Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, the HD consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a novel human stem cell platform for screening new candidate therapeutics.

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 (

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 ⌬ex7/8 knock-in mice and CbCln3 ⌬ex7/8 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.Macroautophagy (herein referred to as autophagy) is a nonselective process by which cytoplasmic constituents are turned over (reviewed in Refs. 1 and 2). Elegant work in yeast has led to the genetic dissection of many of the proteins involved in this pathway (3, 4), which are mostly conserved in mammals (5, 6).The pathway is initiated at times of stress or nutritional deprivation to generate metabolites for cellular survival, but autophagy is also responsible for normal housekeeping. Although the regulatory control of autophagy remains to be fully elucidated, the general process from engulfment to degradation has been delineated (2). Initiation of the pathway involves de novo formation of an isolation membrane that expands to engulf cytoplasmic constituents, including organelles, and encloses to form a double membrane autophagic vacuole. This formed autophagic vacuole is then increasingly acidified, and proteases are delivered through fusion with late endosomes and lysosomes. The inner membrane is degraded to form a single membrane autolysosome, where constituents are finally completely degraded for recycling of metabolites to the cell.Autophagy is emerging as a major pathway involved in a number of neurodegenerative diseases, including Parkinson disease (7, 8), Huntington disease (9 -11), and Alzheimer disease (12, 13). In each case, autophagic vacuoles accumulate, suggesting that activation of autophagy is a common feature ...

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.

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.

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.

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.

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.