Probing the role of synaptic adhesion molecule RTN4RL2 in setting up cochlear connectivity

Reviewer #1 (Public review):

Hearing and balance rely on specialized ribbon synapses that transmit sensory stimuli between hair cells and afferent neurons. Synaptic adhesion molecules that form and regulate transsynaptic interactions between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are crucial for maintaining auditory synaptic integrity and, consequently, for auditory signaling. Synaptic adhesion molecules such as neurexin-3 and neuroligin-1 and -3 have recently been shown to play vital roles in establishing and maintaining these synaptic connections ( doi: 10.1242/dev.202723 and DOI: 10.1016/j.isci.2022.104803). However, the full set of molecules required for synapse assembly remains unclear.

Karagulan et al. highlight the critical role of the synaptic adhesion molecule RTN4RL2 in the development and function of auditory afferent synapses between IHCs and SGNs, particularly regarding how RTN4RL2 may influence synaptic integrity and receptor localization. Their study shows that deletion of RTN4RL2 in mice leads to enlarged presynaptic ribbons and smaller postsynaptic densities (PSDs) in SGNs, indicating that RTN4RL2 is vital for synaptic structure. Additionally, the presence of "orphan" PSDs-those not directly associated with IHCs-in RTN4RL2 knockout mice suggests a developmental defect in which some SGN neurites fail to form appropriate synaptic contacts, highlighting potential issues in synaptic pruning or guidance. The study also observed a depolarized shift in the activation of CaV1.3 calcium channels in IHCs, indicating altered presynaptic functionality that may lead to impaired neurotransmitter release. Furthermore, postsynaptic SGNs exhibited a deficiency in GluA2/3 AMPA receptor subunits, despite normal Gria2 mRNA levels, pointing to a disruption in receptor localization that could compromise synaptic transmission. Auditory brainstem responses showed increased sound thresholds in RTN4RL2 knockout mice, indicating impaired hearing related to these synaptic dysfunctions.

The findings reported here significantly enhance our understanding of synaptic organization in the auditory system, particularly concerning the molecular mechanisms underlying IHC-SGN connectivity. The implications are far-reaching, as they not only inform auditory neuroscience but also provide insights into potential therapeutic targets for hearing loss related to synaptic dysfunction.

Reviewer #2 (Public review):

Summary:

Kargulyan et al. investigate the function of the transsynaptic adhesion molecule RTN4RL2 in the formation and function of ribbon synapses between type I spiral ganglion neurons (SGNs) and inner hair cells. For this purpose, they study constitutive RTN4RL2 knock-out mice. Using immunohistochemistry, they reveal defects in the recruitment of protein to ribbon synapses in the knockouts. Serial block phase EM reveals defects in SGN projections in mutants. Electrophysiological recordings suggest a small but statistically significant depolarized shift in the activation of Cav1.3 Ca2+ channels. Auditory thresholds are also elevated in the mutant mice. The authors conclude that RTN4RL2 contributes to the formation and function of auditory afferent synapses to regulate auditory function.

Strengths:

The authors have excellent tools to analyze ribbon synapses.

Weaknesses:

However, there are several concerns that substantially reduce my enthusiasm for the study.

(1) The analysis of the expression pattern of RTN4RL2 in Figure 1 is incomplete. The authors should show a developmental time course of expression up into maturity to correlate gene expression with major developmental milestones such as axon outgrowth, innervation, and refinement. This would allow the development of models supporting roles in axon outgrowth versus innervation or both.

(2) It would be important to improve the RNAscope data. Controls should be provided for Figure 1B to show that no signal is observed in hair cells from knockouts. The authors apparently already have the sections because they analyzed gene expression in SGNs of the knock-outs (Figure 1C).

(3) It is unclear from the immunolocalization data in Figure 1D if all type I SGNs express RTN4RL2. Quantification would be important to properly document the presence of RTN4RL2 in all or a subset of type I SGNs. If only a subset of SGNs express RTN4RL2, it could significantly affect the interpretation of the data. For example, SGNs selectively projecting to the pillar or modiolar side of hair cells could be affected. These synapses significantly differ in their properties.

(4) It is important to show proper controls for the RTN4RL2 immunolocalization data to show that no staining is observed in knockouts.

(5) The authors state in the discussion that no staining for RTN4RL2 was observed at synaptic sites. This is surprising. Did the authors stain multiple ages? Was there perhaps transient expression during development? Or in axons indicative of a role in outgrowth, not synapse formation?

(6) In Figure 2 it seems that images in mutants are brighter compared to wildtypes. Are exposure times equivalent? Is this a consistent result?

(7) The number of synaptic ribbons for wildtype in Figure 2 is at 10/IHCs, and in Figure 2 Supplementary Figure 2 at 20/IHCs (20 is more like what is normally reported in the literature). The value for mutant similarly drastically varies between the two figures. This is a significant concern, especially because most differences that are reported in synaptic parameters between wild-type and mutants are far below a 2-fold difference.

(8) The authors report differences in ribbon volume between wild-type and mutant. Was there a difference between the modiolar/pillar region of hair cells? It is known that synaptic size varies across the modiolar-pillar axis. Maybe smaller synapses are preferentially lost?

(9) The authors show in Figure 2 - Supplement 3 that GluA2/3 staining is absent in the mutants. Are GluA4 receptors upregulated? Otherwise, synaptic transmission should be abolished, which would be a dramatic phenotype. Antibodies are available to analyze GluA4 expression, the experiment is thus feasible. Did the authors carry out recordings from SGNs?

(10) The authors use SBEM to analyze SGN projections and synapses. The data suggest that a significant number of SGNs are not connected to IHCs. A reconstruction in Figure 3 shows hair cells and axons. It is not clear how the outline of hair cells was derived, but this should be indicated. Also, is this a defect in the formation of synapses and subsequent retraction of SGN projections? Or could RTN4RL2 mutants have a defect in axonal outgrowth and guidance that secondarily affects synapses? To address this question, it would be useful to sparsely label SGNs in mutants, for example with AAV vectors expression GFP, and to trace the axons during development. This would allow us to distinguish between models of RTN4RL2 function. As it stands, it is not clear that RTN4RL2 acts directly at synapses.

(11) The authors observe a tiny shift in the operation range of Ca2+ channels that has no effect on synaptic vesicle exocytosis. It seems very unlikely that this difference can explain the auditory phenotype of the mutant mice.

(12) ABR recordings were conducted in whole-body knockouts. Effects on auditory thresholds could be a secondary consequence of perturbation along the auditory pathway. Conditional knockouts or precisely designed rescue experiments would go a long way to support the authors' hypothesis. I realize that this is a big ask and floxed mice might not be available to conduct the study.

Reviewer #3 (Public review):

In this study, the authors used RNAscope and immunostaining to confirm the expression of RTN4RL2 RNA and protein in hair cells and spiral ganglia. Through RTN4RL2 gene knockout mice, they demonstrated that the absence of RTN4RL2 leads to an increase in the size of presynaptic ribbons and a depolarized shift in the activation of calcium channels in inner hair cells. Additionally, they observed a reduction in GluA2/3 AMPA receptors in postsynaptic neurons and identified additional "orphan PSDs" not paired with presynaptic ribbons. These synaptic alterations ultimately resulted in an increased hearing threshold in mice, confirming that the RTN4RL2 gene is essential for normal hearing. These data are intriguing as they suggest that RTN4RL2 contributes to the proper formation and function of auditory afferent synapses and is critical for normal hearing. However, a thorough understanding of the known or postulated roles of RTN4Rl2 is lacking.

While the conclusions of this paper are generally well supported by the data, several aspects of the data analysis warrant further clarification and expansion.

(1) A quantitative assessment is necessary in Figure 1 when discussing RNA and protein expression. It would be beneficial to show that expression levels are quantitatively reduced in KO mice compared to wild-type mice. This suggestion also applies to Figure 2-supplement 3.D, which examines expression levels.

(2) In Figure 2, the authors present a morphological analysis of synapses and discuss the presence of "orphan PSDs." I agree that Homer1 not juxtaposed with Ctbp2 is increased in KO mice compared to the control group. However, in quantifying this, they opted to measure the number of Homer1 juxtaposed with Ctbp2 rather than directly quantifying the number of Homer1 not juxtaposed with Ctbp2. Quantifying the number of Homer1 not juxtaposed with Ctbp2 would more clearly represent "orphan PSDs" and provide stronger support for the discussion surrounding their presence.

(3) In Figure 2, Supplementary 3, the authors discuss GluA2/3 puncta reduction and note that Gria2 RNA expression remains unchanged. However, there is an issue with the lack of quantification for Gria2 RNA expression. Additionally, it is noted that RNA expression was measured at P4. While the timing for GluA2/3 puncta assessment is not specified, if it was assessed at 3 weeks old as in Figure 2's synaptic puncta analysis, it would be inappropriate to link Gria2 RNA expression with GluA2/3 protein expression at P4. If RNA and protein expression were assessed at P4, please indicate this timing for clarity.

(4) In Figure 3, the authors indicate that RTN4RL2 deficiency reduces the number of type 1 SGNs connected to ribbons. Given that the number of ribbons remains unchanged (Figure 2), it is important to clearly explain the implications of this finding. It is already known that each type I SGN forms a single synaptic contact with a single IHC. The fact that the number of ribbons remains constant while additional "orphan PSDs" are present suggests that the overall number of SGNs might need to increase to account for these findings. An explanation addressing this would be helpful.

(5) In Figure 4F and 5Cii, could you clarify how voltage sensitivity (k) was calculated? Additionally, please provide an explanation for the values presented in millivolts (mV).

(6) In Figure 6, the author measured the threshold of ABR at 2-4 months old. Since previous figures confirming synaptic morphology and function were all conducted on 3-week-old mice, it would be better to measure ABR at 3 weeks of age if possible.