HdhQ111 Mice Exhibit Tissue Specific Metabolite Profiles that Include Striatal Lipid Accumulation.

The HTT CAG expansion mutation causes Huntington's Disease and is associated with a wide range of cellular consequences, including altered metabolism. The mutant allele is expressed widely, in all tissues, but the striatum and cortex are especially vulnerable to its effects. To more fully understand this tissue-specificity, early in the disease process, we asked whether the metabolic impact of the mutant CAG expanded allele in heterozygous B6.HdhQ111/+ mice would be common across tissues, or whether tissues would have tissue-specific responses and whether such changes may be affected by diet. Specifically, we cross-sectionally examined steady state metabolite concentrations from a range of tissues (plasma, brown adipose tissue, cerebellum, striatum, liver, white adipose tissue), using an established liquid chromatography-mass spectrometry pipeline, from cohorts of 8 month old mutant and wild-type littermate mice that were fed one of two different high-fat diets. The differential response to diet highlighted a proportion of metabolites in all tissues, ranging from 3% (7/219) in the striatum to 12% (25/212) in white adipose tissue. By contrast, the mutant CAG-expanded allele primarily affected brain metabolites, with 14% (30/219) of metabolites significantly altered, compared to wild-type, in striatum and 11% (25/224) in the cerebellum. In general, diet and the CAG-expanded allele both elicited metabolite changes that were predominantly tissue-specific and non-overlapping, with evidence for mutation-by-diet interaction in peripheral tissues most affected by diet. Machine-learning approaches highlighted the accumulation of diverse lipid species as the most genotype-predictive metabolite changes in the striatum. Validation experiments in cell culture demonstrated that lipid accumulation was also a defining feature of mutant HdhQ111 striatal progenitor cells. Thus, metabolite-level responses to the CAG expansion mutation in vivo were tissue specific and most evident in brain, where the striatum featured signature accumulation of a set of lipids including sphingomyelin, phosphatidylcholine, cholesterol ester and triglyceride species. Importantly, in the presence of the CAG mutation, metabolite changes were unmasked in peripheral tissues by an interaction with dietary fat, implying that the design of studies to discover metabolic changes in HD mutation carriers should include metabolic perturbations.

Dominant effects of the Huntington's disease HTT CAG repeat length are captured in gene-expression data sets by a continuous analysis mathematical modeling strategy.

In Huntington's disease (HD), the size of the expanded HTT CAG repeat mutation is the primary driver of the processes that determine age at onset of motor symptoms. However, correlation of cellular biochemical parameters also extends across the normal repeat range, supporting the view that the CAG repeat represents a functional polymorphism with dominant effects determined by the longer allele. A central challenge to defining the functional consequences of this single polymorphism is the difficulty of distinguishing its subtle effects from the multitude of other sources of biological variation. We demonstrate that an analytical approach based upon continuous correlation with CAG size was able to capture the modest (∼21%) contribution of the repeat to the variation in genome-wide gene expression in 107 lymphoblastoid cell lines, with alleles ranging from 15 to 92 CAGs. Furthermore, a mathematical model from an iterative strategy yielded predicted CAG repeat lengths that were significantly positively correlated with true CAG allele size and negatively correlated with age at onset of motor symptoms. Genes negatively correlated with repeat size were also enriched in a set of genes whose expression were CAG-correlated in human HD cerebellum. These findings both reveal the relatively small, but detectable impact of variation in the CAG allele in global data in these peripheral cells and provide a strategy for building multi-dimensional data-driven models of the biological network that drives the HD disease process by continuous analysis across allelic panels of neuronal cells vulnerable to the dominant effects of the HTT CAG repeat.

Lack of huntingtin promotes neural stem cells differentiation into glial cells while neurons expressing huntingtin with expanded polyglutamine tracts undergo cell death.

Huntington's disease (HD) is a neurodegenerative disorder that affects muscle coordination and diminishes cognitive abilities. The genetic basis of the disease is an expansion of CAG repeats in the Huntingtin (Htt) gene. Here we aimed to generate a series of mouse neural stem (NS) cell lines that carried varying numbers of CAG repeats in the mouse Htt gene (Hdh CAG knock-in NS cells) or that had Hdh null alleles (Hdh knock-out NS cells). Towards this end, Hdh CAG knock-in mouse ES cell lines that carried an Htt gene with 20, 50, 111, or 140 CAG repeats or that were Htt null were neuralized and converted into self-renewing NS cells. The resulting NS cell lines were immunopositive for the neural stem cell markers NESTIN, SOX2, and BLBP and had similar proliferative rates and cell cycle distributions. After 14 days in vitro, wild-type NS cells gave rise to cultures composed of 70% MAP2(+) neurons and 30% GFAP(+) astrocytes. In contrast, NS cells with expanded CAG repeats underwent neuronal cell death, with only 38%±15% of the MAP2(+) cells remaining at the end of the differentiation period. Cell death was verified by increased caspase 3/7 activity on day 14 of the neuronal differentiation protocol. Interestingly, Hdh knock-out NS cells treated using the same neuronal differentiation protocol showed a dramatic increase in the number of GFAP(+) cells on day 14 (61%±20% versus 24%±10% in controls), and a massive decrease of MAP2(+) neurons (30%±11% versus 64%±17% in controls). Both Hdh CAG knock-in NS cells and Hdh knock-out NS cells showed reduced levels of Bdnf mRNA during neuronal differentiation, in agreement with data obtained previously in HD mouse models and in post-mortem brain samples from HD patients. We concluded that Hdh CAG knock-in and Hdh knock-out NS cells have potential as tools for investigating the roles of normal and mutant HTT in differentiated neurons and glial cells of the brain.

Differential effects of the Huntington's disease CAG mutation in striatum and cerebellum are quantitative not qualitative.

Huntington's disease (HD) involves marked early neurodegeneration in the striatum, whereas the cerebellum is relatively spared despite the ubiquitous expression of full-length mutant huntingtin, implying that inherent tissue-specific differences determine susceptibility to the HD CAG mutation. To understand this tissue specificity, we compared early mutant huntingtin-induced gene expression changes in striatum to those in cerebellum in young Hdh CAG knock-in mice, prior to onset of evident pathological alterations. Endogenous levels of full-length mutant huntingtin caused qualitatively similar, but quantitatively different gene expression changes in the two brain regions. Importantly, the quantitatively different responses in the striatum and cerebellum in mutant mice were well accounted for by the intrinsic molecular differences in gene expression between the striatum and cerebellum in wild-type animals. Tissue-specific gene expression changes in response to the HD mutation, therefore, appear to reflect the different inherent capacities of these tissues to buffer qualitatively similar effects of mutant huntingtin. These findings highlight a role for intrinsic quantitative tissue differences in contributing to HD pathogenesis, and likely to other neurodegenerative disorders exhibiting tissue-specificity, thereby guiding the search for effective therapeutic interventions.

Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis.

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.

Meclizine is neuroprotective in models of Huntington's disease.

Defects in cellular energy metabolism represent an early feature in a variety of human neurodegenerative diseases. Recent studies have shown that targeting energy metabolism can protect against neuronal cell death in such diseases. Here, we show that meclizine, a clinically used drug that we have recently shown to silence oxidative metabolism, suppresses apoptotic cell death in a murine cellular model of polyglutamine (polyQ) toxicity. We further show that this protective effect extends to neuronal dystrophy and cell death in Caenorhabditis elegans and Drosophila melanogaster models of polyQ toxicity. Meclizine's mechanism of action is not attributable to its anti-histaminergic or anti-muscarinic activity, but rather, strongly correlates with its ability to suppress mitochondrial respiration. Since meclizine is an approved drug that crosses the blood-brain barrier, it may hold therapeutic potential in the treatment of polyQ toxicity disorders, such as Huntington's disease.

Cholesterol defect is marked across multiple rodent models of Huntington's disease and is manifest in astrocytes.

Brain cholesterol, which is synthesized locally, is a major component of myelin and cell membranes and participates in neuronal functions, such as membrane trafficking, signal transduction, neurotransmitter release, and synaptogenesis. Here we show that brain cholesterol biosynthesis is reduced in multiple transgenic and knock-in Huntington's disease (HD) rodent models, arguably dependent on deficits in mutant astrocytes. Mice carrying a progressively increased number of CAG repeats show a more evident reduction in cholesterol biosynthesis. In postnatal life, the cholesterol-dependent activities of neurons mainly rely on the transport of cholesterol from astrocytes on ApoE-containing particles. Our data show that mRNA levels of cholesterol biosynthesis and efflux genes are severely reduced in primary HD astrocytes, along with impaired cellular production and secretion of ApoE. Consistently, in CSF of HD mice, ApoE is mostly associated with smaller lipoproteins, indicating reduced cholesterol transport on ApoE-containing lipoproteins circulating in the HD brain. These findings indicate that cholesterol defect is robustly marked in HD animals, implying that strategies aimed at selectively modulating brain cholesterol metabolism might be of therapeutic significance.

Rrs1 is involved in endoplasmic reticulum stress response in Huntington disease.

The induction of Rrs1 expression is one of the earliest events detected in a presymptomatic knock-in mouse model of Huntington disease (HD). Rrs1 up-regulation fulfills the HD criteria of dominance, striatal specificity, and polyglutamine dependence. Here we show that mammalian Rrs1 is localized both in the nucleolus as well as in the endoplasmic reticulum (ER) of neurons. This dual localization is shared with its newly identified molecular partner 3D3/lyric. We then show that both genes are induced by ER stress in neurons. Interestingly, we demonstrate that ER stress is an early event in a presymptomatic HD mouse model that persists throughout the life span of the rodent. We further show that ER stress also occurs in postmortem brains of HD patients.

Widespread disruption of repressor element-1 silencing transcription factor/neuron-restrictive silencer factor occupancy at its target genes in Huntington's disease.

Huntingtin is a protein that is mutated in Huntington's disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.

Discovery of bioactive small-molecule inhibitor of poly adp-ribose polymerase: implications for energy-deficient cells.

Poly (ADP-ribose) polymerase (PARP1) is a nuclear protein that, when overactivated by oxidative stress-induced DNA damage, ADP ribosylates target proteins leading to dramatic cellular ATP depletion. We have discovered a biologically active small-molecule inhibitor of PARP1. The discovered compound inhibited PARP1 enzymatic activity in vitro and prevented ATP loss and cell death in a surrogate model of oxidative stress in vivo. We also investigated a new use for PARP1 inhibitors in energy-deficient cells by using Huntington's disease as a model. Our results showed that insult with the oxidant hydrogen peroxide depleted cellular ATP in mutant cells below the threshold of viability. The protective role of PARP1 inhibitors against oxidative stress has been shown in this model system.

Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Deltaex7/8) knock-in mice and CbCln3(Deltaex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process.

Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice.

Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.

HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism.

The 'expanded' HD CAG repeat that causes Huntington's disease (HD) encodes a polyglutamine tract in huntingtin, which first targets the death of medium-sized spiny striatal neurons. Mitochondrial energetics, related to N-methyl-d-aspartate (NMDA) Ca2+-signaling, has long been implicated in this neuronal specificity, implying an integral role for huntingtin in mitochondrial energy metabolism. As a genetic test of this hypothesis, we have looked for a relationship between the length of the HD CAG repeat, expressed in endogenous huntingtin, and mitochondrial ATP production. In STHdhQ111 knock-in striatal cells, a juvenile onset HD CAG repeat was associated with low mitochondrial ATP and decreased mitochondrial ADP-uptake. This metabolic inhibition was associated with enhanced Ca2+-influx through NMDA receptors, which when blocked resulted in increased cellular [ATP/ADP]. We then evaluated [ATP/ADP] in 40 human lymphoblastoid cell lines, bearing non-HD CAG lengths (9-34 units) or HD-causing alleles (35-70 units). This analysis revealed an inverse association with the longer of the two allelic HD CAG repeats in both the non-HD and HD ranges. Thus, the polyglutamine tract in huntingtin appears to regulate mitochondrial ADP-phosphorylation in a Ca2+-dependent process that fulfills the genetic criteria for the HD trigger of pathogenesis, and it thereby determines a fundamental biological parameter--cellular energy status, which may contribute to the exquisite vulnerability of striatal neurons in HD. Moreover, the evidence that this polymorphism can determine energy status in the non-HD range suggests that it should be tested as a potential physiological modifier in both health and disease.

Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis.

BACKGROUND: JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. RESULTS: To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. CONCLUSIONS: These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.

Specific progressive cAMP reduction implicates energy deficit in presymptomatic Huntington's disease knock-in mice.

Defects in gene transcription and mitochondrial function have been implicated in the dominant disease process that leads to the loss of striatal neurons in Huntington's disease (HD). Here we have used precise genetic HD mouse and striatal cell models to investigate the hypothesis that decreased cAMP responsive element (CRE)-mediated gene transcription may reflect impaired energy metabolism. We found that reduced CRE-signaling in Hdh(Q111) striatum, monitored by brain derived neurotrophic factor and phospho-CRE binding protein (CREB), predated inclusion formation. Furthermore, cAMP levels in Hdh(Q111) striatum declined from an early age (10 weeks), and cAMP was significantly decreased in HD postmortem brain and lymphoblastoid cells, attesting to a chronic deficit in man. Reduced CRE-signaling in cultured STHdh(Q111) striatal cells was associated with cytosolic CREB binding protein that mirrored diminished cAMP synthesis. Moreover, mutant cells exhibited mitochondrial respiratory chain impairment, evidenced by decreased ATP and ATP/ADP ratio, impaired MTT conversion and heightened sensitivity to 3-nitropropionic acid. Thus, our findings strongly suggest that impaired ATP synthesis and diminished cAMP levels amplify the early HD disease cascade by decreasing CRE-regulated gene transcription and altering energy dependent processes essential to neuronal cell survival.

Identification of a presymptomatic molecular phenotype in Hdh CAG knock-in mice.

The hallmark striatal neurodegeneration of Huntington's disease (HD) is first triggered by a dominant property of the expanded glutamine tract in mutant huntingtin that increases in severity with glutamine size. Indeed 111-glutamine murine huntingtin leads to a dominant cascade of phenotypes in Hdh(Q111) mice, although these abnormalities are not manifest in Hdh(Q50) mice, with 50-glutamine mutant protein. Therefore, to identify phenotypes that might reflect events closer to the fundamental trigger mechanism, and that can be measured as a consequence of adult-onset HD mutant huntingtin, we have screened for altered expression of genes conserved in evolution, which are likely to encode essential proteins. Probes generated from Hdh(Q111) homozygote and wild-type striatal RNAs were hybridized to human gene segments on filter arrays, disclosing a mutant-specific increase in hybridization to Rrs1, encoding a ribosomal protein. Subsequent, quantitative RT-PCR assays demonstrated increased Rrs1 mRNA from 3 weeks of age in homozygous and heterozygous Hdh(Q111) striatum and increased Rrs1 mRNA expression with a single copy's worth of 50-glutamine mutant huntingtin in Hdh(Q50) striatum. Moreover, quantitative RT-PCR assays for the human homologue demonstrated elevated Rrs1 mRNA in HD compared with control postmortem brain. These findings, therefore, support a chronic impact of mutant huntingtin on an essential ribosomal regulatory gene to be investigated for its role very early in HD pathogenesis.