Young human alpha synuclein transgenic (BAC-SNCA) mice display sex- and gene-dose-dependent phenotypic disturbances.

Parkinson's disease (PD) is a common neurodegenerative movement disorder, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of aggregated alpha synuclein (aSyn). The disease often presents with early prodromal non-motor symptoms and later motor symptoms. Diagnosing PD based purely on motor symptoms is often too late for successful intervention, as a significant neuronal loss has already occurred. Furthermore, the lower prevalence of PD in females is not well understood, highlighting the need for a better understanding of the interaction between sex and aSyn, the crucial protein for PD pathogenesis. Here, we conducted a comprehensive phenotyping study in 1- to 5-month-old mice overexpressing human aSyn gene (SNCA) in a bacterial artificial chromosome (BAC-SNCA). We demonstrate a SNCA gene-dose-dependent increase of human aSyn and phosphorylated aSyn, as well as a decrease in tyrosine hydroxylase expression in BAC-SNCA mice, with more pronounced effects in male mice. Phosphorylated aSyn was already found in the dorsal motor nucleus of the vagus nerve of 2-month-old mice. This was time-wise associated with significant gait altrations in BAC-SNCA mice as early as 1 and 3 months of age using CatWalk gait analysis. Furthermore, anxiety-related behavioral tests revealed an increase in anxiety levels in male BAC-SNCA mice. Finally, 5-month-old male BAC-SNCA mice exhibited a SNCA gene-dose-dependent elevation in energy expenditure in automated home-cage monitoring. For the first time, these findings describe early-onset, sex- and gene-dose-dependent, aSyn-mediated disturbances in BAC-SNCA mice, providing a model for sex-differences, early-onset neuropathology, and prodromal symptoms of PD.

Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic rodent models, with a focus on analyzing the involvement of TG6 in the age- and genotype-specific pathological features relating to disease progression in HD transgenic mice and a tgHD transgenic rat model using biochemical, histological and functional assays. Our results demonstrate the physical interaction between TG6 and (mutant) huntingtin by co-immunoprecipitation analysis and the contribution of its enzymatic activity for the total aggregate load in SH-SY5Y cells. In addition, we identify that TG6 expression and activity are especially abundant in the olfactory tubercle and piriform cortex, the regions displaying the highest amount of mHTT aggregates in transgenic rodent models of HD. Furthermore, mHTT aggregates were colocalized within TG6-positive cells. These findings point towards a role of TG6 in disease pathogenesis via mHTT aggregate formation.

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.

In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression.

Natural hidden autoantibodies to tissue transglutaminase cross-react with fibrinogen.

INTRODUCTION: Patients with celiac disease display autoantibodies against tissue transglutaminase (TG2), and the high sensitivity and specificity of these autoantibodies render them a reliable tool for diagnosis. However, we found that denatured sera from healthy persons also showed reactivity against TG2. METHODS: To further examine the specificity of this phenomenon, sera of healthy individuals and celiac patients were denatured by heat or pH shift. RESULTS AND DISCUSSION: Denatured sera of all individuals showed autoantibodies against TG2 in ELISA that could be specifically inhibited by TG2, but the biological role of these autoantibodies remains unknown. The alpha fibrinogen precursor could be isolated as serum protein that reacts with TG2 antibodies and treated sera reacted with fibrinogen in Western blotting. Cross-reactivity of TG2 antibodies with fibrinogen and vice versa was observed. CONCLUSION: We hypothesise that denaturation of sera reveals hidden autoantibodies against TG2, which might be normally masked by fibrinogen.

In vitro differentiation of human monocytes into dendritic cells by peptic-tryptic digest of gliadin is independent of genetic predisposition and the presence of celiac disease.

INTRODUCTION: This study was done to further reveal the role of the innate immune system in celiac disease. METHODS: Dendritic cells were matured from venous blood of patients with active or treated celiac disease and DQ2-DQ8-positive or negative controls. Dendritic cells were treated with a peptic-tryptic digest of gliadin (500 microg/ml) and their activation was analyzed by fluorescent-activated cell sorting analysis, cytokine secretion, and their ability to elicit T cell proliferation. RESULTS AND DISCUSSION: Gliadin upregulated interleukin (IL)-6, IL-8, and IL-12 (p40) secretion in dendritic cells and induced strong expression of the maturation markers human leukocyte antigen (HLA)-DR, CD25, CD83, and CD86 of all subjects irrespective of their genotype or the presence of disease, whereas the digest of bovine serum albumin showed no effect. However, gliadin-stimulated dendritic cells from active celiac showed enhanced stimulation of autologous T cells compared to the other groups. CONCLUSION: Further research should be aimed at identifying the mechanisms that control inflammation in healthy individuals.

Halofuginone induces matrix metalloproteinases in rat hepatic stellate cells via activation of p38 and NFkappaB.

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB(NFkappaB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkappaB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkappaB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.

VEGF induces proliferation, migration, and TGF-beta1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-beta1 mRNA, enhanced TGF-beta1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-beta1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.

Hepatitis C virus-replicating hepatocytes induce fibrogenic activation of hepatic stellate cells.

BACKGROUND & AIMS: The mechanism by which hepatitis C virus induces liver fibrosis remains largely obscure. To characterize the profibrogenic potential of hepatitis C virus, we used the hepatitis C virus replicon cell line Huh-7 5-15, which stably expresses the nonstructural hepatitis C virus genes NS3 through NS5B, and hepatic stellate cells as fibrogenic effector cells. METHODS: Rat and human hepatic stellate cells were incubated with conditioned media from replicon cells, and expression of fibrosis-related genes was quantified by using real-time polymerase chain reaction, protein, and functional assays. Transforming growth factor beta1 activity was determined by bioassay. RESULTS: Hepatitis C virus replicon cells release factors that differentially modulate hepatic stellate cell expression of key genes involved in liver fibrosis in a clearly profibrogenic way, up-regulating procollagen alpha1(I) and procollagen alpha1(III) and down-regulating fibrolytic matrix metalloproteinases. Transforming growth factor beta1 expression and bioactivity were increased severalfold in hepatitis C virus-replicating vs mock-transfected hepatoma cells. However, transforming growth factor beta1 activity was responsible for only 50% of the profibrogenic activity. CONCLUSIONS: Hepatitis C virus nonstructural genes induce an increased expression of transforming growth factor beta1 and other profibrogenic factors in infected hepatocytes. The direct induction of profibrogenic mediators by hepatitis C virus in infected hepatocytes explains the frequent observation of progressive liver fibrosis despite a low level of inflammation and suggests novel targets for antifibrotic therapies in chronic hepatitis C.