Seeking Huntington disease biomarkers by multimodal, cross-sectional basal ganglia imaging.

Neurodegeneration of the striatum in Huntington disease (HD) is characterized by loss of medium-spiny neurons, huntingtin nuclear inclusions, reactive gliosis, and iron accumulation. Neuroimaging allows in vivo detection of the macro- and micro-structural changes that occur from presymptomatic stages of the disease (preHD). The aim of our study was to evaluate the reliability of multimodal imaging as an in vivo biomarker of vulnerability and development of the disease and to characterize macro- and micro-structural changes in subcortical nuclei in HD. Macrostructure (T1-weighted images), microstructure (diffusion tensor imaging), and iron content (R 2* relaxometry) of subcortical nuclei and medial temporal lobe structures were evaluated by a 3 T scanner in 17 preHD carriers, 12 early-stage patients and 29 matched controls. We observed a volume reduction and microstructural changes in the basal ganglia (caudate, putamen, and globus pallidus) and iron accumulation in the globus pallidus in both preHD and symptomatic subjects; all these features were significantly more pronounced in patients, in whom degeneration extended to the other subcortical nuclei (i.e., thalamus and accumbens). Mean diffusivity (MD) was the most powerful predictor in models explaining more than 50% of the variability in HD development in the caudate, putamen, and thalamus. These findings suggest that the measurement of MD may further enhance the well-known predictive value of striatal volume to assess disease progression as it is highly sensitive to tissue microimpairment. Multimodal imaging may detect brain changes even in preHD stages.

18F-FDG PET uptake in the pre-Huntington disease caudate affects the time-to-onset independently of CAG expansion size.

PURPOSE: To test in a longitudinal follow-up study whether basal glucose metabolism in subjects with a genetic risk of Huntington disease (HD) may influence the onset of manifest symptoms. METHODS: The study group comprised 43 presymptomatic (preHD) subjects carrying the HD mutation. They underwent a (18)F-FDG PET scan and were prospectively followed-up for at least 5 years using the unified HD rating scale to detect clinical changes. Multiple regression analysis included subject's age, CAG mutation size and glucose uptake as variables in a model to predict age at onset. RESULTS: Of the 43 preHD subjects who manifested motor symptoms, suggestive of HD, after 5 years from the PET scan, 26 showed a mean brain glucose uptake below the cut-off of 1.0493 in the caudate, significantly lower than the 17 preHD subjects who remained symptom-free (P < 0.0001). This difference was independent of mutation size. Measurement of brain glucose uptake improved the CAG repeat number and age-based model for predicting age at onset by 37 %. CONCLUSION: A reduced level of glucose metabolism in the brain caudate may represent a predisposing factor that contributes to the age at onset of HD in preHD subjects, in addition to the mutation size.

Truncated peroxisome proliferator-activated receptor-γ coactivator 1α splice variant is severely altered in Huntington's disease.

BACKGROUND: Reduced peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) gene expression has been observed in striatal cell lines, transgenic mouse models of Huntington's disease (HD), and brain tissue from HD patients. As this protein is a key transcription regulator of the expression of many mitochondrial proteins, these observations strongly support the role of aberrant mitochondrial function in the pathogenesis of HD. The PGC1α protein undergoes posttranslational modifications that affect its transcriptional activity. The N-truncated splice variant of PGC1α (NT-PGC1α) is produced in tissues, but the role of truncated splice variants of PGC1α in HD and in the regulation of mitochondrial gene expression has not been elucidated. OBJECTIVE: To examine the expression and modulation of expression of NT-PGC1α levels in HD. METHODS AND RESULTS: We found that the NT-PGC1α protein, a splice variant of ∼38 kDa, but not full-length PGC1α is severely and consistently altered in human HD brain, human HD myoblasts, mouse HD models, and HD striatal cells. NT-PGC1α levels were significantly upregulated in HD cells and mouse brown fat by physiologically relevant stimuli that are known to upregulate PGC1α gene expression. This resulted in an increase in mitochondrial gene expression and cytochrome c content. CONCLUSION: Our data suggest that NT-PGC1α is an important component of the PGC1α transcriptional network, which plays a significant role in the pathogenesis of HD.

Genotype-, aging-dependent abnormal caspase activity in Huntington disease blood cells.

Huntington's Disease (HD) is caused by trinucleotide CAG repeat expansion >36 in huntingtin (htt), a protein with several documented functions. The elongated polyglutamine (polyQ) stretch in the N-terminal region of htt leads to dysfunctional and degenerative events in neurons and peripheral tissues. In this study, by extending the analysis to several caspase activities (i.e. caspase 2, 3, 6, 8 and 9), we describe genotype- and time- dependent caspase activity abnormalities, decreased cell viability and a large set of alterations in mitochondria morphology, in cultured blood cells from HD patients. Patients homozygous for CAG repeat mutations and heterozygous with high size mutations causing juvenile onset (JHD) presented significantly increased caspase 2, 3, 6, 8 and 9 activities, decreased cell viability and pronounced morphological abnormalities, compared with cells carrying low mutation size and controls. After cyanide treatment, all caspases increased their activities in homozygous and highly expanded heterozygous cells, caspase 8 and 9 increased also in those cells carrying low-size mutations, remarking their key role as 'caspase initiators' in HD. The remarkable ageing-dependent abnormalities in peripheral cells carrying particularly toxic mutations (i.e. homozygotes' and JHD's blood cells) points out the potential dependence of clinical HD development and progression on either mutated htt dosage or missing wild type htt. Peripheral tissues (i.e. blood cells) may theoretically represent an important tool for studying HD mechanisms and searching for new biomarkers, according to the patients' genotype.

Early defect of transforming growth factor ß1 formation in Huntington's disease.

A defective expression or activity of neurotrophic factors, such as brain- and glial-derived neurotrophic factors, contributes to neuronal damage in Huntington's disease (HD). Here, we focused on transforming growth factor-ß (TGF-ß(1) ), a pleiotropic cytokine with an established role in mechanisms of neuroprotection. Asymptomatic HD patients showed a reduction in TGF-ß(1) levels in the peripheral blood, which was related to trinucleotide mutation length and glucose hypometabolism in the caudate nucleus. Immunohistochemical analysis in post-mortem brain tissues showed that TGF-ß(1) was reduced in cortical neurons of asymptomatic and symptomatic HD patients. Both YAC128 and R6/2 HD mutant mice showed a reduced expression of TGF-ß(1) in the cerebral cortex, localized in neurons, but not in astrocytes. We examined the pharmacological regulation of TGF-ß(1) formation in asymptomatic R6/2 mice, where blood TGF-ß(1) levels were also reduced. In these R6/2 mice, both the mGlu2/3 metabotropic glutamate receptor agonist, LY379268, and riluzole failed to increase TGF-ß(1) formation in the cerebral cortex and corpus striatum, suggesting that a defect in the regulation of TGF-ß(1) production is associated with HD. Accordingly, reduced TGF-ß(1) mRNA and protein levels were found in cultured astrocytes transfected with mutated exon 1 of the human huntingtin gene, and in striatal knock-in cell lines expressing full-length huntingtin with an expanded glutamine repeat. Taken together, our data suggest that serum TGF-ß(1) levels are potential biomarkers of HD development during the asymptomatic phase of the disease, and raise the possibility that strategies aimed at rescuing TGF-ß(1) levels in the brain may influence the progression of HD.

Abnormal morphology of peripheral cell tissues from patients with Huntington disease.

We investigated the genotype-dependency of morphological abnormalities in peripheral cells from Huntington disease (HD) patients. Cell cultures derived from skin and muscle biopsies showed a different set of abnormalities depending on the genotype (i.e. heterozygous and homozygous for CAG mutations) and the tissue (i.e. fibroblasts and myoblasts). In general, homozygotes' cell lines showed massive ultrastructural damage of specific cell organelles compared with age matched control. These consist of vacuolization, deranged crests and matrix found within giant mitochondria. In addition, enlarged endoplasmic reticulum and the occurrence of numerous autophagic vacuoles, which were similar to those occurring in neurons within affected brain areas, were described. Despite a comparable dose-dependency on mitochondrial changes, this kind of alterations differ in fibroblasts compared with myoblasts. In fact, the internal mitochondrial structure was merely lost in myoblasts, while it shows pathological re-organization within fibroblasts, where altered crests appear as multilamellar circles. These data indicate that ultrastructural abnormalities from peripheral tissues of HD patients can be used as potential disease markers which are easier to get than autoptic brains. Moreover, the occurrence of ultrastructural cell pathology reminiscent of neuronal degeneration in HD, suggests the use of human peripheral cells as a tool to investigate the pathogenic cascade subsequent to huntingtin dysregulation.

Riluzole protects Huntington disease patients from brain glucose hypometabolism and grey matter volume loss and increases production of neurotrophins.

PURPOSE: Huntington disease (HD) mutation increases gain-of-toxic functions contributing to glutamate-mediated excitotoxicity. Riluzole interferes with glutamatergic neurotransmission, thereby reducing excitotoxicity, enhancing neurite formation in damaged motoneurons and increasing serum concentrations of BDNF, a brain cortex neurotrophin protecting striatal neurons from degeneration. METHODS: We investigated metabolic and volumetric differences in distinct brain areas between 11 riluzole-treated and 12 placebo-treated patients by MRI and (18)F-fluoro-2-deoxy-D-glucose (FDG) PET scanning, according to fully automated protocols. We also investigated the influence of riluzole on peripheral growth factor blood levels. RESULTS: Placebo-treated patients showed significantly greater proportional volume loss of grey matter and decrease in metabolic FDG uptake than patients treated with riluzole in all cortical areas (p<0.05). The decreased rate of metabolic FDG uptake correlated with worsening clinical scores in placebo-treated patients, compared to those who were treated with riluzole. The progressive decrease in metabolic FDG uptake observed in the frontal, parietal and occipital cortex correlated linearly with the severity of motor scores calculated by Unified Huntington Disease Rating Scale (UHDRS-I) in placebo-treated patients. Similarly, the rate of metabolic changes in the frontal and temporal areas of the brain cortex correlated linearly with worsening behavioural scores calculated by UHDRS-III in the placebo-treated patients. Finally, BDNF and transforming growth factor beta-1 serum levels were significantly higher in patients treated with riluzole. CONCLUSION: The linear correlation between decreased metabolic FDG uptake and worsening clinical scores in the placebo-treated patients suggests that FDG-PET may be a valuable procedure to assess brain markers of HD.

Rapid emergence of a viral resistant mutant in WHV chronically infected woodchucks treated with lamivudine and a pre-S/S CHO-derived hepatitis B virus vaccine.

To determine whether the addition of a pre-S/S human vaccine increases the antiviral activity of lamivudine, four woodchucks were treated with a daily dose of 100 mg/kg lamivudine and four 50 microg doses of CHO-derived pre-S/S human vaccine. WHV DNA titres decreased up to two logarithms in three woodchucks. At week 4, in three of the animals, the sequence analysis showed a predominant strain containing a nucleotide change from A to T at position 1696 of domain B of the WHV DNA polymerase. Vaccination did not further suppress WHV DNA, despite anti-HBs production in three animals. The woodchuck remains a useful model for characterising the biology and kinetics of the emergence of drug-resistant variants and could be used for pre-clinical studies of combinations of new antiviral drugs.

A pre-S/S CHO-derived hepatitis B virus vaccine protects woodchucks from WHV productive infection.

We evaluated whether a non-adjuvanted vaccine derived from Chinese hamster ovary cells was capable of providing protection against woodchuck hepatitis virus (WHV). Three woodchucks were vaccinated with four 50-microg doses and challenged with a previously characterized virus isolate (WHV197). In all three animals, titre levels of antibodies against hepatitis B surface antigens (anti-HBs) exceeded 10 mIU/ml, peaking at 150 mIU/ml. Challenge resulted in productive acute infection in the two non-vaccinated woodchucks yet in none of the vaccinated woodchucks. In the vaccinated animals, there was evidence of abortive infection. The results demonstrate that a human vaccine is able to protect woodchucks from WHV infection.