title: Aberrant alternative splicing: the molecular hallmark of sporadic amyotrophic lateral sclerosis and beyond
title: Fehlgeleitetes alternatives Spleißen: das molekulare Kennzeichen der sporadischen Amyotrophen Lateralsklerose und darüber hinaus
Creators: name: Krach, Florian
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2025

description: The results of this thesis (Appendix I) have been published in ‘Acta Neuropathologica’. The respective original publication is attached in the appendix. The other parts, incl. scientific contextualization, are not part of the orignal publication. Citation of original publication: Krach F, Wheeler EC, Regensburger M, Boerstler T, Wend H, Vu AQ, Wang R, Reischl S, Boldt K, Batra R, Aigner S, Ravits J, Winkler J, Yeo GW, Winner B. Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis. Acta Neuropathol. 2022 Sep;144(3):413-435. doi: 10.1007/s00401-022-02450-3. Epub 2022 Jul 1. PMID: 35778567; PMCID: PMC9381448. A note in the Acknowledgements section of the original publication indicates that the work was performed in order to obtain the degree ‘Dr. med’. The original publication was published as an open access article distributed under the terms of the Creative Commons CC BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. All figures within the scientific contextualization were created with BioRender.com.
description: Background and Objectives: Amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative condition, is characterized by the selective loss of motor neurons in the central nervous system, leading to progressive muscle weakness, spasticity, and eventually, paralysis. Most cases of ALS are sporadic. However, some cases of the disease occur in a mendelian pattern of inheritance. On a molecular level, ALS is characterized by aberrant alternative splicing. The primary objective of this study was to identify and analyze RNA-binding proteins (RBPs) affecting this early molecular alteration in ALS. This exploration aims to offer novel perspectives on ALS pathogenesis and potential therapeutic targets. Methods: Employing induced pluripotent stem cell (iPSC)-derived motor neurons from both sporadic and familial ALS patients, we used used mass spectrometry of detergent-resistant protein fractions to identify the insoluble proteome to identify candidate RBPs. We focused on ALS-associated alternative splicing via RNA sequencing (RNA-seq) and analyzed the RNA-binding patterns of the identified candidate RBPs via an advanced technique, enhanced crosslinking and immunoprecipitation (eCLIP). We employed thorough data integration and analysis to identify first which RBPs are involved in aberrant alternative splicing in ALS, and second, how they facilitate the alternative splicing changes. At last, we validated our findings histologically in ALS postmortem spinal cord tissue and employed a pseudo-timing of individual motor neurons in patients by analyzing for the degree of TDP-43 pathology, offering a comprehensive view of the disease's progression. Results and Observations: The study presents compelling evidence of increased insolubility and expression levels of NOVA1 and other RNA-binding proteins (RBPs) in ALS, suggesting a significant disruption in their normal functions. Specifically, NOVA1 was found enriched at sites of alternative splicing, indicating its pivotal role in the dysregulation observed in ALS. This disruption manifests through a complex interplay of loss and gain of function characteristics, underscoring the multifaceted nature of ALS pathology. Remarkably, in the early stages of ALS, increased cytoplasmic accumulation of NOVA1 in motor neurons was observed, even before the onset of TDP-43 pathology, suggesting a critical window for understanding disease initiation. As the disease progresses, a reduction in NOVA1 levels was noted, aligning with advancing TDP-43 pathology and motor neuron degeneration. Conclusions: This comprehensive study emphasizes NOVA1's significant yet complex role in the dysregulation of alternative splicing in ALS, particularly in the disease's early stages. By highlighting the importance of early-stage molecular alterations, the findings offer a new lens through which to view ALS pathogenesis. The insights into NOVA1-mediated alternative splicing not only contribute to our understanding of the disease's molecular origins but also suggest potential therapeutic avenues targeting this dysregulation. The results were published in: Krach F, Wheeler EC, Regensburger M, Boerstler T, Wend H, Vu AQ, Wang R, Reischl S, Boldt K, Batra R, Aigner S, Ravits J, Winkler J, Yeo GW, Winner B. Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis. Acta Neuropathol. 2022 Sep;144(3):413-435. doi: 10.1007/s00401-022-02450-3.