Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.
Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This human pathogen of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. Upon binding to a host receptor on the cell surface, the virus begins a two-step uncoating process, first forming an expanded, altered “A-particle”, which is primed for genome release. In a second step after endocytosis, an unknown trigger leads to RNA expulsion, generating an intact, empty capsid. Cryo-electron microscopy reconstructions of these two capsid states provide insight into the mechanics of genome release. The EV71 A-particle capsid interacts with the genome near the icosahedral two-fold axis of symmetry, which opens to the external environment via a channel ∼10 Å in diameter that is lined with patches of negatively charged residues. After the EV71 genome has been released, the two-fold channel shrinks, though the overall capsid dimensions are conserved. These structural characteristics identify the two-fold channel as the site where a gateway forms and regulates the process of genome release.
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.
We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids. Huntingtin (htt)2 exists predominantly in the cytoplasm as a soluble protein that associates with the plasma membrane and multiple membranous organelles and vesicles (1-3). Many htt binding partners function in membrane trafficking (4). A specific molecular function for htt at membranes has not been demonstrated. The large size of htt (348 kDa) and interactions with numerous membrane-associated proteins suggest that htt may function as a scaffold.A polyglutamine expansion in the N terminus of htt (N-htt) causes neurodegeneration in Huntington disease (HD) and accumulation of htt in neurons. Degradation pathways for htt include endosomal-lysosomal and autophagic pathways and may require targeting to membranes to initiate clearance (5, 6). Therefore, knowledge of the effects of polyglutamine expansion on htt membrane targeting is important for understanding HD pathogenesis.N-htt has a membrane association domain. Membrane fractions prepared from control and HD brains (7) or prepared from cells expressing exogenous htt (5) contain N-htt fragments (ϳ440 -550 aa). In vitro translated normal and mutant htt (aa 1-548) localized to vesicles in extruded squid axoplasm (8). The structural features that mediate membrane association of N-htt are unknown and could include a proline-rich Src homology 3-binding domain that lies immediately distal to the polyglutamine stretch and HEAT domains, which are repeated regions of low homology shared by huntingtin with elongation factor 3, the p65 regulatory A subunit of protein phosphatase 2A, and TOR1 and a host of other proteins (9). The function of HEAT domains is unknown, but these repeated regions are leucine-rich and predicted to be ␣-helical in nature, with each repeat consisting of two helices and a short intervening linker. Crystallography studies of the protein phosphatase 2A p65 regulatory subunit (PR65/A) indicate that its multiple HEAT domains create a superhelical structure (10). htt has three domains at aa 205-329, 745-942, and 1534 -1710 containing a total of 10 HEAT repeats (9).In this study, we identify sequences in N-htt important for binding to membranes, explore mechanisms by which this bindin...
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