Increased tau phosphorylation and cleavage in mouse models of type 1 and type 2 diabetes

Abstract

As the population of the United States ages, the incidence of age-related neurodegenerative and systemic diseases including Alzheimer's disease (AD) and diabetes is increasing rapidly. Multiple studies report that patients with diabetes have a 50-75% increased risk of developing AD compared with age- and gender-matched patients without diabetes. Abnormally phosphorylated tau is a major building block of neurofibrillary tangles, a classic neuropathological characteristic of AD. In addition, proteolytic tau cleavage promotes AD progression due to cleaved tau serving as a nucleation center for the pathological assembly of tau filaments. The current study examines tau modification in type 1 (streptozotocin-injected) and type 2 (db/db) mouse models of diabetes. Tau phosphorylation is increased in the cortex and hippocampus of db/db mice compared with db+ control mouse brain. Interestingly, there is an age-dependent increase in tau cleavage that is not observed in age-matched control db+ animals. Streptozotocin injection also increased tau phosphorylation; however, the increase was less significant compared with the type 2 mouse model, and more importantly, no tau cleavage was detected. Our results suggest tau modification caused by insulin dysfunction and hyperglycemia may contribute to the increased incidence of AD in diabetes. We hypothesize that type 1 and type 2 diabetes may contribute to AD through different mechanisms; in type 2 diabetes, hyperglycemia-mediated tau cleavage may be the key feature, whereas insulin deficiency may be the major contributing factor in type 1 diabetes.

Figure 1

Age-dependent increase of tau phosphorylation in db/db mouse brain. Cortex and hippocampus from db+ and db/db mouse brains at 2, 8, 16 (A), and 24 (B) wk of age were homogenized in T-PER buffer. A and B, Lysates immunoblotted with the indicated antibodies; C, relative density of phosphorylated tau (pTau) over total tau (Tau5) from the same mouse measured after immunoblotting. *, P < 0.05; **, P < 0.01; #, P < 0.001 by t test. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; pT231, phosphorylated threonine 231.

Figure 2

Tau phosphorylation is increased at multiple sites in 24-wk-old db/db mouse brain. Brain slices from db+ (A, C, and D) and db/db (B, E, and F) mouse are stained with antibodies against phosphorylated tau (green) at Ser396 (pS396; A, B, C, and E) or Ser422 (pS422; D and F) and DAPI for nuclear staining (blue). Arrows indicate the increased tau staining in db/db brains in both hippocampus (A and B) and cortex (C–F). Insets show the increased tau phosphorylation in each hippocampal region in higher magnification. Bar, 100 μm.

Figure 3

Increased expression of cleaved tau in older db/db mouse brain. The lysates of cortex or hippocampus from db+ and db/db mouse brains were immunoblotted with TauC3 antibody. There is an age-dependent increase of cleaved tau expression (arrowheads), including a smaller fragment around 25 kDa (arrows), in db/db mouse brains.

Figure 4

Increased TauC3 immunostaining in 24-wk-old db/db mouse hippocampus. Brain slices from 24-wk-old db+ and db/db mice were processed for IHC using TauC3 antibody detecting cleaved tau (green). Nuclei are stained with DAPI (blue). Arrows indicate TauC3-immunopositive cells in db/db mouse hippocampus. Bar, 100 μm.

Figure 5

Tau phosphorylation, but not cleavage, is increased in STZ-injected animals. Mice were injected with STZ at 12 wk of age and killed at 24 wk (12 wk diabetes). The low-STZ group received 50 mg/kg STZ for 5 consecutive days, and the high-STZ group received one injection of 150 mg/kg. A, Cortex lysates are immunoblotted for the phosphorylated (pTau) and total tau. B, The densitometric analysis indicates significant increase in tau phosphorylation at pT231 and pS199/202 for the high-STZ group compared with control or low-STZ group. *, P < 0.01. C, Brain slices are stained with antibody against phosphorylated tau at Ser199/202 (pS199/202). Arrows indicate the increased tau staining in high-dose STZ brains. Bar, 50 μm. D, Cortex lysates are immunoblotted with TauC3 antibody to detect cleaved tau (arrows). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.