Publications by authors named "Leslie M Thompson"

59 Publications

Huntington's disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization.

J Clin Invest 2021 Jun;131(12)

Department of Neurobiology & Behavior.

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.
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http://dx.doi.org/10.1172/JCI140723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203471PMC
June 2021

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.

Proc Natl Acad Sci U S A 2021 01;118(4)

Department of Neurobiology and Behavior, University of California, Irvine, CA 92697;

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.
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http://dx.doi.org/10.1073/pnas.2021836118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848703PMC
January 2021

Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer's disease.

Nat Commun 2020 10 23;11(1):5370. Epub 2020 Oct 23.

Department of Neurobiology & Behavior, University of California Irvine, Irvine, CA, 92697, USA.

The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.
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http://dx.doi.org/10.1038/s41467-020-19227-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584603PMC
October 2020

Calcium Dynamics in Astrocytes During Cell Injury.

Front Bioeng Biotechnol 2020 27;8:912. Epub 2020 Aug 27.

Beckman Laser Institute & Medical Clinic, University of California, Irvine, Irvine, CA, United States.

The changes in intracellular calcium concentration ([Ca]) following laser-induced cell injury in nearby cells were studied in primary mouse astrocytes selectively expressing the Ca sensitive GFAP-Cre Salsa6f fluorescent tandem protein, in an Ast1 astrocyte cell line, and in primary mouse astrocytes loaded with Fluo4. Astrocytes in these three systems exhibit distinct changes in [Ca] following induced death of nearby cells. Changes in [Ca] appear to result from release of Ca from intracellular organelles, as opposed to influx from the external medium. Salsa6f expressing astrocytes displayed dynamic Ca changes throughout the phagocytic response, including lamellae protrusion, cytosolic signaling during vesicle formation, vesicle maturation, and vesicle tract formation. Our results demonstrate local changes in [Ca] are involved in the process of phagocytosis in astrocytes responding to cell corpses and/or debris.
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http://dx.doi.org/10.3389/fbioe.2020.00912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481337PMC
August 2020

Aberrant Development Corrected in Adult-Onset Huntington's Disease iPSC-Derived Neuronal Cultures via WNT Signaling Modulation.

Stem Cell Reports 2020 03 27;14(3):406-419. Epub 2020 Feb 27.

Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA 92697, USA; Department of Neurobiology and Behavior, University of California Irvine, Irvine, CA 96267, USA; Department of Memory Impairment and Neurological Disorders, University of California Irvine, Irvine, CA 92697, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Biological Chemistry, University of California Irvine, Irvine, CA 92617, USA. Electronic address:

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1 neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.
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http://dx.doi.org/10.1016/j.stemcr.2020.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066322PMC
March 2020

Microglial depletion prevents extracellular matrix changes and striatal volume reduction in a model of Huntington's disease.

Brain 2020 01;143(1):266-288

Department of Neurobiology and Behavior, University of California, Irvine (UCI), Irvine, CA, USA.

Huntington's disease is associated with a reactive microglial response and consequent inflammation. To address the role of these cells in disease pathogenesis, we depleted microglia from R6/2 mice, a rapidly progressing model of Huntington's disease marked by behavioural impairment, mutant huntingtin (mHTT) accumulation, and early death, through colony-stimulating factor 1 receptor inhibition (CSF1Ri) with pexidartinib (PLX3397) for the duration of disease. Although we observed an interferon gene signature in addition to downregulated neuritogenic and synaptic gene pathways with disease, overt inflammation was not evident by microglial morphology or cytokine transcript levels in R6/2 mice. Nonetheless, CSF1Ri-induced microglial elimination reduced or prevented disease-related grip strength and object recognition deficits, mHTT accumulation, astrogliosis, and striatal volume loss, the latter of which was not associated with reductions in cell number but with the extracellular accumulation of chondroitin sulphate proteoglycans (CSPGs)-a primary component of glial scars. A concurrent loss of proteoglycan-containing perineuronal nets was also evident in R6/2 mice, and microglial elimination not only prevented this but also strikingly increased perineuronal nets in the brains of naïve littermates, suggesting a new role for microglia as homeostatic regulators of perineuronal net formation and integrity.
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http://dx.doi.org/10.1093/brain/awz363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935750PMC
January 2020

Treatment with JQ1, a BET bromodomain inhibitor, is selectively detrimental to R6/2 Huntington's disease mice.

Hum Mol Genet 2020 01;29(2):202-215

Memory Impairment and Neurological Disorders Research Unit.

Transcriptional and epigenetic alterations occur early in Huntington's disease (HD), and treatment with epigenetic modulators is beneficial in several HD animal models. The drug JQ1, which inhibits histone acetyl-lysine reader bromodomains, has shown promise for multiple cancers and neurodegenerative disease. We tested whether JQ1 could improve behavioral phenotypes in the R6/2 mouse model of HD and modulate HD-associated changes in transcription and epigenomics. R6/2 and non-transgenic (NT) mice were treated with JQ1 daily from 5 to 11 weeks of age and behavioral phenotypes evaluated over this period. Following the trial, cortex and striatum were isolated and subjected to mRNA-seq and ChIP-seq for the histone marks H3K4me3 and H3K27ac. Initially, JQ1 enhanced motor performance in NT mice. In R6/2 mice, however, JQ1 had no effect on rotarod or grip strength but exacerbated weight loss and worsened performance on the pole test. JQ1-induced gene expression changes in NT mice were distinct from those in R6/2 and primarily involved protein translation and bioenergetics pathways. Dysregulation of HD-related pathways in striatum was exacerbated by JQ1 in R6/2 mice, but not in NTs, and JQ1 caused a corresponding increase in the formation of a mutant huntingtin protein-dependent high molecular weight species associated with pathogenesis. This study suggests that drugs predicted to be beneficial based on their mode of action and effects in wild-type or in other neurodegenerative disease models may have an altered impact in the HD context. These observations have important implications in the development of epigenetic modulators as therapies for HD.
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http://dx.doi.org/10.1093/hmg/ddz264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202555PMC
January 2020

A short note for Mike.

J Neurosci Res 2019 Dec;97(12):1748

University of California Irvine.

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http://dx.doi.org/10.1002/jnr.24533DOI Listing
December 2019

IKKβ slows Huntington's disease progression in R6/1 mice.

Proc Natl Acad Sci U S A 2019 05 14;116(22):10952-10961. Epub 2019 May 14.

Department of Psychiatry and Human Behavior, University of California, Irvine, CA 92697;

Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKβ, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKβ on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKβ phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKβ to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKβ knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKβ in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKβ knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKβ is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKβ is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKβ may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.
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http://dx.doi.org/10.1073/pnas.1814246116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561205PMC
May 2019

Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription.

Elife 2019 04 17;8. Epub 2019 Apr 17.

Department of Neurology, University of Texas Medical Branch, Galveston, United States.

How huntingtin (HTT) triggers neurotoxicity in Huntington's disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.
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http://dx.doi.org/10.7554/eLife.42988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529219PMC
April 2019

Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice.

J Huntingtons Dis 2018 ;7(4):321-335

University of California, Irvine, Neurobiology and Behavior, Irvine, CA, USA.

Background: Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions.

Objective: Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation.

Methods: Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers.

Results: Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods.

Conclusions: Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.
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http://dx.doi.org/10.3233/JHD-180329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294605PMC
October 2019

Striatal Mutant Huntingtin Protein Levels Decline with Age in Homozygous Huntington's Disease Knock-In Mouse Models.

J Huntingtons Dis 2018 ;7(2):137-150

Department of Psychiatry and Human Behavior, University of California, Irvine, Irvine, CA, USA.

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder associated with aging, caused by an expanded polyglutamine (polyQ) repeat within the Huntingtin (HTT) protein. In HD, degeneration of the striatum and atrophy of the cortex are observed while cerebellum is less affected.

Objective: To test the hypothesis that HTT protein levels decline with age, which together with HTT mutation could influence disease progression.

Methods: Using whole brain cell lysates, a unique method of SDS-PAGE and western analysis was used to quantitate HTT protein, which resolves as a monomer and as a high molecular weight species that is modulated by the presence of transglutaminase 2. HTT levels were measured in striatum, cortex and cerebellum in congenic homozygous Q140 and HdhQ150 knock-in mice and WT littermate controls.

Results: Mutant HTT in both homozygous knock-in HD mouse models and WT HTT in control striatal and cortical tissues significantly declined in a progressive manner over time. Levels of mutant HTT in HD cerebellum remained high during aging.

Conclusions: A general decline in mutant HTT levels in striatum and cortex is observed that may contribute to disease progression in homozygous knock-in HD mouse models through reduction of HTT function. In cerebellum, sustained levels of mutant HTT with aging may be protective to this tissue which is less overtly affected in HD.
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http://dx.doi.org/10.3233/JHD-170274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002862PMC
October 2019

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene.

Neuron 2018 03;97(6):1268-1283.e6

Department of Neurology, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.
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http://dx.doi.org/10.1016/j.neuron.2018.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867896PMC
March 2018

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species.

J Vis Exp 2018 02 27(132). Epub 2018 Feb 27.

Department of Psychiatry and Human Behavior, University of California Irvine; UCI MIND, University of California Irvine; Department of Neurobiology and Behavior, University of California Irvine;

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution. For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.
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http://dx.doi.org/10.3791/57082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931423PMC
February 2018

Therapeutic effects of stem cells in rodent models of Huntington's disease: Review and electrophysiological findings.

CNS Neurosci Ther 2018 04 6;24(4):329-342. Epub 2018 Mar 6.

Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, CA, USA.

The principal symptoms of Huntington's disease (HD), chorea, cognitive deficits, and psychiatric symptoms are associated with the massive loss of striatal and cortical projection neurons. As current drug therapies only partially alleviate symptoms, finding alternative treatments has become peremptory. Cell replacement using stem cells is a rapidly expanding field that offers such an alternative. In this review, we examine recent studies that use mesenchymal cells, as well as pluripotent, cell-derived products in animal models of HD. Additionally, we provide further electrophysiological characterization of a human neural stem cell line, ESI-017, which has already demonstrated disease-modifying properties in two mouse models of HD. Overall, the field of regenerative medicine represents a viable and promising avenue for the treatment of neurodegenerative disorders including HD.
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http://dx.doi.org/10.1111/cns.12839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489814PMC
April 2018

Human Neural Stem Cell Transplantation Rescues Functional Deficits in R6/2 and Q140 Huntington's Disease Mice.

Stem Cell Reports 2018 01 7;10(1):58-72. Epub 2017 Dec 7.

Institute for Memory Impairment and Neurological Disorders, University of California, Irvine, 3400 Biological Sciences III, Irvine, CA 92697-4545, USA; Department of Neurobiology & Behavior, University of California, Irvine, 3400 Biological Sciences III, Irvine, CA 92697-4545, USA; Department of Psychiatry & Human Behavior, University of California, Irvine, 3400 Biological Sciences III, Irvine, CA 92697-4545, USA; Sue and Bill Gross Stem Cell Center, University of California, Irvine, Gross Hall, Room 3219, 845 Health Sciences Road, Irvine, CA 92697, USA. Electronic address:

Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.
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http://dx.doi.org/10.1016/j.stemcr.2017.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768890PMC
January 2018

The Library of Integrated Network-Based Cellular Signatures NIH Program: System-Level Cataloging of Human Cells Response to Perturbations.

Cell Syst 2018 01 29;6(1):13-24. Epub 2017 Nov 29.

BD2K-LINCS DCIC, Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45220, USA.

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.
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http://dx.doi.org/10.1016/j.cels.2017.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799026PMC
January 2018

A Comparison of mRNA Sequencing with Random Primed and 3'-Directed Libraries.

Sci Rep 2017 11 7;7(1):14626. Epub 2017 Nov 7.

Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Creating a cDNA library for deep mRNA sequencing (mRNAseq) is generally done by random priming, creating multiple sequencing fragments along each transcript. A 3'-end-focused library approach cannot detect differential splicing, but has potentially higher throughput at a lower cost, along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) that correct PCR bias. Here, we compare an implementation of such a 3'-digital gene expression (3'-DGE) approach with "conventional" random primed mRNAseq. Given our particular datasets on cultured human cardiomyocyte cell lines, we find that, while conventional mRNAseq detects ~15% more genes and needs ~500,000 fewer reads per sample for equivalent statistical power, the resulting differentially expressed genes, biological conclusions, and gene signatures are highly concordant between two techniques. We also find good quantitative agreement at the level of individual genes between two techniques for both read counts and fold changes between given conditions. We conclude that, for high-throughput applications, the potential cost savings associated with 3'-DGE approach are likely a reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable many larger scale studies focusing on not only differential expression analysis, but also quantitative transcriptome profiling.
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http://dx.doi.org/10.1038/s41598-017-14892-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676863PMC
November 2017

The ubiquitin conjugating enzyme Ube2W regulates solubility of the Huntington's disease protein, huntingtin.

Neurobiol Dis 2018 Jan 3;109(Pt A):127-136. Epub 2017 Oct 3.

Department of Neurology, University of Michigan, Ann Arbor, MI 48109, USA; Neuroscience Program, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Huntington's disease (HD) is caused by a CAG repeat expansion that encodes a polyglutamine (polyQ) expansion in the HD disease protein, huntingtin (HTT). PolyQ expansion promotes misfolding and aggregation of mutant HTT (mHTT) within neurons. The cellular pathways, including ubiquitin-dependent processes, by which mHTT is regulated remain incompletely understood. Ube2W is the only ubiquitin conjugating enzyme (E2) known to ubiquitinate substrates at their amino (N)-termini, likely favoring substrates with disordered N-termini. By virtue of its N-terminal polyQ domain, HTT has an intrinsically disordered amino terminus. In studies employing immortalized cells, primary neurons and a knock-in (KI) mouse model of HD, we tested the effect of Ube2W deficiency on mHTT levels, aggregation and neurotoxicity. In cultured cells, deficiency of Ube2W activity markedly decreases mHTT aggregate formation and increases the level of soluble monomers, while reducing mHTT-induced cytotoxicity. Consistent with this result, the absence of Ube2W in HdhQ200 KI mice significantly increases levels of soluble monomeric mHTT while reducing insoluble oligomeric species. This study sheds light on the potential function of the non-canonical ubiquitin-conjugating enzyme, Ube2W, in this polyQ neurodegenerative disease.
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http://dx.doi.org/10.1016/j.nbd.2017.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973538PMC
January 2018

Introducing the "Clinical Trials Corner".

J Huntingtons Dis 2017 ;6(3):167

University of California, Irvine, CA, USA.

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http://dx.doi.org/10.3233/JHD-179003DOI Listing
January 2017

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

Proc Natl Acad Sci U S A 2017 06 22;114(23):E4676-E4685. Epub 2017 May 22.

Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.
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http://dx.doi.org/10.1073/pnas.1614943114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468652PMC
June 2017

Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier.

Cell Stem Cell 2017 06 16;20(6):831-843.e5. Epub 2017 May 16.

The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Electronic address:

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy.
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http://dx.doi.org/10.1016/j.stem.2017.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6659720PMC
June 2017

Huntington's Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits.

Cell Rep 2017 05;19(7):1365-1377

Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA; UCI MIND, University of California, Irvine, Irvine, CA 92697, USA; Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA; Psychiatry and Human Behavior, University of California, Irvine, Irvine, CA 92697, USA; Sue and Bill Gross Stem Cell Center, University of California, Irvine, Irvine, CA 92697, USA. Electronic address:

Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.
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http://dx.doi.org/10.1016/j.celrep.2017.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646270PMC
May 2017

Mutant Huntingtin Disrupts the Nuclear Pore Complex.

Neuron 2017 Apr;94(1):93-107.e6

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.
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http://dx.doi.org/10.1016/j.neuron.2017.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595097PMC
April 2017

Human Neural Progenitor Transplantation Rescues Behavior and Reduces α-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.

Stem Cells Transl Med 2017 06 22;6(6):1477-1490. Epub 2017 Feb 22.

Department of Neurobiology and Behavior, Irvine, California, USA.

Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein (α-syn) into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is characterized by the aberrant accumulation of α-syn in cortical, hippocampal, and brainstem neurons, resulting in multiple cellular stressors that particularly impair dopamine and glutamate neurotransmission and related motor and cognitive function. Recent studies show that murine neural stem cell (NSC) transplantation can improve cognitive or motor function in transgenic models of Alzheimer's and Huntington's disease, and DLB. However, examination of clinically relevant human NSCs in these models is hindered by the challenges of xenotransplantation and the confounding effects of immunosuppressant drugs on pathology and behavior. To address this challenge, we developed an immune-deficient transgenic model of DLB that lacks T-, B-, and NK-cells, yet exhibits progressive accumulation of human α-syn (h-α-syn)-laden inclusions and cognitive and motor impairments. We demonstrate that clinically relevant human neural progenitor cells (line CNS10-hNPCs) survive, migrate extensively and begin to differentiate preferentially into astrocytes following striatal transplantation into this DLB model. Critically, grafted CNS10-hNPCs rescue both cognitive and motor deficits after 1 and 3 months and, furthermore, restore striatal dopamine and glutamate systems. These behavioral and neurochemical benefits are likely achieved by reducing α-syn oligomers. Collectively, these results using a new model of DLB demonstrate that hNPC transplantation can impact a broad array of disease mechanisms and phenotypes and suggest a cellular therapeutic strategy that should be pursued. Stem Cells Translational Medicine 2017;6:1477-1490.
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http://dx.doi.org/10.1002/sctm.16-0362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464354PMC
June 2017

TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington's disease.

Proc Natl Acad Sci U S A 2016 09 6;113(38):E5655-64. Epub 2016 Sep 6.

Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093;

Corticostriatal atrophy is a cardinal manifestation of Huntington's disease (HD). However, the mechanism(s) by which mutant huntingtin (mHTT) protein contributes to the degeneration of the corticostriatal circuit is not well understood. We recreated the corticostriatal circuit in microfluidic chambers, pairing cortical and striatal neurons from the BACHD model of HD and its WT control. There were reduced synaptic connectivity and atrophy of striatal neurons in cultures in which BACHD cortical and striatal neurons were paired. However, these changes were prevented if WT cortical neurons were paired with BACHD striatal neurons; synthesis and release of brain-derived neurotrophic factor (BDNF) from WT cortical axons were responsible. Consistent with these findings, there was a marked reduction in anterograde transport of BDNF in BACHD cortical neurons. Subunits of the cytosolic chaperonin T-complex 1 (TCP-1) ring complex (TRiC or CCT for chaperonin containing TCP-1) have been shown to reduce mHTT levels. Both CCT3 and the apical domain of CCT1 (ApiCCT1) decreased the level of mHTT in BACHD cortical neurons. In cortical axons, they normalized anterograde BDNF transport, restored retrograde BDNF transport, and normalized lysosomal transport. Importantly, treating BACHD cortical neurons with ApiCCT1 prevented BACHD striatal neuronal atrophy by enhancing release of BDNF that subsequently acts through tyrosine receptor kinase B (TrkB) receptor on striatal neurons. Our findings are evidence that TRiC reagent-mediated reductions in mHTT enhanced BDNF delivery to restore the trophic status of BACHD striatal neurons.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035849PMC
http://dx.doi.org/10.1073/pnas.1603020113DOI Listing
September 2016

SIRT2- and NRF2-Targeting Thiazole-Containing Compound with Therapeutic Activity in Huntington's Disease Models.

Cell Chem Biol 2016 07 14;23(7):849-861. Epub 2016 Jul 14.

Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address:

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in ex vivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactive oxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.
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http://dx.doi.org/10.1016/j.chembiol.2016.05.015DOI Listing
July 2016

PIAS1 Regulates Mutant Huntingtin Accumulation and Huntington's Disease-Associated Phenotypes In Vivo.

Neuron 2016 05 14;90(3):507-20. Epub 2016 Apr 14.

Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA; Institute of Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA; Department of Psychiatry and Human Behavior, University of California, Irvine, Irvine, CA 92697, USA. Electronic address:

The disruption of protein quality control networks is central to pathology in Huntington's disease (HD) and other neurodegenerative disorders. The aberrant accumulation of insoluble high-molecular-weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated protein corresponds to disease manifestation. We previously identified an HTT-selective E3 SUMO ligase, PIAS1, that regulates HTT accumulation and SUMO modification in cells. Here we investigated whether PIAS1 modulation in neurons alters HD-associated phenotypes in vivo. Instrastriatal injection of a PIAS1-directed miRNA significantly improved behavioral phenotypes in rapidly progressing mutant HTT (mHTT) fragment R6/2 mice. PIAS1 reduction prevented the accumulation of mHTT and SUMO- and ubiquitin-modified proteins, increased synaptophysin levels, and normalized key inflammatory markers. In contrast, PIAS1 overexpression exacerbated mHTT-associated phenotypes and aberrant protein accumulation. These results confirm the association between aberrant accumulation of expanded polyglutamine-dependent insoluble protein species and pathogenesis, and they link phenotypic benefit to reduction of these species through PIAS1 modulation.
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http://dx.doi.org/10.1016/j.neuron.2016.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942306PMC
May 2016

Comparison of phosphodiesterase 10A, dopamine receptors D1 and D2 and dopamine transporter ligand binding in the striatum of the R6/2 and BACHD mouse models of Huntington's disease.

J Huntingtons Dis 2014 ;3(4):333-41

Department of Neuroscience, Amgen Inc., Thousand Oaks, CA, USA.

Background: Phosphodiesterase 10A (PDE10A) is expressed at high levels in the striatum and has been proposed both as a biomarker for Huntington's disease pathology and as a target for intervention.

Objective: PDE10A radiotracers have been successfully used to measure changes in binding density in Huntington's disease patients, but little is known about PDE10A binding in mouse models that are used extensively to model pathology and test therapeutic interventions.

Methods: Our study investigated changes in PDE10A binding using the selective tracer 3H-7980 at specific ages of two Huntington's disease transgenic mouse models: R6/2, a short-lived model carrying exon-1 of mutant HTT and BACHD, a longer-lived model carrying full-length mutant HTT. PDE10A binding was compared to binding of known markers of striatal atrophy in Huntington's disease, e.g. dopamine transporter (DAT) and dopamine receptors D1 and D2.

Results: We found that in the R6/2 model at 6 weeks of age, mice showed high variability of binding, however binding of all ligands was significantly decreased at 8 and 12 weeks of age. In contrast, no changes were detectable in the BACHD model at 8, 10 or 12 month of age.

Conclusions: These findings suggest that radiotracer binding of PDE10A, DAT, D1 and D2 receptor in the R6/2 model may be a good indicator of striatal pathological changes that are observed in Huntington's disease patients, and that the first 12 months in the BACHD model may be more reflective of early stages of the disease.
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http://dx.doi.org/10.3233/JHD-140129DOI Listing
April 2015
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