Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.

While defective α-synuclein homeostasis is central to Parkinson's pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of α-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.

Endocytic sorting and downregulation of the M2 acetylcholine receptor is regulated by ubiquitin and the ESCRT complex.

Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.

Regulation and trafficking of muscarinic acetylcholine receptors.

Fidelity of signal transduction relies on cells expressing the appropriate number of functional receptors. Fluctuation in the total number of muscarinic acetylcholine receptors has been implicated in a range of physiological and pathophysiological processes, and the mechanisms responsible for this regulation represent potential molecular targets for therapeutic intervention. This article will review the current literature on the endocytic trafficking of muscarinic receptors and how knowledge of the trafficking of related receptors might influence future studies. This article is part of the Special Issue entitled 'Neuropharmacology on Muscarinic Receptors'.