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C9orf72 repeat expansion in FTD/ALS - from mechanisms to therapeutic approaches

Final Report Summary - DPR-MODELS (C9orf72 repeat expansion in FTD/ALS - from mechanisms to therapeutic approaches)

An inherited repeat expansion in the C9orf72 gene is responsible for about 10% of all cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related fatal neurodegenerative diseases. We had discovered that the expanded repeat is translated into five aggregating proteins (so called dipeptide repeat proteins, DPR): poly-GA, poly-GP, poly-GR, poly-PA and poly-PR. Here, we analyzed these DPR proteins in patients, cellular models and mice and developed novel therapeutic approaches.

(i) From patient tissue and cerebrospinal fluid (CSF) we could show that DPR proteins start accumulating already many years before disease onset and likely contribute to the subtle prodromal symptoms and trigger secondary pathology and ultimately neuron loss. Surprisingly, DPR distribution in patients poorly correlates with neurodegeneration, but cerebellar DPR levels distinguish ALS and FTD patients suggesting that DPR inclusions in one region may affect other brain regions, for example by transmission from cell to cell.

(ii) We developed various cellular models to study the production and toxicity of the individual DPR species separately. Poly-PR, is most toxic in vitro, but least abundant in patients. In contrast, poly-GA is ~100-fold more abundant in patients but less toxic in cell culture. The in vitro toxicity and in vivo abundance of poly-GR is intermediate. Analyzing poly-GA aggregates at very high resolution revealed a structure resembling the proteins aggregating in other neurodegenerative disease, so called amyloids. Strikingly, the poly-GA aggregates recruit a large number of proteasomes, a molecular machine involved in degrading proteins, and seem to lock them in an otherwise rare confirmation. Our data provide mechanistic insight into the long-standing question of impairment of the proteasome by protein aggregates in neurodegenerative disease by visualizing a direct interaction. By analyzing proteins binding to DPR aggregates we could show that poly-GR/-PR interferes with the protein translation by binding to ribosomes.
To identify the cellular mechanism of the unusual synthesis of DPRs, we developed a high-throughput assay in patient-derived neurons that is still ongoing. Screening a library of FDA-approved drugs may reveal a potential therapeutic compound potent enough for direct repurposing or suitable for further optimization.

(iii) To dissect the role of DPR proteins in vivo and enable preclinical therapeutic studies we developed mouse models expressing individual DPRs. A transgenic mouse expressing low levels of poly-GA shows motor deficits and neuroinflammation a in the absence of neuron loss. These results suggest that presymptomatic DPR expression in patients contribute to prodromal deficits observed in patients. Mouse models with higher DPR expression show far more severe symptoms that need further characterization.

(iv) To investigate the potential utility of antibody therapy targeting DPR proteins, we first used our cellular model systems. We could show that DPR proteins are transmitted from cell to cell. Our monoclonal anti-GA antibodies block DPR transmission and related toxicity. In a first in vivo study, immunotherapy could largely prevent motor deficits in mice. These data sparked several ongoing follow-up studies, resulted in a granted patent and a second patent application. We are currently looking for ways to commercialize these findings to fund further development for clinical trials.

Altogether our data suggest that DPR proteins are the major mechanism driving C9orf72 pathogenesis especially at the early disease stage. We show that DPR expression alters basic cellular processes such a protein synthesis and degradation. In mutation carriers, these effects can apparently be compensated for a long time without overt symptoms. Thus, inhibiting DPR synthesis or promoting DPR clearance (e.g. using antibodies) are promising approaches for therapy and especially for prevention.