Fig 1: Immunostaining displays glial activation and a reduction in the bipolar cells and photoreceptor synapses by five months of age in the Cln7 knockout (KO) mouse. Representative immunostained sections of retinas of two and five month wild-type and Cln7 KO mice. Staining was performed for GS (glutamine synthetase), GFAP (glial fibrillary acidic protein), IBA1 (ionized calcium-binding adaptor molecule 1), RBPMS (RNA binding protein with multiple splicing), PKCa (protein kinase c alpha), PSD95 (post synaptic density protein 95), and VGLUT1 (vesicular glutamine transporter 1) in either red or green fluorescence. DAPI staining shown in blue. N = three mice per group, at least three retinal sections per eye. Scale bar = 25 µm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Fig 2: In vivo induction of miR-567 by agomir inhibits the expression of NEUROD2 and PSD95 (replicate number = 5). Mice were injected with miR-567 agomir along with rTMS treatment of 10 Hz and mild cognitive impairment model induction. (A) Analysis of the results of reverse transcription-quantitative PCR detection of miR-567. (B) Analysis of the results and representative images of Western blot detection of NEUROD2 and PSDD95. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. NC+H rTMS group, mice injected with negative control (NC) agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. Agomir+H rTMS group, mice injected with miR-567 agomir, and subjected to the injection of scopolamine and the treatment of rTMS of 10 Hz. *P<0.05 vs H rTMS group, #P<0.05 vs NC+H rTMS group.
Fig 3: Enhancement of the synaptic plasticity. (A) Typical feature of neurons by tracing analysis. (B) Experimental model for dendritic structure analysis. The number of intersections was calculated per 10 µm. (C) Distance from soma. (D) Number of intersections in total. (E–H) Number of intersections in the range of 0–40 µm, 40–160 µm, 160–210 µm, and more than 210 µm. (I) Typical feature of spines at CA1 apical and basal segments. (J–K) Number of spines at CA1 apical and basal segments was analyzed. (L) Expression of PSD-95 protein in the hippocampus. (n = 5, Data are means ± SEM. Compared with APP/PS1, *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA, followed by Tukey’s multiple comparison test).
Fig 4: RH causes neuronal and dendritic loss in hippocampus under diabetic condition. (A) Representative image for neuronal loss detected by NeuN (neuron) and MAP2 (dendrite) immunostaining in CA1 region (upper) and CA3 region (lower) of hippocampus from indicated groups. The quantitative results showed on the right (n = 6 samples from six mice). Scale bar, 50 µm. DM, type 1 diabetes mellitus; DM-RH, DM mice with recurrent moderate hypoglycemia; TRPC6-/--DM, TRPC6 global knockout mice with DM; TRPC6-/--DM-RH, TRPC6-/--DM mice with recurrent moderate hypoglycemia episodes. (B) Representative image for neuronal apoptosis detected by TUNEL staining in CA1 and CA3 of hippocampus. Quantitative data were shown on the right (n = 6 sample from six mice). Scale bar, 50 µm. (C) Western blot for synapse-associated proteins including PSD95, synaptophysin (SYP), and Synapsin I (Syn I) in hippocampal homogenates (n = 3 samples from three mice). Total protein, the amount of loaded protein detected by Ponceau S. *P < .05, **P < .01. Statistical significance was assessed using a one-way ANOVA or Kruskal-Wallis test
Fig 5: Five month old Cln7 knockout (KO) mice show increased microglial activation and reduced bipolar cells and photoreceptor synapses. Retinal lysates were collected from eyes of two and five month old wild-type and Cln7 KO mice. (a) Representative Western blot membrane for RBPMS (RNA binding protein with multiple splicing), PKCa (protein kinase c alpha), PSD95 (post synaptic density protein 95), PROX1 (prospero homeobox 1), VGLUT1 (vesicular glutamine transporter 1), IBA1 (ionized calcium-binding adaptor molecule 1), B Opsin (blue cone opsin), and R/G Opsin (red/green cone opsin) along with respective GAPDH loading controls. (b) Densitometry of the Western blot results. N = three mice per group, two or more technical replicates per mouse per antibody. Error bars = SEM. One-way ANOVA with Tukey's multiple comparisons test. PKCA (*P = 0.013; **P = 0.0038); PSD95 (****P < 0.0001); IBA1 (**P = 0.0032 for comparison to two month wild-type and 0.0028 for comparison to five month wild-type; ****P < 0.0001); VGLUT1 (*P = 0.028); B Opsin (***P = 0.0004); R/G Opsin (*P = 0.039 for comparison to two month wild-type, 0.038 for five month wild-type compared to two month KO, and 0.049 for comparison of five month wild-type and five month KO).
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