Juergen Gotz

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.

Pronuclear injection for the production of transgenic mice

Transgenic mice have been instrumental in dissecting the role of various neuronal proteins under both physiological and pathological conditions. Pronuclear injection is the most widely used protocol for the generation of transgenic mice. Here, we describe all steps involved from DNA purification to the set up of a mouse colony including vasectomy, injection of the DNA into a donor zygote, transfer of injected zygotes into recipient foster mice, screening of offspring and establishment of transgenic mouse lines. We discuss the use of neuron-specific promoters to express proteins with a role in Alzheimer disease. Transgenic expression of a truncated form of the microtubule-associated protein tau (Δtau) is used as an example for the anticipated results.

P70 S6 kinase mediates tau phosphorylation and synthesis

Currently, we found that the 70‐kDa p70 S6 kinase (p70S6K) directly phosphorylates tau at S262, S214, and T212 sites in vitro. By immunoprecipitation, p‐p70S6K (T421/S424) showed a close association with p‐tau (S262 and S396/404). Zinc‐induced p70S6K activation could only upregulate translation of total S6 and tau but not global proteins in SH‐SY5Y cells. The requirement of p70S6K activation was confirmed in the SH‐SY5Y cells that overexpress wild‐type htau40. Level of p‐p70S6K (T421/S424) was only significantly correlated with p‐tau at S262, S214, and T212, but not T212/S214, in Alzheimers disease (AD) brains. These suggested that p70S6K might contribute to tau related pathologies in AD brains.

Functional Genomics meets neurodegenerative disorders. Part II: application and data integration.

The transcriptomic and proteomic techniques presented in part I (Functional Genomics meets neurodegenerative disorders. Part I: transcriptomic and proteomic technology) of this back-to-back review have been applied to a range of neurodegenerative disorders, including Huntingtons disease (HD), Prion diseases (PrD), Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis (ALS), Alzheimers disease (AD), frontotemporal dementia (FTD) and Parkinsons disease (PD). Samples have been derived either from human brain and cerebrospinal fluid, tissue culture cells or brains and spinal cord of experimental animal models. With the availability of huge data sets it will firstly be a major challenge to extract meaningful information and secondly, not to obtain contradicting results when data are collected in parallel from the same source of biological specimen using different techniques. Reliability of the data highly depends on proper normalization and validation both of which are discussed together with an outlook on developments that can be anticipated in the future and are expected to fuel the field. The new insight undoubtedly will lead to a redefinition and subdivision of disease entities based on biochemical criteria rather than the clinical presentation. This will have important implications for treatment strategies.

Aβ treatment and P301L tau expression in an Alzheimer's disease tissue culture model act synergistically to promote aberrant cell cycle re-entry

Microarrays enable the observation of gene expression in experimental models of Alzheimers disease (AD), with implications for the human pathology. Histopathologically, AD is characterized by Aβ‐containing plaques and tau‐containing neurofibrillary tangles. Here, we used a human SH‐SY5Y neuroblastoma cell system to assess the role of P301L mutant human tau expression, and treatment with or without Aβ on gene regulation. We found that Aβ and P301L tau expression independently affect the regulation of genes controlling cell proliferation and synaptic elements. Moreover, Aβ and P301L tau act synergistically on cell cycle and DNA damage genes, yet influence specific genes within these categories. By using neuronally differentiated P301L tau cells, we can show that Aβ treatment induces an early upregulation of cell cycle control and synaptic genes. At the protein level, by using Kinetworks™ multi‐immunoblotting and BrdU labelling, we found that although P301L tau and Aβ both affected levels of cell cycle proteins, their effects were distinct, in particular concerning DNA damage proteins. Moreover, DNA synthesis was observed only when SH‐SY5Y cells overexpressed human wild‐type or P301L tau and were incubated with Aβ. Thus, our study shows that Aβ treatment and human tau overexpression in an AD cell culture model act synergistically to promote aberrant cell cycle re‐entry, supporting the mitosis failure hypothesis in AD.

Saitohin gene is not associated with Alzheimer’s disease

Background: The deposition of tau protein in neurofibrillary tangles constitutes an important feature of many neurodegenerative disorders, including Alzheimer’s disease. A polymorphic gene, saitohin (STH), nested within the tau gene (microtubule associated protein tau, MAPT), was recently identified and an association of a non-synonymous polymorphism in STH with increased risk for Alzheimer’s disease was suggested. Objective and methods: To test the above hypothesis in a case–control association study of two independent white populations within Switzerland and Greece, comparing genotype and allele frequencies from 225 Alzheimer’s disease patients and 144 healthy control subjects. Results: No differences in allelic or genotypic distributions between Alzheimer’s disease patients and controls was found in the individual samples (Swiss/Greek) or in the combined sample. Stratification for the presence of apolipoprotein E (APOE) ε4 allele, sex, or age did not show significant effects in the populations studied, nor was there an effect on the age of onset. Conclusions: No evidence was found for an association of the non-synonymous polymorphism (Q7R) in STH and Alzheimer’s disease. This finding is in line with earlier studies showing no association between MAPT and Alzheimer’s disease.

Tau physiology and pathomechanisms in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) has been associated with toxic intracellular aggregates of hyperphosphorylated tau (FTLD‐tau). Moreover, genetic studies identified mutations in the MAPT gene encoding tau in familial cases of the disease. In this review, we cover a range of aspects of tau function, both in the healthy and diseased brain, discussing several in vitro and in vivo models. Tau structure and function in the healthy brain is presented, accentuating its distinct compartmentalization in neurons and its role in microtubule stabilization and axonal transport. Furthermore, tau‐driven pathology is discussed, introducing current concepts and the underlying experimental evidence. Different aspects of pathological tau phosphorylation, the proteins genomic and domain organization as well as its spreading in disease, together with MAPT‐associated mutations and their respective models are presented. Dysfunction related to other post‐transcriptional modifications and their effect on normal neuronal functions such as cell cycle, epigenetics and synapse dynamics are also discussed, providing a mechanistic explanation for the observations made in FTLD‐tau cases, with the possibility for therapeutic intervention.

Hyperphosphorylated tau causes reduced hippocampal CA1 excitability by relocating the axon initial segment

Hyperphosphorylated tau has a critical role in tauopathies such as Alzheimer’s disease and frontotemporal dementia, impairing neuronal function and eventually leading to neurodegeneration. A critical role for tau is supported by studies in transgenic mouse models that express the P301L tau mutation found in cases of familial frontotemporal dementia, with the accumulation of hyperphosphorylated tau in the hippocampus causing reductions in hippocampal long-term potentiation and impairments in spatial learning and memory. However, what has remained unexplored is the role of hyperphosphorylated tau in reducing neuronal excitability. Here, we show in two complementary P301L tau transgenic mouse models that hyperphosphorylated tau induces a more depolarized threshold for action potential initiation and reduces firing in hippocampal CA1 neurons, which was rescued by the suppression of transgenic tau. Furthermore, using mutagenesis and primary hippocampal neuronal cultures, we reveal that this reduction in neuronal excitability results from the relocation of the axon initial segment (AIS) down the axon in a tau phosphorylation-dependent manner. We also demonstrate that this effect is microtubule-dependent. In addition, pharmacological stabilization was found to prevent both the structural and functional deficits caused by tau hyperphosphorylation. Finally, we demonstrate that the AIS of neurons from tau transgenic mice is further down the axon, which correlates with a reduction in excitability. We therefore propose that a reduction in hippocampal excitability due to a tau-mediated distal relocalization of the AIS contributes to the hippocampal dysfunction observed in tauopathies.

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.