Comparative Analysis of TDP-43-Controlled RNA Processing in Neuronal and Muscle Cells in Mouse and Human

TDP-43 (TAR DNA-binding protein, encoded by TARDBP gene) is a DNA/RNA-binding protein that participates in various steps of RNA metabolism. First identified as a component of cytoplasmic inclusions in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), its aberrant aggregation later became recogcnized in non-neuronal tissues, in particular skeletal muscles of patients with inclusion body myositis (IBM). Despite its ubiquitous expression and pleiotropic functions, it was unclear to what extent TDP-43 participates in basic cellular processes that are uniform across tissues and whether it exhibits tissue-specific functions in cells of different backgrounds. By silencing TDP-43 in (mouse) neuronal and muscle cell lines we mimicked loss of TDP-43’s function, a pathomechanism commonly believed to underly disease development. RNA-seq allowed transcriptome-wide detection of transcripts regulated by TDP-43 at the level of their overall abundance (differential expression DEG) or splicing (alternative splicing AS). In this work we discuss similarities and differences concerning the activity of TDP-43 across cell-types. We identified subsets of unique cell-type-characteristic mRNA targets and those that are commonly mediated by TDP-43 in muscles and neurons, whose tight regulation might underlie functions crucial for cell survival. Based on this, we investigated functional consequences of TDP-43 loss in either cell-type, linking TDP-43 pathology to previously described hallmarks of neuro- and myodegeneration. We further compared alternative splicing and transcript abundance control as two distinct regulatory mechanisms governed by TDP-43 that are not equally exploited by cells of different types. In addition, we investigated how cell-type-characteristic environments shape TDP-43’s function and render it tissue-specific. Among splicing events that occur in a muscle-specific manner, we started to characterize a TDP-43-dependent switch in Tbc1d1 splicing, as this gene encodes for a GTPase involved in translocation of glucose transporters and thereby mediates glucose uptake by muscle cells. With those results we set the ground for future studies investigating the function of TDP-43 in muscles and a putative contribution of TDP-43 redistribution to IBM development. Furthermore, our findings stress the importance to select an appropriate cell model to study tissue-characteristic features. Finally, we identified two novel TDP-43 targets, PPFIBP1 and ASAP2 consistently detected to undergo TDP-43-dependent isoform switch not only in mouse but also in humans. We have shown that the splicing of those is indeed perturbed in brains and skeletal muscles affected by TDP-43 pathology and we therefore believe these two splicing events could make a universal readout of TDP-43 dysfunction across tissues

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

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease.

TDP-43 (TAR DNA-binding protein 43) aggregation and redistribution are recognised as a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. As TDP-43 inclusions have recently been described in the muscle of inclusion body myositis patients, this highlights the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq, we directly compare TDP-43-mediated RNA processing in muscle (C2C12) and neuronal (NSC34) mouse cells. TDP-43 displays a cell-type-characteristic behaviour targeting unique transcripts in each cell-type, which is due to characteristic expression of RNA-binding proteins, that influence TDP-43's performance and define cell-type specific splicing. Among splicing events commonly dysregulated in both cell lines, we identify some that are TDP-43-dependent also in human cells. Inclusion levels of these alternative exons are altered in tissues of patients suffering from FTLD and IBM. We therefore propose that TDP-43 dysfunction contributes to disease development either in a common or a tissue-specific manner

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease

TDP-43 (TAR DNA-binding protein 43) aggregation and redistribution are recognised as a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. As TDP-43 inclusions have recently been described in the muscle of inclusion body myositis patients, this highlights the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq, we directly compare TDP-43-mediated RNA processing in muscle (C2C12) and neuronal (NSC34) mouse cells. TDP-43 displays a cell-type-characteristic behaviour targeting unique transcripts in each cell-type, which is due to characteristic expression of RNA-binding proteins, that influence TDP-43's performance and define cell-type specific splicing. Among splicing events commonly dysregulated in both cell lines, we identify some that are TDP-43-dependent also in human cells. Inclusion levels of these alternative exons are altered in tissues of patients suffering from FTLD and IBM. We therefore propose that TDP-43 dysfunction contributes to disease development either in a common or a tissue-specific manner

Fasting alters the gut microbiome reducing blood pressure and body weight in metabolic syndrome patients.

Periods of fasting and refeeding may reduce cardiometabolic risk elevated by Western diet. Here we show in the substudy of NCT02099968, investigating the clinical parameters, the immunome and gut microbiome exploratory endpoints, that in hypertensive metabolic syndrome patients, a 5-day fast followed by a modified Dietary Approach to Stop Hypertension diet reduces systolic blood pressure, need for antihypertensive medications, body-mass index at three months post intervention compared to a modified Dietary Approach to Stop Hypertension diet alone. Fasting alters the gut microbiome, impacting bacterial taxa and gene modules associated with short-chain fatty acid production. Cross-system analyses reveal a positive correlation of circulating mucosa-associated invariant T cells, non-classical monocytes and CD4+ effector T cells with systolic blood pressure. Furthermore, regulatory T cells positively correlate with body-mass index and weight. Machine learning analysis of baseline immunome or microbiome data predicts sustained systolic blood pressure response within the fasting group, identifying CD8+ effector T cells, Th17 cells and regulatory T cells or Desulfovibrionaceae, Hydrogenoanaerobacterium, Akkermansia, and Ruminococcaceae as important contributors to the model. Here we report that the high-resolution multi-omics data highlight fasting as a promising non-pharmacological intervention for the treatment of high blood pressure in metabolic syndrome patients