Epigenetic regulation of gene transcription in amyotrophic lateral sclerosis

An aging population has led to a drastic increase in the prevalence of neurodegenerative diseases. Among these disorders, Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive and fatal disease underpinned by degeneration of motor neurons. Most ALS cases are sporadic, while a small proportion is spread in families. As genetics alone cannot explain the etiology of ALS, epigenetic modifications have been considered a key event in the complex genome-environment interactions that work together with the DNA to modulate gene expression programs. In this respect, epigenetic modifications, which are known to accumulate over the lifespan, provide a unique opportunity to identify a common feature among the multitude of events that underlie ALS. In the first part of this work, we generated a genome-scale map of the repressive trimethylation of lysine 27 on histone H3 (H3K27me3) mark in peripheral blood mononuclear cells (PBMCs) of familial ALS patients carrying the most widespread chromosome 9 open reading frame 72 (C9orf72) or superoxide dismutase 1 (SOD1) mutation. While the analysis of C9orf72-specific H3K27me3-associated peaks did not show a specific association with ALS, we identified synaptosome-associated protein 25 (SNAP25) as the core element of the molecular network composed of H3K27me3 peak-associated genes in SOD1-mutant ALS patients. SNAP25 is a component of the soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) complex that is involved in fusing vesicles with membrane-bound compartments in neurons. Its reduction contributes to an impaired neurotransmitter release, highlighting a potentially critical role of H3K27me3-regulated genes in the pathogenesis of ALS. Transcriptome profiling identified the stress-inducible activating transcription factor 3 (ATF3) as a central hub gene of regulatory mechanisms in SOD1 patients. While overexpression of ATF3 is associated with improved motor function in SOD1G93A mice, its expression in PBMCs was down-regulated by H3K27me3 occupancy at its promoter. By integrating chromatin H3K27me3 occupancy data with gene expression data of SOD1-mutated patients, we identified SNAP25 as a direct target of ATF3 through a binding motif in its promoter region. Both genes were also detected at protein level in cerebrospinal fluid and spinal cord of ALS patients, suggesting that an extra/intracellular signal is transmitted systemically by ATF members in different cells to modulate gene expression. However, a single histone modification reflects only a small part of the entire histone code. In contrast, the chromatin accessibility landscape provides a more reliable insight into global changes in chromatin organization induced by modifications of the DNA and/or histones. Therefore, in the second part of this work we used a multi-omic approach combining an assay for transposase accessible chromatin following sequencing (ATAC-seq) and RNA-seq with single-cell sequencing in PBMCs and motor cortex from sporadic ALS patients to find a mechanistic link between the epigenetic regulation and disease pathogenesis. By applying ATAC-seq to PBMCs from sporadic ALS patients and healthy donors, we identified a robust ALS-associated epigenetic signature (‘epiChromALS’) comprising 729 differentially accessible peaks that were annotated to 668 unique genes. Most of these peaks (80 %) were less accessible and enriched in enhancer and promoter regions. Functional enrichment analysis of these differentially accessible peaks revealed an association with neuronal functions and neuronal differentiation, suggesting that neurodevelopmental processes are epigenetically impaired in ALS. The comparison of epiChromALS with genome-wide association study (GWAS) databases and correlation with clinical parameters showed that less accessible regions were indeed associated with important ALS risk loci and positively correlated with age of onset. In addition, epiChromALS was largely independent of the dysregulation of PBMC subtypes found in the transcriptome profile of ALS. Some of the identified epigenetic marks appear to be local and thus specific for the cell where they are expressed (e.g. differentially accessible peaks of a single PBMC subpopulation were mainly related to immune cell functions), while others were detected in different cell types and across tissue. In particular, these common marks were enriched in neuronal terms suggesting that some extra/intracellular stimuli shape the epigenetic landscape of ALS in a systemic manner. Thus, the periphery is able to partially reflect epigenetic alterations of the central nervous system. This opens up new possibilities to monitor diseases where the primarily affected tissue is not accessible over the course of the disease

Impaired ATF3 signaling involves SNAP25 in SOD1 mutant ALS patients

Abstract Epigenetic remodeling is emerging as a critical process for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Genetics alone fails to explain the etiology of ALS, the investigation of the epigenome might therefore provide novel insights into the molecular mechanisms of the disease. In this study, we interrogated the epigenetic landscape in peripheral blood mononuclear cells (PBMCs) of familial ALS (fALS) patients with either chromosome 9 open reading frame 72 (C9orf72) or superoxide dismutase 1 (SOD1) mutation and aimed to identify key epigenetic footprints of the disease. To this end, we used an integrative approach that combines chromatin immunoprecipitation targeting H3K27me3 (ChIP-Seq) with the matching gene expression data to gain new insights into the likely impact of blood-specific chromatin remodeling on ALS-related molecular mechanisms. We demonstrated that one of the hub molecules that modulates changes in PBMC transcriptome in SOD1-mutant ALS patients is ATF3, which has been previously reported in an SOD1 G93A mouse model. We also identified potential suppression of SNAP25, with impaired ATF3 signaling in SOD1-mutant ALS blood. Together, our study shed light on the mechanistic underpinnings of SOD1 mutations in ALS

Increased NF-L levels in the TDP-43G298S ALS mouse model resemble NF-L levels in ALS patients

Elevated levels of neurofilament light chain (NF-L) in CSF and blood are linked to the presymptomatic and symptomatic phase of patients suffering from amyotrophic lateral sclerosis (ALS). However, whether the NF-L level in extracellular liquids like serum or CSF is a marker of destruction or NF-L is secreted actively outside the cell is not known so far. NF-L levels in CSF and blood clearly separate ALS patients and controls [9], serving as a prognostic biomarker for ALS [4]. Also in pre-symptomatic ALS gene mutation carriers NF-L levels are elevated thus allowing prediction for clinical phenoconversion [3, 4]. The picture of NF-L levels in ALS mouse models is less clear. Previous studies report on elevated NF-L plasma levels in SOD1G93Adl and TDP-43 (TAR6/6) mice [6], but the correlation to motor neuron (MN) loss has not been determined. We therefore employed a transgenic TDP-43G298S mouse model to study the interaction of motor neuron pathophysiology, muscle denervation and NF-L levels. TDP-43G298S mutant mice show decreased performance in grip strength and motor activity compared to controls [10]