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No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

Degeneration of the primary motor cortex is a defining feature of amyotrophic lateral sclerosis (ALS), which is associated with the accumulation of microscopic protein aggregates in neurons and glia. However, little is known about the quantitative burden and pattern of motor cortex proteinopathies across ALS genotypes. We combined quantitative digital image analysis with multi-level generalized linear modelling in an independent cohort of 82 ALS cases to explore the relationship between genotype, total proteinopathy load and cellular vulnerability to aggregate formation. Primary motor cortex phosphorylated (p)TDP-43 burden and microglial activation were more severe in sporadic ALS-TDP disease than C9-ALS. Oligodendroglial pTDP-43 pathology was a defining feature of ALS-TDP in sporadic ALS, C9-ALS and ALS with OPTN, HNRNPA1 or TARDBP mutations. ALS-FUS and ALS-SOD1 showed less cortical proteinopathy in relation to spinal cord pathology than ALS-TDP, where pathology was more evenly spread across the motor cortex-spinal cord axis. Neuronal pTDP-43 aggregates were rare in GAD67+ and Parvalbumin+ inhibitory interneurons, consistent with predominant accumulation in excitatory neurons. Finally, we show that cortical microglia, but not astrocytes, contain pTDP-43. Our findings suggest divergent quantitative, genotype-specific vulnerability of the ALS primary motor cortex to proteinopathies, which may have implications for our understanding of disease pathogenesis and the development of genotype-specific therapies.

BackgroundPremorbid body mass index, physical activity, diabetes and cardiovascular disease have been associated with an altered risk of developing amyotrophic lateral sclerosis (ALS). There is evidence of shared genetic risk between ALS and lipid metabolism. A very large prospective longitudinal population cohort permits the study of a range of metabolic parameters and the risk of subsequent diagnosis of ALS.MethodsThe risk of subsequent ALS diagnosis in those enrolled prospectively to the UK Biobank (n=502 409) was examined in relation to baseline levels of blood high and low density lipoprotein (HDL, LDL), total cholesterol, total cholesterol:HDL ratio, apolipoproteins A1 and B (apoA1, apoB), triglycerides, glycated haemoglobin A1c (HbA1c) and creatinine, plus self-reported exercise and body mass index.ResultsControlling for age and sex, higher HDL (HR 0.84, 95% CI 0.73 to 0.96, p=0.010) and apoA1 (HR 0.83, 95% CI 0.72 to 0.94, p=0.005) were associated with a reduced risk of ALS. Higher total cholesterol:HDL was associated with an increased risk of ALS (HR 1.17, 95% CI 1.05 to 1.31, p=0.006). In models incorporating multiple metabolic markers, higher LDL or apoB was associated with an increased risk of ALS, in addition to a lower risk with higher HDL or apoA. Coronary artery disease, cerebrovascular disease and increasing age were also associated with an increased risk of ALS.ConclusionsThe association of HDL, apoA1 and LDL levels with risk of ALS contributes to an increasing body of evidence that the premorbid metabolic landscape may play a role in pathogenesis. Understanding the molecular basis for these changes will inform presymptomatic biomarker development and therapeutic targeting.

A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and the related neurodegenerative disorder frontotemporal dementia, is the cellular mislocalization of transactive response DNA-binding protein 43 kDa (TDP-43). Additionally, multiple mutations in the TARDBP gene (encoding TDP-43) are associated with familial forms of ALS. While the exact role for TDP-43 in the onset and progression of ALS remains unclear, the identification of factors that can prevent aberrant TDP-43 localization and function could be clinically beneficial. Previously, we discovered that the oxidation resistance 1 (Oxr1) protein could alleviate cellular mislocalization phenotypes associated with TDP-43 mutations, and that over-expression of Oxr1 was able to delay neuromuscular abnormalities in the hSOD1G93A ALS mouse model. Here, to determine whether Oxr1 can protect against TDP-43-associated phenotypes in vitro and in vivo, we used the same genetic approach in a newly described transgenic mouse expressing the human TDP-43 locus harbouring an ALS disease mutation (TDP-43M337V). We show in primary motor neurons from TDP-43M337V mice that genetically-driven Oxr1 over-expression significantly alleviates cytoplasmic mislocalization of mutant TDP-43. We also further quantified newly-identified, late-onset neuromuscular phenotypes of this mutant line, and demonstrate that neuronal Oxr1 over-expression causes a significant reduction in muscle denervation and neuromuscular junction degeneration in homozygous mutants in parallel with improved motor function and a reduction in neuroinflammation. Together these data support the application of Oxr1 as a viable and safe modifier of TDP-43-associated ALS phenotypes.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene. All patients have at least one copy of a paralog, SMN2, but a C-to-T transition in this gene results in exon 7 skipping in a majority of transcripts. Approved treatment for SMA involves promoting exon 7 inclusion in the SMN2 transcript or increasing the amount of full-length SMN by gene replacement with a viral vector. Increasing the pool of SMN2 transcripts and increasing their translational efficiency can be used to enhance splice correction. We sought to determine whether the 5′ untranslated region (5′ UTR) of SMN2 contains a repressive feature that can be targeted to increase SMN levels. We found that antisense oligonucleotides (ASOs) complementary to the 5′ end of SMN2 increase SMN mRNA and protein levels and that this effect is due to inhibition of SMN2 mRNA decay. Moreover, use of the 5′ UTR ASO in combination with a splice-switching oligonucleotide (SSO) increases SMN levels above those attained with the SSO alone. Our results add to the current understanding of SMN regulation and point toward a new therapeutic target for SMA., Graphical Abstract, Spinal muscular atrophy (SMA) is caused by SMN protein deficiency. Winkelsas et al. show that antisense oligonucleotides complementary to the 5′ end of SMN transcripts increase SMN protein levels by stabilizing SMN mRNA. In cells, this strategy can be used in combination with splice-switching oligonucleotides (i.e., nusinersen) for SMA treatment.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. Despite this heterogeneity, a key pathological signature is the mislocalization and aggregation of specific proteins in the cytoplasm, suggesting that convergent pathogenic mechanisms focusing on disturbances in proteostasis are important in ALS. In addition, many cellular processes have been identified as potentially contributing to disease initiation and progression, such as defects in axonal transport, autophagy, nucleocytoplasmic transport, ER stress, calcium metabolism, the unfolded protein response and mitochondrial function. Here we review the evidence from in vitro and in vivo models of C9ORF72 and TDP-43-related ALS supporting a central role in pathogenesis for endoplasmic reticulum stress, which activates an unfolded protein response (UPR), and mitochondrial dysfunction. Disruption in the finely tuned signaling between the ER and mitochondria through calcium ions may be a crucial trigger of mitochondrial deficits and initiate an apoptotic signaling cascade, thus acting as a point of convergence for multiple upstream disturbances of cellular homeostasis and constituting a potentially important therapeutic target.

The neurodegenerative disorder amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of upper and lower motor neurons, with pathological involvement of cerebral motor and, additionally, extra-motor areas, in a clinicopathological spectrum with frontotemporal dementia (FTD). A key unresolved question is whether the distribution of pathology in ALS is driven by molecular factors such as regional gene expression, by differential network vulnerability, or is a combination of both. A system of histopathological staging of ALS based on the regional burden of TDP-43 pathology observed in post mortem brains has been supported to some extent by analysis of distribution of in vivo structural MRI changes. In this paper, computational modelling using a Network Diffusion Model (NDM) was used to investigate whether a process of focal pathological ‘seeding’ followed by structural network-based spread recapitulated post mortem histopathological staging and, secondly, whether this had any relationship to the pattern of expression of a panel of genes implicated in ALS across the healthy brain. Regionally parcellated T1-weighted MRI data from ALS patients (baseline n=79) was studied in relation to a healthy control structural connectome and a database of associated regional cerebral gene expression. The NDM provided strong support for a structural network-based basis for regional pathological spread in ALS, but no simple relationship to the spatial distribution of ALS-related genes in the healthy brain. Intriguingly, the critical seed regions for spread within the model were not within the primary motor cortex but basal ganglia, thalamus and insula, where NDM recapitulated aspects of the post mortem histopathological staging system. Within the ALS-FTD clinicopathological spectrum, non-primary motor structures may be among the earliest sites of cerebral pathology.

Polyglutamine (polyQ) diseases comprise Huntington’s disease and several subtypes of spinocerebellar ataxia, including spinocerebellar ataxia type 3 (SCA3). The genomic expansion of coding CAG trinucleotide sequence in disease genes leads to the production and accumulation of misfolded polyQ domain-containing disease proteins, which cause cellular dysfunction and neuronal death. As one of the principal cellular protein clearance pathways, the activity of the ubiquitin–proteasome system (UPS) is tightly regulated to ensure efficient clearance of damaged and toxic proteins. Emerging evidence demonstrates that UPS plays a crucial role in the pathogenesis of polyQ diseases. Ubiquitin (Ub) E3 ligases catalyze the transfer of a Ub tag to label proteins destined for proteasomal clearance. In this study, we identified an E3 ligase, pre-mRNA processing factor 19 (Prpf19/prp19), that modulates expanded ataxin-3 (ATXN3-polyQ), disease protein of SCA3, induced neurodegeneration in both mammalian and Drosophila disease models. We further showed that Prpf19/prp19 promotes poly-ubiquitination and degradation of mutant ATXN3-polyQ protein. Our data further demonstrated the nuclear localization of Prpf19/prp19 is essential for eliciting its modulatory function towards toxic ATXN3-polyQ protein. Intriguingly, we found that exocyst complex component 7 (Exoc7/exo70), a Prpf19/prp19 interacting partner, modulates expanded ATXN3-polyQ protein levels and toxicity in an opposite manner to Prpf19/prp19. Our data suggest that Exoc7/exo70 exerts its ATXN3-polyQ-modifying effect through regulating the E3 ligase function of Prpf19/prp19. In summary, this study allows us to better define the mechanistic role of Exoc7/exo70-regulated Prpf19/prp19-associated protein ubiquitination pathway in SCA3 pathogenesis.

Biallelic mutations in SNORD118, encoding the small nucleolar RNA U8, cause leukoencephalopathy with calcifications and cysts (LCC). Given the difficulty in interpreting the functional consequences of variants in nonprotein encoding genes, and the high allelic polymorphism across SNORD118 in controls, we set out to provide a description of the molecular pathology and clinical spectrum observed in a cohort of patients with LCC. We identified 64 affected individuals from 56 families. Age at presentation varied from 3 weeks to 67 years, with disease onset after age 40 years in eight patients. Ten patients had died. We recorded 44 distinct, likely pathogenic, variants in SNORD118. Fifty two of 56 probands were compound heterozygotes, with parental consanguinity reported in only three families. Forty nine of 56 probands were either heterozygous (46) or homozygous (three) for a mutation involving one of seven nucleotides that facilitate a novel intramolecular interaction between the 5' end and 3' extension of precursor-U8. There was no obvious genotype-phenotype correlation to explain the marked variability in age at onset. Complementing recently published functional analyses in a zebrafish model, these data suggest that LCC most often occurs due to combinatorial severe and milder mutations, with the latter mostly affecting 3' end processing of precursor-U8.

BackgroundAmyotrophic lateral sclerosis is a clinical syndrome with complex biological determinants, but which in most cases is characterized by TDP-43 pathology. The identification in CSF of a protein signature of TDP-43 network dysfunction would have the potential to inform the identification of new biomarkers and therapeutic targets.MethodsWe compared CSF proteomic data from patients with ALS (n = 41), Parkinson’s disease (n = 19) and healthy control participants (n = 20). Weighted correlation network analysis was used to identify modules within the CSF protein network and combined with gene ontology enrichment analysis to functionally annotate module proteins. Analysis of module eigenproteins and differential correlation analysis of the CSF protein network was used to compare ALS and Parkinson’s disease protein co-correlation with healthy controls. In order to monitor temporal changes in the CSF proteome, we performed longitudinal analysis of the CSF proteome in a subset of ALS patients.ResultsWeighted correlation network analysis identified 10 modules, including those enriched for terms involved in gene expression including nucleic acid binding, RNA metabolism and translation; humoral immune system function, including complement pathways; membrane proteins, axonal outgrowth and adherence; and glutamatergic synapses. Immune system module eigenproteins were increased in ALS, whilst axonal module eigenproteins were decreased in ALS. The 19 altered protein correlations in ALS were enriched for gene expression (OR 3.05, p = 0.017) and membrane protein modules (OR 17.48, p = 0.011), including intramodular hub proteins previously identified as TDP-43 interactors. Proteins decreasing over longitudinal analysis ALS were enriched in glutamatergic synapse and axonal outgrowth modules. Protein correlation network disruptions in Parkinson’s disease showed no module enrichment.ConclusionsAlterations in the co-correlation network in CSF samples identified a set of pathways known to be associated with TDP-43 dysfunction in the pathogenesis of ALS, with important implications for therapeutic targeting and biomarker development.

The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

Summary TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9ORF72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9ORF72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9ORF72 iPSC-MNs have higher Ca2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca2+ release. We identify here, by RNA sequencing, that both C9ORF72 and TARDBP iPSC-MNs have upregulation of Ca2+-permeable AMPA and NMDA subunits and impairment of mitochondrial Ca2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca2+ uniporter, indicating that impaired mitochondrial Ca2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9ORF72 or TARDBP mutations., Graphical Abstract, Highlights • Mutations in C9orf72 and TDP-43 reduce Ca2+ buffering capacity in ALS motor neurons • High levels of Ca2+-permeable glutamate receptors link C9orf72 and TDP-43 • Low mitochondrial Ca2+ uptake leads to glutamate-induced cell death • Imbalance of MICU1 and MICU2 reduces mitochondrial Ca2+ uptake, In this study, Dafinca, Talbot, and colleagues provide a mechanistic link between mutations in C9orf72 and TARDBP in induced pluripotent stem cell-derived motor neurons from ALS patients. They show both mutations cause increased expression of Ca2+-permeable receptors, reduced cytosolic Ca2+ buffering, and identify that perturbations in mitochondrial calcium uptake through an imbalance of MICU1 and MICU2 link the mutations.

The carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts a neuroprotective effect in neurodegenerative diseases via the activation of signaling pathways related to neurotrophins, and also through inhibiting apoptotic cell death. Here, we demonstrate that Hc-TeTx preserves motoneurons from chronic excitotoxicity in an in vitro model of amyotrophic lateral sclerosis. Furthermore, we found that PI3-K/Akt pathway, but not p21ras/MAPK pathway, is involved in their beneficial effects under chronic excitotoxicity. Moreover, we corroborate the capacity of the Hc-TeTx to be transported retrogradely into the spinal motor neurons and also its capacity to bind to the motoneuron-like cell line NSC-34. These findings suggest a possible therapeutic tool to improve motoneuron preservation in neurodegenerative diseases such as amyotrophic lateral sclerosis.

The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 and non-homologous end-joining (NHEJ) by deleting the repeat region with the risk of creating indels and genomic instability. In this study we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/FTD patient using CRISPR/Cas9 genome editing and homology directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines, and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC derived motor neurons (iPSMNs).RNA sequencing of the mutant line identified 2220 differentially expressed genes compared to its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the KEGG ALS pathway, as well as new targets of potential relevance to ALS pathophysiology.Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9 mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

ObjectiveTo evaluate the classifier performance, clinical and biochemical correlations of cerebrospinal fluid (CSF) levels of the chitinase proteins Chitotriosidase-1 (CHIT1), Chitinase-3-like protein 1 (CHI3L1) and Chitinase-3-like protein 2 (CHI3L2) in amyotrophic lateral sclerosis (ALS).MethodsCSF levels of CHIT1, CHI3L1, CHI3L2, phosphorylated neurofilament heavy chain (pNFH) and C-reactive protein were measured by ELISA in a longitudinal cohort of patients with ALS (n=82), primary lateral sclerosis (PLS, n=10), ALS-mimic conditions (n=12), healthy controls (n=25) and asymptomatic carriers of ALS-causing genetic mutations (AGC; n=5).ResultsCSF CHIT1, CHI3L1 and CHI3L2 were elevated in patients with ALS compared with healthy controls (pr=0.49, pr=0.42, pr=−0.25, p=0.038). All chitinases correlated with pNFH. CHIT1 levels were associated with survival in multivariate models. Chitinase levels were longitudinally stable.ConclusionsCSF chitinase proteins may have limited value as independent diagnostic and stratification biomarkers in ALS, but offer a window into non-autonomous mechanisms of motor neuronal loss in ALS, specifically in assessing response to therapies targeting neuroinflammatory pathways.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus the pathogenic importance of axonal transport remains to be fully resolved. We therefore analysed the in vivo dynamics of retrogradely transported, neurotrophin-containing signalling endosomes in nerve axons of two new ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43M337V mice, which show neuromuscular pathology without motor neuron loss, displayed perturbations in axonal transport manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in FusΔ14/+ mice. Axonal transport deficiencies are therefore not a common feature to all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.

TDP-43 pathology is a key feature of amyotrophic lateral sclerosis (ALS), but the mechanisms linking TDP-43 to altered cellular function and neurodegeneration remain unclear. We have recently described a mouse model in which human wild-type or mutant TDP-43 are expressed at low levels and where altered stress granule formation is a robust phenotype of TDP-43M337V/− expressing cells. In the present study we use this model to investigate the functional connectivity of human TDP-43 in primary motor neurons under resting conditions and in response to oxidative stress. The interactome of human TDP-43WT or TDP-43M337V was compared by mass spectrometry, and gene ontology enrichment analysis identified pathways dysregulated by the M337V mutation. We found that under normal conditions the interactome of human TDP-43WT was enriched for proteins involved in transcription, translation and poly(A)-RNA binding. In response to oxidative stress, TDP-43WT recruits proteins of the endoplasmic reticulum and endosomal-extracellular transport pathways, interactions which are reduced in the presence of the M337V mutation. Specifically, TDP-43M337V impaired protein-protein interactions involved in stress granule formation including reduced binding to the translation initiation factors Poly(A)-binding protein and Eif4a1 and the endoplasmic reticulum chaperone Grp78. The M337V mutation also affected interactions involved in endosomal-extracellular transport and this this was associated with reduced extracellular vesicle secretion in primary motor neurons from TDP-43M337V/− mice and in human iPSCs-derived motor neurons. Taken together, our analysis highlights a TDP-43 interaction network in motor neurons and demonstrates that an ALS associated mutation may alter the interactome to drive aberrant pathways involved in the pathogenesis of ALS.

Despite the intense research focus on the molecular and cellular mechanisms of motor neuron degeneration using models based on specific genetic mutations, there are good reasons to consider amyotrophic lateral sclerosis (ALS) as a ‘system degeneration’, which arises through multiple biological triggers. ALS experts generally agree that loss of function has a clinically focal onset and a non-random pattern of spread, which favours the ‘vulnerable network’ hypothesis, in which the degenerative process propagates through compartmentalised anatomical and functional networks.1 Furthermore, evidence from neuropathological studies suggests that the burden of TDP-43 proteinopathy reflects an orderly progression of spread through anatomically contiguous pathways.2 All of which raises the critical question of the nature of the anatomical substrate of …

Summary Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus their pathogenetic importance remains to be fully resolved. We therefore analyzed the in vivo dynamics of retrogradely transported, neurotrophin-containing signaling endosomes in nerve axons of two ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43M337V mice, which show neuromuscular pathology without motor neuron loss, display axonal transport perturbations manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in FusΔ14/+ mice. Deficiencies in retrograde axonal transport of signaling endosomes are therefore not shared by all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes., Graphical Abstract, Highlights • Mutant TDP-43 mice display in vivo defects in signaling endosome axonal transport • Despite motor neuron loss, mutant FUS mice display no such deficiency • Axonal transport disruption is thus not common to all ALS mouse models, Sleigh et al. address the importance of disturbances in axonal transport in two mouse models for ALS. They find that deficiencies in the in vivo axonal delivery of target tissue-derived survival factors are not a common feature of all ALS mouse models, suggesting mechanistic distinctions in different ALS-linked genes.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in the basic neuronal process of axonal transport have been identified in several ALS models. However, in vivo evidence of early/pre-symptomatic deficiencies in neuronal cargo trafficking remains limited, thus the pathogenic importance of axonal transport to the ALS disease spectrum remains to be fully resolved. We therefore analysed the in vivo dynamics of retrogradely transported, neurotrophin-containing signalling endosomes in motor neuron axons of two new mouse models of ALS that have mutations in different RNA processing genes (Tardbp and Fus). TDP-43M337V mice, which show neuromuscular pathology but no overt motor neuron loss, displayed in vivo perturbations in axonal transport that manifested between 1.5 and 3 months and preceded motor symptom onset. In contrast, signalling endosome transport remained largely unaffected in mutant FusΔ14/+ mice, despite 20% motor neuron loss. These findings indicate that deficiencies in retrograde neurotrophin signalling and axonal transport are not common to all ALS-linked genes, and that there are inherent and mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of ‘sporadic’ cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

Spinocerebellar ataxia type 14 (SCA14) is a subtype of the autosomal dominant cerebellar ataxias that is characterized by slowly progressive cerebellar dysfunction and neurodegeneration. SCA14 is caused by mutations in the PRKCG gene, encoding protein kinase C gamma (PKCγ). Despite the identification of 40 distinct disease-causing mutations in PRKCG, the pathological mechanisms underlying SCA14 remain poorly understood. Here we report the molecular neuropathology of SCA14 in post-mortem cerebellum and in human patient-derived induced pluripotent stem cells (iPSCs) carrying two distinct SCA14 mutations in the C1 domain of PKCγ, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKCγ in both patient iPSCs and cerebellum. PKCγ aggregates were not efficiently targeted for degradation. Moreover, mutant PKCγ was found to be hyper-activated, resulting in increased substrate phosphorylation. Together, our findings demonstrate that a combination of both, loss-of-function and gain-of-function mechanisms are likely to underlie the pathogenesis of SCA14, caused by mutations in the C1 domain of PKCγ. Importantly, SCA14 patient iPSCs were found to accurately recapitulate pathological features observed in post-mortem SCA14 cerebellum, underscoring their potential as relevant disease models and their promise as future drug discovery tools.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

© 2018 Elsevier Inc., To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca(2+) concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca(2+) dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca(2+) signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca(2+) stores content and delayed Ca(2+) release compared with cells carrying wt-TDP. The deficits in Ca(2+) release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca(2+) oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca(2+) signaling dysregulation.

This scientific commentary refers to ‘Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia’, by Vatsavayai et al. (doi:10.1093/brain/aww250) . The C9orf72 hexanucleotide repeat expansion mutation is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for up to 12% of all patients in an average ALS clinic population. At post-mortem, patients with the C9orf72 hexanucleotide repeat expansion have the typical pathology seen in sporadic ALS and FTD, in which transactive response DNA-binding protein of 43 kDa (TDP-43; encoded by TARDBP ) is translocated from its normal nuclear location to form cytoplasmic aggregates. However, they also show additional pathological features in the form of RNA foci, containing the transcribed hexanucleotide repeats, and aggregates, which stain with antibodies against dipeptide repeat proteins, the product of non-ATG translation. The order in which these distinct molecular abnormalities develop, and whether each of these pathological features is a necessary or sufficient condition for the development of clinical disease, are currently key questions in ALS/FTD research. In this issue of Brain , Vatsavayai et al. describe two strikingly different pathological cases of C9orf72 -positive FTD with excellent ante-mortem characterization that provide novel and provocative contributions to this debate (Vatsavayai et al. , 2016). Transcription of the C9orf72 hexanucleotide repeat expansion can be detected as sense and antisense RNA foci in the nucleus and cytoplasm. Despite being located in the non-coding region of the C9orf72 gene, the repeat sequence is translated through a non-canonical process called ‘repeat associated non-ATG’ (RAN) translation, into five different species of dipeptide …

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Patients with Charcot-Marie-Tooth disease with predominant axonal loss (CMT2) show extensive genetic heterogeneity. Benoy et al. demonstrate a link between CMT2 and histone deacetylase 6 (HDAC6), which controls the acetylation of α-tubulin, and propose that pharmacological inhibition of HDAC6 has therapeutic potential in CMT2 genetic variants., Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Polyglutamine expansions in the ataxin-2 gene (ATXN2) cause autosomal dominant spinocerebellar ataxia type 2 (SCA2), but have recently also been associated with amyotrophic lateral sclerosis (ALS). We present clinical and pathological features of a family in which a pathological ATXN2 expansion led to frontotemporal lobar degeneration with ALS (FTLD-ALS) in the index case, but typical SCA2 in a son, and compare the neuropathology with a case of typical SCA2. The index case shares the molecular signature of SCA2 with prominent polyglutamine and p62-positive intranuclear neuronal inclusions mainly in the pontine nuclei, while harbouring more pronounced neocortical and spinal TDP-43 pathology. We conclude that ATXN2 mutations can cause not only ALS, but also a neuropathological overlap syndrome of SCA2 and FTLD presenting clinically as pure FTLD-ALS without ataxia. The cause of the phenotypic heterogeneity remains unexplained, but the presence of a CAA-interrupted CAG repeat in the FTLD case in this family suggests that one potential mechanism may be variation in repeat tract composition between members of the same family.

Item does not contain fulltext Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

We describe a family with an autosomal dominant familial dyskinesia resembling myoclonus-dystonia associated with a novel missense mutation in ADCY5, found through whole-exome sequencing. A tiered analytical approach was used to analyse whole-exome sequencing data from an affected grandmother-granddaughter pair. Whole-exome sequencing identified 18,000 shared variants, of which 46 were non-synonymous changes not present in a local cohort of control exomes (n = 422). Further filtering based on predicted splicing effect, minor allele frequency in the 1000 Genomes Project and on phylogenetic conservation yielded 13 candidate variants, of which the heterozygous missense mutation c.3086T>G, p. M1029R in ADCY5 most closely matched the observed phenotype. This report illustrates the utility of whole-exome sequencing in cases of undiagnosed movement disorders with clear autosomal dominant inheritance. Moreover, ADCY5 mutations should be considered in cases with apparent myoclonus-dystonia, particularly where SCGE mutations have been excluded. ADCY5-related dyskinesia may manifest variable expressivity within a single family, and affected individuals may be initially diagnosed with differing neurological phenotypes. Electronic supplementary material The online version of this article (doi:10.1007/s10048-017-0510-z) contains supplementary material, which is available to authorized users.

Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene,GARS. The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system in CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted byGarsmutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behaviour concordant with the afferent imbalance, which is present at birth and non-progressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. This suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.Significance StatementCharcot-Marie-Tooth disease (CMT) is a collection of genetically diverse inherited nerve disorders with the unifying feature of peripheral neuron degeneration. The mechanisms triggering this motor and sensory nerve dysfunction remain unresolved, as does the reason for the lack of sensory pathology observed in distal hereditary motor neuropathies, which can be associated with CMT genes. To unravel the mechanisms leading to afferent deterioration, we have studied the sensory nervous system of CMT Type 2D mice. Our work indicates that the specific cellular identity of sensory nerves is perturbed in mutant mice pre-natally. CMT therefore manifests through the complex interplay between malfunctioning developmental, maturation, and survival programs, which has important ramifications for therapeutic timing.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for ligating amino acids to cognate tRNA molecules. Mutations in four genes encoding an ARS have been implicated in inherited peripheral neuropathy with an axonal pathology, suggesting that all ARS genes are relevant candidates for disease in patients with related phenotypes. Here, we present results from a mutation screen of the histidyl-tRNA synthetase (HARS) gene in a large cohort of patients with peripheral neuropathy. These efforts revealed a rare missense variant (c.410G>A/p.Arg137Gln) that resides at a highly conserved amino acid, represents a loss-of-function allele when evaluated in yeast complementation assays, and is toxic to neurons when expressed in a worm model. In addition to the patient with peripheral neuropathy, p.Arg137Gln HARS was detected in three individuals by genome-wide exome sequencing. These findings suggest that HARS is the fifth ARS locus associated with axonal peripheral neuropathy. Implications for identifying ARS alleles in human populations and assessing them for a role in neurodegenerative phenotypes are discussed. © 2012 Wiley Periodicals, Inc.

Dominant mutations in GARS, encoding the essential enzyme glycyl-tRNA synthetase (GlyRS), result in a form of Charcot-Marie-Tooth disease, type 2D (CMT2D), predominantly characterized by lower motor nerve degeneration. GlyRS charges the amino acid glycine with its cognate tRNA and is therefore essential for protein translation. However, the underlying mechanisms linking toxic gain-of-function GARS mutations to lower motor neuron degeneration remain unidentified. The neuromuscular junction (NMJ) appears to be an early target for pathology in a number of peripheral nerve diseases and becomes denervated at later stages in two mouse models of CMT2D. We therefore performed a detailed longitudinal examination of NMJs in the distal lumbrical muscles and the proximal transversus abdominis (TVA) muscles of wild-type and Gars mutant mice. We determined that mutant lumbrical NMJs display a persistent defect in maturation that precedes a progressive, age-dependent degeneration. Conversely, the TVA remains relatively unaffected, with only a subtle, short-lived impairment in pre- and post-synaptic development and no reduction in lower motor neuron connectivity to muscle. Together, these observations suggest that mutant Gars is associated with compromised development of the NMJ prior to synaptic degeneration and highlight the neuromuscular synapse as an important site of early, selective pathology in CMT2D mice.

Mutant superoxide dismutase 1 (SOD1) action within non-neuronal cells is implicated in damage to spinal motor neurons in a genetic form of amyotrophic lateral sclerosis (ALS). Central nervous system glial cells such as astrocytes and microglia drive progression in transgenic mutant SOD1 mice, however, the role of myelinating glia remains unclear. Specifically, peripheral myelinating glial cells are likely candidates for mediating degeneration of distal synapses and axons of motor neurons in ALS. Here, we examine the potential contribution of peripheral axon ensheathing Schwann cells to ALS by constructing transgenic mice expressing dismutase active mutant SOD1(G93A) driven by the myelin protein zero (P0) promoter. In this model, mutant SOD1 accumulation in Schwann cells was comparable to levels in mice ubiquitously expressing a SOD1(G93A) transgene that become paralysed. Growth, locomotion and survival of these P0-SOD1(G93A) mice were indistinguishable from normal animals. There was no evidence for spinal motor neuron loss, distal axonal degeneration and p75 neurotrophin receptor (p75(NTR)) upregulation in the periphery of P0-SOD1(G93A) mice, unlike transgenic SOD1(G93A) mice with presymptomatic p75(NTR) induction and death-signalling. Furthermore, Schwann cells were resistant to mutant SOD1 aggregation in vivo and in transfected primary cultures. Increasing mutant SOD1 synthesis in Schwann cells by cross-breeding transgenic P0-SOD1(G93A) and SOD1(G93A) mice did not affect disease onset or survival. We conclude that dismutase-competent mutant SOD1 accumulation within Schwann cells is not pathological to spinal motor neurons or deleterious to disease course in transgenic ALS model mice, in contrast to astrocytes and microglia.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

Charcot–Marie–Tooth disease type 2D, a hereditary axonal neuropathy, is caused by mutations in glycyl–tRNA synthetase (GARS). The mutations are distributed throughout the protein in multiple functional domains. In biochemical and cell culture experiments, some mutant forms of GARS have been indistinguishable from wild-type protein, suggesting that these in vitro tests might not adequately assess the aberrant activity responsible for axonal degeneration. Recently, mouse and fly models have offered new insights into the disease mechanism. There are still gaps in our understanding of how mutations in a ubiquitously expressed component of the translation machinery result in axonal neuropathy. Here, we review recent reports, weigh the evidence for and against possible mechanisms and suggest areas of focus for future work.

We report two novel mutations in three cases of spinal muscular atrophy (SMA), including two distant cousins who followed an unexpectedly severe course. Diagnosis was confirmed by reduced SMN protein and full-length SMN mRNA levels. Sequencing of the non-deleted SMN1 gene revealed a single G insertion at the end of exon 1 in the two cousins and a novel G275S exon 6 missense mutation in the milder case.

Low levels of full-length survival motor neuron (SMN) protein cause the motor neuron disease, spinal muscular atrophy (SMA). Although motor neurons undoubtedly contribute directly to SMA pathogenesis, the role of muscle is less clear. We demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic severe SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomic data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. Robust upregulation of voltage-dependent anion-selective channel protein 2 (Vdac2) and downregulation of parvalbumin in severe SMA mice was confirmed in a milder SMA mouse model and in human patient muscle biopsies. Molecular pathology of skeletal muscle was ameliorated in mice treated with the FDA-approved histone deacetylase inhibitor, suberoylanilide hydroxamic acid. We conclude that intrinsic pathology of skeletal muscle is an important and reversible event in SMA and also suggest that muscle proteins have the potential to act as novel biomarkers in SMA.

Mutations in the ubiquitously expressed survival motor neuron 1 (SMN1) and superoxide dismutase 1 (SOD1) genes are selectively lethal to motor neurons in spinal muscular atrophy (SMA) and familial amyotrophic lateral sclerosis (ALS), respectively. Genetic association studies provide compelling evidence that SMN1 and SMN2 genotypes encoding lower SMN protein levels are implicated in sporadic ALS, suggesting that SMN expression is a potential determinant of ALS severity. We therefore sought genetic evidence of SMN involvement in ALS by generating transgenic mutant SOD1 mice on an Smn deficient background. Partial genetic disruption of Smn significantly worsened motor performance and survival in transgenic SOD1G93A mice. Furthermore, ALS-linked mutant SOD1 expression severely reduced SMN protein levels, but not transcript, in neuronal culture and mouse models from early presymptomatic disease. SMN protein depletion was linked to the nuclear compartment and a physical interaction between SMN and mutant SOD1 was confirmed in mouse spinal cord. Treatment with the environmental toxin paraquat also depleted SMN protein, implicating oxidative stress in the mechanism underlying SMN deficiency in familial ALS and potentially sporadic disease. In contrast, transgenic SOD1WT overexpression in SMA type I mice was incapable of modulating SMN protein levels or disease progression. These data establish that SMN deficiency accelerates phenotypic severity in transgenic familial ALS mice, consistent with an enhancing genetic modifier role. We therefore propose that SMN replacement and upregulation strategies considered for SMA therapy may have protective potential for ALS.

Reliable markers of diagnosis, disease activity and progression in neurodegenerative diseases are a research priority in the improvement of diagnosis and the development of effective disease-modifying therapies. The fact that neurodegenerative pathology is primarily associated with distinct subsets of cells in discrete areas of the CNS makes the identification of relevant biomarker molecules a challenge. The trafficking of macromolecules from the CNS to the cerebrospinal fluid and blood mediated by extracellular vesicles (EVs) presents a promising source of CNS-specific biomarkers. EVs are released by almost all cell types and carry a cargo of protein and nucleic acid that varies according to the cell of origin. EV output changes according to cell status and reflects intracellular events, so surface marker expression can be used to identify the cell type from which EVs originate. EVs could, therefore, provide an enriched pool of information about core neuropathogenic, cell-specific processes. This Review examines the current knowledge of the biology and function of EVs, discusses the evidence for their involvement in the pathogenesis of neurodegenerative diseases, and considers their potential as biomarkers of disease.

Contains fulltext : 167923.pdf (Publisher’s version ) (Open Access) Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.

An elderly female developed anarthria with prominent emotionality over an 18-month period before specialist neurological assessment. Although tongue electromyography was normal, her corticobulbar signs were consistent with amyotrophic lateral sclerosis (ALS), a pattern that, in the absence of ­functional impairment outside of speech and swallowing, is appropriately termed progressive bulbar palsy. Such patients, often elderly females, may remain ambulant and independent for many months, sometimes years, despite typically rapid anarthria.1 Electromyography may be insensitive to denervation, even when genioglossus is sampled, and this can contribute to diagnostic …

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07521.x Abstract HspB8, a small heat-shock protein implicated in autophagy, is mutated in patients with distal hereditary motor neuropathy type II (dHMNII). Autophagy is essential for maintaining protein homeostasis in the central nervous system, but its role has not been investigated in peripheral motor neurons. We used a novel, multispectral-imaging flow cytometry assay to measure autophagy in cells. This assay revealed that over-expression of wild-type HspB8 in motor neuron-like NSC34 cells led to an increased co-localisation of autophagosomes with the lysosomes. By contrast, over-expression of mutant HspB8 resulted in autophagosomes that co-localised with protein aggregates but failed to co-localise with the lysosomes. A similar impairment of autophagy could also be demonstrated in peripheral blood mononuclear cells from two dHMNII patients with the HspB8K141E mutation. We conclude that defects in HspB8-mediated autophagy are likely to contribute to dHMNII pathology and their detection in peripheral blood mononuclear cells could be a useful, accessible biomarker for the disease.

Spinal muscular atrophy (SMA), which is caused by inactivating mutations in the survival motor neuron 1 (SMN1) gene, is characterized by loss of lower motor neurons in the spinal cord. The gene encoding SMN is very highly conserved in evolution, allowing the disease to be modeled in a range of species. The similarities in anatomy and physiology to the human neuromuscular system, coupled with the ease of genetic manipulation, make the mouse the most suitable model for exploring the basic pathogenesis of motor neuron loss and for testing potential treatments. Therapies that increase SMN levels, either through direct viral delivery or by enhancing full-length SMN protein expression from the SMN1 paralog, SMN2, are approaching the translational stage of development. It is therefore timely to consider the role of mouse models in addressing aspects of disease pathogenesis that are most relevant to SMA therapy. Here, we review evidence suggesting that the apparent selective vulnerability of motor neurons to SMN deficiency is relative rather than absolute, signifying that therapies will need to be delivered systemically. We also consider evidence from mouse models suggesting that SMN has its predominant action on the neuromuscular system in early postnatal life, during a discrete phase of development. Data from these experiments suggest that the timing of therapy to increase SMN levels might be crucial. The extent to which SMN is required for the maintenance of motor neurons in later life and whether augmenting its levels could treat degenerative motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), requires further exploration.

Reduced expression of the survival motor neuron (SMN) gene causes the childhood motor neuron disease spinal muscular atrophy (SMA). Low levels of ubiquitously expressed SMN protein result in the degeneration of lower motor neurons, but it remains unclear whether other regions of the nervous system are also affected. Here we show that reduced levels of SMN lead to impaired perinatal brain development in a mouse model of severe SMA. Regionally selective changes in brain morphology were apparent in areas normally associated with higher SMN levels in the healthy postnatal brain, including the hippocampus, and were associated with decreased cell density, reduced cell proliferation and impaired hippocampal neurogenesis. A comparative proteomics analysis of the hippocampus from SMA and wild-type littermate mice revealed widespread modifications in expression levels of proteins regulating cellular proliferation, migration and development when SMN levels were reduced. This study reveals novel roles for SMN protein in brain development and maintenance and provides the first insights into cellular and molecular pathways disrupted in the brain in a severe form of SMA.

Autosomal recessive hereditary spastic paraplegia with thin corpus callosum (HSP-TCC) maps to the SPG11 locus in the majority of cases. Mutations in the KIAA1840 gene, encoding spatacsin, have been shown to underlie SPG11-linked HSP-TCC. The aim of this study was to perform candidate gene analysis in HSP-TCC subjects from Asian families and to characterize disruption of spatacsin function during zebrafish development. Homozygosity mapping and direct sequencing were used to assess the ACCPN, SPG11, and SPG21 loci in four inbred kindreds originating from the Indian subcontinent. Four novel homozygous SPG11 mutations (c.442+1G>A, c.2146C>T, c.3602_3603delAT, and c.4846C>T) were identified, predicting a loss of spatacsin function in each case. To investigate the role of spatacsin during development, we additionally ascertained the complete zebrafish spg11 ortholog by reverse transcriptase PCR and 5′ RACE. Analysis of transcript expression through whole-mount in situ hybridization demonstrated ubiquitous distribution, with highest levels detected in the brain. Morpholino antisense oligonucleotide injection was used to knock down spatacsin function in zebrafish embryos. Examination of spg11 morphant embryos revealed a range of developmental defects and CNS abnormalities, and analysis of axon pathway formation demonstrated an overall perturbation of neuronal differentiation. These data confirm loss of spatacsin as the cause of SPG11-linked HSP-TCC in Asian kindreds, expanding the mutation spectrum recognized in this disorder. This study represents the first investigation in zebrafish addressing the function of a causative gene in autosomal recessive HSP and identifies a critical role for spatacsin during early neural development in vivo. Electronic supplementary material The online version of this article (doi:10.1007/s10048-010-0243-8) contains supplementary material, which is available to authorized users.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized clinically by rapidly progressive paralysis leading ultimately to death from respiratory failure. There is substantial evidence suggesting that ALS is a heritable disease, and a number of genes have been identified as being causative in familial ALS. In contrast, the genetics of the much commoner sporadic form of the disease is poorly understood and no single gene has been definitively shown to increase the risk of developing ALS. In this review, we discuss the genetic evidence for each candidate gene that has been putatively associated with increased risk of sporadic ALS. We also review whole genome association studies of ALS and discuss the potential of this methodology for identifying genes relevant to motor neuron degeneration.

Murray Valley encephalitis (MVE) virus, a mosquito-borne flavivirus, is the most common cause of viral encephalitis in the tropical 'Top End' of northern Australia. Clinical encephalitis due to MVE virus has a mortality rate of approximately 30%, with a similar proportion of patients being left with significant neurological deficits. We report the case of a 25-year-old man from the UK who acquired MVE while travelling through northern Australia. He required prolonged admission to the Intensive Care Unit and several years later remains partly ventilator-dependent, with flaccid quadriparesis. To our knowledge, this is the first reported case of MVE virus-induced flaccid paralysis in an adult in northern Australia, although it is well described in children. Paralysis was thought to be due to anterior horn cell involvement in the spinal cord and extensive bilateral thalamic destruction, both of which are well recognised complications of infection with MVE virus. Cases of flaccid paralysis with similar pathology have been described following infection with the related flavivirus Japanese encephalitis virus as well as more recently with West Nile virus. Our case highlights the potential severity of flavivirus-induced encephalitis and the importance of avoiding mosquito bites while travelling through endemic areas.