Loss of the spiral ganglion neurons (SGNs) — the primary auditory neurons that relay signals from cochlear hair cells to the brain — causes auditory neuropathy, a form of sensorineural hearing loss for which no biological therapy exists. Li and colleagues (FASEB Journal, 2020) demonstrated that simultaneous ectopic expression of the proneural transcription factors Neurog1 (Ngn1) and Neurod1 converts neonatal cochlear glial cells into SGN-like neurons ("new SGNs") in vivo, but only a subset acquires mature Mafb+/Gata3+ identity, reprogramming efficiency declines with postnatal age (the authors subsequently reported that adult cochlear glia are not responsive), only healthy neonatal cochleae were tested, and functional synapses with hair cells or electrophysiological maturation could not be demonstrated. This problem asks whether and under what conditions reprogrammed glia can become genuinely functional auditory neurons in vivo. It comprises several distinct open sub-questions — functional hair-cell synapses and auditory signaling by new SGNs, the conversion mechanism and the origin of its heterogeneity, the postnatal age restriction, reprogramming in the clinically relevant setting of prior SGN degeneration, and factor combinations that raise the yield of fully converted SGNs — each of which can be investigated and advanced independently; their common theme is turning marker-level glial conversion into functional in situ regeneration of the auditory nerve.

Background

The spiral ganglion neurons (SGNs) are the bipolar primary auditory neurons whose peripheral dendrites receive glutamatergic input from inner hair cells of the cochlea and whose axons form the auditory nerve. In mammals, SGNs are terminally differentiated and do not spontaneously regenerate, so their degeneration — caused by noise, ototoxic drugs, aging, or genetic lesions, and defining auditory neuropathy — is irreversible. Cochlear implants, the main prosthetic intervention, depend on surviving SGNs to carry signals centrally, so SGN loss also limits implant outcomes. Within the spiral ganglion, the neurons are surrounded and their fibers ensheathed by non-neuronal glial cells: satellite glial cells around the somata in Rosenthal's canal and Schwann cells along the fibers. Cochlear glia survive long after neuronal loss and share a developmental lineage with neurons, which motivated attempts to use them as an endogenous replacement source. In vitro, proteolipid-protein-1-positive (Plp1+) glia from neonatal spiral ganglia form neurospheres and can generate neurons (McLean et al., 2016), but whether an in vivo conversion is possible was unknown. Neurog1 (Ngn1) and Neurod1 are basic helix-loop-helix proneural transcription factors that sequentially orchestrate otic neurogenesis during development; Mafb and Gata3 are transcription factors required for SGN maturation and postsynaptic differentiation, so Mafb/Gata3 co-expression marks a fully converted SGN. Li et al. (2020) showed that simultaneous ectopic Ngn1 and Neurod1 expression in neonatal mouse cochlear glia in vivo produces cells expressing the SGN markers Tuj1, Map2, Prox1, Mafb, and Gata3 while losing the glial markers Sox10 and Scn7a; these "new SGNs" are heterogeneous, the well-reprogrammed ones transcriptionally resemble type I SGNs, conversion efficiency scales with factor dosage, and efficiency declines with postnatal age. Functional integration could not be assessed because the EGFP (enhanced green fluorescent protein) reporter signal was insufficient to trace fibers, leaving open whether new SGNs form synapses with hair cells or transmit auditory signals. Contemporaneously, Kempfle et al. (2020) showed that transient Lin28 (an RNA-binding regulator of neural stem-cell programs) overexpression in Plp1+ glia converts them toward neurons in ouabain-damaged cochleae, establishing that a damaged cochlear environment does not block glial conversion by that route, but likewise without demonstrating functional SGN identity. The stage is therefore set by two proof-of-principle in vivo conversion studies whose cells remain at a general-neuronal or partial-SGN stage, with no functional, mechanistic, or clinically realistic demonstration.

Problem Statement

The overarching objective is to determine whether Ngn1/Neurod1-reprogrammed cochlear glial cells can become functional auditory neurons in vivo, and to establish the conditions under which such in situ regeneration succeeds. The following are distinct open sub-questions of that objective; none is a prerequisite for progress on the others. The objective is one compound question — whether reprogrammed glia become genuinely functional auditory neurons in vivo, and under what conditions; sub-question (1) addresses the whether-part, and sub-questions (2)–(5) each address a part of the conditions-part. (1) Functional integration — do the new SGNs form functional synapses with hair cells, extend fibers traceable to the organ of Corti, exhibit the electrophysiological properties of SGNs (e.g., Mafb+ new SGNs), and transmit auditory signals attributable to the converted glia-derived lineage rather than to surviving endogenous SGNs, as required to affect hearing? (2) Mechanism and heterogeneity — by what molecular mechanisms do Ngn1 and Neurod1 specify and execute glial-to-SGN conversion, which glial subpopulation(s) respond (satellite glial cells in Rosenthal's canal versus Schwann cells along the fibers, whose respective roles remain undefined for lack of a method to separate the two), and why does only a subset of new SGNs achieve mature Mafb+ (Mafb+/Gata3+) identity? No particular marker classification is prescribed for identifying the responding population. (3) Age restriction — reprogramming efficiency declines with postnatal age, and the source authors subsequently reported that adult cochlear glial cells are not responsive to ectopic Neurod1/Neurog1; can this age restriction be overcome? (4) Degeneration context — the 2020 study tested healthy, non-degenerated neonatal cochleae; does prior SGN loss promote or impair glial-to-SGN reprogramming, the clinically relevant setting for auditory neuropathy? Ouabain, which damages type I SGNs without selectivity among type I subtypes, is an established lesion for such tests, and the Lypd1-DTR model (Zhang et al., 2023), in which diphtheria-toxin-receptor expression confined to type Ic SGNs permits their selective ablation, is a tool that became available only after the source study. (5) Factor combination — do other factors added to Ngn1/Neurod1 increase the percentage of fully converted (Mafb+/Gata3+) new SGNs in vivo? Mafb and Gata3 are candidate examples, consistent with the source's Mafb+/Gata3+ definition of full conversion and with later reports of Neurog1–Neurod1 and Gata3–Mafb pairing in early SGN development, but no particular factor set is prescribed.

References

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