Publications by authors named "Larry Park"

31 Publications

Rapid and robust patterns of spontaneous locomotor deficits in mouse models of Huntington's disease.

PLoS One 2020 28;15(12):e0243052. Epub 2020 Dec 28.

CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by severe disruption of cognitive and motor functions, including changes in posture and gait. A number of HD mouse models have been engineered that display behavioral and neuropathological features of the disease, but gait alterations in these models are poorly characterized. Sensitive high-throughput tests of fine motor function and gait in mice might be informative in evaluating disease-modifying interventions. Here, we describe a hypothesis-free workflow that determines progressively changing locomotor patterns across 79 parameters in the R6/2 and Q175 mouse models of HD. R6/2 mice (120 CAG repeats) showed motor disturbances as early as at 4 weeks of age. Similar disturbances were observed in homozygous and heterozygous Q175 KI mice at 3 and 6 months of age, respectively. Interestingly, only the R6/2 mice developed forelimb ataxia. The principal components of the behavioral phenotypes produced two phenotypic scores of progressive postural instability based on kinematic parameters and trajectory waveform data, which were shared by both HD models. This approach adds to the available HD mouse model research toolbox and has a potential to facilitate the development of therapeutics for HD and other debilitating movement disorders with high unmet medical need.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243052PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769440PMC
January 2021

NRF2 activation by reversible KEAP1 binding induces the antioxidant response in primary neurons and astrocytes of a Huntington's disease mouse model.

Free Radic Biol Med 2021 Jan 21;162:243-254. Epub 2020 Oct 21.

Department of Translational and Discovery Research, IRBM S.p.A., Via Pontina Km 30,600, 00071, Pomezia, Roma, Italy. Electronic address:

Oxidative stress has been associated with pathogenesis in several diseases including Huntington's disease (HD), a neurodegenerative disorder caused by a mutation in the huntingtin gene. Oxidative stress induced reactive oxygen species (ROS) are normally controlled at the cellular level by the nuclear factor (erythroid-derived 2)-like 2 (NRF2) a transcription factor that regulates the expression of various antioxidants and detoxifying proteins. Normally NRF2 is largely inactivated in the cytoplasm by the Kelch-like ECH-associated protein 1 (KEAP1)/Cullin-3 (CUL3) mediated ubiquitination and subsequent proteosomal degradation. In the presence of ROS, KEAP1 sensor cysteines are directly or indirectly engaged resulting in NRF2 release, nuclear translocation, and activation of its target genes. Consequently the activation of NRF2 by a small-molecule drug may have the therapeutic potential to control oxidative stress by upregulation of the endogenous antioxidant responses. Here we attempted to validate the use of a reversible non-acidic KEAP1 binder (Compound 2) to activate NRF2 with better cellular activity than similar acidic compounds. When tested head to head with sulforaphane, a covalent KEAP1 binder, Compound 2 had a similar ability to induce the expression of genes known to be modulated by NRF2 in neurons and astrocytes isolated from wild-type rat, wild type mouse and zQ175 (an HD mouse model) embryos. However, while sulforaphane also negatively affected genes involved in neurotoxicity in these cells, Compound 2 showed a clean profile suggesting its mode of action has lower off-target activity. We show that Compound 2 was able to protect cells from an oxidative insult by preserving the ATP content and the mitochondrial potential of primary astrocytes, consistent with the hypothesis that neurotoxicity induced by oxidative stress can be limited by upregulation of innate antioxidant response.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.10.022DOI Listing
January 2021

Optimization of linear and cyclic peptide inhibitors of KEAP1-NRF2 protein-protein interaction.

Bioorg Med Chem 2020 11 30;28(21):115738. Epub 2020 Aug 30.

Department of Drug Discovery, IRBM Spa, Via Pontina km 30.600, 00071 Pomezia, Rome, Italy.

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.
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http://dx.doi.org/10.1016/j.bmc.2020.115738DOI Listing
November 2020

Combined Peptide and Small-Molecule Approach toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction.

ACS Med Chem Lett 2020 May 3;11(5):740-746. Epub 2020 Apr 3.

IRBM S.p.A., Via Pontina km 30.600, 00071 Pomezia, Rome, Italy.

The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236277PMC
May 2020

Transcriptional Assessment of Striatal mRNAs as Valid Biomarkers of Disease Progression in Three Mouse Models of Huntington's Disease.

J Huntingtons Dis 2020 ;9(1):13-31

CHDI Management/CHDI Foundation, Inc, Princeton, NJ, USA.

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder that prominently affects the basal ganglia, leading to affective, cognitive, behavioral, and motor decline. The primary site of neuron loss in HD is the striatal part of the basal ganglia, with GABAergic medium size spiny neurons (MSNs) being nearly completely lost in advanced HD.

Objective: Based on the hypothesis that mutant huntingtin (mHTT) protein injures neurons via transcriptional dysregulation, we set out to establish a transcriptional profile of HD disease progression in the well characterized transgenic mouse model, R6/2, and two Knock-in models (KI); zQ175KI (expressing mutant mouse/human chimeric Htt protein) and HdhQ200 HET KI (carrying one allele of expanded mouse CAG repeats).

Methods: In this study, we used quantitative PCR (qPCR) to evaluate striatal mRNA levels of markers of neurotransmission, neuroinflammation, and energy metabolism.

Results: After analyzing and comparing transcripts from pre-symptomatic and symptomatic stages, markers expressed in the basal ganglia MSNs, which are typically involved in maintaining normal neurotransmission, showed a genotype-specific decrease in mRNA expression in a pattern consistent with human studies. In contrast, transcripts associated with neuroinflammation and energy metabolism were mostly unaffected in these animal models of HD.

Conclusion: Our results show that transcripts linked to neurotransmission are significantly reduced and are consistent with disease progression in both zQ175KI and R6/2 transgenic mouse models.
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http://dx.doi.org/10.3233/JHD-190389DOI Listing
January 2020

Improved synthesis of [F] fallypride and characterization of a Huntington's disease mouse model, zQ175DN KI, using longitudinal PET imaging of D2/D3 receptors.

EJNMMI Radiopharm Chem 2019 Aug 5;4(1):20. Epub 2019 Aug 5.

CHDI Management/CHDI Foundation, Los Angeles, CA, USA.

Purpose: Dopamine receptors are involved in pathophysiology of neuropsychiatric diseases, including Huntington's disease (HD). PET imaging of dopamine D2 receptors (D2R) in HD patients has demonstrated 40% decrease in D2R binding in striatum, and D2R could be a reliable quantitative target to monitor disease progression. A D2/3R antagonist, [F] fallypride, is a high-affinity radioligand that has been clinically used to study receptor density and occupancy in neuropsychiatric disorders. Here we report an improved synthesis method for [F]fallypride. In addition, high molar activity of the ligand has allowed us to apply PET imaging to characterize D2/D3 receptor density in striatum of the recently developed zQ175DN knock-in (KI) mouse model of HD.

Methods: We longitudinally characterized in vivo [F] fallypride -PET imaging of D2/D3 receptor densities in striatum of 9 and 12 month old wild type (WT) and heterozygous (HET) zQ175DN KI mouse. Furthermore, we verified the D2/D3 receptor density in striatum with [H] fallypride autoradiography at 12 months of age.

Results: We implemented an improved synthesis method for [F] fallypride to yield high molar activity (MA, 298-360 GBq/μmol) and good reproducibility. In the HET zQ175DN KI mice, we observed a significant longitudinal decrease in binding potential (BP) (30.2%, p < 0.001, 9 months of age and 51.6%, p < 0.001, 12 months of age) compared to WT littermates. No mass effect was observed when the MA of [F] fallypride was > 100 GBq/μmol at the time of injection. Furthermore, the decrease of D2/D3 receptor density in striatum in HET zQ175DN KI was consistent using [H] fallypride autoradiography.

Conclusions: We observed a significant decrease in D2/D3R receptor densities in the striatum of HET zQ175DN KI mice compared to WT mice at 9 and 12 months of age. These results are in line with clinical findings in HD patients, suggesting [F] fallypride PET imaging has potential as a quantitative translational approach to monitor disease progression in preclinical studies.
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http://dx.doi.org/10.1186/s41181-019-0071-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682833PMC
August 2019

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Nat Med 2019 07 1;25(7):1131-1142. Epub 2019 Jul 1.

Sangamo Therapeutics, Inc., Richmond, CA, USA.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
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http://dx.doi.org/10.1038/s41591-019-0478-3DOI Listing
July 2019

Age- and sex-related changes in cortical and striatal nitric oxide synthase in the Q175 mouse model of Huntington's disease.

Nitric Oxide 2019 02 5;83:40-50. Epub 2018 Dec 5.

Department of Neuroscience, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.

In Huntington's disease (HD), corticostriatal and striatopallidal projection neurons preferentially degenerate as a result of mutant huntingtin expression. Pathological deficits in nitric oxide (NO) signaling have also been reported in corticostriatal circuits in HD, however, the impact of age and sex on nitrergic transmission is not well characterized. Thus, we utilized NADPH-diaphorase (NADPH-d) histochemistry and qPCR assays to assess neuronal NO synthase (nNOS) activity/expression in aged male and female Q175 heterozygous mice. Compared to age-matched controls, male Q175 mice exhibited reductions in NADPH-d staining in the motor cortex at 21, but not, 16 months of age. Comparisons across genotypes showed that striatal NADPH-d staining was significantly decreased at both 16 and 21 months of age. Comparisons within sexes in 21 month old mice revealed a decrease in striatal NADPH-d staining in males, but no changes were detected in females. Significant correlations between cortical and striatal NADPH-d staining deficits were also observed in males and females at both ages. To directly assess the role of constitutively active NOS isoforms in these changes, nNOS and endothelial NOS (eNOS) mRNA expression levels were examined in R6/2 (3 month old) and Q175 (11.5 month old) mice using qPCR assays. nNOS transcript expression was decreased in the cortex (40%) and striatum (54%) in R6/2 mice. nNOS mRNA down-regulation in striatum of Q175 animals was more modest (19%), and no changes were detected in cortex. eNOS expression was not changed in the cortex or striatum of Q175 mice. The current findings point to age-dependent deficits in nNOS activity in the HD cortex and striatum which appear first in the striatum and are more pronounced in males. Together, these observations and previous studies indicate that decreases in nitrergic transmission progress with age and are likely to contribute to corticostriatal circuit pathophysiology particularly in male patients with HD.
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http://dx.doi.org/10.1016/j.niox.2018.12.002DOI Listing
February 2019

Impaired Performance of the Q175 Mouse Model of Huntington's Disease in the Touch Screen Paired Associates Learning Task.

Front Behav Neurosci 2018 2;12:226. Epub 2018 Oct 2.

Charles River Discovery Services, Kuopio, Finland.

Cognitive disturbances often predate characteristic motor dysfunction in individuals with Huntington's disease (HD) and place an increasing burden on the HD patients and caregivers with the progression of the disorder. Therefore, application of maximally translational cognitive tests to animal models of HD is imperative for the development of treatments that could alleviate cognitive decline in human patients. Here, we examined the performance of the Q175 mouse knock-in model of HD in the touch screen version of the paired associates learning (PAL) task. We found that 10-11-month-old heterozygous Q175 mice had severely attenuated learning curve in the PAL task, which was conceptually similar to previously documented impaired performance of individuals with HD in the PAL task of the Cambridge Neuropsychological Test Automated Battery (CANTAB). Besides high rate of errors in PAL task, Q175 mice exhibited considerably lower responding rate than age-matched wild-type (WT) animals. Our examination of effortful operant responding during fixed ratio (FR) and progressive ratio (PR) reinforcement schedules in a separate cohort of similar age confirmed slower and unselective performance of mutant animals, as observed during PAL task, but suggested that motivation to work for nutritional reward in the touch screen setting was similar in Q175 and WT mice. We also demonstrated that pronounced sensorimotor disturbances in Q175 mice can be detected at early touch screen testing stages, (e.g., during "Punish Incorrect" phase of operant pretraining), so we propose that shorter test routines may be utilised for more expedient studies of treatments aimed at the rescue of HD-related phenotype.
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http://dx.doi.org/10.3389/fnbeh.2018.00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176131PMC
October 2018

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1).

Arch Biochem Biophys 2017 10 8;631:31-41. Epub 2017 Aug 8.

CHDI Management/CHDI Foundation, Los Angeles, CA, USA. Electronic address:

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a H-N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.
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http://dx.doi.org/10.1016/j.abb.2017.08.003DOI Listing
October 2017

Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington's Disease Models.

Neuron 2016 Dec 1;92(6):1220-1237. Epub 2016 Dec 1.

CHDI Management/CHDI Foundation, 6080 Center Drive, Los Angeles, CA 90045, USA.

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2016.10.064DOI Listing
December 2016

Novel Metabolic Abnormalities in the Tricarboxylic Acid Cycle in Peripheral Cells From Huntington's Disease Patients.

PLoS One 2016 9;11(9):e0160384. Epub 2016 Sep 9.

Weill Cornell Medical College, Brain and Mind Research Institute, Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605, United States of America.

Metabolic dysfunction is well-documented in Huntington's disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160384PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017661PMC
August 2017

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease.

Exp Neurol 2016 08 6;282:99-118. Epub 2016 May 6.

CHDI Foundation/CHDI Management Inc., Los Angeles, USA. Electronic address:

Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntington's disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.
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http://dx.doi.org/10.1016/j.expneurol.2016.05.005DOI Listing
August 2016

Cognitive Training at a Young Age Attenuates Deficits in the zQ175 Mouse Model of HD.

Front Behav Neurosci 2015 11;9:361. Epub 2016 Jan 11.

Psychogenics Inc.Tarrytown, NY, USA; Department of Psychiatry, New York State Psychiatric Institute, Columbia UniversityNew York, NY, USA.

Huntington's Disease (HD) is a progressive neurodegenerative disorder that causes motor, cognitive, and psychiatric symptoms. In these experiments, we tested if operant training at an early age affected adult cognitive deficits in the zQ175 KI Het (zQ175) mouse model of HD. In Experiment 1 we trained zQ175 mice in a fixed-ratio/progressive ratio (FR/PR) task to assay learning and motivational deficits. We found pronounced deficits in response rates and task engagement in naïve adult zQ175 mice (32-33 weeks age), while deficits in zQ175 mice trained from 6-7 weeks age were either absent or less severe. When those mice were re-tested as adults, FR/PR performance deficits were absent or otherwise less severe than deficits observed in naïve adult zQ175 relative to wild type (WT) mice. In Experiment 2, we used a Go/No-go operant task to assess the effects of early cognitive testing on response inhibition deficits in zQ175 mice. We found that zQ175 mice that began testing at 7-8 weeks did not exhibit deficits in Go/No-go testing, but when re-tested at 28-29 weeks age exhibited an initial impairment that diminished with training. These transient deficits were nonetheless mild relative to deficits observed among adult zQ175 mice without prior testing experience. In Experiment 3 we trained mice in a two-choice visual discrimination test to evaluate cognitive flexibility. As in prior experiments, we found performance deficits were mild or absent in mice that started training at 6-9 weeks of age, while deficits in naive mice exposed to training at 28-29 weeks were severe. Re-testing mice at 28-29 weeks age, were previously trained starting at 6-9 weeks, revealed that deficits in learning and cognitive flexibility were absent or reduced relative to effects observed in naive adults. In Experiment 4, we tested working memory deficits with a delayed non-match to position (DNMTP) test. Mice with prior experience exhibited mild working memory deficits, with males zQ175 exhibiting no deficits, and females performing significantly worse than WT mice at a single delay interval, whereas naive zQ175 exhibited severe delay-dependent deficits at all intervals exceeding 1 s. In sum, these experiments indicate that CAG-dependent impairments in motivation, motor control, cognitive flexibility, and working memory are sensitive to the environmental enrichment and experience. These findings are of clinical relevance, as HD carrier status can potentially be detected at an early age.
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http://dx.doi.org/10.3389/fnbeh.2015.00361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707270PMC
January 2016

Abnormalities in the tricarboxylic Acid cycle in Huntington disease and in a Huntington disease mouse model.

J Neuropathol Exp Neurol 2015 Jun;74(6):527-37

From the Weill Cornell Medical College, Brain and Mind Research Institute, Burke Medical Research Institute, White Plains (NNN, HX, JB, GEG); and New York Brain Bank, Taub Institute, Columbia University, New York (JPGV, EPC), New York; and CHDI Management/CHDI Foundation, Inc., Los Angeles, California (LCP, JA).

Glucose metabolism is reduced in the brains of patients with Huntington disease (HD). The mechanisms underlying this deficit, its link to the pathology of the disease, and the vulnerability of the striatum in HD remain unknown. Abnormalities in some of the key mitochondrial enzymes involved in glucose metabolism, including the pyruvate dehydrogenase complex (PDHC) and the tricarboxylic acid (TCA) cycle, may contribute to these deficits. Here, activities for these enzymes and select protein levels were measured in human postmortem cortex and in striatum and cortex of an HD mouse model (Q175); mRNA levels encoding for these enzymes were also measured in the Q175 mouse cortex. The activities of PDHC and nearly all of the TCA cycle enzymes were dramatically lower (-50% to 90%) in humans than in mice. The activity of succinate dehydrogenase increased with HD in human (35%) and mouse (23%) cortex. No other changes were detected in the human HD cortex or mouse striatum. In Q175 cortex, there were increased activities of PDHC (+12%) and aconitase (+32%). Increased mRNA levels for succinyl thiokinase (+88%) and isocitrate dehydrogenase (+64%) suggested an upregulation of the TCA cycle. These patterns of change differ from those reported in other diseases, which may offer unique metabolic therapeutic opportunities for HD patients.
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http://dx.doi.org/10.1097/NEN.0000000000000197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435838PMC
June 2015

Cognitive deficits in transgenic and knock-in HTT mice parallel those in Huntington's disease.

J Huntingtons Dis 2014 ;3(2):145-58

PsychoGenics Inc., Tarrytown, NY, USA Department of Psychiatry, Columbia University, NYSPI, NY, USA.

Background: Huntington's disease (HD) is characterized not only by severe motor deficits but also by early cognitive dysfunction that significantly increases the burden of the disease for patients and caregivers. Considerable efforts have concentrated, therefore, on the assessment of cognitive deficits in some HD mouse models. However, many of these models that exhibit cognitive deficits also have contemporaneous serious motor deficits, confounding interpretation of cognitive decline.

Objective: The BACHD and zQ175 mouse models present a more slowly progressing disease phenotype in both motor and cognitive domains, and might therefore offer a better opportunity to measure cognitive decline over a longer timeframe; such models could be useful in screening therapeutic compounds. In order to better define the cognitive impairments evident in BACHD and zQ175 HD mice, both were tested in an instrumental touchscreen visual discrimination assay designed to assess discrimination learning and cognitive flexibility.

Methods: BACHD and zQ175 mice, as well as their WT controls were tested for their ability to discriminate two complex visual stimuli. Following this discrimination phase, the reinforcement contingencies were reversed and the previously incorrect stimulus became the correct stimulus. In a final, third phase of testing, two novel stimuli were introduced and mice were required to undergo a second round of discrimination testing with these stimuli.

Results: Our results show that learning during the discrimination phase was similar between the WT and BACHD mice. In contrast, the zQ175 at 26 weeks of age showed decreased accuracy over the last 10 days of discrimination, compared to WT controls. During subsequent reversal and novel stimuli phases, both BACHD and zQ175 mice exhibited significant deficits compared to WT controls.

Conclusions: Our results suggest that the BACHD, and for the first time, zQ175 HD models exhibit cognitive inflexibility and psychomotor slowing, a phenotype that is consistent with cognitive symptoms described in HD patients.
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http://dx.doi.org/10.3233/JHD-130061DOI Listing
September 2014

Genetic deletion of transglutaminase 2 does not rescue the phenotypic deficits observed in R6/2 and zQ175 mouse models of Huntington's disease.

PLoS One 2014 23;9(6):e99520. Epub 2014 Jun 23.

CHDI Management/CHDI Foundation, Princeton, New Jersey, United States of America.

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by expansion of CAG repeats in the huntingtin gene. Tissue transglutaminase 2 (TG2), a multi-functional enzyme, was found to be increased both in HD patients and in mouse models of the disease. Furthermore, beneficial effects have been reported from the genetic ablation of TG2 in R6/2 and R6/1 mouse lines. To further evaluate the validity of this target for the treatment of HD, we examined the effects of TG2 deletion in two genetic mouse models of HD: R6/2 CAG 240 and zQ175 knock in (KI). Contrary to previous reports, under rigorous experimental conditions we found that TG2 ablation had no effect on either motor or cognitive deficits, or on the weight loss. In addition, under optimal husbandry conditions, TG2 ablation did not extend R6/2 lifespan. Moreover, TG2 deletion did not change the huntingtin aggregate load in cortex or striatum and did not decrease the brain atrophy observed in either mouse line. Finally, no amelioration of the dysregulation of striatal and cortical gene markers was detected. We conclude that TG2 is not a valid therapeutic target for the treatment of HD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099520PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067284PMC
October 2015

The PDE1/5 Inhibitor SCH-51866 Does Not Modify Disease Progression in the R6/2 Mouse Model of Huntington's Disease.

PLoS Curr 2014 Feb 13;6. Epub 2014 Feb 13.

CHDI Management/CHDI Foundation, Los Angeles, California, USA.

Huntington's disease is a neurodegenerative disorder caused by mutations in the CAG tract of huntingtin. Several studies in HD cellular and rodent systems have identified disturbances in cyclic nucleotide signaling, which might be relevant to pathogenesis and therapeutic intervention. To investigate whether selective phosphodiesterase (PDE) inhibitors can improve some aspects of disease pathogenesis in HD models, we have systematically evaluated the effects of a variety of cAMP and cGMP selective PDE inhibitors in various HD models. Here we present the lack of effect in a variety of endpoints of the PDE subtype selective inhibitor SCH-51866, a PDE1/5 inhibitor, in the R6/2 mouse model of HD, after chronic oral dosing.
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http://dx.doi.org/10.1371/currents.hd.3304e87e460b4bb0dc519a29f4decccaDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923778PMC
February 2014

HDAC4 reduction: a novel therapeutic strategy to target cytoplasmic huntingtin and ameliorate neurodegeneration.

PLoS Biol 2013 Nov 26;11(11):e1001717. Epub 2013 Nov 26.

Department of Medical and Molecular Genetics, King's College London, London, United Kingdom.

Histone deacetylase (HDAC) 4 is a transcriptional repressor that contains a glutamine-rich domain. We hypothesised that it may be involved in the molecular pathogenesis of Huntington's disease (HD), a protein-folding neurodegenerative disorder caused by an aggregation-prone polyglutamine expansion in the huntingtin protein. We found that HDAC4 associates with huntingtin in a polyglutamine-length-dependent manner and co-localises with cytoplasmic inclusions. We show that HDAC4 reduction delayed cytoplasmic aggregate formation, restored Bdnf transcript levels, and rescued neuronal and cortico-striatal synaptic function in HD mouse models. This was accompanied by an improvement in motor coordination, neurological phenotypes, and increased lifespan. Surprisingly, HDAC4 reduction had no effect on global transcriptional dysfunction and did not modulate nuclear huntingtin aggregation. Our results define a crucial role for the cytoplasmic aggregation process in the molecular pathology of HD. HDAC4 reduction presents a novel strategy for targeting huntingtin aggregation, which may be amenable to small-molecule therapeutics.
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http://dx.doi.org/10.1371/journal.pbio.1001717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841096PMC
November 2013

Characterization of neurophysiological and behavioral changes, MRI brain volumetry and 1H MRS in zQ175 knock-in mouse model of Huntington's disease.

PLoS One 2012 20;7(12):e50717. Epub 2012 Dec 20.

Charles River Discovery Research Services, Kuopio, Finland.

Huntington's disease (HD) is an autosomal neurodegenerative disorder, characterized by severe behavioral, cognitive, and motor deficits. Since the discovery of the huntingtin gene (HTT) mutation that causes the disease, several mouse lines have been developed using different gene constructs of Htt. Recently, a new model, the zQ175 knock-in (KI) mouse, was developed (see description by Menalled et al, [1]) in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. Here we confirm the behavioral phenotypes reported by Menalled et al [1], and extend the characterization to include brain volumetry, striatal metabolite concentration, and early neurophysiological changes. The overall reproducibility of the behavioral phenotype across the two independent laboratories demonstrates the utility of this new model. Further, important features reminiscent of human HD pathology are observed in zQ175 mice: compared to wild-type neurons, electrophysiological recordings from acute brain slices reveal that medium spiny neurons from zQ175 mice display a progressive hyperexcitability; glutamatergic transmission in the striatum is severely attenuated; decreased striatal and cortical volumes from 3 and 4 months of age in homo- and heterozygous mice, respectively, with whole brain volumes only decreased in homozygotes. MR spectroscopy reveals decreased concentrations of N-acetylaspartate and increased concentrations of glutamine, taurine and creatine + phosphocreatine in the striatum of 12-month old homozygotes, the latter also measured in 12-month-old heterozygotes. Motor, behavioral, and cognitive deficits in homozygotes occur concurrently with the structural and metabolic changes observed. In sum, the zQ175 KI model has robust behavioral, electrophysiological, and histopathological features that may be valuable in both furthering our understanding of HD-like pathophyisology and the evaluation of potential therapeutic strategies to slow the progression of disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050717PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527436PMC
June 2013

Comprehensive behavioral and molecular characterization of a new knock-in mouse model of Huntington's disease: zQ175.

PLoS One 2012 20;7(12):e49838. Epub 2012 Dec 20.

PsychoGenics Inc., Tarrytown, New York, United States of America.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive and psychiatric manifestations. Since the mutation responsible for the disease was identified as an unstable expansion of CAG repeats in the gene encoding the huntingtin protein in 1993, numerous mouse models of HD have been generated to study disease pathogenesis and evaluate potential therapeutic approaches. Of these, knock-in models best mimic the human condition from a genetic perspective since they express the mutation in the appropriate genetic and protein context. Behaviorally, however, while some abnormal phenotypes have been detected in knock-in mouse models, a model with an earlier and more robust phenotype than the existing models is required. We describe here for the first time a new mouse line, the zQ175 knock-in mouse, derived from a spontaneous expansion of the CAG copy number in our CAG 140 knock-in colony [1]. Given the inverse relationship typically observed between age of HD onset and length of CAG repeat, since this new mouse line carries a significantly higher CAG repeat length it was expected to be more significantly impaired than the parent line. Using a battery of behavioral tests we evaluated both heterozygous and homozygous zQ175 mice. Homozygous mice showed motor and grip strength abnormalities with an early onset (8 and 4 weeks of age, respectively), which were followed by deficits in rotarod and climbing activity at 30 weeks of age and by cognitive deficits at around 1 year of age. Of particular interest for translational work, we also found clear behavioral deficits in heterozygous mice from around 4.5 months of age, especially in the dark phase of the diurnal cycle. Decreased body weight was observed in both heterozygotes and homozygotes, along with significantly reduced survival in the homozygotes. In addition, we detected an early and significant decrease of striatal gene markers from 12 weeks of age. These data suggest that the zQ175 knock-in line could be a suitable model for the evaluation of therapeutic approaches and early events in the pathogenesis of HD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049838PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527464PMC
June 2013

A novel BACHD transgenic rat exhibits characteristic neuropathological features of Huntington disease.

J Neurosci 2012 Oct;32(44):15426-38

Department of Medical Genetics, University of Tuebingen, 72076 Tuebingen, Germany.

Huntington disease (HD) is an inherited progressive neurodegenerative disorder, characterized by motor, cognitive, and psychiatric deficits as well as neurodegeneration and brain atrophy beginning in the striatum and the cortex and extending to other subcortical brain regions. The genetic cause is an expansion of the CAG repeat stretch in the HTT gene encoding huntingtin protein (htt). Here, we generated an HD transgenic rat model using a human bacterial artificial chromosome (BAC), which contains the full-length HTT genomic sequence with 97 CAG/CAA repeats and all regulatory elements. BACHD transgenic rats display a robust, early onset and progressive HD-like phenotype including motor deficits and anxiety-related symptoms. In contrast to BAC and yeast artificial chromosome HD mouse models that express full-length mutant huntingtin, BACHD rats do not exhibit an increased body weight. Neuropathologically, the distribution of neuropil aggregates and nuclear accumulation of N-terminal mutant huntingtin in BACHD rats is similar to the observations in human HD brains. Aggregates occur more frequently in the cortex than in the striatum and neuropil aggregates appear earlier than mutant htt accumulation in the nucleus. Furthermore, we found an imbalance in the striatal striosome and matrix compartments in early stages of the disease. In addition, reduced dopamine receptor binding was detectable by in vivo imaging. Our data demonstrate that this transgenic BACHD rat line may be a valuable model for further understanding the disease mechanisms and for preclinical pharmacological studies.
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http://dx.doi.org/10.1523/JNEUROSCI.1148-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621569PMC
October 2012

Caspase-6 activity in a BACHD mouse modulates steady-state levels of mutant huntingtin protein but is not necessary for production of a 586 amino acid proteolytic fragment.

J Neurosci 2012 May;32(22):7454-65

The Buck Institute for Research on Aging, Novato, CA 94945, USA.

Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.
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http://dx.doi.org/10.1523/JNEUROSCI.6379-11.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454486PMC
May 2012

Effect of the rd1 mutation on motor performance in R6/2 and wild type mice.

PLoS Curr 2012 Feb 13;4:RRN1303. Epub 2012 Feb 13.

Senior Principal Scientist at PsychoGenics, Inc.; Senior Research Scientist, Pfizer Inc.; Data Manager at Psychogenics, Inc; Director, PreClinical Research, CHDI Foundation, Inc; Director of In Vivo Biology, CHDI Foundation Inc and Senior VP Behavioral R&D at PsychoGenics, Inc.

Homozygosis for the rd1 mutation in the Pbe6b gene results in the loss of the rod beta-subunit of the cyclic GMP phosphodiesterase and, eventually, of all rod and cone photoreceptors. The R6/2 mouse line is a widely used model of Huntington's disease (HD). The original line was made available on a mixed background obtained by crossing, via ovarian transplant, female R6/2 (on a B6CBA mixed background) with male B6CBAF1/J mice. As the CBA/J strain used in the US is homozygous for the rd1 mutation and the breeding scheme does not ensure heterozygosis for the mutation, a significant percentage of the offspring on this mixed background is expected to be homozygous for the rd1 mutation. We investigate here the effect of rd1 homozygosis on motor function and examined the effects of the mutation on the R6/2 phenotype. Homozygosis for the rd1 mutation resulted in increased activity in the open field test and reduced rotarod test performance. In addition, rd1 mutation absence or heterozygosis reduced the differences between the R6/2 and the WT mice. Our recommendation for the neurodegeneration field, and for all mouse studies in general, is to carefully control homozygosis for retinal degeneration mutation, even when using tests of motor function.
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http://dx.doi.org/10.1371/currents.RRN1303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278853PMC
February 2012

Pharmacokinetics of memantine in rats and mice.

PLoS Curr 2011 Dec 15;3:RRN1291. Epub 2011 Dec 15.

Director, DMPK, CHDI Management/CHDI Foundation, Princeton, NJ; Director of In Vivo Biology, CHDI Foundation Inc; Director, PreClinical Research, CHDI Foundation, Inc; Pharmacokinetic consultant for the CHDI Foundation, Holland NY; CHDI Foundation, Inc.; VP, Chemistry CHDI Foundation/ CHDI Management Inc. Los Angeles CA; VP Translational Biology, CHDI Management/CHDI Foundation Inc., Los Angeles (USA) and CSO - Drug Discovery & Development, CHDI Foundation / CHDI Management.

To evaluate the potential of memantine as a therapeutic agent for Huntington's disease (HD) we have undertaken a series of in vitro, ex vivo and whole animal studies to characterize its pharmacokinetics (PK) and pharmacodynamics (PD) in rats and mice. Results from these studies will enable determination of memantine exposures needed to engage the related functional PD marker and help predict the dose regimen for clinical trials to test its proposed mechanism of action; the selective blockade of extrasynaptic, but not synaptic, NMDA receptors. The studies reported here describe the PK of memantine in rats and mice at low (1 mg/kg) and high (10 mg/kg) doses. Our studies indicate that the clearance mechanisms of memantine in rats and mice are different from those in human, and that clearance needs to be taken into account when extrapolating to the human. In rats only, there is a significant metabolic contribution to memantine clearance at lower dose levels. While memantine is primarily cleared renally in all three species, the proportion of total systemic clearance above the glomerular filtration rate (GFR) is much higher in rats and mice (~13, 4.5, and 1.4 times higher than GFR in rats, mice, and humans, respectively), suggesting that the contribution of active transport to memantine elimination in rats and mice is more significant than in the human. In rats and mice, memantine had a short half-life (<4 h) and steep Cmax/Cmin ratios (>100). In the human, the half-life of memantine was reported to be very long (60-80 h) with a Cmax/Cmin ratio at steady state concentrations of ~1.5. A small change in the clearance of memantine - for example due to renal impairment or competition for the elimination pathway with a co-administered drug - will likely affect exposure and, therefore, the selectivity of memantine on NMDA receptors . The PK differences observed between these species demonstrate that the PK in mice and rats cannot be directly extrapolated to the human. Further, the relationship between the plasma concentration (and therefore dose) needed to elicit a mechanism-related in vivo functional effect (PD readout) while maintaining the selectivity of the extrasynaptic blockade of the NMDA receptors needs to be established before clinical trials can be appropriately planned.
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http://dx.doi.org/10.1371/currents.RRN1291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269340PMC
December 2011

HD mouse models reveal clear deficits in learning to perform a simple instrumental response.

PLoS Curr 2011 Nov 30;3:RRN1282. Epub 2011 Nov 30.

Principal Scientist at PsychoGenics, Inc.; Research Associate at PsychoGenics Inc; Breeding Manager at PsychoGenics, Inc.; VP, Neurodegenerative Disorders at PsychoGenics, Inc; Director, PreClinical Research, CHDI Foundation, Inc; Director of In Vivo Biology, CHDI Foundation Inc and Senior VP Behavioral R&D at PsychoGenics, Inc.

Mouse models of Huntington's disease (HD) were trained to acquire one of two simple instrumental responses (a lever press or a nosepoke) to obtain food reinforcement. Animals from several HD strains revealed apparently progressive deficits in this task, being significantly less able than littermate controls to perform the required responses, at ages where motor function is only mildly affected. These data could provide a simple way to measure learning deficits in these mouse models, likely related to the characteristic pattern of neural damage observed in HD mouse models.
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http://dx.doi.org/10.1371/currents.RRN1282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327146PMC
November 2011

Comprehensive behavioral testing in the R6/2 mouse model of Huntington's disease shows no benefit from CoQ10 or minocycline.

PLoS One 2010 Mar 22;5(3):e9793. Epub 2010 Mar 22.

PsychoGenics Inc., Tarrytown, New York, United States of America.

Previous studies of the effects of coenzyme Q10 and minocycline on mouse models of Huntington's disease have produced conflicting results regarding their efficacy in behavioral tests. Using our recently published best practices for husbandry and testing for mouse models of Huntington's disease, we report that neither coenzyme Q10 nor minocycline had significant beneficial effects on measures of motor function, general health (open field, rotarod, grip strength, rearing-climbing, body weight and survival) in the R6/2 mouse model. The higher doses of minocycline, on the contrary, reduced survival. We were thus unable to confirm the previously reported benefits for these two drugs, and we discuss potential reasons for these discrepancies, such as the effects of husbandry and nutrition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842438PMC
March 2010

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

Neurobiol Dis 2009 Sep 21;35(3):319-36. Epub 2009 May 21.

PsychoGenics Inc., Tarrytown, NY 10591, USA.

Huntington's 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.
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http://dx.doi.org/10.1016/j.nbd.2009.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728344PMC
September 2009

c-Jun N-terminal kinases mediate reactivation of Akt and cardiomyocyte survival after hypoxic injury in vitro and in vivo.

Circ Res 2006 Jan 23;98(1):111-8. Epub 2005 Nov 23.

Molecular Cardiology, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.

Akt is a central regulator of cardiomyocyte survival after ischemic injury in vitro and in vivo, but the mechanisms regulating Akt activity in the postischemic cardiomyocyte are not known. Furthermore, although much is known about the detrimental role that the c-Jun N-terminal kinases (JNKs) play in promoting death of cells exposed to various stresses, little is known of the molecular mechanisms by which JNK activation can be protective. We report that JNKs are necessary for the reactivation of Akt after ischemic injury. We identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia, apparently by priming Akt for subsequent phosphorylation by 3-phosphoinositide-dependent protein kinase. The reduction in Akt activity that is induced by JNK inhibition may have significant biological consequences, as we find that JNKs, acting via Akt, are critical determinants of survival in posthypoxic cardiomyocytes in culture. Furthermore, in contrast to selective p38-mitogen-activated protein kinase inhibition, which was cardioprotective in vivo, concurrent inhibition of both JNKs and p38-mitogen-activated protein kinases increased ischemia/reperfusion injury in the heart of the intact rat. These studies demonstrate that reactivation of Akt after resolution of hypoxia and ischemia is regulated by JNKs and suggest that this is likely a central mechanism of the myocyte protective effect of JNKs.
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http://dx.doi.org/10.1161/01.RES.0000197781.20524.b9DOI Listing
January 2006