Huntington’s disease is caused by expansion of a polyglutamine tract found in the amino-terminal of the ubiquitously expressed protein huntingtin. Well studied in its mutant form, huntingtin has a wide variety of normal functions, loss of which may also contribute to disease progression. Widespread transcriptional dysfunction occurs in brains of Huntington’s disease patients and in transgenic mouse and cell models of Huntington’s disease.
To identify new transcriptional pathways altered by the normal and/or abnormal function of huntingtin, we probed several nuclear receptors, normally expressed in the brain, for binding to huntingtin in its mutant and wild-type forms.
Wild-type huntingtin could bind to a number of nuclear receptors; LXRα, PPARγ, VDR and TRα1. Over-expression of huntingtin activated, while knockout of huntingtin decreased, LXR mediated transcription of a reporter gene. Loss of huntingtin also decreased expression of the LXR target gene, ABCA1. In vivo, huntingtin deficient zebrafish had a severe phenotype and reduced expression of LXR regulated genes. An LXR agonist was able to partially rescue the phenotype and the expression of LXR target genes in huntingtin deficient zebrafish during early development.
Our data suggest a novel function for wild-type huntingtin as a co-factor of LXR. However, this activity is lost by mutant huntingtin that only interacts weakly with LXR.
OBJECTIVE—The aim of this study was to find an effective treatment for the genetic form of diabetes that is present in some Huntington's disease patients and in Huntington's disease mouse models. Huntington's disease is a neurodegenerative disorder caused by a polyglutamine expansion within the huntingtin protein. Huntington's disease patients exhibit neuronal dysfunction/degeneration, chorea, and progressive weight loss. Additionally, they suffer from abnormalities in energy metabolism affecting both the brain and periphery. Similarly to Huntington's disease patients, mice expressing the mutated human huntingtin protein also exhibit neurodegenerative changes, motor dysfunction, perturbed energy metabolism, and elevated blood glucose levels.
RESEARCH DESIGN AND METHODS—Huntington's disease mice were treated with an FDA-approved antidiabetic glucagon-like peptide 1 receptor agonist, exendin-4 (Ex-4), to test whether euglycemia could be achieved, whether pancreatic dysfunction could be alleviated, and whether the mice showed any neurological benefit. Blood glucose and insulin levels and various appetite hormone concentrations were measured during the study. Additionally, motor performance and life span were quantified and mutant huntingtin (mhtt) aggregates were measured in both the pancreas and brain.
RESULTS—Ex-4 treatment ameliorated abnormalities in peripheral glucose regulation and suppressed cellular pathology in both brain and pancreas in a mouse model of Huntington's disease. The treatment also improved motor function and extended the survival time of the Huntington's disease mice. These clinical improvements were correlated with reduced accumulation of mhtt protein aggregates in both islet and brain cells.
CONCLUSIONS—Targeting both peripheral and neuronal deficits, Ex-4 is an attractive agent for therapeutic intervention in Huntington's disease patients suffering from diabetes.
Huntington’s disease (HD) is a prototypical neurodegenerative disease in which there is selective neuronal degeneration, which leads to progressive disability, manifesting itself as a movement disorder, with both psychiatric and cognitive impairment. The disease is caused by a CAG repeat expansion in the huntingtin gene, which causes an expanded polyglutamine repeat in the huntingtin protein, resulting in a protein with a novel gain of function. The mutant huntingtin protein causes neuronal dysfunction and eventual cell death in which transcriptional impairment, excitotoxicity, oxidative damage, inflammation, apoptosis and mitochondrial dysfunction are all implicated. A critical transcriptional impairment may be impaired expression and function of PGC-1α, a master co-regulator of mitochondrial biogenesis and expression of antioxidant enzymes. A deficiency of PGC-1α leads to increased vulnerability to oxidative stress and to striatal degeneration. The extent and severity of the oxidative damage in HD are features well recognized but perhaps underappreciated. Oxidative damage occurs to lipids, proteins and DNA, and it has been suggested that the latter may contribute to CAG repeat expansion during DNA repair . A marked elevation of oxidized DNA bases occurs in patients’ plasma, which may provide a biomarker of disease progression. Antioxidants are effective in slowing disease progression in transgenic mouse models of HD, and show promise in human clinical trials. Strategies to transcriptionally increase expression of antioxidant enzymes by modulating the Nrf-2/ARE pathway, or by increasing expression of PGC-1α hold great promise for developing new treatments to slow or halt the progression of HD.
Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG) in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntington's disease. hMTH1 transgene expression conferred a dramatic protection against these Huntington's disease–like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntington's disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntington's disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.
The oxidized purine 8-oxo-7,8-dihydroguanine (8-oxodG) is often found at high levels in DNA of post-mortem brains of patients afflicted by Huntington's disease (HD) and other neurodegenerative conditions. It has been unclear, however, whether this damage accumulation is a consequence or a cause of neurodegeneration. We have addressed this problem in a transgenic mouse model in which the human hMTH1 8-oxodGTPase, which hydrolyzes oxidized nucleoside triphosphates to prevent the incorporation of oxidized bases into nucleic acids, was expressed in several organs. We report that high-level hMTH1 expression in the transgenic mouse brain provides robust protection against neurodegeneration as well as HD-like behavioural and neuropathological symptoms in a chemical model of HD (the mitochondrial toxin 3-nitropropionic acid). In a complementary approach, we used a genetic model for HD based on the use of progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene. Also, in this case, hMTH1 provided strong protection against cell death associated with expression of the mutant huntingtin protein. The new findings we report shed important light on the connection between oxidative DNA damage and human neurodegenerative disorders. Our findings go some way towards forging a causative link between neurodegeneration and oxidative DNA/RNA damage and identify the oxidized purine nucleoside triphosphates as important contributors.
The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over ∼37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1α expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdhQ111/Q111 cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1α-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdhQ111/Q111 cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-κB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD.
Huntington's disease (HD) is a tragic neurodegenerative disorder caused by a CAG repeat that specifies the size of a glutamine tract in the huntingtin protein, such that the longer the tract, the earlier the loss of striatal brain cells. A correlation of polyglutamine tract size has also implicated huntingtin in the proper functioning of mitochondria, the cell's energy factories. Here we have tested the prevailing hypothesis, that huntingtin may directly affect the mitochondrion, by using comprehensive gene expression analysis to judge whether the HD mutation may replicate the effects of 3-nitropropionic acid (3-NP), a compound known to inhibit mitochondria, with loss of striatal neurons. We found that, while mutant huntingtin and 3-NP both elicited energy starvation, the gene responses to the HD mutation, unlike the responses to 3-NP, did not highlight damage to mitochondria, but instead revealed effects on huntingtin-dependent processes. Thus, rather than direct inhibition, the polyglutamine tract size appears to modulate some normal activity of huntingtin that indirectly influences the management of the mitochondrion. Understanding the precise nature of this extra-mitochondrial process would critically guide efforts to achieve effective energy-based therapeutics in HD.
Adipose tissue depots originate from distinct precursor cells, are functionally diverse, and modulate disease processes in a depot-specific manner. However, the functional properties of perivascular adipocytes, and their influence on disease of the blood vessel wall, remain to be determined. We show that human coronary perivascular adipocytes exhibit a reduced state of adipocytic differentiation as compared with adipocytes derived from subcutaneous and visceral (perirenal) adipose depots. Secretion of anti-inflammatory adiponectin is markedly reduced, whereas that of pro-inflammatory cytokines IL-6, IL-8, and MCP-1, is markedly increased in perivascular adipocytes. These depot-specific differences in adipocyte function are demonstrable in both freshly isolated adipose tissues and in vitro differentiated adipocytes. Murine aortic arch perivascular adipose tissues likewise express lower levels of adipocyte-associated genes as compared with subcutaneous and visceral adipose tissues. Moreover, two weeks of high fat feeding caused further reductions in adipocyte-associated gene expression, while up-regulating pro-inflammatory gene expression, in perivascular adipose tissues. These changes were observed in the absence of macrophage recruitment to the perivascular adipose depot. We conclude that perivascular adipocytes exhibit reduced differentiation and a heightened pro-inflammatory state, properties that are intrinsic to the adipocytes residing in this depot. Dysfunction of perivascular adipose tissue induced by fat feeding suggests that this unique adipose depot is capable of linking metabolic signals to inflammation in the blood vessel wall.
Perivascular adipose tissue; adipocytes; adventitia; adipokines; cytokines
Preadipocyte factor-1 (Pref-1) is a transmembrane protein highly expressed in preadipocytes. Pref-1 expression is, however, completely abolished in adipocytes. The extracellular domain of Pref-1 undergoes two proteolytic cleavage events that generate 50 and 25 kDa soluble products. To understand the function of Pref-1, we generated transgenic mice that express the full ectodomain corresponding to the large cleavage product of Pref-1 fused to human immunoglobulin-γ constant region. Mice expressing the Pref-1/hFc transgene in adipose tissue, driven by the adipocyte fatty acid–binding protein (aP2, also known as aFABP) promoter, showed a substantial decrease in total fat pad weight. Moreover, adipose tissue from transgenic mice showed reduced expression of adipocyte markers and adipocyte-secreted factors, including leptin and adiponectin, whereas the preadipocyte marker Pref-1 was increased. Pref-1 transgenic mice with a substantial, but not complete, loss of adipose tissue exhibited hypertriglyceridemia, impaired glucose tolerance, and decreased insulin sensitivity. Mice expressing the Pref-1/hFc transgene exclusively in liver under the control of the albumin promoter also showed a decrease in adipose mass and adipocyte marker expression, suggesting an endocrine mode of action of Pref-1. These findings demonstrate the inhibition of adipogenesis by Pref-1 in vivo and the resulting impairment of adipocyte function that leads to the development of metabolic abnormalities.
Huntington’s disease (HD) is an inherited, relentlessly progressive neurodegenerative disease with an invariably fatal outcome. HD is inherited in an autosomal dominant fashion, and is characterized pathologically by the loss of cortical and striatal neurons, and clinically by involuntary choreiform movements accompanied by progressive cognitive impairment and emotional lability. The disorder is caused by an expanded cystosine adenine guanine (CAG) tri-nucleotide repeat encoding polyglutamine (polyQ) in the first exon of the Huntingtin gene. There is a correlation between the number of CAG repeats and disease onset, such that in patients with CAG repeat lengths of 36 to 60, disease symptoms typically manifest after 35 years of age, whereas CAG repeat lengths >60 yield the more severe juvenile form of the disease. Even though mutant huntingtin is expressed throughout the brain, it is characterized by the selective degeneration of medium spiny neurons of the caudate and putamen, which heralds more widespread neuronal degeneration with disease progression. The mechanisms of cell dysfunction and death in HD have been the subjects of a number of studies, which have led to therapeutic strategies largely based on the amelioration of mutant huntingtin-related metabolic impairment and cellular toxicity. Each of these approaches has aimed to delay or stop the preferential degeneration of medium spiny neurons early in the disease course. Yet, in later stages of the disease, after cell death has become prominent, cell replacement therapy (whether by direct cell transplantation or by the mobilization of endogenous progenitors) may comprise a stronger potential avenue for therapy. In this review, we will consider recent progress in the transplantation of fetal striatal cells to the HD brain, as well as emerging alternative sources for human striatal progenitor cells. We will then consider the potential application of gene therapy toward the induction of striatal neurogenesis and neuronal recruitment, with an eye toward its potential therapeutic use in HD.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0075-8) contains supplementary material, which is available to authorized users.
Cell therapy; Neural transplants; Stem cells; Neurogenesis; Medium spiny neuron
Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.
Huntington's disease (HD) is a neurodegenerative disorder for which the mutation results in an extra-long tract of glutamines that causes the huntingtin protein to aggregate. It is characterized by neurological symptoms and brain pathology that is associated with nuclear and cytoplasmic aggregates and with transcriptional dysregulation. Despite the fact that HD has been recognized principally as a neurological disease, there are multiple epidemiological studies showing that HD patients exhibit a high rate of cardiovascular events leading to heart failure. To unravel the cause of cardiac dysfunction in HD models, we employed a wide range of molecular and physiological methods using two well established genetic mouse models of this disease. We found that pre-symptomatic animals developed aberrant gap junction channel expression and a significant deregulation of hypertrophic markers that may predispose them to arrhythmia and an overall change in cardiac function. These changes were accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis in the symptomatic animals. Surprisingly, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation. Therefore, we conclude that the HD-related cardiomyopathy could be driven by altered central autonomic pathways.
Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) repeat in exon-1 in the Huntingtin gene (HTT). This results in misfolding and accumulation of the huntingtin (htt) protein, forming nuclear and cytoplasmic inclusions. HD is associated with dysregulation of gene expression as well as mitochondrial dysfunction. We hypothesized that by improving transcriptional regulation of genes necessary for energy metabolism, the HD motor phenotype would also improve. We therefore examined the protective effects of nicotinamide (NAM), a well-characterized water-soluble B vitamin that is an inhibitor of sirtuin1/class III NAD+-dependent histone deacetylase (HDAC). In this study, both mini-osmotic pumps and drinking water deliveries were tested at 250mg NAM/kg/day, using the B6.HDR6/1 transgenic mouse model. Results were similar for both modes of delivery, and there was no evidence of toxicity. We found that NAM treatment increased mRNA levels of brain-derived neurotrophic factor (BDNF), and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis. Protein levels of BDNF were also significantly increased. In addition, NAM treatment increased PGC-1α activation in HD mice, pointing to a possible mode of action as a therapeutic. Critically, NAM treatment was able to improve motor deficits associated with the HD phenotype, tested as time courses of open field, rotarod, and balance beam activities. These improvements were substantial, despite the fact that NAM did not appear to reduce htt aggregation, or to prevent late-stage weight loss. Our study therefore concludes that NAM or similar drugs may be beneficial in clinical treatment of the motor dysfunctions of HD, while additional therapeutic approaches must be added to combat the aggregation phenotype and overall physiological decline.
Huntington’s Disease; nicotinamide; vitamin; Brain-derived neurotrophic factor; Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; histone deacetylase inhibitor
Huntington's disease is initiated by the expression of a CAG repeat-encoded polyglutamine region in full-length huntingtin, with dominant effects that vary continuously with CAG size. The mechanism could involve a simple gain of function or a more complex gain of function coupled to a loss of function (e.g. dominant negative-graded loss of function). To distinguish these alternatives, we compared genome-wide gene expression changes correlated with CAG size across an allelic series of heterozygous CAG knock-in mouse embryonic stem (ES) cell lines (HdhQ20/7, HdhQ50/7, HdhQ91/7, HdhQ111/7), to genes differentially expressed between Hdhex4/5/ex4/5 huntingtin null and wild-type (HdhQ7/7) parental ES cells. The set of 73 genes whose expression varied continuously with CAG length had minimal overlap with the 754-member huntingtin-null gene set but the two were not completely unconnected. Rather, the 172 CAG length-correlated pathways and 238 huntingtin-null significant pathways clustered into 13 shared categories at the network level. A closer examination of the energy metabolism and the lipid/sterol/lipoprotein metabolism categories revealed that CAG length-correlated genes and huntingtin-null-altered genes either were different members of the same pathways or were in unique, but interconnected pathways. Thus, varying the polyglutamine size in full-length huntingtin produced gene expression changes that were distinct from, but related to, the effects of lack of huntingtin. These findings support a simple gain-of-function mechanism acting through a property of the full-length huntingtin protein and point to CAG-correlative approaches to discover its effects. Moreover, for therapeutic strategies based on huntingtin suppression, our data highlight processes that may be more sensitive to the disease trigger than to decreased huntingtin levels.
Apolipoprotein E (apoE), a key protein in lipid metabolism, is highly expressed in adipose tissues. Studies have shown that human APOE*4 is associated with a lower body mass index but with a greater risk of coronary heart disease compared with other APOE alleles. To define the isoform-specific role of apoE in regulating the expandability and functionality of adipose tissues, we investigated the effects of diet-induced obesity in mice whose endogenous Apoe gene has been replaced by either the human APOE*3 or APOE*4 allele.
After 8 weeks on a Western-type high-fat diet, male APOE4 mice displayed impaired tolerance to glucose and fat overload compared with APOE3 mice. Subcutaneous fat tissues in APOE4 and APOE3 mice after high fat feeding were not different. In contrast, although epididymal fat tissues in APOE4 mice gained 30% less weight during the high fat feeding than in APOE3 mice, they showed impaired insulin-stimulated glucose uptake ex vivo. Epididymal APOE4 adipocytes were larger in size than APOE3 adipocytes, and expressed reduced levels of mRNA for peroxisome proliferator-activated receptor γ2 and adiponectin, important markers of adipocyte functionality. Adenoviral expression of apoE3 in apoE-null culture adipocytes induced adiponectin mRNA in a dose-dependent manner, but the induction was significantly blunted in cells overexpressing apoE4. However, in contrast to the apoE3-expressing cells, Glut1, but not Glut4, expression levels were positively correlated with increased apoE4 mRNA, suggesting that apoE4 expression in adipocyte interferes in insulin-sensing pathways.
Dysfunctional epididymal adipose tissues contribute to the accelerated impairment of glucose tolerance in APOE4 mice fed a Western-type diet. Our results underscore the importance of functionality of individual fat depots rather than total fat mass as a determinant for metabolic disturbance during diet-induced obesity.
apolipoprotein E; apoE3; apoE4; diet-induced obesity; adipocyte functionality
Estrogen receptors (ER) are important regulators of metabolic diseases such as obesity and insulin resistance (IR). While ERα seems to have a protective role in such diseases, the function of ERβ is not clear. To characterize the metabolic function of ERβ, we investigated its molecular interaction with a master regulator of insulin signaling/glucose metabolism, the PPARγ, in vitro and in high-fat diet (HFD)-fed ERβ -/- mice (βERKO) mice. Our in vitro experiments showed that ERβ inhibits ligand-mediated PPARγ-transcriptional activity. That resulted in a blockade of PPARγ-induced adipocytic gene expression and in decreased adipogenesis. Overexpression of nuclear coactivators such as SRC1 and TIF2 prevented the ERβ-mediated inhibition of PPARγ activity. Consistent with the in vitro data, we observed increased PPARγ activity in gonadal fat from HFD-fed βERKO mice. In consonance with enhanced PPARγ activation, HFD-fed βERKO mice showed increased body weight gain and fat mass in the presence of improved insulin sensitivity. To directly demonstrate the role of PPARγ in HFD-fed βERKO mice, PPARγ signaling was disrupted by PPARγ antisense oligonucleotide (ASO). Blockade of adipose PPARγ by ASO reversed the phenotype of βERKO mice with an impairment of insulin sensitization and glucose tolerance. Finally, binding of SRC1 and TIF2 to the PPARγ-regulated adiponectin promoter was enhanced in gonadal fat from βERKO mice indicating that the absence of ERβ in adipose tissue results in exaggerated coactivator binding to a PPARγ target promoter. Collectively, our data provide the first evidence that ERβ-deficiency protects against diet-induced IR and glucose intolerance which involves an augmented PPARγ signaling in adipose tissue. Moreover, our data suggest that the coactivators SRC1 and TIF2 are involved in this interaction. Impairment of insulin and glucose metabolism by ERβ may have significant implications for our understanding of hormone receptor-dependent pathophysiology of metabolic diseases, and may be essential for the development of new ERβ-selective agonists.
In the present study, we demonstrate for the first time a pro-diabetogenic function of the ERβ. Our experiments indicate that ERβ impairs insulin sensitivity and glucose tolerance in mice challenged with a high fat diet (HFD). Loss of ERβ, studied in ERβ -/- mice (βERKO mice), results in increased body weight gain and fat deposition under HFD-treatment. Conversely, absence of ERβ averted accumulation of triglycerides and preserved regular insulin signaling in liver and skeletal muscle. This observation was associated with improved whole-body insulin sensitivity and glucose tolerance. Increased adipose tissue mass in the presence of improved insulin sensitivity and glucose tolerance is usually observed under chronic stimulation of the nuclear hormone receptor PPARγ. In consonance, we show that activation of PPARγ was markedly induced in gonadal fat from βERKO mice and blockade of adipose PPARγ signaling by antisense oligonucleotide injection reversed the metabolic phenotype. Moreover, our cell culture experiments indicate that ERβ is a negative regulator of ligand-induced PPARγ activity in vitro. Finally, we identify SRC1 and TIF2 as key players in the ERβ-PPARγ interaction. In summary, the present study demonstrates that ERβ impairs insulin and glucose metabolism, which may, at least in part, result from a negative cross-talk with adipose PPARγ.
Huntington's disease (HD) is a progressive neurodegenerative disorder characterized clinically by motor and psychiatric disturbances and pathologically by neuronal loss and gliosis (reactive astrocytosis) particularly in the striatum and cerebral cortex. We have recently created HD full-length cDNA transgenic mouse models that may serve as a paradigm for HD. A more detailed characterization of these models is presented here. The transgene encoding normal huntingtin consists of 9417 bp of the huntingtin coding sequences including 16 tandem CAGs coding for polyglutamines as part of exon 1. The transgene is driven by a heterologous cytomegalovirus promoter. Five independent transgenic mouse lines were obtained using this construct. An additional six transgenic lines were obtained using full-length HD constructs that have been modified to include either 48 or 89 CAG repeat expansions. Southern blot and densitometric analyses indicated unique integration sites for the transgene in each of the lines with a copy number ranging from two to 22 copies. Widespread expression of the transgene in brain, heart, spleen, kidney, lung, liver and gonads from each line was determined by Western blot analyses. In the brain, transgene expression was found in cerebral cortex, striatum, hippocampus and cerebellum. Expression of the transgene was as much as five times the endogenous mouse huntingtin level. Phenotypically, only mice expressing 48 or 89 CAG repeats manifested progressive behavioural and motor dysfunction. Early behavioural abnormalities were characterized by trunk curling and clasping of both fore- and hindlimbs when the animals were suspended by their tails. Subsequently, these mice exhibited hyperkinetic movements, including heightened exploratory activities, unidirectional rotational behaviour, backflipping and excessive grooming that lasted for several weeks. Eventually, the animals progressed to a hypokinetic phase consisting of slowed movements and lack of response to sensory stimuli. Urine retention or incontinence was also a prominent feature of the hypokinetic phase. At the end stage of the disease process, HD48(B,D) and HD89(A-C) mice became akinetic just prior to death. Neuropathological examination of mice at various stages indicated that it was only during the hypokinetic phase and thereafter when selective neuronal loss was most apparent. Regions of neurodegeneration and loss included the striatum, cerebral cortex, thalamus and hippocampus. TUNEL staining indicated an apoptotic mode of cell death in these brain regions. Comparative neuronal counts after Nissl staining showed as much as 20% loss of small and medium neurons in the striatum in mice at the hypokinetic and akinetic stages. Reactive astrocytosis accompanied the areas of neurodegeneration and loss. Polyglutamine aggregates in the form of neuronal intranuclear inclusions and diffuse nuclear and perinuclear aggregations were found in a small percentage of neurons, including those in brain regions that are typically spared in HD. This observation suggests that polyglutamine aggregates may not be sufficient to cause neuronal loss in HD. In both behavioural and neuropathological analyses, wild-type and transgenic animals with 16 CAG repeats were indistinguishable from each other and do not exhibit the changes observed for mice carrying the 48 and 89 CAG repeat mutations. Thus, animals expressing the CAG repeat expansions appear to represent clinically analogous models for HD pathogenesis, and may also provide insights into the underlying pathophysiological mechanisms of other triplet repeat disorders.
Psychiatric and metabolic features appear several years before motor disturbances in the neurodegenerative Huntington’s disease (HD), caused by an expanded CAG repeat in the huntingtin (HTT) gene. Although the mechanisms leading to these aspects are unknown, dysfunction in the hypothalamus, a brain region controlling emotion and metabolism, has been suggested. A direct link between the expression of the disease causing protein, huntingtin (HTT), in the hypothalamus and the development of metabolic and psychiatric-like features have been shown in the BACHD mouse model of HD. However, precisely which circuitry in the hypothalamus is critical for these features is not known. We hypothesized that expression of mutant HTT in the ventromedial hypothalamus, an area involved in the regulation of metabolism and emotion would be important for the development of these non-motor aspects. Therefore, we inactivated mutant HTT in a specific neuronal population of the ventromedial hypothalamus expressing the transcription factor steroidogenic factor 1 (SF1) in the BACHD mouse using cross-breeding based on a Cre-loxP system. Effects on anxiety-like behavior were assessed using the elevated plus maze and novelty-induced suppressed feeding test. Depressive-like behavior was assessed using the Porsolt forced swim test. Effects on the metabolic phenotype were analyzed using measurements of body weight and body fat, as well as serum insulin and leptin levels. Interestingly, the inactivation of mutant HTT in SF1-expressing neurons exerted a partial positive effect on the depressive-like behavior in female BACHD mice at 4 months of age. In this cohort of mice, no anxiety-like behavior was detected. The deletion of mutant HTT in SF1 neurons did not have any effect on the development of metabolic features in BACHD mice. Taken together, our results indicate that mutant HTT regulates metabolic networks by affecting hypothalamic circuitries that do not involve the SF1 neurons of the ventromedial hypothalamus.
Recent evidence suggests that mutant huntingtin protein-induced energetic perturbations contribute to neuronal dysfunction in Huntington’s disease (HD). Given the ubiquitous expression of huntingtin, other cell types with high energetic burden may be at risk for HD-related dysfunction. Early-onset cardiovascular disease is the second leading cause of death in HD patients; a direct role for mutant huntingtin in this phenomenon remains unevaluated. Here we tested the hypothesis that expression of mutant huntingtin is sufficient to induce cardiac dysfunction, using a well-described transgenic model of HD (line R6/2). R6/2 mice developed cardiac dysfunction by 8 weeks of age, progressing to severe failure at 12 weeks, assessed by echocardiography. Limited evidence of cardiac remodeling (e.g. hypertrophy, fibrosis, apoptosis, β1 adrenergic receptor downregulation) was observed. Immunogold electron microscopy demonstrated significant elevations in nuclear and mitochondrial polyglutamine presence in the R6/2 myocyte. Significant alterations in mitochondrial ultrastructure were seen, consistent with metabolic stress. Increased cardiac lysine acetylation and protein nitration were observed, and were each significantly associated with impairments in cardiac performance. These data demonstrate that mutant huntingtin expression has potent cardiotoxic effects; cardiac failure may be a significant complication of this important experimental model of HD. Investigation of the potential cardiotropic effects of mutant huntingtin in humans may be warranted.
huntingtin; Huntington’s disease; heart; mitochondria; polyglutamine; cardiovascular; transgenic; nitrotyrosine; acetylation
A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.
Huntington's disease (HD) is an inherited disorder caused by mutation of the gene that encodes the huntingtin protein. The specific mutation that results in disease is an increase in the copies of the amino acid glutamine in a stretch of repeated glutamines at the amino-terminus of the protein. This “expanded polyglutamine” huntingtin acquires toxic properties, presumably through mechanisms that involve its reduced solubility and aberrant interactions with other cellular proteins that do not occur with the normal protein. In this study, we sought to identify cellular processes that were involved in the toxicity conferred by the mutant huntingtin protein. We used RNA interference in order to specifically reduce the levels of individual cellular proteins and identified a number that could reduce mutant huntingtin toxicity. These modifiers clustered into functional pathways know to be involved in HD and other novel pathways. Among these modifiers, we found that the signaling protein RRAS, as well as additional members of its signaling cascade, are involved in mutant huntingtin toxicity. We further showed that a small molecule inhibitor of an enzyme involved in this pathway is effective at reducing this toxicity, indicating that the targeted inhibition of the RRAS pathway may be of therapeutic benefit in Huntington's disease.
We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARγ coactivator-1α (PGC-1α) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1α, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRα mRNA and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA) reduced ATP and PCr levels and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1α, NRF1, Tfam and downstream genes in the striatum and cerebral cortex of wild-type (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1α and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1α, therefore, plays an important role in the behavioral phenotype, metabolic disturbances and pathology of HD, which suggests the possibility that agents that enhance PGC-1α function will exert therapeutic benefits in HD patients.
Adiponectin is an adipocyte hormone that links visceral adiposity with insulin resistance and atherosclerosis. It is unique among adipocyte-derived hormones in that its circulating concentrations are inversely proportional to adiposity, and low adiponectin concentrations predict the development of type 2 diabetes and cardiovascular disease. Consequently, in the decade since its discovery, adiponectin has generated immense interest as a potential therapeutic target for the metabolic syndrome and diabetes.
This review summarizes current research regarding the regulation of circulating adiponectin concentrations by physiological, pharmacological, and nutritional factors, with an emphasis on human studies. In humans, plasma adiponectin concentrations are influenced by age and gender, and are inversely proportional to visceral adiposity. In vitro studies suggest that adiponectin production may be determined primarily by adipocyte size and insulin sensitivity, with larger, insulin-resistant adipocytes producing less adiponectin. While adiponectin concentrations are unchanged after meal ingestion, they are increased by significant weight loss, such as after bariatric surgery. In addition, adiponectin production is inhibited by a number of hormones, including testosterone, prolactin, glucocorticoids and growth hormone, and by inflammation and oxidative stress in adipose tissue. Smoking decreases, while moderate alcohol consumption increases, circulating adiponectin concentrations. Dietary fatty acid composition in rodents influences adiponectin production via ligand-activated nuclear receptors (PPARs); however, current evidence in humans is equivocal. In addition to PPAR agonists (such as thiazolidinediones and fibrates), a number of pharmacological agents (angiotensin receptor type 1 blockers, ACE inhibitors, and cannabinoid receptor antagonists) used in treatment of the metabolic syndrome also increase adiponectin concentrations in humans.
Aims: Increased oxidative stress and mitochondrial dysfunction in obese adipocytes contribute to adipokine dysregulation, inflammation, and insulin resistance. Results: Through an advanced proteomic analysis, we found that peroxiredoxin 3 (Prx3), a thioredoxin-dependent mitochondrial peroxidase, is highly expressed in 3T3-L1 adipocytes compared to preadipocytes. Interestingly, in obese db/db mice and human subjects, adipose Prx3 levels were significantly decreased, indicating its association with obesity. We therefore employed Prx3 knockout (KO) mice and transfected 3T3-L1 cells to examine the role of endogenous Prx3 in adipocyte metabolism. Prx3 KO mice had increased fat mass compared to wild-type due to adipocyte hypertrophy. Increased adipogenic transcription factors and lipogenic gene expression during differentiation of adipose tissue-derived stem cells from Prx3-deficient mice confirmed that these adipocytes are likely to accumulate fat. Mitochondrial protein carbonylation in Prx3 KO adipose tissue and mitochondrial superoxide level in Prx3 knockdown 3T3-L1 cells were increased showing aberrant regulation of oxidative stress. Proteomic analysis and gene expression analysis of Prx3 KO mice adipocytes also showed defect in mitochondria biogenesis along with enzymes involved in glucose/lipid metabolism and oxidative phosphorylation. In addition, expression level of adiponectin was downregulated and plasminogen activator inhibitor-1 was upregulated in Prx3 KO adipocytes. Impaired glucose tolerance and insulin resistance further implied metabolic dysregulation in Prx3 KO mice. Innovation and Conclusion: These data suggest that endogenous Prx3 may play an essential role in maintaining normal characteristics of adipocytes and that defect in Prx3 alters mitochondrial redox state and function, and adipokine expression in adipocytes leading to metabolic alteration. Antioxid. Redox Signal. 16, 229–243.
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 protein. The huntingtin 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 overexpression 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 overexpress BDNF in the forebrain of R6/1 mice which express a fragment of mutant huntingtin 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, 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.
brain-derived neurotrophic factor; Huntington’s disease; motor coordination; brain atrophy; neuronal intranuclear inclusions; gene expression
Obesity-associated diseases account for a large portion of public health challenges. Among obesity-related disorders, a direct and independent relationship has been ascertained for colorectal cancer (CRC). The evidence that adipocyte hypertrophy and excessive adipose tissue accumulation (mainly visceral) can promote pathogenic adipocyte and adipose tissue-related diseases, has led to formulate the concept of “adiposopathy”, defined as adipocyte and adipose tissue dysfunction that contributes to metabolic syndrome. Adipose tissue can, indeed, be regarded as an important and highly active player of the innate immune response, in which cytokine/adipokine secretion is responsible for a paracrine loop between adipocytes and macrophages, thus contributing to the systemic chronic low-grade inflammation associated with visceral obesity, which represents a favorable niche for tumor development. The adipocyte itself participates as a central mediator of this inflammatory response in obese individuals by secreting hormones, growth factors and proinflammatory cytokines, which are of particular relevance for the pathogenesis of CRC. Among adipocyte-secreted hormones, the most relevant to colorectal tumorigenesis are adiponectin, leptin, resistin and ghrelin. All these molecules have been involved in cell growth and proliferation, as well as tumor angiogenesis and it has been demonstrated that their expression changes from normal colonic mucosa to adenoma and adenocarcinoma, suggesting their involvement in multistep colorectal carcinogenesis. These findings have led to the hypothesis that an unfavorable adipokine profile, with a reduction of those with an anti-inflammatory and anti-cancerous activity, might serve as a prognostic factor in CRC patients and that adipokines or their analogues/antagonists might become useful agents in the management or chemoprevention of CRC.
Obesity; Colorectal cancer; Inflammation; Adiponectin; Leptin; Resistin; Ghrelin; Cytokines
Expansion of a polyglutamine repeat at the amino-terminus of huntingtin is the probable cause for Huntington's disease, a lethal progressive autosomal-dominant neurodegenerative disorders characterized by impaired motor performance and severe brain atrophy. The expanded polyglutamine repeat changes the conformation of huntingtin and initiates a range of pathogenic mechanisms in neurons including intracellular huntingtin aggregates, transcriptional dysregulation, energy metabolism deficits, synaptic dystrophy and ultimately neurodegeneration. It is unclear how these events relate to each other or if they can be reversed by pharmacological intervention. Here, we describe neuronal cell lines expressing inducible fragments of normal and mutant huntingtin.
In HN10 cells, the expression of wild type and mutant huntingtin fragments was dependent on the induction time as well as on the concentration of the RheoSwitch® inducing ligand. In order to analyze the effect of mutant huntingtin expression on cellular functions we concentrated on the 72Q exon1 huntingtin expressing cell line and found that upon induction, it was possible to carefully dissect mutant huntingtin-induced phenotypes as they developed over time. Dysregulation of transcription as a result of mutant huntingtin expression showed a transcription signature replicating that reported in animal models and Huntington's disease patients. Crucially, triggering of neuronal differentiation in mutant huntingtin expressing cell resulted in the appearance of additional pathological hallmarks of Huntington's disease including cell death.
We developed neuronal cell lines with inducible expression of wild type and mutant huntingtin. These new cell lines represent a reliable in vitro system for modeling Huntington's disease and should find wide use for high-throughput screening application and for investigating the biology of mutant huntingtin.
Obesity and weight gain are characterized by increased adipose tissue mass due to an increase in the size of individual adipocytes and the generation of new adipocytes. New adipocytes are believed to arise from resident adipose tissue preadipocytes and mesenchymal progenitor cells. However, it is possible that progenitor cells from other tissues, in particular BM, could also contribute to development of new adipocytes in adipose tissue. We tested this hypothesis by transplanting whole BM cells from GFP-expressing transgenic mice into wild-type C57BL/6 mice and subjecting them to a high-fat diet or treatment with the thiazolidinedione (TZD) rosiglitazone (ROSI) for several weeks. Histological examination of adipose tissue or FACS of adipocytes revealed the presence of GFP+ multilocular (ML) adipocytes, whose number was significantly increased by ROSI treatment or high-fat feeding. These ML adipocytes expressed adiponectin, perilipin, fatty acid–binding protein (FABP), leptin, C/EBPα, and PPARγ but not uncoupling protein–1 (UCP-1), the CD45 hematopoietic lineage marker, or the CDllb monocyte marker. They also exhibited increased mitochondrial content. Appearance of GFP+ ML adipocytes was contemporaneous with an increase in circulating levels of mesenchymal and hematopoietic progenitor cells in ROSI-treated animals. We conclude that TZDs and high-fat feeding promote the trafficking of BM-derived circulating progenitor cells to adipose tissue and their differentiation into ML adipocytes.
Obesity contributes to Type 2 diabetes by promoting systemic insulin resistance. Obesity causes features of metabolic dysfunction in the adipose tissue that may contribute to later impairments of insulin action in skeletal muscle and liver; these include reduced insulin-stimulated glucose transport, reduced expression of GLUT4, altered expression of adipokines, and adipocyte hypertrophy. Animal studies have shown that expansion of adipose tissue alone is not sufficient to cause systemic insulin resistance in the absence of adipose tissue metabolic dysfunction. To determine if this holds true for humans, we studied the relationship between insulin resistance and markers of adipose tissue dysfunction in non-obese individuals.
32 non-obese first-degree relatives of Type 2 diabetic patients were recruited. Glucose tolerance was determined by an oral glucose tolerance test and insulin sensitivity was measured with the hyperinsulinaemic-euglycaemic clamp. Blood samples were collected and subcutaneous abdominal adipose tissue biopsies obtained for gene/protein expression and adipocyte cell size measurements.
Our findings show that also in non-obese individuals low insulin sensitivity is associated with signs of adipose tissue metabolic dysfunction characterized by low expression of GLUT4, altered adipokine profile and enlarged adipocyte cell size. In this group, insulin sensitivity is positively correlated to GLUT4 mRNA (R = 0.49, p = 0.011) and protein (R = 0.51, p = 0.004) expression, as well as with circulating adiponectin levels (R = 0.46, 0 = 0.009). In addition, insulin sensitivity is inversely correlated to circulating RBP4 (R = −0.61, 0 = 0.003) and adipocyte cell size (R = −0.40, p = 0.022). Furthermore, these features are inter-correlated and also associated with other clinical features of the metabolic syndrome in the absence of obesity. No association could be found between the hypertrophy-associated adipocyte dysregulation and HIF-1alpha in this group of non-obese individuals.
In conclusion, these findings support the concept that it is not obesity per se, but rather metabolic dysfunction of adipose tissue that is associated with systemic insulin resistance and the metabolic syndrome.
Adipocyte cell size; BMI; Insulin sensitivity; GLUT4; Adiponectin; RBP4