatC9orf72 interact strongly and significantly with SMCR8 (Smith-MagenisChromosomeRegiongene8),whichhasbeenpredictedtocontainanFCLN-likeDENN domain. This interaction is critical for C9orf72 stability (Amick et al.2016). ALShasbeenrelatedtotheloss-of-functionoftheC9orf72gene,gain-of-functionof theC9orf72geneviaRNArepeats,gain-of-functionviaDPRprotein,and finally the formation of G-quadruplexes. In the case of loss-of-functionmutations, reduced levels of C9orf72 transcripts may be a cause ofhypermethylation of the C9orf72 promoter or increased histone methylation.The resulting lower levels of C9orf72 are also identified in ALS cases withoutC9orf72 repeat expansion. This means that loss of C9orf72 could be part of aconventionalpathwayaffectedinthesediseases(Xietal.2015).However,thereare also some arguments against the loss-of-function role. First,intracerebroventricular delivery to adult mice of antisense oligonucleotides(ASOs)targetingC9orf72leadstoknockdownofC9orf72ofthecompletecentralnervous system, but does not produce any motor or behavioural phenotypes.Secondly, nomutationshavebeendiscovered in coding regions of theC9orf72gene.Finally,ararehomozygousC9orf72repeatexpansiondidnotshowclinicalorpathologicalfeaturesoutsidethenormaldiseasespectrum(Mizielinskaetal.2014). Thegainof functionof theC9orf72 genewasproposed to causeALSbytwo mechanisms. The first mechanism associated to the gain-of-functionnoncoding repeat expansion diseases was toxic function of the repeat RNA.There is sequestration of important RNA-binding proteins into aggregates of

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repeat-containing RNA foci. A novel mechanism is discovered, through whichexpandedCAGrepeatsaretranslatedintheabsenceofanATGinitiationcodon,which is called repeat-associated non-ATG dependent translation (RANtranslation)(Mizielinskaetal.2014) ThemannerofactioninALSisbywayofsequestrationofessentialRNA-bindingproteinsintoaggregatesofrepeat-containingRNAfoci,inthenucleusoftheaffectedcell.RNAfociconsistingofrepeatRNAarepresentinfrontalcortex,hippocampus,cerebellumandspinalcordofC9ALSpatients.RNAfociexistedinseveraltypesofglialcells(astrocytes,microglia,andoligodendrocytes),butaremost importantly a neuronal phenotype. Astrocytes derived from familial andsporadicALSpatientscanmakeuseoftoxicitytomotorneurons(Mizielinskaetal.2014).Proteins involved innuclearmRNAexportalsobind toG4C2repeatsandaresequesteredintofocicontainingsenseRNAtranscripts.Therehasbeensuggested that antisense transcripts may also cause transcriptional changes,possiblythroughsequestrationofRNA-bindingproteins(Krameretal.2016). The other new and potentially toxic species in C9ALS are the DPRproteins formed byRAN translation of the expanded repeat. DPR proteins aretranslated fromall framesof theG4C2repeat resulting inpolymersofglycine-alanine(GA),glycine-proline(GP)andglycine-arginine(GR)inthesenseframesandglycine-proline (GP), alanine-proline (AP) andproline-arginine (PR) in theantisense frames. All DPR proteins form extensive neuronal cytoplasmicinclusionsinpatient’sbrain.Poly(GP)andpoly(GA)DPRproteinsdisplaydot-like neuronal inclusions. Not like RNA foci, DPR inclusions appear to be anexclusivelyneuronalphenotype,possibly speculativeof theclearanceabilityofmitoticcells(Mizielinskaetal.2014)(seefigure2).

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Figure2PotentialmechanismsofdiseaseinC9FTD/ALS.AP,alanine-proline;DPR,dipeptiderepeat;GA,glycine-alanine;GP,glycine-proline;GR,glycine-arginine;PR,proline-arginine.Healthyneuron:thereisabalancebetweentheautophagosomesandtheC9orf72LossofC9orf72function:thereisadysregulationofautophagy,meaningalotofC9orf72isphagocytosedGainoffunctionrepeatRNA:thereissequestrationofRNA-bindingproteins The final genetical mechanism in ALS involves G-quadruplexes. TheC9orf72hexanucleotiderepeatexpansionsDNAandRNAsetuptheformationofG-quadruplexes. A G-quadruplex is a guanosine-rich DNA and RNA sequenceswhich easily form a stable four-stranded structure. Two differentmechanismsexplain the involvement of G-quadruplexes in neurological diseases. First,through repeat expansions of G-rich sequences that are predicted to form G-quadruplexesandareknowntocausediseaseasseeninC9ALS.Andsecondly,bymutationsaffectingtheexpressionofG-quadruplexbindingproteins.Inthecaseof ALS, the G- quadruplex of the hexanucleotide repeat is expected to becomprimised in determining the proteins it interacts with. It has beendemonstrated in myotonic dystrophy, where identical non-coding RNA repeatexpansions form nuclear foci, which sequester essential RNA-binding proteinsandbythatcausingfunctionaldefects(Kumaretal.2016). In the next section, I’m going to describe some therapies. One of thesuspectscausingALSaregrowthfactors.Agrowthfactorisasubstance,suchasavitaminorahormone,whichisrequiredforthestimulationofgrowthin livingcells. There are strong indications pointing out that hepatocyte growth factor(HGF)improvedsurvivalinisolatedmotorneuronsfromratembryos.Vascularendothelial growth factor (VEGF) also plays a role in the pathogenesis of ALS.ThenormalfunctionofVEGFistoincreasethevascularpermeability.VEGFalsostimulatesaxonaloutgrowthandneuronalsurvival.InthecaseofALS,(Brownetal. 2006), VEGF protects motor neurons from degeneration in ALS animal models. Therefore,itissuspectedthatVEGFcouldbeusedfortherapy.

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The second group of molecules related to ALS is neurotrophic factors.Neurotrophic factorsare a family ofbiomoleculesthat support the growth,survival, anddifferentiationofbothdevelopingandmatureneurons.Micewithhomozygoustargeteddisruptionof theciliaryneurotrophic factor(CNTF)genedemonstrate a loss of 20%ofmotorneurons. This suggests that CNTFplays asubstantialroleinALS.Ontheotherhand,anotherneurotrophicfactor,i.e.glia-derivedneurotrophicfactor(GDNF),showedapositiveeffectonthesurvivalofmotor neurons. In the study of Oppenheim et al., GDNF was applied to thechorioallantoicmembraneofabird.Thegoalofthestudywastotestiftherewasanymotorneuronsurvival.Indeed,GDNFledtoincreasedmotorneuronsurvivalduringthecriticalperiodofphysiologicalcelldeath.Italsoincreasedthenumberof surviving sympathetic and sensory neurons (Brown et al. 2006). Therefore,GDNFcouldbeapotentialtherapyforALS. 5.Conclusionandfuturereferences ThispaperdescribesthepathophysiologyofALSandtheroleofC9orf72. A lot of different hormones and processes play a role in thepathophysiology. TheC9orf72geneisveryspecialbecauseitliesinanintronicregionandnormallyonlyexonsaretranscribed,notintrons.TheidentificationofC9orf72G4C2repeatexpansionisasignificantdiscoverytowardsunderstandingthe C9ALS. There is only one known medicine, riluzole, which is a glutamateagonist, that increases survival by a few months. Morphologicalandultrastructuraldeformitiesofmitochondriahavebeenfound in autopsies of patients with ALS. In what way is the mitochondriainvolved?Moreresearchhastobedone.

Whether inflammation plays an important role is not yet clear, but theinvolvement ofmicroglial activationwas reported in a transgenicmousewithgeneraloverexpressionofthecytokineinterleukine-3.Further,thereisgrowingevidencethatapoptosiscontributestoneuronallossinmanyacuteandchronicneurologicaldisease.Allthesedifferentprocessesshouldbeinvestigatedfurtherbefore we even can make a medicine that interferes with these processes. G-quadruplexmotifsareinvolvedinALS.Althoughthereisaconsiderableamount of research executed in this field, much remains to be explored yet.Moreover,theloss-of-functionandgain-of-functionalsomightplayaroleinALS. There is an investigation of the role of growth hormones, likeHGF andVEGF, in ALS. Isolated motor neurons from rat embryos revealed improvedsurvival in thepresenceofHGF. Ifwewoulddomore research,maybewecanfind a medicine that interferes with these growth hormones, so that patientswithALScanlivelonger.

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One of the main difficulties in developing new therapies lies in themultipleeventsthatcontributetomotorneurondeathinALS.Anyinterventionaimed at solely reducing any particular pathological condition might not beenoughtotreatthediseaseanditssymptoms.

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