Immune Suppression of Glia Maturation Factor Reverses Behavioral Impairment, Attenuates Amyloid Plaque Pathology and Neuroinflammation in an Alzheimer's Disease Mouse Model

Abstract

Alzheimer's disease (AD) is an irreversible progressive neurodegenerative disorder recognized by accumulation of amyloid-plaques (APs) and neurofibrillary tangles (NFTs) and eventually loss of memory. Glia maturation factor (GMF), a neuroinflammatory protein first time isolated and cloned in our laboratory plays an important role in the pathogenesis of AD. However, no studies have been reported on whether anti-GMF antibody administration could downregulate neuroinflammation and attenuate amyloid pathology in AD brain. We investigated the potential effect of single dose of (2 mg/kg b.wt/mouse) intravenously (iv) injected with anti-GMF antibodyon cognitive function, neuroprotection, neuroinflammation and Aβ load in the brain of 9-month-old 5XFAD mice. Following 4 weeks of anti-GMF antibody delivery in mice, we found reduced expression of GMF, astrocytic glial fibrillary acidic protein (GFAP) and microglial ionizing calcium binding adaptor molecule 1 (Iba1) as well as improvement inneuroinflammatory response via inhibition of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production and amyloid pathology in the cerebral cortex and hippocampal CA1 region of 5XFAD mice. Correspondingly, blockade of GMF function with anti-GMF antibody improved spatial learning, memory, and long-term recognition memory in 5XFAD mice. The present study demonstrates that the immune checkpoint blockade of GMF function with anti-GMF antibody coordinates anti-inflammatory effects to attenuate neurodegeneration in the cortex and hippocampal CA1 region of 5XFAD mouse brain. Further, our data suggest, that pharmacological immune neutralization of GMF is a promising neuroprotective strategy totherapeutically target neuroinflammation and neurodegeneration in AD. Graphical Abstract 5XFAD mice Polyclonal anti-GMF antibody.

Keywords: Alzheimer’s disease; Amyloid pathology; Anti-GMF antibody; Cognition; Neuroinflammation.

Fig. 1

Anti-GMF antibody administration in 5XFAD mice promotes neuroprotection, lowerd Aβ accumulation and inhibits GMF expression in the frontal cortex and hippocampal CA1 region of 5XFAD mice. (a, b) Confocal immunofluorescence staining of MAP2 (green) and NeuN (red) staining following 4 weeks of anti-GMF-antibody delivery. (c, d) Quantitative analysis of MAP2 and NeuN staining. Scale bar= 50 μm. (e, f) Representative immunofluorescence staining of GMF(green) with Aβ (red) in the cortex and CA1 region of hippocampus. (g, h) Quantitative analysis of GMF and Aβ expression in the cortex and CA1 region of hippocampus. Data were represented as mean ± SEM (n=6).Scale bar= 100 μm. Statistical analysis were conducted with student^’s t test. *P < 0.05 versus 5XFAD mice.

Fig. 2

Effect of GMF-antibody administration in 5XFAD mice on glial activation and co-localization with Aβ. (a, b) Representative immunofluorescence staining of Iba1 (red) with Aβ (green) in the cortex and hippocampal CA1 region. (c, d) Quantitative analysis of Iba1 and Aβ. (e, f) Representative confocal images showing reduction in co-localization and expression of GFAP and Aβ in the cortex and CA1 region of hippocampus in the GMF-antibody treated 5XFAD mice compared with untreated 5XFAD mice. (g, h) Quantitative analysis showing significant reduction in expression of GFAP and Aβ in the cortex and CA1 region of hippocampus. Data were represented as mean ± SEM (n= 6) five sections per group. Scale bar= 50 μm. Statistical analysis were performed with student test.*P< 0.05 vs 5XFAD mice.

Fig. 3

Effect of anti-GMF antibody administration in 5XFAD mice on reactive astrogliosis. (a, b) Representative confocal images showing GFAP (green) and Iba1 (red) in the cortex and CA1 region of hippocampus. Intensity associated with GFAP and Iba1 staining were quantified and shown in (c) as percentage change versus WT group. Western blot and quantitative analysis of GFAP and Iba1 from tissue lysates obtained from cortex (d, e) and hippocampus (f, g) following 4 weeks of GMF-antibody injection. Data are presented as mean± SEM (n= 6). Scale bar= 50 μm. *P< 0.05 versus WT, ^#P< 0.05 versus 5XFAD mice.

Fig. 4

Effect of anti-GMF-antibody administration in 5XFAD mice on the expression and co-localization of GMF with reactive astrogliosis in the cortex and hippocampal CA1 region of 5XFAD mice. (a, b) Representative confocal images of GMF (green) and GFAP (red) in cortex and CA1 region of hippocampus shows co-localization and expression of GMF and GFAP is down regulated following injection of GMF-antibody. (c) Quantitative analysis of GMF and GFAP. (d, e) Representative western blots and quantitative analyses of GMF. (f, g) Representative confocal images of GMF (green) and Iba1 (red) in the cortex and CA1 region of hippocampus. The co-localization and expression of GMF (green) and Iba1 (red) is down regulated following injection of GMF-antibody. (h, i) Quantitative analysis of GMF and Iba1. Data are represented as mean ± SEM, (n=6) per group, Scale bar: 50= μm. *P< 0.05 versus WT, ^#P< 0.05 versus 5XFAD mice.

Fig. 5

Effect of GMF-antibody administration in 5XFAD mice on pro-inflammatory cytokines production in the cortex and hippocampal CA1region of 5XFAD mice. ELISA assays of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were performed using cortical homogenates following 4 weeks of anti-GMF-antibody injection (a-c). ELISA assays using hippocampal homogenates following 4 weeks of GMF-antibody injection (d-f). Data are expressed as mean ± SEM (n=6) per group and quantified as percentage changes compared with WT. *P< 0.05 versus WT, ^#P<0.05 versus 5XFAD mice.

Fig. 6

Anti-GMF IgG administration 5XFAD mice exhibit functional improvements, tested using Barnes maze task, novel object recognition and Y-maze test. (a) Escape traces of wild type mice, 5XFAD without anti-GMF-antibody injected mice and anti-GMF-antibody treated mice. Escape latency find the hidden hole from week 1 through week 4 after anti-GMF antibody injection. (b) Anti-GMF-antibody treated mice showed significantly attenuation in escape latency on week 1 through week 4. Probe test was performed following Barnes maze task. (c) Probe traces were presented for WT, 5XFAD and anti-GMF treated mice. Probe test was recorded and statistically showed 5XFAD treated with anti-GMF antibody spent more time in target quadrant compared with untreated 5XFDA mice (d). (e, f) Schematic diagram of training and test phase for the novel object recognition test. The exploration time was analyzed and statistically shown (g). Recognition index (RI) was calculated and statically compared between all groups (h). Schematic diagram of Y-maze (i). Y-maze analysis revealed that anti-GMF antibody treatment improves alternation compared with untreated 5XFAD mice (j). Total entries into each arm represented as index of locomotor activity was lowered in 5XFAD mice. Treatment with anti-GMF antibody promotes more entry of mice in each arm (k). Data are represented as mean ± SEM (n = 6) per group *P< 0.05 versus WT and ^#P< 0.05 versus 5XFAD mice.