Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.     

CGS21680 attenuates symptoms of Huntington's disease in a transgenic mouse model

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.     

Inhibition of lipid signaling enzyme diacylglycerol kinase epsilon attenuates mutant huntingtin toxicity

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease-modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model, Hdh(111Q/111Q) cells. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase-3/7 activity after serum withdrawal in striatal Hdh(111Q/111Q) cells. In addition, R59949 decreased the accumulation of a 513-amino acid N-terminal Htt fragment processed by caspase-3 and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglycerol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain (β, γ, ε, and ζ) using siRNA. Only the knockdown of the family member, DGKε, blocked striatal Hdh(111Q/111Q)-mediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGKε homolog significantly improves Htt-induced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGKε are increased in the striatum of R6/2 HD transgenic mice when compared with littermate controls. Together, these findings indicate that increased levels of kinase DGKε contribute to HD pathogenesis and suggest that reducing its levels or activity is a potential therapy for HD.     

Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.     

Nuclear translocation of AMPK-��1 potentiates striatal neurodegeneration in Huntington��s disease

Adenosine monophosphate–activated protein kinase (AMPK) is a major energy sensor that maintains cellular energy homeostasis. Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (Htt) gene. In this paper, we report that activation of the α1 isoform of AMPK (AMPK-α1) occurred in striatal neurons of humans and mice with HD. Overactivation of AMPK in the striatum caused brain atrophy, facilitated neuronal loss, and increased formation of Htt aggregates in a transgenic mouse model (R6/2) of HD. Such nuclear accumulation of AMPK-α1 was activity dependent. Prevention of nuclear translocation or inactivation of AMPK-α1 ameliorated cell death and down-regulation of Bcl2 caused by mutant Htt (mHtt). Conversely, enhanced expression of Bcl2 protected striatal cells from the toxicity evoked by mHtt and AMPK overactivation. These data demonstrate that aberrant activation of AMPK-α1 in the nuclei of striatal cells represents a new toxic pathway induced by mHtt.     

Inhibition of metabotropic glutamate receptor signaling by the huntingtin-binding protein optineurin

Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca(2+) stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurin-mediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt(Q111/Q111) mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt(Q7/Q7) knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease.     

Mitochondria in Huntington's disease

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder associated with involuntary abnormal movements (chorea), cognitive deficits and psychiatric disturbances. The disease is caused by an abnormal expansion of a CAG repeat located in exon 1 of the gene encoding the huntingtin protein (Htt) that confers a toxic function to the protein. The most striking neuropathological change in HD is the preferential loss of medium spiny GABAergic neurons in the striatum. The mechanisms underlying striatal vulnerability in HD are unknown, but compelling evidence suggests that mitochondrial defects may play a central role. Here we review recent findings supporting this hypothesis. Studies investigating the toxic effects of mutant Htt in cell culture or animal models reveal mitochondrial changes including reduction of Ca²⁺ buffering capacity, loss of membrane potential, and decreased expression of oxidative phosphorylation (OXPHOS) enzymes. Striatal neurons may be particularly vulnerable to these defects. One hypothesis is that neurotransmission systems such as dopamine and glutamate exacerbate mitochondrial defects in the striatum. In particular, mitochondrial dysfunction facilitates impaired Ca²⁺ homeostasis linked to the glutamate receptor-mediated excitotoxicity. Also dopamine receptors modulate mutant Htt toxicity, at least in part through regulation of the expression of mitochondrial complex II. All these observations support the hypothesis that mitochondria, acting as “sensors” of the neurochemical environment, play a central role in striatal degeneration in HD.     

Normal Aging Modulates the Neurotoxicity of Mutant Huntingtin

Aging likely plays a role in neurodegenerative disorders. In Huntington''s disease (HD), a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt), the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or “normal” aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a β-Galactosidase (β-Gal) reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week) and old (15 month) rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that “normal” aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.     

Wild-type huntingtin plays a role in brain development and neuronal survival

While the role of the mutated Huntington's disease (HD) protein in the pathogenesis of HD has been the focus of intensive investigation, the normal protein has received less attention. Nonetheless, the wild-type HD protein appears to be essential for embryogenesis, since deletion of the HD gene in mice results in early embryonic lethality. This early lethality is due to a critical role the HD protein, called huntingtin (Htt), plays in extraembryonic membrane function, presumably in vesicular transport of nutrients. Studies of mutant mice expressing low levels of Htt and of chimeric mice generated by blastocyst injection of Hdh-/- embryonic stem cells show that wildtype Htt plays an important role later in development as well, specifically in forebrain formation. Moreover, various lines of study suggest that normal Htt is also critical for survival of neurons in the adult forebrain. The observation that Htt plays its key developmental and survival roles in those brain areas most affected in HD raises the possibility that a subtle loss of function on the part of the mutant protein or a sequestering of wild-type Htt by mutant Htt may contribute to HD pathogenesis. Regardless of whether this is so, the prosurvival role of Htt suggests that HD therapies that block production of both wild-type and mutant Htt may themselves be harmful.     

Huntington's Disease

Huntington''s disease (HD) is the most common inherited neurodegenerative disease and is characterized by uncontrolled excessive motor movements and cognitive and emotional deficits. The mutation responsible for HD leads to an abnormally long polyglutamine (polyQ) expansion in the huntingtin (Htt) protein, which confers one or more toxic functions to mutant Htt leading to neurodegeneration. The polyQ expansion makes Htt prone to aggregate and accumulate, and manipulations that mitigate protein misfolding or facilitate the clearance of misfolded proteins tend to slow disease progression in HD models. This article will focus on HD and the evidence that it is a conformational disease.     

Selective deficits in the expression of striatal-enriched mRNAs in Huntington's disease

We have identified and cataloged 54 genes that exhibit predominant expression in the striatum. Our hypothesis is that such mRNA molecules are likely to encode proteins that are preferentially associated with particular physiological processes intrinsic to striatal neurons, and therefore might contribute to the regional specificity of neurodegeneration observed in striatal disorders such as Huntington's disease (HD). Expression of these genes was measured simultaneously in the striatum of HD R6/1 transgenic mice using Affymetrix oligonucleotide arrays. We found a decrease in expression of 81% of striatum-enriched genes in HD transgenic mice. Changes in expression of genes associated with G-protein signaling and calcium homeostasis were highlighted. The most striking decrement was observed for a newly identified subunit of the sodium channel, beta 4, with dramatic decreases in expression beginning at 8 weeks of age. A subset of striatal genes was tested by real-time PCR in caudate samples from human HD patients. Similar alterations in expression were observed in human HD and the R6/1 model for the striatal genes tested. Expression of 15 of the striatum-enriched genes was measured in 6-hydroxydopamine-lesioned rats to determine their dependence on dopamine innervation. No changes in expression were observed for any of these genes. These findings demonstrate that mutant huntingtin protein causes selective deficits in the expression of mRNAs responsible for striatum-specific physiology and these may contribute to the regional specificity of degeneration observed in HD.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

Huntington’s Disease: The Past,Present, and Future Search for Disease Modifiers

Huntington’s disease (HD) is an autosomal dominant genetic disorder that specifically causes neurodegeneration of striatal neurons, resulting in a triad of symptoms that includes emotional, cognitive, and motor disturbances. The HD mutation causes a polyglutamine repeat expansion within the N-terminal of the huntingtin (Htt) protein. This expansion causes aggregate formation within the cytosol and nucleus due to the presence of misfolded mutant Htt, as well as altered interactions with Htt’s multiple binding partners, and changes in post-translational Htt modifications. The present review charts efforts toward a therapy that delays age of onset or slows symptom progression in patients affected by HD, as there is currently no effective treatment. Although silencing Htt expression appears promising as a disease modifying treatment, it should be attempted with caution in light of Htt’s essential roles in neural maintenance and development. Other therapeutic targets include those that boost aggregate dissolution, target excitotoxicity and metabolic issues, and supplement growth factors.     

Brain-derived neurotrophic factor over-expression in the forebrain ameliorates Huntington's disease phenotypes in mice

Huntington's disease (HD), a dominantly inherited neurodegenerative disorder characterized by relatively selective degeneration of striatal neurons, is caused by an expanded polyglutamine tract of the huntingtin (htt) protein. The htt mutation reduces levels of brain-derived neurotrophic factor (BDNF) in the striatum, likely by inhibiting cortical BDNF gene expression and anterograde transport of BDNF from cortex to striatum. However, roles of the BDNF reduction in HD pathogenesis have not been established conclusively. We reasoned that increasing striatal BDNF through over-expression would slow progression of the disease if BDNF reduction plays a pivotal role in HD pathogenesis. We employed a Bdnf transgene driven by the promoter for the alpha subunit of Ca²⁺/calmodulin-dependent kinase II to over-express BDNF in the forebrain of R6/1 mice which express a fragment of mutant htt with a 116-glutamine tract. The Bdnf transgene increased BDNF levels and TrkB signaling activity in the striatum, ameliorated motor dysfunction, and reversed brain weight loss in R6/1 mice. Furthermore, it normalized DARPP-32 expression of the 32 kDa dopamine and cAMP-regulated phosphoprotein, increased the number of enkephalin-containing boutons, and reduced formation of neuronal intranuclear inclusions in the striatum of R6/1 mice. These results demonstrate crucial roles of reduced striatal BDNF in HD pathogenesis and suggest potential therapeutic values of BDNF to HD.     

Dithiol-based compounds maintain expression of antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin

Mitochondrial dysfunction and elevated reactive oxygen species are strongly implicated in both aging and various neurodegenerative disorders, including Huntington disease (HD). Because reactive oxygen species can promote the selective oxidation of protein cysteine sulfhydryl groups to disulfide bonds we examined the spectrum of disulfide-bonded proteins that were specifically altered in a HD context. Protein extracts from PC12 cells overexpressing the amino-terminal fragment of the Huntingtin (Htt) protein with either a nonpathogenic or pathogenic polyglutamine repeat (Htt-103Q) were resolved by redox two-dimensional PAGE followed by mass spectrometry analysis. Several antioxidant proteins were identified that exhibited changes in disulfide bonding unique to Htt-103Q expressing cells. In particular, the antioxidant protein peroxiredoxin 1 (Prx1) exhibited both decreased expression and hyperoxidation in response to mutant Htt expressed in either PC12 cells or immortalized striatal cells exposed to 3-nitropropionic acid. Ectopic expression of Prx1 in PC12 cells attenuated mutant Htt-induced toxicity. In contrast, short hairpin RNA-mediated knockdown of Prx1 potentiated mHtt toxicity. Furthermore, treatment with the dithiol-based compounds dimercaptopropanol and dimercaptosuccinic acid suppressed toxicity in both HD cell models, whereas monothiol compounds were relatively ineffective. Dimercaptopropanol treatment also prevented mutant Htt-induced loss of Prx1 expression in both cell models. Our studies reveal for the first time that pathogenic Htt can affect the expression and redox state of antioxidant proteins; an event countered by specific dithiol-based compounds. These findings should provide a catalyst to explore the use of dithiol-based drugs for the treatment of neurodegenerative diseases.