LAUSR

Cognitive impairment is a hallmark of neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease. Growing evidence has demonstrated that cognitive impairment is closely associated with insulin resistance. For instance, the risk of AD is increased in diabetic patients [1]. Moreover, cognitively impaired persons exhibit high levels of fasting glucose and fasting insulin, elevated Homeostasis Model Assessment, an impaired insulin response to glucose, and increased frequency of diabetes. In accord with this, intranasal insulin administration greatly improves the delayed memory and caregiver-rated functional ability of patients with amnestic mild cognitive impairment or AD [2]. Cystatin C (CysC) is a ubiquitously-expressed protein and an inhibitor of cysteine protease. It has been demonstrated that plasma CysC is a positive indicator of central adiposity and insulin resistance [3]. Recently, a clinical survey indicated that a higher titer of plasma CysC impairs insulin sensitivity in patients with type 1 diabetes [4]. This evidence implies that CysC serves as an inducer of insulin resistance. Indeed, we previously showed that CysC impairs the insulin signal pathway in hepatocytes by inducing endoplasmic reticulum (ER) stress [5]. Given that CysC is a causative factor for insulin resistance, it is reasonable to predict that elevated CysC levels lead to cognitive impairment by promoting insulin resistance. To investigate whether CysC affects the actions of insulin, we treated rat primary hippocampal neurons with recombinant CysC at different dosages. As shown in Fig. S1A, phosphorylated Akt (p-Akt) and GSK3b (pGSK3b) were dramatically increased by insulin stimulation in these neurons. However, the increases were decreased by CysC in a dose-dependent manner (Fig. S1A, B). To verify these findings, we also measured insulin-stimulated glucose uptake in primary hippocampal neurons. As expected, we found that the application of insulin greatly stimulated glucose uptake and this stimulation was largely blunted in the presence of CysC (Fig. S1C). These data showed that insulin signal transduction is blocked by CysC in primary hippocampal neurons. Next, we explored the mechanism underlying the CysCmediated blockade of insulin signal transduction in primary hippocampal neurons. It has been shown that CysC attenuates insulin signal transduction in hepatocytes by inducing ER stress [5]. Therefore, we measured the ER stress-related genes Grp78, Chop, and Erdj4 and the results showed that CysC had no effect on their expression (data not shown). Suppressor of cytokine signaling 1/3 (SOCS1/ 3) are negative regulators of insulin signal transduction [6]. Thus, we measured SOCS1/3 expression and found that the mRNA level of SOCS1 was significantly up-regulated by CysC, while SOCS3 did not change (Fig. S2A). The protein level of SOCS1 was also enhanced by CysC (Fig. S2B, C). Lan Luo and Jinyu Ma have contributed equally to this work.