Fig 2: Neuroplastin genetically interacts with Cadherin 23.Phenotypic measures of Nptntm1b mice maintained on a Cdh23-repaired C57BL/6N (Cdh23753A>G) background. (A) ABR threshold measurements recorded from 4-week old mice. Similar to Nptntm1b/tm1b mice on the standard C57BL/6N genetic background, Nptntm1b/tm1b mice on the repaired background (red triangles, n = 6) have profoundly elevated thresholds compared with littermate Nptn+/+ mice (black circles, n = 5). However, on the repaired background Nptn+/tm1b (blue squares, n = 7) have thresholds indistinguishable from control mice, even at the higher frequencies. Data are median ± IQR with individual data points shown. One-way ANOVA comparing against wild type controls using Dunnett’s test for multiple comparisons. (B) DPOAE measurements recorded at 5-6-weeks of age. Response amplitudes were significantly reduced in Nptntm1b/tm1b mice (red triangles, n = 5), whereas Nptn+/tm1b mice (blue squares, n = 5) have amplitudes comparable to that of control mice (Nptn+/+, black circles, n = 5). Data are mean ± SD with individual data points shown. One-way ANOVA comparing against wild type controls using Dunnett’s test for multiple comparisons. (C, D) Click-evoked ABR wave I comparing Nptn+/+ (black, n = 5) and Nptn+/tm1b (blue, n = 7) mice. Waveforms were not significantly different. Amplitude: P = 0.8679, Latency: P = 0.0568 (two-way ANOVA). Inset shows an average of wave I following a click stimulus at 30 dB above threshold. Data are mean ± SD with individual data points shown.

Fig 3: Np65 and Np55 are cochlear expressed.(A) Western blot of membrane-enriched fractions prepared from Nptn+/+ and Nptntm1b/tm1b cochleae. Lysates were treated with PNGase F to remove N-linked glycans before separation. In the wild type membrane fraction, the anti-pan-Np antibody detects bands corresponding to native Np65 (44.4 kDa) and Np55 (31.3 kDa), whereas the anti-Np65-specific antibody detects only a single band corresponding to Np65. No specific bands were detected in the membrane fraction prepared from Nptntm1b/tm1b cochleae using either antibody. Na+/K+-ATPase was used as a marker protein for the plasma membrane. (B) Whole brain protein lysates prepared from Nptntm1b/tm1b (tm1b/tm1b), Nptn?exon2/?exon2 (?2/?2) and their respective wild type controls, were separated by PAGE and Western blotted utilizing an anti-pan-Np antibody and an anti-Np65 antibody. For wild type lysates, both Np55 and Np65 are detected. However, neither protein isoform was detected in Nptntm1b/tm1b lysates. Importantly, only Np55 was detected in Nptn?exon2/?exon2 lysates, confirming the absence of Np65. Western blots were run using native (untreated) and deglycosylated (PNGase F) lysates. Tubulin is used as a loading control. (C) Cochlear immunohistochemistry of cryosections using the anti-pan-Np antibody showed labelling of outer hair cell (OHC) stereocilia, inner hair cells (IHC), support cells lateral to the hair cells, and the spiral ganglion neurons (SGN). No signal was observed in Nptntm1b/tm1b cochlea. Scale = 20 µm. (D) The anti-Np65 specific antibody showed labelling of IHCs and support cells lateral to the hair cells. No signal was observed in Nptntm1b/tm1b cochleae. Scale = 20 µm. (E) ABR threshold measures recorded from 1-month old mice showing profoundly elevated thresholds for all stimuli tested in Nptntm1b/tm1b mice (red triangles, n = 8) compared to wild type littermates (black circle, n = 5). Heterozygous Nptn+/tm1b mice (blue squares, n = 5) exhibit a significant high frequency (=12 kHz) hearing loss compared to control littermates. Data shown as median ± I.Q.R with individual data points. One-way ANOVA comparing to wild type, employing Dunnett’s test for multiple comparisons. (F) Average DPOAE responses recorded from 1-month old mice. Both Nptntm1b/tm1b (n = 10) and Nptn+/tm1b (n = 5) mice have suppressed response amplitudes compared to littermate controls (n = 7). Data are mean ± SD with individual data points shown. One-way ANOVA comparing to wild type controls, employing Dunnett’s test for multiple comparisons.

Fig 4: Age-specific changes in GM1, GD1a, and neuroplastin expression upon chronic stress in the DG hippocampal region. Immunofluorescent staining was analyzed using the shares of single molecules: GD1a, GM1, and Np65; double colocalizations: GD1a/Np65, GM1/Np65, GD1a/GM1; and triple colocalization in absolute pixel counts for all four animal groups. The average ratio of pixel count is shown for each single or combination profile. The Venn diagram defines the expression of combination profiles. Statistical analysis was performed using a two-way analysis of variance (ANOVA) and is shown in Table 2. Abbreviations: CA1, Cornu Ammonis subfield 1; GD1a, ganglioside GD1a; GM1, ganglioside GM1 visualized by cholera toxin beta subunit; NS, nonsignificant; Np65, neuroplastin; OF-C, control group of old females; OF-S, stressed group of old females; YF-C, control group of young females; YF-S, stressed group of young females

Fig 5: Neuroplastin is required for normal IHC maturation.(A, B) Current responses from IHCs of heterozygous (A: Nptn+/tm1b, P18) and homozygous (B: Nptntm1b/tm1b, P19) mice, which is after hearing onset at P12. Current recordings were elicited by using depolarising and hyperpolarising voltage steps (10 mV increments) from the holding potential of –84 mV to the various test potentials shown by some of the traces. (C) An expanded version of the first 10 ms of the current traces from panels A and B, which emphasises the presence of the rapidly activating IK,f. (D) Steady-state current-voltage curves obtained from P18-P25 IHCs from the three genotypes. (E-G) Size of the total outward K+ current IK (E), the isolated IK,f (F) and IK,n (G). The size of IK,f was measured at –25 mV and 1 ms from the onset of the voltage step [24]. The size of IK,n was measured as the difference between the peak and steady-state of the deactivating inward current at –124 mV [21]. The number of IHCs recorded are shown above each column. Average data are plotted as mean ± SD.