Macroglia-derived thrombospondin 2 regulates alterations of presynaptic proteins of retinal neurons following elevated hydrostatic pressure

<div><p>Many studies on retinal injury and repair following elevated intraocular pressure suggest that the survival ratio of retinal neurons has been improved by various measures. However, the visual function recovery is far lower than expected. The homeostasis of retinal synapses in the visual signal pathway is the key structural basis for the delivery of visual signals. Our previous studies found that complicated changes in the synaptic structure between retinal neurons occurred much earlier than obvious degeneration of retinal ganglion cells in rat retinae. The lack of consideration of these earlier retinal synaptic changes in the rescue strategy may be partly responsible for the limited visual function recovery with the types of protective methods for retinal neurons used following elevated intraocular pressure. Thus, research on the modulatory mechanisms of the synaptic changes after elevated intraocular pressure injury may give new light to visual function rescue. In this study, we found that thrombospondin 2, an important regulator of synaptogenesis in central nervous system development, was distributed in retinal macroglia cells, and its receptor α2δ-1 was in retinal neurons. Cell cultures including mixed retinal macroglia cells/neuron cultures and retinal neuron cultures were exposed to elevated hydrostatic pressure for 2 h. The expression levels of glial fibrillary acidic protein (the marker of activated macroglia cells), thrombospondin 2, α2δ-1 and presynaptic proteins were increased following elevated hydrostatic pressure in mixed cultures, but the expression levels of postsynaptic proteins were not changed. SiRNA targeting thrombospondin 2 could decrease the upregulation of presynaptic proteins induced by the elevated hydrostatic pressure. However, in retinal neuron cultures, elevated hydrostatic pressure did not affect the expression of presynaptic or postsynaptic proteins. Rather, the retinal neuron cultures with added recombinant thrombospondin 2 protein upregulated the level of presynaptic proteins. Finally, gabapentin decreased the expression of presynaptic proteins in mixed cultures by blocking the interaction of thrombospondin 2 and α2δ-1. Taken together, these results indicate that activated macroglia cells may participate in alterations of presynaptic proteins of retinal neurons following elevated hydrostatic pressure, and macroglia-derived thrombospondin 2 may modulate these changes via binding to its neuronal receptor α2δ-1.</p></div

Cell compositions in the mixed cultures and retinal neuron cultures.

<p>(A) Double immunofluorescence Map2/GFAP, CD11b/DAPI staining in mixed cultures. (B) Double immunofluorescence Map2/DAPI staining in retinal neuron cultures. Scale bar = 50 μm.</p

Expression of SYN, synapsin, Gephyrin, PSD95 and Homer following EHP in mixed cultures.

<p>Labels are as follows: Control group (Control); 2, 6, 12, and 24 h after EHP (2 h, 6 h, 12 h, and 24 h). (A) Western Blot of SYN, synapsin, PSD95 and Homer following EHP. (B) The statistical analysis of SYN, synapsin, PSD95 and Homer expression by Western Blot following EHP, * compared to Control, P<0.05; ** compared to Control, P<0.01. PSD95 and Homer expression show no statistically significant difference among groups (P>0.05). (C) Immunofluorescence staining of synapsin and Homer following EHP. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels. (D) Double immunofluorescence synapsin/Homer staining and their colocalization (synapse) in the dendrites per 20 μm. (E) Quantification of the number of synapsin, Homer and their colocalization puncta. * compared to Control, P<0.05. The number of Homer positive puncta and synapses show no statistically significant difference among groups (P>0.05) (F) Double immunofluorescence synapsin/Gephyrin staining and their colocalization in the dendrites per 20 μm. (G) Quantification of the number of synapsin, Gephyrin and their colocalization puncta. * compared to Control, P<0.05. The number of Gephyrin positive puncta and synapses show no statistically significant difference among groups (P>0.05).</p

Expression of SYN, synapsin, Gephyrin, PSD95 and Homer under EHP and GBP treatment in mixed cultures.

<p>Labels are as follows: Control group (Control), 12 h after EHP (12 h), GBP + 12 h after EHP (12 h + GBP). (A) Western Blot of SYN, synapsin, PSD95 and Homer expression under EHP and GBP treatment. (B) Statistical analysis of SYN and synapsin expression levels under EHP and GBP treatment, * compared to Control, P<0.05. PSD95 and Homer expression show no statistically significant difference among groups (P>0.05). (C) Immunofluorescence staining of synapsin and Homer following EHP and GBP application. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels. (D) Double immunofluorescence synapsin/Homer staining and their colocalization puncta in the dendrites per 20 μm. (E) Quantification of the number of synapsin, Homer and their colocalization puncta. * compared to 12-h group, P<0.05. The number of Homer positive puncta and synapses show no statistically significant difference among groups (P>0.05) (F) Double immunofluorescence synapsin/Gephyrin staining and their colocalization in the dendrites per 20 μm. (G) Quantification of the number of synapsin, Gephyrin and their colocalization puncta. * compared to 12-h group, P<0.05. The number of Gephyrin positive puncta and synapses show no statistically significant difference among groups (P>0.05).</p

Immunofluorescence for the localization of TSP2 and α2δ-1.

<p>(A) Localization of TSP2 was identified by TSP2/GFAP co-staining. (B) Localization of α2δ-1 was identified by α2δ-1/Map2 co-staining. Scale bar = 50 μm.</p

Expression of SYN, synapsin, Gephyrin, PSD95 and Homer under EHP and with the addition of recombinant TSP2 protein in retinal neuron cultures.

<p>Labels are as follows: Control group (Control), 12 h after EHP (12 h), recombinant TSP2 protein + 12 h after EHP (12 h + TSP2). (A) Western Blot of SYN, synapsin, PSD95 and Homer expression under EHP with the addition of recombinant TSP2 protein. (B) The statistical analysis of SYN and synapsin proteins following EHP and the addition of recombinant TSP2 protein, * compared to Control, P<0.05. PSD95 and Homer expression show no statistically significant difference among groups (P>0.05). (C) Immunofluorescence staining of synapsin and Homer following EHP and TSP2 application. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels. (D) Double immunofluorescence synapsin/Homer staining and their colocalization in the dendrites per 20 μm. (E) Quantification of the number of synapsin, Homer and their colocalization puncta. * compared to 12 h group, P<0.05. The number of Homer positive puncta and synapses show no statistically significant difference among groups (P>0.05) (F) Double immunofluorescence synapsin/Gephyrin staining and their colocalization in the dendrites per 20 μm. (G) Quantification of the number of synapsin, Gephyrin and their colocalization puncta. * compared to 12 h group, P<0.05. The number of Gephyrin positive puncta and synapses show no statistically significant difference among groups (P>0.05).</p

Expression of α2δ-1 under EHP and GBP application.

<p>Labels are as follows: Control group (Control); 2, 6, 12, and 24 h after EHP (2 h, 6 h, 12 h, and 24 h). (A) Immunofluorescence staining of α2δ-1 following EHP. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels. (B) Western Blot of α2δ-1 expression under EHP. (C) Statistical analysis of α2δ-1 expression levels under EHP, * compared to Control, P<0.05. (D) Immunofluorescence staining of α2δ-1, SYN, synapsin, PSD95 and Homer following EHP and GBP treatment. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels.</p

Expression of SYN, synapsin, Gephyrin, PSD95 and Homer under EHP and TSP2 siRNA silencing.

<p>Labels are as follows: Control group (Control), 12 h after EHP (12 h), Reagent + 12 h after EHP (12 h + Reagent), Non-targeting control + 12 h after EHP (12 h + NControl), siRNA + 12 h after EHP (12 h + siRNA). (A) Western Blot of SYN, synapsin, PSD95 and Homer expression following EHP and TSP2 siRNA. (B) The statistical analysis of SYN and synapsin expression following EHP and TSP2 siRNA, * compared to Control, P<0.05. PSD95 and Homer expression show no statistically significant among groups (P>0.05). (C) Immunofluorescence staining of synapsin and Homer following EHP and siRNA knockdown. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels. (D) Double immunofluorescence synapsin/Homer staining and their colocalization in the dendrites per 20 μm. (E) Quantification of the number of synapsin, Homer and their colocalization puncta. * compared to 12 h group, P<0.05. The number of Homer positive puncta and synapses show no statistically significant difference among groups (P>0.05) (F) Double immunofluorescence synapsin/Gephyrin staining and their colocalization in the dendrites per 20 μm. (G) Quantification of the number of synapsin, Gephyrin and their colocalization puncta. * compared to 12-h group, P<0.05. The number of Gephyrin positive puncta and synapses show no statistically significant difference among groups (P>0.05).</p

Expression of TSP2 under EHP and TSP2 siRNA silencing.

<p>Labels are as follows: Control group (Control); 2, 6, 12, and 24 h after EHP (2 h, 6 h, 12 h, and 24 h). (A) Immunofluorescence staining of TSP2 following EHP. The lower panels are the magnified images of the area in the rectangles of the upper panels. (B) Western Blot of TSP2 expression following EHP. (C) Statistical analysis of TSP2 expression following EHP, ** compared to Control, P<0.01. Scale bar = 50 μm. (D) Immunofluorescence staining of TSP2 following EHP and siRNA knockdown. Scale bar = 50 μm. The lower panels are the magnified images of the area in the rectangles of the upper panels. (E) Western Blot of TSP2 expression following EHP and siRNA knockdown. (F) The statistical analysis of TSP2 expression following EHP and TSP2 siRNA, * compared to Control, P<0.05. PSD95 and Homer expression show no statistically significant among groups (P>0.05).</p

Immunobloting of FIS1 in primary refractory AML patients (1–5) and non-refractory AML patients (6–11). *P<0.05.

<p>Immunobloting of FIS1 in primary refractory AML patients (1–5) and non-refractory AML patients (6–11). *P<0.05.</p