Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.

A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6 days, and is still prominent after 12 days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6 days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13 days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.

Protein quality control at the inner nuclear membrane.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

How is alpha-synuclein cleared from the cell?

The levels and conformers of alpha-synuclein are critical in the pathogenesis of Parkinson's Disease and related synucleinopathies. Homeostatic mechanisms in protein degradation and secretion have been identified as regulators of alpha-synuclein at different stages of its intracellular trafficking and transcellular propagation. Here we review pathways involved in the removal of various forms of alpha-synuclein from both the intracellular and extracellular environment. Proteasomes and lysosomes are likely to play complementary roles in the removal of intracellular alpha-synuclein species, in a manner that depends on alpha-synuclein post-translational modifications. Extracellular alpha-synuclein is cleared by extracellular proteolytic enzymes, or taken up by neighboring cells, especially microglia and astrocytes, and degraded within lysosomes. Exosomes, on the other hand, represent a vehicle for egress of excess burden of the intracellular protein, potentially contributing to the transfer of alpha-synuclein between cells. Dysfunction in any one of these clearance mechanisms, or a combination thereof, may be involved in the initiation or progression of Parkinson's disease, whereas targeting these pathways may offer an opportunity for therapeutic intervention. This article is part of the Special Issue "Synuclein".

Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation.

The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t1/2≤30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.

A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation.

The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of ∼45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.

A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis.

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.

A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting.

In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.