Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease

The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.     

ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration

Defects in the endosomal-lysosomal pathway have been implicated in a number of neurodegenerative disorders. A key step in the endocytic regulation of transmembrane proteins occurs in a subset of late-endosomal compartments known as multivesicular bodies (MVBs), whose formation is controlled by endosomal sorting complex required for transport (ESCRT). The roles of ESCRT in dendritic maintenance and neurodegeneration remain unknown. Here, we show that mSnf7-2, a key component of ESCRT-III, is highly expressed in most mammalian neurons. Loss of mSnf7-2 in mature cortical neurons caused retraction of dendrites and neuronal cell loss. mSnf7-2 binds to CHMP2B, another ESCRT-III subunit, in which a rare dominant mutation is associated with frontotemporal dementia linked to chromosome 3 (FTD3). Ectopic expression of the mutant protein CHMP2B(Intron5) also caused dendritic retraction prior to neurodegeneration. CHMP2B(Intron5) was associated more avidly than CHMP2B(WT) with mSnf7-2, resulting in sequestration of mSnf7-2 in ubiquitin-positive late-endosomal vesicles in cortical neurons. Moreover, loss of mSnf7-2 or CHMP2B(Intron5) expression caused the accumulation of autophagosomes in cortical neurons and flies. These findings indicate that ESCRT-III dysfunction is associated with the autophagy pathway, suggesting a novel neurodegeneration mechanism that may have important implications for understanding FTD and other age-dependent neurodegenerative diseases.     

A concentric circle model of multivesicular body cargo sorting

Targeting of ubiquitylated transmembrane proteins into luminal vesicles of endosomal multivesicular bodies (MVBs) depends on their recognition by endosomal sorting complexes required for transport (ESCRTs), which are also required for MVB vesicle formation. The model originally proposed for how ESCRTs function succinctly summarizes much of the protein-protein interaction and genetic data but oversimplifies the coordination of cargo recognition and cannot explain why ESCRTs are required for the budding of MVB vesicles. Recent structural and functional studies of ESCRT complexes suggest an alternative model that might direct the next series of breakthroughs in understanding protein sorting through the MVB pathway.     

The Endosomal Protein CHARGED MULTIVESICULAR BODY PROTEIN1 Regulates the Autophagic Turnover of Plastids in Arabidopsis

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.     

The functional analysis of the CHMP2B missense mutation associated with neurodegenerative diseases in the endo-lysosomal pathway

Endosomal sorting complexes required for transport (ESCRTs) regulate a key sorting step of protein trafficking between endosomal compartments in lysosomal degradation. Interestingly, mutations in charged multivesicular body protein 2B (CHMP2B), which is a core subunit of ESCRT-III, have been identified in some neurodegenerative diseases. However, the cellular pathogenesis resulting from CHMP2B missense mutations is unclear. Furthermore, little is known about their functional analysis in post-mitotic neurons. In order to examine their cellular pathogenesis, we analyzed their effects in the endo-lysosomal pathway in post-mitotic neurons. Interestingly, of the missense mutant proteins, CHMP2B(T104N) mostly accumulated in the Rab5- and Rab7-positive endosomes and caused delayed degradation of EGFR as compared to CHMP2B(WT). Furthermore, CHMP2B(T104N) showed less association with Vps4 ATPase and was avidly associated with Snf7-2, a core component of ESCRT-III, suggesting that it may cause defects in the process of dissociation from ESCRT. Of the missense variants, CHMP2B(T104N) caused prominent accumulation of autophagosomes. However, neuronal cell survival was not dramatically affected by expression of CHMP2B(T104N). These findings suggested that, from among the various missense mutants, CHMP2B(T104N) was associated with relatively mild cellular pathogenesis in post-mitotic neurons. This study provided a better understanding of the cellular pathogenesis of neurodegenerative diseases associated with various missense mutations of CHMP2B as well as endocytic defects.     

Binding to Any ESCRT Can Mediate Ubiquitin‐Independent Cargo Sorting

The ESCRT (endosomal sorting complex required for transport) machinery is known to sort ubiquitinated transmembrane proteins into vesicles that bud into the lumen of multivesicular bodies (MVBs). Although the ESCRTs themselves are ubiquitinated they are excluded from the intraluminal vesicles and recycle back to the cytoplasm for further rounds of sorting. To obtain insights into the rules that distinguish ESCRT machinery from cargo we analyzed the trafficking of artificial ESCRT‐like protein fusions. These studies showed that lowering ESCRT‐binding affinity converts a protein from behaving like ESCRT machinery into cargo of the MVB pathway, highlighting the close relationship between machinery and the cargoes they sort. Furthermore, our findings give insights into the targeting of soluble proteins into the MVB pathway and show that binding to any of the ESCRTs can mediate ubiquitin‐independent MVB sorting.     

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.     

内吞体分选转运复合体 (ESCRT) 及其在包膜病毒出芽中的作用

内吞体分选转运复合体(Endosomal sorting complex required for transport,ESCRT)主要识别泛素化修饰的膜蛋白,介导内吞小泡出芽和多泡体(Multivesicular bodies,MVBs)的形成。此外,以类似的拓扑方式,ESCRT也参与胞质分裂、自体吞噬、以及包膜病毒的出芽等过程。已有的研究表明,大量的反转录病毒和RNA病毒含有晚期结构域(Late-domains),该结构域与ESCRT组分相互作用,将ESCRT-Ⅲ和VPS4等募集在病毒组装与出芽区域,并利用ESCRT-Ⅲ使病毒粒子得以释放。最近,有研究发现,一些DNA包膜病毒、如乙肝病毒、疱疹病毒和杆状病毒等的出芽释放也依赖于宿主细胞ESCRT系统,但其机理尚需深入研究。     

Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen.     

The emerging shape of the ESCRT machinery

The past two years have seen an explosion in the structural understanding of the endosomal sorting complex required for transport (ESCRT) machinery that facilitates the trafficking of ubiquitylated proteins from endosomes to lysosomes via multivesicular bodies (MVBs). A common organization of all ESCRTs is a rigid core attached to flexibly connected modules that recognize other components of the MVB pathway. Several previously unsuspected key links between multiple ESCRT subunits, phospholipids and ubiquitin have now been elucidated, which, together with the detailed morphological analyses of ESCRT-depletion phenotypes, provide new insights into the mechanism of MVB biogenesis.     

ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.     

Targeting of AMSH to endosomes is required for epidermal growth factor receptor degradation

To reach the lysosomes, down-regulated receptors such as the epidermal growth factor receptor must first be sorted into internal vesicles of late endosomes (multivesicular bodies), a ubiquitin-dependent event that requires the coordinated function of the endosome sorting complex required for transport (ESCRT) proteins. Here we report that CHMP3, an ESCRT-III complex component, and associated molecule of SH3 domain of STAM (AMSH), a deubiquitinating enzyme, interact with each other in cells. A dominant-negative version of CHMP3, which specifically prevents targeting of AMSH to endosomes, inhibits degradation but not internalization of EGFR, suggesting that endosomal AMSH is a functional component of the multivesicular body pathway.     

ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.     

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.     

ESCRT proteins and cell signalling

Subunits of the endosomal sorting complex required for transport (ESCRT) were identified as components of a molecular machinery that sorts ubiquitinated membrane proteins into the intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) for subsequent delivery to the lumen of lysosomes or related organelles. As many of the membrane proteins that undergo ESCRT-mediated sorting are signalling receptors that are ubiquitinated in response to ligand binding, ESCRT subunits have been hypothesized to play a crucial role in attenuation of cell signalling by mediating ligand-induced receptor degradation. Here we discuss this concept based on the examples from loss-of-function studies in model organisms and cell lines. The emerging picture is that ESCRTs are indeed involved in downregulation of receptor signalling pathways associated with cell survival, proliferation and polarity. In addition, the recent discovery of a positive role for the ESCRT pathway in Wnt signalling through sequestration of an inhibitory cytosolic component into MVEs illustrates that ESCRTs may also control signalling in ways that are independent of degradative receptor sorting.     

Endosomal sorting complexes required for ESCRTing cells toward death during neurogenesis,neurodevelopment and neurodegeneration

The endosomal sorting complexes required for transport (ESCRT) proteins help in the recognition, sorting and degradation of ubiquitinated cargoes from the cell surface, long‐lived proteins or aggregates, and aged organelles present in the cytosol. These proteins take part in the endo‐lysosomal system of degradation. The ESCRT proteins also play an integral role in cytokinesis, viral budding and mRNA transport. Many neurodegenerative diseases are caused by toxic accumulation of cargo in the cell, which causes stress and ultimately leads to neuronal death. This accumulation of cargo occurs because of defects in the endo‐lysosomal degradative pathway—loss of function of ESCRTs has been implicated in this mechanism. ESCRTs also take part in many survival processes, lack of which can culminate in neuronal cell death. While the role played by the ESCRT proteins in maintaining healthy neurons is known, their role in neurodegenerative diseases is still poorly understood. In this review, we highlight the importance of ESCRTs in maintaining healthy neurons and then suggest how perturbations in many of the survival mechanisms governed by these proteins could eventually lead to cell death; quite often these correlations are not so obviously laid out. Extensive neuronal death eventually culminates in neurodegeneration.      

ESCRT proteins in physiology and disease

As a mechanism of signal attenuation, receptors for growth factors, peptide hormones and cytokines are internalized in response to ligand binding, followed by degradation in lysosomes. Receptor ubiquitination is a key signal for such downregulation, and four protein complexes known as endosomal sorting complex required for transport (ESCRT)-0, -I, -II and -III have been identified as the machinery required for degradative endosomal sorting of ubiquitinated membrane proteins in yeast and metazoans. Three of these complexes contain ubiquitin-binding domains whereas ESCRT-III instead recruits deubiquitinating enzymes. The concerted action of the ESCRTs not only serves to sort ubiquitinated cargo but is also thought to cause inward vesiculation of endosomal membranes, thereby mediating biogenesis of multivesicular endosomes (MVEs). Because ligand-mediated receptor downregulation plays an important role in signal attenuation, it is not surprising that dysfunction of ESCRT components is associated with disease. In this review we discuss the possible roles of ESCRTs in protection against cancer, neurodegenerative diseases and bacterial infections, and we highlight the fact that many RNA viruses exploit the ESCRT machinery for the final abscission step of their budding from cells. We also review the additional functions of ESCRT proteins in cytokinesis and discuss how these may be related to ESCRT-associated pathologies.     

CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.     

Multivesicular Bodies and Autophagy in Erythrocyte Maturation

During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.

Addendum to:

Exosome secretion and red cell maturation: Exploring molecular components involved in the docking and fusion of multivesicular bodies in K562 cells.

Fader CM, Savina A, Sánchez D, Colombo MI. Blood Cells Mol Dis 2005; 35:153-7.

and

Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.

Savina A, Fader CM, Damiani MT, Colombo MI. Traffic 2005; 6:131-43.