Emma Schofield

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.

Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer's Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies.

Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects

AIMS Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. METHODS Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2 , and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. RESULTS In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. CONCLUSIONS In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker.

Quantitation of glycogen synthase kinase-3 sensitive proteins in neuronal membrane rafts

We report a quantitative proteomic study to investigate the changes induced in membrane rafts by the inhibition of glycogen synthase kinase‐3. Sensitive quantitation of membrane raft proteins using isobaric tagging chemistries was enabled by a novel hybrid proteomic method to isolate low‐microgram (10–30 μg) membrane raft protein preparations as unresolved bands in a low‐density acrylamide gel. Samples were in‐gel digested, differentially tagged and combined for 2‐D LC and quantitative MS. Analysis of hippocampal membrane preparations using this approach resulted in a sixfold increase in sensitivity and a threefold increase in the number of quantifiable proteins compared with parallel processing using a traditional in‐solution method. Quantitative analysis of membrane raft preparations from a human neuronal cell line treated with glycogen synthase kinase‐3 inhibitors SB415286 or lithium chloride, that have been reported to modulate processing of the Alzheimer amyloid precursor protein, identified several protein changes. These included decreases in lamin B1 and lamin B receptor, as well as increases in several endosome regulating rab proteins, rab5, rab7 and rab11 that have been implicated in processing of the amyloid precursor protein in Alzheimers disease.

Characterisation of tau and phospho-tau populations within CSF: The relevance to Alzheimer's biomarker development

Background: Cerebral spinal fluid (CSF) measurement of Ab1-42, T-Tau and P-Tau181 are established Alzheimer’s disease (AD) biomarkers. However, previous LC-MS/MS analysis of PHF-Tau from post mortemAD brain revealed 12 novel phosphorylation sites (Hanger et al, 2007) and additionally phosphorylation of Tyr394 (Derkinderen et al, 2005). We have previously reported (ICAD 2011) the development and preclinical application of multiplexed quantitative mass spectrometry (MS) assays (Phospho-Tau SRM) for Tau phosphorylation screening and we now endeavour to extend thesemeasurements into CSF.Methods: Pooled hospital remnant CSF (Seralab Laboratories International Ltd.), depleted of abundant proteins (MARS-6 depletion column, Agilent), was prepared for 2-DE as described by Hanisch et al., 2010, with modifications. Tau and P-Tau specific antibodies (DAKO Tau, BT2, HT7, pT181, pS202, pT231 and pS396) were used to detect spots containing different epitopes. For MS, hospital remnant CSF (1mL) was precipitated with 2.5% perchloric acid prior to immunoprecipitation (IP) with Tau and P-Tau antibodies as described by Portelius et al., 2008, with modifications. Immunoprecipitated proteins were analysed using GeLC-MS/MS (Orbitrap Velos, ThermoFisher Scientific) and the PhosphoTau SRM assay (Proteome Sciences plc). Results: Here we describe the characterisation of numerous Tau and P-Tau moieties present within CSF. 2-DE highlights multiple features with distinct iso-electric points and molecular weights. Low molecular weight species are detected at w14kDa and at least one of these moieties is found to originate from the central region of Tau. A C-terminal fragment of Tau encompassing the full DAKO epitope is also observed. Preliminary LC-MS/MS analysis of Tau species enriched from hospital remnant CSF (500uL) via IP (BT2) identified the Tau peptide TPSLPTPPTR. Analysis via LC-SRM (1mL) identified SGYSSPGSPGTPGSR, STPTAEDVTAPLVDEGAPGK, IGSTENLK, SPVVSGDTSPR and STPTAEAEEAGIGDTPSLEDEAAGHVTQAR (1N Tau isoforms). Experiments with additional IP antibodies and phosphopeptide enrichment techniques will provide further insights. Conclusions: Phospho-Tau SRM assays provide the most comprehensive phospho-site specific array for Tau. With appropriate sample preparation to enrich Tau molecules from CSF, the Phospho-Tau SRM methodology can be extended from preclinical models of AD into clinical materials (CSF). The application of Phospho-Tau SRM assays to support clinical trials will add significant value to AD drug development activities providing further diagnostic utility.