Loss-of-Huntingtin in Medial and Lateral Ganglionic Lineages Differentially Disrupts Regional Interneuron and Projection Neuron Subtypes and Promotes Huntington's Disease-Associated Behavioral, Cellular, and Pathological Hallmarks.

Emerging studies are providing compelling evidence that the pathogenesis of Huntington's disease (HD), a neurodegenerative disorder with frequent midlife onset, encompasses developmental components. Moreover, our previous studies using a hypomorphic model targeting huntingtin during the neurodevelopmental period indicated that loss-of-function mechanisms account for this pathogenic developmental component (Arteaga-Bracho et al., 2016). In the present study, we specifically ascertained the roles of subpallial lineage species in eliciting the previously observed HD-like phenotypes. Accordingly, we used the Cre-loxP system to conditionally ablate the murine huntingtin gene (Httflx) in cells expressing the subpallial patterning markers Gsx2 (Gsx2-Cre) or Nkx2.1 (Nkx2.1-Cre) in Httflx mice of both sexes. These genetic manipulations elicited anxiety-like behaviors, hyperkinetic locomotion, age-dependent motor deficits, and weight loss in both Httflx;Gsx2-Cre and Httflx;Nkx2.1-Cre mice. In addition, these strains displayed unique but complementary spatial patterns of basal ganglia degeneration that are strikingly reminiscent of those seen in human cases of HD. Furthermore, we observed early deficits of somatostatin-positive and Reelin-positive interneurons in both Htt subpallial null strains, as well as early increases of cholinergic interneurons, Foxp2+ arkypallidal neurons, and incipient deficits with age-dependent loss of parvalbumin-positive neurons in Httflx;Nkx2.1-Cre mice. Overall, our findings indicate that selective loss-of-huntingtin function in subpallial lineages differentially disrupts the number, complement, and survival of forebrain interneurons and globus pallidus GABAergic neurons, thereby leading to the development of key neurological hallmarks of HD during adult life. Our findings have important implications for the establishment and deployment of neural circuitries and the integrity of network reserve in health and disease.SIGNIFICANCE STATEMENT Huntington's disease (HD) is a progressive degenerative disorder caused by aberrant trinucleotide expansion in the huntingtin gene. Mechanistically, this mutation involves both loss- and gain-of-function mechanisms affecting a broad array of cellular and molecular processes. Although huntingtin is widely expressed during adult life, the mutant protein only causes the demise of selective neuronal subtypes. The mechanisms accounting for this differential vulnerability remain elusive. In this study, we have demonstrated that loss-of-huntingtin function in subpallial lineages not only differentially disrupts distinct interneuron species early in life, but also leads to a pattern of neurological deficits that are reminiscent of HD. This work suggests that early disruption of selective neuronal subtypes may account for the profiles of enhanced regional cellular vulnerability to death in HD.

Postnatal and adult consequences of loss of huntingtin during development: Implications for Huntington's disease.

The mutation in huntingtin (mHtt) leads to a spectrum of impairments in the developing forebrain of Huntington's disease (HD) mouse models. Whether these developmental alterations are due to loss- or gain-of-function mechanisms and contribute to HD pathogenesis is unknown. We examined the role of selective loss of huntingtin (Htt) function during development on postnatal vulnerability to cell death. We employed mice expressing very low levels of Htt throughout embryonic life to postnatal day 21 (Hdhd•hyp). We demonstrated that Hdhd•hyp mice exhibit: (1) late-life striatal and cortical neuronal degeneration; (2) neurological and skeletal muscle alterations; and (3) white matter tract impairments and axonal degeneration. Hdhd•hyp embryos also exhibited subpallial heterotopias, aberrant striatal maturation and deregulation of gliogenesis. These results indicate that developmental deficits associated with Htt functions render cells present at discrete neural foci increasingly susceptible to cell death, thus implying the potential existence of a loss-of-function developmental component to HD pathogenesis.

Selective expression of mutant huntingtin during development recapitulates characteristic features of Huntington's disease.

Recent studies have identified impairments in neural induction and in striatal and cortical neurogenesis in Huntington's disease (HD) knock-in mouse models and associated embryonic stem cell lines. However, the potential role of these developmental alterations for HD pathogenesis and progression is currently unknown. To address this issue, we used BACHD:CAG-Cre(ERT2) mice, which carry mutant huntingtin (mHtt) modified to harbor a floxed exon 1 containing the pathogenic polyglutamine expansion (Q97). Upon tamoxifen administration at postnatal day 21, the floxed mHtt-exon1 was removed and mHtt expression was terminated (Q97(CRE)). These conditional mice displayed similar profiles of impairments to those mice expressing mHtt throughout life: (i) striatal neurodegeneration, (ii) early vulnerability to NMDA-mediated excitotoxicity, (iii) impairments in motor coordination, (iv) temporally distinct abnormalities in striatal electrophysiological activity, and (v) altered corticostriatal functional connectivity and plasticity. These findings strongly suggest that developmental aberrations may play important roles in HD pathogenesis and progression.

The role of H1 linker histone subtypes in preserving the fidelity of elaboration of mesendodermal and neuroectodermal lineages during embryonic development.

H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers.

Functions of huntingtin in germ layer specification and organogenesis.

Huntington's disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.

Selective roles of normal and mutant huntingtin in neural induction and early neurogenesis.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt) and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt) causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an in vitro mouse embryonic stem cell (ESC) clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs) severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively) during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs), mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2)-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF)-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of Notch, Hes1 and Hes5. These cumulative observations indicate that Htt is required for multiple stages of neural induction, whereas mHtt enhances this process and promotes precocious neurogenesis and oligodendrocyte progenitor cell elaboration.

Corepressor for element-1-silencing transcription factor preferentially mediates gene networks underlying neural stem cell fate decisions.

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) silences neuronal genes in neural stem cells (NSCs) and nonneuronal cells through its role as a dynamic modular platform for recruitment of transcriptional and epigenetic regulatory cofactors to RE1-containing promoters. In embryonic stem cells, the REST regulatory network is highly integrated with the transcriptional circuitry governing self-renewal and pluripotency, although its exact functional role is unclear. The C-terminal cofactor for REST, CoREST, also acts as a modular scaffold, but its cell type-specific roles have not been elucidated. We used chromatin immunoprecipitation-on-chip to examine CoREST and REST binding sites in NSCs and their proximate progenitor species. In NSCs, we identified a larger number of CoREST (1,820) compared with REST (322) target genes. The majority of these CoREST targets do not contain known RE1 motifs. Notably, these CoREST target genes do play important roles in pluripotency networks, in modulating NSC identity and fate decisions and in epigenetic processes previously associated with both REST and CoREST. Moreover, we found that NSC-mediated developmental transitions were associated primarily with liberation of CoREST from promoters with transcriptional repression favored in less lineage-restricted radial glia and transcriptional activation favored in more lineage-restricted neuronal-oligodendrocyte precursors. Clonal NSC REST and CoREST gene manipulation paradigms further revealed that CoREST has largely independent and previously uncharacterized roles in promoting NSC multilineage potential and modulating early neural fate decisions.

Impairment of developmental stem cell-mediated striatal neurogenesis and pluripotency genes in a knock-in model of Huntington's disease.

The pathogenesis of Huntington's disease (HD) remains elusive. The identification of increasingly early pathophysiological abnormalities in HD suggests the possibility that impairments of striatal medium spiny neuron (MSN) specification and maturation may underlie the etiology of HD. In fact, we demonstrate that HD knock-in (Hdh-Q111) mice exhibited delayed acquisition of early striatal cytoarchitecture with aberrant expression of progressive markers of MSN neurogenesis (Islet1, DARPP-32, mGluR1, and NeuN). Hdh-Q111 striatal progenitors also displayed delayed cell cycle exit between E13.5-15.5 (BrdU birth-dating) and an enhanced fraction of abnormal cycling cells in association with expansion of the pool of intermediate progenitors and over expression of the core pluripotency (PP) factor, Sox2. Clonal analysis further revealed that Hdh-Q111 neural stem cells (NSCs) displayed: impaired lineage restriction, reduced proliferative potential, enhanced late-stage self-renewal, and deregulated MSN subtype specification. Further, our analysis revealed that in addition to Sox2, the core PP factor, Nanog is expressed within the striatal generative and mantle regions, and in Hdh-Q111 embryos the fraction of Nanog-expressing MSN precursors was substantially increased. Moreover, compared to Hdh-Q18 embryos, the Hdh-Q111 striatal anlagen exhibited significantly higher levels of the essential PP cofactor, Stat3. These findings suggest that Sox2 and Nanog may play roles during a selective window of embryonic brain maturation, and alterations of these factors may, in part, be responsible for mediating the aberrant program of Hdh-Q111 striatal MSN specification and maturation. We propose that these HD-associated developmental abnormalities might compromise neuronal homeostasis and subsequently render MSNs more vulnerable to late life stressors.

Risk of dementia associated with elevated plasma homocysteine in a latin american population.

The relationship between total homocysteine (tHcy) and dementia risk remains controversial, as the association varies among populations and dementia subtypes. We studied a Venezuelan population that has high prevalence of both elevated tHcy and dementia. We tested the hypotheses that (1) elevated tHcy is associated with increased dementia risk, (2) the risk is greater for vascular dementia (VaD) than for Alzheimer's disease (AD), and (3) a history of stroke may partly explain this association. 2100 participants (>/=55 years old) of the Maracaibo Aging Study underwent standardized neurological, neuropsychiatric, and cardiovascular assessments. Elevated tHcy was significantly associated with dementia, primarily VaD. When history of stroke and other confounding factors were taken into account, elevated tHcy remained a significant risk factor in older (>66 years), but not in younger (55-66 years) subjects. Ongoing studies of this population may provide insight into the mechanism by which tHcy increases risk for dementia.

High prevalence of dementia in a Caribbean population.

BACKGROUND/AIMS: People in Caribbean countries are thought to be at particularly high risk for dementia. Basic descriptive epidemiology of dementia is required for populations in the region to determine the validity of this hypothesis. The main objectives of the study were to assess the prevalence, types and severity of dementia among elderly people (>or=55 years old) in an urban area on the Caribbean coast of Venezuela, and to determine the gender and age distribution of affected people. METHODS: The population-based Maracaibo Aging Study included 3,657 subjects, all of whom underwent a standardized, in-person interview. 2,438 of these subjects underwent neuropsychological, neuropsychiatric, cardiovascular and nutritional assessment. RESULTS: The overall prevalence rate of dementia in elderly subjects was 8.04% and was not significantly different for women and men. Alzheimer's disease was the most frequent type of dementia (50%), followed by vascular dementia (27%). Of all cases of dementia, 41.84% were ranked as mild, 30.10% as moderate and 28.06% as severe. CONCLUSION: The prevalence of dementia in elderly people from the Caribbean coast of Venezuela is much higher than frequencies previously reported for developing countries.

Total plasma homocysteine values among elderly subjects: findings from the Maracaibo Aging Study.

OBJECTIVES: The present study generated baseline data for total homocysteine (tHcy) in elderly Caribbeans of Hispanic ancestry, residing in Venezuela, their country of origin. DESIGN AND METHODS: 2106 participants in the Maracaibo Aging Study (MAS), older than 55 years, underwent standardized clinical and laboratory assessments, including measurement of plasma tHcy levels, folate, and vitamin B12 in fasting samples. RESULTS: tHcy concentration in the healthy, normative group ranged from 4.1 to 31.8 micromol/L, with a median of 11.5+/-4.7 micromol/L. tHcy level increased with age, was significantly higher in men than in women, and exhibited inverse correlations with folate and vitamin B12. CONCLUSIONS: tHcy levels of the MAS participants were generally higher than levels previously reported for community-dwelling elderly populations from other countries. The normative centile curves for tHcy can be used in disease risk analysis for this population, and possibly for other Hispanic populations residing in the Caribbean.

Differential modulation of BMP signaling promotes the elaboration of cerebral cortical GABAergic neurons or oligodendrocytes from a common sonic hedgehog-responsive ventral forebrain progenitor species.

During cerebral cortical development, excitatory glutamatergic projection neurons are generated from neural stem cells intrinsic to the early embryonic cortical ventricular zone by a process of radial migration, whereas most inhibitory gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes (OLs) appear to be elaborated from ventral forebrain stem cells that initially undergo tangential cortical migration before terminal lineage maturation. In contrast to the more compartmentalized developmental organization of the spinal cord, the generation of neurons and OLs from a common ventral forebrain stem cell would expose these cells to the sequential actions of ventral and dorsal gradient morphogens [sonic hedgehog (Shh) and bone morphogenetic proteins (BMPs)] that normally mediate opposing developmental programs. Here we report that Shh promotes GABAergic neuronalOL lineage restriction of forebrain stem cells, in part, by activation of the basic helix-loop-helix transcription factors, Olig2 and Mash1. In mutant mice with a generalized defect in tangential cortical migration (Dlx12--), there is a profound and selective reduction in the elaboration of both cortical GABAergic neurons and OLs. Our studies further demonstrate that the sequential elaboration of cortical GABAergic neurons and OLs from common Shh-responsive ventral forebrain progenitors requires the spatial and temporal modulation of cortical BMP signaling by BMP ligands and the BMP antagonist, noggin, respectively. These findings suggest an integrative model for cerebral cortical GABAergic neuronal and OL lineage maturation that would incorporate the sequential contributions of the ventral and dorsal forebrain, and the potential role of regional developmental cues in modulating transcriptional codes within evolving neural lineage species.

The Maracaibo Aging Study: population and methodological issues.

The Maracaibo Aging Study is a longitudinal, population-based, one-step multidisciplinary study of age-related diseases, with a particular focus on memory-related disorders, among subjects over 55 years living in a neighborhood of the city of Maracaibo, Venezuela. Three phases were scheduled for this study. First, a door-to-door survey was conducted in order to build a registry and to obtain general and sociodemographic characteristics. During the second phase, information regarding changes in the abilities of the subjects was collected. The third phase consisted of a full neuropsychiatric examination, cardiovascular evaluation, nutritional assessment, neuropsychological testing, routine laboratory tests and genetic analysis. 3,657 subjects were surveyed between January and August, 1998. There were more women than men, and the average number of years of formal education was low, particularly among women. The limitations and strengths of the study are discussed.