Huntingtin loss in hepatocytes is associated with altered metabolism, adhesion, and liver zonation.

Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological, and plasma metabolite levels. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.

Huntingtin loss in hepatocytes is associated with altered metabolism, adhesion, and liver zonation.

Huntington's disease arises from a toxic gain of function in the huntingtin ( HTT ) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological and plasma metabolite level. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.

Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease.

Huntington disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid and behavioral assays in HD human cell and Drosophila models, respectively. Validated, disease-relevant mHTT-dependent interactions encompass mediators of synaptic neurotransmission (SNAREs and glutamate receptors) and lysosomal acidification (V-ATPase). Our study provides a resource for understanding mHTT-dependent dysfunction in cortico-striatal cellular networks, partly through impaired synaptic communication and endosomal-lysosomal system. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Uninterrupted CAG repeat drives striatum-selective transcriptionopathy and nuclear pathogenesis in human Huntingtin BAC mice.

In Huntington's disease (HD), the uninterrupted CAG repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed bacterial artificial chromosome (BAC) transgenic mice expressing human mutant huntingtin (mHTT) with uninterrupted, and somatically unstable, CAG repeats that exhibit progressive disease-related phenotypes. Unlike prior mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice show robust striatum-selective nuclear inclusions and transcriptional dysregulation resembling those in murine huntingtin knockin models and HD patients. Importantly, the striatal transcriptionopathy in HD models is significantly correlated with their uninterrupted CAG repeat length but not polyglutamine length. Finally, among the pathogenic entities originating from mHTT genomic transgenes and only present or enriched in the uninterrupted CAG repeat model, somatic CAG repeat instability and nuclear mHTT aggregation are best correlated with early-onset striatum-selective molecular pathogenesis and locomotor and sleep deficits, while repeat RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively.

Huntingtin structure is orchestrated by HAP40 and shows a polyglutamine expansion-specific interaction with exon 1.

Huntington's disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington's disease and illuminate the structural consequences of HTT polyglutamine expansion.

Single-Nucleus RNA-Seq Reveals Dysregulation of Striatal Cell Identity Due to Huntington's Disease Mutations.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Cell death in HD occurs primarily in striatal medium spiny neurons (MSNs), but the involvement of specific MSN subtypes and of other striatal cell types remains poorly understood. To gain insight into cell type-specific disease processes, we studied the nuclear transcriptomes of 4524 cells from the striatum of a genetically precise knock-in mouse model of the HD mutation, HttQ175/+, and from wild-type controls. We used 14- to 15-month-old male mice, a time point at which multiple behavioral, neuroanatomical, and neurophysiological changes are present but at which there is no known cell death. Thousands of differentially expressed genes (DEGs) were distributed across most striatal cell types, including transcriptional changes in glial populations that are not apparent from RNA-seq of bulk tissue. Reconstruction of cell type-specific transcriptional networks revealed a striking pattern of bidirectional dysregulation for many cell type-specific genes. Typically, these genes were repressed in their primary cell type, yet de-repressed in other striatal cell types. Integration with existing epigenomic and transcriptomic data suggest that partial loss-of-function of the polycomb repressive complex 2 (PRC2) may underlie many of these transcriptional changes, leading to deficits in the maintenance of cell identity across virtually all cell types in the adult striatum.SIGNIFICANCE STATEMENT Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder characterized by specific loss of medium spiny neurons (MSNs) in the striatum, accompanied by more subtle changes in many other cell types. It is thought that changes in transcriptional regulation are an important underlying mechanism, but cell type-specific gene expression changes are not well understood, particularly at time points relevant to the onset of disease-related symptoms. Single-nucleus (sn)RNA-seq in a genetically precise mouse model enabled us to identify novel patterns of transcriptional dysregulation because of HD mutations, including bidirectional dysregulation of many cell type identity genes that may be driven by partial loss-of-function of the polycomb repressive complex (PRC). Identifying these regulators of transcriptional dysregulation in HD can be leveraged to design novel disease-modifying therapeutics.

Transcriptional regulatory networks underlying gene expression changes in Huntington's disease.

Transcriptional changes occur presymptomatically and throughout Huntington's disease (HD), motivating the study of transcriptional regulatory networks (TRNs) in HD We reconstructed a genome-scale model for the target genes of 718 transcription factors (TFs) in the mouse striatum by integrating a model of genomic binding sites with transcriptome profiling of striatal tissue from HD mouse models. We identified 48 differentially expressed TF-target gene modules associated with age- and CAG repeat length-dependent gene expression changes in Htt CAG knock-in mouse striatum and replicated many of these associations in independent transcriptomic and proteomic datasets. Thirteen of 48 of these predicted TF-target gene modules were also differentially expressed in striatal tissue from human disease. We experimentally validated a specific model prediction that SMAD3 regulates HD-related gene expression changes using chromatin immunoprecipitation and deep sequencing (ChIP-seq) of mouse striatum. We found CAG repeat length-dependent changes in the genomic occupancy of SMAD3 and confirmed our model's prediction that many SMAD3 target genes are downregulated early in HD.

Early Detection of Apathetic Phenotypes in Huntington's Disease Knock-in Mice Using Open Source Tools.

Apathy is one of the most prevalent and progressive psychiatric symptoms in Huntington's disease (HD) patients. However, preclinical work in HD mouse models tends to focus on molecular and motor, rather than affective, phenotypes. Measuring behavior in mice often produces noisy data and requires large cohorts to detect phenotypic rescue with appropriate power. The operant equipment necessary for measuring affective phenotypes is typically expensive, proprietary to commercial entities, and bulky which can render adequately sized mouse cohorts as cost-prohibitive. Thus, we describe here a home-built, open-source alternative to commercial hardware that is reliable, scalable, and reproducible. Using off-the-shelf hardware, we adapted and built several of the rodent operant buckets (ROBucket) to test HttQ111/+ mice for attention deficits in fixed ratio (FR) and progressive ratio (PR) tasks. We find that, despite normal performance in reward attainment in the FR task, HttQ111/+ mice exhibit reduced PR performance at 9-11 months of age, suggesting motivational deficits. We replicated this in two independent cohorts, demonstrating the reliability and utility of both the apathetic phenotype, and these ROBuckets, for preclinical HD studies.

Peripheral huntingtin silencing does not ameliorate central signs of disease in the B6.HttQ111/+ mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease whose predominant neuropathological signature is the selective loss of medium spiny neurons in the striatum. Despite this selective neuropathology, the mutant protein (huntingtin) is found in virtually every cell so far studied, and, consequently, phenotypes are observed in a wide range of organ systems both inside and outside the central nervous system. We, and others, have suggested that peripheral dysfunction could contribute to the rate of progression of striatal phenotypes of HD. To test this hypothesis, we lowered levels of huntingtin by treating mice with antisense oligonucleotides (ASOs) targeting the murine Huntingtin gene. To study the relationship between peripheral huntingtin levels and striatal HD phenotypes, we utilized a knock-in model of the human HD mutation (the B6.HttQ111/+ mouse). We treated mice with ASOs from 2-10 months of age, a time period over which significant HD-relevant signs progressively develop in the brains of HttQ111/+ mice. Peripheral treatment with ASOs led to persistent reduction of huntingtin protein in peripheral organs, including liver (64% knockdown), brown adipose (66% knockdown), and white adipose tissues (71% knockdown). This reduction was not associated with alterations in the severity of HD-relevant signs in the striatum of HttQ111/+ mice at the end of the study, including transcriptional dysregulation, the accumulation of neuronal intranuclear inclusions, and behavioral changes such as subtle hypoactivity and reduced exploratory drive. These results suggest that the amount of peripheral reduction achieved in the current study does not significantly impact the progression of HD-relevant signs in the central nervous system.

Motivational, proteostatic and transcriptional deficits precede synapse loss, gliosis and neurodegeneration in the B6.HttQ111/+ model of Huntington's disease.

We investigated the appearance and progression of disease-relevant signs in the B6.HttQ111/+ mouse, a genetically precise model of the mutation that causes Huntington's disease (HD). We find that B6.HttQ111/+ mice are healthy, show no overt signs of central or peripheral inflammation, and no gross motor impairment as late as 12 months of age. Behaviorally, we find that 4-9 month old B6.HttQ111/+ mice have normal activity levels and show no clear signs of anxiety or depression, but do show clear signs of reduced motivation. The neuronal density, neuronal size, synaptic density and number of glia is normal in B6.HttQ111/+ striatum, the most vulnerable brain region in HD, up to 12 months of age. Despite this preservation of the synaptic and cellular composition of the striatum, we observe clear progressive, striatal-specific transcriptional dysregulation and accumulation of neuronal intranuclear inclusions (NIIs). Simulation studies suggest these molecular endpoints are sufficiently robust for future preclinical studies, and that B6.HttQ111/+ mice are a useful tool for modeling disease-modifying or neuroprotective strategies for disease processes before the onset of overt phenotypes.

Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo.

N17 Modifies mutant Huntingtin nuclear pathogenesis and severity of disease in HD BAC transgenic mice.

The nucleus is a critical subcellular compartment for the pathogenesis of polyglutamine disorders, including Huntington's disease (HD). Recent studies suggest the first 17-amino-acid domain (N17) of mutant huntingtin (mHTT) mediates its nuclear exclusion in cultured cells. Here, we test whether N17 could be a molecular determinant of nuclear mHTT pathogenesis in vivo. BAC transgenic mice expressing mHTT lacking the N17 domain (BACHD-ΔN17) show dramatically accelerated mHTT pathology exclusively in the nucleus, which is associated with HD-like transcriptionopathy. Interestingly, BACHD-ΔN17 mice manifest more overt disease-like phenotypes than the original BACHD mice, including body weight loss, movement deficits, robust striatal neuron loss, and neuroinflammation. Mechanistically, N17 is necessary for nuclear exclusion of small mHTT fragments that are part of nuclear pathology in HD. Together, our study suggests that N17 modifies nuclear pathogenesis and disease severity in HD mice by regulating subcellular localization of known nuclear pathogenic mHTT species.

Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion leading to an elongated polyglutamine stretch in huntingtin. Mutant huntingtin (mHTT) is ubiquitously expressed in all cells but elicits selective cortical and striatal neurodegeneration in HD. The mechanistic basis for such selective neuronal vulnerability remains unclear. A necessary step toward resolving this enigma is to define the cell types in which mHTT expression is causally linked to the disease pathogenesis. Using a conditional transgenic mouse model of HD, in which the mice express full-length human mHTT from a bacterial artificial chromosome transgene (BACHD), we genetically reduced mHTT expression in neuronal populations in the striatum, cortex or both. We show that reduction of cortical mHTT expression in BACHD mice partially improves motor and psychiatric-like behavioral deficits but does not improve neurodegeneration, whereas reduction of mHTT expression in both neuronal populations consistently ameliorates all behavioral deficits and selective brain atrophy in this HD model. Furthermore, whereas reduction of mHTT expression in cortical or striatal neurons partially ameliorates corticostriatal synaptic deficits, further restoration of striatal synaptic function can be achieved by reduction of mHTT expression in both neuronal cell types. Our study demonstrates distinct but interacting roles of cortical and striatal mHTT in HD pathogenesis and suggests that optimal HD therapeutics may require targeting mHTT in both cortical and striatal neurons.

Genetic manipulations of mutant huntingtin in mice: new insights into Huntington's disease pathogenesis.

This year (2013) marks the 20th anniversary of identification of the causal genetic mutation for Huntington's disease (HD), a landmark discovery that heralded study of the biological underpinnings of this most common dominantly inherited neurodegenerative disorder. Among the variety of model organisms used to study HD pathogenesis, the mouse model is by far the most commonly used mammalian genetic model. Much of our current knowledge regarding mutant huntingtin (mHtt)-induced disease pathogenesis in mammalian models has been obtained by studying transgenic mouse models expressing mHtt N-terminal fragments, full-length murine or human mHtt. In this review, we focus on recent progress in using novel HD mouse models with targeted mHtt expression in specific brain cell types. These models help to address the role of distinct neuronal and non-neuronal cell types in eliciting cell-autonomous or non-cell-autonomous disease processes in HD. We also describe several mHtt transgenic mouse models with targeted mutations in Htt cis-domains to address specific pathogenic hypotheses, ranging from mHtt proteolysis to post-translational modifications. These novel mouse genetic studies, through direct manipulations of the causal HD gene, utilize a reductionist approach to systematically unravel the cellular and molecular pathways that are targeted by mHtt in disease pathogenesis, and to potentially identify novel targets for therapy.

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.

Fate of cajal-retzius neurons in the postnatal mouse neocortex.

Cajal-Retzius (CR) neurons play a critical role in cortical neuronal migration, but their exact fate after the completion of neocortical lamination remains a mystery. Histological evidence has been unable to unequivocally determine whether these cells die or undergo a phenotypic transformation to become resident interneurons of Layer 1 in the adult neocortex. To determine their ultimate fate, we performed chronic in vivo two-photon imaging of identified CR neurons during postnatal development in mice that express the green fluorescent protein (GFP) under the control of the early B-cell factor 2 (Ebf2) promoter. We find that, after birth, virtually all CR neurons in mouse neocortex express Ebf2. Although postnatal CR neurons undergo dramatic morphological transformations, they do not migrate to deeper layers. Instead, their gradual disappearance from the cortex is due to apoptotic death during the second postnatal week. A small fraction of CR neurons present at birth survive into adulthood. We conclude that, in addition to orchestrating cortical layering, a subset of CR neurons must play other roles beyond the third postnatal week.

Common and divergent structural features of a series of corticotropin releasing factor-related peptides.

Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.

Distinct structural and functional roles of conserved residues in the first extracellular domain of receptors for corticotropin-releasing factor and related G-protein-coupled receptors.

The G-protein-coupled receptor B1 family includes corticotropin-releasing factor (CRF), growth hormone-releasing hormone, incretin, and pituitary adenylate cyclase-activating polypeptide receptors. The three-dimensional NMR structure of the first extracellular domain (ECD1) of CRF receptor 2beta (CRF-R2beta), free and complexed with astressin, comprises a Sushi domain. This domain is stabilized in part by a salt bridge between Asp(65) and Arg(101). Analogous residues are conserved in other members of the B1 family. To address the importance of the salt bridge residues within this receptor family, we studied the effects of mutating the residues in full-length CRF-R2beta and isolated ECD1. Mutation D65A or D65R/R101D resulted in loss of the canonical disulfide arrangement, whereas R101A retained the Cys(4)-Cys(6) disulfide bond. The mutations resulted in misfolding within the ECD1 as determined by NMR and 1-anilino-8-naphthalenesulfonate binding but did not prevent cell surface expression. The D65A mutation in CRF-R2beta greatly reduced binding and activation, but the R101A substitution had only a small effect. Similar effects were seen on astressin binding to the ECD1. The different interactions of Asp(65) and Arg(101), deduced from the three-dimensional structure of the complex, are consistent with the differential effects seen in the mutants. The reduction in binding of Asp(65) mutants is a consequence of a distinct Asp(65)-Trp(71) interaction, which stabilizes the ligand-binding loop. Hence, loss of the salt bridge leads to disruption of the overall fold but does not abolish function. Because homologous mutations in other B1 receptors produce similar effects, these conserved residues may play similar roles in the entire receptor family.

Stressin1-A, a potent corticotropin releasing factor receptor 1 (CRF1)-selective peptide agonist.

The potencies and selectivity of peptide CRF antagonists is increased through structural constraints, suggesting that the resulting ligands assume distinct conformations when interacting with CRF1 and CRF2 receptors. To develop selective CRF receptor agonists, we have scanned the sequence -Gln-Ala-His-Ser-Asn-Arg- (residues 30-35 of [DPhe12,Nle21,38]Ac-hCRF4-41) with an i-(i+3) bridge consisting of the Glui-Xaa-Xbb-Lysi+3 scaffold, where residues i=30, 31, and 32. When i=31, stressin1-A, a potent CRF1 receptor-selective agonist was generated. In vitro, stressin1-A was equipotent to h/rCRF to release ACTH. Astressin1-A showed a low nanomolar affinity for CRF1 receptor (Ki=1.7 nM) and greater than 100-fold selectivity versus CRF2 receptor (Ki=222 nM). Stressin1-A released slightly less ACTH than oCRF in adult adrenal-intact male rats, with increased duration of action. Stressin1-A, injected intraperitoneally in rats, induced fecal pellet output (a CRF1 receptor-mediated response) and did not influence gastric emptying and blood pressure (CRF2 receptor-mediated responses).