Huu P. Nguyen

Neurodegeneration and Motor Dysfunction in a Conditional Model of Parkinson's Disease

α-Synuclein (α-syn) has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinsons disease. These disorders are characterized by various neurological and psychiatric symptoms based on progressive neuropathological alterations. Whether the neurodegenerative process might be halted or even reversed is presently unknown. Therefore, conditional mouse models are powerful tools to analyze the relationship between transgene expression and progression of the disease. To explore whether α-syn solely originates and further incites these alterations, we generated conditional mouse models by using the tet-regulatable system. Mice expressing high levels of human wild-type α-syn in midbrain and forebrain regions developed nigral and hippocampal neuropathology, including reduced neurogenesis and neurodegeneration in absence of fibrillary inclusions, leading to cognitive impairment and progressive motor decline. Turning off transgene expression in symptomatic mice halted progression but did not reverse the symptoms. Thus, our data suggest that approaches targeting α-syn-induced pathological pathways might be of benefit rather in early disease stages. Furthermore, α-syn-associated cytotoxicity is independent of filamentous inclusion body formation in our conditional mouse model.

Impaired Regulation of Brain Mitochondria by Extramitochondrial Ca2+ in Transgenic Huntington Disease Rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt51Q), modeling the adult form of HD. batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{Ca}_{mathrm{free}}^{2+}) end{document} up to 2 μm activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{Ca}_{mathrm{free}}^{2+}) end{document} above 2 μm inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not affect the Ca2+-dependent activation of respiration but reduced Ca2+-induced inhibition. Thus, Ca2+ activation was mediated exclusively by extramitochondrial Ca2+, whereas inhibition was promoted also by intramitochondrial Ca2+. In contrast, htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca2+. In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca2+. We suggest that specific regulatory Ca2+ binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt51Q and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.

1H NMR based metabolomics of CSF and blood serum: A metabolic profile for a transgenic rat model of Huntington disease

Huntington disease (HD) is a hereditary brain disease. Although the causative gene has been found, the exact mechanisms of the pathogenesis are still unknown. Recent investigations point to metabolic and energetic dysfunctions in HD neurons. Both univariate and multivariate analyses were used to compare proton nuclear magnetic resonance spectra of serum and cerebrospinal fluid (CSF) taken from presymptomatic HD transgenic rats and their wild-type littermates. N-acetylaspartate (NAA), was found to be significantly decreased in the serum of HD rats compared to wild-type littermates. Moreover, in the serum their levels of glutamine, succinic acid, glucose and lactate are significantly increased as well. An increased concentration of lactate and glucose is also found in CSF. There is a 1:1 stoichiometry coupling glucose utilization and glutamate cycling. The observed increase in the glutamine concentration, which indicates a shutdown in the neuronal-glial glutamate-glutamine cycling, results therefore in an increased glucose concentration. The elevated succinic acid concentration might be due to an inhibition of succinate dehydrogenase, an enzyme linked to the mitochondrial respiratory chain and TCA cycle. Moreover, reduced levels of NAA may reflect an impairment of mitochondrial energy production. In addition, the observed difference in lactate supports a deficiency of oxidative energy metabolism in rats transgenic for HD as well. The observed metabolic alterations seem to be more profound in serum than in CSF in presymptomatic rats. All findings suggest that even in presymptomatic rats, a defect in energy metabolism is already apparent. These results support the hypothesis of mitochondrial energy dysfunction in HD.

Mitochondrial Membrane Fluidity is Consistently Increased in Different Models of Huntington Disease: Restorative Effects of Olesoxime

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT). One prominent target of the mutant huntingtin protein (mhtt) is the mitochondrion, affecting its morphology, distribution, and function. Thus, mitochondria have been suggested as potential therapeutic targets for the treatment of HD. Olesoxime, a cholesterol-like compound, promotes motor neuron survival and neurite outgrowth in vitro, and its effects are presumed to occur via a direct interaction with mitochondrial membranes (MMs). We examined the properties of MMs isolated from cell and animal models of HD as well as the effects of olesoxime on MM fluidity and cholesterol levels. MMs isolated from brains of aged Hdh Q111/Q111 knock-in mice showed a significant decrease in 1,6-diphenyl-hexatriene (DPH) anisotropy, which is inversely correlated with membrane fluidity. Similar increases in MM fluidity were observed in striatal STHdh Q111/Q111 cells as well as in MMs isolated from brains of BACHD transgenic rats. Treatment of STHdh cells with olesoxime decreased the fluidity of isolated MMs. Decreased membrane fluidity was also measured in olesoxime-treated MMs isolated from brains of HD knock-in mice. In both models, treatment with olesoxime restored HD-specific changes in MMs. Accordingly, olesoxime significantly counteracted the mhtt-induced increase in MM fluidity of MMs isolated from brains of BACHD rats after 12xa0months of treatment in vivo, possibly by enhancing MM cholesterol levels. Thus, olesoxime may represent a novel pharmacological tool to treat mitochondrial dysfunction in HD.

Olfactory neuron-specific expression of A30P alpha-synuclein exacerbates dopamine deficiency and hyperactivity in a novel conditional model of early Parkinson's disease stages.

Mutations in the N-terminus of the gene encoding α-synuclein (α-syn) are linked to autosomal dominantly inherited Parkinsons disease (PD). The vast majority of PD patients develop neuropsychiatric symptoms preceding motor impairments. During this premotor stage, synucleinopathy is first detectable in the olfactory bulb (OB) and brain stem nuclei; however its impact on interconnected brain regions and related symptoms is still less far understood. Using a novel conditional transgenic mouse model, displaying region-specific expression of human mutant α-syn, we evaluated effect and reversibility of olfactory synucleinopathy. Our data showed that induction of mutant A30P α-syn expression increased transgenic deposition into somatodendritic compartment of dopaminergic neurons, without generating fibrillar inclusions. We found reversibly reduced levels of dopamine and metabolites in the OB, suggesting an impact of A30P α-syn on olfactory neurotransmitter content. We further showed that mutant A30P expression led to neurodegenerative changes on an ultrastructural level and a behaviorally hyperactive response correlated with novelty, odor processing and stress associated with an increased dopaminergic tone in midbrain regions. Our present data indicate that mutant (A30P) α-syn is directly implicated in reduction of dopamine signaling in OB interneurons, which mediates further alterations in brain regions without transgenic expression leading functionally to a hyperactive response. These modulations of neurotransmission may underlie in part some of the early neuropsychiatric symptoms in PD preceding dysfunction of the nigrostriatal dopaminergic system.

Olesoxime suppresses calpain activation and mutant huntingtin fragmentation in the BACHD rat.

Huntingtons disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntingtons disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntingtons disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoximes effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntingtons disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntingtons disease.

A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.

C05 BRCA mutations are associated with higher CAG numbers found in various polyglutamine disorders

Background The CAG repeat expansion in the HTT gene is a dynamic mutation varying in length in tissues and between generations. The mechanisms behind the instability of CAG expansions are polymerase slippage and DNA repair failure. BRCA1 and BRCA2 are well characterized DNA repair genes and mutations increase the risk of developing breast and ovarian cancer for carriers. Aim Therefore, in our study we analysed CAG lengths in various polyglutamine containing genes to investigate the potential effect of mutations in the DNA repair genes BRCA1 and BRCA2 on CAG length. Methods In a cohort of patients from Department of Obstetrics and Gynecology in Tuebingen carrying BRCA1 or BRCA2 mutations, CAG lengths in 6 different polyglutamine disease genes (HD, SCA1, SCA2, SCA3, SCA6 and SCA7) were determined by fragment length analysis. CAG lengths were classified as non-pathological, intermediate and pathological for each disorder. The findings were compared to data on the general population and to a control group from the Department of Obstetrics and Gynecology in Tuebingen. Results We have found no difference in the overall CAG repeat length in patients carrying BRCA1 or BRCA2 mutations. However, the frequency of patients carrying intermediate CAG lengths was higher in patients carrying BRCA1 and BRCA2 mutations in comparison to the general population. Conclusion The frequency of intermediate alleles was in our cohort higher than reported for the general population. They further link BRCA mutations to defects in DNA repair possibly contributing to the expansion of the CAG repeats with a mechanism common to different polyglutamine disorders. Further studies are needed in order to evaluate the correlation of specific mutations with CAG size and CAG size with disease prognosis of BRCA1 or BRCA2 mutation carriers.

The Alteration of Emotion Regulation Precedes the Deficits in Interval Timing in the BACHD Rat Model for Huntington Disease

Huntington disease (HD) is an autosomal dominantly inherited, progressive neurodegenerative disorder which is accompanied by executive dysfunctions and emotional alteration. The aim of the present study was to assess the impact of emotion/stress on on-going highly demanding cognitive tasks, i.e., temporal processing, as a function of age in BACHD rats (a “full length” model of HD). Middle-aged (4–6 months) and old (10–12 months) rats were first trained on a 2 vs. 8-s temporal discrimination task, and then exposed to a series of bisection tests under normal and stressful (10 mild unpredictable foot-shocks) conditions. The animals were then trained on a peak interval task, in which reinforced fixed-interval (FI) 30-s trials were randomly intermixed with non-reinforced probe trials. After training, the effect of stress upon time perception was again assessed. Sensitivity to foot-shocks was also assessed independently. The results show effects of both age and genotype, with largely greater effects in old BACHD animals. The older BACHD animals had impaired learning in both tasks, but reached equivalent levels of performance as WT animals at the end of training in the temporal discrimination task, while remaining impaired in the peak interval task. Whereas sensitivity to foot-shock did not differ between BACHD and WT rats, delivery of foot-shocks during the test sessions had a disruptive impact on temporal behavior in WT animals, an effect which increased with age. In contrast, BACHD rats, independent of age, did not show any significant disruption under stress. In conclusion, BACHD rats showed a disruption in temporal learning in late symptomatic animals. Age-related modification in stress-induced impairment of temporal control of behavior was also observed, an effect which was greatly reduced in BACHD animals, thus confirming previous results suggesting reduced emotional reactivity in HD animals. The results suggest a staggered onset in cognitive and emotional alterations in HD, with emotional alteration being the earliest, possibly related to different time courses of degeneration in cortico-striatal and amygdala circuits.

Reduced cell size, chromosomal aberration and altered proliferation rates are characteristics and confounding factors in the ST Hdh cell model of Huntington disease

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells’ tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.