INTRODUCTION
Neurodegenerative disorders include a number of diseases caused by progressive loss of neurons. A hallmark of most neurodegenerative diseases is aberrant accumulation and aggregation of misfolded proteins inside neurons, including mutant huntingtin (mHTT) aggregates in Huntington’s disease (HD) (1 ), mutant ataxin-3 in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) (2 ), Amyloid-beta (Aβ or Abeta) in Alzheimer’s disease (which also has extracellular aggregates) (3 ), and α-synuclein in Parkinson’s disease (4 ). HD is an appealing model for pathological study as well as for drug discovery for degenerative diseases because of its simplicity in genetics and its relatively high prevalence (5 ). It is an autosomal dominant neurodegenerative disorder caused by toxic aggregates of mutant HTT proteins (mHTT) in the brain (6 ). HTT exon1 contains a polyglutamine (polyQ) tract that consists of 6–35 glutamine residues in normal neurons. Mutant HTT proteins with expansions greater than 35 glutamine residues induce protein misfolding and are more prone to aggregation, leading to onset of HD. The ability of neuron to target these aggregates for degradation is insufficient, causing proteins accumulation, aggregation and protein dyshomeostasis, a common thread in neurodegenerative diseases (7 ). mHTT abundance and proteostasis are indicators of neurodegeneration and disease severity (8 ). Thus, induction of degradation of disease-causing proteins should prevent aggregate accumulation and neuronal cell death, representing a general therapeutic strategy for HD and other neurodegenerative disorders.
Valosin-containing protein (VCP/p97) is a central and important regulator of proteostasis, which has been implicated in degenerative diseases (9). Chaperone VCP protein (p97 in mouse, TER94 in Drosophila melanogaster, and CDC48 in S. cerevisiae) belongs to the AAA protein family ATPase (10 ). VCP is composed of N terminal domain, followed by tandem D1 and D2 ATPase domain joined by a linker region. VCP forms a hexametric ring in cylindrical structure with the N-terminal domain devoted to allow VCP interaction with adaptor or substrate proteins. VCP regulates degradation of damaged proteins and organelles in proteasome and autophagic pathway (11,12,13 ) and has been linked both directly and indirectly to neurodegenerative disorders. In HD disease as well as other polyQ-related diseases, endogenous VCP co-localizes with the polyQ containing aggregates in cultured polyQ-expressing neuronal cells (14 ). Colocalization of VCP with disease-causing aggregates has been reported in not only Huntington disease but also Machado–Joseph disease (MJD) and Parkinson disease (15,16 ). Furthermore, Missense mutations in the VCP gene has been implicated in pathogenesis of human degenerative disorders including IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), and rare cases of familial amyotrophic lateral sclerosis (ALS) (9 ). These results have emphasized the importance of VCP in the pathology of human neurodegenerative disorders and raised the possibility of targeting VCP to treat HD as well as other neurodegenerative disorders.
One of the crucial mechanisms for clearance of the disease-causing mHTT protein in HD is macroautophagy (hereafter autophagy). Autophagy is a highly conserved cellular process which engulfs cytosolic components for degradation, including damaged organelles, misfolded proteins and aggregates, to maintain cytosolic homeostasis (17 ). It mediates the degradation of large disease proteins or aggregates that cause neurodegenerative diseases, which are mostly inaccessible to proteasome (18 ). In HD, the abnormal accumulation of mHTT proteins arises from insufficiency of the autophagy machinery (19). Strategies that induce moderate autophagy upregulation are believed to be of therapeutic benefit in treatment of HD as well as other neurodegenerative diseases (20,21 ). Microtubule-associated protein 1A/1B-light chain 3 (LC3), the core autophagy component in mammalian cells, plays a pivotal role in the autophagy-lysosome pathway, regulating both autophagosome formation and cargo recognition (22 ). LC3 has been demonstrated to recognize protein aggregates for autophagy removal (23 ). It has been reported that LC3 colocalizes with mHTT aggregates and facilitated mHTT protein degradation by autophagy (24, 25 ). VCP could interacts with LC3 (26) and has also been reported to co-localize with mHTT (14 ), indicating potential involvement of VCP in autophagy-mediated mHTT degradation. However, the role of VCP in autophagy-mediated mHTT clearance remains undefined
To explore the potential treatment of HD by small molecules targeting VCP, we established a high-throughput screening assay using VCP’s ATPase activity as readout and identified gossypol acetate as a novel modulator of VCP. Gossypol acetate acted in a gain-of-function way to induce mHTT-VCP-LC3 complex formation, enabling autophagy-mediated degradation of endogenous mHTT. Gossypol acetate reduced mHTT levels, alleviated neuronal toxicity in HD cells and improved behaviors in Drosophila and mouse HD models, making gossypol a promising lead for the development of new HD therapy.