Fig 2: 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), PKCα (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).

Fig 3: 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), PKCα (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 4: TASK-3 is expressed and mediates outwardly rectifying K+ currents in RGCs.(A) In situ hybridization using RNAscope for TASK-3 (Kcnk9; magenta) and immunohistochemistry (IHC) for RBPMS (green) in C57BL/6J mouse retina (left). The zoom-in view of the white box is on the right. ONL, outer nuclear layer; DAPI (blue), 4′,6-diamidino-2-phenylindole. (B) RNAscope for TASK-3 (Kcnk9; magenta) and melanopsin (Opn4; green) in C57BL/6J mouse retina (left). The zoom-in view of the white box is on the right. White dashed circles illustrate two ipRGCs. (C) Left: Average current-voltage relation (I-V) curve of RGCs in vehicle control [dimethyl sulfoxide (DMSO)] and in PK-THPP (3 μM). Right: Bar graph of the current densities in DMSO and in PK-THPP at +17 mV (n = 11 cells, paired t test). (D) Graphic represents the subtraction trace from (C) representative PK-THPP–sensitive current. (E) Left: Average I-V curve of RGCs from TASK-3 KO and wild-type (WT) mice. Right: Bar graph of the current densities in WT and TASK-3 KO RGCs at 17 mV (n = 12, unpaired t test). (F) The graph illustrates the subtraction trace from (E) representative TASK-3–mediated current. (G) Left: Average I-V curve of TASK-3 KO RGCs in vehicle control (DMSO) and in PK-THPP (3 μM). Right: Bar graph of the current densities in DMSO and in PK-THPP at 17 mV (n = 8 cells, paired t test). (H) The graph shows the subtraction trace from (G) representative PK-THPP–sensitive current in KO. All data are presented as means ± SEM. n.s., not significant; *P < 0.05; ***P < 0.001.

Fig 5: SIRT1 reduces RGCs loss by microglia inhibition. (A) Representative images of Iba1+ area in proximal optic nerve of Vehicle- and Res- group without ONC; (B) Quantitative analysis of Iba1+ area in proximal optic nerve under conditions represented in A (n = 8 mice per group); (C) Representative images of retina Nissl staining of Vehicle- and Res- group without crush injury; (D) Quantitative analysis of RGCs loss of the 2 groups in C (n = 8 mice per group); (E–H) Activation of Sirt1 inhibited Microglial activation (E and H). Co-localization between Iba1 and CD68 was indicated with white arrow. Quantitative analysis of Iba1+ area (F) and co-localization between Iba1 and CD68 (G) under conditions represented in E (n = 8 mice per group); (I)RBPMs+ cell in retina was counted for examining RGCs loss. Representative images of RBPMs+ cell in retina of Vehicle- and Res- group after crush injury; (J) Quantification of RGCs loss by counting RBPMs+ cell in retinas from Vehicle- and Res- group after ONC (n = 8 mice per group); (K) Representative TEM images of axon damage at 0.5 mm behind crush injury site of Vehicle- and Res- group after crush injury. Normal axon was indicated with arrow (>) and axons with abnormal myelin was indicated with (≫); (L and M) Quantification of axon damage of the two groups in K post crush (n = 8 mice per group); (N–Q) Microglia Sirt1 deficiency did not influence microglial activation and RGCs number without injury (N and P). Quantification of microglial activation (O) and RGC number (Q) under conditions in N and P (n = 8/genotype); (R–W) Microglial Sirt1 deficiency increased RGCs (V) loss via aggravating microglial activation (R and U). Co-localization between Iba1 and CD68 was indicated with white arrow. Quantification of microglial activation (S and T) and RGCs loss (W) from the results of R and V (n = 8/genotype). Data are presented as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.