The expression of PHB2 in the cochlea: Possible relation to age-related hearing loss

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly, but its mechanism remains unclear. Scaffold protein prohibitin 2 (PHB2) has been widely involved in aging and neurodegeneration. However, the role of PHB2 in ARHL is undeciphered to date. To investigate the expression pattern and the role of PHB2 in ARHL, we used C57BL/6 mice and HEI-OC1 cell line as models. In our study, we have found PHB2 exists in the cochlea and is expressed in hair cells, spiral ganglion neurons, and HEI-OC1 cells. In mice with ARHL, mitophagy is reduced and correspondingly the expression level of PHB2 is decreased. Moreover, after H₂O₂ treatment the mitophagy is activated and the PHB2 expression is increased. These findings indicate that PHB2 may exert an important role in ARHL through mitophagy. Findings from this study will be helpful for elucidating the mechanism underlying the ARHL and for providing a new target for ARHL treatment.     

Degradation of cochlear Connexin26 accelerate the development of age-related hearing loss

The GJB2 gene, encoding Connexin26 (Cx26), is one of the most common causes of inherited deafness. Clinically, mutations in GJB2 cause congenital deafness or late-onset progressive hearing loss. Recently, it has been reported that Cx26 haploid deficiency accelerates the development of age-related hearing loss (ARHL). However, the roles of cochlear Cx26 in the hearing function of aged animals remain unclear. In this study, we revealed that the Cx26 expression was significantly reduced in the cochleae of aged mice, and further explored the underlying molecular mechanism for Cx26 degradation. Immunofluorescence co-localization results showed that Cx26 was internalized and degraded by lysosomes, which might be one of the important ways for Cx26 degradation in the cochlea of aged mice. Currently, whether the degradation of Cx26 in the cochlea leads directly to ARHL, as well as the mechanism of Cx26 degradation-related hearing loss are still unclear. To address these questions, we generated mice with Cx26 knockout in the adult cochlea as a model for the natural degradation of Cx26. Auditory brainstem response (ABR) results showed that Cx26 knockout mice exhibited high-frequency hearing loss, which gradually progressed over time. Pathological examination also revealed the degeneration of hair cells and spiral ganglions, which is similar to the phenotype of ARHL. In summary, our findings suggest that degradation of Cx26 in the cochlea accelerates the occurrence of ARHL, which may be a novel mechanism of ARHL.     

Augmentation of cellular NAD+ by NQO1 enzymatic action improves age‐related hearing impairment

Age‐related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD⁺/NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP‐1, and PGC‐1α. Nonetheless, the beneficial effect of direct modulation of cellular NAD⁺ levels on aging and age‐related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of β‐lapachone (β‐lap), a known plant‐derived metabolite that modulates cellular NAD⁺ by conversion of NADH to NAD⁺ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD⁺ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro‐inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF‐κB, p53, and IDH2. However, augmentation of NAD⁺ by β‐lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD⁺ levels by pharmacological agents may be a tangible therapeutic option for treating various age‐related diseases, including ARHL.     

Identifying MicroRNAs Involved in Degeneration of the Organ of Corti during Age-Related Hearing Loss

MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA targets, are important regulators of cellular senescence and aging. We questioned which miRNAs are involved in age-related degeneration of the organ of Corti (OC), the auditory sensory epithelium that transduces mechanical stimuli to electrical activity in the inner ear. Degeneration of the OC is generally accepted as the main cause of age-related hearing loss (ARHL), a progressive loss of hearing in individuals as they grow older. To determine which miRNAs are involved in the onset and progression of ARHL, miRNA gene expression in the OC of two mouse strains, C57BL/6J and CBA/J, was compared at three different ages using GeneChip miRNA microarray and was validated by real-time PCR. We showed that 111 and 71 miRNAs exhibited differential expression in the C57 and CBA mice, respectively, and that downregulated miRNAs substantially outnumbered upregulated miRNAs during aging. miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family, which are known regulators of pro-apoptotic pathways. In contrast, miRNAs that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family, which are known to be important for proliferation and differentiation, respectively. The shift of miRNA expression favoring apoptosis occurred earlier than detectable hearing threshold elevation and hair cell loss. Our study suggests that changes in miRNA expression precede morphological and functional changes, and that upregulation of pro-apoptotic miRNAs and downregulation of miRNAs promoting proliferation and differentiation are both involved in age-related degeneration of the OC.     

Effect of SOD1 overexpression on age- and noise-related hearing loss

Reactive oxygen species (ROS) have been implicated in hearing loss associated with aging and noise exposure. Superoxide dismutases (SODs) form a first line of defense against damage mediated by the superoxide anion, the most common ROS. Absence of Cu/Zn SOD (SOD1) has been shown to potentiate hearing loss related to noise exposure and age. Conversely, overexpression of SOD1 may be hypothesized to afford a protection from age- and noise-related hearing loss. This hypothesis may be tested using a transgenic mouse model carrying the human SOD1 gene. Contrary to expectations, here, we report that no protection against age-related hearing loss was observed in mice up to 7 months of age or from noise-induced hearing loss when 8 week old mice were exposed to broadband noise (4-45 kHz, 110 dB for 1 h). Mitochondrial DNA deletion, an index of aging, was elevated in the acoustic nerve of transgenic mice compared to nontransgenic littermates. The results indicate the complexity of oxidative metabolism in the cochlea is greater than previously hypothesized.     

Identifying MicroRNAs Involved in Aging of the Lateral Wall of the Cochlear Duct

Age-related hearing loss is a progressive sensorineural hearing loss that occurs during aging. Degeneration of the organ of Corti and atrophy of the lateral wall of the cochlear duct (or scala media) in the inner ear are the two primary causes. MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA/protein targets, are important regulators of cellular senescence and aging. We examined miRNA gene expression profiles in the lateral wall of two mouse strains, along with exploration of the potential targets of those miRNAs that showed dynamic expression during aging. We show that 95 and 60 miRNAs exhibited differential expression in C57 and CBA mice during aging, respectively. A majority of downregulated miRNAs are known to regulate pathways of cell proliferation and differentiation, while all upregulated miRNAs are known regulators in the pro-apoptotic pathways. By using apoptosis-related gene array and bioinformatic approaches to predict miRNA targets, we identify candidate miRNA-regulated genes that regulate apoptosis pathways in the lateral wall of C57 and CBA mice during aging.     

The JNK inhibitor AS602801 Synergizes with Enzalutamide to Kill Prostate Cancer Cells In Vitro and In Vivo and Inhibit Androgen Receptor Expression

In our previous study, we observed that androgen deprivation therapy (ADT) may induce a compensatory increase in MAPK or JNK signaling. Here, we tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with the AR inhibitor enzalutamide (ENZ) in androgen-sensitive LNCaP cells and androgen-resistant C4-2 and 22Rv1 cells. Enzalutamide combined with AS602801 synergistically killed LNCaP, C4-2, and 22Rv1 cells, and decreased migration and invasion of LNCaP and C4-2 cells. We studied the combination of enzalutamide with AS602801 in vivo using luciferase labeled LNCaP xenografts, and observed that combination of ENZ with AS602801 significantly suppressed tumor growth compared with either drug alone. Importantly, combination therapy resulted in dramatic loss of AR mRNA and protein. Surprisingly, mechanistic studies and Nanostring data suggest that AS602801 likely activates JNK signaling to induce apoptosis. Since AS602801 had sufficient safety and toxicity profile to advance from Phase I to Phase II in clinical trials, repurposing of this compound may represent an opportunity for rapid translation for clinical therapy of CRPC patients.     

Disruption of the autism-related gene Pak1 causes stereocilia disorganization,hair cell loss,and deafness in mice

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases(PAKs)are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout(Pak1à/à) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell(HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of b II-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1à/àmice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.     

Contribution of defective mitophagy to the neurodegeneration in DNA repair-deficient disorders

DNA repair is a prerequisite for life as we know it, and defects in DNA repair lead to accelerated aging. Xeroderma pigmentosum group A (XPA) is a classic DNA repair-deficient disorder with patients displaying sun sensitivity and cancer susceptibility. XPA patients also exhibit neurodegeneration, leading to cerebellar atrophy, neuropathy, and hearing loss, through a mechanism that has remained elusive. Using in silico, in vitro, and in vivo studies, we discovered defective mitophagy in XPA due to PARP1 hyperactivation and NAD⁺ (and thus, SIRT1) depletion. This leads to mitochondrial membrane hyper-polarization, PINK1 cleavage and defective mitophagy. This study underscores the importance of mitophagy in promoting a healthy pool of mitochondria and in preventing neurodegeneration and premature aging.     

Contribution of defective mitophagy to the neurodegeneration in DNA repair-deficient disorders

DNA repair is a prerequisite for life as we know it, and defects in DNA repair lead to accelerated aging. Xeroderma pigmentosum group A (XPA) is a classic DNA repair-deficient disorder with patients displaying sun sensitivity and cancer susceptibility. XPA patients also exhibit neurodegeneration, leading to cerebellar atrophy, neuropathy, and hearing loss, through a mechanism that has remained elusive. Using in silico, in vitro, and in vivo studies, we discovered defective mitophagy in XPA due to PARP1 hyperactivation and NAD⁺ (and thus, SIRT1) depletion. This leads to mitochondrial membrane hyper-polarization, PINK1 cleavage and defective mitophagy. This study underscores the importance of mitophagy in promoting a healthy pool of mitochondria and in preventing neurodegeneration and premature aging.     

Hearing Loss as a Function of Aging and Diabetes Mellitus: A Cross Sectional Study

Background

Although hearing loss may be caused by various factors, it is also a natural phenomenon associated with the aging process. This study was designed to assess the contributions of diabetes mellitus (DM) and hypertension, both chronic diseases associated with aging, as well as aging itself, to hearing loss in health screening examinees.

Methods

This study included 37,773 individuals who underwent health screening examinations from 2009 to 2012. The relationships between hearing threshold and subject age, hearing threshold at each frequency based on age group, the degree of hearing loss and the presence or absence of hypertension and DM were evaluated.

Results

The prevalence of hearing loss increased with age, being 1.6%, 1.8%, 4.6%, 14.0%, 30.8%, and 49.2% in subjects in their twenties, thirties, forties, fifties, sixties, and seventies, respectively (p<0.05). Hearing value per frequency showed aging-based changes, in the order of 6000, 4000, 2000, 1000 and 500 Hz, indicating greater hearing losses at high frequencies. The degree of hearing loss ranged from mild to severe. Aging and DM were correlated with the prevalence of hearing loss (p<0.05). There was no statistically significant association between hearing loss and hypertension after adjusting for age and DM.

Conclusions

The prevalence of hearing loss increases with age and the presence of DM. Hearing loss was greatest at high frequencies. In all age groups, mild hearing loss was the most common form of hearing loss.     

Ups and Downs of Viagra: Revisiting Ototoxicity in the Mouse Model

Sildenafil citrate (Viagra), a phosphodiesterase 5 inhibitor (PDE5i), is a commonly prescribed drug for erectile dysfunction. Since the introduction of Viagra in 1997, several case reports have linked Viagra to sudden sensorineural hearing loss. However, these studies are not well controlled for confounding factors, such as age and noise-induced hearing loss and none of these reports are based on prospective double-blind studies. Further, animal studies report contradictory data. For example, one study (2008) reported hearing loss in rats after long-term and high-dose exposure to sildenafil citrate. The other study (2012) showed vardenafil, another formulation of PDE5i, to be protective against noise-induced hearing loss in mice and rats. Whether or not clinically relevant doses of sildenafil citrate cause hearing loss in normal subjects (animals or humans) is controversial. One possibility is that PDE5i exacerbates age-related susceptibility to hearing loss in adults. Therefore, we tested sildenafil citrate in C57BL/6J, a strain of mice that displays increased susceptibility to age-related hearing loss, and compared the results to those obtained from the FVB/N, a strain of mice with no predisposition to hearing loss. Six-week-old mice were injected with the maximum tolerated dose of sildenafil citrate (10 mg/kg/day) or saline for 30 days. Auditory brainstem responses (ABRs) were recorded pre- and post injection time points to assess hearing loss. Entry of sildenafil citrate in the mouse cochlea was confirmed by qRT-PCR analysis of a downstream target of the cGMP-PKG cascade. ABR data indicated no statistically significant difference in hearing between treated and untreated mice in both backgrounds. Results show that the maximum tolerated dose of sildenafil citrate administered daily for 4 weeks does not affect hearing in the mouse. Our study gives no indication that Viagra will negatively impact hearing and it emphasizes the need to revisit the issue of Viagra related ototoxicity in humans.     

Use of Fixed Dose Combination (FDC) Drugs in India: Central Regulatory Approval and Sales of FDCs Containing Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), Metformin,or Psychotropic Drugs

Background

In 2012, an Indian parliamentary committee reported that manufacturing licenses for large numbers of fixed dose combination (FDC) drugs had been issued by state authorities without prior approval of the Central Drugs Standard Control Organization (CDSCO) in violation of rules, and considered that some ambiguity until 1 May 2002 about states’ powers might have contributed. To our knowledge, no systematic enquiry has been undertaken to determine if evidence existed to support these findings. We investigated CDSCO approvals for and availability of oral FDC drugs in four therapeutic areas: analgesia (non-steroidal anti-inflammatory drugs [NSAIDs]), diabetes (metformin), depression/anxiety (anti-depressants/benzodiazepines), and psychosis (anti-psychotics).

Methods and Findings

This was an ecologic study with a time-trend analysis of FDC sales volumes (2007–2012) and a cross-sectional examination of 2011–2012 data to establish the numbers of formulations on the market with and without a record of CDSCO approval (“approved” and “unapproved”), their branded products, and sales volumes. Data from the CDSCO on approved FDC formulations were compared with sales data from PharmaTrac, a database of national drug sales. We determined the proportions of FDC sales volumes (2011–2012) arising from centrally approved and unapproved formulations and from formulations including drugs banned/restricted internationally. We also determined the proportions of centrally approved and unapproved formulations marketed before and after 1 May 2002, when amendments were made to the drug rules. FDC approvals in India, the United Kingdom (UK), and United States of America (US) were compared.For NSAID FDCs, 124 formulations were marketed, of which 34 (27%) were centrally approved and 90 (73%) were unapproved; metformin: 25 formulations, 20 (80%) approved, five (20%) unapproved; anti-depressants/benzodiazepines: 16 formulations, three (19%) approved, 13 (81%) unapproved; anti-psychotics: ten formulations, three (30%) approved, seven (70%) unapproved. After 1 May 2002, the proportions of approved FDC formulations increased for NSAIDs (26%/28%) and anti-psychotics (0%/38%) and decreased for metformin (100%/75%) and anti-depressants/benzodiazepines (20%/18%), and the overall proportion approved remained similar before and after that date.FDC formulations gave rise to multiple branded products, ranging from 211 anti-psychotic FDC products from ten formulations to 2,739 NSAID FDC products from 124 formulations. The proportions of FDC sales volumes arising from unapproved formulations were as follows: anti-depressants/benzodiazepines, 69%; anti-psychotics, 43%; NSAIDs, 28%; and metformin, 0.4%. Formulations including drugs banned/restricted internationally comprised over 12% of NSAID FDC sales and 53% of anti-psychotic FDC sales. Across the four therapeutic areas, 14 FDC formulations were approved in the UK and 22 in the US.

Conclusions

There was evidence supporting concerns about FDCs. Metformin excepted, substantial numbers of centrally unapproved formulations for NSAID, anti-depressant/benzodiazepine, and anti-psychotic FDCs were marketed; sales volumes were high. The legal need for central approval of new drugs before manufacture has been in place continuously since 1961, including for FDCs meeting the applicable legal test. Proportions of centrally unapproved formulations after 1 May 2002 did not decrease overall, and no ambiguity was found about states’ licensing powers. Unapproved formulations should be banned immediately, prioritising those withdrawn/banned internationally and undertaking a review of benefits and risks for patients in ceasing or switching to other medicines. Drug laws need to be amended to ensure the safety and effectiveness of medicines marketed in India.     

Auditory brainstem responses in aging dark agouti rats

The present study examined auditory function across age in the dark agouti (DA) rat strain. Auditory brainstem responses (ABRs) were measured for frequencies 8, 16, and 32 kHz in male and female DA rats from 3 to 18 months of age. Hearing thresholds and absolute and interpeak latencies (IPLs) were analyzed. Male hearing thresholds remained stable for the first year of life and then significantly increased at 18 months across all frequencies; female hearing remained stable at all tested ages out to 18 months. At 12 months, male DA rats showed significantly longer absolute latencies by age (i.e., compared with 3-month-old males) and sex (compared with 12-month-old females), with no differences in IPLs. At 18 months, female DA rats showed significantly longer absolute latencies with age (compared with 3-month-old females) and sex (compared with 18-month-old males), particularly for the later waves. Female IPLs were also significantly longer with age and by sex for the later waves. This report supports the feasibility of using male DA rats in studies to investigate age-related hearing loss (ARHL; presbycusis).     

A locus on distal chromosome 11 (ahl8) and its interaction with Cdh23 ahl underlie the early onset, age-related hearing loss of DBA/2J mice

The DBA/2J inbred strain of mice is used extensively in hearing research, yet little is known about the genetic basis for its early onset, progressive hearing loss. To map underlying genetic factors we analyzed recombinant inbred strains and linkage backcrosses. Analysis of 213 mice from 31 BXD recombinant inbred strains detected linkage of auditory brain-stem response thresholds with a locus on distal chromosome 11, which we designate ahl8. Analysis of 225 N2 mice from a backcross of (C57BL/6JxDBA/2J) F1 hybrids to DBA/2J mice confirmed this linkage (LOD>50) and refined the ahl8 candidate gene interval. Analysis of 214 mice from a backcross of (B6.CAST-Cdh23 Ahl+ xDBA/2J) F1 hybrids to DBA/2J mice demonstrated a genetic interaction of Cdh23 with ahl8. We conclude that ahl8 is a major contributor to the hearing loss of DBA/2J mice and that its effects are dependent on the predisposing Cdh23 ahl genotype of this strain.     

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.     

有氧运动改善AD模型APP/PS1双转基因小鼠中枢神经元线粒体融合分裂动态平衡

线粒体融合分裂平衡是线粒体动力学的需要。本研究观察12周规律有氧运动对APP/PS1双转基因小鼠中枢神经元线粒体融合分裂动态平衡的影响。本研究采用3月龄雄性APP/PS1小鼠（AD模型）随机分为AD安静组（AS）、AD运动组（AE）,同月龄雄性C57BL/6J小鼠做正常对照组（CS）。AE组进行12周规律跑台运动,5 d/周,60 min/d。前10 min运动速度12 m/min,后50 min运动速度15 m/min,跑台坡度为0°。八臂迷宫实验检测小鼠工作记忆错误频率和参考记忆错误频率;Western印迹检测小鼠皮层、海马组织中线粒体分裂蛋白Drp1和Fis1的含量,以及Drp1的活性（p-Drp1-Ser616）、线粒体融合蛋白Mfn1、Mfn2、Opa1的表达水平;透射电镜观察皮层、海马线粒体形态结构、健康线粒体比率及线粒体平均直径。本研究证实AS组较CS组工作记忆错误频率显著提高（P<0.05）,12周有氧运动显著降低工作记忆错误频率（P<0.05）。AS组小鼠皮层Fis1蛋白和海马脑区Drp1、Fis1蛋白表达水平及皮层、海马脑区Drp1蛋白的活性增加（P<0.05）。而皮层Mfn1和海马Mfn1、Mfn2蛋白表达水平显著降低（P<0.05）。12周有氧运动显著减低Fis1、Drp1蛋白表达及Drp1蛋白的活性,提高Mfn1、Mfn2蛋白表达水平（P<0.05）。AS组小鼠皮层、海马线粒体多呈现球形,部分线粒体膜结构消失,线粒体嵴结构紊乱。且AS组较CS组小鼠健康线粒体比率降低、直径缩短。12周规律有氧运动可明显改善线粒体形态和结构,提高健康线粒体比率及直径。本研究提示,12周规律有氧运动可有效抑制皮层、海马脑区线粒体分裂蛋白Drp1和 Fis1的表达,降低Drp1的活性（p-Drp1-Ser616）,上调线粒体融合蛋白Mfn1、Mfn2的蛋白表达水平,改善线粒体形态和结构以促进线粒体质量控制,是有氧运动改善AD模型空间学习记忆能力的分子机制之一。     

Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice

Summary We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.