Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases

Abstract

Mutations of the human valosin-containing protein gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia. We identified strumpellin as a novel valosin-containing protein binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate that strumpellin is a ubiquitously expressed protein present in cytosolic and endoplasmic reticulum cell fractions. Overexpression or ablation of wild-type strumpellin caused significantly reduced wound closure velocities in wound healing assays, whereas overexpression of the disease-causing strumpellin N471D mutant showed no functional effect. Strumpellin knockdown experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth. Knockdown studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a presynaptic localization. We further identified strumpellin in pathological protein aggregates in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, various myofibrillar myopathies and in cortical neurons of a Huntington's disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that mutant forms of strumpellin and valosin-containing protein may have a concerted pathogenic role in various protein aggregate diseases.

Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo

“…To identify and define novel and pathology-related VCP interactors, we previously identified by coimmunoprecipitation several novel proteins that physically associate with VCP such as WASHC5, PSMF1 or WASHC4 [20]. Whereas the functional roles of WASHC5 and PSMF1 are partially characterized [19,20], the in vivo function of WASHC4 and in particular its role in striated muscle cells is still enigmatic.…”

“…Whereas the functional roles of WASHC5 and PSMF1 are partially characterized [19,20], the in vivo function of WASHC4 and in particular its role in striated muscle cells is still enigmatic. WASHC4 is an evolutionarily highly conserved protein that showed high amino acid identity between human, mouse and zebrafish orthologs (Figure S4).…”

“…We found cytoplasmic staining of WASHC4 which mostly colocalized with Vcp-eGFP (Figure 4(b-b”)), demonstrating that VCP and WASHC4 not only interact but also colocalize in HEK293T cells. To further investigate the subcellular distribution of WASHC4, we coimmunostained HEK293T cells using a WASHC4-specific antibody and an antibody against CANX (calnexin), an established marker of the endoplasmic reticulum (ER) [20]. We found that WASHC4 is present at the endoplasmic reticulum in HEK293T cells (Figure 4(c-c”)).…”

“…To investigate whether loss of Washc4, similar to the situation in Washc5-deficient zebrafish embryos [20], leads to neuronal defects, we conducted immunostaining of Washc4-deficient embryos using antibodies against acetylated tubulin and against the primary motor neuronal marker Syt2b (synaptotagmin IIb). In contrast to Washc5 morphants, Washc4 morphants showed no gross alterations in the development of the caudal neural tube or the ventral and caudal motoneurons compared to control-injected embryos (Figure 6(h) and (i) and Fig.…”

“…MFMs are severe striated muscle disorders often affecting both, heart and skeletal muscle cells [17,18]. The function of VCP critically depends on its association with specific cofactors such as UFD1 (ubiquitin recognition factor in ER associated degradation 1), NPLOC4/NPL4 (NPL4 homolog, ubiquitin recognition factor), PSMF1 (proteasome inhibitor subunit 1) or WASHC5/Strumpellin (WASH complex subunit 5) [19,20]. Recently, Washc4/Swip (WASH complex subunit 4), a member of the WASH (Wiskott-Aldrich syndrome protein and scar homolog) complex that contributes to actin polymerization and endosomal transport processes, has been identified as a novel VCP interactor in Dictyostelium discoideum [20].…”