José J. Lucas

Spatial learning deficit in transgenic mice that conditionally over‐express GSK‐3β in the brain but do not form tau filaments

Deregulation of glycogen synthase kinase‐3 (GSK‐3) activity in neurones has been postulated as a key feature in Alzheimers disease (AD) pathogenesis. This was further supported by our recent characterization of transgenic mice that conditionally over‐express GSK‐3β in hippocampal and cortical neurones. These mice, designated Tet/GSK‐3β, showed many of the biochemical and cellular aspects of AD neuropathology such as tau hyperphosphorylation and somatodendritic localization, decreased nuclear β‐catenin, neuronal death and reactive gliosis. Tet/GSK‐3β mice, however, did not show tau filament formation up to the latest tested age of 3 months at least. Here we report spatial learning deficits of Tet/GSK‐3β mice in the Morris water maze. In parallel, we also measured the increase in GSK‐3 activity while further exploring the possibility of tau filament formation in aged mice. We found a significant increase in GSK‐3 activity in the hippocampus of Tet/GSK‐3β mice whereas no tau fibrils could be found even in very old mice. These data reinforce the hypothesis of GSK‐3 deregulation in AD pathogenesis, and suggest that Tet/GSK‐3β mice can be used as an AD model and, most remarkably, can be used to test the therapeutic potential of the selective GSK‐3 inhibitors that are currently under development. Additionally, these experiments suggest that destabilization of microtubules and alteration of intracellular metabolic pathways contribute to AD pathogenesis independent of toxicity triggered by the aberrant tau deposits.

Glycogen synthase kinase-3 inhibition is integral to long-term potentiation

Glycogen synthase kinase‐3 (GSK‐3) is a serine/threonine kinase regulating diverse cellular functions including metabolism, transcription and cell survival. Numerous intracellular signalling pathways converge on GSK‐3 and regulate its activity via inhibitory serine‐phosphorylation. Recently, GSK‐3 has been involved in learning and memory and in neurodegeneration. Here, we present evidence that implicates GSK‐3 in synaptic plasticity. We show that phosphorylation at the inhibitory Ser9 site on GSK‐3β is increased upon induction of long‐term potentiation (LTP) in both hippocampal subregions CA1 and the dentate gyrus (DG) in vivo. The increase in inhibitory GSK‐3β phosphorylation is robust and persists for at least one hour postinduction. Furthermore, we find that LTP is impaired in transgenic mice conditionally overexpressing GSK‐3β. The LTP deficits can be attenuated/rescued by chronic treatment with lithium, a GSK‐3 inhibitor. These results suggest that the inhibition of GSK‐3 facilitates the induction of LTP and this might explain some of the negative effects of GSK‐3 on learning and memory. It follows that this role of GSK‐3β in LTP might underlie some of the cognitive dysfunction in diseases where GSK‐3 dysfunction has been implicated, including Alzheimers and other dementias.

Full Reversal of Alzheimer's Disease-Like Phenotype in a Mouse Model with Conditional Overexpression of Glycogen Synthase Kinase-3

Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase that is particularly abundant in the CNS. Dysregulation of GSK-3 activity is believed to play a key role in the pathogenesis of CNS chronic disorders such as Alzheimers disease (AD), bipolar disorder, and Huntingtons disease, and of metabolic disorders such as type II diabetes. Accordingly, GSK-3 inhibitors have been postulated as therapeutic tools for these diseases. Interestingly, pathophysiological and pharmacological regulation of GSK-3 is affected by an amplification mechanism that applies both to inhibition and activation. The possibility therefore exists that sustained inhibition or activation might persist after cessation of the initial trigger. Regarding AD, GSK-3 has been shown to accumulate in pretangle neurons. Furthermore, GSK-3 phosphorylates tau in most serine and threonine residues hyperphosphorylated in PHF (paired helical filament)-tau and GSK-3 activity contributes both to β-amyloid production and to β-amyloid-mediated neuronal death. In good agreement, mice with conditional overexpression of GSK-3 in forebrain neurons (Tet/GSK-3β mice) recapitulate aspects of AD neuropathology such as tau hyperphosphorylation, apoptotic neuronal death, and reactive astrocytosis as well as spatial learning deficit. Here, we exploit the conditional system used to generate Tet/GSK-3β mice to explore whether the biochemical, histopathological, and behavioral consequences of increased GSK-3 activity are susceptible to revert after restoration of normal GSK-3 levels. Here, we show that transgene shutdown in symptomatic mice leads to normal GSK-3 activity, normal phospho-tau levels, diminished neuronal death, and suppression of the cognitive deficit, thus further supporting the potential of GSK-3 inhibitors for AD therapeutics.

GSK3: a possible link between beta amyloid peptide and tau protein.

Tau is a neuronal microtubule-associated phosphoprotein that is highly phosphorylated by glycogen synthase kinase 3 (GSK3). Tau phosphorylation by GSK3 regulates tau binding to microtubules, tau degradation and tau aggregation. Tau phosphorylation is important in Alzheimer disease pathology and in other tauopathies. In Alzheimer disease, it has been proposed that the peptide beta amyloid promotes GSK3 activation, resulting in tau phosphorylation. In this work, we review the links between beta amyloid peptide, tau protein and GSK3 that occur in familial Alzheimer disease. We also discuss the possible links between GSK3 and sporadic Alzheimer disease. Finally, we include a brief review of the pathology of animal models overexpressing GSK3.

FTDP-17 Mutations in tau Transgenic Mice Provoke Lysosomal Abnormalities and Tau Filaments in Forebrain

The tauopathies, which include Alzheimers disease (AD) and frontotemporal dementias, are a group of neurodegenerative disorders characterized by filamentous Tau aggregates. That Tau dysfunction can cause neurodegeneration is indicated by pathogenic tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To investigate how Tau alterations provoke neurodegeneration we generated transgenic mice expressing human Tau with four tubulin-binding repeats (increased by FTDP-17 splice donor mutations) and three FTDP-17 missense mutations: G272V, P301L, and R406W. Ultrastructural analysis of mutant Tau-positive neurons revealed a pretangle appearance, with filaments of Tau and increased numbers of lysosomes displaying aberrant morphology similar to those found in AD. Lysosomal alterations were confirmed by activity analysis of the marker acid phosphatase, which was increased in both transgenic mice and transfected neuroblastoma cells. Our results show that Tau modifications can provoke lysosomal aberrations and suggest that this may be a cause of neurodegeneration in tauopathies.

Altered P2X7-receptor level and function in mouse models of Huntington’s disease and therapeutic efficacy of antagonist administration

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntingtons disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP‐gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7‐mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7‐receptor level and altered P2X7‐mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7‐receptor stimulation. Finally, in vivo administration of the P2X7‐antagonist Brilliant Blue‐G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor‐coordination deficits. These in vivo data strongly suggest that altered P2X7‐receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.—Diaz‐Hernandez, M., Diez‐Zaera, M., Sanchez‐Nogueiro, J., Gomez‐Villafuertes, R., Canals, J.M., Alberch, J., Miras‐Portugal, M.T., Lucas, J.J. Altered P2X7‐receptor level and function in mouse models of Huntingtons disease and therapeutic efficacy of antagonist administration. FASEB J. 23, 1893–1906 (2009)

Chronic lithium administration to FTDP‐17 tau and GSK‐3β overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre‐formed neurofibrillary tangles do not revert

Glycogen synthase kinase‐3 (GSK‐3) has been proposed as the main kinase able to aberrantly phosphorylate tau in Alzheimers disease (AD) and related tauopathies, raising the possibility of designing novel therapeutic interventions for AD based on GSK‐3 inhibition. Lithium, a widely used drug for affective disorders, inhibits GSK‐3 at therapeutically relevant concentrations. Therefore, it was of great interest to test the possible protective effects of lithium in an AD animal model based on GSK‐3 overexpression. We had previously generated a double transgenic model, overexpressing GSK‐3β in a conditional manner, using the Tet‐off system and tau protein carrying a triple FTDP‐17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation. This transgenic line shows tau hyperphosphorylation in hippocampal neurones accompanied by neurofibrillary tangles (NFTs). We used this transgenic model to address two issues: first, whether chronic lithium treatment is able to prevent the formation of aberrant tau aggregates that result from the overexpression of FTDP‐17 tau and GSK‐3β; second, whether lithium is able to change back already formed NFTs in aged animals. Our data suggest that progression of the tauopathy can be prevented by administration of lithium when the first signs of neuropathology appear. Furthermore, it is still possible to partially reverse tau pathology in advanced stages of the disease, although NFT‐like structures cannot be changed. The same results were obtained after shut‐down of GSK‐3β overexpression, supporting the possibility that GSK‐3 inhibition is not sufficient to reverse NFT‐like aggregates.

Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntingtons disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntingtons disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntingtons disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntingtons disease, and suggest that activation of these receptors in patients with Huntingtons disease may attenuate disease progression.

N-terminal Cleavage of GSK-3 by Calpain A NEW FORM OF GSK-3 REGULATION

Although GSK-3 activity can be regulated by phosphorylation and through interaction with GSK-3-binding proteins, here we describe N-terminal proteolysis as a novel way to regulate GSK-3. When brain extracts were exposed to calcium, GSK-3 was truncated, generating two fragments of ∼40 and 30 kDa, a proteolytic process that was inhibited by specific calpain inhibitors. Interestingly, instead of inhibiting this enzyme, GSK-3 truncation augmented its kinase activity. When we digested recombinant GSK-3α and GSK-3β protein with calpain, each isoform was cleaved differently, yet the truncated GSK-3 isoforms were still active kinases. We also found that lithium, a GSK-3 inhibitor, inhibits full-length and cleaved GSK-3 isoforms with the same IC50 value. Calpain removed the N-terminal ends of His-tagged GSK-3 isoenzymes, and exposing cultured cortical neurons with ionomycin, glutamate, or N-methyl-d-aspartate led to the truncation of GSK-3. This truncation was blocked by the calpain inhibitor calpeptin, at the same concentration at which it inhibits calpain-mediated cleavage of NMDAR-2B and of p35 (the regulatory subunit of CDK5). Together, our data demonstrate that calpain activation produces a truncation of GSK-3 that removes an N-terminal inhibitory domain. Furthermore, we show that GSK-3α and GSK-3β isoenzymes have a different susceptibility to this cleavage, suggesting a means to specifically regulate these isoenzymes. These data provide the first direct evidence that calpain promotes GSK-3 truncation in a way that has implications in signal transduction, and probably in pathological disorders such as Alzheimer disease.

Cooexpression of FTDP-17 tau and GSK-3β in transgenic mice induce tau polymerization and neurodegeneration

Here we have tested whether tau modification either by point mutation or by hyperphosphorylation can exert maximal pathogenic effects or if, on the contrary, both types of tau modifications can act synergistically to induce neuropathology. For this, we have combined transgenic mice overexpressing the enzyme GSK-3beta (Tet/GSK-3beta mice), with transgenic mice expressing Tau with a triple FTDP-17 mutation which develop prefibrillar tau-aggregates (VLW mice). Tet/GSK-3beta/VLW transgenic mice show tau hyperphosphorylation in hippocampal neurons. This is accompanied by thioflavin-S staining, and formation of filaments similar in width to those found in tauophaties. Finally, the atrophy of the hippocampal dentate gyrus observed in Tet/GSK-3beta mice develops much faster in Tet/GSK-3beta/VLW mice. All these morphological and biochemical data demonstrate that there is a synergistic contribution of both types of tau modifications and that the potential of GSK-3 inhibitors for AD therapeutics also extends to tauopathies caused by point mutations in tau gene.