The gene defective in Huntington's disease encodes a protein, huntingtin, with unknown function. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking.

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.

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.