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Increased platelet GSK3B activity in patients with mild cognitive impairment and Alzheimer’s disease
Journal of Psychiatric Research 45 (2011) 220e224
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Increased platelet GSK3B activity in patients with mild cognitive impairment and Alzheimer’s disease Orestes V. Forlenza*, Carolina A. Torres, Leda L. Talib, Vanessa J. de Paula, Helena P.G. Joaquim, Breno S. Diniz, Wagner F. Gattaz Laboratory of Neuroscience (LIM 27), Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, Rua Dr. Ovídio Pires de Campos 785, 05403-010 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2010 Received in revised form 25 May 2010 Accepted 1 June 2010
The disruption of glycogen synthase kinase 3-beta (GSK3B) homeostasis has implications in the pathophysiology of neuropsychiatric disorders, namely Alzheimer’s disease (AD). GSK3B activity is increased within the AD brain, favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. Such abnormality has also been detected in leukocytes of patients with cognitive disorders. The aim of the present study was to determine the expression of total and phosphorylated GSK3B at protein level in platelets of older adults with varying degrees of cognitive impairment, and to compare GSK3B activity in patients with AD, mild cognitive impairment (MCI) and healthy controls. Sixty-nine older adults were included (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 elderly controls). The expression of platelet GSK3B (total- and Ser-9 phosphorylated GSK3B) was determined by Western blot. GSK3B activity was indirectly assessed by means of the proportion between phospho-GSK3B to total GSK3B (GSK3B ratio), the former representing the inactive form of the enzyme. Ser-9 phosphorylated GSK3B was significantly reduced in patients with MCI and AD as compared to controls (p ¼ 0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p ¼ 0.04), and positively correlated with scores on memory tests (r ¼ 0.298, p ¼ 0.01). In conclusion, we corroborate previous evidence of increased GSK activity in peripheral tissues of patients with MCI and AD, and further propose that platelet GSK may be an alternative peripheral biomarker of this abnormality, provided samples are adequately handled in order to preclude platelet activation. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: GSK3B Alzheimer’s disease Mild cognitive impairment Platelets
1. Introduction Glycogen synthase kinase 3-beta (GSK3B) is a serineethreonine kinase identified in the early 80s as a key enzyme in the regulation of the glycogen synthesis, given its ability to phosphorylate and inactivate glycogen synthase (Embi et al., 1980; Chin et al., 2005; Balaraman et al., 2006). In addition to glucose metabolism, GSK3B is also involved in the regulation of critical intracellular signaling pathways, including cell cycle, gene expression and apoptosis (Jope et al., 2007; Peineau et al., 2008; Muyllaert et al., 2008). Two widely distributed isoforms, alpha and beta (GSK3A and GSK3B) have been described (Gould and Manji, 2005; Forde and Dale, 2007), the latter being the most abundant in the brain (Grimes and Jope, 2001; Planel et al., 2002). In neurons, GSK3B plays a major role in cytoskeletal organization and remodeling, being thus involved in
* Corresponding author. E-mail address: [email protected] (O.V. Forlenza). 0022-3956/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2010.06.002
mechanisms of synaptic plasticity, neurogenesis and resilience to neuronal injury (Grimes and Jope, 2001; Gould and Manji, 2005; Muyllaert et al., 2008). GSK3B is the most important Tau kinase in neurons (Lovestone et al., 1994). The phosphorylation state of Tau is determinant of its ability to stabilize microtubules. In the pathophysiology of AD, hyperactive GSK3B has been associated with the formation of paired helicoidal filaments (PHF-Tau) and, therefore, of neurofibrillary tangles (Frame and Cohen, 2001; Lovestone and McLoughlin, 2002; Kar et al., 2004; Balaraman et al., 2006). In experimental models of AD, GSK3B has been shown to hyperphosphorylate Tau, leading to microtubule disassembly and loss of function (Lovestone et al., 1994). In addition, the activation of GSK3B inhibits the secretory cleavage of the amyloid precursor protein (APP), increasing the production of the amyloid-beta (Ab42) peptide (Rockenstein et al., 2007), and leads to memory impairment in animal models (Grimes and Jope, 2001). Therefore, the deregulation of GSK3B activity has major effects in key pathological features of AD and its abnormal activation may be
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implicated in the early and primary event in the physiopathology of AD (Hooper et al., 2008). Conversely, the phosphorylation of GSK3B at Serine 9 leads to its inactivation (Klein and Melton, 1996; Doble and Woodgett, 2003), which may prevent Tau hyperphosphorylation and also have potential neuroprotective effects against Ab42 toxicity (Koh et al., 2008; Noble et al., 2005). Naturalistic and observational studies carried out in patients with bipolar disorder showed that chronic intake of lithium salts, a potent GSK3B inhibitor, may reduce the prevalence of AD (Nunes et al., 2007; Kessing et al., 2008). In the search for peripheral correlates of intracerebral GSK3B, Hye et al. (2005) found a marked increase in GSK3B activity in leukocytes of patients with AD and mild cognitive impairment (MCI), as compared to age-matched controls. GSK3B is also expressed in human platelets, in amounts even higher than in leukocytes (personal observation; data available upon request). The precise physiological role of platelet GSK3B has not yet been fully determined, but there is evidence of involvement in the regulation of platelet activation, since both GSK3A and GSK3B are inhibited by phosphorylation at Ser-21 and Ser-9 (respectively) by platelet agonists (Barry et al., 2003). In addition to these mechanisms, platelet GSK3B is subject to similar regulatory mechanisms as those observed in the brain, such as phosphoinositide 3-kinase (PI3K) and PKB signaling. Finally, platelet GSK3B is inhibited by lithium (manuscript in preparation) and other specific inhibitors that have been validated in neuronal models (Martinez et al., 2002; Mendes et al., 2009). In our group, we have accumulated over the past years a reasonable expertise handling human platelets on experiments dedicated to ascertain biochemical abnormalities in patients with MCI and AD (Gattaz et al., 1996; Forlenza et al., 2005). In the present study, we address the feasibility and potential advantages of using human platelets to determine GSK3B activity for clinicalelaboratorial explorations. Therefore, the objective of the present study is to determine GSK3B expression and activity in platelets of patients with AD and MCI as compared to cognitively unimpaired controls. 2. Methods Sixty-nine older adults were recruited to this study (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 healthy elderly controls as comparison group). All patients and subjects were recruited at the Memory Clinic of the Laboratory of Neuroscience (LIM 27), Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, Brazil. Written informed consent was obtained after the procedures had been fully explained to all patients. The study was approved by the local ethics committee and conducted according to the principles of the Helsinki Declaration. Patients and controls were examined by geriatric psychiatrists with the aid of the Brazilian version of the CAMDEX (Cambridge Mental Disorders of the Elderly Examination) (Roth et al., 1986), which yields scores for the Cambridge Cognitive Test (CAMCOG) (Nunes et al., 2008) and the Mini-Mental State Examination (MMSE) (Folstein et al., 1975). All participants were further assessed at baseline with a comprehensive neuropsychological battery in order to ascertain the cognitive state, which was discussed at expert consensus meetings in the light of all available clinical data (for details on the assessment protocol, please see Diniz et al., 2008a). Laboratorial exams were carried out for every patient, encompassing thyroid function, complete blood count, blood chemistry serum levels, folic acid and vitamin B12, blood lipid profile, and syphilis tests to rule out potentially reversible causes of cognitive impairment. Neuroimaging studies (computed tomography scans or magnetic resonance imaging) were completed in patients with suspected dementing or cerebrovascular disorders.
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Exclusion criteria were illiteracy, visual and hearing disabilities, and other relevant health conditions that could either affect cognition or limit the administration of neuropsychological tests. Patients with severe dementia, concomitant psychiatric diseases, and clinical evidence of cerebrovascular disease (as the main cause of cognitive decline) were also excluded. Diagnoses of AD and MCI were established in accordance to standard diagnostic criteria (McKhann et al., 1984; Petersen et al., 2001). In the presence of common medical comorbidities such as systemic hypertension, diabetes mellitus or hypothyroidism, subjects were included only if clinically stable on the respective treatments. One patient in the AD group and one healthy control were using oral hypoglycemiants, and a statistically similar number across the three groups (AD, 11; MCI, 10; controls, 18) were receiving more than one drug for the treatment of hypertension, gastrointestinal symptoms, thyroid replacement or sleeping aids. All patients with AD (but none in the other groups) were on stable doses of cholinesterase inhibitors when included in this study. The concomitant prescription of antidepressants and antipsychotic drugs was significantly more frequent for patients in the AD group (11 and 5) as compared to MCI (2 and 0) and control subjects (2 and 0). 2.1. Determination of GSK3B protein levels Blood samples were collected in 0.1 M sodium citrate-coated tubes from a peripheral vein of the forearm of all participants between 8AM and 9AM and observing a 10e12-h fasting. Samples were homogenized in 1 ml of acid citrate dextrose solution (ACDNIH-formel-A solution) and centrifuged at 1600 rpm for 15 min at 20 C (room temperature e RT). Platelet-rich plasma (PRP) was pH adjusted to 6.5 and re-centrifuged for 10 min at 2400 rpm (RT). Pellets were resuspended in 5 ml of wash-solution (sodium citrate 30 mM pH 6.5, potassium chloride 5 mM, calcium chloride 2 mM, magnesium chloride 1 mM, glucose 5 mM, albumin 500 mg/ml, apyrase 50 mg/ml) and centrifuged for 8 min at 2400 rpm (RT). Platelet-rich pellets were then resuspended in 500 ml of Trisesucrose solution (Tris 50 mM pH 7.4, sucrose 233 mM) and stored at 70 C. Protein levels were determined for each aliquot by a modified Lowry method (Bio-Rad DC Protein Assay). A pool of platelets from healthy adults was prepared following the same protocol and was used as internal standard (IS) to correct analytical variation. The IS was analyzed in each assay. The IS densitometry was used to calculate the intra- and inter-day coefficient of variation (CV). 2.2. Western-blot analysis For each sample, 24 mg of protein (prepared in Laemmli sample buffer) was separated by electrophoresis in 10% polyacrylamide gels (150 V, 90 min) and transferred to nitrocellulose membranes (250 mA, 90 min); unspecific binding was blocked with 5% nonfat milk. For the assessment of total GSK3B, membranes were incubated for 1 h with a monoclonal, primary antibody raised against GSK3B (anti-GSK3B, Clone GSK-4B, Sigma), diluted 1:3000 in TBS-T; membranes were then washed and incubated for 1 h with the secondary antibody (Anti-mouse Ig Biotinylated, GE), diluted 1:400 in TBS-T; and subsequently incubated for 1 h with horseradish peroxidase-conjugated (GE), diluted 1:3000 in TBS-T. For the assessment of phospho-GSK3B, membranes were incubated overnight at 4 C, with a polyclonal, primary antibody raised against the phosphorylated Ser-9 epitope of GSK3B (phospho-ser-9, Cell Signaling), diluted 1:5000 in a 5% (w/v) solution of milk in TBS-T; membranes were then incubated for 1 h with a polyclonal, secondary antibody (Anti-goat Ig Biotinylated, GE) diluted 1:400 in
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5% (w/v) milk protein in TBS-T; and finally incubated for 1 h with horseradish peroxidase-conjugated (GE), 1:3000 in TBS-T. Imaging was performed in the ChemiimagerÔ 4000 equipment (Alpha Innotech), which captures the chemiluminescent light emission from the reaction of the peroxidase-conjugated antibody with the ECL reagent (GE). Light emission was continuously captured for 15 min, and the densitometry of distinct bands was performed with specific software tools. Each sample was analyzed in duplicates. The intra-assay CVs were 5.00% for total GSK3B and 5.26% for ser-9 phospho-GSK3B; the inter-assay CVs were 4.92% for total GSK3B and 3.96% for ser-9 phospho-GSK3B. GSK3B activity was indirectly inferred by the ratio between phosphorylated (inactive) to total GSK3B (GSK3B ratio ¼ phosphoGSK/total GSK) (Fig. 1). A higher GSK3B ratio indicates a higher proportion of its phosphorylated form; thus, lower enzymatic activity. 2.3. Statistical analysis KolmogoroveSmirnoff test was carried out to ascertain the normality of data distribution. As GSK3B (total and phosphorylated) and data on socio-demographic and cognitive variables showed a normal distribution, parametric tests were further carried out. Analyses of variance (ANOVA) were done to assess mean difference of GSK3B (total and phosphorylated), data on socio-demographic and cognitive variables among the diagnostic group. Pearson correlation analyses were done to assess the correlation between GSK3B (total and phosphorylated) and data on socio-demographic and cognitive variables in the total sample and in the diagnostic groups individually. As age and gender have a significant impact on cognitive performance, partial correlation analyses were carried out to control for potential effects of the former variables on the correlation between GSK3B levels (total and phosphorylated) and the GSK3B ratio and cognitive performance. All statistical analyses were done with the software Statistical Package for the Social Sciences (SPSS, Chicago, Il) v14.0 for Windows and statistical significance was set at p < 0.05.
Table 1 Socio-demographic and scores on cognitive tests according to the diagnostic groups.
Gender (M/W) Age (years) Schooling (years) CAMCOG (total score) CAMCOG (memory sub-score) MMSE
Controls (n ¼ 23)
MCI (n ¼ 22)
AD (n ¼ 24)
p
7/16 71.2 (5.3) 12.7 (5.5) 96.5 (3.6) 23.5 (2.1) 28.0 (3.3)
4/18 73,5 (6.8) 9.0 (5.7) 85.5 (6.7) 19.8 (3.8) 26.4 (2.2)
10/14 76.1 (4.2) 5.8 (4.6) 62.9 (12.0) 10.8 (4.9) 18.5 (4.1)
0.2 0.1 <0.001 <0.001 <0.001 <0.001
M: man; W: woman; CAMCOG: Cambridge Cognitive Test; MMSE: Mini-Mental State Examination. All data is depicted as mean (standard deviation).
patients with MCI, but age differences between the latter two groups were not statistically significant. One-way ANOVA showed significant differences in education level (which was accounted for by the difference between MCI and AD) and gender distribution across diagnostic groups (Table 1). There was no significant difference in total GSK3B levels between the diagnostic group (Table 2). Nevertheless, patients with MCI and AD showed a significant lower ser-9 phosphoGSK3B levels and GSK3B ratio (thus, indicating higher GSK3B activity). Such differences remained statistically significant after controlling for age, educational level and the current use of antidepressants or antipsychotics by means of analysis of covariance (ANCOVA, F ¼ 3.3, p ¼ 0.04). Ser-9 phospho-GSK3B levels and GSK3B ratio showed a positive and significant correlation with total CAMCOG scores (r ¼ 0.28, p ¼ 0.01 and r ¼ 0.29, p ¼ 0.01, respectively), CAMCOG memory subscore (r ¼ 0.27, p ¼ 0.02 and r ¼ 0.30, p ¼ 0.01, respectively) and MMSE scores (r ¼ 0.24, p ¼ 0.03 and r ¼ 0.26, p ¼ 0.02, respectively). In the partial correlation analysis, GSK3B ratio remained significantly correlated to total CAMCOG scores (r ¼ 0.28, p ¼ 0.02) and CAMCOG memory sub-score (r ¼ 0.28, p ¼ 0.02). 4. Discussion
3. Results As expected, all groups had significantly different performances on cognitive tests (Table 1). Significant age differences were observed across the three diagnostic groups. Post-hoc tests indicated that AD patients were significantly older than controls and
We developed a new protocol to estimate GSK3B activity in human platelets. The rationale for using platelets to determine GSK3B in peripheral blood relies on the fact that the expression of this enzyme is substantially higher in platelets than in leukocytes. However, platelets require careful handling in view of the possibility of activation through experimentation (Barry et al., 2003). Provided this interference is controlled for during preparation of aliquots, by using protease and phosphatase inhibitors and preserving membrane integrity, the proportion of inactive to active GSK3B adequately probably reflects the resting state in the peripheral blood of patients and controls. We found a significant increase in the proportion of active forms of the enzyme in platelets of patients with MCI and AD, as compared to cognitively healthy older adults, as indicated by the lower GSK3B ratio. In addition, we showed that increased GSK3B activity was significantly correlated with a worse performance on memory tests. To the best of our knowledge, this is the first study
Table 2 Platelet levels of total GSK3B, ser-9 phospho-GSK3B and GSK3B ratio according to the diagnostic groups.
Fig. 1. (A) Representative Western Blots showing the immunolabeling of total GSK3B and phosphor-ser-9-GSK3B of platelets from one subject and internal standard (IS). (B) Mean GSK3B ratio of platelets obtained from controls, MCI and AD.
Total GSK3B ser-9 phospho-GSK3B GSK3B ratio
Controls (n ¼ 23)
MCI (n ¼ 22)
AD (n ¼ 24)
p
0.94 (0.45) 0.62 (0.36) 0.67 (0.22)
0.81 (0.43) 0.42 (0.24) 0.54 (0.19)
0.97 (0.59) 0.46 (0.24) 0.53 (0.19)
0.3 0.04 0.04
GSK3B: glycogen synthase kinase 3-beta.
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to report increased GSK3B activity in platelets of patients within MCI and AD. The present results are in line with the findings of increased GSK3B activity in leukocytes of patients with MCI and AD (Hye et al., 2005), and suggest that the peripheral determination of GSK3B activity may be a useful diagnostic biomarker for early AD and a surrogate marker of early pathophysiological changes in pivotal cascades in the MCIeAD continuum (Diniz et al., 2008b). Platelets and neurons share several homeostatic functions, such as accumulation and release of neurotransmitters, responsiveness to variations in calcium concentration, and expression of membrane-bound compounds (e.g., receptors and enzymes) (Mohammed El and Rosado, 2009). In addition, recent evidence suggest that other peripheral correlates of the AD process, such as platelet APP ratio (Di Luca et al., 2000; Padovani et al., 2001; Zainaghi et al., 2007) and platelet phospholipase A2 activity (Gattaz et al., 2004; Forlenza et al., 2005) correlate with markers of the intracerebral pathology. Thus, if one considers the premise that the regulation of platelet GSK3B activity depends on systemic mechanisms that also influence CNS homeostasis, the present findings may indicate that hyperactive platelet GSK3B parallels intracerebral abnormalities in this system. GSK3B is inactivated by the phosphorylation of the Serine 9 epitope, which is modulated by several signal transduction pathways, including Akt/PKB, canonical Wnt pathway and the insulin cascade (Peineau et al., 2008). In addition to an abnormal regulatory shift reducing the inactivation of the enzyme, the failure to balance phosphorylated and nonphosphorylated GSK3B may suggest that upstream cascades responsible for the enzyme regulation are dysfunctional in the MCIeAD continuum. Regarding the small differences observed between MCI and AD patients, one must consider that the use of antidementia and/or other psychoactive drugs by the latter patients may have altered the balance between active and inactive GSK3B, since many such drugs (e.g., lithium, olanzapine and fluoxetine) have been shown to affect GSK3B activity (Kozlovsky et al., 2006); yet no studies addressing the effect of the treatment with cholinesterase inhibitors on GSK3B activity have been performed so far. In conclusion, we propose that the determination of platelet GSK3B ratio may be useful to ascertain biological changes in critical processes related to the pathophysiology of AD, and perhaps to monitor the effect of disease-modifying antidementia drugs aiming at GSK3B inhibition. Acknowledgments The Laboratory of Neuroscience receives financial support from Associação Beneficente Alzira Denise Hertzog da Silva (ABADHS). Contributors Orestes V. Forlenza: responsible for the study design, intellectual inputs and manuscript revision. Carolina A. Torres, Leda L. Talib, Vanessa J. de Paula, Helena P.G. Joaquim: responsible for laboratorial analysis, manuscript draft. Breno S. Diniz: responsible for statistical analysis and manuscript revision. Wagner F. Gattaz: responsible for study design, intellectual inputs and final manuscript revision. Role of the funding source The present work was supported by FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo (Project 02/13633-7) and the Alzheimer’s Association (NIRG-08-90688).
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Conflict of interest The authors declare no conflict of interest in the preparation of this manuscript. References Balaraman Y, Limaye ER, Levey AI, Srinivasan S. Glycogen synthase kinase 3B and Alzheimer’s disease: pathophysiological and therapeutic significance. Cellular and Molecular Life Science 2006;63:1226e35. Barry FA, Graham GJ, Fry MJ, Gibbins JM. Regulation of glycogen synthase kinase 3 in humans platelets: a possible role in platelet function? FEBS Letters 2003;553:173e8. Chin PC, Majdzadeh N, D’Mello SR. Inhibition of GSK3b is a common event in neuroprotection by different survival factors. Molecular Brain Research 2005;137:193e201. Di Luca M, Colciaghi F, Pastorino L, Borroni B, Padovani A, Cattabeni F. Platelets as a peripheral district were to study pathogenetic mechanisms of Alzheimer disease: the case of amyloid precursor protein. European Journal of Pharmacology 2000;405:277e83. Diniz BS, Nunes PV, Yassuda MS, Pereira FS, Flaks MK, Viola LF, et al. Mild cognitive impairment: cognitive screening or neuropsychological assessment? Revista Brasileira de Psiquiatria 2008a;30:316e21. Diniz BS, Pinto Júnior JA, Forlenza OV. Do CSF total tau, phosphorylated tau, and beta-amyloid 42 help to predict progression of mild cognitive impairment to Alzheimer’s disease? A systematic review and meta-analysis of the literature. World Journal of Biological Psychiatry 2008b;9:172e82. Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. Journal of Cell Science 2003;116:1175e86. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. European Journal of Biochemistry 1980;107:519e27. Folstein MF, Folstein SE, Mchugh PR. Mini-mental state: a practical method for grading the cognitive state of patients for the clinician. Journal of Psychiatric Research 1975;12:189e98. Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cell fate. Cellular and Molecular Life Science 2007;64:1930e44. Forlenza OV, Wacker P, Nunes PV, Yacubian J, Castro CC, Otaduy MCG, et al. Reduced phospholipid breakdown in Alzheimer’s brains: a 31P spectroscopy study. Psychopharmacology 2005;180:359e65. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. The Biochemical Journal 2001;359:1e16. Gattaz WF, Cairns NJ, Levy R, Förstl H, Braus DF, Maras A. Decreased phospholipase A2 activity in the brain and in platelets of patients with Alzheimer’s disease. European Archives of Psychiatry and Clinical Neuroscience 1996;246:129e31. Gattaz WF, Forlenza OV, Talib LL, Barbosa NR, Bottino CM. Platelet phospholipase A2 activity in Alzheimer’s disease and mild cognitive impairment. Journal of Neural Transmission 2004;111:591e601. Gould TD, Manji HK. Glycogen synthase kinase-3: a putative molecular target for lithium mimetic drugs. Neuropsychopharmacology 2005;30:1223e37. Grimes CA, Jope RS. The multifaceted roles of GSK3b in cellular signaling. Progress in Neurobiology 2001;65:391e426. Hooper C, Killick R, Lovestone S. The GSK3 hypothesis of Alzheimer’s disease. Journal of Neurochemistry 2008;104:1433e9. Hye A, Kerr F, Archer N, Foy C, Poppe M, Brown R, et al. Glycongen synthase kinase 3 is increased in white cells early in Alzheimer’s Disease. Neuroscience Letters 2005;373:1e4. Jope RS, Yuskaitis CJ, Beurel E. Glycogen synthase kinase-3 (GSK3): inflammation, diseases, and therapeutics. Neurochemistry Research 2007;32:577e95. Kar S, Slowikowski SPM, Westaway D, Mount HTJ. Interactions between b amyloid and central cholinergic neurons: implications for Alzheimer’s disease. Journal of Psychiatry and Neuroscience 2004;29:427e41. Klein PS, Melton DA. A molecular mechanism for the effect of lithium on development. Proceedings of the National Academy of Sciences of the United States of America 1996;93:8455e9. Kessing LV, Søndergård L, Forman JL, Andersen PK. Lithium treatment and risk of dementia. Archives of General Psychiatry 2008;65:1331e5. Koh S, Noh MY, Kim SY. Amyloid-beta-induced neurotoxicity is reduced by inhibition of glycogen synthase kinase-3. Brain Research 2008;1188:254e62. Kozlovsky N, Amar S, Belmaker RH, Agam G. Psychotropic drugs affect Ser9-phosphorylated GSK-3 beta protein levels in rodent frontal cortex. International Journal of Neuropsychopharmacology 2006;9:337e42. Lovestone S, Reynolds CH, Latimer D, Davis DR, Anderton BH, Gallo JM, et al. Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Current Biology 1994;4:1077e86. Lovestone S, McLoughlin DM. Protein aggregates and dementia: is there a common toxicity? Journal of Neurology Neurosurgery and Psychiatry 2002;72:152e61. Martinez A, Alonso M, Castro A, Perez C, Moreno FJ. First non-ATP competitive glycogen synthase kinase 3b (GSK-3b) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease. Journal of Medicinal Chemistry 2002;45:1292e9. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer disease: report of NINCDS-ADRDA work group under the
224
O.V. Forlenza et al. / Journal of Psychiatric Research 45 (2011) 220e224
auspices of Department of Health and Human Service Task Force on Alzheimer Disease. Neurology 1984;34:939e44. Mendes CT, Mury FB, Moreira ES, Alberto FL, Forlenza OV, Dias-Neto E, et al. Lithium reduces Gsk3b mRNA levels: implications for Alzheimer disease. European Archives of Psychiatry and Clinical Neuroscience 2009;259:16e22. Mohammed El H, Rosado JA. Platelet function in hypertension. Blood Cells, Molecules and Diseases 2009;42:38e43. Muyllaert D, Kremer A, Jaworski T, Borghgraef P, Devijver H, Croes S, et al. Glycogen synthase kinase-3b, or a link between amyloid and tau pathology? Genes, Brain and Behavior 2008;7:57e66. Nunes PV, Forlenza OV, Gattaz WF. Lithium and risk for Alzheimer’s disease in elderly patients with bipolar disorder. British Journal of Psychiatry 2007;190: 359e60. Nunes PV, Diniz BS, Radanovic M, Abreu ID, Borelli DT, Yassuda MS, et al. CAMcog as a screening tool for diagnosis of mild cognitive impairment and dementia in a Brazilian clinical sample of moderate to high education. International Journal of Geriatric Psychiatry 2008;23:1127e33. Noble W, Planel E, Zehr C, Olm V, Meyerson J, Suleman J, et al. Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proceedings of the National Academy of Sciences of the United States of America 2005;102:6990e5.
Padovani A, Pastorino L, Borroni B, Colciaghi F, Rozzini L, Monastero R, et al. Amyloid precursor protein in platelets. A peripheral marker for the diagnosis of sporadic AD. Neurology 2001;57:2243e8. Peineau S, Bradley C, Taghibiglou C, Doherty A, Bortolotto ZA, Wang YT, et al. The role of GSK-3 in synaptic plasticity. British Journal of Pharmacology 2008;153:S428e37. Petersen RC, Doody R, Kurz A, Mohs RC, Morris JC, Rabins PV, et al. Current concepts in mild cognitive impairment. Archives of Neurology 2001;58:1985e92. Planel E, Sun X, Takashima A. Role of GSK-3b in Alzheimer’s disease pathology. Drug Development Research 2002;56:491e510. Rockenstein E, Torrance M, Adame A, Mante M, Bar-on P, Rose JB, et al. Neuroprotective effects of regulators of the glycogen synthase kinase-3b signaling pathway in a transgenic model of Alzheimer’s disease are associated with reduced amyloid precursor protein phosphorylation. Journal of Neuroscience 2007;27:1981e91. Roth M, Tym E, Mountjoy CQ, Huppert FA, Hendrie H, Verma S, et al. A standardized instrument for the diagnosis of mental disorders in the elderly with special reference to early detection of dementia. British Journal of Psychiatry 1986;149:698e709. Zainaghi IA, Forlenza OV, Gattaz WF. Abnormal APP processing in platelets of patients with Alzheimer's disease: correlations with membrane fluidity and cognitive decline. Psychopharmacology 2007;192:547e53.
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Increased platelet GSK3B activity in patients with mild cognitive impairment and Alzheimer’s disease Orestes V. Forlenza*, Carolina A. Torres, Leda L. Talib, Vanessa J. de Paula, Helena P.G. Joaquim, Breno S. Diniz, Wagner F. Gattaz Laboratory of Neuroscience (LIM 27), Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, Rua Dr. Ovídio Pires de Campos 785, 05403-010 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2010 Received in revised form 25 May 2010 Accepted 1 June 2010
The disruption of glycogen synthase kinase 3-beta (GSK3B) homeostasis has implications in the pathophysiology of neuropsychiatric disorders, namely Alzheimer’s disease (AD). GSK3B activity is increased within the AD brain, favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. Such abnormality has also been detected in leukocytes of patients with cognitive disorders. The aim of the present study was to determine the expression of total and phosphorylated GSK3B at protein level in platelets of older adults with varying degrees of cognitive impairment, and to compare GSK3B activity in patients with AD, mild cognitive impairment (MCI) and healthy controls. Sixty-nine older adults were included (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 elderly controls). The expression of platelet GSK3B (total- and Ser-9 phosphorylated GSK3B) was determined by Western blot. GSK3B activity was indirectly assessed by means of the proportion between phospho-GSK3B to total GSK3B (GSK3B ratio), the former representing the inactive form of the enzyme. Ser-9 phosphorylated GSK3B was significantly reduced in patients with MCI and AD as compared to controls (p ¼ 0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p ¼ 0.04), and positively correlated with scores on memory tests (r ¼ 0.298, p ¼ 0.01). In conclusion, we corroborate previous evidence of increased GSK activity in peripheral tissues of patients with MCI and AD, and further propose that platelet GSK may be an alternative peripheral biomarker of this abnormality, provided samples are adequately handled in order to preclude platelet activation. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: GSK3B Alzheimer’s disease Mild cognitive impairment Platelets
1. Introduction Glycogen synthase kinase 3-beta (GSK3B) is a serineethreonine kinase identified in the early 80s as a key enzyme in the regulation of the glycogen synthesis, given its ability to phosphorylate and inactivate glycogen synthase (Embi et al., 1980; Chin et al., 2005; Balaraman et al., 2006). In addition to glucose metabolism, GSK3B is also involved in the regulation of critical intracellular signaling pathways, including cell cycle, gene expression and apoptosis (Jope et al., 2007; Peineau et al., 2008; Muyllaert et al., 2008). Two widely distributed isoforms, alpha and beta (GSK3A and GSK3B) have been described (Gould and Manji, 2005; Forde and Dale, 2007), the latter being the most abundant in the brain (Grimes and Jope, 2001; Planel et al., 2002). In neurons, GSK3B plays a major role in cytoskeletal organization and remodeling, being thus involved in
* Corresponding author. E-mail address: [email protected] (O.V. Forlenza). 0022-3956/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2010.06.002
mechanisms of synaptic plasticity, neurogenesis and resilience to neuronal injury (Grimes and Jope, 2001; Gould and Manji, 2005; Muyllaert et al., 2008). GSK3B is the most important Tau kinase in neurons (Lovestone et al., 1994). The phosphorylation state of Tau is determinant of its ability to stabilize microtubules. In the pathophysiology of AD, hyperactive GSK3B has been associated with the formation of paired helicoidal filaments (PHF-Tau) and, therefore, of neurofibrillary tangles (Frame and Cohen, 2001; Lovestone and McLoughlin, 2002; Kar et al., 2004; Balaraman et al., 2006). In experimental models of AD, GSK3B has been shown to hyperphosphorylate Tau, leading to microtubule disassembly and loss of function (Lovestone et al., 1994). In addition, the activation of GSK3B inhibits the secretory cleavage of the amyloid precursor protein (APP), increasing the production of the amyloid-beta (Ab42) peptide (Rockenstein et al., 2007), and leads to memory impairment in animal models (Grimes and Jope, 2001). Therefore, the deregulation of GSK3B activity has major effects in key pathological features of AD and its abnormal activation may be
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implicated in the early and primary event in the physiopathology of AD (Hooper et al., 2008). Conversely, the phosphorylation of GSK3B at Serine 9 leads to its inactivation (Klein and Melton, 1996; Doble and Woodgett, 2003), which may prevent Tau hyperphosphorylation and also have potential neuroprotective effects against Ab42 toxicity (Koh et al., 2008; Noble et al., 2005). Naturalistic and observational studies carried out in patients with bipolar disorder showed that chronic intake of lithium salts, a potent GSK3B inhibitor, may reduce the prevalence of AD (Nunes et al., 2007; Kessing et al., 2008). In the search for peripheral correlates of intracerebral GSK3B, Hye et al. (2005) found a marked increase in GSK3B activity in leukocytes of patients with AD and mild cognitive impairment (MCI), as compared to age-matched controls. GSK3B is also expressed in human platelets, in amounts even higher than in leukocytes (personal observation; data available upon request). The precise physiological role of platelet GSK3B has not yet been fully determined, but there is evidence of involvement in the regulation of platelet activation, since both GSK3A and GSK3B are inhibited by phosphorylation at Ser-21 and Ser-9 (respectively) by platelet agonists (Barry et al., 2003). In addition to these mechanisms, platelet GSK3B is subject to similar regulatory mechanisms as those observed in the brain, such as phosphoinositide 3-kinase (PI3K) and PKB signaling. Finally, platelet GSK3B is inhibited by lithium (manuscript in preparation) and other specific inhibitors that have been validated in neuronal models (Martinez et al., 2002; Mendes et al., 2009). In our group, we have accumulated over the past years a reasonable expertise handling human platelets on experiments dedicated to ascertain biochemical abnormalities in patients with MCI and AD (Gattaz et al., 1996; Forlenza et al., 2005). In the present study, we address the feasibility and potential advantages of using human platelets to determine GSK3B activity for clinicalelaboratorial explorations. Therefore, the objective of the present study is to determine GSK3B expression and activity in platelets of patients with AD and MCI as compared to cognitively unimpaired controls. 2. Methods Sixty-nine older adults were recruited to this study (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 healthy elderly controls as comparison group). All patients and subjects were recruited at the Memory Clinic of the Laboratory of Neuroscience (LIM 27), Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, Brazil. Written informed consent was obtained after the procedures had been fully explained to all patients. The study was approved by the local ethics committee and conducted according to the principles of the Helsinki Declaration. Patients and controls were examined by geriatric psychiatrists with the aid of the Brazilian version of the CAMDEX (Cambridge Mental Disorders of the Elderly Examination) (Roth et al., 1986), which yields scores for the Cambridge Cognitive Test (CAMCOG) (Nunes et al., 2008) and the Mini-Mental State Examination (MMSE) (Folstein et al., 1975). All participants were further assessed at baseline with a comprehensive neuropsychological battery in order to ascertain the cognitive state, which was discussed at expert consensus meetings in the light of all available clinical data (for details on the assessment protocol, please see Diniz et al., 2008a). Laboratorial exams were carried out for every patient, encompassing thyroid function, complete blood count, blood chemistry serum levels, folic acid and vitamin B12, blood lipid profile, and syphilis tests to rule out potentially reversible causes of cognitive impairment. Neuroimaging studies (computed tomography scans or magnetic resonance imaging) were completed in patients with suspected dementing or cerebrovascular disorders.
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Exclusion criteria were illiteracy, visual and hearing disabilities, and other relevant health conditions that could either affect cognition or limit the administration of neuropsychological tests. Patients with severe dementia, concomitant psychiatric diseases, and clinical evidence of cerebrovascular disease (as the main cause of cognitive decline) were also excluded. Diagnoses of AD and MCI were established in accordance to standard diagnostic criteria (McKhann et al., 1984; Petersen et al., 2001). In the presence of common medical comorbidities such as systemic hypertension, diabetes mellitus or hypothyroidism, subjects were included only if clinically stable on the respective treatments. One patient in the AD group and one healthy control were using oral hypoglycemiants, and a statistically similar number across the three groups (AD, 11; MCI, 10; controls, 18) were receiving more than one drug for the treatment of hypertension, gastrointestinal symptoms, thyroid replacement or sleeping aids. All patients with AD (but none in the other groups) were on stable doses of cholinesterase inhibitors when included in this study. The concomitant prescription of antidepressants and antipsychotic drugs was significantly more frequent for patients in the AD group (11 and 5) as compared to MCI (2 and 0) and control subjects (2 and 0). 2.1. Determination of GSK3B protein levels Blood samples were collected in 0.1 M sodium citrate-coated tubes from a peripheral vein of the forearm of all participants between 8AM and 9AM and observing a 10e12-h fasting. Samples were homogenized in 1 ml of acid citrate dextrose solution (ACDNIH-formel-A solution) and centrifuged at 1600 rpm for 15 min at 20 C (room temperature e RT). Platelet-rich plasma (PRP) was pH adjusted to 6.5 and re-centrifuged for 10 min at 2400 rpm (RT). Pellets were resuspended in 5 ml of wash-solution (sodium citrate 30 mM pH 6.5, potassium chloride 5 mM, calcium chloride 2 mM, magnesium chloride 1 mM, glucose 5 mM, albumin 500 mg/ml, apyrase 50 mg/ml) and centrifuged for 8 min at 2400 rpm (RT). Platelet-rich pellets were then resuspended in 500 ml of Trisesucrose solution (Tris 50 mM pH 7.4, sucrose 233 mM) and stored at 70 C. Protein levels were determined for each aliquot by a modified Lowry method (Bio-Rad DC Protein Assay). A pool of platelets from healthy adults was prepared following the same protocol and was used as internal standard (IS) to correct analytical variation. The IS was analyzed in each assay. The IS densitometry was used to calculate the intra- and inter-day coefficient of variation (CV). 2.2. Western-blot analysis For each sample, 24 mg of protein (prepared in Laemmli sample buffer) was separated by electrophoresis in 10% polyacrylamide gels (150 V, 90 min) and transferred to nitrocellulose membranes (250 mA, 90 min); unspecific binding was blocked with 5% nonfat milk. For the assessment of total GSK3B, membranes were incubated for 1 h with a monoclonal, primary antibody raised against GSK3B (anti-GSK3B, Clone GSK-4B, Sigma), diluted 1:3000 in TBS-T; membranes were then washed and incubated for 1 h with the secondary antibody (Anti-mouse Ig Biotinylated, GE), diluted 1:400 in TBS-T; and subsequently incubated for 1 h with horseradish peroxidase-conjugated (GE), diluted 1:3000 in TBS-T. For the assessment of phospho-GSK3B, membranes were incubated overnight at 4 C, with a polyclonal, primary antibody raised against the phosphorylated Ser-9 epitope of GSK3B (phospho-ser-9, Cell Signaling), diluted 1:5000 in a 5% (w/v) solution of milk in TBS-T; membranes were then incubated for 1 h with a polyclonal, secondary antibody (Anti-goat Ig Biotinylated, GE) diluted 1:400 in
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5% (w/v) milk protein in TBS-T; and finally incubated for 1 h with horseradish peroxidase-conjugated (GE), 1:3000 in TBS-T. Imaging was performed in the ChemiimagerÔ 4000 equipment (Alpha Innotech), which captures the chemiluminescent light emission from the reaction of the peroxidase-conjugated antibody with the ECL reagent (GE). Light emission was continuously captured for 15 min, and the densitometry of distinct bands was performed with specific software tools. Each sample was analyzed in duplicates. The intra-assay CVs were 5.00% for total GSK3B and 5.26% for ser-9 phospho-GSK3B; the inter-assay CVs were 4.92% for total GSK3B and 3.96% for ser-9 phospho-GSK3B. GSK3B activity was indirectly inferred by the ratio between phosphorylated (inactive) to total GSK3B (GSK3B ratio ¼ phosphoGSK/total GSK) (Fig. 1). A higher GSK3B ratio indicates a higher proportion of its phosphorylated form; thus, lower enzymatic activity. 2.3. Statistical analysis KolmogoroveSmirnoff test was carried out to ascertain the normality of data distribution. As GSK3B (total and phosphorylated) and data on socio-demographic and cognitive variables showed a normal distribution, parametric tests were further carried out. Analyses of variance (ANOVA) were done to assess mean difference of GSK3B (total and phosphorylated), data on socio-demographic and cognitive variables among the diagnostic group. Pearson correlation analyses were done to assess the correlation between GSK3B (total and phosphorylated) and data on socio-demographic and cognitive variables in the total sample and in the diagnostic groups individually. As age and gender have a significant impact on cognitive performance, partial correlation analyses were carried out to control for potential effects of the former variables on the correlation between GSK3B levels (total and phosphorylated) and the GSK3B ratio and cognitive performance. All statistical analyses were done with the software Statistical Package for the Social Sciences (SPSS, Chicago, Il) v14.0 for Windows and statistical significance was set at p < 0.05.
Table 1 Socio-demographic and scores on cognitive tests according to the diagnostic groups.
Gender (M/W) Age (years) Schooling (years) CAMCOG (total score) CAMCOG (memory sub-score) MMSE
Controls (n ¼ 23)
MCI (n ¼ 22)
AD (n ¼ 24)
p
7/16 71.2 (5.3) 12.7 (5.5) 96.5 (3.6) 23.5 (2.1) 28.0 (3.3)
4/18 73,5 (6.8) 9.0 (5.7) 85.5 (6.7) 19.8 (3.8) 26.4 (2.2)
10/14 76.1 (4.2) 5.8 (4.6) 62.9 (12.0) 10.8 (4.9) 18.5 (4.1)
0.2 0.1 <0.001 <0.001 <0.001 <0.001
M: man; W: woman; CAMCOG: Cambridge Cognitive Test; MMSE: Mini-Mental State Examination. All data is depicted as mean (standard deviation).
patients with MCI, but age differences between the latter two groups were not statistically significant. One-way ANOVA showed significant differences in education level (which was accounted for by the difference between MCI and AD) and gender distribution across diagnostic groups (Table 1). There was no significant difference in total GSK3B levels between the diagnostic group (Table 2). Nevertheless, patients with MCI and AD showed a significant lower ser-9 phosphoGSK3B levels and GSK3B ratio (thus, indicating higher GSK3B activity). Such differences remained statistically significant after controlling for age, educational level and the current use of antidepressants or antipsychotics by means of analysis of covariance (ANCOVA, F ¼ 3.3, p ¼ 0.04). Ser-9 phospho-GSK3B levels and GSK3B ratio showed a positive and significant correlation with total CAMCOG scores (r ¼ 0.28, p ¼ 0.01 and r ¼ 0.29, p ¼ 0.01, respectively), CAMCOG memory subscore (r ¼ 0.27, p ¼ 0.02 and r ¼ 0.30, p ¼ 0.01, respectively) and MMSE scores (r ¼ 0.24, p ¼ 0.03 and r ¼ 0.26, p ¼ 0.02, respectively). In the partial correlation analysis, GSK3B ratio remained significantly correlated to total CAMCOG scores (r ¼ 0.28, p ¼ 0.02) and CAMCOG memory sub-score (r ¼ 0.28, p ¼ 0.02). 4. Discussion
3. Results As expected, all groups had significantly different performances on cognitive tests (Table 1). Significant age differences were observed across the three diagnostic groups. Post-hoc tests indicated that AD patients were significantly older than controls and
We developed a new protocol to estimate GSK3B activity in human platelets. The rationale for using platelets to determine GSK3B in peripheral blood relies on the fact that the expression of this enzyme is substantially higher in platelets than in leukocytes. However, platelets require careful handling in view of the possibility of activation through experimentation (Barry et al., 2003). Provided this interference is controlled for during preparation of aliquots, by using protease and phosphatase inhibitors and preserving membrane integrity, the proportion of inactive to active GSK3B adequately probably reflects the resting state in the peripheral blood of patients and controls. We found a significant increase in the proportion of active forms of the enzyme in platelets of patients with MCI and AD, as compared to cognitively healthy older adults, as indicated by the lower GSK3B ratio. In addition, we showed that increased GSK3B activity was significantly correlated with a worse performance on memory tests. To the best of our knowledge, this is the first study
Table 2 Platelet levels of total GSK3B, ser-9 phospho-GSK3B and GSK3B ratio according to the diagnostic groups.
Fig. 1. (A) Representative Western Blots showing the immunolabeling of total GSK3B and phosphor-ser-9-GSK3B of platelets from one subject and internal standard (IS). (B) Mean GSK3B ratio of platelets obtained from controls, MCI and AD.
Total GSK3B ser-9 phospho-GSK3B GSK3B ratio
Controls (n ¼ 23)
MCI (n ¼ 22)
AD (n ¼ 24)
p
0.94 (0.45) 0.62 (0.36) 0.67 (0.22)
0.81 (0.43) 0.42 (0.24) 0.54 (0.19)
0.97 (0.59) 0.46 (0.24) 0.53 (0.19)
0.3 0.04 0.04
GSK3B: glycogen synthase kinase 3-beta.
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to report increased GSK3B activity in platelets of patients within MCI and AD. The present results are in line with the findings of increased GSK3B activity in leukocytes of patients with MCI and AD (Hye et al., 2005), and suggest that the peripheral determination of GSK3B activity may be a useful diagnostic biomarker for early AD and a surrogate marker of early pathophysiological changes in pivotal cascades in the MCIeAD continuum (Diniz et al., 2008b). Platelets and neurons share several homeostatic functions, such as accumulation and release of neurotransmitters, responsiveness to variations in calcium concentration, and expression of membrane-bound compounds (e.g., receptors and enzymes) (Mohammed El and Rosado, 2009). In addition, recent evidence suggest that other peripheral correlates of the AD process, such as platelet APP ratio (Di Luca et al., 2000; Padovani et al., 2001; Zainaghi et al., 2007) and platelet phospholipase A2 activity (Gattaz et al., 2004; Forlenza et al., 2005) correlate with markers of the intracerebral pathology. Thus, if one considers the premise that the regulation of platelet GSK3B activity depends on systemic mechanisms that also influence CNS homeostasis, the present findings may indicate that hyperactive platelet GSK3B parallels intracerebral abnormalities in this system. GSK3B is inactivated by the phosphorylation of the Serine 9 epitope, which is modulated by several signal transduction pathways, including Akt/PKB, canonical Wnt pathway and the insulin cascade (Peineau et al., 2008). In addition to an abnormal regulatory shift reducing the inactivation of the enzyme, the failure to balance phosphorylated and nonphosphorylated GSK3B may suggest that upstream cascades responsible for the enzyme regulation are dysfunctional in the MCIeAD continuum. Regarding the small differences observed between MCI and AD patients, one must consider that the use of antidementia and/or other psychoactive drugs by the latter patients may have altered the balance between active and inactive GSK3B, since many such drugs (e.g., lithium, olanzapine and fluoxetine) have been shown to affect GSK3B activity (Kozlovsky et al., 2006); yet no studies addressing the effect of the treatment with cholinesterase inhibitors on GSK3B activity have been performed so far. In conclusion, we propose that the determination of platelet GSK3B ratio may be useful to ascertain biological changes in critical processes related to the pathophysiology of AD, and perhaps to monitor the effect of disease-modifying antidementia drugs aiming at GSK3B inhibition. Acknowledgments The Laboratory of Neuroscience receives financial support from Associação Beneficente Alzira Denise Hertzog da Silva (ABADHS). Contributors Orestes V. Forlenza: responsible for the study design, intellectual inputs and manuscript revision. Carolina A. Torres, Leda L. Talib, Vanessa J. de Paula, Helena P.G. Joaquim: responsible for laboratorial analysis, manuscript draft. Breno S. Diniz: responsible for statistical analysis and manuscript revision. Wagner F. Gattaz: responsible for study design, intellectual inputs and final manuscript revision. Role of the funding source The present work was supported by FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo (Project 02/13633-7) and the Alzheimer’s Association (NIRG-08-90688).
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Conflict of interest The authors declare no conflict of interest in the preparation of this manuscript. References Balaraman Y, Limaye ER, Levey AI, Srinivasan S. Glycogen synthase kinase 3B and Alzheimer’s disease: pathophysiological and therapeutic significance. Cellular and Molecular Life Science 2006;63:1226e35. Barry FA, Graham GJ, Fry MJ, Gibbins JM. Regulation of glycogen synthase kinase 3 in humans platelets: a possible role in platelet function? FEBS Letters 2003;553:173e8. Chin PC, Majdzadeh N, D’Mello SR. Inhibition of GSK3b is a common event in neuroprotection by different survival factors. Molecular Brain Research 2005;137:193e201. Di Luca M, Colciaghi F, Pastorino L, Borroni B, Padovani A, Cattabeni F. Platelets as a peripheral district were to study pathogenetic mechanisms of Alzheimer disease: the case of amyloid precursor protein. European Journal of Pharmacology 2000;405:277e83. Diniz BS, Nunes PV, Yassuda MS, Pereira FS, Flaks MK, Viola LF, et al. Mild cognitive impairment: cognitive screening or neuropsychological assessment? Revista Brasileira de Psiquiatria 2008a;30:316e21. Diniz BS, Pinto Júnior JA, Forlenza OV. Do CSF total tau, phosphorylated tau, and beta-amyloid 42 help to predict progression of mild cognitive impairment to Alzheimer’s disease? A systematic review and meta-analysis of the literature. World Journal of Biological Psychiatry 2008b;9:172e82. Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. Journal of Cell Science 2003;116:1175e86. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. European Journal of Biochemistry 1980;107:519e27. Folstein MF, Folstein SE, Mchugh PR. Mini-mental state: a practical method for grading the cognitive state of patients for the clinician. Journal of Psychiatric Research 1975;12:189e98. Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cell fate. Cellular and Molecular Life Science 2007;64:1930e44. Forlenza OV, Wacker P, Nunes PV, Yacubian J, Castro CC, Otaduy MCG, et al. Reduced phospholipid breakdown in Alzheimer’s brains: a 31P spectroscopy study. Psychopharmacology 2005;180:359e65. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. The Biochemical Journal 2001;359:1e16. Gattaz WF, Cairns NJ, Levy R, Förstl H, Braus DF, Maras A. Decreased phospholipase A2 activity in the brain and in platelets of patients with Alzheimer’s disease. European Archives of Psychiatry and Clinical Neuroscience 1996;246:129e31. Gattaz WF, Forlenza OV, Talib LL, Barbosa NR, Bottino CM. Platelet phospholipase A2 activity in Alzheimer’s disease and mild cognitive impairment. Journal of Neural Transmission 2004;111:591e601. Gould TD, Manji HK. Glycogen synthase kinase-3: a putative molecular target for lithium mimetic drugs. Neuropsychopharmacology 2005;30:1223e37. Grimes CA, Jope RS. The multifaceted roles of GSK3b in cellular signaling. Progress in Neurobiology 2001;65:391e426. Hooper C, Killick R, Lovestone S. The GSK3 hypothesis of Alzheimer’s disease. Journal of Neurochemistry 2008;104:1433e9. Hye A, Kerr F, Archer N, Foy C, Poppe M, Brown R, et al. Glycongen synthase kinase 3 is increased in white cells early in Alzheimer’s Disease. Neuroscience Letters 2005;373:1e4. Jope RS, Yuskaitis CJ, Beurel E. Glycogen synthase kinase-3 (GSK3): inflammation, diseases, and therapeutics. Neurochemistry Research 2007;32:577e95. Kar S, Slowikowski SPM, Westaway D, Mount HTJ. Interactions between b amyloid and central cholinergic neurons: implications for Alzheimer’s disease. Journal of Psychiatry and Neuroscience 2004;29:427e41. Klein PS, Melton DA. A molecular mechanism for the effect of lithium on development. Proceedings of the National Academy of Sciences of the United States of America 1996;93:8455e9. Kessing LV, Søndergård L, Forman JL, Andersen PK. Lithium treatment and risk of dementia. Archives of General Psychiatry 2008;65:1331e5. Koh S, Noh MY, Kim SY. Amyloid-beta-induced neurotoxicity is reduced by inhibition of glycogen synthase kinase-3. Brain Research 2008;1188:254e62. Kozlovsky N, Amar S, Belmaker RH, Agam G. Psychotropic drugs affect Ser9-phosphorylated GSK-3 beta protein levels in rodent frontal cortex. International Journal of Neuropsychopharmacology 2006;9:337e42. Lovestone S, Reynolds CH, Latimer D, Davis DR, Anderton BH, Gallo JM, et al. Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Current Biology 1994;4:1077e86. Lovestone S, McLoughlin DM. Protein aggregates and dementia: is there a common toxicity? Journal of Neurology Neurosurgery and Psychiatry 2002;72:152e61. Martinez A, Alonso M, Castro A, Perez C, Moreno FJ. First non-ATP competitive glycogen synthase kinase 3b (GSK-3b) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease. Journal of Medicinal Chemistry 2002;45:1292e9. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer disease: report of NINCDS-ADRDA work group under the
224
O.V. Forlenza et al. / Journal of Psychiatric Research 45 (2011) 220e224
auspices of Department of Health and Human Service Task Force on Alzheimer Disease. Neurology 1984;34:939e44. Mendes CT, Mury FB, Moreira ES, Alberto FL, Forlenza OV, Dias-Neto E, et al. Lithium reduces Gsk3b mRNA levels: implications for Alzheimer disease. European Archives of Psychiatry and Clinical Neuroscience 2009;259:16e22. Mohammed El H, Rosado JA. Platelet function in hypertension. Blood Cells, Molecules and Diseases 2009;42:38e43. Muyllaert D, Kremer A, Jaworski T, Borghgraef P, Devijver H, Croes S, et al. Glycogen synthase kinase-3b, or a link between amyloid and tau pathology? Genes, Brain and Behavior 2008;7:57e66. Nunes PV, Forlenza OV, Gattaz WF. Lithium and risk for Alzheimer’s disease in elderly patients with bipolar disorder. British Journal of Psychiatry 2007;190: 359e60. Nunes PV, Diniz BS, Radanovic M, Abreu ID, Borelli DT, Yassuda MS, et al. CAMcog as a screening tool for diagnosis of mild cognitive impairment and dementia in a Brazilian clinical sample of moderate to high education. International Journal of Geriatric Psychiatry 2008;23:1127e33. Noble W, Planel E, Zehr C, Olm V, Meyerson J, Suleman J, et al. Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proceedings of the National Academy of Sciences of the United States of America 2005;102:6990e5.
Padovani A, Pastorino L, Borroni B, Colciaghi F, Rozzini L, Monastero R, et al. Amyloid precursor protein in platelets. A peripheral marker for the diagnosis of sporadic AD. Neurology 2001;57:2243e8. Peineau S, Bradley C, Taghibiglou C, Doherty A, Bortolotto ZA, Wang YT, et al. The role of GSK-3 in synaptic plasticity. British Journal of Pharmacology 2008;153:S428e37. Petersen RC, Doody R, Kurz A, Mohs RC, Morris JC, Rabins PV, et al. Current concepts in mild cognitive impairment. Archives of Neurology 2001;58:1985e92. Planel E, Sun X, Takashima A. Role of GSK-3b in Alzheimer’s disease pathology. Drug Development Research 2002;56:491e510. Rockenstein E, Torrance M, Adame A, Mante M, Bar-on P, Rose JB, et al. Neuroprotective effects of regulators of the glycogen synthase kinase-3b signaling pathway in a transgenic model of Alzheimer’s disease are associated with reduced amyloid precursor protein phosphorylation. Journal of Neuroscience 2007;27:1981e91. Roth M, Tym E, Mountjoy CQ, Huppert FA, Hendrie H, Verma S, et al. A standardized instrument for the diagnosis of mental disorders in the elderly with special reference to early detection of dementia. British Journal of Psychiatry 1986;149:698e709. Zainaghi IA, Forlenza OV, Gattaz WF. Abnormal APP processing in platelets of patients with Alzheimer's disease: correlations with membrane fluidity and cognitive decline. Psychopharmacology 2007;192:547e53.