Claudie Hooper

The GSK3 hypothesis of Alzheimer's disease

J. Neurochem. (2008) 104, 1433–1439.

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.

Deletion of Irs2 reduces amyloid deposition and rescues behavioural deficits in APP transgenic mice.

As impaired insulin signalling (IIS) is a risk factor for Alzheimer’s disease we crossed mice (Tg2576) over-expressing human amyloid precursor protein (APP), with insulin receptor substrate 2 null (Irs2−/−) mice which develop insulin resistance. The resulting Tg2576/Irs2−/− animals had increased tau phosphorylation but a paradoxical amelioration of Aβ pathology. An increase of the Aβ binding protein transthyretin suggests that increased clearance of Aβ underlies the reduction in plaques. Increased tau phosphorylation correlated with reduced tau-phosphatase PP2A, despite an inhibition of the tau-kinase glycogen synthase kinase-3. Our findings demonstrate that disruption of IIS in Tg2576 mice has divergent effects on pathological processes—a reduction in aggregated Aβ but an increase in tau phosphorylation. However, as these effects are accompanied by improvement in behavioural deficits, our findings suggest a novel protective effect of disrupting IRS2 signalling in AD which may be a useful therapeutic strategy for this condition.

Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt–PCP–JNK pathway

Although the mechanism of Aβ action in the pathogenesis of Alzheimer’s disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt–planar cell polarity (PCP)–c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt–PCP–JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt–PCP–JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.

p53 is upregulated in Alzheimer's disease and induces tau phosphorylation in HEK293a cells

p53 and tau are both associated with neurodegenerative disorders. Here, we show by Western blotting that p53 is upregulated approximately 2-fold in the superior temporal gyrus of Alzheimers patients compared to healthy elderly control subjects. Moreover, p53 was found to induce phosphorylation of human 2N4R tau at the tau-1/AT8 epitope in HEK293a cells. Confocal microscopy revealed that tau and p53 were spatially separated intracellularly. Tau was found in the cytoskeletal compartment, whilst p53 was located in the nucleus, indicating that the effects of p53 on tau phosphorylation are indirect. Collectively, these findings have ramifications for neuronal death associated with Alzheimers disease and other tauopathies.

GSK3α exhibits β‐catenin and tau directed kinase activities that are modulated by Wnt

In the presence of a Wnt signal β‐catenin is spared from proteasomal degradation through a complex mechanism involving GSK3β, resulting in the transcription of Wnt target genes. In this study we have explored whether GSK3α, a related isoform, can also regulate nuclear β‐catenin levels and whether this and the tau‐directed kinase activity of GSK3α are modulated by Wnt. GSK3α or GSK3β and their substrates, β‐catenin and tau, were transiently expressed in mammalian cells. Immunoblotting revealed that GSK3α reduces nuclear levels of β‐catenin, whilst reporter gene assays demonstrated that GSK3α inhibits β‐catenin‐directed Tcf/Lef‐dependent transcription. Moreover, activation of the Wnt pathway was found to attenuate both the β‐catenin‐ and the tau‐directed kinase activities of GSK3α and GSK3β. By immunoprecipitation we also found that axin‐1, the β‐catenin destruction complex scaffold protein, binds GSK3α. In the light of these findings GSK3α warrants further investigation regarding its involvement in Wnt signalling and tauopathies such as Alzheimers disease.

Wnt3a induces exosome secretion from primary cultured rat microglia

BackgroundMicroglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a.ResultsWnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including β-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia.ConclusionThese findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo.

Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood-brain barrier damage

J. Neurochem. (2009) 109, 694–705.

TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones.

p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter‐gene assays, we demonstrate that C‐promoter binding factor‐1 (CBF‐1)‐dependent gene transcription driven by the Notch‐1 intracellular domain (N1ICD) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH‐SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c‐Abl or cisplatin treatment also potently antagonized N1ICD/CBF‐1‐dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split‐1 (HES‐1) protein levels and antagonized the increase in HES‐1 protein induced by exogenous N1ICD expression. Evidence of a direct physical interaction between N1ICD and TAp73α was demonstrated by co‐immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73α binds the N1ICD in a region C‐terminal of aa 2094. Interestingly, ΔNp73α and TAp73αR292H also co‐purified with N1ICD, but neither inhibited N1ICD/CBF‐1‐dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73α reversed the N1ICD‐mediated repression of retinoic acid‐induced differentiation of SH‐SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73α on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis.

Chromogranin A activates diverse pathways mediating inducible nitric oxide expression and apoptosis in primary microglia

Chromogranin A (CgA) is associated with microglial activation cascades implicated in neurodegeneration in Alzheimers, Picks and Parkinsons diseases. In primary rat microglia, CgA-mediated inducible nitric oxide (iNOS) expression, nitric oxide (NO) production, mitochondrial depolarisation and apoptosis were inhibited by PP2 (Src kinase inhibitor). CgA-mediated iNOS expression and NO production were also inhibited by U0126 (MEK inhibitor), but mitochondrial depolarisation and apoptosis were not. PP2 inhibited ERK phosphorylation; therefore, Src mediates CgA-induced ERK phosphorylation leading to iNOS expression and NO production. Glutamate release induced by CgA was independent of both pathways. These findings provide insights into the way microglia are activated by CgA and the microglial signalling mechanisms associated with neurological disorders such as Alzheimers disease.