ARTICLE OPEN ACCESS

Absence of NEFL in patient-specific neurons inearly-onset Charcot-Marie-Tooth neuropathyMarkus T Sainio MSc Emil Ylikallio MD PhD Laura Maenpaa MSc Jenni Lahtela PhD Pirkko Mattila PhD

Mari Auranen MD PhD Johanna Palmio MD PhD and Henna Tyynismaa PhD

Neurol Genet 20184e244 doi101212NXG0000000000000244

Correspondence

Dr Tyynismaa

hennatyynismaahelsinkifi

AbstractObjectiveWe used patient-specific neuronal cultures to characterize the molecular genetic mechanism ofrecessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease

MethodsMotor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL Quantitative PCRprotein analytics immunocytochemistry electron microscopy and single-cell transcriptomicswere used to investigate patient and control neurons

ResultsWe show that the recessive nonsense mutation causes a nearly total loss of NEFL messengerRNA (mRNA) leading to the complete absence of NEFL protein in patientrsquos cultured neuronsYet the cultured neurons were able to differentiate and form neuronal networks and neuro-filaments Single-neuron gene expression fingerprinting pinpointed NEFL as the most down-regulated gene in the patient neurons and provided data of intermediate filament transcriptabundancy and dynamics in cultured neurons Blocking of nonsense-mediated decay partiallyrescued the loss of NEFL mRNA

ConclusionsThe strict neuronal specificity of neurofilament has hindered the mechanistic studies of re-cessive NEFL nonsense mutations Here we show that such mutation leads to the absence ofNEFL causing childhood-onset neuropathy through a loss-of-function mechanism We pro-pose that the neurofilament accumulation a common feature of many neurodegenerativediseases mimics the absence of NEFL seen in recessive CMT if aggregation prevents the properlocalization of wild-type NEFL in neurons Our results suggest that the removal of NEFL asa proposed treatment option is harmful in humans

From the Research Programs Unit (MTS EY LM MA HT) Molecular Neurology University of Helsinki Clinical Neurosciences Neurology (EY MA) University of Helsinki andHelsinki University Hospital Institute for Molecular Medicine Finland (FIMM) (JL PM) University of Helsinki Neuromuscular Research Center (JP) Tampere University Hospitaland University of Tampere and Department of Medical and Clinical Genetics (HT) University of Helsinki Finland

Funding information and disclosures are provided at the end of the article Full disclosure form information provided by the authors is available with the full text of this article atNeurologyorgNG

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2018 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Neurofilaments are 10-nm-wide intermediate filaments ex-clusive to neurons and crucial for the maintenance of neu-rite structure and integrity1 Neurofilament light chain(NEFL) is among the core subunits of neurofilamentusually forming heterodimers with medium (NEFM) andheavy chains (NEFH) sometimes supplemented with ei-ther α-internexin (INA) or peripherin (PRPH) The toxicaccumulation of neurofilament is a hallmark of many neu-rodegenerative disorders2ndash4 and thus the removal of NEFLhas been investigated as a treatment option5 In additionthe potential of NEFL as a serum biomarker of neuronalinjury in a number of neurologic disorders is currentlyinvestigated6ndash9

Genemutations inNEFL have been found to underlie Charcot-Marie-Tooth disease (CMT) either the demyelinatingCMT1F4 or axonal CMT2E10 form1011 Most disease-causingNEFL mutations are dominantly inherited missense variantsfunctioning through a gain-of-function mechanism1213 inwhich the missense mutant NEFL protein disrupts neurofila-ment assembly and organelle transport in the axons by formingaggregates1415 All reported recessive NEFL mutations havebeen homozygous nonsense variants131617 Elucidation of thediseasemechanism of the recessive variants in humans has beencomplicated by the restricted neuronal expression of NEFL18

Thus it is not known if the homozygous nonsense variantscause disease through aggregation effects mediated by a trun-cated protein or by the loss of NEFL protein throughnonsense-mediated messenger RNA (mRNA) decay (NMD)Here we used patient-specific induced pluripotent stem cell(iPSC)-derived neuronal cultures and report that the completeabsence of NEFL in patients with a homozygous NEFL non-sense mutation causes early-onset CMT

MethodsStandard protocol approvals registrationsand patient consentsPatient and control samples were taken according to theDeclaration of Helsinki with informed consent The studywas approved by the Institutional Review Board of the Hel-sinki University Hospital

PatientsTwo siblings patients P1 and P2 now aged 30 and 27 yearsrespectively who were born to healthy unrelated parents ofFinnish origin were investigated for motor and de-velopmental delay in early childhood The skin fibroblastsused in the study were derived from patient P1 Experimental

details are described in the e-Methods (appendix e-1 link-slwwcomNXGA54)

ResultsClinical findingsP1 was first examined as an infant because of nystagmus andtremor Her feet were deformed resembling clubfoot andher movements were clumsy She learned to walk at the ageof 20 months Electrophysiology showed strongly de-creased nerve conduction velocities (NCVs) correspondingto demyelinating sensorimotor neuropathy At the age of 8years there was no response from the sensory mediannerve Ulnar sensory NCVs from the wrist to finger and theelbow to wrist were 9 and 19 ms respectively Median andulnar motor NCVs from the elbow to wrist were 21 and 16ms respectively The only response in the lower leg wasrecorded from tibialis anterior Four years later sensoryNCVs were absent in all upper limb nerves (ie radial ulnarand median) Median motor response was also absent andulnar NCVs were markedly reduced (ie 11 ms from theelbow to wrist no response from the wrist to finger)Needle examination showed denervation There were un-specific mild white matter lesions on her brain MRI thatwere not progressive Her younger brother P2 had similarsymptoms and electrophysiologic findings although lesssevere and his brain MRI was normal During their schoolyears the patients were estimated to be approximately 2years behind their peers in cognitive development How-ever both managed to finish supported elementary schooldespite difficulties

As neuropathy slowly progressed distal muscle weaknessbecame apparent in upper and lower extremities Muscle at-rophy was evident in the legs and intrinsic hand muscles Bothpatients underwent orthopedic surgery for feet deformitiesand tight Achilles tendon Distal weakness and waddling gaitwere observed P2 aged 27 years can still walk 50 m withoutaid whereas P1 lost ambulation at the age of 25 years Forboth patients grip strength was reduced pinching impossibleand fine motor skills decreased Weakness in finger extensionand flexion was severe (1-25 Medical Research Council[MRC] scale) All movements were essentially lost in ankleplantar and dorsiflexion There was also proximal muscleweakness in the upper and lower limbs but to a lesser extent(3-45 MRC) Tendon reflexes were absent No clear sensorydisturbances were found In both patients articulation wasslow but there were no dysarthria facial weakness or otherbulbar symptoms

GlossaryCMT = Charcot-Marie-Tooth INA = internexin iPSC = induced pluripotent stem cell mRNA = messenger RNA NCV =nerve conduction velocity NEFH = neurofilament heavy NEFL = neurofilament light NEFM = neurofilament mediumNMD = nonsense-mediated mRNA decay PRPH = peripherin qPCR = quantitative PCRUMI = unique molecular identifier

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P1 was also diagnosed with ventricle septum defect in child-hood and later aortic valve stenosis and regurgitation sleepapnea and severe obesity The parents were healthy bothunderwent electrophysiologic studies as part of their child-renrsquos investigations with normal NCVs

Genetic findingsTargeted next-generation sequencing panel for knownCMT disease genes19 for the DNA sample of P1 revealeda novel homozygous c1099CgtT (g24811765CgtT) variantin exon 2 of the NEFL gene predicting a nonsense changepArg367 The patients were homozygous for the variantand parents were heterozygous carriers (figure 1A) TheGnomAD20 database (277044 alleles) lists 15 heterozygouscarriers of the variant in Finland indicating an enrichment ofthe variant with a carrier frequency of 000058 The locali-zation of the variant together with previously reporteddisease-causing variants in NEFL domains is summarized infigure 1B

Absence of NEFL in patient-specificcultured neuronsTo investigate the consequences of the NEFL nonsense var-iant we differentiated patient-specific iPSC reprogrammedfrom P1 skin fibroblasts into neurons Two iPSC clones wereused from P1 and 1 clone each from 3 unrelated controliPSCs We used the motor neuron differentiation protocolmodified from reference 21 as summarized in figure 2A Weverified the neuronal differentiation by quantitative PCR(qPCR) of microtubule-associated protein 2 (MAP2) andβIII-tubulin (TUBB3) mRNA expression (figure 2B) and byimmunocytochemistry with TUBB3 (TUJ1) and MAP2antibodies (figure 2C) Approximately 90 of DAPI-positivecells were also MAP2 andor TUJ1 positive in each imagedframe To validate the motor neuronal identity of the differ-entiated neurons we analyzed the expression levels of ISLLIM homeobox 1 (ISL1) motor neuron and pancreashomeobox 1 (MNX1) and acetylcholine transferase (CHAT)by qPCR (figure 2D) Although some variation was detected

Figure 1 Dominant and recessive missense and nonsense variants in neurofilament light (NEFL)

(A) Sequencing traces of the c1099CgtT variant in the family members show that both parents of the patients are heterozygous carriers of the mutation (B)NEFL protein domains are depicted and the localization of the reported missense and nonsense variants is indicated (modified from references 17 and 25)The nonsense variant A367 identified in this study is shown in red

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Figure 2 Neuron differentiation and validation

(A)Work-flowof fibroblast-derived inducedpluripotent stemcell (iPSC) differentiation intomotor neuronsWnt signaling pathway activator (WNTact) retinoicacid (RA) Sonic hedgehog (SHH) growth factors (GF BDNF IGF-1 andCNTF) Poly-D-lysine (PDL) (B) Validation of the expression of neural transcriptsMAP2 andTUBB3 against GAPDH by quantitative PCR (qPCR) in total culture RNA of patient 1 clones 1 (Pt C1) and 2 (Pt C2) and controls 1-3 (ctr 1-3) after motor neurondifferentiation (C) Immunocytochemical analysis of MAP2 (green) and TUBB3 (red) proteins in patient 1 and control neuronal cultures (D) Validation of theexpression of motor neural transcripts ISL1 MNX1 and CHAT by qPCR as in B (E) Immunocytochemical analysis of ISL1 (red) and NEFM (green) protein inpatient 1 and control neuronal cultures ISL1-positive neurons are shown in larger cell clusters in the final differentiation stage (day 14 of in PDL + laminin-coated plates) (F) Expression of intermediate filament subunits neurofilament medium (NEFM) neurofilament heavy (NEFH) and neurofilament light (NEFL)by qPCR as in B The bars in each graph representmean levels plusmn SD n = 3 for each cell line All scale bars 50μm 496-diamidino-2-phenylindole (DAPI) indicatesnuclear staining

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between the different clones for the motor neuron markersno decrease in differentiation potential was observed in pa-tient lines in comparison with control lines ISL1 expressionwas confirmed by immunocytochemistry in the neuronalcultures (figure 2E)

We then analyzed the mRNA levels of NEFL NEFM andNEFH and observed that the patient neuronal cultures hada markedly decreased NEFL mRNA with a residual level ofabout 5 in patient vs control samples whereas patientNEFM and NEFH mRNA levels were comparable with con-trol levels (figure 2F) This suggested that the nonsensemutant NEFL transcript was degraded by NMD We nextinvestigated whether NEFL protein could be detected in pa-tient neurons by immunoblotting or immunocytochemistryRelatively even neuronal differentiation of lysed samples wasvalidated by immunoblotting for TUJ1 NEFM and CHATproteins (figure 3A) The NEFL nonsense variant had pre-dicted a potential C-terminally truncated protein of 366amino acids (approximately 45 kDa) However the Westernblots of patient neuron lysates showed no full-length NEFLpolypeptide (68 kDa) or signs of a truncated NEFL proteinusing either an N-terminal monoclonal (recognizing residues6ndash25) or a polyclonal pan-NEFL antibody (figure 3A) in-dicating that patient neurons were absent of NEFL Usingimmunocytochemistry highly similar neurite structures andneuronal networks were seen in patient and control motorneurons by NEFM immunostaining (figure 3B) The patient

neurons did not show any staining with NEFL antibodyconfirming that they were devoid of NEFL However theywere still able to form as long branching projections as thecontrol neurons suggesting that the intermediate filamentnetwork in the absence of NEFL was sufficient for axonalmaintenance in culture

Intermediate filament transcript dynamics incultured neuronsTo examine the gene expression fingerprints in single culturedneurons we used the differentiated neuronal cultures of P1clone 1 and control 1 for single-cell transcriptomics by theMacosko22 method with 10X Genomics Single Cell Plat-form23 After quality control 1336 cells could be profiledfrom P1 clone 1 and 418 cells from control 1 The expressionof 17318 genes could be reliably detected in these cellsClustering of the individual cells based on their transcriptomeprofiles resulted in 5 clusters Neuronal cells clustered dis-tinctly from other cells driven by the expression of neuron-specific transcripts (figure 4A) Clustering revealed that 261of the captured cells from P1 clone 1 (349 of 1336 cells) and230 from control 1 (96 of 418 cells) had a neural identitydepicted in t-SNE projections colored by the expression ofMAP2 microtubule-associated protein tau (MAPT) growth-associated protein 43 (GAP43) and synaptophysin (SYP)(figure 4B) The captured neurons also expressed motorneuronal markers as depicted in figure 4C in which CHATSLC18A3 ISL1MNX1 LHX1 LHX3DCCONECUT1 and

Figure 3 Complete loss of neurofilament light (NEFL) protein in cultured patient neurons

(A) Immunoblotting of whole cell lysates of patient 1 clones 1 and 2 (Pt C1 and C2) and controls 1-3 (ctr 1-3) after motor neuronal differentiation with an N-terminal monoclonal or a polyclonal pan-NEFL antibody Protein levels of neuronal markers ChAT TUJ1 and neurofilament medium (NEFM) and the loadingcontrol glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as well as the stain-free blot are shown (B) Immunocytochemical analysis of NEFM (green) andNEFL (orange) of neurite architecture in patient 1 and control neurons after motor neural differentiation 496-diamidino-2-phenylindole (DAPI) indicatesnuclear staining Scale bars 50 μm

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Figure 4 Transcriptome dynamics of patient and control neurons

(A) Clustering of the single cells derived from patient 1 and control iPSCs in the tSNE-plot based on their gene expression fingerprints Different clusters are colorcoded In the combined single-cell sequencingdata patient cells are shownas filleddots and control cells asdiamondsNeurons are clusteredasa clearly separategroup of cells (cluster 1 red) (B)MAP2 MAPT SYP andGAP43 expression is high in the neuronal cluster cells Red indicates high expression and gray indicates low(C) Cells in the neural cluster express motor neuron lineage-specific transcripts CHAT SLC18A3 ISL1MNX1 LHX1 LHX3 DCC ONECUT1 and ONECUT2 summed inthe tSNE-plot Purple indicates high expression and gray indicates low (D) Themost significantly upregulated and downregulated transcripts (adjusted p lt 0001and absolute fold change ge15) in the neural cluster between patient and control cells Neurofilament light (NEFL) is the most downregulated transcript in thepatient neurons (E) In the violin plots each individual cell is shown with its specific transcript level depicting the most downregulated transcript NEFL in patientneurons and evenly expressed intermediate filament subunit transcripts INANEFMNEFH PRPH and VIM Expression refers to normalized log(e) expression scale(F) Expression of NEFL against GAPDH by qPCR from total culture RNA of patient clones 1 (Pt C1) and 2 (Pt C2) and controls 1-3 (Ctr 1-3) after motor neuronaldifferentiation nontreated (NT) or treatedwith 200 μgmL cycloheximide (CHX) for 18 hours The comparisons weremade individually between each cell line withand without CHX treatment n = 3 for each cell line and treatment (unpaired 2-tailed t test p lt 0001 p lt 001) Bars represent mean levels plusmn SD

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ONECUT2 are summed indicating that these neurons arepart of the motor neuronal lineage The neuronal differ-entiation efficiency in cultures was much greater than thepercentage of captured neurons which reflects the difficultyto capture neuronal cells with long extensions in contrast tomorphologically more favorable cell types However largenumbers of neurons were successfully captured from pa-tient and control cultures and the proportion of neuronalcells of all captured cells was comparable in patient andcontrol data

To gain an overall appreciation of the abundancy of the cap-tured mRNAs per gene in the cultured neurons and of theratios of the different intermediate filament transcripts weanalyzed the mean unique molecular identifier (UMI) countsfor each gene in the control neuronal cell cluster The mosthighly expressed gene was MALAT1 (metastasis associatedlung adenocarcinoma transcript 1) followed by a number ofgenes for cytoskeletal proteins such as tubulin and actin ri-bosome subunits and mitochondrial-DNA encoded oxidativephosphorylation complex subunits (figure e-1 linkslwwcomNXGA51)NEFMwas the most highly captured intermediatefilament transcript (46th in abundance) closely followed byNEFL (66th) whereas INA (238) VIM (2728) PRPH(2991) and NEFH (3456) were much less frequent (figuree-1) The UMI counts per gene thus indicated that bothNEFLand NEFM were very highly expressed transcripts in controlcultured neurons

At a single-cell level when comparing the autosomal tran-script levels in the patient neurons with control neurons(adjusted p lt 0001 and absolute fold change ge15)NEFLwasthe most significantly downregulated transcript (10-fold) inthe patient neurons (figure 4D) Violin plots in figure 4Edemonstrate the reduced level of NEFL transcripts in in-dividual neurons of patient identity in comparison to controlidentity whereas other intermediate filaments were not sig-nificantly altered indicating no transcriptional compensationAlthough we found no evidence of stable NEFL protein incultured patient neurons the violin plot in figure 4E showsthat some neurons still expressed a low level ofNEFLmRNATo study NEFL transcript dynamics we blocked NMD bytreating the neuronal cultures with cycloheximide (CHX) aninhibitor of protein synthesis CHX significantly increased theamount of nonsense NEFL mRNA in patient neuronal cul-tures but at the same time CHX dramatically decreased theamount of wild-type NEFLmRNA in control neurons (figure4F) These results indicated that NMD machinery was re-sponsible for the nonsense mRNA degradation but could notcompletely abolish it because of its high abundancy In ad-dition the NEFL mRNA levels appear to be tightly regulatedin relation to protein synthesis

In this study we did not investigate in detail the potentialother transcriptional alterations that were associated withNEFL loss in patientrsquos cultured neurons as these findingsrequire substantial additional studies For example urotensin

2 (UTS2) a small cyclic peptide shown previously to regulateintracellular calcium in rat spinal cord neurons24 was themostupregulated gene in patient vs control neurons but its ex-pression profile showed high variation within the individualpatient neurons (figure e-2 linkslwwcomNXGA52)Therefore its specific role in association with NEFL loss is notclear

Neurite architectureBecauseNEFL is believed to be a fundamental building block inthe intermediate filament network of axons and dendrites1 weexamined neurites in our cultures By immunocytochemistrywe did not detect defects in neurite morphology in the patientrsquoscultured neurons in comparison with control neurites Theneurite signals for TUJ1 MAP2 and NEFM were not reducedin patient cell lines (figure e-3A linkslwwcomNXGA53)We also performed electron microscopic analysis to furtherexamine neurite structureNeurite areas varied in cross sectionsfrom 7000 to 170000 nm2 but did not significantly differbetween control and patient samples (figure e-3B) Un-expectedly cross sections of patient neurites showed in additionto microtubules a clear presence of intermediate filaments(figure 5)We counted the percentages of neurite cross sectionsthat contained microtubules or filament bundles and observedsimilar numbers in control and patient samples (figure e-3C)Furthermore the longitudinal sections of patient neurites dis-played no signs of neurofilament accumulation or abnormalitiesindicating dysregulation in the microtubule network or axonaltransport Collectively these results showed that cultured hu-man neurons can form neurofilaments and maintain axonalstructure in the absence of NEFL

DiscussionWe describe here CMT1F patients with a novel homozygousnonsense mutation in NEFL and demonstrate that the mu-tation leads to the absence of NEFL in patient-derived cul-tured neurons Both patients had the disease onset at infancyand presented with severely reduced NCVs and slowly pro-gressive distal muscle weakness in lower and upper extremi-ties The low NCVs suggested that myelin was lost in theperipheral neurons but nerve biopsies were not available fromthe patients to investigate whether the reduced NCVs weredue to the dramatic loss of axonal caliber in the absence ofNEFL or the loss of myelin Intermediate to severe reductionin NCVs has been previously reported in association withcertain NEFL mutations411 In addition to peripheral nerveinvolvement both of our patients had mild intellectual dis-ability possibly also resulting from the NEFL defect sinceabnormalities in cognitive development have been previouslyreported in a few patients with dominant or recessive NEFLmutations1325

Recessively inherited NEFL nonsense mutations typicallycause an early-onset CMT131617 Homozygous pGlu140mutation was described in 1 patient with gait disturbance andprogressive muscle weakness since school age16 pGlu210 in

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4 siblings with slowly progressive distal muscle weakness andatrophy starting at approximately 15 years13 and pGlu163in an adolescent girl with muscle weakness and gait disturbancesduring the first decade17 Although neurofilament aggregation iswell documented for dominant NEFL mutations121326 as wellas in other neurodegenerative disorders23 the molecular con-sequences of recessive nonsense mutations in NEFL have notbeen fully investigated Neuronal specificity of NEFL has pre-viously prevented studying the nonsensemutations in detail andespecially in cells with endogenous levels of mutant NEFLmRNA Using neurons differentiated from patient-specific iPSCwe unexpectedly observed that the recessive NEFL nonsensemutation led to a complete absence of NEFL protein throughNMD of the nonsense mutant mRNA

In this study we demonstrate the loss of NEFL mRNA andprotein in human neurons In the literature NEFL is largelyconsidered as an essential component of neurofilament inmatureneurons together with NEFM andNEFH15 The composition ofneurofilaments is also dependent on the neuronal type and de-velopmental stage15 Our single-neuron transcriptomics showedthat NEFL and NEFM were highly abundant transcripts in thecultured neurons whereasNEFHwas not LowNEFH transcriptcapture is consistent with its expression increasing only as a resultof axonal maturation concomitant with myelination27 INA andPRPH may also contribute to neurofilament formation but aremostly expressed during early embryonic neuronal differentia-tion or in early postnatal brain respectively2829 or following

neuronal injury3031 In the cultured neurons of this study wefound the intermediate filaments expressed in the following or-der of abundance NEFMgtNEFLgtINAgtVIMgtNEFHgtPRPHIn the patient neurons lacking NEFL we found no indication oftranscriptional compensation of other neurofilament poly-peptides althoughwe could detect neurofilaments in the neuritesby electron microscopy This suggests that the intermediate fil-ament formation in cultured neurons does not require NEFLHowever a recent study reported that a CMT patient withrecessive NEFL nonsense mutations had no neurofilament inaxons in a nerve biopsy as detected by electron microscopy17

Combined with our demonstration of NEFL nonsense muta-tions leading to NEFL absence their result indicates that inhuman peripheral axons the lack of NEFL protein indeed leadsto neurofilament loss It is possible that the transport of neuro-filaments to the long distal sural nerve may be impaired inpatients and this cannot be reproduced by the current in vitromodel It is important that the attempts to remove NEFL asa therapeutic intervention to its toxic accumulation5 should takeinto account that its loss is equally harmful to peripheral neuronsand caused a severe early-onset disease in our patients It is alsonoteworthy that the full Nefl mouse knockout only displayeda phenotype following nerve injury32 suggesting major differ-ences in the neurofilament biology between humans and micewhich may be connected to axon length

Previous study of iPSC-derived neurons from CMT indi-viduals carrying a NEFL missense variant found NEFL

Figure 5 Neurite structure is not disrupted by the lack of neurofilament light (NEFL)

Representative electron microscopy images ofneurite architecture in patient 1 and control neu-rons Intermediate filaments (outlined arrow) andmicrotubules (filled arrow) are indicated in crosssections Normal neurofilament network is seen inlongitudinal sections of patient neurites Scale bars500 nm

8 Neurology Genetics | Volume 4 Number 3 | June 2018 NeurologyorgNG

aggregate retention in the perikarya of neurons possiblydisrupting the neurofilament network and axonal mainte-nance33 Our results indicate that CMT can be caused byboth the loss of NEFL and its toxic accumulation12 Wetherefore speculate that in the cases of NEFL accumulationthe toxicity is at least partly caused by the aggregates pre-venting the proper localization and function of wild-typeNEFL as well as disrupting the maintenance and turnover ofintermediate filaments in the axon This could result inNEFL loss in critical parts of the axons similar to the situ-ation in patients with recessive NEFL nonsense mutationsIndeed reduced neurofilament has been detected in cuta-neous nerve fibers of patients with dominant CMT2Esuggesting that aggregates in cell bodies led to neurofilamentdisruption distally34

Here we demonstrated that the absence of NEFL in humanneurons causes early-onset CMT As a limitation of our studyskin fibroblasts of only 1 patient from the family were availablefor iPSC generation The lack of an obvious defect in neu-rofilament formation in cultured patient-specific neuronschallenges the use of the current model system in studies ofpathogenic mechanisms In addition we presented a case inwhich single-neuron transcriptomics could be used to identifythe genetic defect based on the consequent gene expressionalteration

Author contributionsAll authors acquired and analyzed data and contributed to thewriting of the manuscript MT Sainio E Ylikallio J LahtelaP Mattila M Auranen and H Tyynismaa designed theexperiments L Maenpaa performed bioinformatic analysisJ Palmio performed clinical investigations E Ylikallio andH Tyynismaa supervised the study

AcknowledgmentThe authors thank Riitta Lehtinen for technical help Theyacknowledge the Electron Microscopy Unit of the Institute ofBiotechnology University of Helsinki for providing labora-tory facilities and electron microscopy-sample preparationand the Biomedicum Stem Cell Center University ofHelsinki for iPSC generation and technical help

Study fundingThis work was supported by the Academy of Finland SigridJuselius Foundation University of Helsinki Helsinki Uni-versity Hospital Doctoral Programme in Biomedicine andFinska Lakaresallskapet

DisclosureMarkus T Sainio reports no disclosures Emil Ylikallio hasreceived research support from the Academy of FinlandUniversity of Helsinki and Emil Aaltonen Foundation LauraMaenpaa Jenni Lahtela Pirkko Mattila Mari Auranen andJohanna Palmio report no disclosures Henna Tyynismaa hasserved on the editorial board of Scientific Reports and hasreceived research support from the Academy of Finland and

European Research Council Full disclosure form informationprovided by the authors is available with the full text of thisarticle at NeurologyorgNG

Received January 17 2018 Accepted in final form April 19 2018

References1 Brown HG Troncoso JC Hoh JH Neurofilament-L homopolymers are less

mechanically stable than native neurofilaments J Microsc 1998191229ndash2372 Hirano A Nakano I Kurland LT Mulder DW Holley PW Saccomanno G Fine

structural study of neurofibrillary changes in a family with amyotrophic lateral scle-rosis J Neuropathol Exp Neurol 198443471ndash480

3 Israeli E Dryanovski DI Schumacker PT et al Intermediate filament aggregates causemitochondrial dysmotility and increase energy demands in giant axonal neuropathyHum Mol Genet 2016252143ndash2157

4 Jordanova A De Jonghe P Boerkoel CF et al Mutations in the neurofilament lightchain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease Brain2003126590ndash597

5 Yadav P Selvaraj BT Bender FL et al Neurofilament depletion improves microtu-bule dynamics via modulation of Stat3stathmin signaling Acta Neuropathol 201613293ndash110

6 Meeter LH Dopper EG Jiskoot LC et al Neurofilament light chain a biomarker forgenetic frontotemporal dementia Ann Clin Transl Neurol 20163623ndash636

7 Disanto G Barro C Benkert P et al Serum neurofilament light a biomarker ofneuronal damage in multiple sclerosis Ann Neurol 201781857ndash870

8 Weydt P Oeckl P Huss A et al Neurofilament levels as biomarkers in asymptomaticand symptomatic familial amyotrophic lateral sclerosis Ann Neurol 201679152ndash158

9 Byrne LM Rodrigues FB Blennow K et al Neurofilament light protein in blood asa potential biomarker of neurodegeneration in huntingtonrsquos disease a retrospectivecohort analysis Lancet Neurol 201716601ndash609

10 Mersiyanova IV Perepelov AV Polyakov AV et al A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-lightgene Am J Hum Genet 20006737ndash46

11 De Jonghe P Mersivanova I Nelis E et al Further evidence that neurofilament lightchain gene mutations can cause Charcot-Marie-Tooth disease type 2E Ann Neurol200149245ndash249

12 Sasaki T Gotow T Shiozaki M et al Aggregate formation and phosphorylation ofneurofilament-L Pro22 Charcot-Marie-Tooth disease mutants Hum Mol Genet200615943ndash952

13 Yum SW Zhang J Mo K Li J Scherer SS A novel recessive nefl mutation causesa severe early-onset axonal neuropathy Ann Neurol 200966759ndash770

14 Gentil BJ Minotti S BeangeM Baloh RH Julien JP DurhamHD Normal role of thelow-molecular-weight neurofilament protein in mitochondrial dynamics and disrup-tion in Charcot-Marie-Tooth disease FASEB J 2012261194ndash1203

15 Gentil BJ Tibshirani M Durham HD Neurofilament dynamics and involvement inneurological disorders Cell Tissue Res 2015360609ndash620

16 Abe A Numakura C Saito K et al Neurofilament light chain polypeptide genemutations in Charcot-Marie-Tooth disease nonsense mutation probably causesa recessive phenotype J Hum Genet 20095494ndash97

17 Fu J Yuan Y A novel homozygous nonsense mutation in NEFL causes autosomalrecessive Charcot-Marie-Tooth disease Neuromuscul Disord 20182844ndash47

18 Shy ME Patzko A Axonal Charcot-Marie-Tooth disease Curr Opin Neurol 201124475ndash483

19 Ylikallio E Johari M Konovalova S et al Targeted next-generation sequencing revealsfurther genetic heterogeneity in axonal Charcot-Marie-Tooth neuropathy and a mu-tation in HSPB1 Eur J Hum Genet 201422522ndash527

20 Lek M Karczewski KJ Minikel EV et al Analysis of protein-coding genetic variationin 60706 humans Nature 2016536285ndash291

21 Du ZW Chen H Liu H et al Generation and expansion of highly pure motor neuronprogenitors from human pluripotent stem cells Nat Commun 201566626

22 Macosko EZ Basu A Satija R et al Highly parallel genome-wide expression profilingof individual cells using nanoliter droplets Cell 20151611202ndash1214

23 Zheng GX Terry JM Belgrader P et al Massively parallel digital transcriptionalprofiling of single cells Nat Commun 2017814049

24 Filipeanu CM Brailoiu E Le Dun S Dun NJ Urotensin-II regulates in-tracellular calcium in dissociated rat spinal cord neurons J Neurochem 200283879ndash884

25 Horga A Laura M Jaunmuktane Z et al Genetic and clinical characteristics of NEFL-related Charcot-Marie-Tooth disease J Neurol Neurosurg Psychiatry 201788575ndash585

26 Leung CL Nagan N Graham TH Liem RK A novel duplicationinsertion mutationof NEFL in a patient with Charcot-Marie-Tooth disease Am J Med Genet A 20061401021ndash1025

27 Haynes RL Borenstein NS Desilva TM et al Axonal development in the ce-rebral white matter of the human fetus and infant J Comp Neurol 2005484156ndash167

28 Escurat M Djabali K Gumpel M Gros F Portier MM Differential expression of twoneuronal intermediate-filament proteins peripherin and the low-molecular-massneurofilament protein (NF-L) during the development of the rat J Neurosci 199010764ndash784

NeurologyorgNG Neurology Genetics | Volume 4 Number 3 | June 2018 9

29 Kaplan MP Chin SS Fliegner KH Liem RK Alpha-internexin a novel neuronalintermediate filament protein precedes the low molecular weight neurofilamentprotein (NF-L) in the developing rat brain J Neurosci 1990102735ndash2748

30 Beaulieu JM Kriz J Julien JP Induction of peripherin expression in subsets of brainneurons after lesion injury or cerebral ischemia Brain Res 2002946153ndash161

31 Troy CM Muma NA Greene LA Price DL Shelanski ML Regulation of peripherinand neurofilament expression in regenerating rat motor neurons Brain Res 1990529232ndash238

32 Zhu Q Couillard-Despres S Julien JP Delayed maturation of regeneratingmyelinated axons in mice lacking neurofilaments Exp Neurol 1997148299ndash316

33 Saporta MA Dang V Volfson D et al Axonal Charcot-Marie-Tooth disease patient-derived motor neurons demonstrate disease-specific phenotypes including abnormalelectrophysiological properties Exp Neurol 2015263190ndash199

34 Pisciotta C Bai Y Brennan KM et al Reduced neurofilament expression in cutaneousnerve fibers of patients with CMT2E Neurology 201585228ndash234

10 Neurology Genetics | Volume 4 Number 3 | June 2018 NeurologyorgNG

DOI 101212NXG000000000000024420184 Neurol Genet

Markus T Sainio Emil Ylikallio Laura Maumlenpaumlauml et al neuropathy

Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth

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reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

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References httpngneurologyorgcontent43e244fullhtmlref-list-1

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httpngneurologyorgcgicollectionperipheral_neuropathyPeripheral neuropathy

httpngneurologyorgcgicollectiongene_expression_studiesGene expression studies

httpngneurologyorgcgicollectionall_geneticsAll Geneticsfollowing collection(s) This article along with others on similar topics appears in the

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httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

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httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet