BACE1-cleavage of Sez6 and Sez6L is elevated in Niemann-Pick type C disease mouse brains

It is intriguing that a rare, inherited lysosomal storage disorder Niemann-Pick type C (NPC) shares similarities with Alzheimer’s disease (AD). We have previously reported an enhanced processing of β-amyloid precursor protein (APP) by β-secretase (BACE1), a key enzyme in the pathogenesis of AD, in NPC1-null cells. In this work, we characterized regional and temporal expression and processing of the recently identified BACE1 substrates seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), and APP, in NPC1-/- (NPC1) and NPC1+/+ (wt) mouse brains. We analysed 4-weeks old brains to detect the earliest changes associated with NPC, and 10-weeks of age to identify changes at terminal disease stage. Sez6 and Sez6L were selected due to their predominant cleavage by BACE1, and their potential role in synaptic function that may contribute to presentation of seizures and/or motor impairments in NPC patients. While an enhanced BACE1-cleavage of all three substrates was detected in NPC1 vs. wt-mouse brains at 4- weeks of age, at 10-weeks increased proteolysis by BACE1 was observed for Sez6L in the cortex, hippocampus and cerebellum of NPC1-mice. Interestingly, both APP and Sez6L were found to be expressed in Purkinje neurons and their immunostaining was lost upon Purkinje cell neurodegeneration in 10-weeks old NPC1 mice. Furthermore, in NPC1- vs. wt-mouse primary cortical neurons, both Sez6 and Sez6L showed increased punctuate staining within the endolysosomal pathway as well as increased Sez6L and BACE1-positive puncta. This indicates that a trafficking defect within the endolysosomal pathway may play a key role in enhanced BACE1-proteolysis in NPC disease. Overall, our findings suggest that enhanced proteolysis by BACE1 could be a part of NPC disease pathogenesis. Understanding the basic biology of BACE1 and the functional impact of cleavage of its substrates is important to better evaluate the therapeutic potential of BACE1 against AD and, possibly, NPC disease

Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer’s Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies

Molekulare Mechanismen zur Steuerung tangentialer Zellwanderungen im embryonalen Gehirn der Maus

Während der embryonalen Entwicklung des Gehirns der Maus erreichen neuronale Vorläuferzellen entweder über radiale oder tangentiale Wanderung ihre Zielgebiete. Bei der tangentialen Wanderung ziehen die Zellen entlang der pialen Oberfläche, wobei sie weite Strecken zurücklegen können. Das Ziel dieser Arbeit war es, Mechanismen tangentialer Wanderungen im embryonalen Gehirn der Maus aufzuklären. Dabei wurden die Untersuchungen exemplarisch am posterioren extramuralen Wanderungsstrom (pes) des Rhombencephalons durchgeführt. Zur grundlegenden Untersuchung des pes stand die YT-PhPax6-tTA-transgene Mäuselinie zur Verfügung, mit der eine gezielte GFP-Markierung der Pax6-positiven Zellen dieser Wanderung realisiert werden konnte. Mit Hilfe dieser transgenen Mäuselinie wurden Transplantations-, Kollagenkokulturexperimente sowie Überexpressionsstudien am pes durchgeführt. Zusätzlich wurden Expressionstudien zur Identifizierung von Proteinen erstellt, die Einfluß auf die Wanderungen des Rhombencephalons nehmen könnten. Über den Verlauf des pes konnte gezeigt werden, daß die Zellen nicht entlang von andersartig gebildeten Fortsätzen wandern. Zudem hat die homogen strukturierte Bodenplatte, vermutlich über Netrin-1, eine anziehende Wirkung auf die Zellen. Die anziehende Wirkung wird jedoch aufgehoben, sobald diese Zellen die Bodenplatte gekreuzt haben. Nach der Kreuzung wirkt entweder die Flügelplatte anziehend oder/und die Bodenplatte abstoßend auf die wandernden Zellen, so daß diese ihre kontralateralen Zielgebiete erreichen können. Ferner konnte für diese Wanderung der Unc5H3 Rezeptor als „dependence receptor“ bestätigt werden. Daneben kommt dem Rezeptor während des Einwanderns der Zellen in das Parenchym möglicherweise eine Funktion bei der Zielerkennung zu. Außerdem konnten EphA3, EphA7 und EphrinB1 als mögliche regulatorische Proteine der tangentialen Wanderungen des Rhombencephalons identifiert werden. Zusätzlich wurde erstmals ein direkter Vergleich der Expressionsmuster von Netrin-1 und seinen Rezeptoren DCC, Neogenin und Unc5H1-4 durchgeführt, der eine wahrscheinliche Aufgabe dieser Proteine nicht nur für die tangentialen Wanderungen im Rhombencephalon sondern auch für tangentiale und radiale Wanderungen im Telencephalon belegt. Durch diese Untersuchungen konnten erstmals grundlegende Mechanismen des pes geklärt, sowie Proteine, die in tangentialen Wanderungen involviert sind, identifiziert werden

A novel approach to selectively target neuronal subpopulations reveals genetic pathways that regulate tangential migration in the vertebrate hindbrain.

International audienceVertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications

Impact of ApoB-100 expression on cognition and brain pathology in wild-type and hAPPsl mice

During their lifetime, people are commonly exposed to several vascular risk factors that may affect brain ageing and cognitive function. In the last few years, increasing evidence suggests that pathological plasma lipid profiles contribute to the pathogenesis of late-onset Alzheimer's disease. Importantly, hypercholesterolemia, especially elevated low-density lipoprotein cholesterol values, that is, increased apolipoprotein B-100 (ApoB-100) levels, represents an independent risk factor. In this study, the effects of ApoB-100 overexpression, either alone or in combination with cerebral expression of human amyloid precursor protein (hAPP), on cognitive functions and brain pathology were assessed. Our results show that ApoB-100 overexpression induces memory decline and increases cerebral lipid peroxidation and amyloid beta levels compared to those in wild-type animals. Although double-transgenic ApoBxAPP animals did not develop more distinct behavioral deficits than single-transgenic hAPP littermates, hApoB-100 expression caused additional pathophysiological features, such as high LDL and low HDL-cholesterol levels, increased lipid peroxidation, and pronounced ApoB-100 accumulation in cerebral vessels. Thus, our results indicate that ApoBxAPP mice might better reflect the situation of elderly humans than hAPPsl overexpression alone. (C) 2013 Elsevier Inc. All rights reserved

Brain cortical cholesterol metabolism is highly affected by human APP overexpression in mice

Processing of the amyloid precursor protein (APP) and amyloid beta (A beta) has been for decades in the center of Alzheimer's disease (AD) research. Beside many other variables, lipids, especially cholesterol and its derivatives, are discussed to contribute to AD pathogenesis. Several studies show that cholesterol affects APP metabolism. Also the converse mechanism, the direct influence of A beta on cholesterol metabolism, has been described. To further investigate this crosstalk between cholesterol- and APP metabolism, a high-fat feeding study was conducted with animals overexpressing human APP(SL) and/or human ApoB-100. The impact of diet and genotype on cerebral cholesterol metabolism and content as well as spatial learning and memory was examined. While behavioral performance was not influenced by this high fat diet (HFD), reduction of cortical free cholesterol levels and mRNA expression patterns under normal diet and HFD conditions in human APP(SL) overexpressing mice argue for an important role of APP in cerebral lipid metabolism. From our results we conclude that increased APP metabolism in ApoBxAPP and APP(SL) mice induces mechanisms to reduce free cholesterol levels. (C) 2016 Elsevier Inc. All rights reserved

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

Non-selective calcium channel blocker bepridil decreases secondary pathology in mice after photothrombotic cortical lesion.

Experimental studies have identified a complex link between neurodegeneration, β-amyloid (Aβ) and calcium homeostasis. Here we asked whether early phase β-amyloid pathology in transgenic hAPPSL mice exaggerates the ischemic lesion and remote secondary pathology in the thalamus, and whether a non-selective calcium channel blocker reduces these pathologies. Transgenic hAPPSL (n = 33) and non-transgenic (n = 30) male mice (4-5 months) were subjected to unilateral cortical photothrombosis and treated with the non-selective calcium channel blocker bepridil (50 mg/kg, p.o., once a day) or vehicle for 28 days, starting administration 2 days after the operation. Animals were then perfused for histological analysis of infarct size, Aβ and calcium accumulation in the thalamus. Cortical photothrombosis resulted in a small infarct, which was associated with atypical Aβ and calcium accumulation in the ipsilateral thalamus. Transgenic mice had significantly smaller infarct volumes than non-transgenic littermates (P<0.05) and ischemia-induced rodent Aβ accumulation in the thalamus was lower in transgenic mice compared to non-transgenic mice (P<0.01). Bepridil decreased calcium load in the thalamus (P<0.01). The present data suggest less pronounced primary and secondary pathology in hAPPSL transgenic mice after ischemic cortical injury. Bepridil particularly decreased calcium pathology in the thalamus following ischemia

In vitro assessment of dietary bioactives for TFEB activation as a possible target to support cognitive and emotional wellbeing

Aging and chronic stress are associated with altered brain plasticity and an increased risk of developing cognitive and emotional health problems. A key cellular mechanism involved is autophagy, which is known to be important for memory encoding and information processing, and is also thought to be important for psychiatric and emotional health. Activation of the autophagic system can be established by the transcription factor EB (TFEB), which regulates both autophagosome formation and lysosomal biogenesis and function. As well-known activators of autophagy like caloric restriction, fasting, or regular exercise require intervention programs that are hard to adhere to, the search for nutritional solutions with similar effects, is increasing. We therefore evaluated pro-autophagic dietary bioactives for effects specifically through TFEB activation and assessed whether combining bioactives can lead to synergistic or additive effects. We also discuss current evidence supporting the use of these natural dietary compounds to promote cognitive and emotional wellbeing. Methods: Primary cortical astrocytes were treated with 13 different ingredients and analyzed for their effect on TFEB nuclear translocation by high content imaging. Effective ingredients were then evaluated at different concentrations for their effect on TFEB downstream signaling by analyzing mRNA levels of Beclin1, LC3, Lamp-1 and Cathepsin D. Furthermore, the synergistic effects of sub-optimal concentrations of different ingredient combinations were evaluated on the same TFEB downstream signaling markers. Potential signs of toxicity of all ingredients and their combinations were evaluated by analysis of ATP production and nuclear counts. Results: DHA, EPA, 7,8-DHF, ellagic acid, curcumin and spermidine increased TFEB nuclear localization. DHA and spermidine exhibited the strongest effects when measuring mRNA levels of TFEB downstream signaling markers. The most significant synergies were observed when combining spermidine with ellagic acid or when combining DHA with curcumin. None of the ingredients alone or in combination presented any sign of toxicity at the concentrations tested. Discussion/Conclusion: The results of our study show for the first time a synergistic effect of spermidine and ellagic acid and of DHA and curcumin on TFEB signaling and thus its ability to act on the autophagic system in brain cells. These combinations may hold potential as dietary promoters of cognitive and emotional wellbeing