Curcumin enhances the polyglutamine-expanded truncated N-terminal huntingtin-induced cell death by promoting proteasomal malfunction

Formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various proteasome components is the major hallmark of the polyglutamine diseases. Curcumin is a polyphenolic compound having anti-inflammatory, anti-tumor, and anti-oxidative properties. Recently, curcumin has been reported to suppress the amyloid-beta accumulation, oxidative damage, and inflammation in the transgenic mice model of Alzheimer's disease. Here, we found that the treatment of curcumin increases the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-dependent cell death. Curcumin also causes rapid proteasomal malfunction in the mutant huntingtin expressing cells in comparison with normal glutamine repeat expressing cells. Finally, we show that N-acetyl cysteine (NAC), a potent antioxidant, reverted the curcumin-induced mutant huntingtin aggregation and proteasomal malfunction in the mutant huntingtin expressing cells. NAC also protects curcumin-induced cell death. Our result suggests that curcumin promotes mutant huntingtin-induced cell death by mimicking proteasomal dysfunction.     

Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by mimicking proteasomal malfunction

Huntington's disease (HD) is a familial neurodegenerative disorder caused by an abnormal expansion of CAG repeats in the coding region of huntingtin gene. A major hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. However, the mechanism by which the mutant huntingtin causes neurodegeneration is not well understood. Here, we found that oxidative stimuli enhance the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-induced cell death. Oxidative stimuli also lead to rapid proteasomal dysfunction in the mutant huntingtin expressing cells as compared to normal glutamine repeat expressing cells. Overexpression of Cu/Zn superoxide dismutase (SOD1), Hsp40 or Hsp70 reverses the oxidative stress-induced proteasomal malfunction, mutant huntingtin aggregation, and death of the mutant huntingtin expressing cells. Finally, we show the higher levels of expression of SOD1 and DJ-1 in the mutant huntingtin expressing cells. Our result suggests that oxidative stress-induced proteasomal malfunction might be linked with mutant huntingtin-induced cell death.     

Brain-derived neurotrophic factor modulates dopaminergic deficits in a transgenic mouse model of Huntington's disease

Dysfunction of dopaminergic neurons may contribute to motor impairment in Huntington's disease. Here, we study the role of brain-derived neurotrophic factor (BDNF) in alterations of the nigrostriatal system associated with transgenics carrying mutant huntingtin. Using huntingtin-BDNF+/- double-mutant mice, we analyzed the effects of reducing the levels of BDNF expression in a model of Huntington's disease (R6/1). When compared with R6/1 mice, these mice exhibit an increased number of aggregates in the substantia nigra pars compacta. In addition, reduction of BDNF expression exacerbates the dopaminergic neuronal dysfunction seen in mutant huntingtin mice, such as the decrease in retrograde labelling of dopaminergic neurons and striatal dopamine content. However, mutant huntingtin mice with normal or lowered BDNF expression show the same decrease in the anterograde transport, number of dopaminergic neurons and nigral volume. In addition, reduced BDNF expression causes decreased dopamine receptor expression in mutant huntingtin mice. Examination of changes in locomotor activity induced by dopamine receptor agonists revealed that, in comparison with R6/1 mice, the double mutant mice exhibit lower activity in response to amphetamine, but not to apomorphine. In conclusion, these findings demonstrate that the decreased BDNF expression observed in Huntington's disease exacerbates dopaminergic neuronal dysfunction, which may participate in the motor disturbances associated with this neurodegenerative disorder.     

Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity

Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin-mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease.     

Induction of autophagy with catalytic mTOR inhibitors reduces huntingtin aggregates in a neuronal cell model

Huntington's disease is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. This expansion produces a mutant form of the huntingtin protein, which contains an elongated polyglutamine stretch at its amino-terminus. Mutant huntingtin may adopt an aberrant, aggregation-prone conformation predicted to start the pathogenic process leading to neuronal dysfunction and cell death. Thus, strategies reducing mutant huntingtin may lead to disease-modifying therapies. We investigated the mechanisms and molecular targets regulating huntingtin degradation in a neuronal cell model. We first found that mutant and wild-type huntingtin displayed strikingly diverse turn-over kinetics and sensitivity to proteasome inhibition. Then, we show that autophagy induction led to accelerate degradation of mutant huntingtin aggregates. In our neuronal cell model, allosteric inhibition of mTORC1 by everolimus, a rapamycin analogue, did not induce autophagy or affect aggregate degradation. In contrast, this occurred in the presence of catalytic inhibitors of both mTOR complexes mTORC1 and mTORC2. Our data demonstrate the existence of an mTOR-dependent but everolimus-independent mechanism regulating autophagy and huntingtin-aggregate degradation in cells of neuronal origin.     

Arfaptin 2 regulates the aggregation of mutant huntingtin protein

Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.     

GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway

Increased protein aggregation and altered cell signaling accompany many neurodegenerative diseases including Huntington's disease (HD). Cell stress is counterbalanced by signals mediating cell repair but the identity of these are not fully understood. We show here that the mammalian target of rapamycin (mTOR) pathway is inhibited and cytoprotective autophagy is activated in neuronal PC6.3 cells at 24 h after expression of mutant huntingtin proteins. Tuberous sclerosis complex (TSC) 1/2 interacted with growth arrest and DNA damage protein 34 (GADD34), which caused TSC2 dephosphorylation and induction of autophagy in mutant huntingtin expressing cells. However, GADD34 and autophagy decreased at later time points, after 48 h of transfection with the concomitant increase in mTOR activity. Overexpression of GADD34 counteracted these effects and increased cytoprotective autophagy and cell survival. These results show that GADD34 plays an important role in cell protection in mutant huntingtin expressing cells. Modulation of GADD34 and the TSC pathway may prove useful in counteracting cell degeneration accompanying HD and other neurodegenerative diseases.     

Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease

Neurodegenerative disorders like Huntington's disease (HD) are characterized by progressive and putative irreversible clinical and neuropathological symptoms, including neuronal protein aggregates. Conditional transgenic models of neurodegenerative diseases therefore could be a powerful means to explore the relationship between mutant protein expression and progression of the disease. We have created a conditional model of HD by using the tet-regulatable system. Mice expressing a mutated huntingtin fragment demonstrate neuronal inclusions, characteristic neuropathology, and progressive motor dysfunction. Blockade of expression in symptomatic mice leads to a disappearance of inclusions and an amelioration of the behavioral phenotype. We thus demonstrate that a continuous influx of the mutant protein is required to maintain inclusions and symptoms, raising the possibility that HD may be reversible.     

Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity

Although NH2-terminal mutant huntingtin (htt) fragments cause neurological disorders in Huntington's disease (HD), it is unclear how toxic htt fragments are generated and contribute to the disease process. Here, we report that complex NH2-terminal mutant htt fragments smaller than the first 508 amino acids were generated in htt-transfected cells and HD knockin mouse brains. These fragments constituted neuronal nuclear inclusions and appeared before neurological symptoms. The accumulation and aggregation of these htt fragments were associated with an age-dependent decrease in proteasome activity and were promoted by inhibition of proteasome activity. These results suggest that decreased proteasome activity contributes to late onset htt toxicity and that restoring the ability to remove NH2-terminal fragments will provide a more effective therapy for HD than inhibiting their production.     

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.     

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.     

Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.     

Mutant huntingtin causes metabolic imbalance by disruption of hypothalamic neurocircuits

In Huntington's disease (HD), the mutant huntingtin protein is ubiquitously expressed. The disease was considered to be limited to the basal ganglia, but recent studies have suggested a more widespread pathology involving hypothalamic dysfunction. Here we tested the hypothesis that expression of mutant huntingtin in the hypothalamus causes metabolic abnormalities. First, we showed that bacterial artificial chromosome-mediated transgenic HD (BACHD) mice developed impaired glucose metabolism and pronounced insulin and leptin resistance. Selective hypothalamic expression of a short fragment of mutant huntingtin using adeno-associated viral vectors was sufficient to recapitulate these metabolic disturbances. Finally, selective hypothalamic inactivation of the mutant gene prevented the development of the metabolic phenotype in BACHD mice. Our findings establish a causal link between mutant huntingtin expression in the hypothalamus and metabolic dysfunction, and indicate that metabolic parameters are powerful readouts to assess therapies aimed at correcting dysfunction in HD by silencing huntingtin expression in the brain.