X. William Yang

Full-Length Human Mutant Huntingtin with a Stable Polyglutamine Repeat Can Elicit Progressive and Selective Neuropathogenesis in BACHD Mice

To elucidate the pathogenic mechanisms in Huntingtons disease (HD) elicited by expression of full-length human mutant huntingtin (fl-mhtt), a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) was developed expressing fl-mhtt with 97 glutamine repeats under the control of endogenous htt regulatory machinery on the BAC. BACHD mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuron degeneration. Power analyses reveal the robustness of the behavioral and neuropathological phenotypes, suggesting BACHD as a suitable fl-mhtt mouse model for preclinical studies. Additional analyses of BACHD mice provide novel insights into how mhtt may elicit neuropathogenesis. First, unlike previous fl-mhtt mouse models, BACHD mice reveal that the slowly progressive and selective pathogenic process in HD mouse brains can occur without early and diffuse nuclear accumulation of aggregated mhtt (i.e., as detected by immunostaining with the EM48 antibody). Instead, a relatively steady-state level of predominantly full-length mhtt and a small amount of mhtt N-terminal fragments are sufficient to elicit the disease process. Second, the polyglutamine repeat within fl-mhtt in BACHD mice is encoded by a mixed CAA-CAG repeat, which is stable in both the germline and somatic tissues including the cortex and striatum at the onset of neuropathology. Therefore, our results suggest that somatic repeat instability does not play a necessary role in selective neuropathogenesis in BACHD mice. In summary, the BACHD model constitutes a novel and robust in vivo paradigm for the investigation of HD pathogenesis and treatment.

FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains

A major challenge in systems neuroscience is to perform precise molecular genetic analyses of a single neuronal population in the context of the complex mammalian brain. Existing technologies for profiling cell type–specific gene expression are largely limited to immature or morphologically identifiable neurons. In this study, we developed a simple method using fluorescent activated cell sorting (FACS) to purify genetically labeled neurons from juvenile and adult mouse brains for gene expression profiling. We identify and verify a new set of differentially expressed genes in the striatonigral and striatopallidal neurons, two functionally and clinically important projection neuron subtypes in the basal ganglia. We further demonstrate that Ebf1 is a lineage-specific transcription factor essential to the differentiation of striatonigral neurons. Our study provides a general approach for profiling cell type–specific gene expression in the mature mammalian brain and identifies a set of genes critical to the function and dysfunction of the striatal projection neuron circuit.

Serines 13 and 16 Are Critical Determinants of Full-length Human Mutant Huntingtin-Induced Disease Pathogenesis in HD Mice

The N-terminal 17 amino acids of huntingtin (NT17) can be phosphorylated on serines 13 and 16; however, the significance of these modifications in Huntingtons disease pathogenesis remains unknown. In this study, we developed BAC transgenic mice expressing full-length mutant huntingtin (fl-mhtt) with serines 13 and 16 mutated to either aspartate (phosphomimetic or SD) or alanine (phosphoresistant or SA). Both mutant proteins preserve the essential function of huntingtin in rescuing knockout mouse phenotypes. However, fl-mhtt-induced disease pathogenesis, including motor and psychiatric-like behavioral deficits, mhtt aggregation, and selective neurodegeneration are abolished in SD but preserved in SA mice. Moreover, modification of these serines in expanded repeat huntingtin peptides modulates aggregation and amyloid fibril formation in vitro. Together, our findings demonstrate that serines 13 and 16 are critical determinants of fl-mhtt-induced disease pathogenesis in vivo, supporting the targeting of huntingtin NT17 domain and its modifications in HD therapy.

Systematic behavioral evaluation of Huntington’s disease transgenic and knock-in mouse models

Huntingtons disease (HD) is one of the few neurodegenerative diseases with a known genetic cause, knowledge that has enabled the creation of animal models using genetic manipulations that aim to recapitulate HD pathology. The study of behavioral and neuropathological phenotypes of these HD models, however, has been plagued by inconsistent results across laboratories stemming from the lack of standardized husbandry and testing conditions, in addition to the intrinsic differences between the models. We have compared different HD models using standardized conditions to identify the most robust phenotypic differences, best suited for preclinical therapeutic efficacy studies. With a battery of tests of sensory-motor function, such as the open field and prepulse inhibition tests, we replicate previous results showing a strong and progressive behavioral deficit in the R6/2 line with an average of 129 CAG repeats in a mixed CBA/J and C57BL/6J background. We present the first behavioral characterization of a new model, an R6/2 line with an average of 248 CAG repeats in a pure C57BL/6J background, which also showed a progressive and robust phenotype. The BACHD in a FVB/N background showed robust and progressive behavioral phenotype, while the YAC128 full-length model on either an FVB/N or a C57BL/6J background generally showed milder deficits. Finally, the Hdh(Q111) knock-in mouse on a CD1 background showed very mild deficits. This first extensive standardized cross-characterization of several HD animal models under standardized conditions highlights several behavioral outcomes, such as hypoactivity, amenable to standardized preclinical therapeutic drug screening.

Neuroprotective role of Sirt1 in mammalian models of Huntington's disease through activation of multiple Sirt1 targets

Huntingtons disease is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in huntingtin (HTT) protein. We previously showed that calorie restriction ameliorated Huntingtons disease pathogenesis and slowed disease progression in mice that model Huntingtons disease (Huntingtons disease mice). We now report that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT–mediated metabolic abnormalities in Huntingtons disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT–induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntingtons disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT–induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntingtons disease models and open new avenues for the development of neuroprotective strategies in Huntingtons disease.

Pathological Cell-Cell Interactions Elicited by a Neuropathogenic Form of Mutant Huntingtin Contribute to Cortical Pathogenesis in HD Mice

Expanded polyglutamine (polyQ) proteins in Huntingtons disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.

Network Organization of the Huntingtin Proteomic Interactome in Mammalian Brain

We used affinity-purification mass spectrometry to identify 747 candidate proteins that are complexed with Huntingtin (Htt) in distinct brain regions and ages in Huntingtons disease (HD) and wild-type mouse brains. To gain a systems-level view of the Htt interactome, we applied Weighted Correlation Network Analysis to the entire proteomic data set to unveil a verifiable rank of Htt-correlated proteins and a network of Htt-interacting protein modules, with each module highlighting distinct aspects of Htt biology. Importantly, the Htt-containing module is highly enriched with proteins involved in 14-3-3 signaling, microtubule-based transport, and proteostasis. Top-ranked proteins in this module were validated as Htt interactors and genetic modifiers in an HD Drosophila model. Our study provides a compendium of spatiotemporal Htt-interacting proteins in the mammalian brain and presents an approach for analyzing proteomic interactome data sets to build in vivo protein networks in complex tissues, such as the brain.

Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism

Mice carrying a genetic mutation that causes autistic symptoms show improved sociability after being treated with oxytocin, a hormone promoting mothering and trust. Going Social Oxytocin—a hormone that promotes mothering, trust, and social bonding in animals—seems a likely treatment for the social isolation of individuals on the autism spectrum, but tests in humans have generally proved disappointing. To delve deeper into how oxytocin affects autism symptoms, Peñagarikano et al. created a mouse mimic of one type of genetic autism, cortical dysplasia and focal epilepsy (CDFE) syndrome, by deleting the gene that is mutated in human patients. Unlike normal mouse-loving mice, CDFE mice were asocial, showing no preference for other mice over objects, but this deficit was reversed by giving them oxytocin. Further, revving up the sluggish production of their own oxytocin in the paraventricular nucleus in the hypothalamus also improved sociability. Most hopeful for patients, the authors found that giving young CDFE mice multiple doses of oxytocin just after birth produces a long-lasting improvement in oxytocin brain levels and sociability. Mouse models of neuropsychiatric diseases provide a platform for mechanistic understanding and development of new therapies. We previously demonstrated that knockout of the mouse homolog of CNTNAP2 (contactin-associated protein-like 2), in which mutations cause cortical dysplasia and focal epilepsy (CDFE) syndrome, displays many features that parallel those of the human disorder. Because CDFE has high penetrance for autism spectrum disorder (ASD), we performed an in vivo screen for drugs that ameliorate abnormal social behavior in Cntnap2 mutant mice and found that acute administration of the neuropeptide oxytocin improved social deficits. We found a decrease in the number of oxytocin immunoreactive neurons in the paraventricular nucleus (PVN) of the hypothalamus in mutant mice and an overall decrease in brain oxytocin levels. Administration of a selective melanocortin receptor 4 agonist, which causes endogenous oxytocin release, also acutely rescued the social deficits, an effect blocked by an oxytocin antagonist. We confirmed that oxytocin neurons mediated the behavioral improvement by activating endogenous oxytocin neurons in the paraventricular hypothalamus with Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Last, we showed that chronic early postnatal treatment with oxytocin led to more lasting behavioral recovery and restored oxytocin immunoreactivity in the PVN. These data demonstrate dysregulation of the oxytocin system in Cntnap2 knockout mice and suggest that there may be critical developmental windows for optimal treatment to rectify this deficit.

Deletion of Astroglial Dicer Causes Non-Cell-Autonomous Neuronal Dysfunction and Degeneration

The endoribonuclease, Dicer, is indispensable for generating the majority of mature microRNAs (miRNAs), which are posttranscriptional regulators of gene expression involved in a wide range of developmental and pathological processes in the mammalian CNS. Although functions of Dicer-dependent miRNA pathways in neurons and oligodendrocytes have been extensively investigated, little is known about the role of Dicer in astrocytes. Here, we report the effect of Cre-loxP-mediated conditional deletion of Dicer selectively from postnatal astroglia on brain development. Dicer-deficient mice exhibited normal motor development and neurological morphology before postnatal week 5. Thereafter, mutant mice invariably developed a rapidly fulminant neurological decline characterized by ataxia, severe progressive cerebellar degeneration, seizures, uncontrollable movements, and premature death by postnatal week 9–10. Integrated transcription profiling, histological, and functional analyses of cerebella showed that deletion of Dicer in cerebellar astrocytes altered the transcriptome of astrocytes to be more similar to an immature or reactive-like state before the onset of neurological symptoms or morphological changes. As a result, critical and mature astrocytic functions including glutamate uptake and antioxidant pathways were substantially impaired, leading to massive apoptosis of cerebellar granule cells and degeneration of Purkinje cells. Collectively, our study demonstrates the critical involvement of Dicer in normal astrocyte maturation and maintenance. Our findings also reveal non-cell-autonomous roles of astrocytic Dicer-dependent pathways in regulating proper neuronal functions and implicate that loss of or dysregulation of astrocytic Dicer-dependent pathways may be involved in neurodegeneration and other neurological disorders.

DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation

DNA methylation is a major epigenetic factor regulating genome reprogramming, cell differentiation and developmental gene expression. To understand the role of DNA methylation in central nervous system (CNS) neurons, we generated conditional Dnmt1 mutant mice that possess approximately 90% hypomethylated cortical and hippocampal cells in the dorsal forebrain from E13.5 on. The mutant mice were viable with a normal lifespan, but displayed severe neuronal cell death between E14.5 and three weeks postnatally. Accompanied with the striking cortical and hippocampal degeneration, adult mutant mice exhibited neurobehavioral defects in learning and memory in adulthood. Unexpectedly, a fraction of Dnmt1(-/-) cortical neurons survived throughout postnatal development, so that the residual cortex in mutant mice contained 20-30% of hypomethylated neurons across the lifespan. Hypomethylated excitatory neurons exhibited multiple defects in postnatal maturation including abnormal dendritic arborization and impaired neuronal excitability. The mutant phenotypes are coupled with deregulation of those genes involved in neuronal layer-specification, cell death and the function of ion channels. Our results suggest that DNA methylation, through its role in modulating neuronal gene expression, plays multiple roles in regulating cell survival and neuronal maturation in the CNS.