An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.

Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.

A polyglutamine expansion located in the N terminus of huntingtin (N-htt) causes Huntington's disease (HD). How the mutation causes cell death is unknown. Several recent observations implicate altered huntingtin (htt) processing in the pathogenesis of HD. In the HD brain, mutant N-htt fragments aggregate in the nucleus and cytoplasm (1); the expression of mutant N-htt fragments in vitro causes cell death (2, 3). These findings suggest that proteolysis in the N-terminal region of htt may be important in HD pathogenesis. Furthermore, htt cleavage by caspase 3 could contribute to neurodegeneration in HD. Htt can serve as a substrate for caspase activity. Two proximate caspase 3-sensitive sites and one caspase 6-sensitive site are distal to the polyglutamine tract at aspartate residues 513, 552, and 589, respectively, in the wild-type (wt) protein (4-6). Caspase 3-cleaved N-htt products have been observed in vitro after exogenous expression of human htt in HEK 293 cells (4) and clonal striatal neurons (7). Treatment with the broad acting caspase inhibitor Z-VAD-FMK attenuates caspase 3 cleavage of htt and increases cell survival (6, 7). Enhanced immunoreactivity for caspases has been reported in HD striatal neurons compared with control brain, § supporting the involvement of caspase activation in HD pathogenesis. However, there is no evidence for htt proteolysis by caspases in the brain. No non-caspase proteases have been identified that produce a limited proteolysis of htt. Thus, despite the presence of N-htt fragments in adult and juvenile HD brain (1), the proteolytic pathway involved in the production of mutant N-htt fragments in the brain is unknown. That caspase 3 or other proteases cleave the N terminus of mutant htt in the HD brain would provide strong support that N-terminal htt proteolysis is a critical factor in HD pathogenesis.Here, we demonstrate that caspase 3-cleaved N-htt fragments occur in the control and HD brain and are similar in size to fragments produced in vitro after exogenous expression of human wt and mutant htt in mouse clonal striatal cells (7,8). The wt and mutant caspase 3-cleaved N-htt fragments in brain varied in size with polyglutamine length and were preferentially enriched in membrane fractions. Partial proteolysis of the caspase 3-cleaved N-htt fragments by calpain produced smaller Nterminal fragments. We speculate that caspase 3 cleavage regulates the proteolysis of wt and mutant htt in the brain and increases the association of N-terminal htt with membranes. Calpain-induced proteolysis of the caspase 3-cleaved mutant N-htt fragment may lead to the formation of mutant N-htt fragments that can aggregate and form inclusions. Materials and MethodsCell Culture and Transfections. The culturing and transfection of mouse clonal striatal cells (X57 cells) have been described in our recent publications (7,8,9). MCF-7 cells were grown according to the suppliers' recommendations [American Tissue Culture Collection (ATCC)]. DNA was introduced by using Superfect Transfection Reagent (Qiagen, ...

The N-terminus of mutant huntingtin (htt) has a polyglutamine expansion and forms neuronal aggregates in the brain of Huntington's disease (HD) patients. Htt expression in vitro activates autophagy, but it is unclear whether autophagic/lysosomal pathways process htt, especially N-terminal htt fragments. We explored the role of autophagy in htt processing in three cell lines, clonal striatal cells, PC12 cells and rodent embryonic cells lacking cathepsin D. Blocking autophagy raised levels of exogenously expressed htt1-287 or 1-969, reduced cell viability and increased the number of cells bearing mutant htt aggregates. Stimulating autophagy promoted htt degradation, including breakdown of caspase cleaved N-terminal htt fragments. Htt expression increased levels of the lysosomal enzyme cathepsin D by an autophagy-dependent pathway. Cells without cathepsin D accumulated more N-terminal htt fragments and cells with cathepsin D were more efficient in degrading wt htt than mutant htt in vitro. These results suggest that autophagy plays a critical role in the degradation of N-terminal htt. Altered processing of mutant htt by autophagy and cathepsin D may contribute to HD pathogenesis.