Fig 1: deaf14 cochlea is histologically normal. Mid-modiolar sections of Aspa+/+ (A,C) and Aspadeaf14/deaf14 (B,D) cochleae stained with hematoxylin and eosin. Insets show location of higher-power images in the middle cochlear turn. (A,B) One row of inner hair cells (arrow) and three rows of outer hair cells (arrowheads) were visible in wild-type and deaf14 cochleae. (C,D) Neurons (arrows) and Schwann cells (arrowheads) in Rosenthal’s canal had normal morphology. (E) Neuron and Schwann cell densities were normal in deaf14 mice (P>0.05). n=10–12 cochleae per genotype. Mice were 8 weeks of age. Scale bars: 50 µm in A–D and 500 µm in insets.
Fig 2: deaf14 brain is extensively vacuolated. Cresyl-violet-stained serial sections of immersion-fixed (A–F) or perfusion-fixed (G–N) brains of 10- to 12-week-old Aspa+/+ (A–C,G–J) and Aspadeaf14/deaf14 (D–F,K–N) mice. (A–F) Overview at the level of the frontal (A,D), parietal (B,E) and occipital cortex (C,F). Note the extensive vacuolization in layer V of the frontal cortex (arrow, D), the septal nuclei (arrowhead, D) and dorsolateral aspects of the caudate-putamen (asterix, D), the thalamus (arrowhead, E), the pyramidal layer of the hippocampus (arrow, E), the intermediate layers of the superior colliculi (arrow, F) and the peri-aqueductal region (arrowhead, F). (G–N) The auditory pathway, including the cochlear nucleus (G,K), the superior olivary complex (H,L), the inferior colliculus (I,M) and the medial geniculate nucleus (J,N). Note the extensive vacuolization (arrowheads, K,M,N) and the occasional distended blood vessel in the control (arrow, G–J). AC, anterior commissure; Aq, aqueduct; bCP, basal cerebral peduncle; CA1, 2, 3, fields of the hippocampus; fCtx, pCtx, oCtx, frontal, parietal and occipital cortex; CP, caudate putamen; Hi, hippocampus; HT, hypothalamus; PBP, parabrachial pigmented nuclei; Pi, piriform cortex; PN, pontine nuclei; SC, superior colliculus; Se, septum; Th thalamus. Scale bar in A: 1.4 mm in A-F and 92 µm in G–N.
Fig 3: deaf14 mice have a low acoustic startle response. Startle response of (A) male and (B) female mice to white-noise pulses between 85 and 115 dB SPL. Aspadeaf14/deaf14 mice had a lower magnitude startle response than Aspa+/+ or Aspa+/deaf14 mice. n=10 mice per group. * indicates a significant difference (P<0.05) in one-way ANOVA and Dunnett’s multiple comparison test versus Aspa+/+. Weights of (C) male and (D) female mice at 3 to 12 weeks of age. Mean Aspadeaf14/deaf14 body weights were not significantly different from Aspa+/+ mean body weights. A cut-off for statistical significance of P<0.01 was used to adjust for multiple testing. n=10 mice per group. Bars=s.e.m.
Fig 4: deaf14 mice harbor a nonsense mutation in Aspa. (A) Manhattan plot showing probability of linkage to deaf14 locus across the genome. A LOD score above 3 is indicative of linkage. This was observed for a single peak on chromosome 11, indicating that the deaf14 mutation is on this chromosome. (B) Haplotypes of 140 N1F1 mice for SNPs on chromosome 11. Mice were deemed affected or unaffected based on their click ABR. SNP identity is indicated on the left and chromosomal location is indicated on the right. The number of mice with each haplotype is indicated below. SNP locations were taken from Genome Reference Consortium Mouse Build 38 patch release 1 (GRCm38.p1). (C) DNA sequence of Aspa exon 3 in +/+ (upper panel) and deaf14/deaf14 mouse (lower panel). A c.516T>A mutation (*) is predicted to encode a p.Y172X premature termination of translation in the deaf14/deaf14 mouse. (D) Western blot of p26 whole brain lysates probed with anti-ASPA (upper panel) and anti-actin (lower panel) antibodies. The upper panel shows a band at 35 kDa, the predicted molecular weight of ASPA, in Aspa+/+ and Aspa+/deaf14 brain that is not present in Aspadeaf14/deaf14 brain. The faint band in the Aspadeaf14/deaf14 lane (*) is of a slightly higher molecular weight and is also present in the two other lanes, albeit almost obscured by the ASPA band. This is likely to be a non-specific band. The lower panel demonstrates that equal amounts of protein were loaded in each lane. NS, non-specific band; 14, deaf14.
Fig 5: deaf14 mice have impaired motor coordination. (A) deaf14 mice fell off a rotating rod before wild-type mice in four consecutive trials. (B) deaf14 mice made more moves but travelled less distance than wild-type mice in an open field, suggesting ataxia. (C) deaf14 mice were indistinguishable from wild-type mice in the Y-maze, which measures memory, and (D) the light dark test, which measures anxiety. n=9–10 mice per group. All mice were 9- to 10-weeks of age. 14, deaf14. *P<0.05 versus Aspa+/+.
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