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bioRxiv · 10.1101/2025.11.25.690484

The molecular mechanism of uptake and cell-to-cell transmission of arginine-containing dipeptide repeat proteins.

Sutter, A. B.·Buksh, B. F.·Mojsilovic-Petrovic, J.·Dalton, C.·Till, N. A.·Morgan, D. C.·MacMillan, D. W. C.·Kalb, R. G.
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Abstract

Micro-satellite repeat expansion of the 5 GGGGCC 3 sequence in the C9orf72 gene is the most common monogenic form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) translated from the mutant allele can be detected in postmortem brains of afflicted individuals. The arginine containing peptides, poly-PR and poly-GR, are particularly noxious to cells. Both have been shown to undergo cell-cell transmission, but the underlying mechanisms are not understood. We found rapid internalization and nucleolar localization of bath-applied hemagglutinin (HA) tagged poly-PR with twenty repeats (HA-PR20) in cell lines and neurons. Small molecule and RNAi approaches implicated a temperature-dependent, fluid phase endocytosis mechanism in HA-PR20 uptake. We sought to identify DPR-related cell surface uptake factors using a high-resolution proximity labeling technique developed in the MacMillan group, termed {micro}Map. DPR-iridium conjugates identified candidate cell-surface proteins which were interrogated in an RNAi screen. Focusing on our strongest candidate, chondroitin sulfate proteoglycan 4 (CSPG4), we showed that cellular uptake of HA-PR20 is blocked by inhibition of glycosaminoglycan chain synthesis (using drugs or RNAi) and knockdown or ablation of CSPG4 (using RNAi or CRISPR editing). Reduction of CSPG4 protected PR20-induced neuronal toxicity. We used a dual reporter system to interrogate in vitro neuron-to-neuron transmission of PR50 and found that PR50 synthesized by one neuron readily spread to neighboring neurons. Transmission was significantly reduced when CSPG4 was knocked down. These results suggest CSPG4 is an important factor in poly-PR internalization and transmission and therefore may be a therapeutic target to slow DPR transmission and disease progression. Significance StatementA GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common monogenic form of ALS/FTD. This expansion leads to dipeptide repeat protein (DPR) production through non-canonical translation of repeat-containing RNA. DPRs have been shown to transmit between cells, but how this occurs is not well understood. We identified the cell surface protein chondroitin sulfate proteoglycan 4 (CSPG4) as a mediator of the uptake and intercellular spread of toxic arginine-rich DPRs. Targeting CSPG4 may provide a strategy to block DPR transmission and slow disease progression.

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Sutter, A. B., Buksh, B. F., Mojsilovic-Petrovic, J., Dalton, C., Till, N. A., Morgan, D. C., MacMillan, D. W. C., Kalb, R. G.. 2025-11-28. The molecular mechanism of uptake and cell-to-cell transmission of arginine-containing dipeptide repeat proteins.. https://doi.org/10.1101/2025.11.25.690484

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