Amplification of neurotoxic HTTex1 assemblies in human neurons.

Huntington's disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.

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.

DJ-1 modulates aggregation and pathogenesis in models of Huntington's disease.

The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimer's disease and Huntington's disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.

Gut Microbiota as a Modifier of Huntington's Disease Pathogenesis.

Huntingtin (HTT) protein is expressed in most cell lineages, and the toxicity of mutant HTT in multiple organs may contribute to the neurological and psychiatric symptoms observed in Huntington's disease (HD). The proteostasis and neurotoxicity of mutant HTT are influenced by the intracellular milieu and responses to environmental signals. Recent research has highlighted a prominent role of gut microbiota in brain and immune system development, aging, and the progression of neurological disorders. Several studies suggest that mutant HTT might disrupt the homeostasis of gut microbiota (known as dysbiosis) and impact the pathogenesis of HD. Dysbiosis has been observed in HD patients, and in animal models of the disease it coincides with mutant HTT aggregation, abnormal behaviors, and reduced lifespan. This review article aims to highlight the potential toxicity of mutant HTT in organs and pathways within the microbiota-gut-immune-central nervous system (CNS) axis. Understanding the functions of Wild-Type (WT) HTT and the toxicity of mutant HTT in these organs and the associated networks may elucidate novel pathogenic pathways, identify biomarkers and peripheral therapeutic targets for HD.

IKKß signaling mediates metabolic changes in the hypothalamus of a Huntington disease mouse model.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Metabolic changes are associated with HD progression, but underlying mechanisms are not fully known. As the IKKß/NF-κB pathway is an essential regulator of metabolism, we investigated the involvement of IKKß, the upstream activator of NF-κB in hypothalamus-specific HD metabolic changes. We expressed amyloidogenic N-terminal fragments of mutant HTT (mHTT) in the hypothalamus of mice with brain-specific ablation of IKKß (Nestin/IKKßlox/lox) and control mice (IKKßlox/lox). We assessed effects on body weight, metabolic hormones, and hypothalamic neuropathology. Hypothalamic expression of mHTT led to an obese phenotype only in female mice. CNS-specific inactivation of IKKß prohibited weight gain in females, which was independent of neuroprotection and microglial activation. Our study suggests that mHTT in the hypothalamus causes metabolic imbalance in a sex-specific fashion, and central inhibition of the IKKß pathway attenuates the obese phenotype.

Gut Bacteria Regulate the Pathogenesis of Huntington's Disease in Drosophila Model.

Changes in the composition of gut microbiota are implicated in the pathogenesis of several neurodegenerative disorders. Here, we investigated whether gut bacteria affect the progression of Huntington's disease (HD) in transgenic Drosophila melanogaster (fruit fly) models expressing full-length or N-terminal fragments of human mutant huntingtin (HTT) protein. We find that elimination of commensal gut bacteria by antibiotics reduces the aggregation of amyloidogenic N-terminal fragments of HTT and delays the development of motor defects. Conversely, colonization of HD flies with Escherichia coli (E. coli), a known pathobiont of human gut with links to neurodegeneration and other morbidities, accelerates HTT aggregation, aggravates immobility, and shortens lifespan. Similar to antibiotics, treatment of HD flies with small compounds such as luteolin, a flavone, or crocin a beta-carotenoid, ameliorates disease phenotypes, and promotes survival. Crocin prevents colonization of E. coli in the gut and alters the levels of commensal bacteria, which may be linked to its protective effects. The opposing effects of E. coli and crocin on HTT aggregation, motor defects, and survival in transgenic Drosophila models support the involvement of gut-brain networks in the pathogenesis of HD.

Corrigendum: Gut bacteria regulate the pathogenesis of Huntington's disease in Drosophila model.

[This corrects the article DOI: 10.3389/fnins.2022.902205.].

The role of IκB kinase complex in the neurobiology of Huntington's disease.

The IκB kinase ß (IKKß) is a prominent regulator of neuroinflammation, which is implicated in the pathogenesis of Huntington's disease (HD). Inflammatory mediators accumulate in the serum and CNS of premanifest and manifest HD patients, and cytokine levels correlate with disease progression. IKKß may also directly regulate the neurotoxicity of huntingtin (Htt). Activation of IKKß by DNA damage triggers caspase-dependent cleavage of WT and mutant Htt and enhances the accumulation of oligomeric fragments. Moreover, the N-terminal fragments of mutant Htt (HDx1) directly bind to and activate IKKß. Thus, the IKKß-dependent cleavage of full-length mutant Htt and the buildup of HDx1 could form a deleterious feed-forward loop. Elevated IKKß activity is present throughout the CNS in a symptomatic mouse model of HD expressing HDx1, whereas in asymptomatic mice with full-length mutant Htt, it is confined to the striatum. IKKß could also influence the phosphorylation of Htt at Ser13 and Ser16, which is linked to HD pathology. IKKß inhibitors ameliorate the toxicity of mutant Htt in striatal neurons and prevent DNA damage-induced Htt cleavage. Inhibition of IKKß in the CNS also reduces neuroinflammation and imparts neuroprotection in a chemical model of HD. These findings support an active role for IKKß in HD pathogenesis and represent an example of how gene-environment (exemplified by DNA damage and inflammation) interactions can influence Htt neurotoxicity. We will summarize these findings and describe the therapeutic potentials of IKKß for HD.

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington's Disease Knock-in Mice.

The deposition of mutant huntingtin (mHTT) protein aggregates in neurons of patients is a pathological hallmark of Huntington's disease (HD). Previous investigations in cell-free and cell-based disease models showed mHTT exon-1 (mHTTex1) fragments with pathogenic polyglutamine (polyQ) tracts (>40 glutamines) to self-assemble into highly stable, ß-sheet-rich protein aggregates with a fibrillar morphology. HD knock-in mouse models have not been extensively studied with regard to mHTT aggregation. They endogenously produce full-length mHTT with a pathogenic polyQ tract as well as mHTTex1 fragments. Here, we demonstrate that seeding-competent, fibrillar mHTT aggregates can be readily detected in brains of zQ175 knock-in HD mice. To do this, we applied a highly sensitive FRET-based protein amplification assay that is capable of detecting seeding-competent mHTT aggregate species down to the femtomolar range. Furthermore, we show that fibrillar structures with an average length of ∼200 nm can be enriched with aggregate-specific mouse and human antibodies from zQ175 mouse brain extracts through immunoprecipitations, confirming that such structures are formed in vivo. Together these studies indicate that small, fibrillar, seeding-competent mHTT structures are prominent aggregate species in brains of zQ175 mice.

Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity.

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as V(L)12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which V(L)12.3 does not change. In contrast, expression of V(L)12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.

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.

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.

Elevated IKKα accelerates the differentiation of human neuronal progenitor cells and induces MeCP2-dependent BDNF expression.

The IκB kinase α (IKKα) is implicated in the differentiation of epithelial and immune cells. We examined whether IKKα also plays a role in the differentiation and maturation of embryonic human neuronal progenitor cells (NPCs). We find that expression of an extra copy of IKKα (IKKα+) blocks self-renewal and accelerates the differentiation of NPCs. This coincides with reduced expression of the Repressor Element Silencing Transcription Factor/Neuron-Restrictive Silencing Factor (REST/NRSF), which is a prominent inhibitor of neurogenesis, and subsequent induction of the pro-differentiation non-coding RNA, miR-124a. However, the effects of IKKα on REST/NRSF and miR-124a expression are likely to be indirect. IKKα+ neurons display extensive neurite outgrowth and accumulate protein markers of neuronal maturation such as SCG10/stathmin-2, postsynaptic density 95 (PSD95), syntaxin, and methyl-CpG binding protein 2 (MeCP2). Interestingly, IKKα associates with MeCP2 in the nuclei of human neurons and can phosphorylate MeCP2 in vitro. Using chromatin immunoprecipitation assays, we find that IKKα is recruited to the exon-IV brain-derived neurotrophic factor (BDNF) promoter, which is a well-characterized target of MeCP2 activity. Moreover, IKKα induces the transcription of BDNF and knockdown expression of MeCP2 interferes with this event. These studies highlight a role for IKKα in accelerating the differentiation of human NPCs and identify IKKα as a potential regulator of MeCP2 function and BDNF expression.

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.

IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome.

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species.

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.