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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 1H-15N 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.
•Antioxidant monotherapies failed in neurodegenerative diseases.•Activation of NRF2 mediated endogenous antioxidant response is a viable alternative.•KEAP1 reversible binding is a sound strategy to activate NRF2.•Informative and robust biochemical methods are needed to discover new KEAP1 binders.•A compendium of HTS, kinetics and structural assays for this purpose is described.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
•Age and sex impact on NOS activity in corticostriatal circuits in HD mouse models.•Deficits in NOS-1 activity/expression appear first in striatum in aged Q175 mice.•Age-related deficits in NOS-1 activity are more pronounced in male Q175 mice.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract 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 HdhQ111 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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
Huntington’s disease (HD) results from an expansion mutation in the polyglutamine tract in huntingtin. Although huntingtin is ubiquitously expressed in the body, the striatum suffers the most severe ...pathology. Rhes is a Ras-related small GTP-binding protein highly expressed in the striatum that has been reported to modulate mTOR and sumoylation of mutant huntingtin to alter HD mouse model pathogenesis. Reports have varied on whether Rhes reduction is desirable for HD. Here we characterize multiple behavioral and molecular endpoints in the Q175 HD mouse model with genetic Rhes knockout (KO). Genetic RhesKO in the Q175 female mouse resulted in both subtle attenuation of Q175 phenotypic features, and detrimental effects on other kinematic features. The Q175 females exhibited measurable pathogenic deficits, as measured by MRI, MRS and DARPP32, however, RhesKO had no effect on these readouts. Additionally, RhesKO in Q175 mixed gender mice deficits did not affect mTOR signaling, autophagy or mutant huntingtin levels. We conclude that global RhesKO does not substantially ameliorate or exacerbate HD mouse phenotypes in Q175 mice.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
