Analysis of huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons

Abstract

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin‐associated protein 1 (HAP‐1), a brain‐enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP‐1 is unknown, but recent evidence supports a role in microtubule‐dependent organelle transport. We examined the subcellular localization of HAP‐1 with an antibody made against the NH2‐terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP‐1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP‐1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP‐1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP‐1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP‐1 localized to membrane‐bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP‐1 was also strongly associated with an unusual large “dense” organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP‐1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP‐1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons. J. Comp. Neurol. 403:421–430, 1999. © 1999 Wiley‐Liss, Inc.

Huntingtin-associated Protein 1 Interacts with Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate and Functions in Endosomal Trafficking

“…By comparison, the subcellular localization of HAP1 has not yet been fully characterized. Previous electron microscopic studies demonstrate that HAP1 is associated with endosomes and synaptic vesicles as well as other types of tubulovesicular structures and membranous organelles (8,9). Consistent with these observations, our results of immunofluorescence and subcellular fractionation studies reveal that in PC12 cells, endogenous HAP1 was primarily associated with early endosomes and a significant percentage of HAP1 co-localizes with Hrs.…”

“…Biochemically, HAP1 has been shown to interact with p150 glued , a dynactin subunit that participates in microtubule-based transport (4,5), and with Duo/Kalirin, a Rac1 guanine nucleotide exchange factor that regulates actin cytoskeleton dynamics (6,7), although the functional role of these interactions has not yet been examined. Electron microscopic studies reveal that HAP1 is associated with many types of membranous organelles, including endosomes, tubulovesicular structures, and synaptic vesicles (8,9). Based on these observations, it has been speculated that HAP1 may have a role in vesicular trafficking and/or organelle transport (4,6,8).…”

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A receptor membrane trafficking

“…HAP1 exhibited clusters in the dendrites and axons, in addition to substantial intracellular somatic staining, including large perinuclear structures consistent with the localization of this protein to intracellular compartments such as endosomes and tubulovesicular structures (Fig. 2G), as demonstrated (23)(24)(25)(26). The GABA A R ␥2 subunit appeared as membrane clusters in addition to abundant intracellular aggregates (Fig.…”

“…GABA A Rs undergo clathrin-mediated endocytosis and can be detected in endocytic structures (1,8,10,11,27). HAP1 has been localized to clathrin-coated vesicles and the endocytic pathway (23)(24)(25)(26) and has been implicated in endocytic protein trafficking (26), suggesting that HAP1 may be involved in the endocytic sorting of GABA A Rs. To further examine the effects of HAP1 on the endocytic sorting of GABA A Rs, cultured cortical neurons were nucleofected with a HAP1 cDNA to modify HAP1 expression levels.…”

DYRK1A regulates Hap1–Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome

“…Hap1 was initially recognized for its binding to polyglutamine-expanded huntingtin (Htt), the protein responsible for Huntington's disease (3). Because Hap1 is expressed at various levels in different types of neurons (1,(4)(5)(6)(7)(8), it is likely involved in cell type-specific functions of the brain. For example, Hap1 is abundantly expressed in the hypothalamus, and its expression in the hypothalamus influences the central control of feeding (9); as a direct result, mice lacking Hap1 are malnourished and die in the early postnatal period (5, 10, 11).…”