Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis

The primary cause of Huntington's disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild-type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.     

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.     

Mitochondrial DNA damage Is associated with reduced mitochondrial bioenergetics in Huntington's disease

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.     

Maintenance of Basal Levels of Autophagy in Huntington’s Disease Mouse Models Displaying Metabolic Dysfunction

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin protein. Neuropathology in the basal ganglia and in the cerebral cortex has been linked to the motor and cognitive symptoms whereas recent work has suggested that the hypothalamus might be involved in the metabolic dysfunction. Several mouse models of HD that display metabolic dysfunction have hypothalamic pathology, and expression of mutant huntingtin in the hypothalamus has been causally linked to the development of metabolic dysfunction in mice. Although the pathogenic mechanisms by which mutant huntingtin exerts its toxic functions in the HD brain are not fully known, several studies have implicated a role for the lysososomal degradation pathway of autophagy. Interestingly, changes in autophagy in the hypothalamus have been associated with the development of metabolic dysfunction in wild-type mice. We hypothesized that expression of mutant huntingtin might lead to changes in the autophagy pathway in the hypothalamus in mice with metabolic dysfunction. We therefore investigated whether there were changes in basal levels of autophagy in a mouse model expressing a fragment of 853 amino acids of mutant huntingtin selectively in the hypothalamus using a recombinant adeno-associate viral vector approach as well as in the transgenic BACHD mice. We performed qRT-PCR and Western blot to investigate the mRNA and protein expression levels of selected autophagy markers. Our results show that basal levels of autophagy are maintained in the hypothalamus despite the presence of metabolic dysfunction in both mouse models. Furthermore, although there were no major changes in autophagy in the striatum and cortex of BACHD mice, we detected modest, but significant differences in levels of some markers in mice at 12 months of age. Taken together, our results indicate that overexpression of mutant huntingtin in mice do not significantly perturb basal levels of autophagy.     

Allele-specific silencing of mutant Huntington's disease gene

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a poly-glutamine expansion in huntingtin, the protein encoded by the HD gene. PolyQ-expanded huntingtin is toxic to neurons, especially the medium spiny neurons of the striatum. At the same time, wild-type huntingtin has important – indeed essential – protective functions. Any effective molecular therapy must preserve the expression of wild-type huntingtin, while silencing the mutant allele. We hypothesized that an appropriate siRNA molecule would display the requisite specificity and efficacy. As RNA interference is incapable of distinguishing among alleles with varying numbers of CAG (glutamine) codons, another strategy is needed. We used HD fibroblasts in which the pathogenic mutation is linked to a polymorphic site: the Δ2642 deletion of one of four tandem GAG triplets. We silenced expression of the harmful Δ2642-marked polyQ-expanded huntingtin without compromising synthesis of its wild-type counterpart. Following this success in HD fibroblasts, we obtained similar results with neuroblastoma cells expressing both wild-type and mutant HD genes. As opposed to the effect of depleting wild-type huntingtin, specifically silencing the mutant species actually lowered caspase-3 activation and protected HD cells under stress conditions. These findings have therapeutic implications not only for HD, but also for other autosomal dominant diseases. This approach has great promise: it may lead to personalized genetic therapy, a holy grail in contemporary medicine.     

Azadiradione Restores Protein Quality Control and Ameliorates the Disease Pathogenesis in a Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by expansion of CAG repeats in the coding area of huntingtin gene. In the HD brain, mutant huntingtin protein goes through proteolysis, and its amino-terminal portion consisting of polyglutamine repeats accumulate as inclusions that result in progressive impairment of cellular protein quality control system. Here, we demonstrate that partial rescue of the defective protein quality control in HD model mouse by azadiradione (a bioactive limonoids found in the seed of Azadirachta indica) could potentially improve the disease pathology. Prolonged treatment of azadiradione to HD mice significantly improved the progressive deterioration in body weight, motor functioning along with extension of lifespan. Azadiradione-treated HD mice brain also exhibited considerable decrease in mutant huntingtin aggregates load and improvement of striatal pathology in comparison with age-matched saline-treated HD controls. Biochemical analysis further revealed upregulation and activation of not only HSF1 (master regulator of protein folding) but also Ube3a (an ubiquitin ligase involved in the clearance of mutant huntingtin) in azadiradione-treated mice. Our results indicate that azadiradione-mediated enhanced folding and clearance of mutant huntingtin might underlie improved disease pathology in HD mice and suggests that it could be a potential therapeutic molecule to delay the progression of HD.     

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.     

Increased Levels of Rictor Prevent Mutant Huntingtin-Induced Neuronal Degeneration

Rictor associates with mTOR to form the mTORC2 complex, which activity regulates neuronal function and survival. Neurodegenerative diseases are characterized by the presence of neuronal dysfunction and cell death in specific brain regions such as for example Huntington’s disease (HD), which is characterized by the loss of striatal projection neurons leading to motor dysfunction. Although HD is caused by the expression of mutant huntingtin, cell death occurs gradually suggesting that neurons have the capability to activate compensatory mechanisms to deal with neuronal dysfunction and later cell death. Here, we analyzed whether mTORC2 activity could be altered by the presence of mutant huntingtin. We observed that Rictor levels are specifically increased in the striatum of HD mouse models and in the putamen of HD patients. Rictor-mTOR interaction and the phosphorylation levels of Akt, one of the targets of the mTORC2 complex, were increased in the striatum of the R6/1 mouse model of HD suggesting increased mTORC2 signaling. Interestingly, acute downregulation of Rictor in striatal cells in vitro reduced mTORC2 activity, as shown by reduced levels of phospho-Akt, and increased mutant huntingtin-induced cell death. Accordingly, overexpression of Rictor increased mTORC2 activity counteracting cell death. Furthermore, normalization of endogenous Rictor levels in the striatum of R6/1 mouse worsened motor symptoms suggesting an induction of neuronal dysfunction. In conclusion, our results suggest that increased Rictor striatal levels could counteract neuronal dysfunction induced by mutant huntingtin.     

Depletion of wild-type huntingtin in mouse models of neurologic diseases

Huntington's disease (HD) is caused by a mutation in the gene encoding for huntingtin resulting in selective neuronal degeneration. Because HD is an autosomal dominant disorder, affected individuals have one copy of the mutant and one copy of the wild-type allele. Huntingtin has antiapoptotic properties and is critical for cell survival. However, the important role of wild-type huntingtin in both HD and other neurological diseases has not been fully recognized. We demonstrate disease-associated decreased levels of full-length huntingtin in brains of transgenic mouse models of HD, ischemia, trauma, and in spinal cord after injury. In addition, overexpression of wild-type huntingtin confers in vivo protection of neurodegeneration after ischemia. We propose that in HD, in addition to a toxic gain-of-function of mutant huntingtin, a parallel depletion of wild-type huntingtin results in a detrimental loss-of-function, playing an important role in disease progression.     

D-β-hydroxybutyrate is protective in mouse models of Huntington's disease

Abnormalities in mitochondrial function and epigenetic regulation are thought to be instrumental in Huntington's disease (HD), a fatal genetic disorder caused by an expanded polyglutamine track in the protein huntingtin. Given the lack of effective therapies for HD, we sought to assess the neuroprotective properties of the mitochondrial energizing ketone body, D-β-hydroxybutyrate (DβHB), in the 3-nitropropionic acid (3-NP) toxic and the R6/2 genetic model of HD. In mice treated with 3-NP, a complex II inhibitor, infusion of DβHB attenuates motor deficits, striatal lesions, and microgliosis in this model of toxin induced-striatal neurodegeneration. In transgenic R6/2 mice, infusion of DβHB extends life span, attenuates motor deficits, and prevents striatal histone deacetylation. In PC12 cells with inducible expression of mutant huntingtin protein, we further demonstrate that DβHB prevents histone deacetylation via a mechanism independent of its mitochondrial effects and independent of histone deacetylase inhibition. These pre-clinical findings suggest that by simultaneously targeting the mitochondrial and the epigenetic abnormalities associated with mutant huntingtin, DβHB may be a valuable therapeutic agent for HD.     

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.     

Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin

Mutant huntingtin accumulates in the neuronal nuclei and processes, which suggests that its subcellular localization is critical for the pathology of Huntington's disease (HD). However, the contribution of cytoplasmic mutant huntingtin and its aggregates in neuronal processes (neuropil aggregates) has not been rigorously explored. We generated an intracellular antibody (intrabody) whose binding to a unique epitope of human huntingtin is enhanced by polyglutamine expansion. This intrabody decreases the cytotoxicity of mutant huntingtin and its distribution in neuronal processes. When expressed in the striatum of HD mice via adenoviral infection, the intrabody reduces neuropil aggregate formation and ameliorates neurological symptoms. Interaction of the intrabody with mutant huntingtin increases the ubiquitination of cytoplasmic huntingtin and its degradation. These findings suggest that the intrabody reduces the specific neurotoxicity of cytoplasmic mutant huntingtin and its associated neurological symptoms by preventing the accumulation of mutant huntingtin in neuronal processes and promoting its clearance in the cytoplasm.     

The Q175 Mouse Model of Huntington’s Disease Shows Gene Dosage- and Age-Related Decline in Circadian Rhythms of Activity and Sleep

Sleep and circadian disruptions are commonly reported by patients with neurodegenerative diseases, suggesting these may be an endophenotype of the disorders. Several mouse models of Huntington’s disease (HD) that recapitulate the disease progression and motor dysfunction of HD also exhibit sleep and circadian rhythm disruption. Of these, the strongest effects are observed in the transgenic models with multiple copies of mutant huntingtin gene. For developing treatments of the human disease, knock-in (KI) models offer advantages of genetic precision of the insertion and control of mutation copy number. Therefore, we assayed locomotor activity and immobility-defined sleep in a new model of HD with an expansion of the KI repeats (Q175). We found evidence for gene dose- and age-dependent circadian disruption in the behavior of the Q175 line. We did not see evidence for loss of cells or disruption of the molecular oscillator in the master pacemaker, the suprachiasmatic nucleus (SCN). The combination of the precise genetic targeting in the Q175 model and the observed sleep and circadian disruptions make it tractable to study the interaction of the underlying pathology of HD and the mechanisms by which the disruptions occur.     

Enhancement of brain-type creatine kinase activity ameliorates neuronal deficits in Huntington's disease

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Brain-type creatine kinase (CKB) is an enzyme involved in energy homeostasis via the phosphocreatine–creatine kinase system. Although downregulation of CKB was previously reported in brains of HD mouse models and patients, such regulation and its functional consequence in HD are not fully understood. In the present study, we demonstrated that levels of CKB found in both the soma and processes were markedly reduced in primary neurons and brains of HD mice. We show for the first time that mutant HTT (mHTT) suppressed the activity of the promoter of the CKB gene, which contributes to the lowered CKB expression in HD. Exogenous expression of wild-type CKB, but not a dominant negative CKB mutant, rescued the ATP depletion, aggregate formation, impaired proteasome activity, and shortened neurites induced by mHTT. These findings suggest that negative regulation of CKB by mHTT is a key event in the pathogenesis of HD and contributes to the neuronal dysfunction associated with HD. In addition, besides dietary supplementation with the CKB substrate, strategies aimed at increasing CKB expression might lead to the development of therapeutic treatments for HD.     

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.