Onderzoek naar de pathogenese van sporadische ALS: Inzichten uit hiPSC-afgeleide motorneuronen en microglia

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective and progressive loss of motor neurons in the motor cortex, brainstem and ventral horn of the spinal cord. This results in paralysis and mortality of the patient usually within 2 to 5 years after the diagnosis. While 10% of ALS patients have one or more affected family members (familial ALS; fALS), approximately 90% of ALS patients suffer from sporadic ALS (sALS) with no known family disease history. While 5 to 10% of sALS cases can be explained by mutations in the same genes that are responsible for fALS, the heritability of sALS is estimated to be approximately 60%. However, the genetic variants involved in sALS remain largely unknown, posing a significant challenge for modelling and understanding this form of the disease. The discovery of the human induced pluripotent stem cell (hiPSC) technology has provided a valuable tool for studying sALS. This technology enables the generation of patient-specific motor neurons with the correct genetic background, thereby facilitating the investigation of sALS disease mechanisms. We utilized hiPSCs to differentiate motor neurons from sALS patients to investigate early disease mechanisms. Our studies revealed profound axonal defects in sALS motor neurons, including impaired axonal transport, defective axonal outgrowth, and reduced neuromuscular junction (NMJ) formation with myotubes. Transcriptomic analysis identified an enrichment of pathways related to axonal guidance and an upregulation of genes associated with inhibiting axonal regeneration in motor neurons from sALS patients. These findings suggest that dysregulation of axonal guidance pathways could play a role in the sALS pathogenesis. A hallmark of ALS pathology, observed in 97% of patients, is the mislocalization and aggregation of the TDP-43 protein. Our studies using hiPSC-derived sALS motor neurons revealed axonal defects without observing TDP-43 mislocalization or motor neuron death. This suggests that additional triggers are necessary to obtain disease-related phenotypes such as protein aggregation, DNA damage and cell death, which are observed in postmortem material of sALS patients. Since aging is a major risk factor for ALS, we investigated the effects of various aging associated stressors on hiPSC-derived motor neurons in an attempt to induce these defects. We observed that telomerase inhibition with BIBR1532 could cause DNA damage in hiPSCs, however, it did not lead to differences in DNA damage levels or neurite lengths in the hiPSC-derived motor neurons. Upon Camptothecin (CPT)-induced DNA damage, hiPSC-derived motor neurons from sALS patients exhibited fewer γ-H2AX foci and a higher cytoplasmic-to-nuclear ratio of TDP-43 protein. The response to arsenite-induced stress and subsequent recovery were similar between control and sALS motor neurons. However, proteasome inhibition resulted in increased cytoplasmic TDP-43 accumulation in sALS motor neurons, indicating an increased vulnerability to a disrupted protein homeostasis. These results support aging-related changes underlying ALS pathogenesis, and highlight the potential contributions of DNA damage and impaired proteostasis in disease development. Finally, we explored the role of microglia in ALS pathology and their interaction with motor neurons using induced microglia-like cells (iMGLs) differentiated from hiPSCs. Due to the complexity of sALS pathogenesis, we focused this part of the study on known fALS mutations as well as their corresponding isogenic controls in order to increase the likelihood of capturing disease-specific phenotypes. Therefore, we examined FUS gene mutations (P525L and R521H) and their CRISPR-Cas9 gene-edited isogenic controls (P525P and R521R). Through an established protocol, we successfully generated high-purity, functional iMGLs via a hematopoietic progenitor cell (HPC) state from hiPSC lines. These iMGLs demonstrated key microglial functions, including phagocytosis, inflammatory gene regulation in response to lipopolysaccharide (LPS), and neuronal support by enhancing axonal transport and reducing debris accumulation. Cytoplasmic mislocalization of the FUS protein was observed in the P525L line, while R521H microglia exhibited pro-inflammatory gene expression following LPS stimulation. However, no differences in phagocytic ability and cytokine release were observed among the different cell lines. These findings suggest that FUS mutations did not lead to significant alterations in microglia function of phagocytosis or cytokine release under the tested conditions.