Fig 2: The BXD32 retina exhibits cell death by apoptosis and proper function of microglia. Using TUNEL labeling (red) for apoptotic nuclei (arrowheads, >) and immunolabeling for monocytes (IBA1, green) and photoreceptors (RHO, blue) at p63 (A), p105 (B) and p168 (C), presumed microglia can be observed migrating to the ONL and phagocytosing apoptotic photoreceptor nuclei (arrows) the normal function of microglial cells in the retina under degenerative conditions. Nuclei stained with DAPI (white/grey). RPE, retinal pigment epithelium; ROS/IS, rod outer segments/inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm.

Fig 3: Role of GLP-1 and TRF on induced decrease in Aβ, GFAP, and IBA1 expression. (A–C) Representative hippocampus and cortex images from three groups stained for Aβ (MOAB-2), GFAP, and (IBA1), including a 4× field and a 20× field. Scale bars are 200 and 50 μm respectively. (D–F) The quantitative analysis of the positive areas of the three proteins in the hippocampal and cortical regions, respectively. Hippocampus: Averages of 4 fields per slice, n = 4 animals per group were quantified. Cortex: Averages of 5 fields per slice were quantified. n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Aβ: The Kruskal-Wallis test. GFAP: The Kruskal-Wallis test was used for quantitative analysis of hippocampal regions and Brown-Forsythe and Welch ANOVA tests for cortical regions. IBA1 of hippocampus: One-Way ANOVA followed by Dunnett’s test. IBA1 of cortex: The Kruskal-Wallis test.

Fig 4: Resident microglia and recruited macrophage numbers in the optic nerve with fingolimod treatment. (A) Resident microglia and recruited macrophages were labeled with Iba1+ TMEM119+ and Iba1+ TMEM119- in the optic nerve, respectively. (B) Comparison of microglia and macrophage numbers among groups in the optic nerve (area = 5 × 103 μm2). (C) Comparison of microglia numbers among groups in RGL (area = 5 × 103 μm2). (D) Comparison of macrophage numbers among groups in RGL (area = 5 × 103 μm2). (E) Iba1 mRNA expression in retina. Values are mean ± SD. One-way ANOVA plus Tukey’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 5 μm.

Fig 5: Extraction and characterization of synaptosomes.a Brief workflow of the current study. b Immunoblotting analysis of synaptic (SNAP25, synaptophysin and PSD95) and cytosolic (elF1a and PCNA) proteins in cytosolic fraction, synaptosomal fraction and leftover tissue debris of unaffected control postmortem brain tissues. c Densitometry analysis of synaptic and cytosolic proteins. Synaptic proteins levels (PSD95; P = 0.003), (SNAP25; P = 0.0061), (Synaptophysin; P = 0.026) were significantly higher in synaptosomes and cytosolic proteins (elF1a; P = 0.012) and (PCNA; P = 0.018) levels were significantly lower in synaptosomes relative to cytosol. d qRT-PCR analysis for mRNA fold change analysis of synaptic and cytosolic genes in cytosolic and synaptosomal fractions (n = 5). e TEM analysis of synapse assembly in synaptosomal fraction from unaffected control and AD patients’ postmortem brains (scale bar 500 nm magnification). Electron micrograph shows synapse components: Mt mitochondria, SV synaptic vesicles, PSD postsynaptic density, SC synaptic cleft. f Immunoblotting analysis of brain cells markers (Neuron-NeuN; Microglia-Iba1), excitatory synapse marker (VGLUT1) and inhibitory synapse marker (GABARA1) proteins in unaffected controls (n = 4) and AD (n = 4) synaptosomes. g Densitometry analysis of NeuN, Iba1, VGLUT1, and GABARA1 proteins in unaffected controls and AD synaptosomes. All blots are driven from the same experiment and were proceed parallelly (b, f). Values in the bar diagrams are mean ± SEM and error bars are equivalent throughout the figure (c, d, g).