Targeted spiral ganglion neuron degeneration in parvalbumin-Cre neonatal mice

Abstract

The spiral ganglion neurons (SGNs) are the primary afferent neurons in the cochlea; damage to the SGNs leads to irreversible hearing impairment. Mouse models that allow selective SGN degeneration while sparing other cell types in the cochlea are lacking. Here, we investigated a genetic ablation method of the SGN using a Cre-responsive adeno-associated virus (AAV) vector expressing diphtheria toxin subunit-A (DTA). We microinjected AAV2-retro-FLEX-DTA-mCherry driven by the EF1a or hSYN promoter in neonatal parvalbumin-Cre (PV^Cre) and wild-type strains via the posterior semicircular canal. Apoptotic markers were observed in the degenerating SGNs as early as 3 days. After 1 week, we assessed the SGN cell density, revealing an average degeneration of 60% for AAV-DTA driven by the EF1a promoter and 61% for that driven by the hSYN promoter. By 1 month, injected ears demonstrated a nearly complete loss of SGN, while hair cell morphology was intact. The auditory brain stem response result showed significantly elevated threshold shifts at 1 month, while the distortion-product otoacoustic emissions function remained intact. Furthermore, we show that our method did not effectively ablate SGN in adult PV^Cre mice. We generated a neonatal mouse model with primary SGN degeneration in PV^Cre mice, mimicking auditory neuropathy phenotype using an AAV Cre-dependent expression of DTA.

Keywords: AAV; DTA; auditory neuropathy; hearing loss; spiral ganglion neuron degeneration.

Graphical abstract

Figure 1

AAV2-retro-CMV-EGFP leads to robust SGN transduction without transducing HCs (A) Representative low-magnification (10×) image of the cochlear mid-modiolar cross-section 7 dpi after AAV2-retro-CMV-EGFP injection. The insert demonstrates a magnified view (63×) of the organ of Corti in the dotted box. GFP is expressed in neurites but not in phalloidin-positive hair cells (HCs). GFP expression is observed in the stria vascularis (white arrow) and in the lateral wall. Of note, cochleae were stained for phalloidin alone. (B) Schematic of FLEX-DTA-mCherry plasmid, which was cloned into AAV2-retro capsid and driven under EF1a or hSYN promoter. (C) Images of representative cryosection of WT and PV^Cre, demonstrating the Rosenthal’s canal of the basal turn of cochlea on 3 dpi following injection of AAV-DTA. Cochleae were stained for TUJ1. White arrowheads point to colocalization of mCherry and TUJ1 in the SGN in WT mice, which is not observed in PV^Cre mice.

Figure 2

AAV-DTA-induced apoptosis of SGN cells (A) The injected WT control (7 dpi) retains a healthy population of SGN in the Rosenthal’s canal (RC) with negligible expression of cleaved caspase-3. Cleaved caspase-3 expression (white arrowheads) is upregulated in PV^Cre mice both 3 and 7 days after AAV-DTA injection. There is a noticeable degeneration of TUJ1⁺ SGN by 7 days from AAV-DTA injection. (B) AAV-DTA-injected WT control mice uniformly express cytochrome c in the cytoplasm of SGN 7 dpi. Inserts demonstrate high magnification of cytochrome⁺ SG cells; the cytoplasm (yellow arrowheads) becomes disorganized in PV^Cre mice 3 and 7 dpi.

Figure 3

AAV-DTA leads to Cre-dependent SGN degeneration After cross-breeding tdTomato/Ai9^+/+ with PV^Cre strain, we were able to confirm the expression of Cre recombinase in the HCs and SGN. (A) Robust tdTomato expression is observed in TUJ1⁺ SGN cells in control Td mice at P9 (n = 3). (B and C) P2 Td/PV^Cre mice were injected with either AAV-hSYN-DTA (n = 5) or AAV-EF1a-DTA (n = 5), both demonstrating substantial loss of both TUJ1 and tdTomato signals consistent with SGN damage. At far right, higher magnification of the respective dashed boxes in the mid-turn (A–C). (D) Quantification of SGN density showed a significant SGN degeneration of 60%–61% after 7 dpi in both AAV-hSYN-DTA- and AAV-EF1a-DTA-injected mice compared to control. No significant difference was observed between the efficacy of AAV-EF1a-DTA and AAV-hSYN-DTA in selective SGN ablation. Student’s t test confirmed that there was a statistically significant degeneration of SGN in the apex, mid-, and basal turn at 7 dpi (p < 0.05).

Figure 4

Time-dependent progressive degeneration of SGN Cochleae were examined at 28 days after AAV-hSYN-DTA injections into P2 Td/PV^Cre mice. (A) Age-matched control Td mice in apex, mid-, and basal turns. (B) AAV-hSYN-DTA-injected Td/PV^Cre mice demonstrated near-total loss of SGN cells in the injected left ear. We observed crossing fibers in the Rosenthal’s canal that likely represent efferent fibers (white arrowheads). (C) Contralateral ear demonstrates significant SGN damage, with few surviving SGN. (D) Quantification of SGN density showed nearly complete ablation of TUJ1 immunopositive SGN in all three turns of the injected left ear and less but significant degeneration in the contralateral right ear. In the contralateral ear, quantification of SGN density showed 92% ablation in the apex and basal turn and 94% ablation in the mid-turn (n = 3) (p < 0.05).

Figure 5

AAV-DTA injected mice are profoundly deaf, with elevated ABR but normal DPOAE amplitude Functional hearing tests are performed on ≥P30 uninjected control mice and AAV-DTA 28 dpi mice (n = 3). (A) ABR showed profound hearing loss, above the 90-dB threshold, in the injected mice. (B) DPOAE showed the mean amplitude vs. level function at 16 kHz in the injected mice is comparable to controls. Our results demonstrate elevated ABR thresholds with an intact function of outer HCs (OHCs), which is consistent with auditory neuropathy.

Figure 6

The morphology of the HCs remains intact (A) We demonstrate representative whole-mount tissue at 10%–20%, 40%–50%, and 60%–70% distance from the apical tip at P30 control mice. (B) Representative whole-mount tissue at 10%–20%, 40%–50%, and 60%–70% distance from the apical tip 28 days after AAV-DTA injection in Td/PV^Cre mice. HCs were preserved in all turns. Colocalization of tdTomato and myo7A confirmed the expression of Cre recombinase in HCs.

Figure 7

Afferent nerve fiber degeneration after AAV-DTA injection (A) Representative low magnification (20×) of the mid-modiolar cochlear section of age-matched Td/PV^Cre control and 28 dpi Td/PV^Cre mice. tdTomato is expressed in the afferent fibers crossing the OSL and RC. The inserts demonstrate the magnified view of tdTomato⁺ HCs in the organ of Corti. The MOC terminal at the OHCs (yellow arrows) and ISBs (violet circle) are immunopositive for TUJ1 in the dashed box. In the AAV-DTA-injected Td/PV^Cre mice, the tdTomato⁺ afferent fibers are not visible while TUJ1 expression in the MOC terminal is present. (B) In adult WT whole-mount tissue, we observed the TUJ1⁺ ISB and the thin spiral fibers of type II SGN (white arrowheads) of the OSB. In the AAV-DTA-injected PV^Cre mice, we observed significant thinning of the ISB and neurites in the osseous spiral lamina (OSL) and near-total loss of the OSB, while the MOC terminals appear intact, but the ISB is reduced in size. (C) The efferent fibers for the olivocochlear system are immunopositive for ChAT and TUJ1 in the organ of Corti (WT). The AAV-DTA-injected PV^Cre mice maintain a comparable number of ChAT and TUJ1⁺ radial efferent fibers forming the MOC terminals, but the ChAT signal in the ISB is reduced compared to the WT.