C9orf72 poly GA RAN-translated protein plays a key role in amyotrophic lateral sclerosis via aggregation and toxicity.

An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.

The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis.

Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by TARDBP) neuronal cytoplasmic inclusions are the pathological hallmark in ∼95% of amyotrophic lateral sclerosis and ∼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a (encoded by DNAJB2, and also known as HSJ1a) as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with sporadic amyotrophic lateral sclerosis. This implies that the HSF1-mediated DNAJB2a/HSP70 heat shock response pathway is compromised in amyotrophic lateral sclerosis. Defective refolding of TDP-43 is predicted to aggravate the TDP-43 proteinopathy. The finding that the pathological accumulation of insoluble TDP-43 can be reduced by the activation of HSF1/HSP pathways presents an exciting opportunity for the development of novel therapeutics.

Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species.

TAR DNA-binding protein (TDP-43, also known as TARDBP) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients post mortem. TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.

ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules.

Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein-protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.

Mutations in FUS lead to synaptic dysregulation in ALS-iPSC derived neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder characterized by progressive muscular weakness due to the selective loss of motor neurons. Mutations in the gene Fused in Sarcoma (FUS) were identified as one cause of ALS. Here, we report that mutations in FUS lead to upregulation of synaptic proteins, increasing synaptic activity and abnormal release of vesicles at the synaptic cleft. Consequently, FUS-ALS neurons showed greater vulnerability to glutamate excitotoxicity, which raised neuronal swellings (varicose neurites) and led to neuronal death. Fragile X mental retardation protein (FMRP) is an RNA-binding protein known to regulate synaptic protein translation, and its expression is reduced in the FUS-ALS lines. Collectively, our data suggest that a reduction of FMRP levels alters the synaptic protein dynamics, leading to synaptic dysfunction and glutamate excitotoxicity. Here, we present a mechanistic hypothesis linking dysregulation of peripheral translation with synaptic vulnerability in the pathogenesis of FUS-ALS.

Generation of an Open-Access Patient-Derived iPSC Biobank for Amyotrophic Lateral Sclerosis Disease Modelling.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting the upper and lower motor neurons, causing patients to lose control over voluntary movement, and leading to gradual paralysis and death. There is no cure for ALS, and the development of viable therapeutics has proved challenging, demonstrated by a lack of positive results from clinical trials. One strategy to address this is to improve the tool kit available for pre-clinical research. Here, we describe the creation of an open-access ALS iPSC biobank generated from patients carrying mutations in the TARDBP, FUS, ANXA11, ARPP21, and C9ORF72 genes, alongside healthy controls. To demonstrate the utilisation of these lines for ALS disease modelling, a subset of FUS-ALS iPSCs were differentiated into functionally active motor neurons. Further characterisation revealed an increase in cytoplasmic FUS protein and reduced neurite outgrowth in FUS-ALS motor neurons compared to the control. This proof-of-principle study demonstrates that these novel patient-derived iPSC lines can recapitulate specific and early disease-related ALS phenotypes. This biobank provides a disease-relevant platform for discovery of ALS-associated cellular phenotypes to aid the development of novel treatment strategies.

Optineurin inclusions occur in a minority of TDP-43 positive ALS and FTLD-TDP cases and are rarely observed in other neurodegenerative disorders.

Optineurin (OPTN) is a multifunctional protein involved in vesicular trafficking, signal transduction and gene expression. OPTN mutations were described in eight Japanese patients with familial and sporadic amyotrophic lateral sclerosis (FALS, SALS). OPTN-positive inclusions co-localising with TDP-43 were described in SALS and in FALS with SOD-1 mutations, potentially linking two pathologically distinct pathways of motor neuron degeneration. We have explored the abundance of OPTN inclusions using a range of antibodies in postmortem tissues from 138 cases and controls including sporadic and familial ALS, frontotemporal lobar degeneration (FTLD) and a wide range of neurodegenerative proteinopathies. OPTN-positive inclusions were uncommon and detected in only 11/32 (34%) of TDP-43-positive SALS spinal cord and 5/15 (33%) of FTLD-TDP. Western blot of lysates from FTLD-TDP frontal cortex and TDP-43-positive SALS spinal cord revealed decreased levels of OPTN protein compared to controls (p < 0.05), however, this correlated with decreased neuronal numbers in the brain. Large OPTN inclusions were not detected in FALS with SOD-1 and FUS mutation, respectively, or in FTLD-FUS cases. OPTN-positive inclusions were identified in a few Alzheimer's disease (AD) cases but did not co-localise with tau and TDP-43. Occasional striatal neurons contained granular cytoplasmic OPTN immunopositivity in Huntington's disease (HD) but were absent in spinocerebellar ataxia type 3. No OPTN inclusions were detected in FTLD-tau and α-synucleinopathy. We conclude that OPTN inclusions are relatively rare and largely restricted to a minority of TDP-43 positive ALS and FTLD-TDP cases. Our results do not support the proposition that OPTN inclusions play a central role in the pathogenesis of ALS, FTLD or any other neurodegenerative disorder.

Nuclear import impairment causes cytoplasmic trans-activation response DNA-binding protein accumulation and is associated with frontotemporal lobar degeneration.

Trans-activation response DNA-binding protein (TDP-43) accumulation is the major component of ubiquitinated protein inclusions found in patients with amyotrophic lateral sclerosis, and frontotemporal lobar degeneration with TDP-43 positive ubiquitinated inclusions, recently relabelled the 'TDP-43 proteinopathies'. TDP-43 is predominantly located in the nucleus, however, in disease it mislocalizes to the cytoplasm where it aggregates to form hallmark pathological inclusions. The identification of TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis cases confirms its pathogenic role; but it is wild-type TDP-43 that is deposited in the vast majority of TDP-43 proteinopathies, implicating other unknown factors for its mislocalization and aggregation. One such mechanism may be defective nuclear import of TDP-43 protein, as a disruption of its nuclear localization signal leads to mislocalization and aggregation of TDP-43 in the cytoplasm. In order to explore the factors that regulate the nuclear import of TDP-43, we used a small interfering RNA library to silence 82 proteins involved in nuclear transport and found that knockdowns of karyopherin-beta1 and cellular apoptosis susceptibility protein resulted in marked cytoplasmic accumulation of TDP-43. In glutathione S-transferase pull-down assays, TDP-43 bound to karyopherin-alphas, thereby confirming the classical nuclear import pathway for the import of TDP-43. Analysis of the expression of chosen nuclear import factors in post-mortem brain samples from patients with TDP-43 positive frontotemporal lobar degeneration, and spinal cord samples from patients with amyotrophic lateral sclerosis, revealed a considerable reduction in expression of cellular apoptosis susceptibility protein in frontotemporal lobar degeneration. We propose that cellular apoptosis susceptibility protein associated defective nuclear transport may play a mechanistic role in the pathogenesis of the TDP-43 positive frontotemporal lobar degeneration.

Synaptopathy Mechanisms in ALS Caused by C9orf72 Repeat Expansion.

Amyotrophic Lateral Sclerosis (ALS) is a complex neurodegenerative disease caused by degeneration of motor neurons (MNs). ALS pathogenic features include accumulation of misfolded proteins, glutamate excitotoxicity, mitochondrial dysfunction at distal axon terminals, and neuronal cytoskeleton changes. Synergies between loss of C9orf72 functions and gain of function by toxic effects of repeat expansions also contribute to C9orf72-mediated pathogenesis. However, the impact of haploinsufficiency of C9orf72 on neurons and in synaptic functions requires further examination. As the motor neurons degenerate, the disease symptoms will lead to neurotransmission deficiencies in the brain, spinal cord, and neuromuscular junction. Altered neuronal excitability, synaptic morphological changes, and C9orf72 protein and DPR localization at the synapses, suggest a potential involvement of C9orf72 at synapses. In this review article, we provide a conceptual framework for assessing the putative involvement of C9orf72 as a synaptopathy, and we explore the underlying and common disease mechanisms with other neurodegenerative diseases. Finally, we reflect on the major challenges of understanding C9orf72-ALS as a synaptopathy focusing on integrating mitochondrial and neuronal cytoskeleton degeneration as biomarkers and potential targets to treat ALS neurodegeneration.

Generation of six induced pluripotent stem cell lines from patients with amyotrophic lateral sclerosis with associated genetic mutations in either FUS or ANXA11.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons, causing gradual paralysis, and resulting in death 3-5 years from diagnosis. ALS causative mutations have been identified in multiple genes, including Fused in sarcoma (FUS), and recently characterized Annexin A11 (ANXA11). We have derived induced pluripotent stem cell (iPSC) lines from six ALS patient lymphoblastoid cell lines, three with mutations in FUS (Q519E, R521H, R522G), and three with mutations in ANXA11 (G38R, D40G, R235Q). These lines have been characterized and provide a novel resource for investigation into ALS pathology.

A recessive S174X mutation in Optineurin causes amyotrophic lateral sclerosis through a loss of function via allele-specific nonsense-mediated decay.

Loss of function (LoF) mutations in Optineurin can cause recessive amyotrophic lateral sclerosis (ALS) with some heterozygous LoF mutations associated with dominant ALS. The molecular mechanisms underlying the variable inheritance pattern associated with OPTN mutations have remained elusive. We identified that affected members of a consanguineous Middle Eastern ALS kindred possessed a novel homozygous p.S174X OPTN mutation. Analysis of these primary fibroblast lines from family members identified that the p.S174X mutation reduces OPTN mRNA expression in an allele-dependent fashion by nonsense mediated decay. Western blotting correlated a reduced expression in heterozygote carriers but a complete absence of OPTN protein in the homozygous carrier. This data suggests that the p.S174X truncation mutation causes recessive ALS through LoF. However, functional analysis detected a significant increase in mitophagy markers TOM20 and COXIV, and higher rates of mitochondrial respiration and ATP levels in heterozygous carriers only. This suggests that heterozygous LoF OPTN mutations may not be causative in a Mendelian manner but may potentially behave as contributory ALS risk factors.

ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function.

Mutations in TANK binding kinase 1 (TBK1) have been linked to amyotrophic lateral sclerosis. Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency; however, many other mutations are missense with unknown functional effects. We exome sequenced 699 familial amyotrophic lateral sclerosis patients and identified 16 TBK1 novel or extremely rare protein-changing variants. We characterized a subset of these: p.G217R, p.R357X, and p.C471Y. Here, we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate 2 of its kinase targets, IRF3 and optineurin, and to undergo phosphorylation. They both inhibit binding to optineurin and the p.G217R, within the TBK1 kinase domain, reduces homodimerization, essential for TBK1 activation and function. Finally, we show that the proportion of TBK1 that is active (phosphorylated) is reduced in 5 lymphoblastoid cell lines derived from patients harboring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations.

C9ORF72 repeat expansion causes vulnerability of motor neurons to Ca2+-permeable AMPA receptor-mediated excitotoxicity.

Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.

The Use of Stem Cells to Model Amyotrophic Lateral Sclerosis and Frontotemporal Dementia: From Basic Research to Regenerative Medicine.

In recent years several genes have linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as a spectrum disease; however little is known about what triggers their onset. With the ability to generate patient specific stem cell lines from somatic cells, it is possible to model disease without the need to transfect cells with exogenous DNA. These pluripotent stem cells have opened new avenues for identification of disease phenotypes and their relation to specific molecular pathways. Thus, as never before, compounds with potential applications for regenerative medicine can be specifically tailored in patient derived cultures. In this review, we discuss how patient specific induced pluripotent stem cells (iPSCs) have been used to model ALS and FTD and the most recent drug screening targets for these diseases. We also discuss how an iPSC bank would improve the quality of the available cell lines and how it would increase knowledge about the ALS/FTD disease spectrum.

Monoamine oxidase a polymorphism in Brazilian patients: risk factor for late-onset Alzheimer's disease?

Different studies have attempted to find polymorphisms involved in the serotonergic pathway that could be involved in mood disorders and late-onset Alzheimer's disease (LOAD) symptoms. Here, we compared the frequency of two polymorphisms: monoamine oxidase A (MAOA) and serotonin transporter in LOAD patients versus controls. No evidence of association was observed when these polymorphisms were compared separately; however, the combination of the MAOA allele 1+the short allele of 5-HTTLPR+ApoE-epsilon4 was significantly more frequent in patients than in controls. It reinforces the hypothesis that different genes acting together might play a role in AD susceptibility. Based on these data, we suggest replicating these studies in larger samples of LOAD patients belonging to different ethnic groups.

Analysis of IL-1alpha, IL-1beta, and IL-1RA [correction of IL-RA] polymorphisms in dysthymia.

Investigators of independent studies reported alterations in cytokine serum levels in patients with different mood disorders. Several polymorphisms associated with neuropsychiatric disorders such as schizophrenia and Alzheimer's disease have been reported at the interleukin-1 (IL-1) panel. Here we report the results of three specific polymorphisms at the IL-1alpha, IL-1beta, and IL-1RA genes, which were analyzed in 128 Brazilian subjects: 59 dysthymic patients and 69 normal controls. We found a statistically significant difference (p = 0.002) in the frequency of haplotypes with alleles 2+ (IL-1RA), T+ (IL-1alpha), and C+ (IL-1beta) in patients as compared to controls. We also observed that haplotype IL-1RA1.2/IL-1alpha CT/IL-1beta CC, present in 6 dysthymic patients (10%) was absent in the normal control group (p = 0.012). These results suggest that these polymorphisms might confer a greater susceptibility to develop dysthymia in Brazilian patients. However, to validate these data it will be of great interest to repeat this study in larger samples and other ethnic groups.

Lack of association between the brain-derived neurotrophin factor (C-270T) polymorphism and late-onset Alzheimer's disease (LOAD) in Brazilian patients.

After the identification of the apolipoprotein E gene isoform (APOE-epsilon4) as a risk factor for late-onset Alzheimer's disease (LOAD), the search for other polymorphisms associated with AD has been undertaken by many groups of investigators around the world. These studies have shown controversial results in many populations. More recently, a single nucleotide polymorphism in the promoter region of the brain-derived neurotrophin factor (BDNF) was found to be a risk factor for AD in two independent population studies. Here we report the analysis of this polymorphism in a group of 188 LOAD Brazilian patients compared to matched normal controls. A strong association between the APOE-epsilon4 polymorphism and LOAD was observed, but there was no significant association between this BNDF polymorphism and affected patients. The possibility that other polymorphisms or mutations in this gene play a role in the development of AD cannot be ruled out. However, the results of the present study suggest that in opposition to the two reported studies, this polymorphism does not seem to be implicated in LOAD Brazilian patients. It also shows the importance of replication studies in different populations, as susceptibility loci might differ in different ethnic groups; this will have important implications in future treatments with pharmacological agents.

Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells.

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.

Downregulation of microRNA-9 in iPSC-derived neurons of FTD/ALS patients with TDP-43 mutations.

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43, and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization, two to three iPSC lines from each subject were selected, differentiated into postmitotic neurons, and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First, TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second, the total TDP-43 level was lower in patient neurons with the A90V mutation. Third, the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43, as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation, suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts.