The ESCRT complexes are involved in sorting of endocytosed ubiquitinated integral membrane proteins into the ILVs of MVBs. Their depletion in mammalian cells has been found to inhibit degradation of proteins like the EGF-R and results in MVBs with abnormal morphology (Slagsvold et al., 2006
). However, little is known about the role of MVBs, and the consequence of depleting ESCRT subunits, for autophagic degradation. In this study we show that siRNA-mediated depletion of ESCRT subunits inhibits autophagic degradation, leading to accumulation of large ubiquitin-positive protein aggregates, also containing Alfy and p62, proteins known to closely associate with cytoplasmic ubiquitin-positive structures and autophagic membranes (Simonsen et al., 2004
; Bjorkoy et al., 2005
). We show that degradation of the autophagic membrane protein Atg8/LC3-II and the autophagic substrate p62 is inhibited and that there is a shift toward p62 insolubility, indicative of a more aggregated conformation, in cells depleted of Tsg101 and Vps24. Autophagosomes and amphisomes are formed in ESCRT-depleted cells, but the formation of autolysosomes is inhibited. This is in line with previous studies, showing that mutations in Vps class C proteins (Rieder and Emr, 1997
; Lindmo et al., 2006
) and the class E Vps proteins Vps4p/SKD1 (Shirahama et al., 1997
; Nara et al., 2002
; Besteiro et al., 2006
) and CeVPS-27 (Roudier et al., 2005
) impede the formation of autolysosomes. In addition, we observed clusters of small vesicular–tubular elements and membrane-free dense p62-positive cytosolic aggregates in ESCRT-depleted cells. Although we cannot exclude the possibility that ESCRTs also have non-MVB related functions in autophagy, our data strongly indicate that dysfunctional MVBs inhibit autophagic degradation, leading to formation of large aggregates that eventually may cause neurodegenerative disease.
The ESCRT-III subunit CHMP2B, forming a complex with Vps24 (Babst et al., 2002
; von Schwedler et al., 2003
), was recently found to be mutated in a large Danish family with familial FTD (Skibinski et al., 2005
) and in patients with ALS (Parkinson et al., 2006
). CHMP2B mutations are not a common cause of FTD, as several studies have failed to identify CHMP2B mutations in FTD patients (Cannon et al., 2006
; Rizzu et al., 2006
). We show here that cells expressing CHMP2B mutants corresponding to the mutation found in the Danish FTD patients are characterized by accumulation of ubiquitin, p62, and LC3, indicating that autophagic degradation is impeded in these cells. Our data thus suggest a possible explanation to the observed neurodegenerative phenotypes seen in CHMP2B mutant patients. Ubiquitin and p62 are common components of protein inclusions associated with neurodegenerative disease (Talbot and Ansorge, 2006
) and are found in the brains of the Danish FTD patients (Holm et al., 2007
). However, the ubiquitin- and p62-positive inclusions observed in the Danish FTD patient brains with the CHMP2B mutation occur at low frequency as compared with other cases of FTLD-U and are generally observed mostly in the hippocampus, which is not a site of the neurodegenerative pathology (Holm et al., 2007
). This suggests that although autophagy may be impaired globally, leading to cell death, the formation of ubiquitin- and p62-positive inclusions occurs only in a subset of cells in vivo. This could be because the majority of cells with such inclusions have degenerated by the end stage of the disease that is observed in post mortem
material, or that the inclusions are a protective mechanism against impaired autophagy.
Ubiquitin-positive tau-negative neuronal cytoplasmic inclusions are common pathological features in FTLD-U and ALS, and TDP-43 was recently identified as the major ubiquitinated protein in these disorders (Arai et al., 2006
; Neumann et al., 2006
). We here show that TDP-43 accumulates in cells depleted of Tsg101 and Vps24, suggesting that impaired MVB function could have a role in TDP-43 aggregate formation in FTLD-U and ALS. It is not clear from our experiments if TDP-43 itself is degraded via autophagy, although this could be one explanation for its accumulation. In contrast to other cases of FTLD-U, the ubiquitin-positive inclusions found in patients from the Danish CHMP2B mutant family are TDP-43 negative (Holm et al., 2007
). We also failed to detect TDP-43 in the cytoplasmic ubiquitin-, p62-positive structures that accumulate in cells expressing mutant CMHP2B, suggesting that the molecular mechanisms responsible for accumulation of p62 and TDP-43 differ.
Using a cell-based system for HD, we show that ESCRTs (Vps24) are required also for efficient clearance of the Htt polyQ aggregates, both in human HeLa and mouse neuronal cells. It has previously been shown that the autophagic pathway is responsible for clearance of Htt polyQ aggregates (Ravikumar et al., 2004
; Iwata et al., 2005
; Ravikumar and Rubinsztein, 2006
; Yamamoto et al., 2006
), but our results show, for the first time, that functional MVBs and a Vps class E protein are required for efficient clearance of Htt inclusions.
Recently, it was found that mice with neuronal-specific deficiencies for Atg5 or Atg7, proteins known to be essential for autophagy, are characterized by accumulation of cytoplasmic inclusion bodies and a neurodegenerative phenotype (Hara et al., 2006
; Komatsu et al., 2006
). Our results indicate that depletion of ESCRT proteins likely will result in a similar phenotype, but do not allow us to conclude about when these proteins become ubiquitinated. There is little or no evidence to date showing that polyubiquitinated proteins are normally degraded by autophagy and it is probably more likely that proteins that are normally turned over by autophagy become polyubiquitinated when autophagic degradation is inhibited. However, it was recently found that induction of autophagy leads to enhanced delivery of ubiquitin to lysosomes and that this correlates with enhanced lysosomal bactericidal capacity (Alonso et al., 2007
), which might support the idea that polyubiquitinated proteins are autophagic substrates. p62, containing a ubiquitin-binding UBA domain (Vadlamudi et al., 1996
), would be an excellent candidate for recognition and targeting of polyubiquitin linked proteins to the autophagic pathway ().
Figure 10. Model for autophagic degradation in control and ESCRT-depleted cells. In control cells, cytoplasmic cargo (proteins and organelles) is sequestered by an isolation membrane/ phagophore, forming double-membrane autophagosomes that can fuse with MVBs, forming (more ...)
In conclusion, we have shown that depletion of ESCRT subunits or overexpression of CHMP2B mutant proteins inhibit autophagic degradation, leading to accumulation of ubiquitin-positive aggregates that contain proteins associated with neurodegenerative disease. Our data indicate that functional MVBs are required to prevent accumulation of abnormal proteins that can disrupt neural function and ultimately lead to neurodegeneration.