PERIPHERAL BIOMARKERS AND THERAPEUTIC MODULATION OF PROTEOSTASIS PATHWAYS IN AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic lateral sclerosis (ALS) is a fatal and clinically heterogeneous neurodegenerative disorder characterized by progressive motor neuron degeneration and abnormal accumulation of misfolded TDP-43 protein aggregates. Despite significant advances in the understanding of ALS genetics and molecular underpinnings, effective treatments and reliable diagnostic biomarkers remain critically lacking. This PhD thesis sought to address these gaps by combining patient-derived cellular models to screen for candidate pharmacological compounds, and biomarker discovery approaches in peripheral tissues from living patients, with the dual objective to explore phenotype-specific susceptibility to stress-modulating drugs and to identify novel diagnostic tools. In the first part of the project (Research Chapter 1), we established an in vitro ALS model by generating human induced pluripotent stem cells (iPSCs)-derived motor neurons from bulbar- or spinal-onset ALS patients and healthy controls. We evaluated the biological effects of three therapeutic compounds – riluzole (the current standard of care for ALS), guanabenz (an integrated stress response [ISR] modulator), and IFB-088 (or Sephin1, a selective GADD34-PP1c inhibitor of the ISR) – across multiple readouts, including gene expression profiling, tubulin composition, electrophysiological activity, and cytokine/chemokine release. Interestingly, motor neurons from bulbar-onset ALS patients displayed distinctive stress-related abnormalities at baseline when compared to both spinal-onset cultures and controls. Moreover, IFB-088 treatment consistently reversed several key pathological features in bulbar-onset cultures, as evidenced by transcriptional rebalancing of ISR- and autophagy-related markers, restoration of cytoskeletal integrity and neuronal firing, and a shift toward a neuroprotective secretory phenotype. Of note, these effects were not observed in spinal-onset or control-derived cultures, emphasizing the therapeutic relevance of phenotype-specific vulnerability in ALS and supporting the potential of proteostasis-targeting strategies in bulbar-onset phenotypes. In the second part of the project (Research Chapter 2), we implemented a Seed Amplification Assay (SAA) to detect pathological TDP-43 seeding activity in olfactory mucosa samples obtained from living ALS patients with either genetic or sporadic disease. The assay efficiently revealed seeding activity in a subset of cases, providing proof-of-concept that ALS-related proteinopathy can be captured in vivo. Although we found no correlation with clinical, neuropsychological, or genetic features in our cohort, these results support the feasibility of using olfactory mucosa as a minimally invasive tissue source for detecting ALS pathology. Together, these findings offer new insights into ALS pathogenesis and provide experimental evidence supporting both phenotype-specific therapeutic approaches and peripheral biomarker development. By integrating iPSC-based disease models and protein-based assays, this work yields mechanistic and translational insights that may guide future research toward more personalized medicine strategies for this devastating disorder.