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. 2015;43(3):967-76.
doi: 10.3233/JAD-141281.

Increased tau phosphorylation and aggregation in the hippocampus of mice overexpressing corticotropin-releasing factor

Shannon N Campbell  1 , Cheng Zhang  1 , Louise Monte  1 , Allyson D Roe  1 , Kenner C Rice  2 , Yvette Taché  3 , Eliezer Masliah  4 , Robert A Rissman  1
Affiliations

Increased tau phosphorylation and aggregation in the hippocampus of mice overexpressing corticotropin-releasing factor

Shannon N Campbell et al. J Alzheimers Dis. 2015.

Abstract

Clinical and basic science research suggests that stress and/or changes in central stress signaling intermediates may be involved in Alzheimer's disease (AD) pathogenesis. Although the links between stress and AD remain unsettled, data from our group and others have established that stress exposure in rodents may confer susceptibility to AD pathology by inducing hippocampal tau phosphorylation (tau-P). Work in our laboratory has shown that stress-induced tau-P requires activation of the type-1 corticotropin-releasing factor receptor (CRFR1). CRF overexpressing (CRF-OE) mice are a model of chronic stress that display cognitive impairment at 9-10 month of age. In this study we used 6-7 month old CRF-OE mice to examine whether sustained exposure to CRF and stress steroids would impact hippocampal tau-P and kinase activity in the presence or absence of the CRFR1-specific antagonist, R121919, given daily for 30 days. CRF-OE mice had significantly elevated tau-P compared to wild type (WT) mice at the AT8 (S202/T204), PHF-1 (S396/404), S262, and S422 sites. Treating CRF-OE mice with R121919 blocked phosphorylation at the AT8 (S202/T204) and PHF-1 (S396/404) sites, but not at the S262 and S422 sites and reduced phosphorylation of c-Jun N Terminal Kinase (JNK). Examination of hippocampal extracts from CRF-OE mice at the ultrastructural level revealed negatively stained round/globular aggregates that were positively labeled by PHF-1. These data suggest critical roles for CRF and CRFR1 in tau-P and aggregation and may have implications for the development of AD cognitive decline.

Keywords: Alzheimer's disease; corticotropin-releasing factor (CRF); corticotropin-releasing factor receptor (CRFR); electron microscopy; hippocampus; immunohistochemistry; stress; tau phosphorylation (tau-P); western blot.

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Figures

Fig. 1
Fig. 1
Increased tau-P in CRF-OE mice and involvement of CRFR1. A) CRF-OE mice had significantly elevated basal levels of tau-P in the hippocampus compared to WT cohorts at all phosphoepitopes examined. B) Treatment with the selective CRFR1 antagonist, R121919, significantly reduced tau-P at the AT8 and PHF-1 sites to at or below WT levels. C) R121919 treatment did not impact phosphorylation of the S262 or S422 sites. With the exception of S262 that had no WT baseline signal for comparison all data are presented as Mean ± SEM percentage of WT values, n = 10/group (*p < 0.05 Vehicle versus R121919; +p < 0.05 CRF-OE versus WT control).
Fig. 2
Fig. 2
Cellular localization of PHF-1 site tau-P in vehicle or CRF-antagonist treated CRF-OE mice. A) Bands of phospho-tau positive axons in stratum lacunosum-moleculare. B) Treatment with R121919 blocked −1 labeling in cells in CRF-OE mice (CRF-OE + R121919), leaving unlabeled neuronal profiles starkly visible against the neuropil. C) CRF-OE mice had widespread tau-P (PHF-1) labeling in the dentate gyrus and CA3 areas, and included perikarya scattered throughout the pyramidal and proximal dendritic zones. D) No PHF-1 immunoreactivity was observed in the dentate/hilar areas in CRF-OE mice regardless of drug treatment. Scale bar = 50 μm.
Fig. 3
Fig. 3
Modulation of tau kinases in vehicle or CRFR1 antagonist treated CRF-OE mice. A) In the hippocampus, CRF-OE had significantly elevated levels of both inhibited and activated forms of GSK-3 compared to WT animals. B) GSK-3β Y216 phosphorylation levels were unaffected by drug treatment. C) Phosphorylation levels of JNK in CRF-OE were not significantly different from WT, but were significantly reduced by R121919 treatment. This is in line with our tau-P data showing dramatically reduced AT8 and PHF-1 reactivities in the hippocampus. No significant modulations of ERK1/2, CDK5, or MAPK p38 kinases were seen in the hippocampus of CRF-OE mice regardless of treatment. All data are expressed as mean ± SEM percentage of WT values, n = 10/group (*p < 0.05 Vehicle versus R121919).
Fig. 4
Fig. 4
Immunoelectron microscopy of tau-P aggregates in the hippocampal extracts from CRF-OE mice. A) CRF-OE mice had negatively stained round/globular aggregates (~50 nm diameter) which were not present with R121919 treatment. B) Aggregates in CRF-OE mice could be decorated with PHF-1 immunogold labeling. C) Negative controls included trials without primary or secondary antibodies. Scale bar = 20 nm.
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