Hepatic and neuronal phenotype of NPC1−/− mice

Niemann-Pick type C disease (NPC) is a fatal autosomal recessive disorder characterized by a defect in the intracellular transport of lipoproteins leading to the accumulation of lipids in diverse tissues. A visceral and neuronal phenotype mimicking human NPC1 disease has been described in NPC1 mutant mice. These mice are by now the most widely used NPC1 rodent model to study NPC and developmental compounds against this devastating disease. Here we characterized NPC1−/− mice for their hepatic and neuronal phenotype to confirm the stability of the phenotype, provide a characterization of disease progression and pinpoint the age of robust phenotype onset. Animals of 4–10 weeks of age were analyzed for general health, motor deficits as well as hepatic and neuronal alterations with a special focus on cerebellar pathology.Our results show that NPC1−/− mice have a reduced general health at the age of 9–10 weeks. Robust motor deficits can be observed even earlier at 8 weeks of age. Hepatic changes included increased organ weight and cholesterol levels at 6 weeks of age accompanied by severely increased liver enzyme levels. Analysis of NPC1−/− brain pathology showed decreased cholesterol and increased Aβ levels in the hippocampus at the age of 6 weeks. Further analysis revealed a decrease of the cytokine IL-12p70 in the cerebellum along with a very early increase of astrocytosis. Hippocampal IL-12p70 levels were increased at the age of 6 weeks followed by increased activated microglia levels. By the age of 10 weeks, also cerebellar Aβ levels were increased along with strongly reduced Calbindin D-28k levels.Our results validate and summarize the progressive development of the hepatic and neuronal phenotype of NPC1−/− mice that starts with cerebellar astrocytosis, making this mouse model a valuable tool for the development of new compounds against NPC

Administration of oral methylphenidate during adolescence prevents suppressive development of dopamine projections into prefrontal cortex and amygdala after an early pharmacological challenge in gerbils

Grund T, Teuchert-Noodt G, Busche A, et al. Administration of oral methylphenidate during adolescence prevents suppressive development of dopamine projections into prefrontal cortex and amygdala after an early pharmacological challenge in gerbils. BRAIN RESEARCH. 2007;1176:124-132.The enduring effects of postweaning subchronic methylphenidate (MP) treatment and/or previous early preweaning methamphetamine (MA) application on dopamine (DA) fiber density were investigated in multiple cortical and subcortical areas of the gerbil brain. The study aimed to explore three questions: (1) is the development of DA fiber innervation in control animals sensitive to a clinically relevant subchronic treatment with MP? (2) Is the development of DA fiber innervation in the forebrain altered by a single early MA challenge? (3) if so, might the subsequent institution of a therapeutically relevant MP application scheme interfere with such early induced alternative developmental trajectories for DA fiber innervation? For this purpose, gerbils pretreated both with saline and MA (50 mg/kg, i. p.) on day 14 received either H2O or MP (5 mg/kg) orally on days 30 to 60. On day 90, DA fibers were immunohistochemically detected and quantified. As a result, MP on its own did not have any significant influence on the postnatal development of the DA fiber systems, whereas it prevented a previously MA triggered suppressive development of DA fiber innervation in the prefrontal cortex and amygdala complex (30% less fiber innervation in both areas). Thus, MP prevented previously initiated miswiring of DA fibers from actually being implemented in the gerbil forebrain. During earlier studies, rather complex miswiring has been documented in response to an early preweaning MA challenge. This miswiring was associated with functional deficits resembling some of the symptoms of patients with ADHD. Therefore, morphogenetic properties of MP need further attention. (C) 2007 Elsevier B.V. All rights reserved

Correlation of pyroglutamate amyloid β and ptau Ser202/Thr205 levels in Alzheimer's disease and related murine models.

Senile plaques frequently contain Aβ-pE(3), a N-terminally truncated Aβ species that is more closely linked to AD compared to other Aβ species. Tau protein is highly phosphorylated at several residues in AD, and specifically phosphorylation at Ser202/Thr205 is known to be increased in AD. Several studies suggest that formation of plaques and tau phosphorylation might be linked to each other. To evaluate if Aβ-pE(3) and ptau Ser202/Thr205 levels correlate in human and transgenic AD mouse models, we analyzed human cortical and hippocampal brain tissue of different Braak stages as well as murine brain tissue of two transgenic mouse models for levels of Aβ-pE(3) and ptau Ser202/Thr205 and correlated the data. Our results show that Aβ-pE(3) formation is increased at early Braak stages while ptau Ser202/Thr205 mostly increases at later stages. Further analyses revealed strongest correlations between the two pathologies in the temporal, frontal, cingulate, and occipital cortex, however correlation in the hippocampus was weaker. Evaluation of murine transgenic brain tissue demonstrated a slow but steady increase of Aβ-pE(3) from 6 to 12 months of age in the cortex and hippocampus of APPSL mice, and a very early and strong Aβ-pE(3) increase in 5xFAD mice. ptau Ser202/Thr205 levels increased at the age of 9 months in APPSL mice and at 6 months in 5xFAD mice. Our results show that Aβ-pE(3) and ptau Ser202/Thr205 levels strongly correlate in human as well as murine tissues, suggesting that tau phosphorylation might be amplified by Aβ-pE(3)

Phosphorylation of different tau sites during progression of Alzheimer’s disease

Abstract Alzheimer’s disease is characterized by accumulation of amyloid plaques and tau aggregates in several cortical brain regions. Tau phosphorylation causes formation of neurofibrillary tangles and neuropil threads. Phosphorylation at tau Ser202/Thr205 is well characterized since labeling of this site is used to assign Braak stage based on occurrence of neurofibrillary tangles. Only little is known about the spatial and temporal phosphorylation profile of other phosphorylated tau (ptau) sites. Here, we investigate total tau and ptau at residues Tyr18, Ser199, Ser202/Thr205, Thr231, Ser262, Ser396, Ser422 as well as amyloid-β plaques in human brain tissue of AD patients and controls. Allo- and isocortical brain regions were evaluated applying rater-independent automated quantification based on digital image analysis. We found that the level of ptau at several residues, like Ser199, Ser202/Thr205, and Ser422 was similar in healthy controls and Braak stages I to IV but was increased in Braak stage V/VI throughout the entire isocortex and transentorhinal cortex. Quantification of ThioS-stained plaques showed a similar pattern. Only tau phosphorylation at Tyr18 and Thr231 was already significantly increased in the transentorhinal region at Braak stage III/IV and hence showed a progressive increase with increasing Braak stages. Additionally, the increase in phosphorylation relative to controls was highest at Tyr18, Thr231 and Ser199. By contrast, Ser396 tau and Ser262 tau showed only a weak phosphorylation in all analyzed brain regions and only minor progression. Our results suggest that the ptau burden in the isocortex is comparable between all analyzed ptau sites when using a quantitative approach while levels of ptau at Tyr18 or Thr231 in the transentorhinal region are different between all Braak stages. Hence these sites could be crucial in the pathogenesis of AD already at early stages and therefore represent putative novel therapeutic targets

Early and progressive microstructural brain changes in mice overexpressing human α-Synuclein detected by diffusion kurtosis imaging

Diffusion kurtosis imaging (DKI) is sensitive in detecting α-Synuclein (α-Syn) accumulation-associated microstructural changes at late stages of the pathology in α-Syn overexpressing TNWT-61 mice. The aim of this study was to perform DKI in young TNWT-61 mice when α-Syn starts to accumulate and to compare the imaging results with an analysis of motor and memory impairment and α-Syn levels. Findings indicate that α-Syn accumulation-associated changes may start in areas with a high density of dopaminergic nerve terminals. We also found TBSS changes in white matter only at 6mo, suggesting α-Syn accumulation-associated changes start in the gray matter and later progress to the white matter. MK is able to detect microstructural changes induced by α-Syn overexpression in TNWT-61 mice and could be a useful clinical tool for detecting early-stage Parkinson's disease in human patients

Characterization of the visceral and neuronal phenotype of 4L/PS-NA mice modeling Gaucher disease.

Gaucher disease is caused by a deficiency in glucocerebrosidase that can result in non-neuronal as well as neuronal symptoms. Common visceral symptoms are an increased organ size, specifically of the spleen, and glucosylceramide as well as glucosylsphingosine substrate accumulations as a direct result of the glucocerebrosidase deficiency. Neuronal symptoms include motor deficits and strong alterations in the cerebellum. To evaluate the effect of new compounds for the treatment of this devastating disease, animal models are needed that closely mimic the human phenotype. The 4L/PS-NA mouse as model of Gaucher disease is shown to present reduced glucocerebrosidase activity similar to human cases but an in-depth characterization of the model was still not performed. We therefore analyzed 4L/PS-NA mice for visceral alterations, motor deficits and also neuronal changes like glucocerebrosidase activity, substrate levels and neuroinflammation. A special focus was set at pathological changes of the cerebellum. Our results show that 4L/PS-NA mice have strongly enlarged visceral organs that are infiltrated by enlarged leukocytes and macrophages. Furthermore, animals present strong motor deficits that are accompanied by increased glucosylceramide and glucosylsphingosine levels in the brain, astrocytosis and activated microglia in the cortex and hippocampus as well as reduced calbindin levels in the cerebellum. The latter was directly related to a strong Purkinje cell loss. Our results thus provide a detailed characterization of the 4L/PS-NA mouse model over age showing the translational value of the model and validating its usefulness for preclinical efficiency studies to evaluate new compounds against Gaucher disease

Additional file 1: of Phosphorylation of different tau sites during progression of Alzheimer’s disease

Online Source 9 Double labeling of pSer262 and pSer202/Thr205 tau in the temporal cortex at Braak stage V/VI. Images show different labeling pattern of pSer262 (arrows) and pS202 (white arrowheads) as well as their overlay (yellow arrowheads) (a1) and single fluorescence images (a2,3,4) of case 17. AF: autofluorescence. Scale bar: 20 μm. Online Source 10 Example of measurement procedure of tau pSer262. Objects in the unlabeled autofluorescence channel were detected by thresholding (red in a1). The resulting mask images (a2) were then subtracted from tau pSer262 images to remove autofluorescence (a3). The resulting images were Edge+ filtered (a4) to facilitate threshold-based detection of tau pSer262-positive objects (red outline in a5). These outlines were then loaded onto the raw images to quantify original tau pSer262 signal (red outline in a6). AF: autofluorescence. Scale bar: 20 μm. Online Source 11 Example of detecting ThioS-positive amyloid-β but not NFTs. Image a displays the co-labeling of ThioS (green) and HT7 (red), while images b and c, respectively, show single channel images. ThioS shows intense labeling of plaque-associated β-sheets (b, asterisk) whereas tangles are only weakly labeled (c, arrows) (c). A combination of threshold-based identification of ThioS and size restriction (d‘, green rectangle) enables quantification of ThioS+ plaque labeling (red highlighted) but not tangles (d). ThioS: ThioflavinS. Scale bar: 20 μm. (PDF 599 kb

mTh1 driven expression of hTDP-43 results in typical ALS/FTLD neuropathological symptoms

<div><p>Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models.</p></div

Quantification of hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.

<p>Quantitative hTDP-43 expression in hypothalamus (A), medulla oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS samples. (D, E) Representative overview image and magnifications of hTDP-43 labeling of a brain section (olfactory bulb (1), cortex (2), hippocampus (3), thalamus (4), hypothalamus (5) and medulla oblongata (6)) of 3 months old TAR6/6 (D) and ntg (E) mice. Scale bars: Overview images = 1000 μm; magnification images = 20 μm. (A, B) 1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5; TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (C) n as in A, B exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between age groups. *p<0.05, **p<0.01, ***p<0.001.</p