Inside out: the role of nucleocytoplasmic transport in ALS and FTLD

Neurodegenerative diseases are characterized by the presence of protein inclusions with a different protein content depending on the type of disease. Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are no exceptions to this common theme. In most ALS and FTLD cases, the predominant pathological species are RNA-binding proteins. Interestingly, these proteins are both depleted from their normal nuclear localization and aggregated in the cytoplasm. This key pathological feature has suggested a potential dual mechanism with both nuclear loss of function and cytoplasmic gain of function being at play. Yet, why and how this pathological cascade is initiated in most patients, and especially sporadic cases, is currently unresolved. Recent breakthroughs in C9orf72 ALS/FTLD disease models point at a pivotal role for the nuclear transport system in toxicity. To address whether defects in nuclear transport are indeed implicated in the disease, we reviewed two decades of ALS/FTLD literature and combined this with bioinformatic analyses. We find that both RNA-binding proteins and nuclear transport factors are key players in ALS/FTLD pathology. Moreover, our analyses suggest that disturbances in nucleocytoplasmic transport play a crucial initiating role in the disease, by bridging both nuclear loss and cytoplasmic gain of functions. These findings highlight this process as a novel and promising therapeutic target for ALS and FTLD.status: publishe

Lancet Neurol

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease. The discovery of genes associated with amyotrophic lateral sclerosis, commencing with SOD1 in 1993, started fairly gradually. Recent advances in genetic technology have led to the rapid identification of multiple new genes associated with the disease, and to a new understanding of oligogenic and polygenic disease risk. The overlap of genes associated with amyotrophic lateral sclerosis with those of other neurodegenerative diseases is shedding light on the phenotypic spectrum of neurodegeneration, leading to a better understanding of genotype-phenotype correlations. A deepening knowledge of the genetic architecture is allowing the characterisation of the molecular steps caused by various mutations that converge on recurrent dysregulated pathways. Of crucial relevance, mutations associated with amyotrophic lateral sclerosis are amenable to novel gene-based therapeutic options, an approach in use for other neurological illnesses. Lastly, the exposome-the summation of lifetime environmental exposures-has emerged as an influential component for amyotrophic lateral sclerosis through the gene-time-environment hypothesis. Our improved understanding of all these aspects will lead to long-awaited therapies and the identification of modifiable risks factors.R01 TS000327/TS/ATSDR CDC HHSUnited States/K23 ES027221/ES/NIEHS NIH HHSUnited States/MR/L501529/1/MRC_/Medical Research CouncilUnited Kingdom/R01 ES030049/ES/NIEHS NIH HHSUnited States/R01 NS120926/NS/NINDS NIH HHSUnited States/R01 NS127188/NS/NINDS NIH HHSUnited States/MR/R024804/1/MRC_/Medical Research CouncilUnited Kingdom/R01 TS000289/TS/ATSDR CDC HHSUnited States

Integrated transcriptome landscape of ALS identifies genome instability linked to TDP-43 pathology

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response

Understanding Neuromuscular Health and Disease: Advances in Genetics, Omics, and Molecular Function

This compilation focuses on recent advances in the molecular and cellular understandingof neuromuscular biology, and the treatment of neuromuscular disease.These advances are at the forefront of modern molecular methodologies, oftenintegrating across wet-lab cell and tissue models, dry-lab computational approaches,and clinical studies. The continuing development and application ofmultiomics methods offer particular challenges and opportunities in the field,not least in the potential for personalized medicine

Identification of Novel Genetic Variations for Amyotrophic Lateral Sclerosis (ALS)

A list of genes have been identified to carry mutations causing familial ALS such as SOD1, TARDBP, C9orf72. But for sporadic ALS, which is 90% of all ALS cases, the underlying genetic variants are still largely unknown. There are multiple genome-wide association study (GWAS) for sporadic ALS, but usually a large number nominated SNP can hardly be replicated in larger cohort analysis. Also majority of GWAS SNP lie within noncoding region of genome, imposing a huge challenge to study their biological role in ALS pathology. With the rapid development of next-generation sequencing technology, we are able to sequence exome and whole-genome of a large number of ALS patients to search for novel genetic variants and their potential biological function. Here by analyzing exam data, we discovered two novel or extremely rare missense mutations of DPP6 from a Mestizo Mexican ALS family. We showed the two mutations could exert loss-of-function effect by affecting electrophysiological properties of Potassium channels as well as the membrane localization of DPP6. To our knowledge this is the first report of DPP6 nonsynonymous mutations in familial ALS patients. In addition, by analyzing whole-genome data, we discovered strong linkage disequilibrium between SNP rs12608932, a repeatedly significant ALS GWAS signal, and one polymorphic TGGA tetra-nucleotide tandem repeat, which is further flanked by large TGGA repetitive sequences. We also demonstrated rs12608932 risk allele is associated with reduced UNC13A expression level in human cerebellum and UNC13A knockout could lead to shorter survival in SOD1-G93A ALS mice. Thus the TGGA repeat might be the real underlying genetic variation that confer risk to sporadic ALS

Why TDP-43? Why not? Mechanisms of metabolic dysfunction in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder for which there is no effective curative treatment available and minimal palliative care. Mutations in the gene encoding the TAR DNA-binding protein 43 (TDP-43) are a well-recognized genetic cause of ALS, and an imbalance in energy homeostasis correlates closely to disease susceptibility and progression. Considering previous research supporting a plethora of downstream cellular impairments originating in the histopathological signature of TDP-43, and the solid evidence around metabolic dysfunction in ALS, a causal association between TDP-43 pathology and metabolic dysfunction cannot be ruled out. Here we discuss how TDP-43 contributes on a molecular level to these impairments in energy homeostasis, and whether the protein’s pathological effects on cellular metabolism differ from those of other genetic risk factors associated with ALS such as superoxide dismutase 1 (SOD1), chromosome 9 open reading frame 72 (C9orf72) and fused in sarcoma (FUS)

ALS Linked Mutations in Matrin 3 Alter Protein-Protein Interactions and Impede mRNA Nuclear Export

abstract: Exome sequencing was used to identify novel variants linked to amyotrophic lateral sclerosis (ALS), in a family without mutations in genes previously linked to ALS. A F115C mutation in the gene MATR3 was identified, and further examination of other ALS kindreds identified an additional three mutations in MATR3; S85C, P154S and T622A. Matrin 3 is an RNA/DNA binding protein as well as part of the nuclear matrix. Matrin 3 interacts with TDP-43, a protein that is both mutated in some forms of ALS, and found in pathological inclusions in most ALS patients. Matrin 3 pathology, including mislocalization and rare cytoplasmic inclusions, was identified in spinal cord tissue from a patient carrying a mutation in Matrin 3, as well as sporadic ALS patients. In an effort to determine the mechanism of Matrin 3 linked ALS, the protein interactome of wild-type and ALS-linked MATR3 mutations was examined. Immunoprecipitation followed by mass spectrometry experiments were performed using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify ALS-causing mutations in the gene MATR3, as well as a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS.Dissertation/ThesisDoctoral Dissertation Neuroscience 201

Investigating the molecular etiologies of sporadic ALS (sALS) using RNA-Sequencing

ALS is an often lethal disease involving degeneration of motor neurons in the brain and spinal cord. Current treatments only extend life by several months, and novel therapies are needed. We combined RNA-Sequencing, systems biology analyses, and molecular biology assays to elucidate sporadic ALS group-specific differences in postmortem cervical spinal sections (7 sALS and 8 control samples) that may be relevant to disease pathology. \u3e55 million 2X150 RNA-sequencing reads per sample were generated and processed. In Chapter 2, we used bioinformatics tools to identify nuclear differentially expressed genes (DEGs) between our two groups. Further, we used Weighted Gene Co-Expression Network Analysis to identify gene co-expression networks associated with disease status. Qiagen’s Ingenuity Pathway Analysis revealed our sALS group-specific DEGs and a sALS group-specific gene co-expression network were associated with inflammatory processes and TNF-α signaling. Further, TNFAIP2 was identified as a sALS group-specific upregulated DEG and a network hub gene within that network. We hypothesized TNFAIP2’s upregulation in our ALS samples reflected increased TNF-α signaling and that TNFAIP2 promoted motor neuron death via TNF superfamily apoptotic pathways. Transient overexpression of TNFAIP2 decreased cell viability in both neural stem cells and induced pluripotent stem cell-derived motor neurons. Further, inhibition of activated caspase 9 (a protein necessary for TNF superfamily mitochondrial-mediated apoptosis) reversed this effect in neural stem cells. In Chapters 3 and 4, we used bioinformatics tools to identify sALS group-specifc mitochondrial DEGs and differentially used exons (DUEs). Qiagen’s Ingenuity Pathway Analysis revealed our sALS group-specific DUEs were associated with cholesterol biosynthesis

Protein homeostasis regulation in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disorder that affects motor neurons in the motor cortex, brainstem and spinal cord. The lack of an effective treatment indicates the need for a deeper understanding of the pathogenesis underlying this disease. ALS, as well as the majority of neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) are characterised by dysfunctions in protein homeostasis (proteostasis). The endoplasmic reticulum (ER) plays an important role in proteostasis through the unfolded protein response (UPR). We therefore started this thesis by characterising the UPR signalling pathways in human post-mortem spinal cord from sporadic ALS (SALS) and in frontal and temporal cortex from FTLD and AD cases and compared with healthy controls. In ALS, UPR activation was confirmed by a substantial expression increase of both known and novel target genes involved particularly in ER-associated degradation (ERAD), while in AD a distinct pattern emerged, with a predominant involvement of protein folding genes, such as Protein Disulphide Isomerases (PDIs). Similarly, in human motor cortex of SALS cases we found an increased expression of PDIs and other specific UPR target genes which correlated with oligodendrocyte markers. Moreover, we found that the heat shock response (HSR), a major proteostasis regulatory pathway, and ERAD genes were activated predominately in the spinal cord and strongly correlated with the motor neuron marker VAPB. Finally, we performed a meta-analysis of publicly available RNA-Seq studies derived from the spinal cord of healthy and ALS cases. We identified cholesterol metabolism, cell adhesion and regulation of vesicle-mediated transport as top disease-associated processes and 21 hub genes as central nodes in these networks. We conclude that proteostasis is strongly and selectively activated specific cell types in SALS motor cortex and spinal cord. Hence, these results provide novel insights into the pathophysiology of ALS and other neurodegenerative disorders.Open Acces