Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V L 12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken -actin promoter. V L 12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAVHapp1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD.
Amyloid aggregates of the amyloid- (A) peptide are implicated in the pathology of Alzheimer's disease. Anti-A monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-A mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize A monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the A(1-8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to A(1-8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target A with improved specificity and higher affinity.amyloid ͉ crystal structure ͉ EFRH ͉ monoclonal antibody ͉ EFRHD
We have generated eight mAbs (MW1-8) that bind the epitopes polyglutamine (polyQ), polyproline (polyP), or the C terminus of exon 1 in huntingtin (htt) protein. In the brains of Huntington's disease (HD) mouse models, the anti-polyQ mAbs bind to various cytoplasmic compartments, whereas the anti-polyP and anti-C terminus mAbs bind nuclear inclusions containing htt. To use these mAbs as intracellular perturbation agents, we have cloned and expressed the antigen-binding domains of three of the mAbs as single-chain variable region fragment Abs (scFvs). In 293 cells cotransfected with htt exon 1 containing an expanded polyQ domain, MW1, MW2, and MW7 scFvs colocalize with htt exon 1. Moreover, these scFvs coimmunoprecipitate with htt exon 1 in cell extracts. In perturbation experiments, MW7 scFv, recognizing the polyP domains of htt, significantly inhibits aggregation as well as the cell death induced by mutant htt protein. In contrast, MW1 and MW2 scFvs, recognizing the polyQ stretch, stimulate htt aggregation and apoptosis. Therefore, these anti-htt scFvs can be used to investigate the role of the polyP and polyQ domains in HD pathogenesis, and antibody binding to the polyP domain has potential therapeutic value in HD. H untington's disease (HD), a fatal neurodegenerative disorder, is caused by abnormal expansion of CAG repeats that translate into an extended polyglutamine (polyQ) stretch in exon 1 of the protein huntingtin (htt) (1). Mutant htt with Ͼ40 CAG repeats gains a toxic function and induces death in subpopulations of neurons in the striatum and cortex (2-4). A hallmark of HD and other polyQ diseases is the formation of insoluble protein aggregates in affected neurons (5, 6). A major component of the aggregates in HD is the N terminus exon 1 of mutant htt (2,(5)(6)(7)(8). Abnormal behavior and aggregate formation are also seen in transgenic mice expressing htt exon 1 with an expanded polyQ stretch (9-11).Neuronal death in HD has been variously attributed to polyQ toxicity, activation of caspases, interference with transcriptional machinery, and sequestration͞inactivation of wild-type htt and other important cellular factors (12-17). Several proteins that interact with exon 1 of htt have been identified (14,(18)(19)(20)(21)(22)(23), and although the function of these proteins in the etiology of HD is unclear, the transcriptional coactivator CREB-binding protein (CBP) as well as proteins with WW domains have been implicated in the HD pathology (18-21). Binding of htt to CBP has been shown to repress CBP-mediated gene expression (18,19). Moreover, ectopic expression of CBP appears to block httmediated toxicity, indicating that transcriptional dysregulation may contribute to HD pathogenesis (19,24). Several different WW-containing proteins have been shown to interact with proline-rich domains in the C terminus of htt exon 1 (20, 21). These interactors include spliceosomes (HYPA and HYPC) and transcription factors (HYPB), which appear to have a higher affinity for expanded polyQ htt (20). By using specific anti...
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