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

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-PR ₂₀ ) in cell lines and neurons. Small molecule and RNAi approaches implicated a temperature-dependent, fluid phase endocytosis mechanism in HA-PR ₂₀ uptake. We sought to identify DPR-related cell surface uptake factors using a high-resolution proximity labeling technique developed in the MacMillan group, termed µMap. 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-PR ₂₀ 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 PR ₂₀ -induced neuronal toxicity. We used a dual reporter system to interrogate in vitro neuron-to-neuron transmission of PR ₅₀ and found that PR ₅₀ 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.