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GGGGCC repeat expansion in C9ORF72 , which can be translated in both sense and antisense directions into five dipeptide repeat (DPR) proteins, including poly(GP), poly(GR), and poly(GA), is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we developed sensitive assays that can detect poly(GA) and poly(GR) in the cerebrospinal fluid (CSF) of patients with C9ORF72 mutations. CSF poly(GA) and poly(GR) levels did not correlate with age at disease onset, disease duration, or rate of decline of ALS Functional Rating Scale, and the average levels of these DPR proteins were similar in symptomatic and pre-symptomatic patients with C9ORF72 mutations. However, in a patient with C9ORF72 -ALS who was treated with antisense oligonucleotide (ASO) targeting the aberrant C9ORF72 transcript, CSF poly(GA) and poly(GR) levels decreased approximately 50% within 6 weeks, indicating they may serve as sensitive fluid-based biomarkers in studies directed against the production of GGGGCC repeat RNAs or DPR proteins. The GGGGCC repeat expansion in C9ORF72 -ALS/FTD can be translated into five dipeptide repeat (DPR) proteins, including poly(GR) and poly(GA). Here, the authors develop assays to detect the levels of these DPR proteins in the CSF of individuals with ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that causes paralysis and death. TDP-43 is a protein that regulates gene expression. TDP-43 aggregation and depletion from cell nucleus are found in ALS. Therefore, TDP-43 may cause neurodegeneration by generating toxicity from its aggregation or by a loss of its function. Our experiments test the consequence of a partial loss of TDP-43 function in mice. The results demonstrate that a partial loss of TDP-43 function is sufficient to cause neurodegeneration and ALS symptoms. In addition, we have found evidence for TDP-43 dysfunction in human ALS. Therefore, we propose that TDP-43 dysfunction causes neurodegeneration in the human disease, and future therapy should aim to restore the normal function of TDP-43. Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease that causes motor neuron degeneration, progressive motor dysfunction, paralysis, and death. Although multiple causes have been identified for this disease, >95% of ALS cases show aggregation of transactive response DNA binding protein (TDP-43) accompanied by its nuclear depletion. Therefore, the TDP-43 pathology may be a converging point in the pathogenesis that originates from various initial triggers. The aggregation is thought to result from TDP-43 misfolding, which could generate cellular toxicity. However, the aggregation as well as the nuclear depletion could also lead to a partial loss of TDP-43 function or TDP-43 dysfunction. To investigate the impact of TDP-43 dysfunction, we generated a transgenic mouse model for a partial loss of TDP-43 function using transgenic RNAi. These mice show ubiquitous transgene expression and TDP-43 knockdown in both the periphery and the central nervous system (CNS). Strikingly, these mice develop progressive neurodegeneration prominently in cortical layer V and spinal ventral horn, motor dysfunction, paralysis, and death. Furthermore, examination of splicing patterns of TDP-43 target genes in human ALS revealed changes consistent with TDP-43 dysfunction. These results suggest that the CNS, particularly motor neurons, possess a heightened vulnerability to TDP-43 dysfunction. Additionally, because TDP-43 knockdown predominantly occur in astrocytes in the spinal cord of these mice, our results suggest that TDP-43 dysfunction in astrocytes is an important driver for motor neuron degeneration and clinical phenotypes of ALS.

Two patients with amyotrophic lateral sclerosis caused by SOD1 mutations received an intrathecal infusion of adeno-associated virus containing microRNA targeting SOD1 . SOD1 protein levels did not change in the patients’ cerebrospinal fluid, but one patient had reduced spinal cord SOD1 expression.

Expansions of a G 4 C 2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G 4 C 2 repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the G 4 C 2 repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations. An antisense oligonucleotide targets mutant transcripts of the ALS gene C9orf72, suppressing the poly(GP) dipeptide in tissues in mice and in the spinal fluid of a single patient harboring the C9orf72 gene mutation

Could similar changes in SOD1 underlie both familial and sporadic ALS? Here, Bosco et al . find that wild-type SOD1 from sporadic ALS tissues shows conformational changes similar to those seen in familial ALS and that aberrant wild-type SOD1 can be pathogenic, potentially as a result of the same SOD1-dependent mechanism seen in familial ALS. Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.

Objective Dysferlin is a large transmembrane protein that functions in critical processes of membrane repair and vesicle fusion. Dysferlin‐deficiency due to mutations in the dysferlin gene leads to muscular dystrophy (Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD2B), distal myopathy with anterior tibial onset (DMAT)), typically with early adult onset. At least 416 pathogenic dysferlin mutations are known, but for approximately 17% of patients, one or both of their pathogenic variants remain undefined following standard exon sequencing methods that interrogate exons and nearby flanking intronic regions but not the majority of intronic regions. Methods We sequenced RNA from myogenic cells to identify a novel dysferlin pathogenic variant in two affected siblings that previously had only one disease‐causing variant identified. We designed antisense oligonucleotides (AONs) to bypass the effects of this mutation on RNA splicing. Results We identified a new pathogenic point mutation deep within dysferlin intron 50i. This intronic variant causes aberrant mRNA splicing and inclusion of an additional pseudoexon (PE, we term PE50.1) within the mature dysferlin mRNA. PE50.1 inclusion alters the protein sequence, causing premature translation termination. We identified this mutation in 23 dysferlinopathy patients (seventeen families), revealing it to be one of the more prevalent dysferlin mutations. We used AON‐mediated exon skipping to correct the aberrant PE50.1 splicing events in vitro, which increased normal mRNA production and significantly restored dysferlin protein expression. Interpretation Deep intronic mutations can be a common underlying cause of dysferlinopathy, and importantly, could be treatable with AON‐based exon‐skipping strategies.

Objective Mutations in dysferlin (DYSF), a Ca2+‐sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T‐tubule function, cause Miyoshi myopathy, limb‐girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon–intron junctions. In ~17% of patients who lack normal DYSF, only a single disease‐causing mutation has been identified. We studied one family with one known mutant allele to identify both the second underlying genetic defect and potential therapeutic approaches. Methods We sequenced the full DYSF cDNA and investigated antisense oligonucleotides (AONs) as a tool to modify splicing of the mRNA transcripts in order to process out mutant sequences. Results We identified a novel pseudoexon between exons 44 and 45, (pseudoexon 44.1, PE44.1), which inserts an additional 177 nucleotides into the mRNA and 59 amino acids within the conserved C2F domain of the DYSF protein. Two unrelated dysferlinopathy patients were also found to carry this mutation. Using AONs targeting PE44.1, we blocked the abnormal splicing event, yielding normal, full‐length DYSF mRNA, and increased DYSF protein expression. Interpretation This is the first report of a deep intronic mutation in DYSF that alters mRNA splicing to include a mutant peptide fragment within a key DYSF domain. We report that AON‐mediated exon‐skipping restores production of normal, full‐length DYSF in patients' cells in vitro, offering hope that this approach will be therapeutic in this genetic context, and providing a foundation for AON therapeutics targeting other pathogenic DYSF alleles.

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10⁻⁸) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

A MYOTROPHIC lateral sclerosis, often referred to as Lou Gehrig's disease, motor neuron disease, or Charcot's syndrome, is a devastating paralytic disorder with onset in adulthood, caused by degeneration of large motor neurons of the brain and spinal cord. 1 It causes generalized and progressive wasting and weakness of skeletal muscles 2 and usually results in death within five years. 2 Currently there is no treatment to prevent this disease or to alter its unremitting course. Its annual incidence is similar to that of multiple sclerosis and more than five times that of Huntington's disease. 3 Since the average duration of life after the . . .