Andreas Schild

Transgenic animal models of Alzheimer's disease and related disorders: histopathology, behavior and therapy

Alzheimers disease (AD) is a devastating neurodegenerative disease that affects more than 15 million people worldwide. Within the next generation, these numbers will more than double. To assist in the elucidation of pathogenic mechanisms of AD and related disorders, such as frontotemporal dementia (FTDP-17), genetically modified mice, flies, fish and worms were developed, which reproduce aspects of the human histopathology, such as β-amyloid-containing plaques and tau-containing neurofibrillary tangles (NFT). In mice, the tau pathology caused selective behavioral impairment, depending on the distribution of the tau aggregates in the brain. β-Amyloid induced an increase in the numbers of NFT, whereas the opposite was not observed in mice. In β-amyloid-producing transgenic mice, memory impairment was associated with increased levels of β-amyloid. Active and passive β-amyloid-directed immunization caused the removal of β-amyloid plaques and restored memory functions. These findings have since been translated to human therapy. This review aims to discuss the suitability and limitations of the various animal models and their contribution to an understanding of the pathophysiology of AD and related disorders.

Coordinate activation of autophagy and the proteasome pathway by FoxO transcription factor

The rapid loss of muscle mass, which occurs with disuse and systemically with fasting, cancer and many other diseases, results primarily from accelerated breakdown of muscle proteins. In atrophying muscles, the ubiquitin-proteasome pathway catalyzes the accelerated degradation of myofibrillar proteins, but the possible importance of the autophagic/lysosomal pathway in atrophy has received little attention. Our prior studies demonstrate that activation of FoxO transcription factors is essential for muscle atrophy, and that activated FoxO3 by itself causes dramatic atrophy of muscles and cultured myotubes via transcription of a set of atrophy-related genes (“atrogenes”) including critical ubiquitin ligases. Using selective inhibitors, we measured isotopically the actual contribution of proteasomes and lysosomes to the FoxO3-induced increase in protein breakdown in myotubes and found that FoxO3 coordinately activates both proteolytic systems, but especially lysosomal proteolysis. Activated FoxO3 stimulates autophagy through a transcription-dependent mechanism and increases the transcription of many autophagy-related genes, which are also induced in mouse muscles atrophying due to denervation or fasting. Thus, in atrophying muscles, decreased IGF1-PI3K-Akt signaling stimulates autophagy, not only through mTOR, but also more slowly by FoxO3-dependent transcription. These findings on muscle provide the first evidence for coordinate regulation of proteasomal and lysosomal systems, although in neuronal and hepatic cells, FoxO3 stimulates the autophagic process selectively. Addendum to: Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 2007; 6:472-83.

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

Of all forms of dementia, Alzheimers disease is the most prevalent. It is histopathologically characterized by β‐amyloid‐containing plaques, tau‐containing neurofibrillary tangles, reduced synaptic density and neuronal loss in selected brain areas. For the rare familial forms of Alzheimers disease, pathogenic mutations have been identified in both the gene encoding the precursor of the Aβ peptide, APP, itself and in the presenilin genes which encode part of the APP‐protease complex. For the more frequent sporadic forms of Alzheimers disease, the pathogenic trigger has not been unambiguously identified. Whether Aβ 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 β‐amyloid deposition, mutations have been identified in tau which also lead to neurodegeneration and dementia.

Animal models reveal role for tau phosphorylation in human disease.

Many proteins that are implicated in human disease are posttranslationally modified. This includes the microtubule-associated protein tau that is deposited in a hyperphosphorylated form in brains of Alzheimers disease patients. The focus of this review article is on the physiological and pathological phosphorylation of tau; the relevance of aberrant phosphorylation for disease; the role of kinases and phosphatases in this process; its modeling in transgenic mice, flies, and worms; and implications of phosphorylation for therapeutic intervention.

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 Alzheimers 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/Bβ 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/Bα and Bβ protein expression varies significantly in areas of the brain affected by neurodegenerative diseases such as the hippocampus or cerebellum. At the cellular level, PR55/Bβ protein expression was confined to neurons, whereas PR55/Bα 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.

Transgenic and knockout models of PP2A.

Publisher Summary This chapter discusses the transgenic and knockout models of PP2A. Considering the putative role of PP2A in the pathogenesis of human diseases, the development of more transgenic and knockout models of PP2A may provide insight in the regulation of PP2A. This may eventually lead to the discovery of therapeutic agents that can specifically counteract PP2A dysfunction. This chapter reviews various experimental approaches in mice, aimed to dissect PP2A function in vivo such as PP2A knockout mice, PP2A regulatory subunit transgenic mice, and PP2A Cα dominant negative mutant mice. The advantages and limitations of these approaches are discussed along with their implications for the understanding of human disease. Lethality can be overcome by more sophisticated gene targeting approaches using either tissue-specific or inducible promoters. Redundancies, on the other hand, can be overcome by creating multiple knockouts, or by analyzing the mice even more carefully to detect subtle phenotypic alterations. However, overexpression of regulatory subunits as in the case of PP2A may be complicated by the fact that total levels of PP2A are tightly regulated.

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.

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 Alzheimers 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 PR55α mRNA levels are highest in both HEK293 cells and SH‐SY5Y neuroblastoma cells whereas PR55β levels are lowest. Stepwise neuronal differentiation of SH‐SY5Y cells causes the selective upregulation of PR55β, and to some extent PR55γ and PR55δ, but not PR55α mRNAs. In agreement with the qRT‐PCR analysis, neuronal differentiation does not alter PR55α protein levels, whereas interestingly, PR55β and PR55γ 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 PR55α being the major isoform.