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Background Neuronal function depends heavily on the ability to transport various molecules along axons. The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington’s disease (HD), we studied the biology of vesicle-associated HTT. Given that HTT interacts with several arginine methyltransferases and that arginine methylation is particularly abundant in neurons, we wondered whether HTT itself is methylated and whether this influences HTT’s roles in axonal transport. Methods We immunopurified HTT from vesicular fractions isolated from brains of wild-type mice and analyzed HTT arginine methylation by mass spectrometry. We validated the presence of HTT arginine methylation by in vitro methylation assay and immunoblotting experiments in cellular models. Finally, we specifically modulated arginine methylation in vitro and in vivo to assess its effect on HTT-mediated vesicular trafficking, on mutant HTT (mHTT) toxicity and the HD phenotype. Results We found that HTT is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mHTT toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Conclusions Arginine methylation regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD.

SUMMARY The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington’s disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD., Graphical abstract, In brief Migazzi et al. identify arginine methylation as a new post-translational modification in huntingtin (HTT) that modulates its function in axonal transport. In Huntington’s disease models, enhancement of HTT methylation by PRMT6, a class I arginine methyltransferase, rescues axonal transport defects and neuronal health.

The Huntingtin (HTT) protein is centrally involved in bidirectional axonal trafficking. To better understand how HTT-mediated transport is regulated, we studied vesicle-associated HTT and found that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT’s association with vesicles is markedly reduced along with anterograde trafficking, and neuronal death ensues — very similar to what is observed in Huntington’s disease (HD), which is caused by polyglutamine (polyQ) expansions in mutant HTT (mHTT). Inhibiting PRMT6 in HD cells exacerbated mutant HTT-induced toxicity, whereas overexpressing PRMT6 rescued axonal transport and neuronal death, except in the presence of a methylation-defective version of mHTT. Overexpressing PRMT6 also rescued axonal defects and eclosion of HD flies. Arginine methylation thus regulates HTT’s activity in vesicular axonal transport, and increasing HTT methylation could be of therapeutic interest for HD.

International audience; Most of the cellular or molecular studies in HD used so far separated cultures of striatal or cortical neurons. However, in the brain these neurons are connected and form a particular network that is defective in HD. The polarised nature of neurons and the size and density of synapses complicates the manipulation and visualisation of specific events taking place in axons or dendrites and of specific synaptic transmission within the cortico-striatal network.To overcome these limitations, we developed several microfluidic systems compatible with high-resolution videomicroscopy and connected to microelectrode arrays (MEA) to reconstitute and identify each component of the corticostriatal network. The microfluidic system directs the formation of identified synapses separately between cortical axons and striatal dendrites and soma. In parallel, a multielectrode substrate monitors and controls presynaptic and postsynaptic activity independently. Using this multicomplex system we are investigating how the trafficking of synaptic vesicles or mitochondria along axons is regulated by presynaptic and postsynaptic patterns in the corticostriatal network in health and HD. In addition, the system allows modifying the genetic status of the cortical or striatal neurons as a way to selectively investigate how disease neurons differentially affect pre or post-synaptic events in HD and overall alter synapse function.