Unravelling the role of patient-derived dipeptide repeat proteins in the C9orf72 expansion-related pathogenesis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two different disorders characterized by a distinctive pattern of neurodegeneration. However, ALS and FTLD also display overlapping clinical features (e.g. cognitive and motor impairment), neuropathological hallmarks (e.g. TDP-43 pathology), and monogenetic causes in a subset of the patients. With respect to the latter, the hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene is the most common mutation underlying both diseases. Since no disease-modifying treatments are currently available for ALS or FTLD, the in-depth understanding of the C9orf72 expansion-related pathogenesis, which will contribute to the development of new therapeutic strategies, is of utmost importance. Based on current knowledge, the unconventional translation of the repeat expansion into five distinct dipeptide repeat proteins (DPRs), which form protein aggregates in neurons, might be a predominant contributing mechanism to the C9orf72 expansion-related pathogenesis. However, observations in disease models and in post-mortem central nervous system (CNS) tissue are not consistently concordant. This might be due to the different repeat length, nature and levels of DPRs used in most disease models in comparison with DPRs observed in patient's CNS (i.e. patient-derived DPRs). The aim of this PhD project was to investigate the actual level of toxicity of patient-derived DPRs as a part of the C9orf72 expansion-related pathogenesis ongoing in the CNS of C9orf72 ALS and/or FTLD patients. In particular, we analyzed an extensive number of CNS regions of post-mortem cases to investigate (i) the minimum repeat length required to trigger neuropathological hallmarks (i.e. DPR and TDP-43 pathology), (ii) the potential cell-autonomous toxicity of DPRs in extra-motor brain regions, and (iii) the process of DPR aggregation whether or not driven by DPR spreading along neuronal networks. Moreover, we evaluated non-cell-autonomous toxicity in control- and C9orf72 patient-derived human motor neurons exposed to C9orf72 patient cerebrospinal fluid (CSF) and cerebellar homogenates. First, we showed that a repeat length of 38 repeats in the C9orf72 gene in blood as well as in brain and spinal cord resulted in sparse DPR pathology and typical TDP-43 pathology, combined with the clinical phenotype of ALS, in the absence of somatic instability. These findings contribute to the establishment of a valid pathogenic repeat threshold and have important implications for genetic counselling of ALS and FTLD cases with intermediate repeat lengths. Second, we observed DPR pathology together with limited or absent TDP-43 pathology in suprachiasmatic nucleus-related neurons, the pineal gland, and along the hypothalamic-pituitary axis. These DPR lesions in the absence of florid TDP-43 pathology might indicate cell-autonomous DPR toxicity resulting in extra-motor symptoms (i.e. sleep and endocrinal disturbances). Our data are an important first step to more focused studies on the hormone-producing and -secreting capacity of the pineal and pituitary gland in C9orf72 repeat expansion carriers to further elucidate the clinical impact of our findings. Third, the DPR neuroanatomical distribution pattern, here analyzed in a wide range of CNS regions, indicates a minor and non-exclusive role for DPR spreading along neuronal networks in the development of widespread DPR pathology. In addition, we showed the presence of poly(GA) - one of the five DPR species - in every cytoplasmic DPR inclusion in several brain regions, whether or not co-localized with one or multiple other DPR species, suggesting a maturation aspect in the process of DPR aggregation. This implicates differences in DPR aggregate composition within and between patients, which might potentially affect DPR toxicity profiles and might play a role in selective neuronal vulnerability. Finally, we did not observe additional cytotoxicity in human motor neurons upon exposure to C9orf72 patient CSF and cerebellar homogenates containing patient-derived DPRs compared with exposure to nonC9orf72-derived counterparts devoid of patient-derived DPRs. This might be due to limited patient-derived DPR uptake, since we did not observe convincing uptake of patient-derived poly(GA) in motor neurons. Our findings suggest the absence of a fulminant and fast acting non-cell-autonomous cytotoxic contribution of patient-derived DPRs to the impairment of motor neurons, as opposed to the prominently and quickly observed DPR toxicity and axonal DPR transmission in disease models. In conclusion, our findings provide new insights into the actual role of patient-derived DPRs in the C9orf72 expansion-related pathogenesis and into the process of DPR aggregation ongoing in patients. With this study, we shed light on candidate treatment approaches to combat C9orf72 diseases and support the use of poly(GA) immunotherapy as a part of a multi-targeted approach. Importantly, future studies investigating DPR toxicity may rely on our findings to develop better disease models most relevant to the pathogenesis in patients in order to accurately position patient-derived DPRs in the C9orf72 expansion-related pathogenic cascade.

Publication year:2021

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