Fig 1: Expression and localization of GluA3 in 12- and 24-month-old App knock-in mice. (A, B) Brain sections from 12- and 24-month-old Appwt, AppNL-F, AppNL-G-F mice show wide distribution of GluA3 in CA3, CA1 and dentate gyrus (DG) region of the hippocampus. Representative images show an overall decrease of GluA3 immunoreactivity throughout the hippocampal structures in both App knock-in models at 12 months of age and in 24-month-old AppNL-F mice. (C) In the CA1 region, GluA3 immunofluorescence was predominantly located in the stratum piramidale (SP) with moderate immunofluorescence detected also in the stratum radiatum (SR) and stratum lacunosum (SL). Throughout the whole CA1 region, GluA3 immunoreactivity was decreased in 24-month-old App knock-in models when compared with Appwt mice. (D) Decreased GluA3 immunoreactivity was observed in the piriform cortex (PC) of 24-month-old AppNL-F mice. (E, F) Representative images of Iba1 and Glu3A reveal co-staining of microglia and GluA3 in AppNL-G-F mouse brain with strong GluA3 immunoreactivity around FSB-positive Aß plaques. (G) GFAP and GluA3 immunostaining reveals absence of GluA3 immunoreactivity in astrocytes. Scale bars represent 200 µm or 50 µm. Data represent mean ± SEM. (n = 4–6 mice per group, and a minimum of two sections per mouse were used for quantification). In the case of Appwt, AppNL-F three male and one female mouse were included, while for AppNL-G-F four male and two female mice were analysed. Kruskal–Wallis`s test followed by Dunn`s multiple comparison`s test has been used, * denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001.
Fig 2: CSF alterations correlate significantly with brain regional changes in App knock-in mice. CSF concentrations of all three synaptic proteins correlated with hippocampal concentrations of the respective proteins (C, B, C), moreover CSF concentrations of GluA3 further correlated with PFC concentrations (C) Pearson’s correlation was used to determine associations. Abbreviations: CSF, cerebrospinal fluid; PFC, prefrontal cortex.
Fig 3: Altered concentration of ZnT3, Dyn1 and GluA3 in the brains of App knock-in mice. Violin plots represent synaptic protein concentrations measured by ELISA showing median, quartiles and individual values; differences between the groups were assessed by Kruskal–Wallis`s test followed by Dunn`s multiple comparison test. Overall hippocampal decrease in ZnT3 (A) and GluA3 (C) concentrations, with additional cortical drop in GluA3 concentrations were found in both 12-month-old App knock-in mice. (B) No major changes in Dyn1 concentrations were detected at 12 months. (D) Concentrations of ZnT3 in 24-month-old animals showed similar levels throughout all the genotypes. Reduction of both Dyn1 (E) and GluA3 (F) was observed in the hippocampi of both 24-month-old App knock-in models when compared with Appwt mice. Age-related concentration changes in the 12- (transparent shape) and 24-month-old (filled shape) App knock-in mice (G, H, I). In all cases *denotes significance with *P = 0.05 **P = 0.01, *** P = 0.001. Abbreviations: CSF, cerebrospinal fluid; PFC, prefrontal cortex.
Fig 4: Increased CSF concentrations of synaptic proteins in dementia patients (A–C) and App knock-in mice (D–F). Violin plots represent synaptic protein concentrations measured by ELISA showing median, quartiles and individual values (Kruskal–Wallis’s test followed by Dunn’s multiple comparison) (A–C) All three synaptic proteins show significantly elevated levels in Alzheimer’s disease compared with control cases. Additionally, ZnT3 (A) and GluA3 (C) concentrations are elevated in MCI patients. When MCI group was further stratified based on the available clinical follow-up data, MCI Cv group show elevated ZnT3 concentrations in comparison to MCI NCv group. Also, MCI nCv concentrations are significantly lower than Alzheimer’s disease cases. (D–F) CSF concentrations of GluA3 (F) were significantly increased in AppNL-G-F knock-in mice compared with Appwt mice. All values are shown as the mean ± SD. In all cases * denotes significance with P = 0.05, **P = 0.01, *** P = 0.001 level; # denotes comparison including C, MCI Cv, MCI nCv and Alzheimer’s disease cases only. Abbreviations: C, control; Cv, converter; CSF, cerebrospinal fluid; MCI mild cognitive impairment; nCv, non-converter.
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