Unconventional protein secretion-mediated protein quality control in health and diseases

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Proteinaceous inclusions termed Lewy bodies (LBs) is a classical hallmark of Parkinsons disease (PD). The primary component of these inclusions is alpha-synuclein (a-syn), a protein with an intrinsic propensity to misfold and aggregate. In PD patients, alpha-syn inclusions first observed in the olfactory bulb and the dorsal motor nucleus, progressively spread throughout the brain. Further findings that healthy embryonic dopamine neurons transplanted into PD patients developed LBs points suggest the tantalizing possibility of neuron-to-neuron transmission of a-syn. Subsequent work comfirmed that synthetic a-syn pre-formed fibrils (PFFs) can be taken up by neurons, eliciting the misfolding of endogenous -syn into insoluble Lewy-like inclusions. Collectively, these studies led to the prion hypothesis of PD, wherein misfolded -syn provides a template for seeding new aggregates, propogating misfolded a-syn and associated cytotoxicity. Thus, that propagation of -syn is a viable new target to be explored in the development of new PD therapies. The intercellular transmission of synuclien consists of two key steps: the secretion of synuclein from a donor neuron and its uptake by a recipient neuron. Misfolding-associated protein secretion (MAPS) is a recently discovered protein quality control process that selectively exports misfolded cytosolic proteins including a-syn. Secretion through MAPS requires the membrane localized deubiquitinase USP19, which recruits aberrant polypeptides to endoplasmic reticulum (ER) surface to facilitate their incorporation into to late endosomes that are in tight association with the ER. Misfolded proteins are secreted to the extracellular milieu when late endosomes fuse with the plasma membrane. The fate of the released misfolded proteins is current unknown. Our recent studies suggest that mammalian cells can internalize misfolded proteins via endocytosis, but it is unclear whether they possess one or more receptors for misfolded proteins. Whether internalized proteins can impose damage prior to degradation by the lysosome is also unclear. The proposal is to elucidate the physiological relevance of the MAPS pathway by characterizing the interplay between secreted misfolded proteins and target cells. Using a proximity based ligation approach, we have used purified Tau as a bait to identify candidate Protein aggregates formed by microtubule-associated protein Tau can be transmitted in a stereotypic pattern in human brains, which correlates with the progression of several neurodegenerative diseases collectively termed Tauopathies. This process requires Tau, released from neurons, to interact with a cell surface receptor on a target cell, but little is known about the underlying mechanisms and the downstream pathophysiological consequences particularly for Tau that engages glial cells. Using a spatially resolved proteomic mapping strategy, we identify integrin V/1, a heterodimer of the integrin family, as a receptor that not only mediates Tau fibril entry into astrocytes, but also activates integrin signaling upon Tau binding. We show that distinct Tau species differentially activates integrin signaling, with filamentous Tau being a more robust stimulator. This leads to NFB activation and differential upregulation of pro-inflammatory cytokines and chemokines. Additionally, a sub-group of neurotoxic astrocyte markers are also induced in an integrin-dependent manner preferentially by filamentous Tau, causing astrocytes to release a neurotoxic factor(s). Together, these findings establish a paradigm that astrocytes can be directly converted into a neurotoxic state by filamentous Tau via an integrin receptor, which as a sensor transducing unfolded protein pathology, may provide a new therapeutic target for Tauopathies. In Parkinsons disease, cell-to-cell transmission of synapse specific, misfolding-prone protein -synuclein (-Syn) comprises two poorly characterized cellular processes release of -Syn via unconventional protein secretion and uptake of -Syn-bearing fibrils via Clathrin-dependent endocytosis. Using CRISPR-mediated genetic screen, we identify SLC35B2 as a critical regulator for endocytosis of preformed -Syn fibril (PFF) in both non-neuronal cells and neurons. SLC35B2 mediates the synthesis of cell surface heparan sulfate proteoglycans (HSPG), whose sugar moiety, bearing linearized negative charges, can insert into a groove formed by clustered Lysine residues in oligomerized -Syn. Intriguingly, the uptake of -Syn fibrils and other polycation-carrying HSPG cargos also involves a Myosin motor. This Myosin acts in conjunction with actin filaments maintain membrane dynamics at plasma membrane domains enriched in Clathrin. Without Myosin, a fraction of Clathrin-coated pits fail to be severed from the plasma membranes, causing accumulation of giant Clathrin positive scars on the cell surface. Our data suggest that actin-mediated membrane dynamics facilitates maturation and function of Clathrin-coated pits at the cell surface to promote endocytosis of a collection of HSPG cargos.