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 = 0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p = 0.04), and positively correlated with scores on memory tests (r = 0.298, p = 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.
Glycogen synthase kinase 3-beta (GSK3B) is a serine–threonine 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 and 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 and Muyllaert et al., 2008). Two widely distributed isoforms, alpha and beta (GSK3A and GSK3B) have been described (Gould and Manji, 2005 and Forde and Dale, 2007), the latter being the most abundant in the brain (Grimes and Jope, 2001 and Planel et al., 2002). In neurons, GSK3B plays a major role in cytoskeletal organization and remodeling, being thus involved in mechanisms of synaptic plasticity, neurogenesis and resilience to neuronal injury (Grimes and Jope, 2001, Gould and Manji, 2005 and 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 and 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 (Aβ42) 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 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 and Doble and Woodgett, 2003), which may prevent Tau hyperphosphorylation and also have potential neuroprotective effects against Aβ42 toxicity (Koh et al., 2008 and 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 and 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 and Forlenza et al., 2005). In the present study, we address the feasibility and potential advantages of using human platelets to determine GSK3B activity for clinical–laboratorial 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.
