Delivery of gene therapy through a cerebrospinal fluid conduit to rescue hearing in adult mice.

Inner ear gene therapy has recently effectively restored hearing in neonatal mice, but it is complicated in adulthood by the structural inaccessibility of the cochlea, which is embedded within the temporal bone. Alternative delivery routes may advance auditory research and also prove useful when translated to humans with progressive genetic-mediated hearing loss. Cerebrospinal fluid flow via the glymphatic system is emerging as a new approach for brain-wide drug delivery in rodents as well as humans. The cerebrospinal fluid and the fluid of the inner ear are connected via a bony channel called the cochlear aqueduct, but previous studies have not explored the possibility of delivering gene therapy via the cerebrospinal fluid to restore hearing in adult deaf mice. Here, we showed that the cochlear aqueduct in mice exhibits lymphatic-like characteristics. In vivo time-lapse magnetic resonance imaging, computed tomography, and optical fluorescence microscopy showed that large-particle tracers injected into the cerebrospinal fluid reached the inner ear by dispersive transport via the cochlear aqueduct in adult mice. A single intracisternal injection of adeno-associated virus carrying solute carrier family 17, member 8 (Slc17A8), which encodes vesicular glutamate transporter-3 (VGLUT3), rescued hearing in adult deaf Slc17A8-/- mice by restoring VGLUT3 protein expression in inner hair cells, with minimal ectopic expression in the brain and none in the liver. Our findings demonstrate that cerebrospinal fluid transport comprises an accessible route for gene delivery to the adult inner ear and may represent an important step toward using gene therapy to restore hearing in humans.

Differential effects of noise exposure between substrains of CBA mice.

Noise trauma involves a plethora of mechanisms including reactive oxygen species, apoptosis, tissue damage, and inflammation. Recently, circadian mechanisms were also found to contribute to the vulnerability to noise trauma in mice, with greater damage occurring during their active phase (nighttime), when compared to similar noise exposures during their inactive phase (daytime). These effects seem to be regulated by mechanisms involving Bdnf responses to noise trauma and circulating levels of corticosterone (CORT). However, recent studies using different noise paradigms show contradicting results and it remains unclear how universal these findings are. Here we show that these findings differ even between substrains of mice and are restricted to a narrow window of noise intensity. We found that CBA/Sca mice exposed to 103 dB SPL display differential day/night noise sensitivity as measured by auditory brainstem responses (ABRs), but not at 100 (where full recovery is observed in day or night exposed mice) or 105 dB SPL (where permanent damage is found in both groups). In contrast, neither CBA/CaJ or CBA/JRj displayed such differences in day/night noise sensitivity, whatever noise intensity used. These effects appeared to be independent from outer hair cell function, as distortion product otoacoustic emissions appeared equally affected by day or night noise exposure, in all strains and in all noise conditions. Minor differences in ribbon counts or synaptic pairing were found in CBA/Sca mice, which were inconsistent with ABR wave 1 amplitude changes. Interestingly, CORT levels peaked in CBA/Sca mice at the onset of darkness at zeitgeber time 12 reaching levels of 43.8 ng/ml, while in the CBA/CaJ and the CBA/JRj, levels were 11.9 and 15.6 ng/ml respectively and peaking 4 h earlier (zeitgeber time 8). These findings were consistent with higher period of daily rhythm in CBA/Sca mice when measured in complete darkness using running wheels (23.7 h), than in CBA/CaJ (23.45 h) or CBA/JRj (23.13 h). In conclusion, our study suggests that the differential vulnerability to noise trauma between inactive and active phase is not universal and is as sensitive as substrain differences that might be governed by the circadian amplitude of the circulating CORT profiles.

Auditory synaptopathy in mice lacking the glutamate transporter GLAST and its impact on brain activity.

Neurotransmission of acoustic signals from the hair cells to the auditory nerve relies on a tightly controlled communication between pre-synaptic ribbons and post-synaptic glutamatergic terminals. After noise overexposure, de-afferentation occurs as a consequence of excessive glutamate release. What maintains synaptic integrity in the cochlea is poorly understood. The objective of this study is to evaluate the role of GLAST in maintaining synaptic integrity in the cochlea in absence or presence of noise, and its impact on sound-evoked brain activity using manganese-enhanced MRI (MeMRI). The glutamate aspartate transporter GLAST is present in supporting cells near the afferent synapse and its genetic deletion leads to greater synaptic swelling after noise overexposure. At baseline, GLAST knockout (GLAST KO) mice displayed two-fold lower wave 1 amplitude of the auditory brainstem response (ABR) when compared to their wild-type littermates in spite of similar ABR and distortion product otoacoustic emissions (DPOAE) thresholds. While the abundance of ribbons was not affected by the loss of GLAST function, the number of paired synapses was halved in GLAST KO mice, suggestive of a pre-existing auditory synaptopathy. Immediately after the noise exposure ABR thresholds rose by 41-62dB to a similar degree in GLAST WT and KO mice and DPOAE remained unaffected. In the acute phase following noise exposure, GLAST KO mice showed near complete de-afferentation unlike WT mice which maintained four to seven paired synapses per IHC. Brain activity using MeMRI found noise exposure to cause greater activity in the inferior colliculus in GLAST KO but not in WT mice. No changes in brain activity was found in GLAST KO mice at baseline in spite of affected afferent synapses, suggesting that auditory synaptopathy may not be sufficient to alter brain activity in the absence of noise exposure.

Circadian vulnerability of cisplatin-induced ototoxicity in the cochlea.

The chemotherapeutic agent cisplatin is renowned for its ototoxic effects. While hair cells in the cochlea are established targets of cisplatin, less is known regarding the afferent synapse, which is an essential component in the faithful temporal transmission of sound. The glutamate aspartate transporter (GLAST) shields the auditory synapse from excessive glutamate release, and its loss of function increases the vulnerability to noise, salicylate, and aminoglycosides. Until now, the involvement of GLAST in cisplatin-mediated ototoxicity remains unknown. Here, we test in mice lacking GLAST the effects of a low-dose cisplatin known not to cause any detectable change in hearing thresholds. When administered at nighttime, a mild hearing loss in GLAST KO mice was found but not at daytime, revealing a potential circadian regulation of the vulnerability to cisplatin-mediated ototoxicity. We show that the auditory synapse of GLAST KO mice is more vulnerable to cisplatin administration during the active phase (nighttime) when compared to WT mice and treatment during the inactive phase (daytime). This effect was not related to the abundance of platinum compounds in the cochlea, rather cisplatin had a dose-dependent impact on cochlear clock rhythms only after treatment at nighttime suggesting that cisplatin can modulate the molecular clock. Our findings suggest that the current protocols of cisplatin administration in humans during daytime may cause a yet undetectable damage to the auditory synapse, more so in already damaged ears, and severely impact auditory sensitivity in cancer survivors.

Impact of noise exposure on the circadian clock in the auditory system.

Circadian rhythms control the timing of all bodily functions, and misalignment in the rhythms can cause various diseases. Moreover, circadian rhythms are highly conserved and are regulated by a transcriptional-translational feedback loop of circadian genes that has a periodicity of approximately 24 h. The cochlea and the inferior colliculus (IC) have been shown to possess an autonomous and self-sustained circadian system as demonstrated by recording, in real time, the bioluminescence from PERIOD2::LUCIFERASE (PER2::LUC) mice. The cochlea and IC both express the core clock genes, Per1, Per2, Bmal1, and Rev-Erbα, where RNA abundance is rhythmically distributed with a 24 h cycle. Noise exposure alters clock gene expression in the cochlea and the IC after noise stimulation, although in different ways. These findings highlight the importance of circadian responses in the cochlea and the IC and emphasize the importance of circadian mechanisms for understanding the differences in central and peripheral auditory function and the subsequent molecular changes that occur after daytime (inactive phase) or nighttime (active phase) noise trauma.

Circadian Regulation of Cochlear Sensitivity to Noise by Circulating Glucocorticoids.

The cochlea possesses a robust circadian clock machinery that regulates auditory function. How the cochlear clock is influenced by the circadian system remains unknown. Here, we show that cochlear rhythms are system driven and require local Bmal1 as well as central input from the suprachiasmatic nuclei (SCN). SCN ablations disrupted the circadian expression of the core clock genes in the cochlea. Because the circadian secretion of glucocorticoids (GCs) is controlled by the SCN and GCs are known to modulate auditory function, we assessed their influence on circadian gene expression. Removal of circulating GCs by adrenalectomy (ADX) did not have a major impact on core clock gene expression in the cochlea. Rather it abolished the transcription of clock-controlled genes involved in inflammation. ADX abolished the known differential auditory sensitivity to day and night noise trauma and prevented the induction of GABA-ergic and glutamate receptors mRNA transcripts. However, these improvements were unrelated to changes at the synaptic level, suggesting other cochlear functions may be involved. Due to this circadian regulation of noise sensitivity by GCs, we evaluated the actions of the synthetic glucocorticoid dexamethasone (DEX) at different times of the day. DEX was effective in protecting from acute noise trauma only when administered during daytime, when circulating glucocorticoids are low, indicating that chronopharmacological approaches are important for obtaining optimal treatment strategies for hearing loss. GCs appear as a major regulator of the differential sensitivity to day or night noise trauma, a mechanism likely involving the circadian control of inflammatory responses.

Circadian integration of inflammation and glucocorticoid actions: Implications for the cochlea.

Auditory function has been shown to be influenced by the circadian system. Increasing evidence point towards the regulation of inflammation and glucocorticoid actions by circadian rhythms in the cochlea. Yet, how these three systems (circadian, immune and endocrine) converge to control auditory function remains to be established. Here we review the knowledge on immune and glucocorticoid actions, and how they interact with the circadian and the auditory system, with a particular emphasis on cochlear responses to noise trauma. We propose a multimodal approach to understand the mechanisms of noise-induced hearing loss by integrating the circadian, immune and endocrine systems into the bearings of the cochlea. Considering the well-established positive impact of chronotherapeutic approaches in the treatment of cardiovascular, asthma and cancer, an increased knowledge on the mechanisms where circadian, immune and glucocorticoids meet in the cochlea may improve current treatments against hearing disorders.

The genetic vulnerability to cisplatin ototoxicity: a systematic review.

Ototoxicity is one of the major side-effects of platinum-based chemotherapy, in particular cisplatin (cis-diammine dichloroplatinum II). To our knowledge, no systematic review has previously provided a quantitative summary estimate of the impact of genetics upon the risk of developing hearing loss. We searched Embase, Medline, ASSIA, Pubmed, Scopus, and Web of Science, for studies documenting the genetic risk of ototoxicity in patients with cancer treated with cisplatin. Titles/abstracts and full texts were reviewed for inclusion. Meta-analytic estimates of risk (Odds Ratio) from the pooled data were calculated for studies that have been repeated twice or more. The search identified 3891 papers, of which 30 were included. The majority were retrospective (44%), ranging from n = 39 to n = 317, some including only patients younger than 25 years of age (33%), and some on both genders (80%). The most common cancers involved were osteosarcoma (53%), neuroblastoma (37%), prostate (17%) and reproductive (10%). Most studies performed genotyping, though only 5 studies performed genome-wide association studies. Nineteen single-nucleotide polymorphisms (SNPs) from 15 genes were repeated more than twice. Meta-analysis of group data indicated that rs1872328 on ACYP2, which plays a role in calcium homeostasis, increases the risk of ototoxicity by 4.61 (95% CI: 3.04-7.02; N = 696, p < 0.0001) as well as LRP2 rs4668123 shows a cumulated Odds Ratio of 3.53 (95% CI: 1.48-8.45; N = 118, p = 0.0059), which could not be evidenced in individual studies. Despite the evidence of heterogeneity across studies, these meta-analytic results from 30 studies are consistent with a view of a genetic predisposition to platinum-based chemotherapy mediated ototoxicity. These new findings are informative and encourage the genetic screening of cancer patients in order to identify patients with greater vulnerability of developing hearing loss, a condition having a potentially large impact on quality of life. More studies are needed, with larger sample size, in order to identify additional markers of ototoxic risk associated with platinum-based chemotherapy and investigate polygenic risks, where multiple markers may exacerbate the side-effects.

CO2-evoked release of PGE2 modulates sighs and inspiration as demonstrated in brainstem organotypic culture.

Inflammation-induced release of prostaglandin E2 (PGE2) changes breathing patterns and the response to CO2 levels. This may have fatal consequences in newborn babies and result in sudden infant death. To elucidate the underlying mechanisms, we present a novel breathing brainstem organotypic culture that generates rhythmic neural network and motor activity for 3 weeks. We show that increased CO2 elicits a gap junction-dependent release of PGE2. This alters neural network activity in the preBötzinger rhythm-generating complex and in the chemosensitive brainstem respiratory regions, thereby increasing sigh frequency and the depth of inspiration. We used mice lacking eicosanoid prostanoid 3 receptors (EP3R), breathing brainstem organotypic slices and optogenetic inhibition of EP3R(+/+) cells to demonstrate that the EP3R is important for the ventilatory response to hypercapnia. Our study identifies a novel pathway linking the inflammatory and respiratory systems, with implications for inspiration and sighs throughout life, and the ability to autoresuscitate when breathing fails.