Le Clemens

A Novel BACHD Transgenic Rat Exhibits Characteristic Neuropathological Features of Huntington Disease

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.

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.

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.

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.

Activation of AMPK-induced autophagy ameliorates Huntington disease pathology in vitro

The expansion of a polyglutamine repeat in huntingtin (HTT) causes Huntington disease (HD). Although the exact pathogenesis is not entirely understood, mutant huntingtin (mHTT) causes disruption of various cellular functions, formation of aggregates and ultimately cell death. The process of autophagy is the main degradation pathway for mHTT, and various studies have demonstrated that the induction of autophagy leads to an amelioration of aggregate formation and an increase in cell viability. Commonly, this is achieved by inhibition of the mammalian target of rapamycin (mTOR), a prominent regulator of cell metabolism. Alternatively, non-canonical AMPK or mTOR-independent autophagy regulation has been recognized. Given mTORs involvement in major cellular pathways besides autophagy, its inhibition may come with potentially detrimental effects. Here, we investigated if AMPK activation may provide a target for the induction of autophagy in an mTOR-independent manner. We demonstrate that activation of AMPK by A769662 and overexpression of a constitutively active form of AMPKα in STHdh cells and mouse embryonic fibroblasts (MEFs), leads to increased expression of the autophagosomal markers LC3 and p62, suggesting efficient autophagy induction. The induction of autophagy was independent of mTOR, and accompanied by a decrease of mHTT-containing aggregates as well as improved cell viability. Therefore, we validated AMPK as a promising therapeutic target to treat HD, and identified A769662 as a potential therapeutic compound to facilitate the clearance of mHTT.

A behavioral comparison of the common laboratory rat strains Lister Hooded, Lewis, Fischer 344 and Wistar in an automated homecage system

Behavioral characterization is an important part of establishing novel animal models, but classical behavioral tests struggle to reveal conclusive results due to problems with both reproducibility and validity. On the contrary, automated homecage observations are believed to produce robust outcomes that relate more to natural animal behavior. However, information on the behavior of background strains from such observations, which could provide important reference material, is rare. For this reason, we compared the behavior of the commonly used Lister Hooded, Lewis, Fischer 344 and Wistar rats during 70 h of exposure to an automated homecage system at 2, 4 and 6 months of age. We found considerable strain differences in metabolic parameters, novelty‐induced and baseline activity‐related behavior as well as differences in the development of these parameters with age. The results are discussed in terms of advantages and disadvantages of the system compared to classical behavioral tests, as well as the systems ability to recreate common findings in literature.

The calpain-suppressing effects of olesoxime in Huntington's disease

ABSTRACT Olesoxime, a small molecule drug candidate, has recently attracted attention due to its significant beneficial effects in models of several neurodegenerative disorders including Huntingtons disease. Olesoximes neuroprotective effects have been assumed to be conveyed through a direct, positive influence on mitochondrial function. In a long-term treatment study in BACHD rats, the latest rat model of Huntingtons disease, olesoxime revealed a positive influence on mitochondrial function and improved specific behavioral and neuropathological phenotypes. Moreover, a novel target of the compound was discovered, as olesoxime was found to suppress the activation of the calpain proteolytic system, a major contributor to the cleavage of the disease-causing mutant huntingtin protein into toxic fragments, and key player in degenerative processes in general. Results from a second model of Huntingtons disease, the HdhQ111 knock-in mouse, confirm olesoximes calpain-suppressing effects and support the therapeutic value of olesoxime for Huntingtons disease and other disorders involving calpain overactivation.

P06 Olesoxime improves specific features of the HD pathology

Background Olesoxime, a cholesterol-oxime, is a neuroprotective compound initially developed for the treatment of ALS (Bordet et al 2007). It interacts with proteins on the outer membrane of mitochondria (OMM) and inhibits the opening of the mitochondrial permeability transition pore (mPTP). Furthermore, it was found to accelerate oligodendrocyte maturation, thereby enhancing myelination (Magalon et al 2012). Since both mitochondrial function and myelination are impaired in HD (Lin & Beal, 2006; Xiang et al 2010), we evaluated the effect of olesoxime on the behavioural and neuropathological phenotype of the BACHD rat. Methods BACHD rats and their wild type littermates were fed ad libitum with either an olesoxime-containing or a control diet beginning at 5 weeks of age (n=15/group). Behavioural observations were carried out during a 12 months study period and neuropathology and mitochondrial function were investigated subsequently (n=4–6/group). Results Olesoxime treatment improved the cognitive and psychiatric phenotype in BACHD rats, which might be associated with a reduced mhtt accumulation found in the prelimbic cortex that is involved in learning and emotionality. It further increased the width of axon bundles in the striatum, which was significantly decreased in BACHD rats compared to wild types, possibly due to an improved myelination. Olesoxime was further capable of restoring mitochondrial respiratory chain function, rescuing a deficit in the expression of OMM proteins and normalise mitochondrial membrane fluidity. Conclusions Olesoxime did not improve motor and metabolic function but it ameliorated the cognitive, psychiatric and mitochondrial pathology in the BACHD rat.

C3 Reduced cell size and enhanced cell proliferation are characteristics of sthdhq111/111 cells and should be considered as possible confounding factors

Background The STHdh cell lines are immortalised striatal precursor cells from wild type and Hdh Q111 knock-in mice and are commonly used to study the molecular aspects of HD. Morphological differences between the wild type and mutant cell lines exist, but are rarely described or clearly considered in published reports. Aims The aim was to characterise cell size and proliferation differences in the STHdh cells, to investigate the importance of these phenotypes for HD and their possible confounding nature. Methods Cell size, cell proliferation, mTOR-related cell signalling and chromosome content were assessed in wild type STHdh Q7/7 and HD mutant STHdh Q111/111 cell lines as well as in primary cultures of mouse embryonic fibroblasts (MEF) established from the same mouse model (MEFHdh Q7/7 and MEFHdh Q111/111 cells). Cell viability and cell death were measured in STHdh cells using standard fluorometric assays and flow cytometry. Results STHdh Q111/111 as well as MEFHdh Q111/111 cells were smaller, showed higher proliferation rates and higher levels of phosphorylated mTOR pathway components compared to their wild type counterpart. Both STHdh cell lines displayed chromosome multiplications already at early passages, although the phenotype was more severe in STHdh Q7/7 cells. No marked chromosome abnormalities were found in the MEF cells. Results from fluorometric cell viability assays indicated that STHdh Q111/111 cells had reduced cell viability and increased cell mortality. This was not supported by the results from flow cytometry, the readouts of which are likely to be unaffected by cell size and proliferation. Conclusions Differences in cell size and proliferation are characteristics of STHdh Q111/111 cells and might be caused by altered mTOR-related signalling. These phenotypes appear to be a general feature of HD, as they are also found in the second cell model. The different degree of genomic instability in wild type and mutant STHdh cells puts the usefulness of the wild type cells as controls in question. Furthermore, cell size and proliferation phenotypes are likely to confound test results and lead to inaccurate conclusions. Thus, our observations suggest that careful experimental design and well-considered data analysis are crucial when using this cell model.

C8 Metabolic properties of hypothalamic primary neuron cultures from bachd rats

Background The BACHD rat overexpresses a bacterial artificial chromosome (BAC) with the full length human mutant huntingtin (mHTT) with 97 alternating CAA/CAG repeats, which are the sole cause of disease onset. BACHD rats display mHTT aggregates and nuclear accumulation of mHTT throughout the brain and develop behavioural and neuropathological- and metabolic abnormalities, growth deficits, mitochondrial dysfunction as well as obesity and a lack of body weight loss that are not typical for human HD. Aims The aim of this study was to investigate different metabolic properties and possible mitochondrial deficits in primary neurons of BACHD rats. Methods BACHD rat pups were dissected at embryonic day 19 and primary neurons of the hypothalamus, striatum and cortex were cultivated and used for following assays: MTT- and LDH-assays and IGF-1 levels for general health and growth. MitoTracker measurements and JC-1-assay to assess the mitochondrial membrane potential YO-PRO-1-Assay for the rate of mitochondrial apoptosis Results Hypothalamic neuron cultures had a lower overall metabolic activity than primary neurons of WT controls. These cells also showed an increased survival rate compared to control cells. The same effects were observed in the striatum, whereas cells from the cortex showed no significant reduction of the metabolic activity, but a decreased survival rate. Since the experiments are still onging at the date of the abstract submission no further results can be shown. Conclusion Our preliminary data and upcoming results will establish BACHD primary neurons as a valuable tool for the investigation of metabolic dysfunction in HD.