Nonsteroidal Anti-Inflammatory Drugs and Ectodomain Shedding of the Amyloid Precursor Protein

Background: Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer's disease (AD). Several mechanisms have been proposed to explain these findings including increased shedding of the soluble ectodomain of the amyloid precursor protein (sAPP), which functions as a neurotrophic and neuroprotective factor in vitro and in vivo. Objective: To clarify whether NSAIDs consistently stimulate sAPP secretion. Methods: 293-EBNA cells with stable overexpression of an APP-alkaline phosphatase fusion protein (APP-AP), SH-SY5Y and PC12 cells or primary telencephalic chicken neurons were treated with ibuprofen or indomethacin. APP shedding was then determined by measuring AP activity in conditioned media, Western blot analysis with antibodies against total sAPP or specific for sAPP-alpha, or in a pulse-chase paradigm. Results: AP activity in conditioned media was not increased after NSAID treatment of 293-EBNA cells whereas it was elevated by phorbol ester. Surprisingly, ibuprofen or indomethacin treatment of SH-SY5Y and PC12 cells expressing endogenous APP did not cause changes in sAPP or sAPP-alpha secretion or downregulation of cellular APP. These findings were further corroborated in primary chicken neuronal cultures. Conclusions: Using various experimental settings, we were unable to confirm sAPP or sAPP-alpha stimulation with the NSAIDs ibuprofen and indomethacin in transfected and nontransfected cells of neuronal and nonneuronal origin. Importantly, these findings seem to rule out chronic sAPP stimulation as an alternative mechanism of NSAID action in AD. Copyright (C) 2008 S. Karger AG, Base

Claudin-12 is not required for blood-brain barrier tight junction function

Background The blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. Methods We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. Results Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12(lacZ/lacZ) C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12(lacZ/lacZ) C57BL/6J mice. Conclusions Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12(lacZ/lacZ) C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease

Entwicklung eines stringent kontrollierbaren Toxingensystems zur Analyse degenerativer Prozesse im Nervensystem der Maus

Leuchtenberger S. Entwicklung eines stringent kontrollierbaren Toxingensystems zur Analyse degenerativer Prozesse im Nervensystem der Maus. Bielefeld (Germany): Bielefeld University; 2003.To analyse molecular events in pathological processes and cell interactions underlying degenerative diseases, a genetic approach of conditional cell ablation is desirable. In my thesis, conditional cell ablation was optimized for applications in post-mitotic and slowly proliferative cells by modification of the tetracycline (tet)-dependent expression system in combination with the bacterial ribonuclease barnase. The regulated barnase expression and the cytotoxicity of the gene product were examined in cell culture, in primary cells of transgenic mice and in the nervous system of transgenic mice. Taken together the analysis of this cell ablation approach demonstrates that in cell culture and to a limited extent in transgenic mice, a conditional cell ablation is feasible. By its universal application, e.g. by crossing barnase-transgenic mice with cell-specific transactivator mice, this system represents an alternative to existing methods for the investigation of pathomechanisms of degenerative diseases, also in the CNS.Viele degenerative Erkrankungen werden auf den Verlust bestimmter Zelltypen im Organismus zurückgeführt. Um Erkrankungen im Tiermodell zu simulieren, ist es erforderlich, mithilfe genetischer Methoden einen kontrollierten Zelltod ohne invasiven Eingriff herbeizuführen. In einem derartigen Tiermodell kann eine detaillierte Analyse von pathologischen Prozessen und Zellinteraktionen vorgenommen werden. In dieser Arbeit wurde eine konditionale Toxingenexpression durch Modifikation des Tetrazyklin (Tet)-abhängigen Expressionssystems in Kombination mit der bakteriellen Ribonuklease Barnase für Anwendungen in postmitotischen und wenig proliferativen Zellen optimiert. Die regulierte barnase-Expression und die Zytotoxizität des Genprodukts wurden in Zellkultur, in Primärzellen transgener Mäuse und beispielhaft im Nervensystem von transgenen Mäusen untersucht. Die durchgeführten Untersuchungen zeigen, dass sowohl in Zellkultur als auch eingeschränkt im transgenen Organismus eine regulierte Toxingenexpression durch das modifizierte Tet/Barnase-System erreichbar war und damit eine Alternative zu bestehenden Methoden für die Untersuchung von Pathomechanismen degenerativer Erkrankungen im ZNS darstellt

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P(TF), was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P(TF) to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P(NSE)) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed

Nonsteroidal Anti-Inflammatory Drugs and Ectodomain Shedding of the Amyloid Precursor Protein

Background: Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer’s disease (AD). Several mechanisms have been proposed to explain these findings including increased shedding of the soluble ectodomain of the amyloid precursor protein (sAPP), which functions as a neurotrophic and neuroprotective factor in vitro and in vivo. Objective: To clarify whether NSAIDs consistently stimulate sAPP secretion. Methods: 293-EBNA cells with stable overexpression of an APP-alkaline phosphatase fusion protein (APP-AP), SH-SY5Y and PC12 cells or primary telencephalic chicken neurons were treated with ibuprofen or indomethacin. APP shedding was then determined by measuring AP activity in conditioned media, Western blot analysis with antibodies against total sAPP or specific for sAPP- _ , or in a pulse-chase paradigm. Results: AP activity in conditioned media was not increased after NSAID treatment of 293-EBNA cells whereas it was elevated by phorbol ester. Surprisingly, ibuprofen or indomethacin treatment of SH-SY5Y and PC12 cells expressing endogenous APP did not cause changes in sAPP or sAPP- _ secretion or downregulation of cellular APP. These findings were further corroborated in primary chicken neuronal cultures. Conclusions: Using various experimental settings, we were unable to confirm sAPP or sAPP- _ stimulation with the NSAIDs ibuprofen and indomethacin in transfected and nontransfected cells of neuronal and nonneuronal origin. Importantly, these findings seem to rule out chronic sAPP stimulation as an alternative mechanism of NSAID action in AD

Insensitivity to a beta 42-lowering nonsteroidal anti-inflammatory drugs and gamma-secretase inhibitors is common among aggressive presenilin-1 mutations

A beta 42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to A beta 42-lowering NSAIDs. Of particular interest is the PS1-Delta Exon9 mutation, which provokes a pathogenic increase in the A beta 42/A beta 40 ratio and dramatically reduces the cellular response to the A beta 42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-Delta Exon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the A beta 42/A beta 40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to A beta 42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble A beta 42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors

Alpha-secretase mediated conversion of the amyloid precursor protein derived membrane stub c99 to c83 limits a beta generation

The Swedish mutation within the amyloid precursor protein (APP) causes early-onset Alzheimer's disease due to increased cleavage of APP by BACE1. While beta-secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild-type APP occurs mainly in endocytic compartments. However, we show that liberation of A beta from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall beta-secretase cleaved soluble APP released from APP(swe) secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased A beta secretion. We point out that alpha-secretase activity-mediated conversion of C99 to C83 is the main cause of this A beta reduction. Furthermore, we demonstrate that alpha-secretase cleavage of C99 even contributes to the reduction of A beta secretion of internalization deficient wild-type APP. Therefore, inhibition of alpha-secretase cleavage increased A beta secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells