Figure 7 - available from: Scientific Reports
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Dissection of the cochlea. (a) External aspect of the intact bony cochlea. (b) Bony wall of the cochlea partly removed. Cochlear duct: →; osseus spiral lamina: *. (c) Removal of the apical part of the cochlear duct (→). (d) Cochlea broken into two parts for gaining access to the basal part of the cochlear duct (→). (e) The main part with the modiolus after removal of the cochlear duct. Osseus spiral lamina: *. (f) The other part of the cochlea with the cochlear duct (→). Subsequently this part of the cochlear duct will also be dissected (not shown).
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Pituitary adenylate cyclase activating polypeptide (PACAP) is a regulatory and cytoprotective neuropeptide, its deficiency implies accelerated aging in mice. It is present in the auditory system having antiapoptotic effects. Expression of Ca²⁺-binding proteins and its PAC1 receptor differs in the inner ear of PACAP-deficient (KO) and wild-type (WT)...
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... was continuously oxygenated 74 . Dissection of the cochlea occurred under operating microscope in the perilymph-like solution. The outer bony wall of the cochlea was removed and the apical part of the cochlear duct was separated from the modiolus. Then the cochlea was broken into two parts and the remaining part of cochlear duct was dissected (Fig. 7). The cochlear ducts were placed in Eppendorf tubes containing PBS buffer with protease inhibitor, homogenized with a handheld homogenizer, sonicated on ice for 2 min and centrifuged for 5 min with 14,000 rpm. Until further work, the samples were stored at −20 °C in the protease inhibitor containing solution. The protein concentrations ...
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Citations
... PACAP plays a role in neuronal excitability (May et al. 2021), urinary bladder activity (Girard et al. 2021), gastrointestinal motility and secretion (Karpiesiuk and Palus 2021;Reglodi et al. 2018;Rytel et al. 2021), cartilage and bone formation (Jozsa et al. 2021), and reproduction and embryonal growth (Koves et al. 2020;Ross et al. 2018;Shan et al. 2021) and it also has immunomodulatory functions (Abad and Tan 2018). Several studies show that PACAP plays important roles in numerous pathological conditions such as tumor growth and proliferation (D'Amico et al. 2021b, c;Maugeri et al. 2018aMaugeri et al. , 2021Moody et al. 2016) and nervous system disorders like migraine, schizophrenia, anxiety, and depression (Eslami et al. 2021;Kormos et al. 2016;Martelle et al. 2021;Ross et al. 2020;Tiihonen et al. 2021) as well as inflammatory conditions Tamas et al. 2021), sudden infant death syndrome , and hearing loss (Fulop et al. 2019;Ruel et al. 2021). ...
Pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide with widespread distribution and diverse biological functions. Several studies show that PACAP has strong cytoprotective effects mediated mostly through its specific PAC1 receptor (PAC1-R) and it plays important roles in several pathological conditions. Its distribution and altered expression are known in various human tissues, but there is no descriptive data about PACAP and its receptors in the human eyebulb. Since PACAP38 is the dominant form of the naturally occurring PACAP, our aim was to investigate the distribution of PACAP38-like immunoreactivity in the human eye and to describe the presence of PAC1-R. Semiquantitative evaluation was performed after routine histology and immunohistochemical labeling on human eye sections. Our results showed high level of immunopositivity in the corneal epithelium and endothelium. Within the vascular layer, the iris and the ciliary body had strong immunopositivity for both PACAP and PAC1-R. Several layers of the retina showed immunoreactivity for PACAP and PAC1-R, but the ganglion cell layer had a special pattern in the immunolabeling. Labeling was observed in the neuropil within the optic nerve in both cases and glial cells displayed immunoreactivity for PAC1-R. In summary, our study indicates the widespread occurrence of PACAP and its specific receptor in the human eye, implying that the results from in vitro and animal studies have translational value and most probably are also present in the human eye.
... The detected decrease in PACAP levels of PD patients may contribute to the earlier appearance of aging processes and the aggravation of neurodegenerative alterations. This hypothesis is also supported by our animal experiments, where early aging signs were detected in PACAP knockout (KO) animals [89][90][91]. The reduced protective effects of PACAP with aging lead to increased neuronal vulnerability and systemic degeneration (increased apoptosis, inflammation, and oxidative stress) [92] which may contribute to age-related symptoms. ...
The neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (PACAP) have been shown in numerous in vitro and in vivo models of Parkinson’s disease (PD) supporting the theory that PACAP could have an important role in the pathomechanism of the disorder affecting mostly older patients. Earlier studies found changes in PACAP levels in neurological disorders; therefore, the aim of our study was to examine PACAP in plasma samples of PD patients. Peptide levels were measured with ELISA and correlated with clinical parameters, age, stage of the disorder based on the Hoehn and Yahr (HY) scale, subtype of the disease, treatment, and specific scores measuring motor and non-motor symptoms, such as movement disorder society-unified Parkinson’s disease rating scale (MDS-UPDRS), Epworth sleepiness scale (ESS), Parkinson’s disease sleep scale (PDSS-2), and Beck depression inventory (BDI). Our results showed significantly decreased PACAP levels in PD patients without deep brain stimulation (DBS) therapy and in akinetic-rigid subtype; additionally we also observed a further decrease in the HY stage 3 and 4. Elevated PACAP levels were found in patients with DBS. There were no significant correlations between PACAP level with MDS-UPDRS, type of pharmacological treatment, PDSS-2 sleepiness, or depression (BDI) scales, but we found increased PACAP level in patients with more severe sleepiness problems based on the ESS scale. Based on these results, we suggest that following the alterations of PACAP with other frequently used clinical biomarkers in PD patients might improve strategic planning of further therapeutic interventions and help to provide a clearer prognosis regarding the future perspective of the disease.
... Recent studies have shown that PACAP KO animals display earlyonset and a more generalized form of systemic amyloid deposition throughout the body in various organs (Reglodi et al. 2018b), and PACAP deficiency makes the articular cartilage structure more prone to degenerate Lauretta et al. 2020). PACAP-deficient mice also display accelerated hearing impairment compared to wild-type mice (Fulop et al. 2019a). The protective effect of endogenous PACAP has also been described in retinal toxic, ischemic, metabolic, and inflammatory lesions (Kawaguchi et al. 2010;Szabadfi et al. 2012;Vaczy et al. 2018). ...
Pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues, including the retina. Previously, we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Wild-type and PACAP-knockout (KO) mouse pups at postnatal day (PD) 7 were maintained at 75% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15, animals underwent electroretinography (ERG) to assess visual function. On PD16, eyes were harvested for either immunohistochemistry to determine the percentage of the central avascular retinal area or molecular analysis to assess angiogenesis proteins by array kit and anti-apoptotic protein kinase B (Akt) change by western blot. Retinas of PACAP-deficient OIR mice showed a greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated due to OIR, and 11 different proteins markedly increased in PACAP-deficient mice, whereas western blot analysis revealed a reduction in Akt phosphorylation, suggesting an advanced cell death in the lack of PACAP. This is the first study to examine the endogenous effect of PACAP in the OIR model. Previously, we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings, we suggest that PACAP could be a novel retinoprotective agent in ROP.
... Using noise-exposed mice with PACAP deletion or over-expression of the human PACAP transgene, we identified a protective effect of endogenous PACAP against acute noise-induced hearing loss. Interestingly, a recent study demonstrated that endogenous PACAP is essential in the maintenance of hearing during aging in mice (Fulop et al., 2019). Also, previous results from the same lab have already demonstrated an anti-apoptotic effect of PACAP against oxidative stress-induced chicken cochlear cell death in vitro (Racz et al., 2010). ...
Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)-the secretin-glucagon family of neuropeptides. They act through two classes of receptors: PACAP type 1 (PAC1) and type 2 (VPAC1 and VPAC2). Among their pleiotropic effects throughout the body, PACAP functions as neuromodulators and neuroprotectors, rescuing neurons from apoptosis, mostly through the PAC1 receptor. To explore the potential protective effect of endogenous PACAP against Noise-induced hearing loss (NIHL), we used a knockout mouse model lacking PAC1 receptor expression (PACR1−/−) and a transgenic humanized mouse model expressing the human PAC1 receptor (TgHPAC1R). Based on complementary approaches combining electrophysiological, histochemical, and molecular biological evaluations, we show PAC1R expression in spiral ganglion neurons and in cochlear apical cells of the organ of Corti. Wild-type (WT), PAC1R−/−, and TgHPAC1R mice exhibit similar auditory thresholds. For most of the frequencies tested after acute noise damage, however, PAC1R−/− mice showed a larger elevation of the auditory threshold than did their WT counterparts. By contrast, in a transgene copy number-dependent fashion, TgHPAC1R mice showed smaller noise-induced elevations of auditory thresholds compared to their WT counterparts. Together, these findings suggest that PACAP could be a candidate for endogenous protection against noise-induced hearing loss.
... PACAP is a pleiotropic neuropeptide which is involved in a variety physiological function, including thermogenesis, locomotor activity, mobilization of energy stores, auditory role, and appetite [6][7][8]. Although it has been proposed that PACAP and its receptors are strongly expressed in the hypothalamus, and responsible for appetite regulation [4], reports are discrepant. ...
... Interestingly, we showed that the PACAP PVH mice decreased the time spent in the center region and the distance travelled in the open-field test, suggesting that PACAP signaling in the PVH might be involved in anxiety-related behaviors. A recent study has demonstrated the impairment of auditory function in PACAP (−/−) mice [7]. However, auditory function may not play an important role regarding the above-mentioned behavioral alterations, because selective overexpression of PACAP in PVH showed the anxiety-like behavior. ...
Pituitary adenylate cyclase-activating polypeptide (PACAP) is abundantly expressed in the hypothalamus and contributes to hypothalamic functions, including appetite regulation. Although food intake is suggested to be decreased in PACAP (−/−) mice, the detailed mechanisms are still being discussed. We sought to investigate this link. The food consumption at 8 h after refeeding in the (−/−) mice who had fasted for 2 days was significantly lower than in the PACAP (+/+) mice. The nocturnal and daily food intake of (−/−) mice was significantly lower than those of (+/+) mice, but the diurnal food intake showed a tendency to increase. mRNA expression levels of agouti-related peptide (AgRP) were decreased, but those of proopiomelanocortin (POMC) were increased in the hypothalamus of (−/−) mice 4 h after refeeding. Furthermore, intracerebroventricular administration of a PACAP receptor antagonist, PACAP6–38 (1 nmol/4 μL/mouse), decreased food intake and body weight 1, 2, and 4 h after refeeding, as well as expression levels of AgRP at 4 h after refeeding in (+/+) mice. The selective overexpression of PACAP by the infection of an adeno-associated virus in the ventromedial hypothalamus (VMH) resulted in an increase in food intake and AgRP expression in the nocturnal period in addition to the increased food intake at 8 h after refeeding. These results suggest that food intake behavior in mice is triggered by the increase in PACAP expression in the VMH via modulation of AgRP expression in the hypothalamus, pointing to PACAP inhibition as a potential strategy for the development of anti-obesity drugs.
The prevalence of hearing loss-related diseases caused by different factors is increasing worldwide year by year. Currently, however, the patient’s hearing loss has not been effectively improved. Therefore, there is an urgent need to adopt new treatment measures and treatment techniques to help improve the therapeutic effect of hearing loss. G protein-coupled receptors (GPCRs), as crucial cell surface receptors, can widely participate in different physiological and pathological processes, particularly play an essential role in many disease occurrences and be served as promising therapeutic targets. However, no specific drugs on the market have been found to target the GPCRs of the cochlea. Interestingly, many recent studies have demonstrated that GPCRs can participate in various pathogenic process related to hearing loss in the cochlea including heredity, noise, ototoxic drugs, cochlear structure, and so on. In this review, we comprehensively summarize the functions of 53 GPCRs known in the cochlea and their relationships with hearing loss, and highlight the recent advances of new techniques used in cochlear study including cryo-EM, AI, GPCR drug screening, gene therapy vectors, and CRISPR editing technology, as well as discuss in depth the future direction of novel GPCR-based drug development and gene therapy for cochlear hearing loss. Collectively, this review is to facilitate basic and (pre-) clinical research in this area, and provide beneficial help for emerging GPCR-based cochlear therapies.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution and diverse biological functions. Several studies show that PACAP has strong cytoprotective effects mediated mostly through its specific PAC1 receptor (PAC1-R) and it plays important roles in several pathological conditions. Its distribution and altered expression are known in various human tissues, but there is no descriptive data about PACAP and its receptors in the human eyebulb. Since the PACAP38 is the dominant form of the naturally occurring PACAP, our aim was to investigate the distribution of PACAP38-like immunoreactivity in the human eye and to describe the presence of PAC1-R. Semiquantitative evaluation was performed after routine histology and immunohistochemical labeling on human eye sections. Our results showed high level of immunopositivity in the corneal epithelium and endothelium. Within the vascular layer the iris and the ciliary body had strong immunopositivity for both PACAP and the PAC1-R. Several layers of the retina showed immunoreactivity for PACAP and PAC1-R, but the ganglion cell layer had a special pattern in the immunolabeling. Labeling was observed in the neuropil within the optic nerve in both cases and glial cells displayed immunoreactivity for PAC1-R. In summary, our study indicates the widespread occurrence of PACAP and its specific receptor in the human eye, implying that the results from in vitro and animal studies have translational value and most probably are also present in the human eye.
Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues including the retina. Previously we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP.
Methods: Wild-type and PACAP-KO mouse pups at postnatal day (PD) 7 were maintained at 80% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15 animals underwent electroretinography (ERG) to assess visual function. On PD16 eyes were harvested for either immunohistochemistry to determine the percentage of central avascular retinal area and neovascular tuft formation or molecular analysis to assess angiogenesis proteins by array kit and antiapoptotic protein kinase B (Akt) change by western blot.
Results: The retina of PACAP deficient OIR mice showed greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated as a results of OIR and 11 further proteins markedly increased in PACAP deficient mice.
Conclusion: This is the first study to examine the endogenous effect of PACAP in OIR model. Previously we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings we suppose that PACAP could be a novel retinoprotective agent in ROP.
PACAP is a neuropeptide with widespread distribution and diverse biological functions. It has strong cytoprotective effects mediated mainly through specific PAC1 receptors. Experimental data show protective effects of PACAP in the retina and cornea in several pathological conditions. Although intravitreal injections are a common practice in some ocular diseases, delivery of therapeutic agents in the form of eye drops would be more convenient and would lead to fewer side effects. We have previously shown that PACAP, in the form of eye drops, is able to pass through the ocular barriers and can exert retinoprotective effects. As eye drops represent a promising form of administration of PACAP in ocular diseases, it is important to investigate the stability of PACAP in solutions used in eye drops. In this study, the stability of PACAP1-27 and PACAP1-38 in eye drops was measured in four common media and a commercially available artificial tear solution at both room temperature and +4 °C. Mass spectrometry results show that the highest stability was gained with PACAP1-38 in water and 0.9% saline solution at +4 °C, representing 80–90% drug persistence after 2 weeks. PACAP1-38 in the artificial tear showed very fast degradation at room temperature, but was stable at +4 °C. In summary, PACAP1-38 has higher stability than PACAP1-27, with highest stability at +4 °C in water solution, but both peptides in each medium can be stored for relatively longer periods without significant degradation. These data can provide reference for future therapeutic use of PACAP in eye drops.
Sensory processing abnormalities are frequently associated with autism spectrum disorders, but the underlying mechanisms are unclear. Here we studied auditory processing in a mouse model of Fragile X Syndrome (FXS), a leading known genetic cause of autism and intellectual disability. Both humans with FXS and the Fragile X mental retardation gene (Fmr1) knock-out (KO) mouse model show auditory hypersensitivity, with the latter showing a strong propensity for audiogenic seizures (AGS) early in development. Because midbrain abnormalities cause AGS, we investigated whether the inferior colliculus (IC) of the Fmr1 KO mice shows abnormal auditory processing compared to wild-type (WT) controls at specific developmental time points. Using antibodies against neural activity marker c-Fos, we found increased density of c-Fos+ neurons in the IC, but not auditory cortex, of Fmr1 KO mice at P21 and P34 following sound presentation. In vivo single-unit recordings showed that IC neurons of Fmr1 KO mice are hyper-responsive to tone bursts and amplitude-modulated tones during development, and show broader frequency tuning curves. There were no differences in rate-level responses or phase locking to amplitude-modulated tones in IC neurons between genotypes. Taken together, these data provide evidence for the development of auditory hyper-responsiveness in the IC of Fmr1 KO mice. Although most human and mouse work in autism and sensory processing has centered on the forebrain, our new findings, along with recent work on the lower brainstem, suggest that abnormal subcortical responses may underlie auditory hypersensitivity in autism spectrum disorders.
