PFN1 phosphorylation marks protein aggregation and white matter pathology in ALS

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is the most common form of motor neuron disease. In familial ALS, Multiple mutations of, PFN1 gene a well-known actin-binding protein have been linked to ALS disease recently. Phosphorylation in many degenerative conditions plays an important role in disease mechanism but its potential role in ALS remains not fully understood. We sought to look further into not previously studied phosphorylation of PFN1 as a potential contributor to aggregation and toxicity in ALS. Using different histochemistry and cytochemistry and molecular biology approaches, we observed that phosphorylation on Profilin shows a very distinctive pattern in PFN1C71G andSOD1G93A disease models. This modification is abundantly found in both astrocytes and white matter which latter indeed marks a staining pattern that is indistinguishable between two ALS mice model compared to controls. Interestingly, pPFN1 reactive areas colocalized with Myelin in the spinal cord are frequently found in the proximity of CD68 positive macrophages. Moreover, biochemical fractionation using ultracentrifugation detects endogenous pPFN1 in the highly insoluble fraction of protein lysate from both PFN1C71G andSOD1G93A model. Finally, a similar staining pattern to the ALS mice model was also observed in human sporadic ALS cases. Overall, our results suggest for the first time a role for phosphorylation of PFN1 in protein aggregation and white matter pathology in ALS that will shed more light on the mechanism of disease and developing potential therapeutics in near future

Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis.

Oligodendrocytes associate with axons to establish myelin and provide metabolic support to neurons. In the spinal cord of amyotrophic lateral sclerosis (ALS) mice, oligodendrocytes downregulate transporters that transfer glycolytic substrates to neurons and oligodendrocyte progenitors (NG2(+) cells) exhibit enhanced proliferation and differentiation, although the cause of these changes in oligodendroglia is unknown. We found extensive degeneration of gray matter oligodendrocytes in the spinal cord of SOD1 (G93A) ALS mice prior to disease onset. Although new oligodendrocytes were formed, they failed to mature, resulting in progressive demyelination. Oligodendrocyte dysfunction was also prevalent in human ALS, as gray matter demyelination and reactive changes in NG2(+) cells were observed in motor cortex and spinal cord of ALS patients. Selective removal of mutant SOD1 from oligodendroglia substantially delayed disease onset and prolonged survival in ALS mice, suggesting that ALS-linked genes enhance the vulnerability of motor neurons and accelerate disease by directly impairing the function of oligodendrocytes

NOS1AP is a novel molecular target and critical factor in TDP-43 pathology

Cappelli et al. reported that Nitric Oxide Synthase 1 Adaptor Protein is a co-regulated transcript of the TAR DNA-binding protein 43 kDa, reduced in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients with TAR DNA-binding protein 43 kDa pathology. Overall, their results highlight Nitric Oxide Synthase 1 Adaptor Protein as a novel druggable disease-relevant gene in TAR DNA-binding protein 43 kDa-related proteinopathies.Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies

Rare variant analyses validate known ALS genes in a multi-ethnic population and identifies ANTXR2 as a candidate in PLS

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting over 300,000 people worldwide. It is characterized by the progressive decline of the nervous system that leads to the weakening of muscles which impacts physical function. Approximately, 15% of individuals diagnosed with ALS have a known genetic variant that contributes to their disease. As therapies that slow or prevent symptoms continue to develop, such as antisense oligonucleotides, it is important to discover novel genes that could be targets for treatment. Additionally, as cohorts continue to grow, performing analyses in ALS subtypes, such as primary lateral sclerosis (PLS), becomes possible due to an increase in power. These analyses could highlight novel pathways in disease manifestation.MethodsBuilding on our previous discoveries using rare variant association analyses, we conducted rare variant burden testing on a substantially larger multi-ethnic cohort of 6,970 ALS patients, 166 PLS patients, and 22,524 controls. We used intolerant domain percentiles based on sub-region Residual Variation Intolerance Score (subRVIS) that have been described previously in conjunction with gene based collapsing approaches to conduct burden testing to identify genes that associate with ALS and PLS.ResultsA gene based collapsing model showed significant associations with SOD1, TARDBP, and TBK1 (OR = 19.18, p = 3.67 × 10–39; OR = 4.73, p = 2 × 10–10; OR = 2.3, p = 7.49 × 10–9, respectively). These genes have been previously associated with ALS. Additionally, a significant novel control enriched gene, ALKBH3 (p = 4.88 × 10–7), was protective for ALS in this model. An intolerant domain-based collapsing model showed a significant improvement in identifying regions in TARDBP that associated with ALS (OR = 10.08, p = 3.62 × 10–16). Our PLS protein truncating variant collapsing analysis demonstrated significant case enrichment in ANTXR2 (p = 8.38 × 10–6).ConclusionsIn a large multi-ethnic cohort of 6,970 ALS patients, collapsing analyses validated known ALS genes and identified a novel potentially protective gene, ALKBH3. A first-ever analysis in 166 patients with PLS found a candidate association with loss-of-function mutations in ANTXR2

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

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.Peer reviewe

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p

Transcriptional profiling of HERV-K(HML-2) in amyotrophic lateral sclerosis and potential implications for expression of HML-2 proteins

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. About 90% of ALS cases are without a known genetic cause. The human endogenous retrovirus multi-copy HERV-K(HML-2) group was recently reported to potentially contribute to neurodegeneration and disease pathogenesis in ALS because of transcriptional upregulation and toxic effects of HML-2 Envelope (Env) protein. Env and other proteins are encoded by some transcriptionally active HML-2 loci. However, more detailed information is required regarding which HML-2 loci are transcribed in ALS, which of their proteins are expressed, and differences between the disease and non-disease states. Methods For brain and spinal cord tissue samples from ALS patients and controls, we identified transcribed HML-2 loci by generating and mapping HML-2-specific cDNA sequences. We predicted expression of HML-2 env gene-derived proteins based on the observed cDNA sequences. Furthermore, we determined overall HML-2 transcript levels by RT-qPCR and investigated presence of HML-2 Env protein in ALS and control tissue samples by Western blotting. Results We identified 24 different transcribed HML-2 loci. Some of those loci are transcribed at relatively high levels. However, significant differences in HML-2 loci transcriptional activities were not seen when comparing ALS and controls. Likewise, overall HML-2 transcript levels, as determined by RT-qPCR, were not significantly different between ALS and controls. Indeed, we were unable to detect full-length HML-2 Env protein in ALS and control tissue samples despite reasonable sensitivity. Rather our analyses suggest that a number of HML-2 protein variants other than full-length Env may potentially be expressed in ALS patients. Conclusions Our results expand and refine recent publications on HERV-K(HML-2) and ALS. Some of our results are in conflict with recent findings and call for further specific analyses. Our profiling of HML-2 transcription in ALS opens up the possibility that HML-2 proteins other than canonical full-length Env may have to be considered when studying the role of HML-2 in ALS disease

Genome-wide structural variant analysis identifies risk loci for non-Alzheimer’s dementias

We characterized the role of structural variants, a largely unexplored type of genetic variation, in two non-Alzheimer’s dementias, namely Lewy body dementia (LBD) and frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS). To do this, we applied an advanced structural variant calling pipeline (GATK-SV) to short-read whole-genome sequence data from 5,213 European-ancestry cases and 4,132 controls. We discovered, replicated, and validated a deletion in TPCN1 as a novel risk locus for LBD and detected the known structural variants at the C9orf72 and MAPT loci as associated with FTD/ALS. We also identified rare pathogenic structural variants in both LBD and FTD/ALS. Finally, we assembled a catalog of structural variants that can be mined for new insights into the pathogenesis of these understudied forms of dementia

Deficient Sarcolemma Repair in ALS: A Novel Mechanism with Therapeutic Potential

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS

Biomarker Qualification for Neurofilament Light Chain in Amyotrophic Lateral Sclerosis: Theory and Practice

OBJECTIVE: To explore whether the utility of neurofilament light chain (NfL), as a biomarker to aid amyotrophic lateral sclerosis (ALS) therapy development, would be enhanced by obtaining formal qualification from the US Food and Drug Administration for a defined context-of-use. METHODS: Consensus discussion among academic, industry, and patient advocacy group representatives. RESULTS: A wealth of scientific evidence supports the use of NfL as a prognostic, response, and potential safety biomarker in the broad ALS population, and as a risk/susceptibility biomarker among the subset of SOD1 pathogenic variant carriers. Although NfL has not yet been formally qualified for any of these contexts-of-use, the US Food and Drug Administration has provided accelerated approval for an SOD1-lowering antisense oligonucleotide, based partially on the recognition that a reduction in NfL is reasonably likely to predict a clinical benefit. INTERPRETATION: The increasing incorporation of NfL into ALS therapy development plans provides evidence that its utility-as a prognostic, response, risk/susceptibility, and/or safety biomarker-is already widely accepted by the community. The willingness of the US Food and Drug Administration to base regulatory decisions on rigorous peer-reviewed data-absent formal qualification, leads us to conclude that formal qualification, despite some benefits, is not essential for ongoing and future use of NfL as a tool to aid ALS therapy development. Although the balance of considerations for and against seeking NfL biomarker qualification will undoubtedly vary across different diseases and contexts-of-use, the robustness of the published data and careful deliberations of the ALS community may offer valuable insights for other disease communities grappling with the same issues. ANN NEUROL 2023