While there has been a guideline for laboratory/genetic diagnosis of Huntington Disease (HD) since 1998, no such statement exists for the diagnosis of clinical HD. Informally, the most frequently used criteria for diagnosis of clinical HD is ‘Motor 4’ within the Unified Huntington Disease Rating Scale ’99 (motor), made when the rater is highly confident that ‘motor abnormalities observed are unequivocal signs of HD’. Recent studies involving pre-manifest individuals illustrated the shortcomings of this motor-only diagnostic approach. For instance, PREDICT-HD found cognitive changes decades before the expected date of motor diagnosis. Using a number of case studies, we highlight some of the subtleties involved in diagnosing clinical HD, in the absence of unequivocal motor signs for HD. New, broader, criteria for the diagnosis of clinical HD would be helpful in many ways. However its formulation will need to flexible rather than prescriptive, and will require extensive consultation with clinicians and families with HD.
Metabolic dysfunction and mitochondrial involvement are recognised as part of the pathology in Huntington’s Disease (HD). Post-mortem examinations of the striatum from end-stage HD patients have shown a decrease in the in vitro activity of complexes II, III and IV of the electron transport system (ETS). In different models of HD, evidence of enzyme defects have been reported in complex II and complex IV using enzyme assays. However, such assays are highly variable and results have been inconsistent.
We investigated the integrated ETS function ex vivo using a sensitive high-resolution respirometric (HRR) method. The O2 flux in a whole-cell sample combined with the addition of mitochondrial substrates, uncouplers and inhibitors enabled us to accurately quantitate the function of individual mitochondrial complexes in intact mitochondria, while retaining mitochondrial regulation and compensatory mechanisms.
We used HRR to examine the mitochondrial function in striata from 12-week old R6/2 mice expressing exon 1 of human HTT with 130 CAG repeats. A significant reduction in complex II and complex IV flux control ratios was found in the R6/2 mouse striatum at 12 weeks of age compared to controls, confirming previous findings obtained with spectrophotometric enzyme assays.
This report represents a detailed description of experiments designed to replicate and extend the findings of a published study on the effects of treating the R6/2 Huntington’s disease (HD) mouse model with ~300 CAG repeats using the pimelic diphenylamide histone deacetylase (HDAC) inhibitor, HDACi 4b (Thomas et al., 2008). In addition to testing the R6/2 mice, similar experiments examined the effects of the drug on a second transgenic HD mouse model, the N171-82Q mice. As in the original study, the drug was delivered in the drinking water. In the present study we tested larger groups of mice than in the original study. The results indicated that we were unable to replicate the significant behavioral effects of oral HDACi 4b treatment in the R6/2 mice. There were however, non-significant trends for the treated R6/2 mice to be less affected on some of the measures and there were instances of phenotype progression being delayed in these treated mice. In contrast, we did replicate the protection from striatal atrophy in the R6/2 mice. We also did not observe any beneficial effects of HDACi 4b treatment in the N171-82Q mice. Although the behavioral procedures were replicated and an automated activity assessment was added, there were several unexpected complications in terms of solubility of the drug, CAG repeat length differences and gender differences in progression of the phenotype that could have affected outcomes. Clearly more studies will have to be performed using other methods of delivery as well as assessing effects in more slowly progressing HD models to better evaluate the effects of this HDAC inhibitor.
Background
Abnormalities in brain cholesterol homeostasis have been reported in Huntington’s disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein.
Results
Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol.
Conclusions
Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner.
*Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it
Juvenile Huntington’s disease (JHD) is usually defined as Huntington’s disease with an onset ≤ 20 years. The proportion of JHD cases reported in studies of Huntington’s disease (HD) varies. A review of the literature found 62 studies that reported the proportion of JHD cases amongst all HD cases. The proportion of JHD cases in these studies ranged from 1% to 15%, and in a meta-analysis the pooled proportion of JHD cases was 4.92% (95% confidence interval of 4.07% to 5.84%). Limiting the analysis to the 25 studies which used multiple methods of ascertainment resulted in a similar pooled proportion of 5.32%, (95% confidence interval 4.18% to 6.60%).
A small difference was observed when the meta-analysis was restricted to studies from countries defined by the World Bank as high income, that used multiple methods of ascertainment, and that were conducted since 1980 (4.81%, 95% confidence interval 3.31% to 6.58%, n=11). This contrasts with the pooled result from three post 1980 studies using multiple methods of ascertainment from South Africa and Venezuela, defined by the World Bank as upper middle income, where the estimated mean proportion was 9.95%, (95% confidence interval 6.37% to 14.22%).
These results, which are expected to be more robust than those from a single study alone, may be helpful in estimating the proportion of JHD cases in a given population.
Key Words: Juvenile Huntington’s disease, prevalence, epidemiology
ABSTRACT Huntington’s disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown [...]
Stem cell-based treatment for Huntington’s disease (HD) is an expanding field of research. Although various stem cells have been shown to be beneficial in vivo, no long standing clinical effect has been demonstrated. To address this issue, we are developing a stem cell-based therapy designed to improve the microenvironment of the diseased tissue via delivery of neurotrophic factors (NTFs). Previously, we established that bone marrow derived human mesenchymal stem cells (MSCs) can be differentiated using medium based cues into NTF-secreting cells (NTF+ cells) that express astrocytic markers. NTF+ cells were shown to alleviate neurodegeneration symptoms in several disease models in vitro and in vivo, including the model for excitotoxicity.
In the present study, we explored if the timing of intrastriatal transplantation of hNTF+ cells into the R6/2 transgenic mouse model for HD influences motor function and survival. One hundred thousand cells were transplanted bilaterally into the striatum of immune-suppressed mice at 4.5, 5.5 and 6.5 weeks of age.
Contrary to our expectations, early transplantation of NTF+ cells did not improve motor function or overall survival. However, late (6.5 weeks) transplantation resulted in a temporary improvement in motor function and an extension of life span relative to that observed for PBS treated mice.
We conclude that late transplantation of NTF+ cells induces a beneficial effect in this transgenic model for HD. Since no transplanted NTF+ cells could be detected in vivo, we suspect that the temporary nature of the beneficial effect is due to poor survival of transplanted cells. In general, we submit that NTF+ cells should be further evaluated for the therapy of HD.
The physiological role of huntingtin and the pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin changes its normal localization and produces cytoplasmic and intranuclear inclusions, changes gene transcription, alters synaptic transmission, impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules, promotes excitotoxicity, energy deficits, synthesis and release reduction of neurotrophic factors and oxidative stress. Previous studies confirm that the mutant huntingtin difficult neurotrophic function of astrocytes leading to neuronal dysfunction in Huntington’s disease. Our objective was to study the neuroprotective potential role of glia-conditioned medium (GCM) in an in vitro model of Huntington’s disease. We used conditionally-immortalized striatal neuronal progenitor cell lines (STHdhQ7/Q7 and STHdhQ111/Q111) expressing endogenous levels of normal and mutant huntingtin with 7 and 111 glutamines, respectively. We studied the protection of fetal and postnatal glia conditioned medium (GCM) on H2O2 (2 µM), glutamate (5 mM) and 3-nitropropionic acid (2.5 mM) related toxicity. We also compared the neuroprotective effects of GCM versus that of the growth factors bFGF, BDNF and GDNF.
Fetal GCM protects from every toxin, reducing the cell death and increasing the cell survival. Fetal GCM reduces the caspases fragmentation of the protein PARP, the expression of chaperone Hsp70 and the accumulation of ROS and polyubiquitinated proteins. In addition, in Q111 striatal cells treated with H2O2 (2 µM) for 24 hours, the intracellular GSH levels are higher in the presence of GCM. Notably, the 13-day and 2-month postnatal GCM, totally protects from H2O2 induced cell death in mutant striatal cells. GCM neuroprotective effects are more potent than those of the already identified neurotrophic factors.
We conclude that GCM protects Q111 cells from neuronal neurotoxins and the effects of GCM are more potent than those of any known neurotrophic factor. GCM may contain new and more potent, as yet unidentified, neurotrophic molecules, potentially useful in patients with Huntington’s disease.
Apathy, characterized by generally reduced interest in and likelihood to perform goal-directed actions, is a recognized symptom of Huntington’s disease (HD), a devastating neurological disorder caused by a CAG repeat expansion of the Htt gene located on chromosome 4. The present experiments used a modified progressive ratio task that incorporated a fixed-ratio schedule of reinforcement component to assess consummatory behavior, and a progressive-ratio schedule component that required increasing numbers of lever-presses for successive reinforcers (0.01 ml of evaporated milk). The studies revealed an apathetic phenotype in two mouse models of HD, with decreased response rates either overall or only at higher ratio requirements in the progressive-ratio component relative to wild-type controls. Based on the procedure used (within-session fixed- and progressive-ratio components), it is proposed that an observed phenotype can be ascribed either specifically to reduced motivation to work for food reinforcement or more generally to deficits in consummatory behavior. This procedure provides a simple means to assess this type of phenotype in rodents, with issues in consummatory vs. incentive motivation reflected in general alterations in fixed- versus progressive alterations on an escalating-ratio schedules respectively, providing translational measures of the amotivation/apathy construct of the human realm to the homologous construct of incentive motivation in preclinical models of human disease.
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 (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.