br Discussion Firstly we find that activation of GSK

Discussion Firstly, we find that activation of GSK-3β in peripheral circulating platelet is diagnostic of cognitive impairment in T2DM patients. Accumulating laboratory evidence suggest that GSK-3β activation is involved in both T2DM and AD, such as disturbed glycogen metabolism (Rayasam et al., 2009), tau hyperphosphorylation (Martin et al., 2013), Aβ production (Phiel et al., 2003), and neurodegeneration (Hooper et al., 2008). Clinical studies also show that level of GSK-3β mRNA and protein in leucocytes is increased (Hye et al., 2005; Sheng et al., 2012), and the GSK-3β gene variants are associated with cognitive function in AD patients (Kettunen et al., 2015). However, it is not reported whether GSK-3β activity is different in human T2DM patients with or without cognitive impairment. In the present study, we found that the GSK-3β activity was much higher in T2DM-MCI patients than the cognitively healthy T2DM patients, indicating that GSK-3β activation could be an indicator of cognitive impairment in T2DM patients. This finding is of great significance because it provides a feasible tool to screen among the rapidly increasing T2DM populations who may develop into AD, by which we can also establish efficient strategies to arrest the conversion of T2DM into AD. Secondly, we find that expression of ApoE ε4 gene is strongly associated with the cognitive impairment in T2DM patients. ApoE gene has three major Mifepristone (ɛ2, ɛ3 and ɛ4) based on the amino-acid substitutions (Arg or Cys) at 112 and 158 of the protein (Sutton et al., 2015), in which the ApoE ɛ4 allele has a reduced ability to repair neuronal damage and a decreased antioxidant activity compared with the ɛ2 and ɛ3 (Ma et al., 1994). Growing genetic evidence suggests that ApoE ɛ4 allele is the major genetic risk factor for AD (Bertram et al., 2010) and is associated with an increased risk of MCI conversion into AD (Devanand et al., 2005). ~70% of AD patients have one or two copies of ApoE ɛ4. The presence of ApoE ɛ4 allele confers greater amyloid burden (Fleisher et al., 2011), changes of amyloid and tau in cerebrospinal fluids (Vemuri et al., 2010), higher degree of neurodegeneration (Caroli et al., 2010), and alterations in brain function and glucose metabolism (Langbaum et al., 2009). The T2DM patients carrying ApoE ɛ4 allele had a higher number of hippocampal neuritic plaques and neurofibrillary tangles in the cortex and hippocampus, and they had a higher risk of cerebral amyloid angiopathy (Peila et al., 2002). In the current study, we are the first to show that the expression of ApoE ɛ4 allele is strongly associated with the cognitive impairment in T2DM patients, which provides a viable genetic factor for screening the high AD risk individuals in T2DM patients. Thirdly, we find that reduction of olfactory score is indicative for cognitive impairment in T2DM patients. Olfactory dysfunction is a common and early symptom of many neurodegenerative diseases, particularly of AD and MCI (Attems et al., 2014; Lehrner et al., 2009; Christen-Zaech et al., 2003). ~86% of normal elderly participants present with Alzheimer-type neurofibrillary tangles in the olfactory bulbs, and one third of them also have amyloid deposition (Kovacs, 2004). Studies show that the identification of olfactory impairment has clinical utility as an early diagnostic biomarker for MCI and AD (Devanand et al., 2000; Djordjevic et al., 2008; Vyhnalek et al., 2015). In the present study, we find a strong association of smell identification deficit with cognitive impairment in T2DM patients, which provides an applicable clinical tool in screening the potential AD conversion in T2DM patients. We also find that combination of ApoE ε4 gene, olfactory score and the ratio of tGSK-3β/pS9GSK-3β can improve the AUC and accuracy of MCI diagnosis in T2DM patients. Additionally, we developed a simple protocol (dot blot) for measurement of total and pS9GSK-3β in human platelets. As this method is simple handling and has good reproducibility, we believe that the method may be suitable for application in clinical laboratories. Platelets and neurons have several homeostatic functions in common, such as accumulation and release of neurotransmitters, expression of membrane-bound compounds, and responsiveness to variations in calcium concentration (El Haouari and Rosado, 2009).