Molecular and temporal underpinnings of non-cell autonomous pathology in amyotrophic lateral sclerosis

Abstract

Amyotrophic Lateral Sclerosis (ALS) is an incurable and rapidly progressing neurodegenerative disease with limited treatment and diagnostic options. These constraints result in diagnostic delays, hindering early intervention. These theragnostic challenges are indicative of our limited understanding of the fundamental pathogenic mechanisms of ALS, especially during the initial stages of the disease. Here I propose to understand the temporal and non-cell autonomous molecular drivers of disease to address this gap in knowledge. ALS is a genetically, pathologically, and clinically heterogeneous disease, making molecular markers essential for clinical trial stratification and target engagement. Whilst most ALS cases are sporadic (sALS), approximately 10% are familial (fALS). Familial ALS cases implicate over 20 genes in the pathogenesis of the disease, with the most common being C9orf72. Although prior research has primarily focused on the impact of ALS on neuronal cells, emerging evidence indicates that non-cell-autonomous effects of non-neuronal cells play a significant role in ALS pathogenesis. Evidence suggests that neuroinflammatory astrogliosis is a common pathology in ALS, with studies showing that modulation of gliosis in mouse models can prolong lifespan. However, the contribution of glial pathology to ALS pathogenesis and the temporal nature of this contribution are poorly understood.

To address this knowledge gap, I have employed three interconnected approaches. First, I re-stratified existing datasets based on the extent of glial pathology. Specifically, I compared ALS cases (both fALS and sALS) categorized by the presence or absence of glial pathology. This approach aimed to generate data that highlight the impact of glial burden on pathology, potentially revealing theragnostic targets obscured by the inherent heterogeneity when comparing healthy controls and ALS cases. Secondly, I developed novel tools to accurately probe TDP-43 pathology in these cases. To do this, I used specific TDP-43 markers that are indicative of loss-of-function (cryptic exons) and gain-of-function (TDP-43 aptamers) to stratify cases. Finally , these tools were also used to link my findings to temporally stratify induced pluripotent stem cell (iPSC)-based models of fALS models. This approach aimed to enhance our comprehension of the temporal progression of pathogenic mechanisms in ALS. By leveraging these models, we can pinpoint and characterise the earliest stages of disease pathogenesis during critical developmental milestones, documenting initial disease-associated alterations and key molecular changes.

The findings provided compelling evidence that astrocytic TDP-43 pathology significantly associates with genetic and regional vulnerability, and molecular phenotype variability, suggesting that astrocytes actively contribute to disease progression through both gain- and loss-of-function mechanisms. Specifically, the inclusion of cryptic exons, indicative of TDP-43 nuclear loss of function, further confirmed this notion. Additionally, a systematic review of the literature revealed significant astrocyte involvement in ALS pathogenesis, demonstrating that dysfunctional astrocytes induce neurotoxicity and substantially affect motor neuron survival. Using patient-derived iPSC models, it was established that early pathogenic features, including altered TDP-43 and normal STMN2 expression, manifest early in the differentiation process, providing robust platforms for temporal mapping and therapeutic screening. Moreover, by employing NanoString sequencing in key developmental stages of motor neuron development, genes governing early neurodevelopment, including those involved in progenitor proliferation, axon elongation, and RNA metabolism, were downregulated in C9orf72 hiPSCs-derived cells relative to controls. Additionally, overlapping molecular signatures from post-mortem ALS tissues and hiPSC-derived models highlighted dysregulation in pathways related to myelination, metabolism, and neuroinflammation. Finally, novel single-cell transcriptomics methods developed during this research, including optimized hydrogel droplet sequencing (Hydrops), allowed for the capture of previously undetectable heterogeneity within ALS samples, thus greatly enhancing the resolution of pathological and therapeutic insights.

This thesis significantly advances our understanding of ALS pathogenesis by defining the temporal and cell-specific contributions of TDP-43 dysfunction and glial pathology, highlighting astrocyte involvement, and offering methodological innovations. These insights provide a foundation for developing targeted early-stage interventions and robust stratification strategies for future clinical trials, ultimately driving progress toward more personalised and effective ALS therapies.