Huntington's Disease Induced Cardiac Amyloidosis Is Reversed by Modulating Protein Folding and Oxidative Stress Pathways in the Drosophila Heart

Amyloid-like inclusions have been associated with Huntington''s disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients.     

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington''s disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.     

Primary cilia and autophagic dysfunction in Huntington's disease

Huntington''s disease (HD) is an inherited, neurodegenerative disorder caused by a single-gene mutation: a CAG expansion in the huntingtin (HTT) gene that results in production of a mutated protein, mutant HTT, with a polyglutamine tail (polyQ-HTT). Although the molecular pathways of polyQ-HTT toxicity are not fully understood, because protein misfolding and aggregation are central features of HD, it has long been suspected that cellular housekeeping processes such as autophagy might be important to disease pathology. Indeed, multiple lines of research have identified abnormal autophagy in HD, characterized generally by increased autophagic induction and inefficient clearance of substrates. To date, the origin of autophagic dysfunction in HD remains unclear and the search for actors involved continues. To that end, recent studies have suggested a bidirectional relationship between autophagy and primary cilia, signaling organelles of most mammalian cells. Interestingly, primary cilia structure is defective in HD, suggesting a potential link between autophagic dysfunction, primary cilia and HD pathogenesis. In addition, because polyQ-HTT also accumulates in primary cilia, the possibility exists that primary cilia might play additional roles in HD: perhaps by disrupting signaling pathways or acting as a reservoir for secretion and propagation of toxic, misfolded polyQ-HTT fragments. Here, we review recent research suggesting potential links between autophagy, primary cilia and HD and speculate on possible pathogenic mechanisms and future directions for the field.     

Huntingtin’s Function in Axonal Transport Is Conserved in Drosophila melanogaster

Huntington’s disease (HD) is a devastating dominantly inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-terminal part of the huntingtin (HTT) protein. HTT is a large scaffold protein that interacts with more than a hundred proteins and is probably involved in several cellular functions. The mutation is dominant, and is thought to confer new and toxic functions to the protein. However, there is emerging evidence that the mutation also alters HTT’s normal functions. Therefore, HD models need to recapitulate this duality if they are to be relevant. Drosophila melanogaster is a useful in vivo model, widely used to study HD through the overexpression of full-length or N-terminal fragments of mutant human HTT. However, it is unclear whether Drosophila huntingtin (DmHTT) shares functions similar to the mammalian HTT. Here, we used various complementary approaches to analyze the function of DmHTT in fast axonal transport. We show that DmHTT interacts with the molecular motor dynein, associates with vesicles and co-sediments with microtubules. DmHTT co-localizes with Brain-derived neurotrophic factor (BDNF)-containing vesicles in rat cortical neurons and partially replaces mammalian HTT in a fast axonal transport assay. DmHTT-KO flies show a reduced fast axonal transport of synaptotagmin vesicles in motoneurons in vivo. These results suggest that the function of HTT in axonal transport is conserved between flies and mammals. Our study therefore validates Drosophila melanogaster as a model to study HTT function, and its dysfunction associated with HD.     

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.     

miR-196a Ameliorates Phenotypes of Huntington Disease in Cell,Transgenic Mouse,and Induced Pluripotent Stem Cell Models

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder characterized by dysregulation of various genes. Recently, microRNAs (miRNAs) have been reported to be involved in this dysregulation, suggesting that manipulation of appropriate miRNA regulation may have a therapeutic benefit. Here, we report the beneficial effects of miR-196a (miR196a) on HD in cell, transgenic mouse models, and human induced pluripotent stem cells derived from one individual with HD (HD-iPSCs). In the in vitro results, a reduction of mutant HTT and pathological aggregates, accompanying the overexpression of miR-196a, was observed in HD models of human embryonic kidney cells and mouse neuroblastoma cells. In the in vivo model, HD transgenic mice overexpressing miR-196a revealed the suppression of mutant HTT in the brain and also showed improvements in neuropathological progression, such as decreases of nuclear, intranuclear, and neuropil aggregates and late-stage behavioral phenotypes. Most importantly, miR-196a also decreased HTT expression and pathological aggregates when HD-iPSCs were differentiated into the neuronal stage. Mechanisms of miR-196a in HD might be through the alteration of ubiquitin-proteasome systems, gliosis, cAMP response element-binding protein pathway, and several neuronal regulatory pathways in vivo. Taken together, these results show that manipulating miR-196a provides beneficial effects in HD, suggesting the potential therapeutical role of miR-196a in HD.     

Expression of Mutant Huntingtin in Leptin Receptor-Expressing Neurons Does Not Control the Metabolic and Psychiatric Phenotype of the BACHD Mouse

Metabolic and psychiatric disturbances occur early on in the clinical manifestation of Huntington’s disease (HD), a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. Hypothalamus has emerged as an important site of pathology and alterations in this area and its neuroendocrine circuits may play a role in causing early non-motor symptoms and signs in HD. Leptin is a hormone that controls energy homeostasis by signaling through leptin receptors in the hypothalamus. Disturbed leptin action is implicated in both obesity and depression and altered circulating levels of leptin have been reported in both clinical HD and rodent models of the disease. Pathological leptin signaling may therefore be involved in causing the metabolic and psychiatric disturbances of HD. Here we tested the hypothesis that expression of mutant HTT in leptin receptor carrying neurons plays a role in the development of the non-motor phenotype in the BACHD mouse model. Our results show that inactivation of mutant HTT in leptin receptor-expressing neurons in the BACHD mouse using cross-breeding based on a cre-loxP system did not have an effect on the metabolic phenotype or anxiety-like behavior. The data suggest that mutant HTT disrupts critical hypothalamic pathways by other mechanisms than interfering with intracellular leptin signaling.     

Deregulated Splicing Is a Major Mechanism of RNA-Induced Toxicity in Huntington's Disease

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into an elongated polyglutamine stretch. In addition to the neurotoxic mutant HTT protein, the mutant CAG repeat RNA can exert toxic functions by trapping RNA-binding proteins. While few examples of proteins that aberrantly bind to mutant HTT RNA and execute abnormal function in conjunction with the CAG repeat RNA have been described, an unbiased approach to identify the interactome of mutant HTT RNA is missing. Here, we describe the analysis of proteins that preferentially bind mutant HTT RNA using a mass spectrometry approach. We show that (I) the majority of proteins captured by mutant HTT RNA belong to the spliceosome pathway, (II) expression of mutant CAG repeat RNA induces mis-splicing in a HD cell model, (III) overexpression of one of the splice factors trapped by mutant HTT ameliorates the HD phenotype in a fly model and (VI) deregulated splicing occurs in human HD brain. Our data suggest that deregulated splicing is a prominent mechanism of RNA-induced toxicity in HD.     

The cell biology of disease: cellular mechanisms of cardiomyopathy

The heart exhibits remarkable adaptive responses to a wide array of genetic and extrinsic factors to maintain contractile function. When compensatory responses are not sustainable, cardiac dysfunction occurs, leading to cardiomyopathy. The many forms of cardiomyopathy exhibit a set of overlapping phenotypes reflecting the limited range of compensatory responses that the heart can use. These include cardiac hypertrophy, induction of genes normally expressed during development, fibrotic deposits that replace necrotic and apoptotic cardiomyocytes, and metabolic disturbances. The compensatory responses are mediated by signaling pathways that initially serve to maintain normal contractility; however, persistent activation of these pathways leads to cardiac dysfunction. Current research focuses on ways to target these specific pathways therapeutically.     

A Large Scale Huntingtin Protein Interaction Network Implicates Rho GTPase Signaling Pathways in Huntington Disease

Huntington disease (HD) is an inherited neurodegenerative disease caused by a CAG expansion in the HTT gene. Using yeast two-hybrid methods, we identified a large set of proteins that interact with huntingtin (HTT)-interacting proteins. This network, composed of HTT-interacting proteins (HIPs) and proteins interacting with these primary nodes, contains 3235 interactions among 2141 highly interconnected proteins. Analysis of functional annotations of these proteins indicates that primary and secondary HIPs are enriched in pathways implicated in HD, including mammalian target of rapamycin, Rho GTPase signaling, and oxidative stress response. To validate roles for HIPs in mutant HTT toxicity, we show that the Rho GTPase signaling components, BAIAP2, EZR, PIK3R1, PAK2, and RAC1, are modifiers of mutant HTT toxicity. We also demonstrate that Htt co-localizes with BAIAP2 in filopodia and that mutant HTT interferes with filopodial dynamics. These data indicate that HTT is involved directly in membrane dynamics, cell attachment, and motility. Furthermore, they implicate dysregulation in these pathways as pathological mechanisms in HD.     

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD.     

Attenuation of beta2-adrenergic receptors and homocysteine metabolic enzymes cause diabetic cardiomyopathy

Although adrenergic receptors (AR) and hyperhomocysteinemia (HHcy) are implicated in heart failure, their role in diabetic cardiomyopathy is not completely understood. We tested the hypothesis that glucose mediated depletion of beta2-AR and HHcy impair contractile function of cardiomyocytes leading to diabetic cardiomyopathy. To prove the hypothesis, cardiac function was assessed in 12 week male diabetic Ins2+/− Akita and C57BL/6 J mice by echocardiography, pressure-volume loop, and contractile function of cardiomyocytes. The results revealed cardiac dysfunction in Akita. To investigate the mechanism, the levels of beta2-AR, GLUT4, sarcoplasmic reticulum calcium ATP-ase-isoform 2 (SERCA-2) and homocysteine (Hcy) metabolic enzymes-cystathionine beta synthase (CBS), cystathionine gamma lyase (CTH), and methyl tetrahydrofolate reductase (MTHFR) were determined in the heart. It revealed down-regulation of beta2-AR, GLUT4, SERCA-2, CBS, CTH, and MTHFR in Akita. Attenuation of beta2-AR in hyperglycemic condition was also confirmed in cardiomyocytes at in vitro level. Interestingly, the ex vivo treatment of cardiomyocytes with beta2-AR antagonist deteriorated whereas beta-AR agonist ameliorated contractile function. It points to the involvement of beta2-AR in diabetic cardiomyopathy. We conclude that degradation of beta2-AR and impairment of Hcy metabolism is implicated in diabetic cardiomyopathy.