Ritchie Williamson

Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimers disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimers disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimers disease.

Membrane-bound β-amyloid oligomers are recruited into lipid rafts by a fyn-dependent mechanism

Recently published research indicates that soluble oligomers of β‐amyloid (Aβ) may be the key neurotoxic species associated with the progression of Alzheimers disease (AD) and that the process of Aβ aggregation may drive this event. Furthermore, soluble oligomers of Aβ and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Aβ on neuronal plasma membranes where exogenously applied Aβ in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL‐induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Aβ accumulation and redistribution on the neuronal mem brane and alterations in the protein composition of lipid rafts. In addition, our data from fyn−/− transgenic mice show that accumulation of Aβ on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Aβ and subsequent cell death. These results identify fyn‐depen dent Aβ redistribution and accumulation in lipid rafts as being key to ADDL‐induced cell death and defines a mechanism by which oligomers of Aβ and tau accumu late in lipid rafts.—Williamson, R., Usardi, A., Hanger, D. P., Anderton, B. H. Membrane‐bound β‐amyloid oligomers are recruited into lipid rafts by a fyn‐depen dent mechanism. FASEB J. 22, 1552–1559 (2008)

Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain

J. Neurochem. (2010) 115, 974–983.

The antidepressant clomipramine regulates cortisol intracellular concentrations and glucocorticoid receptor expression in fibroblasts and rat primary neurones.

Incubation of LMCAT fibroblasts cells with antidepressants potentiates glucocorticoid receptor (GR)-mediated gene transcription in the presence of cortisol, but not of corticosterone. We have suggested that antidepressants do so by inhibiting the LMCAT cells membrane steroid transporter and thus by increasing cortisol intracellular concentrations. We now confirm and extend this model to primary neuronal cultures. Clomipramine, a tricyclic antidepressant, increased the intracellular accumulation of 3H-cortisol, but not 3H-corticosterone, in LMCAT cells (+80%) and primary rat neurones (+20%). The latter finding is the first demonstration that a membrane steroid transporter is present in neurones. Moreover, verapamil, a membrane steroid transporter inhibitor, reduced the effects of clomipramine on the intracellular accumulation of 3H-cortisol in LMCAT cells. Finally, clomipramine also decreased GR expression (whole-cell Western blot) in LMCAT cells (50% reduction) and primary rat neurones (80% reduction). This GR downregulation can explain the reduced GR-mediated gene transcription previously described under experimental conditions that do not elicit the effects on the LMCAT cells steroid transporter. This work further supports the hypothesis that membrane steroid transporters regulating the access of glucocorticoids to the brain in vivo are a fundamental target for antidepressant action.

High fat feeding promotes simultaneous decline in insulin sensitivity and cognitive performance in a delayed matching and non-matching to position task.

Obesity is the single greatest risk factor for the development of Type 2 diabetes mellitus (T2DM), with the prevalence of both dramatically increasing in recent years. These conditions are associated with medical complications such as hypertension, neuropathy and cardiovascular disease. Recent evidence also suggests a greater risk of developing dementia including Alzheimers disease. The molecular mechanisms governing these changes remain obscure, although epidemiological evidence suggests that reduced insulin sensitivity (a characteristic of T2DM) is an independent risk factor for Alzheimers disease. Here we examine the effects of diet-induced insulin resistance on cognitive ability in an animal model not predisposed to develop Alzheimers pathology. Following 12 weeks on a high fat diet (45% of calories as crude fat) male Wistar rats were overweight and insulin resistant but not frankly diabetic. High fat fed animals were consistently poorer in all aspects of an operant based delayed matching to position task, yet were not impaired in spatial working memory as judged by the open field watermaze test. The cognitive deficit of the HF fed animals was most apparent when the task was switched from matching to non-matching to position, suggestive of an inability to change contingency. Performance in this task was negatively correlated with whole body insulin sensitivity but not weight gain. In conclusion this study has shown that insulin resistant animals exhibit impairments in an operant measure of behavioural flexibility which precede the development of diabetes.

The Microtubule-Associated Protein Tau is Also Phosphorylated on Tyrosine

Tau protein is the principal component of the neurofibrillary tangles found in Alzheimers disease (AD), where it is hyperphosphorylated on serine and threonine residues. It is hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilization of microtubules. There is now evidence that phosphorylation of tau can also occur on tyrosine residues. Human tau has five tyrosines numbered 18, 29, 197, 310, and 394, according to the sequence of the longest CNS isoform. Tyrosines 18, 197, and 394 have been shown to be phosphorylated in the brain of patients with AD whereas tyrosine 394 is the only residue that has been described to date that is phosphorylated in physiological conditions. Src family kinases and spleen tyrosine kinase (Syk) have been shown to phosphorylate tyrosine 18 while c-Abl is capable of phosphorylating tyrosine 394. Recently, a dual specificity kinase termed TTBK1 has been characterized in human brain and shown to be able to phosphorylate residue 197 of tau. Data about the role of tau tyrosine phosphorylation in neuronal physiology are still scarce and preliminary. In contrast, there is mounting evidence suggesting that tau tyrosine phosphorylation is an early event in the pathophysiology of AD and that Fyn and c-Abl are critical in the neurodegenerative process which occurs in tauopathies.

Tyrosine phosphorylation of tau regulates its interactions with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau

Recent reports have demonstrated that interactions between the microtubule‐associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to β‐amyloid (Aβ) in models of Alzheimer’s disease. Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Aβ‐induced neurotoxicity. Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains. Tau is known to bind to Fyn via its Src‐homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau. Here, we show that Pro216 within the PXXP(213–216) motif in tau plays an important role in mediating the interaction of tau with Fyn‐SH3. We also show that tau interacts with the SH2 domain of Fyn, and that this association, unlike that of Fyn‐SH3, is influenced by Fyn‐mediated tyrosine phosphorylation of tau. In particular, phosphorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn‐SH2–tau interactions. Finally, we show that tyrosine phosphorylation influences the localization of tau to detergent‐resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn‐dependent. These findings may have implications for the development of novel therapeutic strategies aimed at disrupting the tau/Fyn‐mediated synaptic dysfunction that occurs in response to elevated Aβ levels in neurodegenerative disease.

CRMP2 Hyperphosphorylation is Characteristic of Alzheimer's Disease and not a Feature Common to Other Neurodegenerative Diseases

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that regulates neurite outgrowth. It is phosphorylated by Cdk5 and GSK3, and these modifications are abnormally high in the brains of Alzheimers disease (AD) patients. Increased phosphorylation of CRMP2 is also apparent in mouse models of AD that express mutated AβPP and PSEN1, but not AβPP or tau alone, where it is detectable before the appearance of amyloid plaques and neurofibrillary tangles, suggesting it is an early event in AD pathogenesis. Here, we have extended these observations by showing that CRMP2 is not hyperphosphorylated in mice overexpressing mutated PSEN1 alone, or in cultured neurons treated with soluble, oligomeric Aβ42 peptide. Similarly, CRMP2 phosphorylation was not increased in a mouse model of severe neurodegeneration (PMSC-1 knockout) or in cultured neurons subjected to neurotoxic concentrations of NMDA or staurosporine. Most interestingly, CRMP2 phosphorylation was not increased in frontal cortex from patients with frontotemporal lobar degeneration associated with mutations in MAPT or with Pick bodies. Together, these observations are consistent with the hypothesis that abnormal phosphorylation of CRMP2 is specific to AD and occurs downstream of excessive processing of AβPP, but that neither excessive Aβ42 peptide nor neurotoxicity alone are sufficient to promote hyperphosphorylation.

Tyrosine Phosphorylation of Tau by the Src Family Kinases Lck and Fyn

BackgroundTau protein is the principal component of the neurofibrillary tangles found in Alzheimers disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimers disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease.ResultsIn this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others.ConclusionsFyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.