GLT-1 Knockdown Inhibits Ceftriaxone-Mediated Improvements on Cognitive Deficits, and GLT-1 and xCT Expression and Activity in APP/PS1 AD Mice

Abstract

Objective Glutamate transporter-1 (GLT-1) and system x c – mediate glutamate uptake and release, respectively. Ceftriaxone has been reported to upregulate GLT-1 expression and improve cognitive decline in APP/PS1 mice. The aim of the present study was to elucidate the role of GLT-1 in ceftriaxone-mediated improvement on cognitive deficits and associated changes in xCT (catalytic subunit of system x c – ) expression and activity using GLT-1 knockdown APP/PS1 mice. Methods GLT-1 knockdown (GLT-1 ± ) mice were generated in C57BL/6J mice using the CRISPR/Cas9 technique and crossed to APP/PS1 mice to generate GLT-1 ± APP/PS1 mice. The cognition was evaluated by novel object recognition and Morris water maze tests. GLT-1 and xCT expression, GLT-1 uptake for glutamate, and glutathione levels of hippocampus were assayed using Western blot and immunohistochemistry, 3 H-glutamate, and glutathione assay kit, respectively. Results In comparison with wild-type mice, APP/PS1 mice exhibited significant cognitive deficits, represented with poor performance in novel object recognition and Morris water maze tests, downregulated GLT-1 expression and glutamate uptake. Ceftriaxone treatment significantly improved the above impairments in APP/PS1 mice, but had negligible impact in GLT-1 ± APP/PS1 mice. The xCT expression increased in APP/PS1 and GLT-1 ± APP/PS1 mice. This upregulation might be a compensatory change against the accumulated glutamate resulting from GLT-1 impairment. Ceftriaxone treatment restored xCT expression in APP/PS1 mice, but not in GLT-1 ± APP/PS1 mice. Glutathione levels decreased in APP/PS1 mice in comparison to the wild-type group. After ceftriaxone administration, the decline in glutathione level was restored in APP/PS1 mice, but not in GLT-1 ± APP/PS1 mice. Conclusion Ceftriaxone improves cognitive impairment of APP/PS1 mice by upregulating GLT-1-mediated uptake of glutamate and co-regulation of GLT-1 and xCT in APP/PS1 mice.

Ceftriaxone improves impairments in synaptic plasticity and cognitive behavior in APP/PS1 mouse model of Alzheimer's disease by inhibiting extrasynaptic NMDAR‐STEP61 signaling

“…Abnormal activation of extrasynaptic NMDAR is one of the mechanisms involved in the occurrence and development of synaptic plasticity damage and cognitive impairment in AD (Bordji et al, 2010; Sun et al, 2016; Talantova et al, 2013; Wang & Reddy, 2017). Our recent study has shown that Cef plays an anti‐AD role by upregulating the GLT‐1 expression, promoting glutamate–glutamine cycle, and improving cognitive impairment in APP/PS1 AD mice (Fan et al, 2018; Gao et al, 2020). This study demonstrated that Cef improved cognitive impairment and synaptic plasticity in APP/PS1 AD mice by inhibiting extrasynaptic NMDAR and increasing STEP 61 expression, and provided new evidence for elucidating the mechanism by which Cef plays an anti‐AD role.…”

“…Briefly, Cef was administered to the mice via an intraperitoneal injection once daily for 14 days at a dose of 200 mg/kg in all groups subjected to the Cef treatment. This Cef dose and protocol significantly upregulated the GLT‐1 expression and its glutamate uptake (Gao et al, 2020; Rothstein et al, 2005). The mice in the wild‐type and APP/PS1 groups received normal saline as vehicle control of Cef with the same protocols.…”

“…The MWM test was performed to evaluate spatial learning and memory cognition according to previous reports (Fan et al, 2018; Gao et al, 2020). The apparatus consisted of a plastic pool (diameter: 120 cm, height: 50 cm), an escape platform (diameter: 9 cm) placed in the first quadrant 1 cm below the water surface, and a monitoring camera focused on the pool.…”

“…Several studies have demonstrated the beneficial effects of Cef treatment in animal models of human diseases associated with extracellular glutamate accumulation. Our previous studies showed that Cef upregulated the GLT‐1 expression, enhanced its glutamate uptake, and improved cognitive impairment in APPswe/PS1dE9 (APP/PS1) AD mice at an early stage (Fan et al, 2018; Gao et al, 2020). Therefore, to further prove whether Cef improves cognitive impairment in AD by inhibiting extrasynaptic NMDAR activation, this study investigated its effect on the expression of extrasynaptic NMDAR and its downstream signals, including STEP 61 and p38 MAPK, the impact of STEP 61 modulation on the Cef‐induced effects, and the relevance of these changes to the beneficial effects of Cef on impaired synaptic plasticity and cognitive behavior in APP/PS1 AD mice.…”

Asymmetric dysregulation of glutamate dynamics across the synaptic cleft in a mouse model of Alzheimer’s disease

“…After blocking, slides were incubated in primary antibody (guinea pig anti-GLT-1, 1:500, Abcam, AB1783) overnight at 4 ℃. The specificity of this particular antibody is nicely demonstrated in a recent study using Western blot and immunohistochemistry showing clear GLT-1 reductions in GLT-1 ± mice and significant GLT-1 upregulation following ceftriaxone treatment [ 33 ]. Slides were then washed three times in 0.01 M PBS for 10 min each and incubated in secondary antibody (Alexa fluor 647-conjugated donkey anti-guinea pig, 1:500, Jackson Immunoresearch Labs) at room temperature for 2 h. After the final washes in 3 times in 0.01 M PBS for 10 min each, slides were cover slipped using Dako mounting medium containing DAPI.…”