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

Huntingtin, the protein product of the Huntington's disease (HD) gene, is e xpressed with an expanded polyglutamine domain in the brain and in nonneuro nal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin an d thus may be important in HD pathogenesis. The function of HAP-1 is unknow n, but recent evidence supports a role in microtubule-dependent organelle t ransport. We examined the subcellular localization of HAP-1 with an antibod y made against the NH2-terminus of the protein. In immunoblot assays of mou se 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 a pparatus, especially at spindle poles and on vesicles and microtubules of t he spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling t hroughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clo nal striatal cells showed that HAP-1B was preferentially enriched in membra ne fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including lar ge 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 MAP-I in vesicle trafficking and organelle movement in mitotic c ells and differentiated neurons and implicate HAP-1B as the predominant mol ecular subtype associated with vesicle membranes in striatal neurons. (C) 1 999 Wiley-Liss, Inc.