Auditory sensory deprivation induced by noise exposure exacerbates cognitive decline in a mouse model of Alzheimer’s disease

Essential revisions:

(1) The ABR analysis did not include important information, especially which wave or waves were used for determining thresholds. This can affect the interpretation of results because changes in specific ABR waves could indicate where along the auditory pathway the noise-induced hearing loss occurs. For instance, a threshold shift in the later waves could not be simply attributed to cochlear injury, as suggested in the manuscript. In fact, the study of noise induced hearing loss in C57BL6 mice, cited above, reported that physiological effects were not necessarily associated with peripheral injury.

As requested, more information on ABR procedure has been added in the revised manuscript. Specifically, hearing threshold levels were determined as the stimulus intensity at which a Wave I peak could be identified. We agree with the Reviewer that the later waves do not reflect cochlear injury. In fact, the mouse ABR is composed of four components, reflecting neural activity chiefly from the auditory nerve (Wave I), the cochlear nucleus (Wave II), the superior olivary complex (Wave III), and the lateral lemniscus and/or inferior colliculus (Wave IV) (Alvarado et al., Neurosci Res 2012; Scimemi et al., Acta Oto Ital 2014, Willot, Handbook of Mouse Auditory Research: From Behavior to Molecular Biology: Taylor and Francis, 2001). To measure auditory thresholds we used Wave I, that in mice is the largest and usually the last wave to disappear as the sound stimulus decreases (Willot, Handbook of Mouse Auditory Research: From Behavior to Molecular Biology: Taylor and Francis, 2001). It has been documented that Wave I is affected by noise exposure in several models and it correlated with damage to inner hair cells and loss of auditory nerve fibers (Kujawa and Liberman, J Neurosci 2009; Lavinsky et al., PLOS Genet 2015; Mehraei et al., J Neurosci 2016; Jongkamonwiwat et al., Cell Rep 2020). In mice most studies use Wave I to determine thresholds (Willot, Handbook of Mouse Auditory Research: From Behavior to Molecular Biology: Taylor and Francis, 2001). Nonetheless, in the revised manuscript we added the analysis of Wave I and Wave II latency-intensity curves (reflecting the speed of neural transmission) to provide further electrophysiological data supporting noise-induced cochlear damage.

In addition, the methods state that the tone bursts were "…presented monaurally in an open field" and then states, "ABRs were assessed bilaterally in all animals…". Are the thresholds presented in Figure 2 an average of ABR thresholds for each ear?

In the revised manuscript we clarified the procedure used (see pages 20-21, lines 483-488). Specifically, to verify normal hearing before noise exposure, ABRs were recorded bilaterally to ensure no consistent left-right ear ABR asymmetry due to ear pathologies. After noise exposure, we do not expect inter-aural differences, considering that acoustic trauma delivered in open field induces bilateral and symmetric hearing loss. Thus the ABRs were recorded from the right ear only to increase the efficiency of data acquisition.

(2) There is a disconnect between the auditory cortex physiology results and the behavior. The memory task does not rely on auditory processing or auditory plasticity. Therefore, the relationship between auditory cortex measures and an AD-associated behavioral phenotype should be addressed.

To further investigate connections between alterations of the auditory cortex physiology and AD-associated behavioral phenotype, we performed an animal by animal analysis. Our data revealed no correlation between hearing thresholds and memory performance in NOR test. However, in noise exposed AD mice a high correlation between spine loss in auditory cortex and memory performance in NOR test was found (r² = 0.96; P = 0.019; see page10, lines 226-239 and Figure 7- supplement 2). These results suggest a correlation between structural plasticity of the auditory cortex and memory performance. Literature data suggest a relationship between auditory processes and cognitive functions (Griffiths et al., Neuron 2020; Johnson et al., Brain 2020; Slade et al., Trends Neurosci 2020); nevertheless, more in depth analyses of the anatomical substrates linking alterations of auditory cortex plasticity and neural circuits underlying memory deserve further investigations.

(3) The difference in ROS is dramatic between control and sensory-deprived AD mice. Could the authors confirm these findings by other tools? Does such a dramatic increase in ROS cause apoptosis of neurons, in addition to the reduction in spine density?

To address these issues we performed additional Western blot experiments further investigating oxidative stress and apoptosis.

Specifically, our analyses in the hippocampus of 3×Tg-AD mice revealed an increase of Nitrotyrosine in transgenic mice exposed to noise, reflecting an enhancement of reactive nitrogen species (RNS) formation and confirming oxidative/nitrosative stress in AD mice exposed to noise. Moreover, increased expression of active Caspase-3 and the pro-apoptotic protein Bax were observed, suggesting activation of apoptotic pathways in the AD mice exposed to noise.

The results of these experiments are reported at pages 12-13, lines 287-298 and shown in Figure 10- supplement 1.

(4) Changes in ROS and in HO1 expression are only correlative and not linked to functional and structural impairments observed in AD model mice. In the previous studies, the authors show that cochlear impairments can be reversed by Q10. A possible causal link between oxidative stress and impairments in CA3-CA1 synaptic transmission / spine density can be tested by local injections of antioxidants to the hippocampus. Otherwise, these results remain purely correlative and do not imply that it is ROS that impairs homeostatic capabilities of hippocampus and auditory cortex in AD model.

We agree with the reviewer that additional experiments would be required to unequivocally demonstrate the cause-effect relationship between changes in ROS/HO1 expression and functional/structural impairments observed in AD model mice. However, we would like to draw the reviewer’s attention on the fact that, according to Italian laws, new experiments including local injection of antioxidants to the hippocampus require a specific approval from the Ethics Committee of our University followed by additional authorization by the Italian Ministry of Health. These administrative procedures require many months, that would delay much the submission of our manuscript revision. We would appreciate very much if this Reviewer accepted that such control experiment is postponed to a follow-up study. In the revised manuscript, we acknowledged that in our experimental model changes in ROS/HO1 expression are potentially responsible for functional and structural impairments observed in AD model mice and that further experimental evidence is required to establish a cause-effect relationship (see page 17, lines 414-420).

(5) The study used only male mice, but did not provide a rationale for doing so. There is a broad literature that indicates sex-specific differences in age-related hearing loss, noise-induced hearing loss, and progression of AD /cognitive decline. Therefore, it would be particularly important for a study of this sort to consider sex as a critical biological variable.

We agree with the Reviewers on the crucial role of gender differences. We have considered this potential criticism in planning our experiments. A rationale for the use of male animals has been added to the revised version of the manuscript (see page 19, lines 444-451). In fact, sex differences in the prevalence, risk and severity of AD have been demonstrated in numerous clinical and epidemiological studies (Zhu et al., Cell and Mol Life Sci 2021). Animal research with mouse models of AD also supports the greater susceptibility to AD in females (Koran et al., Brain Imaging Behav 2017; Laws et al., Curr Opin Psychiatry 2018;Yang et al., Neurosci. Bull 2018).

On the other hand, sexual hormones can influence noise-induced hearing loss in rodents (Milon et al., Biol Sex Differ 2018). Specifically, estrogens may have neuroprotective function in the inner ear (Meltser et al., J Clin Invest 2008; Shuster et al., J Acoust Soc Am 2019; Delhez et al., Cell Mol Life Sci 2020).

Taken together, females are more susceptible to AD pathology but they are less susceptible to noise-induced hearing loss. Based on this evidence and taken into account that male mice are more susceptible to noise-induced cochlear damage, we opted to focus our study on male animals.

(6) There is a potential concern regarding the statistical approach: For example,

In Figure 4, the groups in Figure 4C and 4D appear to have been analyzed using two separate ANOVAs. Since the dependent variable (i.e., number of spines) is the same for the different groups/conditions, one ANOVA should be used to establish whether or not a main effect is observed.

A two-way ANOVA comparing all groups has been performed. Statistical results (F values and level of significance) have been added to the revised manuscript (see page 7, lines 151-152 and 155-156).

In Figure 3D, individual data points between Ctrl and Noise are largely overlapping, so significance may be influenced by the 'outlier' in the Ctrl group. Please clarify, and also if all assumptions for a Student's t-test are met here.

To identify outliers in our data set, we used the Outlier calculator in GraphPad Prism Software. This analysis identified no outliers from the curve fit and, therefore, no records were excluded from data analysis. However, we checked if statistical significance was affected by the highest data point in the Ctrl group and, after its removal, the level of significance was still > 0.05.

Statistical analysis has been revised and two-way ANOVA has been performed rather than Student’s t-test (see page 6, lines 129-130 and 132-133).

(7) In Figure 1, it seems that a large number of mice was included in this study (74 3xTg and 67 WT) but it is difficult to understand how these mice were distributed to individual experiments. Do the authors have individual mice that underwent all (or most) of the experimental steps, and if so an analysis on an animal-by-animal basis would be appreciated.

Sample size used for different experimental procedure is specified in each figure legend. Several animals underwent more than one experimental procedure (i.e., ABR, NOR, spine density evaluations). In the revised manuscript, we identified the subgroup of animals undergoing NOR test, ABR measurements and spine density and we performed an additional linear regression analysis to evaluate correlation index among these variables. Results of animal-by-animal analysis have been added in the revised manuscript (see page 10, lines 226-239) and shown in Figure 7- supplement 2.

(8) More information regarding animals would be helpful, e.g. were mutant and wild-type mice housed together? Were any mice single housed? This may affect behavior. Were experimenters blinded for the behavioral experiments?

WT and AD mice and, within each group, noise-exposed and no-noise mice, were housed separately, in cages containing from 3 to 5 animals (see page 19, lines 455-456).

(9) It should be discussed / justified more explicitly how well the noise-induced hearing loss represents the type of midlife hearing loss that is present in humans.

Noise-induced hearing loss caused by single exposure to very loud sounds or repeated exposures to lower intensity sounds over an extended period is a major source of hearing disability worldwide. Moreover, noise for occupational or leisure exposures is considered one of the major risk factors for hearing loss in midlife (Nelson et al., 2005; Dobie, 2008; European Agency for Safety and Health at Work, 2002; Lancet committee 2020). Furthermore, in experimental studies, noise-induced hearing loss represents a reliable and replicable model of sensorineural hearing loss.

Our paradigm of acoustic trauma of repeated and high intensity stimulation can be representative of dangerous exposure in humans, in whom exposure to intensities > 85-87 dB are generally considered limit safe values (ISO 1990; https://www.who.int; https://www.cdc.gov/niosh/topics/noise/default.html). Furthermore, based on the mouse lifespan, the age of 3-6 months can be considered has a mature adult age (https://www.jax.org/news-and-insights/jax-blog/2017/november/when-are-mice-considered-old) and considering that 3×Tg-AD mice at the age of 2-3 months can be considered a model of preclinical AD, whereas the onset of AD phenotype manifests at 7 to 9 months, we focused our study on 6 months of age animals. Details have been added in the revised manuscript (see page 19, lines 440-441 and page 20, lines 468-470).