Early postnatal behavioral, cellular, and molecular changes in models of Huntington disease are reversible by HDAC inhibition.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene (HTT). Although mutant HTT is expressed during embryonic development and throughout life, clinical HD usually manifests later in adulthood. A number of studies document neurodevelopmental changes associated with mutant HTT, but whether these are reversible under therapy remains unclear. Here, we identify very early behavioral, molecular, and cellular changes in preweaning transgenic HD rats and mice. Reduced ultrasonic vocalization, loss of prepulse inhibition, and increased risk taking are accompanied by disturbances of dopaminergic regulation in vivo, reduced neuronal differentiation capacity in subventricular zone stem/progenitor cells, and impaired neuronal and oligodendrocyte differentiation of mouse embryo-derived neural stem cells in vitro. Interventional treatment of this early phenotype with the histone deacetylase inhibitor (HDACi) LBH589 led to significant improvement in behavioral changes and markers of dopaminergic neurotransmission and complete reversal of aberrant neuronal differentiation in vitro and in vivo. Our data support the notion that neurodevelopmental changes contribute to the prodromal phase of HD and that early, presymptomatic intervention using HDACi may represent a promising novel treatment approach for HD.

Early Alterations in Operant Performance and Prominent Huntingtin Aggregation in a Congenic F344 Rat Line of the Classical CAGn51trunc Model of Huntington Disease.

The transgenic rat model of Huntington disease expressing a fragment of mutant HTT (tgHD rat) has been thoroughly characterized and reproduces hallmark symptoms of human adult-onset HD. Pursuing the optimization of this model for evaluation of translational therapeutic approaches, the F344 inbred rat strain was considered as advantageous genetic background for the expression of the HD transgenic construct. In the present study, a novel congenic line of the SPRDtgHD transgenic model of HD, carrying 51 CAG repeats, was generated on the F344 rat genetic background. To assess the behavioral phenotype, classical assays investigating motor function, emotion, and sensorimotor gating were applied, along with automated screening of metabolic and activity parameters as well as operant conditioning tasks. The neuropathological phenotype was analyzed by immunohistochemistry and ex vivo magnetic resonance imaging. F344tgHD rats displayed markedly reduced anxiety-like behavior in the social interaction test and elevated impulsivity traits already at 3 months of age. Neuropathologically, reduced striatal volume and pronounced aggregation of mutant huntingtin in several brain regions were detected at later disease stage. In conclusion, the congenic F344tgHD model reproduces key aspects of the human HD phenotype, substantiating its value for translational therapeutic approaches.

Automated phenotyping and advanced data mining exemplified in rats transgenic for Huntington's disease.

BACKGROUND: The need for improving throughput, validity, and reliability in the behavioral characterization of rodents may benefit from integrating automated intra-home-cage-screening systems allowing the simultaneous detection of multiple behavioral and physiological parameters in parallel. NEW METHOD: To test this hypothesis, transgenic Huntington's disease (tgHD) rats were repeatedly screened within phenotyping home-cages (PhenoMaster and IntelliCage for rats), where spontaneous activity, feeding, drinking, temperature, and metabolic performance were continuously measured. Cognition and emotionality were evaluated within the same environment by means of operant learning procedures and refined analysis of the behavioral display under conditions of novelty. This investigator-independent approach was further correlated with behavioral display of the animals in classical behavioral assays. Multivariate analysis (MVA) including Principle Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) was used to explore correlation patterns of variables within and across the two genotypes. RESULTS: The automated systems traced previously undetected aspects in the phenotype of tgHD rats (circadian activity, energy metabolism, rearing), and out of those spontaneous free rearing correlated with individual performance in the accelerod test. PCA revealed a segregation by genotype in juvenile tgHD rats that differed from adult animals, being further resolved by PLS-DA detecting "temperature" (juvenile) and "rearing" (adult) as phenotypic key variables in the tgHD model. CONCLUSIONS: Intra-home-cage phenotyping in combination with MVA, is capable of characterizing a complex phenotype by detecting novel physiological and behavioral markers with high sensitivity and standardization using fewer human resources. A broader application of automated systems for large-scale screening is encouraged.

Effects of dipeptidyl peptidase-4 inhibition in an animal model of experimental asthma: a matter of dose, route, and time.

The CD26-associated enzymatic activity of dipeptidyl peptidase-4 (DPP4) as well as the recruitment of CD26(+) T cells increase under allergic airway inflammation. Furthermore, genetic deficiency of CD26/DPP4 exerts protective effects in experimental asthma. Therefore, CD26/DPP4 might represent a novel therapeutic target in asthma. To study the effects of pharmacological inhibition of DPP4 on allergic airway inflammation the DPP4-inhibitor isoleucine thiazolidide was tested using different doses at different time points (at sensitization, immediately before and simultaneously with the allergen challenge, as well as continuously via drinking water), and different routes (intraperitoneal, oral, and by inhalation). Allergic-like airway inflammation was induced in Fischer 344 rats (Charles River) sensitized against ovalbumin (OVA) using OVA aerosols. Intraperitoneal application of the DPP4 inhibitor showed effects neither at sensitization nor at challenge, whereas a continuous application via drinking water using high doses of the inhibitor led to an aggravation of the histomorphological signs of airway inflammation. In contrast, aerosolization of the DPP4 inhibitor simultaneously with the allergen significantly reduced airway hyperresponsiveness and ameliorated histopathological signs compared to controls. In addition, this treatment resulted in increased mRNA levels of surfactant proteins, suggesting an involvement of DPP4 inhibitors in surfactant metabolism in OVA-challenged rats. Continuous systemic inhibition of DPP4 via the oral route aggravates allergic airway inflammation. In contrast, topical inhibition of DPP4 exerts potential protective effects, and further research in humans is needed.

Altered hypothalamic protein expression in a rat model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg) rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP), heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4), glycogen synthase1 (Gys1) and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1). In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.

Identification and characterization of Huntington related pathology: an in vivo DKI imaging study.

An important focus of Huntington Disease (HD) research is the identification of symptom-independent biomarkers of HD neuropathology. There is an urgent need for reproducible, sensitive and specific outcome measures, which can be used to track disease onset as well as progression. Neuroimaging studies, in particular diffusion-based MRI methods, are powerful probes for characterizing the effects of disease and aging on tissue microstructure. We report novel diffusional kurtosis imaging (DKI) findings in aged transgenic HD rats. We demonstrate altered diffusion metrics in the (pre)frontal cerebral cortex, external capsule and striatum. Presence of increased diffusion complexity and restriction in the striatum is confirmed by an increased fiber dispersion in this region. Immunostaining of the same specimens reveals decreased number of microglia in the (pre)frontal cortex, and increased numbers of oligodendrocytes in the striatum. We conclude that DKI allows sensitive and specific characterization of altered tissue integrity in this HD rat model, indicating a promising potential for diagnostic imaging of gray and white matter pathology.

Microstructural changes observed with DKI in a transgenic Huntington rat model: evidence for abnormal neurodevelopment.

Huntington Disease (HD) is a fatal neurodegenerative disorder, caused by a mutation in the Huntington gene. Although HD is most often diagnosed in mid-life, the key to its clinical expression may be found during brain maturation. In the present work, we performed in vivo diffusion kurtosis imaging (DKI) in order to study brain microstructure alterations in developing transgenic HD rat pups. Several developing brain regions, relevant for HD pathology (caudate putamen, cortex, corpus callosum, external capsule and anterior commissure anterior), were examined at postnatal days 15 (P15) and 30 (P30), and DKI results were validated with histology. At P15, we observed higher mean (MD) and radial (RD) diffusivity values in the cortex of transgenic HD rat pups. In addition, at the age of P30, lower axial kurtosis (AK) values in the caudate putamen of transgenic HD pups were found. At the level of the external capsule, higher MD values at P15 but lower MD and AD values at P30 were detected. The observed DKI results have been confirmed by myelin basic protein immunohistochemistry, which revealed a reduced fiber staining as well as less ordered fibers in transgenic HD rat pups. These results indicate that neuronal development in young transgenic HD rat pups occurs differently compared to controls and that the presence of mutant huntingtin has an influence on postnatal brain development. In this context, various diffusivity parameters estimated by the DKI model are a powerful tool to assess changes in tissue microstructure and detect developmental changes in young transgenic HD rat pups.

Selective hippocampal neurodegeneration in transgenic mice expressing small amounts of truncated Aß is induced by pyroglutamate-Aß formation.

Posttranslational amyloid-ß (Aß) modification is considered to play an important role in Alzheimer's disease (AD) etiology. An N-terminally modified Aß species, pyroglutamate-amyloid-ß (pE3-Aß), has been described as a major constituent of Aß deposits specific to human AD but absent in normal aging. Formed via cyclization of truncated Aß species by glutaminyl cyclase (QC; QPCT) and/or its isoenzyme (isoQC; QPCTL), pE3-Aß aggregates rapidly and is known to seed additional Aß aggregation. To directly investigate pE3-Aß toxicity in vivo, we generated and characterized transgenic TBA2.1 and TBA2.2 mice, which express truncated mutant human Aß. Along with a rapidly developing behavioral phenotype, these mice showed progressively accumulating Aß and pE3-Aß deposits in brain regions of neuronal loss, impaired long-term potentiation, microglial activation, and astrocytosis. Illustrating a threshold for pE3-Aß neurotoxicity, this phenotype was not found in heterozygous animals but in homozygous TBA2.1 or double-heterozygous TBA2.1/2.2 animals only. A significant amount of pE3-Aß formation was shown to be QC-dependent, because crossbreeding of TBA2.1 with QC knock-out, but not isoQC knock-out, mice significantly reduced pE3-Aß levels. Hence, lowering the rate of QC-dependent posttranslational pE3-Aß formation can, in turn, lower the amount of neurotoxic Aß species in AD.

Genotype specific age related changes in a transgenic rat model of Huntington's disease.

We aimed to characterize the transgenic Huntington rat model with in vivo imaging and identify sensitive and reliable biomarkers associated with early and progressive disease status. In order to do so, we performed a multimodality (DTI and PET) longitudinal imaging study, during which the same TgHD and wildtype (Wt) rats were repetitively scanned. Surprisingly, the relative ventricle volume was smaller but increased faster in TgHD compared to Wt animals. DTI (mean, axial, radial diffusivity) revealed subtle genotype-specific aging effects in the striatum and its surrounding white matter, already in the presymptomatic stage. Using ¹8F-FDG and ¹8F-Fallypride PET imaging, we were not able to demonstrate genotype-specific aging effects within the striatum. The outcome of this longitudinal study was somewhat surprising as it demonstrated a significant differential aging pattern in TgHD versus Wt animals. Although it seems that the TgHD rat model does not have a sufficient expression of disease yet at the age of 12 months, further validation of this model is highly beneficial since there is still an incomplete understanding of the early disease mechanisms of Huntington's disease.

Stem cell quiescence in the hippocampal neurogenic niche is associated with elevated transforming growth factor-beta signaling in an animal model of Huntington disease.

Cellular proliferation, differentiation, integration, and survival within the adult neural stem cell niche are altered under pathological conditions, but the molecular cues regulating the biology of this niche are mostly unknown. We examined the hippocampal neural stem cell niche in a transgenic rat model of Huntington disease. In this model, progressive cognitive deficits develop at the age of 9 months, suggesting possible hippocampal dysfunction. We found a disease-associated progressive decline in hippocampal progenitor cell proliferation accompanied by an expansion of the pool of 5-bromo-2-deoxyuridine label-retaining Sox-2-positive quiescent stem cells in the transgenic animals. Increments in quiescent stem cells occurred at the expense of cAMP-responsive element-binding protein-mediated neuronal differentiation and survival. Because elevated levels of transforming growth factor-beta1 (TGF-beta1) impair neural progenitor proliferation, we investigated hippocampal TGF-beta signaling and determined that TGF-beta1 induces the neural progenitors to exit the cell cycle. Although phospho-Smad2, an effector of TGF-beta signaling, is normally absent in subgranular stem cells, it accumulated progressively in Sox2/glial fibrillary acidic protein-expressing cells of the subgranular zone in the transgenic rats. These results indicate that alterations in neurogenesis in transgenic Huntington disease rats occur in successive phases that are associated with increasing TGF-beta signaling. Thus, TGF-beta1 signaling seems to be a crucial modulator of neurogenesis in Huntington disease and may represent a target for future therapy.

Dipeptidyl peptidase expression during experimental colitis in mice.

BACKGROUND: We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection. MATERIALS AND METHODS: Wildtype (WT) and DPIV(-/-) mice consumed 2% DSS in drinking water for 6 days to induce colitis. Mice were treated with saline or the DP inhibitors Ile-Pyrr-(2-CN)*TFA or Ile-Thia. DP mRNA and enzyme levels were measured in the colon. Glucagon-like peptide (GLP)-2 and GLP-1 concentrations were determined by radioimmunoassay, regulatory T-cells (Tregs) by fluorescence activated cell sorting (FACS) on FOXp3+T cells in blood, and neutrophil infiltration assessed by myeloperoxidase (MPO) assay. RESULTS: DP8 and DP2 mRNA levels were increased (P < 0.05) in WT+saline mice compared to untreated WT mice with colitis. Cytoplasmic DP enzyme activity was increased (P < 0.05) in DPIV(-/-) mice at day 6 of DSS, while DP2 activity was increased (P < 0.05) in WT mice with colitis. GLP-1 (63%) and GLP-2 (50%) concentrations increased in WT+Ile-Pyrr-(2-CN)*TFA mice compared to day-0 controls. MPO activity was lower in WT+Ile-Thia and WT+Ile-Pyrr-(2-CN)*TFA treated mice compared to WT+saline (P < 0.001) at day 6 colitis. CONCLUSIONS: DP expression and activity are differentially regulated during DSS colitis, suggesting a pathophysiological role for these enzymes in human inflammatory bowel disease (IBD). DP inhibitors impaired neutrophil recruitment and maintenance of the Treg population during DSS-colitis, providing further preclinical evidence for the potential therapeutic use of these inhibitors in IBD. Finally, DPIV appears to play a critical role in mediating the protective effect of DP inhibitors.

Phenotyping of congenic dipeptidyl peptidase 4 (DP4) deficient Dark Agouti (DA) rats suggests involvement of DP4 in neuro-, endocrine, and immune functions.

BACKGROUND: Treatment of diabetes type 2 using chronic pharmacological inhibition of dipeptidyl peptidase 4 (DP4) still requires an in-depth analysis of models for chronic DP4 deficiency, because adverse reactions induced by some DP4 inhibitors have been described. METHODS: In the present study, a novel congenic rat model of DP4 deficiency on a "DP4-high" DA rat genetic background was generated (DA.F344-Dpp4(m)/ SvH rats) and comprehensively phenotyped. RESULTS: Similar to chronic pharmacological inhibition of DP4, DP4 deficient rats exhibited a phenotype involving reduced diet-induced body weight gain and improved glucose tolerance associated with increased levels of glucagon-like peptide-1 (GLP-1) and bound leptin as well as decreased aminotransferases and triglycerides. Additionally, DA.F344-Dpp4(m)/SvH rats showed anxiolytic-like and reduced stress-like responses, a phenomenon presently not targeted by DP4 inhibitors. However, several immune alterations, such as differential leukocyte subset composition at baseline, blunted natural killer cell and T-cell functions, and altered cytokine levels were observed. CONCLUSIONS: While this animal model confirms a critical role of DP4 in GLP-1-dependent glucose regulation, genetically induced chronic DP4 deficiency apparently also affects stress-regulatory and immuneregulatory systems, indicating that the use of chronic DP4 inhibitors might have the potential to interfere with central nervous system and immune functions in vivo.

Sex differences in a transgenic rat model of Huntington's disease: decreased 17beta-estradiol levels correlate with reduced numbers of DARPP32+ neurons in males.

Recent clinical studies have highlighted that female sex hormones represent potential neuroprotective mediators against damage caused by acute and chronic brain diseases. This evidence has been confirmed by experimental studies documenting the protective role of female sex hormones both in vitro and in vivo, although these studies did not specifically focus on Huntington's disease (HD). We therefore investigated the onset and course of HD in female and male transgenic (tg) HD (CAG(n51)) and control rats across age and focused on three aspects: (i) behavioral and physiological alterations (energy expenditure, home-cage activity, emotional disturbance and motor dysfunction), (ii) morphological markers (numbers and characteristics of striatal DARPP32(+) medium-sized spiny neurons (MSNs) and dopamine receptor autoradiography) and (iii) peripheral sex hormone levels as well as striatal estrogen receptor expression. Independent of their sex, tgHD rats exhibited increased levels of food intake, elevated home-cage activity scores and anxiolytic-like behavior, whereas only males showed an impairment of motor function. In line with the latter finding, loss and atrophy of DARPP32(+) MSNs were apparent only in male tgHD rats. This result was associated with a decreased striatal dopamine D1 receptor density and lower plasma levels of 17beta-estradiol at the age of 14 months. As DARPP32(+) MSNs expressed both alpha- and beta-estrogen receptors and showed a correlation between cell numbers and 17beta-estradiol levels, our findings suggest sex-related differences in the HD phenotype pointing to a substantial neuroprotective effect of sex hormones and opening new perspectives on the therapy of HD.

Neuropeptide Y receptor-specifically modulates human neutrophil function.

Despite a continuously growing body of evidence highlighting the role of NPY in the immune system, surprisingly little is known about its ability to alter human leukocyte function. We therefore set out to examine NPY receptor expression and functional effects of NPY in freshly isolated human neutrophils. Our results not only demonstrate for the first time the presence of specific NPY receptors on human neutrophils, but also unveil of how these receptors differentially modulate critical functions of neutrophils such as phagocytosis of bacteria as well as the release of reactive oxygen species.

Postnatal life events affect the severity of asthmatic airway inflammation in the adult rat.

Genetic and hygienic factors influence susceptibility to asthma. In autoimmune and inflammatory diseases, additional effects of the psychosocial environment have been demonstrated that might also play a role in asthma. In this study, the impact of different early postnatal stressors on an OVA-induced model of asthma was tested in adulthood. Fischer 344 rats were subjected to either repeated handling stimulation (HA), maternal separation (MS), or were left undisturbed in their first 4 wk of life. Behavioral differences were characterized at the age of 4 mo. At 5 mo of age, immunological cellular and serologic changes were investigated and experimental asthma was induced. Results show significantly increased exploratory behavior and reduced anxiety in HA rats compared with MS and controls. Without further behavioral or immunological challenges, HA animals exhibited an increased ex vivo NK cell cytotoxicity but no other obvious immunological differences. After induction of asthma, in contrast, MS animals exhibited proinflammatory effects in leukocyte subset composition including increased eosinophil numbers, whereas levels of IgE and the allergy-specific cytokine IL-13 were reduced compared with HA. There was a most remarkable increase of adrenocorticotropin in HA animals, comparing pre- to postchallenge plasma levels. These data demonstrate for the first time that early postnatal stimulative or adverse experiences exert long-lasting changes of the "neuroendocrinoimmune" interface in adulthood, resulting in either protective or aggravating mechanisms in allergic airway disease. Thus, in addition to genetic and hygienic factors, nongenetically acquired individual differences contribute to the pathobiology of asthma.

Regulation of expression and function of dipeptidyl peptidase 4 (DP4), DP8/9, and DP10 in allergic responses of the lung in rats.

The expression of dipeptidyl peptidase 4 (DP4, CD26) affects T-cell recruitment to lungs in an experimental rat asthma model. Furthermore, the gene of the structural homologous DP10 represents a susceptibility locus for asthma in humans, and the functional homologous DP8/9 are expressed in human leukocytes. Thus, although several mechanisms may account for a role of DP4-like peptidases in asthma, detailed information on their anatomical sites of expression and function in lungs is lacking. Therefore, bronchi and lung parenchyma were evaluated using immunohistochemistry and histochemical/enzymatic activity assays, as well as quantitative real-time PCR for this family of peptidases in naïve and asthmatic rat lungs derived from wild-type F344 and DP4-deficient F344 rat strains. Surprisingly, results show not only that the induction of experimental asthma increases DP4 enzymatic activity in the bronchoalveolar lavage fluid and parenchyma, but also that DP8/9 enzymatic activity is regulated and, as well as the expression of DP10, primarily found in the bronchial epithelium of the airways. This is the first report showing a differential and site-specific DP4-like expression and function in the lungs, suggesting a pathophysiologically significant role in asthma.

Selective striatal neuron loss and alterations in behavior correlate with impaired striatal function in Huntington's disease transgenic rats.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by selective striatal neuron loss and motor, cognitive and affective disturbances. The present study aimed to test the hypothesis of adult-onset neuron loss in striatum and frontal cortical layer V as well as alterations in behavior pointing to impaired striatal function in a recently developed transgenic rat model of HD (tgHD rats) exhibiting enlarged ventricles, striatal atrophy and pycnotic pyramidal cells in frontal cortical layer V. High-precision design-based stereological analysis revealed a reduced mean total number of neurons in the striatum but not in frontal cortical layer V of 12-month-old tgHD rats compared with age-matched wild-type controls. No alterations in mean total numbers of striatal neurons were found in 6-month-old animals. Testing 14-month-old animals in a choice reaction time task indicated impaired striatal function of tgHD rats compared with controls.