Herman Devijver

Amyloid Activates GSK-3β to Aggravate Neuronal Tauopathy in Bigenic Mice

The hypothesis that amyloid pathology precedes and induces the tau pathology of Alzheimers disease is experimentally supported here through the identification of GSK-3 isozymes as a major link in the signaling pathway from amyloid to tau pathology. This study compares two novel bigenic mouse models: APP-V717I x Tau-P301L mice with combined amyloid and tau pathology and GSK-3beta x Tau-P301L mice with tauopathy only. Extensive and remarkable parallels were observed between these strains including 1) aggravation of tauopathy with highly fibrillar tangles in the hippocampus and cortex; 2) prolonged survival correlated to alleviated brainstem tauopathy; 3) development of severe cognitive and behavioral defects in young adults before the onset of amyloid deposition or tauopathy; and 4) presence of pathological phospho-epitopes of tau, including the characteristic GSK-3beta motif at S396/S404. Both GSK-3 isozymes were activated in the brain of parental APP-V717I amyloid mice, even at a young age when cognitive and behavioral defects are evident but before amyloid deposition. The data indicate that amyloid induces tauopathy through activation of GSK-3 and suggest a role for the kinase in maintaining the functional integrity of adult neurons.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

ABSTRACT The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Glycogen synthase kinase‐3β, or a link between amyloid and tau pathology?

Phosphorylation is the most common post‐translational modification of cellular proteins, essential for most physiological functions. Deregulation of phosphorylation has been invoked in disease mechanisms, and the case of Alzheimer’s disease (AD) is no exception: both in the amyloid pathology and in the tauopathy are kinases deeply implicated. The glycogen synthase kinase‐3 (GSK‐3) isozymes participate in diverse cellular processes and important signalling pathways and have been implicitly linked to diverse medical problems, i.e. from diabetes and cancer to mood disorders and schizophrenia, and in the neurodegeneration of AD. Here, we review specific aspects of GSK‐3 isozymes in the framework of recent data that we obtained in novel transgenic mouse models that robustly recapitulate the pathology and mechanistical problems of AD.

Efficacy and Safety of A Liposome-Based Vaccine against Protein Tau, Assessed in Tau.P301L Mice That Model Tauopathy

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimers disease. The unmet need of effective therapy for Alzheimers disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimers disease.

Deregulation of NMDA-receptor function and down-stream signaling in APP(V717I) transgenic mice

Evidence is accumulating for a role for amyloid peptides in impaired synaptic plasticity and cognition, while the underlying mechanisms remain unclear. We here analyzed the effects of amyloid peptides on NMDA-receptor function in vitro and in vivo. A synthetic amyloid peptide preparation containing monomeric and oligomeric A beta (1-42) peptides was used and demonstrated to bind to synapses expressing NMDA-receptors in cultured hippocampal and cortical neurons. Pre-incubation of primary neuronal cultures with A beta peptides significantly inhibited NMDA-receptor function, albeit not by a direct pharmacological inhibition of NMDA-receptors, since acute application of A beta peptides did not change NMDA-receptor currents in autaptic hippocampal cultures nor in xenopus oocytes expressing recombinant NMDA-receptors. Pre-incubation of primary neuronal cultures with A beta peptides however decreased NR2B-immunoreactive synaptic spines and surface expression of NR2B containing NMDA-receptors. Furthermore, we extended these findings for the first time in vivo, demonstrating decreased concentrations of NMDA-receptor subunit NR2B and PSD-95 as well as activated alpha-CaMKII in postsynaptic density preparations of APP[V717I] transgenic mice. This was associated with impaired NMDA-dependent LTP and decreased NMDA- and AMPA-receptor currents in hippocampal CA1 region in APP[V717I] transgenic mice. In addition, induction of c-Fos following cued and contextual fear conditioning was significantly impaired in the basolateral amygdala and hippocampus of APP[V717I] transgenic mice. Our data demonstrate defects in NMDA-receptor function and learning dependent signaling cascades in vivo in APP[V717I] transgenic mice and point to decreased surface expression of NMDA-receptors as a mechanism involved in early synaptic defects in APP[V717I] transgenic mice in vivo.

Upper Airway Dysfunction of Tau-P301L Mice Correlates with Tauopathy in Midbrain and Ponto-Medullary Brainstem Nuclei

Tauopathy comprises hyperphosphorylation of the microtubule-associated protein tau, causing intracellular aggregation and accumulation as neurofibrillary tangles and neuropil treads. Some primary tauopathies are linked to mutations in the MAPT gene coding for protein tau, but most are sporadic with unknown causes. Also, in Alzheimers disease, the most frequent secondary tauopathy, neither the cause nor the pathological mechanisms and repercussions are understood. Transgenic mice expressing mutant Tau-P301L suffer cognitive and motor defects and die prematurely from unknown causes. Here, in situ electrophysiology in symptomatic Tau-P301L mice (7–8 months of age) revealed reduced postinspiratory discharges of laryngeal motor outputs that control laryngeal constrictor muscles. Under high chemical drive (hypercapnia), postinspiratory discharge was nearly abolished, whereas laryngeal inspiratory discharge was increased disproportionally. The latter may suggest a shift of postinspiratory laryngeal constrictor activity into inspiration. In vivo double-chamber plethysmography of Tau-P301L mice showed significantly reduced respiratory airflow but significantly increased chest movements during baseline breathing, but particularly in hypercapnia, confirming a significant increase in inspiratory resistive load. Histological analysis demonstrated hyperphosphorylated tau in brainstem nuclei, directly or indirectly involved in upper airway motor control (i.e., the Kölliker–Fuse, periaqueductal gray, and intermediate reticular nuclei). In contrast, young Tau-P301L mice did not show breathing disorders or brainstem tauopathy. Consequently, in aging Tau-P301L mice, progressive upper airway dysfunction is linked to progressive tauopathy in identified neural circuits. Because patients with tauopathy suffer from upper airway dysfunction, the Tau-P301L mice can serve as an experimental model to study disease-specific synaptic dysfunction in well defined functional neural circuits.

AAV-Tau Mediates Pyramidal Neurodegeneration by Cell-Cycle Re-Entry without Neurofibrillary Tangle Formation in Wild-Type Mice

In Alzheimers disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions. Transgenic mouse models do not resolve this problem conclusively, i.e. the relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here, we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers. We present novel AAV-based models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies, including Alzheimers disease.

GSK-3α/β kinases and amyloid production in vivo.

Arising from C. J. Phiel, C. A. Wilson, V. M.-Y. Lee & P. S. Klein 423, 435–439 (2003)10.1038/nature01640A major unresolved issue in Alzheimer’s disease is identifying the mechanisms that regulate proteolytic processing of amyloid precursor protein (APP)—glycogen synthase kinase-3 (GSK-3) isozymes are thought to be important in this regulation. Phiel et al. proposed that GSK-3α, but not GSK-3β, controls production of amyloid. We analysed the proteolytic processing of mouse and human APP in mouse brain in vivo in five different genetic and viral models. Our data do not yield evidence for either GSK-3α-mediated or GSK-3β-mediated control of APP processing in brain in vivo.

5-HT4 receptor agonists increase sAPPα levels in the cortex and hippocampus of male C57BL/6j mice

A strategy to treat Alzheimers disease (AD) is to increase the soluble form of amyloid precursor protein (sAPPα), a promnesic protein, in the brain. Because strong evidence supports beneficial effects of 5‐hydroxytryptamine 5‐HT4 receptor agonists in memory and learning, we investigated the role of 5‐HT4 receptors on APP processing in 8 weeks‐old male C57BL/6j mice.

Neurodegeneration and neuroinflammation in cdk5/p25-inducible mice: a model for hippocampal sclerosis and neocortical degeneration.

The cyclin-dependent kinase cdk5 is atypically active in postmitotic neurons and enigmatic among the kinases proposed as molecular actors in neurodegeneration. We generated transgenic mice to express p25, the N-terminally truncated p35 activator of cdk5, in forebrain under tetracycline control (TET-off). Neuronal expression of p25 (p25(ON)) caused high mortality postnatally and early in life. Mortality was completely prevented by administration of doxycycline in the drinking water of pregnant dams and litters until P42, allowing us to study the action of p25 in adult mouse forebrain. Neuronal p25 triggered neurodegeneration and also microgliosis, rapidly and intensely in hippocampus and cortex. Progressive neurodegeneration was severe with marked neuron loss, causing brain atrophy (40% loss at age 5 months) with nearly complete elimination of the hippocampus. Neurodegeneration did not involve phosphorylation of protein tau or generation of amyloid peptide. Degenerating neurons did not stain for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling or activated caspase-3 but were marked by FluoroJadeB in early stages. Diseased neurons were always closely associated with activated microglia already very early in the disease process. Primary neurons derived from p25 embryos were more prone to apoptosis than wild-type neurons, and they activated microglial cells in co-culture. The inducible p25 mice present as a model for neurodegeneration in hippocampal sclerosis and neocortical degeneration, with important contributions of activated microglia.