Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin.

The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1-4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.

IKKß and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD.

Neuronal interleukin-34 (IL-34) promotes the expansion of microglia in the central nervous system-microglial activation and expansion are in turn implicated in the pathogenesis of Huntington's disease (HD). We thus examined whether the accumulation of an amyloidogenic exon-1 fragment of mutant huntingtin (mHTTx1) modulates the expression of IL-34 in dopaminergic neurons derived from a human embryonic stem cell line. We found that mHTTx1 aggregates induce IL-34 production selectively in post-mitotic neurons. Exposure of neurons to DNA damaging agents or the excitotoxin NMDA elicited similar results suggesting that IL-34 induction may be a general response to neuronal stress including the accumulation of misfolded mHTTx1. We further determined that knockdown or blocking the activity of IκB kinase beta (IKKß) prevented the aggregation of mHTTx1 and subsequent IL-34 production. While elevated IL-34 itself had no effect on the aggregation or the toxicity of mHTTx1 in neuronal culture, IL-34 expression in a rodent brain slice model with intact neuron-microglial networks exacerbated mHTTx1-induced degeneration of striatal medium-sized spiny neurons. Conversely, an inhibitor of the IL-34 receptor reduced microglial numbers and ameliorated mHTTx1-mediated neurodegeneration. Together, these findings uncover a novel function for IKKß/mHTTx1 interactions in regulating IL-34 production, and implicate a role for IL-34 in non-cell-autonomous, microglial-dependent neurodegeneration in HD.

Antibodies and intrabodies against huntingtin: production and screening of monoclonals and single-chain recombinant forms.

Antibodies can be extremely useful tools for the field of triplet repeats diseases. These reagents are important for localizing proteins in tissues and they can be used in the isolation and characterization of the components of protein complexes. In the context of huntingtin (Htt), antibodies can distinguish Htt with normal or an expanded polyglutamine (polyQ) repeats, and they can identify distinct conformations of Htt. Htt is the protein that, when mutated to contain an expanded polyQ motif, causes Huntington's disease (HD). Our group has produced monoclonal and recombinant single-chain antibodies (intrabodies) that can be used for these purposes and to perturb the function of Htt in living cells. Studies with anti-Htt intrabodies have led to identification of novel pathogenic epitopes. Moreover, some of the isolated intrabodies can reduce the neurotoxicity of mutant Htt in cell culture and animal models of HD. Thus, the production of antibodies and intrabodies has made a significant contribution to the understanding of HD pathogenesis and has introduced a novel strategy to treat this debilitating neurodegenerative disorder.

Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease.

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 beta-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 AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD.

IKKalpha and IKKbeta regulation of DNA damage-induced cleavage of huntingtin.

BACKGROUND: Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington's disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IkappaB kinase (IKK), which can influence neurodegeneration in HD. METHODOLOGY/PRINCIPAL FINDINGS: We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or gamma-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80-90 kDa. This event requires IKKbeta and is suppressed by IKKalpha. Elevated levels of IKKalpha, or inhibition of IKKbeta expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKbeta phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKbeta expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKbeta, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis. CONCLUSIONS/SIGNIFICANCE: These results indicate that DNA damage triggers cleavage of Htt and identify IKKbeta as a prominent regulator. Moreover, IKKbeta-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKbeta may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders.

Monoclonal antibodies recognize distinct conformational epitopes formed by polyglutamine in a mutant huntingtin fragment.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt). PolyQ expansion above 35-40 results in disease associated with htt aggregation into inclusion bodies. It has been hypothesized that expanded polyQ domains adopt multiple potentially toxic conformations that belong to different aggregation pathways. Here, we used atomic force microscopy to analyze the effect of a panel of anti-htt antibodies (MW1-MW5, MW7, MW8, and 3B5H10) on aggregate formation and the stability of a mutant htt-exon1 fragment. Two antibodies, MW7 (polyproline-specific) and 3B5H10 (polyQ-specific), completely inhibited fibril formation and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These results suggest that expanded polyQ domains adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies, which has important implications for understanding the structural basis for polyQ toxicity and the development of intrabody-based therapeutics for HD.

Structures of Abeta-related peptide--monoclonal antibody complexes.

Passive immunotherapy (PI) is being explored as a potential therapeutic against Alzheimer's disease. The most promising antibodies (Abs) used in PI target the EFRH motif of the Abeta N-terminus. The monoclonal anti-Abeta Ab PFA1 recognizes the EFRH epitope of Abeta. PFA1 has a high affinity for Abeta fibrils and protofibrils (0.1 nM), as well as good affinity for Abeta monomers (20 nM). However, PFA1 binds the toxic N-terminally modified pyroglutamate peptide pyro-Glu3-Abeta with a 77-fold loss in affinity compared to the WT Abeta(1-8). Furthermore, our earlier work illustrated PFA1's potential for cross-reactivity. The receptor tyrosine kinase Ror2, which plays a role in skeletal and bone formation, possesses the EFRH sequence. PFA1 Fab binds the Ror2(518-525) peptide sequence REEFRHEA with a 3-fold enhancement over WT Abeta(1-8). In this work, the crystal structures of the hybridoma-derived PFA1 Fab in complex with pyro-Glu3-Abeta peptide and with a cross-reacting peptide from Ror2 have been determined at resolutions of 1.95 and 2.7 A, respectively. As with wild-type Abeta, these peptides bind to the Fab via a combination of charge- and shape-complementarity, hydrogen-bonding, and hydrophobic interactions. Comparison of the structures of the four peptides Abeta(1-8), Grip1, pyro-Glu3-Abeta(3-8), and Ror2 in complex with PFA1 shows that the greatest conformational flexibility occurs at residues 2 to 3 and 8 of the peptide. These structures provide a molecular basis of the specificity tolerance of PFA1 and its ability to recognize Abeta N-terminal heterogeneity. The structures provide clues to improving mAb specificity and affinity for pyroglutamate Abeta.

Molecular basis for passive immunotherapy of Alzheimer's disease.

Amyloid aggregates of the amyloid-beta (Abeta) peptide are implicated in the pathology of Alzheimer's disease. Anti-Abeta 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-Abeta mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize Abeta monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the Abeta(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 Abeta(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 Abeta with improved specificity and higher affinity.

Activation of the IkappaB kinase complex and nuclear factor-kappaB contributes to mutant huntingtin neurotoxicity.

Transcriptional dysregulation by mutant huntingtin (Htt) protein has been implicated in the pathogenesis of Huntington's disease (HD). We find that cultured cells expressing mutant Htt and striatal cells from HD transgenic mice have elevated nuclear factor-kappaB (NF-kappaB) activity. Furthermore, NF-kappaB is concentrated in the nucleus of neurons in the brains of HD transgenic mice. In inducible PC12 cells and in HD transgenic mice, mutant Htt activates the IkappaB kinase complex (IKK), a key regulator of NF-kappaB. Activation of IKK is likely mediated by direct interaction with mutant Htt, because the expanded polyglutamine stretch and adjacent proline-rich motifs in mutant Htt interact with IKKgamma, a regulatory subunit of IKK. Activation of IKK may also influence the toxicity of mutant Htt, because expression of IKKgamma promotes aggregation and nuclear localization of mutant Htt exon-1. Moreover, in acute striatal slice cultures, inhibition of IKK activity with an N-terminally truncated form of IKKgamma blocks mutant Htt-induced toxicity in medium-sized spiny neurons (MSNs). In addition, blocking degradation of NF-kappaB inhibitors with a dominant-negative ubiquitin ligase beta-transducin repeat-containing protein also reduces the toxicity of mutant Htt in MSNs. Therefore, aberrant NF-kappaB activation may contribute to the neurodegeneration induced by mutant Htt.

Antibodies against huntingtin: production and screening of monoclonals and single-chain recombinant forms.

Antibodies can be extremely useful tools for the field of triplet repeat diseases. These reagents are important for localizing proteins in tissues, and within cells, they can be used in the isolation and characterization of the components of protein complexes, they can distinguish proteins with normal or an expanded polyglutamine repeat, they may be able to distinguish distinct conformations of a protein, and they can be used to perturb the function of proteins in living cells. Our group has produced monoclonal and recombinant single-chain antibodies that can be used for each of these purposes with huntingtin. This is the protein that, when mutated to contain an expanded polyQ motif, causes Huntington's disease.

Neonatal impact of leukemia inhibitory factor on neurobehavioral development in rats.

Cytokines have been implicated in the etiology or pathology of various psychiatric diseases of developmental origin such as autism and schizophrenia. Leukemia inhibitory factor (LIF) is induced by a variety of brain insults and known to have many influences on mature and immature nervous system. Here, we assessed the neurobehavioral and pathological consequences of peripheral administration of LIF in newborn rats. Subcutaneous LIF injection induced STAT3 phosphorylation in many brain regions and increased glial fibrillary acidic protein (GFAP) immunoreactivity in the neocortex, suggesting that LIF had direct effects in the central nervous system. The LIF-treated rats displayed decreased motor activity during juvenile stages, and developed abnormal prepulse inhibition in the acoustic startle test during and after adolescence. They displayed normal learning ability in active avoidance test, however. Brain neuronal structures and startle responses were grossly normal, except for the cortical astrogliosis during neonatal LIF administration. These results indicate that LIF induction in the periphery of the infant has a significant, but discrete impact on neurobehavioral development.

Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity.

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.