The Molecular Link Between TDP-43, Endogenous Retroviruses and Inflammatory Neurodegeneration in Amyotrophic Lateral Sclerosis: a Potential Target for Triumeq, an Antiretroviral Therapy.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a progressive neurological disorder, characterised by the death of upper and lower motor neurons. The aetiology of ALS remains unknown, and treatment options are limited. Endogenous retroviruses (ERVs), specifically human endogenous retrovirus type K (HERV-K), have been proposed to be involved in the propagation of neurodegeneration in ALS. ERVs are genomic remnants of ancient viral infection events, with most being inactive and not retaining the capacity to encode a fully infectious virus. However, some ERVs retain the ability to be activated and transcribed, and ERV transcripts have been found to be elevated within the brain tissue of MND patients. A hallmark of ALS pathology is altered localisation of the transactive response (TAR) DNA binding protein 43 kDa (TDP-43), which is normally found within the nucleus of neuronal and glial cells and is involved in RNA regulation. In ALS, TDP-43 aggregates within the cytoplasm and facilitates neurodegeneration. The involvement of ERVs in ALS pathology is thought to occur through TDP-43 and neuroinflammatory mediators. In this review, the proposed involvement of TDP-43, HERV-K and immune regulators on the onset and progression of ALS will be discussed. Furthermore, the evidence supporting a therapy based on targeting ERVs in ALS will be reviewed.

Intronic NEFH variant is associated with reduced risk for sporadic ALS and later age of disease onset.

Neurofilament heavy (NEFH) is one of the critical proteins required for the formation of the neuronal cytoskeleton and polymorphisms in NEFH are reported as a rare cause of sporadic ALS (sALS). In the current study, a candidate tetranucleotide (TTTA) repeat variant in NEFH was selected using an in-silico short structural variant (SSV) evaluation algorithm and investigated in two cohorts of North American sALS patients, both separately and combined (Duke cohort n = 138, Coriell cohort n = 333; combined cohort n = 471), compared to a group of healthy controls from the Coriell Institute biobank (n = 496). Stratification according to site of disease onset revealed that the 9 TTTA allele was associated with reduced disease risk, specifically confined to spinal-onset sALS patients in the Duke cohort (p = 0.001). Furthermore, carriage of the 10 TTTA allele was associated with a 2.7 year later age of disease onset in the larger combined sALS cohort (p = 0.02). These results suggest that the 9 and 10 TTTA motif length may have a protective advantage for potentially lowering the risk of sALS and delaying the age of disease onset, however, these results need to be replicated in larger multicenter and multi-ethnic cohorts.

Synucleinopathy in Amyotrophic Lateral Sclerosis: A Potential Avenue for Antisense Therapeutics?

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease classified as both a neurodegenerative and neuromuscular disorder. With a complex aetiology and no current cure for ALS, broadening the understanding of disease pathology and therapeutic avenues is required to progress with patient care. Alpha-synuclein (αSyn) is a hallmark for disease in neurodegenerative disorders, such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. A growing body of evidence now suggests that αSyn may also play a pathological role in ALS, with αSyn-positive Lewy bodies co-aggregating alongside known ALS pathogenic proteins, such as SOD1 and TDP-43. This review endeavours to capture the scope of literature regarding the aetiology and development of ALS and its commonalities with "synucleinopathy disorders". We will discuss the involvement of αSyn in ALS and motor neuron disease pathology, and the current theories and strategies for therapeutics in ALS treatment, as well as those targeting αSyn for synucleinopathies, with a core focus on small molecule RNA technologies.

Short structural variants as informative genetic markers for ALS disease risk and progression.

There is considerable variability in disease progression for patients with amyotrophic lateral sclerosis (ALS) including the age of disease onset, site of disease onset, and survival time. There is growing evidence that short structural variations (SSVs) residing in frequently overlooked genomic regions can contribute to complex disease mechanisms and can explain, in part, the phenotypic variability in ALS patients. Here, we discuss SSVs recently characterized by our laboratory and how these discoveries integrate into the current literature on ALS, particularly in the context of application to future clinical trials. These markers may help to identify and differentiate patients for clinical trials that have a similar ALS disease mechanism(s), thereby reducing the impact of participant heterogeneity. As evidence accumulates for the genetic markers discovered in SQSTM1, SCAF4, and STMN2, we hope to improve the outcomes of future ALS clinical trials.

New perspectives on cytoskeletal dysregulation and mitochondrial mislocalization in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington's disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.

Novel STMN2 Variant Linked to Amyotrophic Lateral Sclerosis Risk and Clinical Phenotype.

OBJECTIVE: There is a critical need to establish genetic markers that explain the complex phenotypes and pathogenicity of ALS. This study identified a polymorphism in the Stathmin-2 gene and investigated its association with sporadic ALS (sALS) disease risk, age-of onset and survival duration. METHODS: The candidate CA repeat was systematically analyzed using PCR, Sanger sequencing and high throughput capillary separation for genotyping. Stathmin-2 expression was investigated using RT-PCR in patient olfactory neurosphere-derived (ONS) cells and RNA sequencing in laser-captured spinal motor neurons. RESULTS: In a case-control analysis of a combined North American sALS cohort (n = 321) and population control group (n = 332), long/long CA genotypes were significantly associated with disease risk (p = 0.042), and most strongly when one allele was a 24 CA repeat (p = 0.0023). In addition, longer CA allele length was associated with earlier age-of-onset (p = 0.039), and shorter survival duration in bulbar-onset cases (p = 0.006). In an Australian longitudinal sALS cohort (n = 67), ALS functional rating scale scores were significantly lower in carriers of the long/long genotype (p = 0.034). Stathmin-2 mRNA expression was reduced in sporadic patient ONS cells. Additionally, sALS patients and controls exhibited variable expression of Stathmin-2 mRNA according to CA genotype in laser-captured spinal motor neurons. CONCLUSIONS: We report a novel non-coding CA repeat in Stathmin-2 which is associated with sALS disease risk and has disease modifying effects. The potential value of this variant as a disease marker and tool for cohort enrichment in clinical trials warrants further investigation.

The TOMM40 '523' polymorphism in disease risk and age of symptom onset in two independent cohorts of Parkinson's disease.

Abnormal mitochondrial function is a key process in the pathogenesis of Parkinson's disease (PD). The central pore-forming protein TOM40 of the mitochondria is encoded by the translocase of outer mitochondrial membrane 40 homologue gene (TOMM40). The highly variant '523' poly-T repeat is associated with age-related cognitive decline and age of onset in Alzheimer's disease, but whether it plays a role in modifying the risk or clinical course of PD it yet to be elucidated. The TOMM40 '523' allele length was determined in 634 people with PD and 422 healthy controls from an Australian cohort and the Parkinson's Progression Markers Initiative (PPMI) cohort, using polymerase chain reaction or whole genome sequencing analysis. Genotype and allele frequencies of TOMM40 '523' and APOE ε did not differ significantly between the cohorts. Analyses revealed TOMM40 '523' allele groups were not associated with disease risk, while considering APOE ε genotype. Regression analyses revealed the TOMM40 S/S genotype was associated with a significantly later age of symptom onset in the PPMI PD cohort, but not after correction for covariates, or in the Australian cohort. Whilst variation in the TOMM40 '523' polymorphism was not associated with PD risk, the possibility that it may be a modifying factor for age of symptom onset warrants further investigation in other PD populations.

Single Nucleotide Polymorphisms Associated With Gut Homeostasis Influence Risk and Age-at-Onset of Parkinson's Disease.

Research is increasingly focusing on gut inflammation as a contributor to Parkinson's disease (PD). Such gut inflammation is proposed to arise from a complex interaction between various genetic, environmental, and lifestyle factors, however these factors are under-characterized. This study investigated the association between PD and single-nucleotide polymorphisms (SNPs) in genes responsible for binding of bacterial metabolites and intestinal homeostasis, which have been implicated in intestinal infections or inflammatory bowel disease. A case-control analysis was performed utilizing the following cohorts: (i) patients from the Australian Parkinson's Disease Registry (APDR) (n = 212); (ii) a Caucasian subset of the Parkinson's Progression Markers Initiative (PPMI) cohort (n = 376); (iii) a combined control group (n = 404). The following SNPs were analyzed: PGLYRP2 rs892145, PGLYRP4 rs10888557, TLR1 rs4833095, TLR2 rs3804099, TLR4 rs7873784, CD14 rs2569190, MUC1 rs4072037, MUC2 rs11825977, CLDN2 rs12008279 and rs12014762, and CLDN4 rs8629. PD risk was significantly associated with PGLYRP4 rs10888557 genotype in both cohorts. PGLYRP2 rs892145 and TLR1 rs4833095 were also associated with disease risk in the APDR cohort, and TLR2 rs3804099 and MUC2 rs11825977 genotypes in the PPMI cohort. Interactive risk effects between PGLYRP2/PGLYRP4 and PGLYRP4/TLR2 were evident in the APDR and PPMI cohorts, respectively. In the APDR cohort, the PGLYRP4 GC genotype was significantly associated with age of symptom onset, independently of gender, toxin exposure or smoking status. This study demonstrates that genetic variation in the bacterial receptor PGLYRP4 may modulate risk and age-of-onset in idiopathic PD, while variants in PGLYRP2, TLR1/2, and MUC2 may also influence PD risk. Overall, this study provides evidence to support the role of dysregulated host-microbiome signaling and gut inflammation in PD, and further investigation of these SNPs and proteins may help identify people at risk of developing PD or increase understanding of early disease mechanisms.

Disease-modifying effects of an SCAF4 structural variant in a predominantly SOD1 ALS cohort.

OBJECTIVE: To test the hypothesis that rs573116164 will have disease-modifying effects in patients with superoxide dismutase 1 (SOD1) familial amyotrophic lateral sclerosis (fALS), we characterized rs573116164 within a cohort of 190 patients with fALS and 560 healthy age-matched controls to assess the variant for association with various measures of disease. METHODS: Using a previously described bioinformatics evaluation algorithm, a polymorphic short structural variant associated with SOD1 was identified according to its theoretical effect on gene expression. An 12-18 poly-T repeat (rs573116164) within the 3' untranslated region of serine and arginine rich proteins-related carboxy terminal domain associated factor 4 (SCAF4), a gene that is adjacent to SOD1, was assessed for disease association and influence on survival and age at onset in an fALS cohort using PCR, Sanger sequencing, and capillary separation techniques for allele detection. RESULTS: In a North American cohort of predominantly SOD1 fALS patients (n =190) and age-matched healthy controls (n = 560), we showed that carriage of an 18T SCAF4 allele was associated with disease within this cohort (odds ratio [OR] 6.6; 95% confidence interval [CI] 3.9-11.2; p = 4.0e-11), but also within non-SOD1 cases (n = 27; OR 5.3; 95% CI 1.9-14.5; p = 0.0014). This finding suggests genetically SOD1-independent effects of SCAF4 on fALS susceptibility. Furthermore, carriage of an 18T allele was associated with a 26-month reduction in survival time (95% CI 6.6-40.8; p = 0.014), but did not affect age at onset of disease. CONCLUSIONS: The findings in this fALS cohort suggest that rs573116164 could have SOD1-independent and broader relevance in ALS, warranting further investigation in other fALS and sporadic ALS cohorts, as well as studies of functional effects of the 18T variant on gene expression.

Association of a structural variant within the SQSTM1 gene with amyotrophic lateral sclerosis.

OBJECTIVE: As structural variations may underpin susceptibility to complex neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the objective of this study was to investigate a structural variant (SV) within sequestosome 1 (SQSTM1). METHODS: A candidate insertion/deletion variant within intron 5 of the SQSTM1 gene was identified using a previously established SV evaluation algorithm and chosen according to its subsequent theoretical effect on gene expression. The variant was systematically assessed through PCR, polyacrylamide gel fractionation, Sanger sequencing, and reverse transcriptase PCR. RESULTS: A reliable and robust assay confirmed the polymorphic nature of this variant and that the variant may influence SQSTM1 transcript levels. In a North American cohort of patients with familial ALS (fALS) and sporadic ALS (sALS) (n = 403) and age-matched healthy controls (n = 562), we subsequently showed that the SQSTM1 variant is associated with fALS (p = 0.0036), particularly in familial superoxide dismutase 1 mutation positive patients (p = 0.0005), but not with patients with sALS (p = 0.97). CONCLUSIONS: This disease association highlights the importance and implications of further investigation into SVs that may provide new targets for cohort stratification and therapeutic development.

Structural Variants May Be a Source of Missing Heritability in sALS.

The underlying genetic and molecular mechanisms that drive amyotrophic lateral sclerosis (ALS) remain poorly understood. Structural variants within the genome can play a significant role in neurodegenerative disease risk, such as the repeat expansion in C9orf72 and the tri-nucleotide repeat in ATXN2, both of which are associated with familial and sporadic ALS. Many such structural variants reside in uncharacterized regions of the human genome, and have been under studied. Therefore, characterization of structural variants located in and around genes associated with ALS could provide insight into disease pathogenesis, and lead to the discovery of highly informative genetic tools for stratification in clinical trials. Such genomic variants may provide a deeper understanding of how gene expression can affect disease etiology, disease severity and trajectory, patient response to treatment, and may hold the key to understanding the genetics of sporadic ALS. This article outlines the current understanding of amyotrophic lateral sclerosis genetics and how structural variations may underpin some of the missing heritability of this disease.