Fig 2: 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.

Fig 3: (a) Representative Western blots showing Neuroplastin 65 (Np65), Neuroplastin 55 (Np55) and PMCAs distribution in lipid raft (LR) and non-LR fractions isolated from cortical tissue of WT and GM2/GD2 synthase-deficient mice. (b) Box and whisker plots representing the percentage of immunoreactivity intensity in LR and non-LR fractions calculated using ImageJ. (* p < 0.05, Student’s t-test). WT = wild-type mice; pPMCA = pan PMCA encompassing all PMCA isoforms.

Fig 4: Heterozygous Nptn+/tm1b mice exhibit reduced ABR wave I amplitudes and increased latencies.(A) Maximum intensity projections of whole-mount mid-apical cochleae from Nptn+/+, Nptn+/tm1b, and Nptntm1b/tm1b mice labelled with the pre-synaptic ribbon marker Ribeye (red) and post-synaptic density marker GluR2/3 (cyan). (B) Counts of Ribeye and GluR2/3 puncta were made from ten mid-apical IHCs from Nptn+/+ (black), Nptn+/tm1b (blue) and Nptntm1b/tm1b (red) cochleae (two independent regions per mouse, three mice per genotype). Ribeye and GluR2/3 puncta were considered matched when directly juxtaposed to one another. A larger number of unmatched GluR2/3 puncta and fewer Ribeye puncta were observed in Nptntm1b/tm1b cochleae. (C) Maximum intensity projections of whole-mount basal cochleae from Nptn+/+ and Nptn+/tm1b mice labelled with the pre-synaptic ribbon marker Ribeye (red) and post-synaptic density marker GluR2/3 (cyan). (D) Counts of Ribeye and GluR2/3 puncta were made from 5–10 IHCs from Nptn+/+ (black) and Nptn+/tm1b (blue) cochleae (two independent regions per mouse, three mice per genotype). Nptn+/tm1b mice show a decrease in the number of unmatched GluR2/3 puncta compared with Nptn+/+. Insets in (A) and (C) are an enlarged view of a 5 µm x 5 µm region highlighted by the dashed box in the corresponding above image. Scale bars in (A) and (C) = 5 µm. Data are mean ± SD with individual data points shown. Data are compared against wild type controls using unpaired t-tests with a Holm-Sidak correction for multiple comparisons. (E) Averaged ABR wave I in response to a click stimulus recorded at 30 dB above hearing threshold for Nptn+/tm1b (blue line, n = 5) and Nptn+/+ (black line, n = 5) mice. Across all intensities, Nptn+/tm1b mice had reduced amplitudes (F) and increased latencies (G) compared with Nptn+/+ mice. Data are mean ± SD with individual data points shown. Analysed using a two-way ANOVA.

Fig 5: Neuroplastin gene schematic and alleles.(A) Schematic of the wild type Neuroplastin gene locus consisting of eight exons that encode: three extracellular Ig domains; a transmembrane region (TM); and, a short intracellular region. Alternative splicing involving the inclusion, or skipping, of exon 2 leads to the production of transcripts encoding the larger Np65, or smaller Np55, protein isoform, respectively. Previously reported mutant alleles audio-1 [13] and pitch [12] are point mutations present in exons 3 and 6, respectively, affecting both isoforms. (B) the Nptntm1a(EUCOMM)Hmgu (Nptntm1a) is a knockout first, conditional-ready allele, that was used to generate both the Nptntm1b(EUCOMM)Hmgu (Nptntm1b) knockout allele lacking exons 5 and 6, and the Nptntm1c(EUCOMM)Hmgu (Nptnfl) floxed allele. Subsequent exposure of the Nptnfl allele to cre recombinase excises exons 5 and 6, resulting in a null allele (Nptntm1d) in cells expressing cre. (C) An isoform-specific Np65 knockout mutant (Nptn?exon2) was generated using CRISPR/Cas9-mediated deletion of 941 nucleotides encompassing exon 2. Mice homozygous for the Nptn?exon2 are unable to express the larger Np65 isoform, while expression of Np55 remains.