Deciphering Axonal Dysfunction in ALS Using Spatial Multi-Omics in FUS-ALS hiPSC Models

• Applicant: Dr. Marina Chekulaeva
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Biochemistry ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545044131

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition marked by the progressive degeneration of motor neurons, ultimately resulting in paralysis and respiratory failure. Present therapeutic development approaches for neurodegenerative diseases are typically mutation-specific, leveraging antisense oligonucleotides or small molecules to target aberrant mRNA. Such an approach demands personalized treatments for each patient. The complexity increases due to heterogenous etiology of ALS, which has been linked to mutations in over 40 genes. Moreover, more than 90% of ALS cases are sporadic, with no traceable genetic cause, making targeted treatments unfeasible. However, a common feature among ALS cases is the early loss of axons, accompanied by defects in axonal transport and protein synthesis. Our current proposal aims to explore these defects in ALS in-depth. By doing so, we hope to formulate conceptually novel therapeutic strategies that could be applied to a range of neurodegenerative conditions. In our prior research, we laid the groundwork for the proposed study. We created human-induced pluripotent stem cell (hiPSC)-based FUS-ALS models that mimic the cell survival and myotube contractility defects seen in patients. We have also developed a method to isolate neuronal subcellular compartments – cell bodies and axons - for omics analysis, including proteomic, RNA-seq, SLAM-seq, and Ribo-seq (referred to as spatial omics). Additionally, we developed a technique to map RNA localization elements by integrating a massively parallel reporter assay with the isolation of neuronal compartments (N-zip for neuronal zipcode identification protocol). Building on this foundation, we plan to apply spatial omics and N-zip to FUS-ALS models derived from patient hiPSCs. This will enable us to identify proteins and RNAs with varying localization, stability, and translation across the subcellular compartments of ALS models and their isogenic controls, and to pinpoint elements responsible for mRNA localization to axons and their dysfunction in FUS-ALS. For validation and comprehensive mechanistic studies, we will focus on 3-5 selected candidates. These candidates will be chosen based on their potential role in neurodegeneration and their observed changes in axonal localization. To assess the impact of validated candidates on neurodegeneration, we will use overexpression and knockdown techniques coupled with functional tests, such as assessing motor neuron survival and contractility in myotube/motor neuron co-cultures. Our research aims to provide a comprehensive and integrated understanding of spatial gene expression in FUS-ALS, offering new insights into the complex and multifaceted pathogenesis of ALS. This could pave the way for the development of innovative therapeutic strategies that are applicable to multiple forms of ALS.

• DFG Programme: Research Grants