KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS.

Sirtuin 2 (SIRT2) is one of seven known mammalian protein deacetylases homologous to the yeast master lifespan regulator Sir2. In recent years, the sirtuin protein deacetylases have emerged as candidate therapeutic targets for many human diseases, including metabolic and age-dependent neurological disorders. In non-neuronal cells, SIRT2 has been shown to function as a tubulin deacetylase and a key regulator of cell division and differentiation. However, the distribution and function of the SIRT2 microtubule (MT) deacetylase in differentiated, postmitotic neurons remain largely unknown. Here, we show abundant and preferential expression of specific isoforms of SIRT2 in the mammalian central nervous system and find that a previously uncharacterized form, SIRT2.3, exhibits age-dependent accumulation in the mouse brain and spinal cord. Further, our studies reveal that focal areas of endogenous SIRT2 expression correlate with reduced α-tubulin acetylation in primary mouse cortical neurons and suggest that the brain-enriched species of SIRT2 may function as the predominant MT deacetylases in mature neurons. Recent reports have demonstrated an association between impaired tubulin acetyltransferase activity and neurodegenerative disease; viewed in this light, our results showing age-dependent accumulation of the SIRT2 neuronal MT deacetylase in wild-type mice suggest a functional link between tubulin acetylation patterns and the aging brain.

Cysteine oxidation within N-terminal mutant huntingtin promotes oligomerization and delays clearance of soluble protein.

Huntington disease (HD) is a progressive neurodegenerative disorder caused by expression of polyglutamine-expanded mutant huntingtin protein (mhtt). Most evidence indicates that soluble mhtt species, rather than insoluble aggregates, are the important mediators of HD pathogenesis. However, the differential roles of soluble monomeric and oligomeric mhtt species in HD and the mechanisms of oligomer formation are not yet understood. We have shown previously that copper interacts with and oxidizes the polyglutamine-containing N171 fragment of huntingtin. In this study we report that oxidation-dependent oligomers of huntingtin form spontaneously in cell and mouse HD models. Levels of these species are modulated by copper, hydrogen peroxide, and glutathione. Mutagenesis of all cysteine residues within N171 blocks the formation of these oligomers. In cells, levels of oligomerization-blocked mutant N171 were decreased compared with native N171. We further show that a subset of the oligomerization-blocked form of glutamine-expanded N171 huntingtin is rapidly depleted from the soluble pool compared with "native " mutant N171. Taken together, our data indicate that huntingtin is subject to specific oxidations that are involved in the formation of stable oligomers and that also delay removal from the soluble pool. These findings show that inhibiting formation of oxidation-dependent huntingtin oligomers, or promoting their dissolution, may have protective effects in HD by decreasing the burden of soluble mutant huntingtin.

Discovery of bioactive small-molecule inhibitor of poly adp-ribose polymerase: implications for energy-deficient cells.

Poly (ADP-ribose) polymerase (PARP1) is a nuclear protein that, when overactivated by oxidative stress-induced DNA damage, ADP ribosylates target proteins leading to dramatic cellular ATP depletion. We have discovered a biologically active small-molecule inhibitor of PARP1. The discovered compound inhibited PARP1 enzymatic activity in vitro and prevented ATP loss and cell death in a surrogate model of oxidative stress in vivo. We also investigated a new use for PARP1 inhibitors in energy-deficient cells by using Huntington's disease as a model. Our results showed that insult with the oxidant hydrogen peroxide depleted cellular ATP in mutant cells below the threshold of viability. The protective role of PARP1 inhibitors against oxidative stress has been shown in this model system.

Sp1 is up-regulated in cellular and transgenic models of Huntington disease, and its reduction is neuroprotective.

Interactions between mutant huntingtin (Htt) and a variety of transcription factors including specificity proteins (Sp) have been suggested as a central mechanism in Huntington disease (HD). However, the transcriptional activity induced by Htt in neurons that triggers neuronal death has yet to be fully elucidated. In the current study, we characterized the relationship of Sp1 to Htt protein aggregation and neuronal cell death. We found increased levels of Sp1 in neuronal-like PC12 cells expressing mutant Htt, primary striatal neurons, and brain tissue of HD transgenic mice. Sp1 levels were also elevated when 3-nitropropionate (3-NP) was used to induce cell death in PC12 cells. To assess the effects of knocking down Sp1 in HD pathology, we used Sp1 siRNA, a heterozygous Sp1 knock-out mouse, and mithramycin A, a DNA-intercalating agent that inhibits Sp1 function. The three approaches consistently yielded reduced levels of Sp1 which ameliorated toxicity caused by either mutant Htt or 3-NP. In addition, when HD mice were crossed with Sp1 heterozygous knock-out mice, the resulting offspring did not experience the loss of dopamine D2 receptor mRNA characteristic of HD mice, and survived longer than their HD counterparts. Our data suggest that enhancement of transcription factor Sp1 contributes to the pathology of HD and demonstrates that its suppression is beneficial.

Cystamine increases L-cysteine levels in Huntington's disease transgenic mouse brain and in a PC12 model of polyglutamine aggregation.

Cystamine, a small disulfide-containing chemical, is neuroprotective in a transgenic mouse and a Drosophila model of Huntington's disease (HD) and decreases huntingtin aggregates in an in vitro model of HD. The mechanism of action of cystamine in these models is widely thought to involve inhibition of transglutaminase mediated cross-linking of mutant huntingtin in the process of aggregate formation/stabilization. In this study we show that cystamine, both in vitro and in a transgenic mouse model of HD (R6/2), increases levels of the cellular antioxidant L-cysteine. Several oxidative stress markers increase in HD brain. We provide further evidence of oxidative stress in mouse HD by demonstrating compensatory responses in R6/2 HD brains. We found age-dependent increases in forebrain glutathione (GSH), and increased levels of transcripts coding for proteins involved in GSH synthesis and detoxification pathways, as revealed by quantitative PCR analysis. Given the general importance of oxidative stress as a mediator of neurodegeneration we propose that an increase in brain L-cysteine levels could be protective in HD. Furthermore, cystamine was dramatically protective against 3-nitropropionic acid-induced striatal injury in mice. We suggest that cystamine's neuroprotective effect in HD transgenic mice results from pleiotropic effects that include transglutaminase inhibition and antioxidant activity.

Huntington's disease: prospects for neuroprotective therapy 10 years after the discovery of the causative genetic mutation.

PURPOSE OF REVIEW: Ten years of intensive research are now beginning to bring candidate neuroprotective therapies to clinical trials. This review describes recent progress in basic, preclinical, and clinical research that underlies current and potential neuroprotective trials. RECENT FINDINGS: Basic research continues to elucidate the proteolytic processing of huntingtin into toxic fragments and has examined the toxic potential of huntingtin monomers versus oligomers versus insoluble aggregates. Energy depletion has been reinvigorated as a therapeutic target by studies identifying very early mitochondrial alterations. Toxic interactions between mutant huntingtin and a variety of transcription factors have emerged as a major focus with a variety of studies suggesting transcriptional dysfunction to be a central mechanism in Huntington's disease. Progress in preclinical research included therapeutic leads identified by compound library screens, by designing polypeptides that can interact with huntingtin, and by testing compounds in transgenic mice with the potential for affecting some of the mechanisms thought to underlie neurodegeneration. While early results of neurotransplantation are generating increasing controversy, a variety of compounds discovered to benefit transgenic mice are working their way into clinical trials in symptomatic patients. Studies in presymptomatic individuals at risk for developing Huntington's disease are underway to enable the testing of agents with the potential for delaying or preventing onset of symptoms. SUMMARY: While laboratory research continues to advance and provide therapeutic leads, clinical trials are needed to test existing leads and guide further progress. With any luck, some of these tests will begin to identify treatments that make a difference for families with the disease.

Creatine therapy provides neuroprotection after onset of clinical symptoms in Huntington's disease transgenic mice.

While there have been enormous strides in the understanding of Huntington's disease (HD) pathogenesis, treatment to slow or prevent disease progression remains elusive. We previously reported that dietary creatine supplementation significantly improves the clinical and neuropathological phenotype in transgenic HD mice lines starting at weaning, before clinical symptoms appear. We now report that creatine administration started after onset of clinical symptoms significantly extends survival in the R6/2 transgenic mouse model of HD. Creatine treatment started at 6, 8, and 10 weeks of age, analogous to early, middle, and late stages of human HD, significantly extended survival at both the 6- and 8-week starting points. Significantly improved motor performance was present in both the 6- and 8-week treatment paradigms, while reduced body weight loss was only observed in creatine-supplemented R6/2 mice started at 6 weeks. Neuropathological sequelae of gross brain and neuronal atrophy and huntingtin aggregates were delayed in creatine-treated R6/2 mice started at 6 weeks. We show significantly reduced brain levels of both creatine and ATP in R6/2 mice, consistent with a bioenergetic defect. Oral creatine supplementation significantly increased brain concentrations of creatine and ATP to wild-type control levels, exerting a neuroprotective effect. These findings have important therapeutic implications, suggesting that creatine therapy initiated after diagnosis may provide significant clinical benefits to HD patients.

HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis.

Huntington disease (HD) is caused by polyglutamine [poly(Q)] expansion in the protein huntingtin (htt). Although the exact mechanism of disease progression remains to be elucidated, altered interactions of mutant htt with its protein partners could contribute to the disease. Using the yeast two-hybrid system, we have isolated a novel htt interacting protein, HIP14. HIP14's interaction with htt is inversely correlated to the poly(Q) length in htt. mRNAs of 9 and 6 bp are transcribed from the HIP14 gene, with the 6 kb transcript being predominantly expressed in the brain. HIP14 protein is enriched in the brain, shows partial co-localization with htt in the striatum, and is found in medium spiny projection neurons, the subset of neurons affected in HD. HIP14 localizes to the Golgi, and to vesicles in the cytoplasm. The HIP14 protein has sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae. Expression of human HIP14 results in rescue of the temperature-sensitive lethality in akr1 Delta yeast cells and, furthermore, restores their defect in endocytosis, demonstrating a role for HIP14 in intracellular trafficking. Our findings suggest that decreased interaction between htt and HIP14 could contribute to the neuronal dysfunction in HD by perturbing normal intracellular transport pathways in neurons.

Therapeutic effects of cystamine in a murine model of Huntington's disease.

The precise cause of neuronal death in Huntington's disease (HD) is unknown. Proteolytic products of the huntingtin protein can contribute to toxic cellular aggregates that may be formed in part by tissue transglutaminase (Tgase). Tgase activity is increased in HD brain. Treatment in R6/2 transgenic HD mice, using the transglutaminase inhibitor cystamine, significantly extended survival, improved body weight and motor performance, and delayed the neuropathological sequela. Tgase activity and N(Sigma)-(gamma-L-glutamyl)-L-lysine (GGEL) levels were significantly altered in HD mice. Free GGEL, a specific biochemical marker of Tgase activity, was markedly elevated in the neocortex and caudate nucleus in HD patients. Both Tgase and GGEL immunoreactivities colocalized to huntingtin aggregates. Cystamine treatment normalized transglutaminase and GGEL levels in R6/2 mice. These findings are consistent with the hypothesis that transglutaminase activity may play a role in the pathogenesis of HD, and they identify cystamine as a potential therapeutic strategy for treating HD patients.

Caspase cleavage of mutant huntingtin precedes neurodegeneration in Huntington's disease.

Huntington's disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspase-cleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD.

Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. Here, we report that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms.