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Biomedical subjects

Andreas Schild

Publications and source records attributed to Andreas Schild.

8 recordsLinked to original sources
PubMed2006

Altered levels of PP2A regulatory B/PR55 isoforms indicate role in neuronal differentiation.

The ubiquitously expressed serine/threonine-specific protein phosphatase 2A (PP2A) is prominent in brain where it serves a wide range of functions under both physiological and pathological conditions. PP2A holoenzymes are composed of a catalytic subunit and a tightly complexed scaffolding subunit. This core enzyme associates with regulatory subunits of the B/PR55, B'/PR56/PR61, B''/PR72 and B'''/PR93/PR110 families. We previously determined distribution and expression levels of the four members of the B/PR55 family in brain, as dysregulation of this subunit family has been specifically implicated in neurodegenerative disorders including Alzheimer's disease. In the present study, we used cell lines widely used in neuroscience research to determine levels of the four PR55 isoforms by qRT-PCR under different experimental conditions. We show that PR55alpha mRNA levels are highest in both HEK293 cells and SH-SY5Y neuroblastoma cells whereas PR55beta levels are lowest. Stepwise neuronal differentiation of SH-SY5Y cells causes the selective upregulation of PR55beta, and to some extent PR55gamma and PR55delta, but not PR55alpha mRNAs. In agreement with the qRT-PCR analysis, neuronal differentiation does not alter PR55alpha protein levels, whereas interestingly, PR55beta and PR55gamma protein levels are reduced when compared to undifferentiated cells. Our data point at specific roles for distinct regulatory B/PR55 subunits of PP2A in neuron-like cells with PR55alpha being the major isoform.

Blotting, Western↗
PubMed2006

Impaired development of the Harderian gland in mutant protein phosphatase 2A transgenic mice.

Although Harderian glands are especially large in rodents, many features of this retroocular gland, including its development and function, are not well established. Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes expressed in this gland. PP2A substrate specificity is determined by regulatory subunits with leucine 309 of the catalytic subunit playing a crucial role in the recruitment of regulatory subunits into the complex in vitro. Here we expressed an L309A mutant catalytic subunit in Harderian gland of transgenic mice. We found a delayed postnatal development and hypoplasia of the gland, causing enophthalmos. To determine why expression of the L309A mutant caused this phenotype, we determined the PP2A subunit composition. We found an altered subunit composition in the transgenic gland that was accompanied by pronounced changes of proteins regulating cell adhesion. Specifically, cadherin and beta-catenin were dramatically reduced and shifted to the cytosol. Furthermore, we found an inactivating phosphorylation of the cadherin-directed glycogen synthase kinase-3beta. In conclusion, the carboxy-terminal leucine L309 of the PP2A catalytic subunit determines PP2A heterotrimer composition in vivo. Moreover, our data demonstrate that PP2A subunit composition plays a crucial role in regulating cell adhesion and as a consequence in the development of the Harderian gland.

Animals↗
PubMed2006

Altered phosphorylation of cytoskeletal proteins in mutant protein phosphatase 2A transgenic mice.

Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes with diverse functions under physiologic and pathologic conditions such as Alzheimer's disease. All PP2A holoenzymes have in common a catalytic subunit C and a structural scaffolding subunit A. These core subunits assemble with various regulatory B subunits to form heterotrimers with distinct functions in the cell. Substrate specificity of PP2A in vitro is determined by regulatory subunits with leucine 309 of the catalytic subunit C playing a crucial role in the recruitment of regulatory subunits into the complex. Here we expressed a mutant form of Calpha, L309A, in brain and Harderian (lacrimal) gland of transgenic mice. We found an altered recruitment of regulatory subunits into the complex, demonstrating a role for the carboxyterminal leucine of Calpha in regulating holoenzyme assembly in vivo. This was associated with an increased phosphorylation of tau in brain and an impaired dephosphorylation of vimentin demonstrating that both cytoskeletal proteins are in vivo substrates of distinct PP2A holoenzyme complexes.

Animals↗
PubMed2004

Reference genes identified in SH-SY5Y cells using custom-made gene arrays with validation by quantitative polymerase chain reaction.

Transcriptomic methods are widely used as an initial approach to gain a mechanistic insight into physiological and pathological processes. Because differences in gene regulation to be assessed by RNA screening methods (e.g., SAGE, Affymetrix GeneChips) can be very subtle, these techniques require stable reference genes for accurate normalization. It is widely known that housekeeping genes, which are routinely used for normalization, can vary significantly depending on the tissue, and experimental test. In this study, we aimed at identifying stable reference genes for a fibrillar Abeta(42) peptide-treated, human tau-expressing SH-SY5Y neuroblastoma cell line derived to model aspects of Alzheimer's disease in tissue culture. We selected genes exhibiting potential normalization characteristics from public databases to create a custom-made microarray allowing the identification of reference genes for low, intermediate, and abundant mRNAs. A subset of these candidates was subjected to quantitative real-time polymerase chain reaction and was analyzed with geNorm software. By doing so, we were able to identify GAPD, M-RIP, and POLR2F as stable and usable reference genes irrespective of differentiation status and Abeta(42) treatment.

Alzheimer Disease↗
PubMed2004

Amyloid-induced neurofibrillary tangle formation in Alzheimer's disease: insight from transgenic mouse and tissue-culture models.

Of all forms of dementia, Alzheimer's disease is the most prevalent. It is histopathologically characterized by beta-amyloid-containing plaques, tau-containing neurofibrillary tangles, reduced synaptic density and neuronal loss in selected brain areas. For the rare familial forms of Alzheimer's disease, pathogenic mutations have been identified in both the gene encoding the precursor of the Abeta peptide, APP, itself and in the presenilin genes which encode part of the APP-protease complex. For the more frequent sporadic forms of Alzheimer's disease, the pathogenic trigger has not been unambiguously identified. Whether Abeta is again the main cause remains to be heavily discussed. In a related disorder termed frontotemporal dementia, which is characterized by tangles in the absence of beta-amyloid deposition, mutations have been identified in tau which also lead to neurodegeneration and dementia. For Alzheimer's disease the existence of familial forms lead to the proposition of the amyloid cascade hypothesis, which claims that beta-amyloid causes or enhances the tangle pathology. In this review, we describe tau transgenic mouse models in which aspects of the tau-associated pathology, including tangle formation, has been achieved. Moreover, tau transgenic mouse and tissue-culture models were used to test the amyloid cascade hypothesis. In addition, we discuss alternative hypotheses to explain the sporadic forms. The animal and tissue-culture models will provide insight into the underlying biochemical mechanisms of tau aggregation and nerve cell degeneration. These mechanisms may be partially shared between sporadic Alzheimer's disease, the familial forms and frontotemporal dementia. Eventually, Alzheimer's disease may be redefined based on biochemical events rather than phenotype.

Alzheimer Disease↗
PubMed2004

Adeno-associated viral-mediated insulin-like growth factor delivery protects motor neurons in vitro.

Recent work has demonstrated that adeno-associated viral (AAV) vector-mediated delivery of the insulin-like growth factor (IGF-I) gene through retrograde axonal transport can prolong survival and delay disease onset in the superoxide dismutase mutant mouse model of motor neuron (MN) disease. The present experiment examines IGF-I gene transfer in vitro. Adenoviral and AAV vectors for IGF-I infect neurons triggering expression and secretion of biologically active IGF-I. AAV-mediated IGF-I expression in SH-SY5Y neurons protects both cells expressing the transgene, and bystanders without transgene expression from glutamate-induced apoptosis. Similarly, AAV-mediated IGF-I delivery in primary E15 MN culture provides a titer-dependent neuroprotection from glutamate-induced DNA fragmentation. Both infected and noninfected neurons are equally protected. These observations argue that vector-mediated IGF-I gene transfer induces secretion of active IGF-I that acts through direct effects on spinal cord MNs. This mechanism may explain the therapeutic effects observed in vivo despite relatively low affinity AAV spinal cord uptake.

Animals↗
PubMed2002

Diversity, developmental regulation and distribution of murine PR55/B subunits of protein phosphatase 2A.

Protein phosphatase (PP2A) 2A is a hetero-trimeric holoenzyme that consists of a core dimer composed of a catalytic subunit that is tightly complexed with the scaffolding subunit PR65/A. This core dimer associates with variable regulatory subunits of the PR55/B, PR61/B', PR72/B" and PR93/PR110/B"' families. As PP2A holoenzymes containing PR55/B have been shown to be involved in the pathogenesis of Alzheimer's disease, we characterized the PR55/B family with particular emphasis on its distribution and expression in the brain. We determined the genomic organization of all members of the PR55/B family and cloned their murine cDNAs. Thereby, two novel splice variants of PR55/Bbeta were identified. In addition, Northern blot analysis revealed multiple transcripts for the different PR55 subunits, suggesting a higher variability within the PR55 family. In situ hybridization analysis revealed that all PR55/B subunits were widely expressed in the brain. PR55/Balpha and Bbeta protein expression varies significantly in areas of the brain affected by neurodegenerative diseases such as the hippocampus or cerebellum. At the cellular level, PR55/Bbeta protein expression was confined to neurons, whereas PR55/Balpha was also expressed in activated astrocytes indicating that the PR55 isoforms confer a different function to the holoenzyme complex. As PP2A dysfunction has been demonstrated to contribute to various human diseases, dissecting the PP2A holoenzyme and its particular function in different cell types will assist in the development of novel therapeutic strategies.

Alternative Splicing↗