Fig 1: Differentiation and lineage of NEC from different regions. DCX: (a) The SPL of the TLobe shows a band of NEC and processes but DCX is confined to occasional small immature cells. (b) In cortical layer II occasional small tufted immature DCX+ neurons were seen (arrowhead) but nestin is confined to the endothelium. (c) In the amygdala paralaminar nucleus, small DCX and nestin positive cells form distinct, intermingled populations. Mushashi: (d) Small cells in layer I of the cortex co-localized with nestin and (e) in the PVWM (arrowheads). (f). NeuN: Expression was mainly restricted to mature, large neurons but occasional expression in small NEC was observed (arrowheads). GFAP: (g) Expression was more extensive in all compartments than nestin and in this field of the SPL, NEC are mainly GFAP negative. (h) In the SGZ multipolar NEC co-expressing GFAP were seen (arrowhead). GFAP-delta: (i) There was more extensive co-expression with nestin compared with GFAP, particularly in CA1 astrocytes (arrowhead) but (j) NEC-negative cells were also observed. (k) Nestin-positive bipolar cells and threads in the PVWM were GFAP (shown) and delta negative. Aq4: (l–n). Aq4 showed extensive labeling of astrocytic processes through all regions (shown here for the SPL, SGZ, and PVWM) but in all regions NEC lacking Aq4 expression were noted (arrowheads). GS: (o) In the SPL (shown here) and cortex numerous astroglial cell bodies were positive for GS forming an extensive plexus. A striking proportion of NEC were GS negative and also observed in the (p) SGZ (arrowhead) as well as (q) fibers. (r) Excitatory amino acid 1 (EAAT1) showed labeling of astroglial like cells (arrows), including perivascular foot processes; NEC in all regions including bipolar cells (arrowhead) were EAAT1 negative. pS6: (s) Small NEC show frequent co-labeling with pS6, shown here in the SPL. CD34: (t) CD34 labeling was confined to the capillary endothelium in the PVWM and other regions including (u) the SVZ and not in NEC or elongated threads and bipolar cells. PDGFRbeta: (v) Labeled pericytes around nestin-positive endothelium (arrow) were noted in addition to scatted small multipolar cells, a small number of which co-labeled with nestin (arrowhead). Bar is equivalent to 35 microns (based on original magnifications)
Fig 2: JWA regulates GLT-1 expression via ERK/MAPK and PI3K/Akt signaling pathways.a C8D1A cells were transfected with RFP-con or RFP-JWA. After 48 h, immunofluorescence for GLT-1 (green) was immunostained with anti-GLT-1 antibody. Scale bars = 200 μm. b C8D1A cells were transfected with si-con or si-JWA for various concentration (50 pmol or 100 pmol), followed by treatment with or without MPP+ 100 µM for 24 h. Expression levels of JWA and GLT-1 were detected by western blotting, GAPDH was used as a loading control to confirm that equal amounts of protein were loaded in each line (n = 3). c [3H]-Glutamate uptake assay was performed after C8D1A cells were transfected with si-JWA or Flag-JWA (n = 3). d C8D1A cells were transfected with si-JWA or Flag-JWA, followed by western blotting analysis to detect JWA, GLAST, GLT-1, phospho- and total-ERK, P38, JNK, and Akt proteins. The quantification for analysis of JWA, GLT-1, p-ERK, p-Akt protein (n = 3). Data are presented as mean ± SEM, one-way ANOVA, *P < 0.05, **P < 0.01
Fig 3: Astrocytic JWA deficiency induces astrocyte activation and reduced the expression of GLT-1 in vivo.a Immunohistochemical staining and quantitative data for GFAP-positive neurons in the SNc (n = 4). Scale bars = 200 µm (low magnification) and 50 µm (high magnification). b, c Quantitative real-time PCR analyses for GLT-1 and GLAST are shown in the SNc. GAPDH was used as a control to normalize the differences in the amount of total RNA in each sample (n = 6). d Western blotting and quantification for analysis of GLT-1 and GLAST protein expression in midbrain extracts. GAPDH was used as a loading control to confirm that equal amounts of protein were loaded in each line (n = 7). Data are presented as mean ± SEM, one-way ANOVA, *P < 0.05, **P < 0.01
Fig 4: IL-17A deficiency mitigates RMC activation and dysfunction in diabetic mice.The design of the mice was similar to that in Fig. 1. A Expression of GFAP and VEGF in the retina. The data in the statistical histogram were from six mice for each group. B, C GFAP and VEGF contents in the retina. The numbers in the statistical histogram indicate that eight or six mice were used in the groups. D Glutamate content in the retina detected by HPLC. The numbers in the statistical histogram represent that six mice were used in each group. E Retinal expression of GS and EAAT1. The data from six mice for each group were statistically analyzed. **P < 0.01, versus WT mice; ##P < 0.01, versus Akita mice.
Fig 5: Transcription factor CREB is responsible for JWA-mediated GLT-1 expression.a, b C8D1A cells were transfected with si-JWA or Flag-JWA. After 48 h, the mRNA levels of GLAST and GLT-1 were measured by quantitative real-time PCR. GAPDH was used as a control to normalize the differences in the amount of total RNA in each sample (n = 3). c C8D1A cells were transfected with si-JWA or Flag-JWA, followed by western blotting analysis to detect transcription factor p-P65, YY1, phospho- and total-CREB proteins (n = 3). d, e C8D1A cells were incubated with 50 µM of U0126 and/or 20 µM of LY for 3 h after transfect with Flag-con or Flag-JWA for 48 h, followed by western blotting analysis to detect p-ERK, p-Akt, p-CREB and GLT-1 (n = 3). [3H]-Glutamate uptake assay was also performed (n = 3). f C8D1A cells were co-transfected with either Flag-con or Flag-JWA, together with the si-con or si-CREB. After 48 h, expression levels of GLT-1 were detected by western blotting, GAPDH was used as a loading control to confirm that equal amounts of protein were loaded in each line (n = 3). g SH-SY5Y cells were treated for 24 h with astrocyte CM from astrocytes transfected with si-con or from astrocytes transfected with si-JWA. After the treatment, SH-SY5Y cells were exposed to MPP+ (50 µM or 100 µM) for 24 h. The expression levels of TH were detected by western blotting, GAPDH was used as a loading control to confirm that equal amounts of protein were loaded in each line (n = 3). Data are presented as mean ± SEM, one-way ANOVA, *P < 0.05, **P < 0.01
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