Regulator of G protein signaling 17 represents a novel target for treating cisplatin induced hearing loss

Regulators of G protein signaling (RGS) accelerate the GTPase activity of G proteins to enable rapid termination of the signals triggered by G protein-coupled receptors (GPCRs). Activation of several GPCRs, including cannabinoid receptor 2 (CB2R) and adenosine A1 receptor (A1AR), protects against noise and drug-induced ototoxicity. One such drug, cisplatin, an anticancer agent used to treat various solid tumors, produces permanent hearing loss in experimental animals and in a high percentage of cancer patients who undergo treatments. In this study we show that cisplatin induces the expression of the RGS17 gene and increases the levels of RGS17 protein which contributes to a significant proportion of the hearing loss. Knockdown of RGS17 suppressed cisplatin-induced hearing loss in male Wistar rats, while overexpression of RGS17 alone produced hearing loss in vivo. Furthermore, RGS17 and CB2R negatively regulate the expression of each other. These data suggest that RGS17 mediates cisplatin ototoxicity by uncoupling cytoprotective GPCRs from their normal G protein interactions, thereby mitigating the otoprotective contributions of endogenous ligands of these receptors. Thus, RGS17 represents a novel mediator of cisplatin ototoxicity and a potential therapeutic target for treating hearing loss

The endocannabinoid/cannabinoid receptor 2 system protects against cisplatin-induced hearing loss

Previous studies have demonstrated the presence of cannabinoid 2 receptor (CB2R) in the rat cochlea which was induced by cisplatin. In an organ of Corti-derived cell culture model, it was also shown that an agonist of the CB2R protected these cells against cisplatin-induced apoptosis. In the current study, we determined the distribution of CB2R in the mouse and rat cochleae and examined whether these receptors provide protection against cisplatin-induced hearing loss. In a knock-in mouse model expressing the CB2R tagged with green fluorescent protein, we show distribution of CB2R in the organ of Corti, stria vascularis, spiral ligament and spiral ganglion cells. A similar distribution of CB2R was observed in the rat cochlea using a polyclonal antibody against CB2R. Trans-tympanic administration of (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH015), a selective agonist of the CB2R, protected against cisplatin-induced hearing loss which was reversed by blockade of this receptor with 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630), an antagonist of CB2R. JWH015 also reduced the loss of outer hair cells (OHCs) in the organ of Corti, loss of inner hair cell (IHC) ribbon synapses and loss of Na+/K+-ATPase immunoreactivity in the stria vascularis. Administration of AM630 alone produced significant hearing loss (measured by auditory brainstem responses) which was not associated with loss of OHCs, but led to reductions in the levels of IHC ribbon synapses and strial Na+/K+-ATPase immunoreactivity. Furthermore, knock-down of CB2R by trans-tympanic administration of siRNA sensitized the cochlea to cisplatin-induced hearing loss at the low and middle frequencies. Hearing loss induced by cisplatin and AM630 in the rat was associated with increased expression of genes for oxidative stress and inflammatory proteins in the rat cochlea. In vitro studies indicate that JWH015 did not alter cisplatin-induced killing of cancer cells suggesting this agent could be safely used during cisplatin chemotherapy. These data unmask a protective role of the cochlear endocannabinoid/CB2R system which appears tonically active under normal conditions to preserve normal hearing. However, an exogenous agonist is needed to boost the activity of endocannabinoid/CB2R system for protection against a more traumatic cochlear insult, as observed with cisplatin administration.</p

Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity

AbstractCisplatin-induced ototoxicity is one of the major factors limiting cisplatin chemotherapy. Ototoxicity results from damage to outer hair cells (OHCs) and other regions of the cochlea. At the cellular level, cisplatin increases reactive oxygen species (ROS) leading to cochlear inflammation and apoptosis. Thus, ideal otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin's chemotherapeutic efficacy. In this study, we show that epigallocatechin-3-gallate (EGCG) is a prototypic agent exhibiting these properties of an effect otoprotective agent. Rats administered oral EGCG demonstrate reduced cisplatin-induced hearing loss, reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. EGCG also protected against the loss of ribbon synapses associated with inner hair cells and Na+/K+ ATPase α1 in the stria vascularis and spiral ligament. In vitro studies showed that EGCG reduced cisplatin-induced ROS generation and ERK1/2 and signal transducer and activator of transcription-1 (STAT1) activity, but preserved the activity of STAT3 and Bcl-xL. The increase in STAT3/STAT1 ratio appears critical for mediating its otoprotection. EGCG did not alter cisplatin-induced apoptosis of human-derived cancer cells or cisplatin antitumor efficacy in a xenograft tumor model in mice because of its inability to rescue the downregulation of STAT3 in these cells. These data suggest that EGCG is an ideal otoprotective agent for treating cisplatin-induced hearing loss without compromising its antitumor efficacy.</jats:p

Assessing Sex-Specific Circadian, Metabolic, and Cognitive Phenotypes in the AβPP/PS1 and APPNL-F/NL-F Models of Alzheimer\u27s Disease.

BACKGROUND: Circadian disruption has long been recognized as a symptom of Alzheimer\u27s disease (AD); however, emerging data suggests that circadian dysfunction occurs early on in disease development, potentially preceding any noticeable cognitive deficits. OBJECTIVE: This study compares the onset of AD in male and female wild type (C57BL6/J), transgenic (AβPP/PS1), and knock-in (APPNL-F/NL-F) AD mouse models from the period of plaque initiation (6 months) through 12 months. METHODS: Rhythmic daily activity patterns, glucose sensitivity, cognitive function (Morris water maze, MWM), and AD pathology (plaques formation) were assessed. A comparison was made across sexes. RESULTS: Sex-dependent hyperactivity in AβPP/PS1 mice was observed. In comparison to C57BL/6J animals, 6-month-old male AβPP/PS1 demonstrated nighttime hyperactivity, as did 12-month-old females. Female AβPP/PS1 animals performed significantly worse on a MWM task than AβPP/PS1 males at 12 months and trended toward increased plaque pathology. APPNL-F/NL-F 12-month-old males performed significantly worse on the MWM task compared to 12-month-old females. Significantly greater plaque pathology occurred in AβPP/PS1 animals as compared to APPNL-F/NL-F animals. Female AβPP/PS1 animals performed significantly worse than APPNL-F/NL-F animals in spatial learning and memory tasks, though this was reversed in males. CONCLUSION: Taken together, this study provides novel insights into baseline sex differences, as well as characterizes baseline diurnal activity variations, in the AβPP/PS1 and APPNL-F/NL-F AD mouse models

Regulator of G-protein Signaling 17 – A key modulator in Cisplatin-induced Hearing Loss

Regulators of G-protein signaling (RGS) are a multifunctional and highly diverse group of proteins that negatively regulate G-protein coupled receptor (GPCR) signaling pathways. The common mechanism of RGS is to act as GTPase accelerating proteins (GAP) to accelerate the hydrolysis of active GTP bound G proteins and terminate the actions of the associated GPCR. In addition to the traditional function of inhibiting G-protein signaling, recent studies have highlighted the role of RGS proteins in modulating GPCRs in GAP-independent way. There are more than 30 RGS proteins, and depending upon cell/tissue type, they interact and associate with different G proteins and GPCRs to modulate various physiological functions. RGS17, a member of the RGS-RZ subfamily, commonly targets GTP bound Gαz, Gαi, and Gαo for hydrolysis and signal termination. RGS17 is abundantly expressed in the central nervous system and is highly associated with opioid, dopamine and cannabinoid receptors in the brain. RGS17 is also upregulated in many malignant tumors such as lung, prostate and breast cancers. Analysis of whole cochlea transcriptome data from our lab revealed higher levels of RGS17 in the cochlea after cisplatin treatment. This highlights a possible role of RGS17 (and probably other RGS proteins) in cisplatin ototoxicity. Activation of endogenous, otoprotective GPCRs such as adenosine (A1AR) and cannabinoid (CB2) receptor is beneficial for promoting protection against cisplatin-induced hearing loss. Taking all this together, the underlying hypothesis for this study is that cisplatin could possibly mediate ototoxicity by increasing the expression of RGS17, which reduces the otoprotective effect of endogenous receptors such as cannabinoid receptor 2. The main objective of the study is to examine the expression and function of RGS17 in the cochlea and determine if inhibition of RGS17 could protect against cisplatin ototoxicity.The expression of RGS17 was observed in the both in vitro and in vivo models of cochlear cell types. Immunofluorescence study, western blot analysis, and RT-qPCR results showed the presence of RGS17 in UB\OC-1, as well as Wistar rat cochlea; expression levels increased after cisplatin treatment. To determine the role of RGS17 in hearing, first, it was overexpressed in the cochlea using adenoviral vector that was found to significantly increase ABR threshold shifts and decrease ABR Wave I amplitude. Conversely, knockdown of RGS17 (by siRGS17) decreased cisplatin-induced elevations in ABR thresholds along with increased wave I amplitude and latency. Furthermore, siRGS17 pretreatment prevented cisplatin-mediated synapse loss at inner hair cells. This indicates inhibition of RGS17 can preserve the functional and physiological integrity of the cochlea, which is essential for hearing. Cochleae that were treated with siRGS17, followed by cisplatin, showed fewer TUNEL-positive cells and reduced loss of Outer hair cells (OHC) as compared to cisplatin-treated rats. Moreover, overexpression of RGS17 increased the ratio of the transcription factors, pSTAT1/pSTAT3, which may indicate initiation of the apoptotic pathway. Moreover, UB\OC-1 cells treated with Celastrol, a RGS17 inhibitor, showed an increase in cell viability against cisplatin toxicity. In addition to apoptosis, overexpression of RGS17 also elevated ROS production and oxidative stress. But, the inhibition of RGS17 attenuated the cisplatin-induced increase in transcripts for oxidative and inflammatory stress markers, such as NOX3, iNOS, KIM1, TNF-α, and COX2, whereas the mRNA level of antioxidant genes such as Nrf2 and SOD2 were increased. Activation of CB2 via JWH-015 (a CB2 agonist) prior to cisplatin administration significantly reduced the cisplatin-induced elevated levels of RGS17, while knockdown of CB2 increased RGS17 expression in the cochlea. siRGS17 treatment boosted endogenous CB2R-Gα expression. Additionally, cisplatin decreased the expression of Gαi/o and Gαz in vitro, but the activation of CB2 increased the expression of these G proteins. Furthermore, JWH-015 treatment alleviated RGS17-dependent cell death. This study suggests that RGS17 could serve as a mediator of cisplatin ototoxicity by reducing the duration of active CB2R-G protein signaling, which normally suppresses cochlear oxidative stress, inflammation and hair cell apoptosis, and thereby preserves normal hearing. These data also indicate the existence of tonic reciprocal inhibition between RGS17 and CB2 mediated via the G proteins. Thus, we propose that RGS17 inhibitors could serve as an effective treatment against cisplatin ototoxicity when used alone and can potentiate the actions of CB2 agonists when used in combination therapy against cisplatin-induced hearing loss

Oxidative Stress and Inflammation Caused by Cisplatin Ototoxicity

Hearing loss is a significant health problem that can result from a variety of exogenous insults that generate oxidative stress and inflammation. This can produce cellular damage and impairment of hearing. Radiation damage, ageing, damage produced by cochlear implantation, acoustic trauma and ototoxic drug exposure can all generate reactive oxygen species in the inner ear with loss of sensory cells and hearing loss. Cisplatin ototoxicity is one of the major causes of hearing loss in children and adults. This review will address cisplatin ototoxicity. It includes discussion of the mechanisms associated with cisplatin-induced hearing loss including uptake pathways for cisplatin entry, oxidative stress due to overpowering antioxidant defense mechanisms, and the recently described toxic pathways that are activated by cisplatin, including necroptosis and ferroptosis. The cochlea contains G-protein coupled receptors that can be activated to provide protection. These include adenosine A1 receptors, cannabinoid 2 receptors (CB2) and the Sphingosine 1-Phosphate Receptor 2 (S1PR2). A variety of heat shock proteins (HSPs) can be up-regulated in the cochlea. The use of exosomes offers a novel method of delivery of HSPs to provide protection. A reversible MET channel blocker that can be administered orally may block cisplatin uptake into the cochlear cells. Several protective agents in preclinical studies have been shown to not interfere with cisplatin efficacy. Statins have shown efficacy in reducing cisplatin ototoxicity without compromising patient response to treatment. Additional clinical trials could provide exciting findings in the prevention of cisplatin ototoxicity