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.

Neural stem (NS) cells are a limitless resource, and thus superior to primary neurons for drug discovery provided they exhibit appropriate disease phenotypes. Here we established NS cells for cellular studies of Huntington's disease (HD). HD is a heritable neurodegenerative disease caused by a mutation resulting in an increased number of glutamines (Q) within a polyglutamine tract in Huntingtin (Htt). NS cells were isolated from embryonic wild-type (Htt(7Q/7Q)) and "knock-in" HD (Htt(140Q/140Q)) mice expressing full-length endogenous normal or mutant Htt. NS cells were also developed from mouse embryonic stem cells that were devoid of Htt (Htt(-/-)), or knock-in cells containing human exon1 with an N-terminal FLAG epitope tag and with 7Q or 140Q inserted into one of the mouse alleles (Htt(F7Q/7Q) and Htt(F140Q/7Q)). Compared to Htt(7Q/7Q) NS cells, HD Htt(140Q/140Q) NS cells showed significantly reduced levels of cholesterol, increased levels of reactive oxygen species (ROS), and impaired motility. The heterozygous Htt(F140Q/7Q) NS cells had increased ROS and decreased motility compared to Htt(F7Q/7Q). These phenotypes of HD NS cells replicate those seen in HD patients or in primary cell or in vivo models of HD. Huntingtin "knock-out" NS cells (Htt(-/-)) also had impaired motility, but in contrast to HD cells had increased cholesterol. In addition, Htt(140Q/140Q) NS cells had higher phospho-AKT/AKT ratios than Htt(7Q/7Q) NS cells in resting conditions and after BDNF stimulation, suggesting mutant htt affects AKT dependent growth factor signaling. Upon differentiation, the Htt(7Q/7Q) and Htt(140Q/140Q) generated numerous Beta(III)-Tubulin- and GABA-positive neurons; however, after 15 days the cellular architecture of the differentiated Htt(140Q/140Q) cultures changed compared to Htt(7Q/7Q) cultures and included a marked increase of GFAP-positive cells. Our findings suggest that NS cells expressing endogenous mutant Htt will be useful for study of mechanisms of HD and drug discovery.

Huntington's disease (HD) is caused by an autosomal dominant polyglutamine expansion mutation of Huntingtin (HTT). HD patients suffer from progressive motor, cognitive, and psychiatric impairments, along with significant degeneration of the striatal projection neurons (SPNs) of the striatum. HD is widely accepted to be caused by a toxic gain-of-function of mutant HTT. However, whether loss of HTT function, because of dominant-negative effects of the mutant protein, plays a role in HD and whether HTT is required for SPN health and function are not known. Here, we delete Htt from specific subpopulations of SPNs using the Cre-Lox system and find that SPNs require HTT for motor regulation, synaptic development, cell health, and survival during aging. Our results suggest that loss of HTT function in SPNs could play a critical role in HD pathogenesis.

No disease modifying treatment currently exists for Huntington's disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays an essential role in cellular and systemic O(2) homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1alpha in the brain, we exposed adult mice with late-stage brain deletion of HIF-1alpha to hypoxic injuries. Contrary to expectations, the brains from the HIF-1alpha-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1alpha can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1alpha-deficient mice. Although the role of HIF-1alpha has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1alpha in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1alpha-deficient mice is mechanistically consistent with a predominant role of HIF-1alpha as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1alpha, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.