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.     

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.     

Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW-polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.     

Nuclear localization of a non-caspase truncation product of atrophin-1, with an expanded polyglutamine repeat,increases cellular toxicity

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder similar to Huntington's disease, with clinical manifestations including chorea, incoordination, ataxia, and dementia. It is caused by an expansion of a CAG trinucleotide repeat encoding polyglutamine in the atrophin-1 gene. Both patients and DRPLA transgenic mice have nuclear accumulation of atrophin-1, especially an approximately 120-kDa fragment, which appears to represent a cleavage product. We now show that this is an N-terminal fragment that does not correspond to the previously described caspase-3 fragment, or any other known caspase cleavage product. The atrophin-1 sequence contains a putative nuclear localization signal in the N terminus of the protein and a putative nuclear export signal in the C terminus. We have tested the hypothesis that endogenous localization signals are functional in atrophin-1, and that nuclear localization and proteolytic cleavage contribute to atrophin-1 cell toxicity. In transient cell transfection experiments using a neuroblastoma cell line, full-length atrophin-1 with 26 (normal) or 65 (expanded) glutamines localized to both nucleus and cytoplasm, with no significant difference in toxicity between the normal and mutant proteins. A construct with 65 glutamine repeats encoding an N-terminal fragment (which removes an NES) of atrophin-1 similar in size to the truncation product in DRPLA patient tissue, showed increased nuclear labeling, and an increase in cellular toxicity, compared with a similar fragment with 26 glutamines. Full-length atrophin-1 with 65 polyglutamine repeats and mutations inactivating the NES also yielded increased nuclear localization and increased toxicity. These data suggest that truncation enhances cellular toxicity of the mutant protein, and that the NES is a relevant region deleted during truncation. Furthermore, mutating the NLS in the truncated protein shifted atrophin-1 more to the cytoplasm and eliminated the increased toxicity, consistent with the idea that nuclear localization enhances toxicity. In none of the experiments were inclusions visible in the nucleus or cytoplasm suggesting that inclusion formation is unrelated to cell death. These data indicate that truncation of atrophin-1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear interactions and cell toxicity.     

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.     

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.     

Modeling Huntington disease in Drosophila

Huntington disease (HD) is an inherited neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the huntingtin (Htt) gene. Despite years of research, there is no treatment that extends life for patients with the disorder. Similarly, little is known about which cellular pathways that are altered by pathogenic Huntingtin (Htt) protein expression are correlated with neuronal loss. As part of a longstanding effort to gain insights into HD pathology, we have been studying the protein in the context of the fruitfly Drosophila melanogaster. We generated transgenic HD models in Drosophila by engineering flies that carry a 12-exon fragment of the human Htt gene with or without the toxic trinucleotide repeat expansion. We also created variants with a monomeric red fluorescent protein (mRFP) tag fused to Htt that allows in vivo imaging of Htt protein localization and aggregation. While wild-type Htt remains diffuse throughout the cytoplasm of cells, pathogenic Htt forms insoluble aggregates that accumulate in neuronal soma and axons. Aggregates can physically block transport of numerous organelles along the axon. We have also observed that aggregates are formed quickly, within just a few hours of mutant Htt expression. To explore mechanisms of neurodegeneration in our HD model, we performed in vivo and in vitro screens to search for modifiers of viability and pathogenic Htt aggregation. Our results identified several novel candidates for HD therapeutics that can now be tested in mammalian models of HD. Furthermore, these experiments have highlighted the complex relationship between aggregates and toxicity that exists 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.     

Mechanisms of neuronal cell death in Huntington's disease

Huntington's disease (HD) is a genetically dominant neurodegenerative condition caused by an unique mutation in the disease gene huntingtin. Although the Huntington protein (Htt) is ubiquitously expressed, expansion of the polyglutamine tract in Htt leads to the progressive loss of specific neuronal subpopulations in HD brains. In this article, we will summarize the current understanding on mechanisms of how mutant Htt can elicit cytotoxicity, as well as how the selective sets of neuronal cell death occur in HD brains.     

Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington's disease

Huntington's disease (HD) is a progressive, fatal neurodegenerative disease caused by expanded polyglutamine repeats in the HD gene. HD is characterized by chorea, seizures, involuntary movements, dystonia, cognitive decline, intellectual impairment and emotional disturbances. Research into mutant huntingtin (Htt) and mitochondria has found that mutant Htt interacts with the mitochondrial protein dynamin-related protein 1 (Drp1), enhances GTPase Drp1 enzymatic activity, and causes excessive mitochondrial fragmentation and abnormal distribution, leading to defective axonal transport of mitochondria and selective synaptic degeneration. This article summarizes latest developments in HD research and focuses on the role of abnormal mitochondrial dynamics and defective axonal transport in HD neurons. This article also discusses the therapeutic strategies that decrease mitochondrial fragmentation and neuronal damage in 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.     

Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington's disease

Huntington's disease (HD) is a progressive, fatal neurodegenerative disease caused by expanded polyglutamine repeats in the HD gene. HD is characterized by chorea, seizures, involuntary movements, dystonia, cognitive decline, intellectual impairment and emotional disturbances. Research into mutant huntingtin (Htt) and mitochondria has found that mutant Htt interacts with the mitochondrial protein dynamin-related protein 1 (Drp1), enhances GTPase Drp1 enzymatic activity, and causes excessive mitochondrial fragmentation and abnormal distribution, leading to defective axonal transport of mitochondria and selective synaptic degeneration. This article summarizes latest developments in HD research and focuses on the role of abnormal mitochondrial dynamics and defective axonal transport in HD neurons. This article also discusses the therapeutic strategies that decrease mitochondrial fragmentation and neuronal damage in HD.     

Mass spectrometric identification of novel lysine acetylation sites in huntingtin

Huntingtin (Htt) is a protein with a polyglutamine stretch in the N-terminus and expansion of the polyglutamine stretch causes Huntington's disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that post-translational modifications of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization, and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase inhibitors ameliorates HD-like symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1-612) in a cell culture model using mass spectrometry. Myc-tagged Htt23Q (1-612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDS-PAGE, digested and subjected to high performance liquid chromatography tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys-178, Lys-236, Lys-345 and two previously described sites Lys-9 and Lys-444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring MS assay was developed to compare quantitatively the Lys-178 acetylation level between wild-type Htt23Q and mutant Htt148Q (1-612). This report represents the first comprehensive mapping of lysine acetylation sites in N-terminal region of Htt.