Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea.

Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL

Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea

<div><p><i>Neuropilin-1 (Nrp1)</i> encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific <i>Nrp1</i> conditional knockout (CKO) mouse line because <i>Nrp1</i> constitutive knockouts are embryonic lethal. <i>In situ</i> hybridization demonstrated weak <i>Nrp1</i> mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, <i>Nrp1</i> CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old <i>Nrp1</i> CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old <i>Nrp1</i> CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of <i>Nrp1</i> in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of <i>Nrp1</i> show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons <i>in vitro</i>. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that <i>Nrp1</i> may play a role in ARHL.</p></div

Presynaptic marker CtBP2 counts of the IHCs and OHCs.

<p>Cochlear whole mounts of WT, <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice at P5 (n = 4 per group) and 4 months (n = 5 per group) were immunostained with presynaptic marker CtBP2. Panel A and B shows CtBP2 immunostaining of the 4-month-old mice (A, B). The number of CtBP2 puncta was compared separately to the number of inner and outer hair cells per section. CtBP2 ratios of IHCs did not differ significantly between WT and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutants from P5 to maturity (C). OHC synaptic ribbon ratios of 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> demonstrated a significant decrease in CtBP2 counts in the basal turn (p<0.05). CtBP2 counts of the IHCs showed two cochlea out of five with nearly 50% decreased numbers of the puncta when compared to WT controls; however, the average of the counts (five cochlea) did not show a statistically significant difference (D). *p<0.05. Scale bar = 10 μm.</p

<i>In situ</i> hybridization of <i>Nrp1</i> and <i>Sema3a</i>.

<p><i>In situ</i> hybridization showing the expression pattern of <i>Nrp1</i> (A-C) and <i>Sema3a</i> (D-F) in mice at time points E13.5, E16.5, E18.5, and P1. <i>Nrp1</i> was not expressed at E16.5 (A), weakly expressed in the SG and SV at E18.5 (B), and more highly expressed in the SG, greater epithelial region (GER), and SV at P1 (C). <i>Sema3a</i> expression pointed by arrows on the abneural side of the cochlear epithelium was observed around E13.5 (D) and continued at E16.5 (E) and E18.5 (F) on the same region of the cochlear epithelium and spiral ganglion neurons. (G, H) Negative controls for <i>Nrp1</i> (E18.5) and <i>Sema3a</i> (E16.5) using sense probes. (I) Panel I shows the plane of sections. SG: spiral ganglion. SV: stria vascularis. Scale bar = 50 μm.</p

SGN projections in the basal turns of cochleae of WT and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice at P5 and 4-month-old age.

<p>Disorganized outer spiral bundles were evident in cochleae of the <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice (P5) in the basal turn (n = 3 per group). The arrow shows disorganized outer spiral bundles (B). TUJ1 immunostaining of the SGN fibers extending into the inner and outer hair cell regions in 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutants revealed aberrant axons with abnormal innervation of the OHCs. Arrow shows abnormal innervation of the OHCs (D). WT mice did not show aberrant axon migration or innervation. Scale bar = 20 μm.</p

<i>Nrp 1</i> mediates the semaphorin-3A-induced inhibition of neurite outgrowth.

<p>SGN explants were transfected with either 50nM <i>Nrp1</i> siRNA or scrambled siRNA, and treated with semaphorin-3A (250 ng/ml). Neurons were stained with β-tubulin III monoclonal antibodies. Control (A); Semaphorin-3A alone (B); <i>Nrp1</i> siRNA alone (C); D. <i>Nrp1</i> siRNA and semaphorin-3A (D); Scrambled siRNA alone (E); Scrambled siRNA and semaphorin-3A (F). Scale bar = 200 μm. (G,H, J) Recombinant Sema3a inhibits SGN outgrowth in whole cochlear cultures. (G, H) Three-dimensional confocal z-stacks from E17.5 cochlear cultures treated with 20nM of either control IgG-Fc or Sema3a-Fc. Tissue samples were stained with anti TUJ1 to mark SGNs (white) and Myo6 for HCs (blue). Note the dramatically diminished presence of fibers in the sample treated with Sema3a-Fc (arrowheads). Scale bar = 10um. (I) Average length of neurites grown from the SGN explants showing statistically significant decreased neurite outgrowth for Sama3a, <i>Nrp1</i> siRNA, and scrambled siRNA/Sema3a when compared to the control group. * p<0.001. (J) Panel J showing average percent neurite coverage of sensory epithelium. Error bars, SEM. * p<0.0001. (K) Western immunoblots of Nrp1 protein from SGN explants after transfection with either Nrp1 siRNA or scrambled siRNA control as indicated. β- Actin was used as a loading control.</p

Stria vascularis abnormalities in <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutant mice at P5 and 4 months of age.

<p>Panel A showing neuropilin-1 expression (green) on vascular tissue of the cochlea. Arrows show neuropilin-1 stained vessels (A). Lectin immunostaining of WT and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> cochleae at P5 and 4 months of age were performed to assess the morphology of the micro-vessels of the SV at the basal turn of the cochlea (n = 3 per group) (B-G). Grossly enlarged SV microvessels were apparent in both ages of <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutants. The minimum and maximum microvessel diameter of the <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice were 4.17μm and 43.67μm at P5, and 3.22 and 95μm in 4-month-old mice, respectively. The minimum and maximum microvessel diameters of the WT mice were 5.32μm and 23.90μm at P5, and 5.2μm and 26.15μm in 4-month-old mice, respectively. Panel B and C showing normal morphology of the microvessels of the WT mice at P5 and 4-month-old (B). <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutants at P5 showed some enlarged microvessels when compared to WT controls (D). Panel E-G showing vascular abnormalities of 3 different 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutants (E, G). Overall, the maximum microvessel diameter in 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice was 3.6 fold higher than WT controls. Scale bar = 30 μm.</p

SGN density and caspase-3 expression in 4-month-old WT and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutant mice.

<p>Mid-modiolar sections of the cochlea (basal turn) showing lower density of the SGNs in 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice compared to WT controls (A, B). Cochlear SGN of 4-month-old WT and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice were immunostained with antibodies to Caspase-3 (Red) to ascertain the fate of the SGN at cochlear maturity (C, D). Caspase-3 positive SGNs pointed by arrows in panel D (red staining) showing apoptosis of neurons in 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice compared to WT controls on panel C. (E) There was no statistically significant difference between SGNs count of the <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice at P5 when compared to WT controls (n = 4 per group). SGNs count at the apical, middle, and basal (n = 5 per group) turns of the cochlea was consistently lower in the 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutant group compared to WT controls (F). *p<0.05. Scale bar = 40 μm for A, and B. Scale bar = 10 μm for C, and D.</p

Wave I P-N amplitude for WT, <i>Nrp1</i>^<i>fl/+</i>;<i>Pax2</i>^<i>Cre</i>, and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice at 2 and 4 months of age.

<p>Analysis of the wave I P-N amplitudes at 80dB suprathreshold did not show significant difference between <i>Nrp1</i>^<i>fl/+</i><i>Pax2</i>^<i>Cre</i> mice and WT controls at 2-month-old (A); however, statistically significant higher amplitudes at 8kHz, 12kHz, and 24kHz were shown in 4-month-old <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mutants compared to controls (B). *p<0.05.</p

ABR for WT, <i>Nrp1</i>^<i>fl/+</i>;<i>Pax2</i>^<i>Cre</i>, and <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice at 2 and 4 months of age.

<p>ABR results of the 2-month-old mice (Panel A) showed that the hearing thresholds of the <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> group (n = 8) were significantly higher than WT controls (n = 11) at 4kHz, 8kHz, 16kHz, 24kHz, and 32kHz (A). At 4 months of age (Panel B), <i>Nrp1</i>^<i>fl/fl</i>;<i>Pax2</i>^<i>Cre</i> mice (n = 8) showed elevated hearing thresholds at all tested frequencies except 12 kHz when compared to WT mice (n = 11) (B).</p