Impaired Protein Aggregate Handling and Clearance Underlie the Pathogenesis of p97/VCP-associated Disease

Abstract

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA؉ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.Ubiquitinated inclusions (UBIs) 2 are a common pathology in many degenerative disorders associated with brain and muscle (1). In many cases the molecular constituents of the UBIs are known and are a result of the misfolding of an aggregate prone or mutant protein such as ␣-synuclein in autosomal dominant Parkinson disease or expanded polyglutamine containing proteins in Huntington disease. UBIs in other degenerative disorders may be due to a global impairment in protein degradation. For example, after a misfolded substrate overwhelms the ubiquitin proteasome system (UPS) or as a result of mutations in proteins involved in specific protein degradation pathways. One example is the autosomal dominantly inherited disorder inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD) associated with mutations in the UPS chaperone p97/VCP (2). Affected tissue in IBMPFD contains prominent cytoplasmic and intranuclear UBIs (3-5). In some cases these UBIs contain tubulofilamentous inclusions and insoluble protein aggregates (4). p97/VCP belongs the AAAϩ (ATPases associated with a variety of activities) protein family and participates in the degradation of proteins via the UPS (6). p97/VCP has a clear role in endoplasmic reticulumassociated degradation (ERAD) of misfolded proteins (6). In association with co-factors derlin-1, Ufd1, and Npl4, p97/VCP participates in the retrotranslocation of misfolded endoplasmic reticulum lumen and transmembrane proteins facilitating their delivery to the UPS. We have previously shown that IBMPFD mutant p97/VCP fails to degrade the misfolded ERAD substrate ⌬F508-CFTR as efficiently as wild type p97/VCP in cultured myoblasts (7). ...

The VCP/p97 system at a glance: connecting cellular function to disease pathogenesis

“…Therefore, it can be surmised that the function of VCP/p97 in mammalian development is preserved with these mutations. This mirrors cell culture studies, in which the expression of VCP/p97 protein carrying disease-associated mutations does not appear to affect cell cycle control or cellular division (Ju et al, 2008). Although the cellular expression of disease-associated mutant VCP/p97 can recapitulate some of the features that are seen with chemical inhibition or small interfering (si)RNA-mediated knockdown of VCP/p97 activity (e.g.…”

“…Although the cellular expression of disease-associated mutant VCP/p97 can recapitulate some of the features that are seen with chemical inhibition or small interfering (si)RNA-mediated knockdown of VCP/p97 activity (e.g. defects in autophagy and endolysosomal sorting), some functions appear to be preserved or are affected to a lesser degree by these mutations (see below) (Weihl et al, 2006;Ju et al, 2008;Ju et al, 2009;Ritz et al, 2011). However, which of these functions are involved in distinct tissue-specific pathogenesis and subsequent pathogenic phenotypes remains uncertain.…”

“…For example, inclusions containing ubiquitylated proteins are a common pathologic feature found in all mutant VCP/p97 disease-affected tissues . Indeed, VCP disease mutations affect the consolidation of aggregateprone proteins into inclusion bodies and disrupt the autophagic degradation of ubiquitylated proteins, resulting in the accumulation of non-degradative autophagosomes, another common pathologic feature (Ju et al, 2008;Ju et al, 2009;Tresse et al, 2010). Interestingly, mutations in two other proteins that are necessary for the targeting of autophagic substrates to the autophagosome, the autophagy adaptors p62/SQSTM1 and optineurin, are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Paget's disease of the bone (PDB), or ALS and PDB, further suggesting that a defect in this process is the underlying basis of VCP/p97-mediated disease pathogenesis (Laurin et al, 2002;Fecto et al, 2011;Rubino et al, 2012).…”

Valosin-containing Protein (VCP) in Novel Feedback Machinery between Abnormal Protein Accumulation and Transcriptional Suppression

“…Ufd1 and Npl4) and ubiquitinated proteins, but also showed enhanced aggregate-forming activities (Refs. 28,49). 3 Regarding with these characteristics, we could not observe clear difference between wild-type VCP and VCP(DEQ).…”

Interaction of SQSTM1 with the motor protein dynein: SQSTM1 is required for normal dynein function and trafficking

“…However, in either case, once associated with HDAC6, the polyubiquitylated proteins are relocated to aggresomes through the interaction of HDAC6 with the motor protein dynein. Loss of HDAC6 has been reported to result in the failure to colocalize aggregated proteins to the aggresome for degradation leading to aggregate buildup, neurodegeneration (Ju et al, 2008;Lee et al, 2010) and inclusion body formation (Guthrie and Kraemer, 2011;Richter-Landsberg and Leyk, 2013). SQSTM1 also binds ubiquitylated substrates, again with a preference for K63-polyubiquitin, through its C-terminal UBA domain.…”