A massive and widespread increase in the phosphorylation of the microtubule-binding protein tau was found in mouse brain after depletion of insulin by administration of streptozotocin. Tau phosphorylation is a key regulator of the ability of tau to bind and stabilize microtubules (12
), suggesting that this function is impaired by insulin insufficiency. Additionally, tau hyperphosphorylation is a key early event in the pathogenesis of Alzheimer’s disease, raising the possibility that the reported association between diabetes and Alzheimer’s disease (6
) could in part be due to increased phosphorylation of tau caused by insulin deficiency. Although tau hyperphosphorylation alone is not sufficient to cause the neuropathological changes that occur in Alzheimer’s disease, preexisting tau hyperphosphorylation may sensitize neurons to subsequent or concomitant insults associated with Alzheimer’s disease to promote progressive neurodegeneration.
Our finding that mouse brain tau is hyperphosphorylated on multiple residues in this streptozotocin model of type 1 insulin-dependent diabetes extends previous reports of increases in tau phosphorylation in mouse brain in other conditions where insulin signaling was experimentally depressed. The many site-specific phosphorylation-dependent tau antibodies that are available allow precise measurements of the phosphorylation state of tau on individual residues, providing the means for identification of individual and multisite phosphorylation changes in tau.
These antibodies have previously been used in studies of mouse models of deficient insulin signaling, and the findings are all indicative that hyperphosphorylation of tau results from deficient insulin signaling in the brain. Increased brain tau phosphorylation specifically on threonine-231 (AT180 antibody) was found in adult mice with neuron-specific knockout of the insulin receptor, and this was attributed to increased activity of GSK3, which is known to phosphorylate this site on tau (19
). Similarly, Thr-231-tau was selectively hyperphosphorylated in the brains of 36-h-old insulin receptor knockout mice (20
). In insulin receptor substrate-2 (IRS-2) knockout mice, brain tau phosphorylation was increased on serine-202 (AT8 antibody), and it was suggested that this may result from a decrease in PP2A activity (21
). These previous findings indicated that eliminating insulin receptors or IRS-2 in the brain caused increased site-specific phosphorylation of tau. Our results support and extend this conclusion, showing that modeling diabetes by streptozotocin treatment causes large increases (fivefold average increase) in the phosphorylation of tau at multiple residues in both the cerebral cortex and the hippocampus. Clearly, streptozotocin-induced insulin deficiency has a more profound effect on tau phosphorylation than elimination of insulin receptors or IRS-2, suggesting that more than just deficient insulin receptor–mediated signaling is involved in the diabetic outcome of tau hyperphosphorylation on multiple residues. Thus, insulin deficiency has a much greater effect on tau phosphorylation than was previously known.
The phosphorylation state of tau results from a coordinated balance between kinase-mediated phosphorylations of tau and dephosphorylation by protein phosphatases (22
). Many kinases have been shown to phosphorylate tau but invariably on only a limited number of residues (12
). Considering that streptozotocin treatment–increased tau phosphorylation was detected with eight different phosphorylation-dependent tau antibodies, we postulated that this multisite tau hyperphosphorylation may be due to deficient protein phosphatase activity. We particularly focused on PP2A because previous studies have shown that it is the major protein phosphatase acting on tau (18
) and PP2A is decreased in Alzheimer’s disease (22
). Specifically, the activity of PP2A and PP2B toward tau was decreased ~30% in Alzheimer’s disease brain compared with matched controls (33
). In mouse brain after streptozotocin treatment, the activity of PP2A was decreased to an even greater extent, by 44% in the cerebral cortex and by 55% in the hippocampus. This was a specific effect because the activity of PP2B was not changed by streptozotocin treatment. These results support the hypothesis that PP2A, rather than PP2B, is a major regulator of tau phosphorylation in the cerebral cortex and hippocampus of type 1 insulin-dependent diabetic mice in accordance with previous reports that PP2A is the major tau phosphatase in the brain (18
). Thus, the large decrease in PP2A activity is likely to account for a majority of the large multisite increases in tau phosphorylation on multiple residues caused by streptozotocin treatment. However, these experiments do not rule out the possibility that one or more kinases may contribute to the increased tau phosphorylation because increases in the phosphorylation of p38 and JNK were detected concurrently with the decreased PP2A activity.
These findings show that streptozotocin-induced insulin deficiency shares with Alzheimer’s disease two common outcomes: reduced PP2A activity and increased tau phosphorylation. Tau hyperphosphorylation is an early event in the pathogenesis of Alzheimer’s disease, eventually aggregating into filamentous polymers and neurofibrillary tangles that parallel the progression of neuronal loss in Alzheimer’s disease. Thus, the decreased PP2A activity and tau hyperphosphorylation associated with insulin deficiency may increase the susceptibility of diabetic brain to insults associated with Alzheimer’s disease, thereby contributing to the recently recognized association between diabetes and heightened susceptibility to Alzheimer’s disease.