Fig 2: Reduction of glutamatergic synapses in the CNS of nsfst53 mutants (5 dpf).Representative z-projections of the optic tectum and cerebellar region from a top view are shown. Wild-type (A), nsfst53 mutant (B), and nsfst53 mutant in Tg(-5neurod:nsf-GFP)vo1 background (C), which expresses Nsf-GFP (Blue) in various neurons, were labeled with Vglut1 and MAGUK antibody labeling. Scale bar: 100 µm; z-projection of 15 confocal planes (10 µm each). D-E', Magnified views from the boxed region in panel A'. Activated Caspase 3 (Casp3) antibody was used to label apoptotic cells (arrow heads) in the cerebellar region, and GFP signals from TgBAC(neurod:EGFP)nl1 fish were used to identify neurons, likely the Purkinje cells, in the cerebellum. F, Cell density (3 dpf, 41875.0±3287.4, n = 4; 4 dpf, 68500.0±3840.6, n = 5; 5 dpf, 59687.5±1856.3, n = 8 in wild-type and 3 dpf, 44285.7±1867.2, n = 7; 4 dpf, 54375.0±3264.7, n = 8; 5 dpf, 53750.0±2116.4, n = 6 in nsfst53) and (G) the rate of apoptosis in the cerebellum of wild-type (0% for 3 dpf to 5 dpf, n = 8) and nsfst53 mutants (3 dpf, 4.3±1.3%; 4 dpf, 3.2±0.9%; 5 dpf, 3.1±1.0%, n = 8). P values compare nsfst53 mutants to their wild-type siblings at the same developmental stage.

Fig 3: Expression of Nsf in the CNS and lateral line organ (5 dpf). A, An antibody against human NSF protein labels the larval nervous system (5 dpf). B, Labeling is absent in nsfst53 mutants. Images are projections of 5 optical sections of 10 µm thickness. Scale bar, 100 µm. C-F, Confocal projections (10×1 µm sections) of anti-NSF (red) and Ribeye b (Blue) in TgBAC(neurod:EGFP)nl1 transgenic fish. Nsf signal colocalized with anti-GFP (green) in the afferent nerve, and was also in Ribeye b-positive hair cells. Scale bar, 10 µm.

Fig 4: Nsf function is required in both pre- and postsynaptic cells to stabilize afferent innervation of hair cells. A-E″, Representative z-projections of a single neuromast from a top-down view (5 dpf). Wild-type (A), nsfst53 mutants (B), and nsfst53 mutants with Nsf-GFP expressed in hair cells (C), pLLG (D), or in both hair cells and pLLG (E) were labeled by antibodies against GFP (pseudo-colored light blue), MAGUK (green), and Ribeye b (red). Scale bar: 10 µm. F, The average number of MAGUK puncta per hair cell in wild-type (3.8 ±0.1, n = 7), nsfst53 mutants (0.4±0.1, n = 7), and nsfst53 mutants with hair-cell Nsf-GFP (1.2±0.4, n = 5), pLLG Nsf-GFP (2.2±0.2, n = 6), or both hair-cells and pLLG Nsf-GFP (3. 6±0.1, n = 7). G, Zebrafish larvae were stained with Zn12 and Ribeye b antibody. The fluorescent intensity (A.U.) of Zn12 was quantified in wild-type (1.813e6±147069, n = 6), nsfst53 mutants (311769±43541, n = 6), and nsfst53 mutants with Nsf-GFP rescued in hair cells (507787±97798, n = 5), pLLG (659674±85851, n = 5), or both hair cells and pLLG (1.335e6±223319, n = 8). The p-values were generated comparing the data from the nsfst53 mutant to each transgenic mutant line.

Fig 5: Rapamycin alleviated disturbances in the levels of SNARE chaperones in hippocampal synaptosomes caused by surgery. (A) Representative immunoblot of SNARE chaperones in hippocampal synaptosomes. (B) Munc18-1, (C) complexin 1, (D) complexin 2, (E) CSPa, and (F) NSF were quantified by densitometry and normalized to GAPDH. Values represent the mean ± SEM, n = 4 per group. ** p < 0.01 surgery vs. sham group or surgery+rapamycin vs. surgery group. RAP: rapamycin.