ABSTRACT
Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.
Subject(s)
CRISPR-Associated Proteins/administration & dosage , Gene Editing/methods , Genes, Dominant/genetics , Genetic Therapy/methods , Hearing Loss/genetics , Acoustic Stimulation , Alleles , Animals , Animals, Newborn , Auditory Threshold , Base Sequence , CRISPR-Associated Proteins/metabolism , CRISPR-Associated Proteins/therapeutic use , CRISPR-Cas Systems , Cell Survival , Cochlea/cytology , Cochlea/metabolism , Disease Models, Animal , Evoked Potentials, Auditory, Brain Stem , Female , Fibroblasts , Hair Cells, Auditory/cytology , Hearing Loss/physiopathology , Hearing Loss/prevention & control , Humans , Liposomes , Male , Membrane Proteins/genetics , Mice , Reflex, StartleABSTRACT
BACKGROUND/AIMS: According to recent studies, central auditory impairments are closely related to neurodegenerative diseases. However, the mechanism of central presbycusis remains unclear. Ubiquitin C-terminal hydrolase L1 (UCHL1) is important in maintaining proteasomal activity; however, the detailed mechanism has not yet been fully elucidated. This study aims to investigate the molecular alterations involved in UCHL1 regulation during auditory cortex aging. METHODS: D-Galactose (D-gal) induces oxidative stress and senescence in the auditory cortex, as reported in our previous studies. Primary auditory cortex cells were treated with D-gal for 72 h or 5 days. The proteins related to the ubiquitin proteasome system (UPS) and proteasomal activities were evaluated. UCHL1 was overexpressed, and the effects of UCHL1 on the UPS and proteasomal activity were analyzed. RESULTS: Proteasomal activity was elevated at 72 h and decreased at 5 days in D-gal-treated primary auditory cortex cells. We also found that overexpression of UCHL1 increased the UPS-related proteins UBE1, PSMA7, ubiquitinated proteins, and monoubiquitin, and proteasomal activity. CONCLUSION: The results suggest that UCHL1 may modify the aging process in the auditory cortex by regulating UPS- related proteins.
Subject(s)
Aging/metabolism , Auditory Cortex/metabolism , Reactive Oxygen Species/metabolism , Ubiquitin Thiolesterase/metabolism , Analysis of Variance , Animals , Biomarkers/analysis , Blotting, Western , Cells, Cultured , Disease Models, Animal , Random Allocation , Rats , Rats, Sprague-Dawley , Sensitivity and SpecificityABSTRACT
BACKGROUND: With the increasing popularity of mobile phones, the potential hazards of radiofrequency electromagnetic radiation (RF-EMR) on the auditory system remain unclear. Apart from RF-EMR, humans are also exposed to various physical and chemical factors. We established a lipopolysaccharide (LPS)-induced inflammation in vitro model to investigate whether the possible sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation (at specific absorption rates: 2, 4 W/kg) will increase. METHODS: Spiral ganglion neurons (SGN) were obtained from neonatal (1- to 3-day-old) Sprague Dawley® (SD) rats. After the SGN were treated with different concentrations (0, 20, 40, 50, 100, 200, and 400 µg/ml) of LPS, the Cell Counting Kit-8 (CCK-8) and alkaline comet assay were used to quantify cellular activity and DNA damage, respectively. The SGN were treated with the moderate LPS concentrations before RF-EMR exposure. After 24 h intermittent exposure at an absorption rate of 2 and 4 W/kg, DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autophagy markers LC3-II and Beclin1 were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay. RESULTS: LPS (100 µg/ml) induced DNA damage and suppressed cellular activity (P < 0.05). LPS (40 µg/ml) did not exhibit cellular activity changes or DNA damage (P > 0.05); therefore, 40 µg/ml was used to pretreat the concentration before exposure to RF-EMR. RF-EMR could not directly induce DNA damage. However, the 4 W/kg combined with LPS (40 µg/ml) group showed mitochondria vacuoles, karyopyknosis, presence of lysosomes and autophagosome, and increasing expression of LC3-II and Beclin1. The ROS values significantly increased in the 4 W/kg exposure, 4 W/kg combined with LPS (40 µg/ml) exposure, and H2O2 groups (P < 0.05, 0.01). CONCLUSIONS: Short-term exposure to radiofrequency electromagnetic radiation could not directly induce DNA damage in normal spiral ganglion neurons, but it could cause the changes of cellular ultrastructure at special SAR 4.0 W/kg when cells are in fragile or micro-damaged condition. It seems that the sensitivity of SGN to damage caused by mobile phone electromagnetic radiation will increase in a lipopolysaccharide-induced inflammation in vitro model.
Subject(s)
Cell Phone , Electromagnetic Phenomena , Inflammation/etiology , Lipopolysaccharides/adverse effects , Lipopolysaccharides/pharmacology , Spiral Ganglion/drug effects , Spiral Ganglion/physiology , Animals , Animals, Newborn , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Cells, Cultured , DNA Damage/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , In Vitro Techniques , Inflammation/metabolism , Inflammation/physiopathology , Microtubule-Associated Proteins/metabolism , Neurons/cytology , Neurons/drug effects , Neurons/physiology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Spiral Ganglion/cytology , Time FactorsABSTRACT
Aging has been associated with mitochondrial DNA (mtDNA) common deletion (CD). Age changes in the central auditory system are well known to affect speech perception. Base excision repair (BER) is the major type of DNA repair in mitochondria. The current study was designed to investigate potential causative mechanisms of central presbycusis by using a rat mimetic aging model induced by subcutaneous administration of D-galactose (D-gal). Quantitative real-time PCR and Western blotting analyses were performed to identify the mtDNA 4834 bp deletion and selected mitochondrial DNA repair enzymes, DNA polymerase γ (pol γ) and 8-oxoguanine DNA glycosylase (OGG1). Cell apoptosis in the auditory cortex was detected using terminal deoxynucleotidyltransferase mediated UTP nick-end labeling (TUNEL). Our data showed that mtDNA 4834 bp deletion and TUNEL-positive cells were significantly increased and the expression of pol γ and OGG1 were remarkably down-regulated in the auditory cortex in D-gal-treated rats compared to control rats. During aging, increased mtDNA damage likely results from decreased DNA repair capacity in the auditory cortex. DNA repair enzymes such as pol γ and OGG1 may provide novel pharmacological targets to promote DNA repair and rescue the central auditory system in patients with degenerative diseases.
Subject(s)
Aging/drug effects , Auditory Cortex/drug effects , Auditory Cortex/metabolism , DNA Damage , DNA Repair/drug effects , DNA, Mitochondrial/metabolism , Galactose/pharmacology , Aging/metabolism , Animals , Apoptosis/drug effects , Auditory Cortex/pathology , Base Pairing/drug effects , Blotting, Western , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA, Mitochondrial/drug effects , Densitometry , Galactose/administration & dosage , Gene Expression Regulation/drug effects , In Situ Nick-End Labeling , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Sequence Deletion/geneticsABSTRACT
Mitochondrial DNA (mtDNA) mutations, especially deletions, have been suggested to play an important role in aging and degenerative diseases. In particular, the common deletion in humans and rats (4977bp and 4834bp deletion, respectively) has been shown to accumulate with age in post-mitotic tissues with high energetic demands. Among numerous deletions, the common deletion has been proposed to serve as a molecular marker for aging and play a critical role in presbyacusis. However, so far no previous publication has quantified the contribution of common deletion to the total burden of mtDNA deletions in tissues during aging process. In the present study, we established a rat model with various degrees of aging in inner ear induced by three different doses of d-galactose (d-gal) administration. Firstly, multiple mtDNA deletions in inner ear were detected by nested PCR and long range PCR. In addition to the common deletion, three novel mtDNA deletions were identified. All four deletions, located in the major arc of mtDNA, are flanked by direct repeats and involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. Additionally, absolute quantitative real-time PCR assay was used to detect the level of common deletion and total deletion burden of mtDNA. The quantitative data show that the common deletion is the most frequent type of mtDNA deletions, exceeding 67.86% of the total deletion burden. Finally, increased mtDNA copy number, reduced COX activity and mosaic ultrastructural impairments in inner ear were identified in d-gal-induced aging rats. The increase of mtDNA replication may contribute to the accelerated accumulation of mtDNA deletions, which may result in impairment of mitochondrial function in inner ear. Taken together, these findings suggest that the common deletion may serve as an ideal molecular marker to assess the mtDNA damage in inner ear during aging.
Subject(s)
Aging/genetics , DNA, Mitochondrial , Ear, Inner/physiology , Sequence Deletion , Animals , Base Sequence , Ear, Inner/ultrastructure , Electron Transport Complex IV/genetics , Female , Galactose , Hearing , Molecular Sequence Data , Rats , Rats, Sprague-DawleyABSTRACT
Presbycusis is the cumulative effect of aging on hearing. Recent studies have shown that common mitochondrial gene deletions are closely related to deafness caused by degenerative changes in the auditory system, and some of these nuclear factors are proposed to participate in the regulation of mitochondrial function. However, the detailed mechanisms involved in age-related degeneration of the auditory systems have not yet been fully elucidated. In this study, we found that FOXG1 plays an important role in the auditory degeneration process through regulation of macroautophagy/autophagy. Inhibition of FOXG1 decreased the autophagy activity and led to the accumulation of reactive oxygen species and subsequent apoptosis of cochlear hair cells. Recent clinical studies have found that aspirin plays important roles in the prevention and treatment of various diseases by regulating autophagy and mitochondria function. In this study, we found that aspirin increased the expression of FOXG1, which further activated autophagy and reduced the production of reactive oxygen species and inhibited apoptosis, and thus promoted the survival of mimetic aging HCs and HC-like OC-1 cells. This study demonstrates the regulatory function of the FOXG1 transcription factor through the autophagy pathway during hair cell degeneration in presbycusis, and it provides a new molecular approach for the treatment of age-related hearing loss.Abbreviations: AHL: age-related hearing loss; baf: bafilomycin A1; CD: common deletion; D-gal: D-galactose; GO: glucose oxidase; HC: hair cells; mtDNA: mitochondrial DNA; RAP: rapamycin; ROS: reactive oxygen species; TMRE: tetramethylrhodamine, ethyl ester.
Subject(s)
Autophagy , Presbycusis , Aging/metabolism , Apoptosis/genetics , Autophagy/genetics , Cell Survival , Forkhead Transcription Factors/metabolism , Hair Cells, Auditory , Humans , Nerve Tissue Proteins/metabolism , Presbycusis/genetics , Presbycusis/metabolismABSTRACT
Inflammation is a self-defense response to protect individuals from infection and tissue damage, but excessive or persistent inflammation can have adverse effects on cell survival. Many individuals become especially susceptible to chronic-inflammation-induced sensorineural hearing loss as they age, but the intrinsic molecular mechanism behind aging individuals' increased risk of hearing loss remains unclear. FoxG1 (forkhead box transcription factor G1) is a key transcription factor that plays important roles in hair cell survival through the regulation of mitochondrial function, but how the function of FoxG1 changes during aging and under inflammatory conditions is unknown. In this study, we first found that FoxG1 expression and autophagy both increased gradually in the low concentration lipopolysaccharide (LPS)-induced inflammation model, while after high concentration of LPS treatment both FoxG1 expression and autophagy levels decreased as the concentration of LPS increased. We then used siRNA to downregulate Foxg1 expression in hair cell-like OC-1â¯cells and found that cell death and apoptosis were significantly increased after LPS injury. Furthermore, we used d-galactose (D-gal) to create an aging model with hair cell-like OC-1â¯cells and cochlear explant cultures in vitro and found that the expression of Foxg1 and the level of autophagy were both decreased after D-gal and LPS co-treatment. Lastly, we knocked down the expression of Foxg1 under aged inflammation conditions and found increased numbers of dead and apoptotic cells. Together these results suggest that FoxG1 affects the sensitivity of mimetic aging hair cells to inflammation by regulating autophagy pathways.
Subject(s)
Aging/genetics , Forkhead Transcription Factors/genetics , Hair Cells, Auditory/drug effects , Lipopolysaccharides/adverse effects , Nerve Tissue Proteins/genetics , Animals , Autophagy , Cell Death , Cell Line , Forkhead Transcription Factors/metabolism , Galactose/metabolism , Gene Expression Regulation , Hair Cells, Auditory/cytology , Humans , Mice , Models, Biological , Nerve Tissue Proteins/metabolism , Rats , Signal TransductionABSTRACT
OBJECTIVE: To determine which of the two, recombinant adeno-associated viral vector 2 (rAAV2) and recombinant adenovirus vector 5 (rAd5), is more suitable for gene transfer in rodent cochlea. METHODS: The rAAV2-EGFP and rAd5-EGFP particles were injected into the perilymph through round window membrane. The target tissue accessibility, time course of expression, tissue toxicity of gene transfer and effects on hearing were evaluated. RESULTS: The expression of EGFP was detected in spiral ligament, strial vascukarises, Reissner membrane, basilar membrane, spiral ganglion, and contralateral cochlea. EGFP expression in the rAAV2 lasted over 60 days, with peak expression between days 14 and 60. EGFP in the rAd5 was detected within 24 h of transfection, and peak expression was observed between days 1 to 21. EGFP activity decreased sharply on day 30 after transfection with rAd5, while high EGFP expression was observed 60 days after transfection with rAAV2. CONCLUSION: AAV has significant advantages for long-term transgene expression and no ototoxicity in the cochlea compared to adenovirus vectors.
Subject(s)
Cochlea/metabolism , Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors , Animals , Gene Expression , Gene Transfer Techniques , Male , Rats , Rats, WistarABSTRACT
Since,D-galactose (D-gal) overload model has been used as a premature aging model, we hypothesized that it may also lead to accelerated aging in the inner ear. Furthermore, though the mitochondrial DNA (mtDNA) 4834 bp deletion mutation has been considered as the marker of aging, there is no information available in the literature concerning the mtDNA 4834 bp deletion mutation condition of the D-gal induced premature aging model. We investigate the changes in inner ear enzymatic activity, the occurring of mtDNA 4834 bp deletion in inner ear and other tissues and the relating hearing thresholds after the administration of high dosage (150 mg/kg per day) and low dosage (50 mg/kg per day) of D-gal to rats. Furthermore, the incidence of the mtDNA 4834 bp deletion in different tissues as well as in blood sample was compared. The results showed that daily subcutaneous injections of D-gal into rats for 8 weeks could lead to the biochemical defects and mtDNA 4834 bp deletion in the inner ear tissue and other tissues, which represent the typical aging animals, but the relating hearing threshold shifts (TS) were nearly identical in the three groups. This study also indicates that using of blood samples to detect mtDNA 4834 bp deletion in clinical research might lead to a 'false negative' result. A higher sensitive result could be gained using tissue biopsy to examine mtDNA 4834 bp deletion.
Subject(s)
Aging, Premature/chemically induced , Aging, Premature/genetics , DNA, Mitochondrial/genetics , Ear, Inner/physiopathology , Galactose/adverse effects , Aging/genetics , Aging/physiology , Animals , Dose-Response Relationship, Drug , Ear, Inner/enzymology , False Negative Reactions , Genetic Markers , Injections, Subcutaneous , Models, Animal , Mutation , Rats , Rats, WistarABSTRACT
BACKGROUND: Mitochondrial DNA mutations have been found in sensorineural deafness. The aim of this study was to compare three methods for extraction of nucleic acid from membranate inner ear tissue of rats. METHODS: Alkaline denaturation, a conventional phenol-chloroform method and Trizol reagent were respectively used to extract the slight nucleic acid from membranate inner ear tissue of rats. We assessed the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR). RESULTS: The yield and purity (OD260/OD280) of DNA from inner ear tissue using the phenol-chloroform method was the highest of the three methods. Mitochondrial DNA (mtDNA) fragment can be amplified by PCR from nucleic acid prepared by all methods, while no nuclear DNA (nDNA) fragment can be amplified by method of alkaline denaturation. Both nuclear and mitochondrial genes could be amplified by reverse transcriptional PCR from the RNA prepared by Trizol reagent. CONCLUSION: Adequate amount and high-quality of mtDNA, nDNA and RNA were obtained from unilateral membranate inner ear tissue of rats. Method of alkaline denaturation could be chosen when mtDNA without nDNA was needed, while phenol-chloroform method was suitable for extracting total DNA (including nDNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.
Subject(s)
DNA, Mitochondrial/isolation & purification , DNA/isolation & purification , Ear, Inner/chemistry , RNA/isolation & purification , Animals , Polymerase Chain Reaction , Rats , Rats, WistarABSTRACT
OBJECTIVE: To evaluate the clinical results, the selection of time and procedures of surgery and preoperative evaluation of the phacoemulsification or small incision cataract surgery with intraocular lens implantation (PSI) in senile cataract patients with cardiovascular disease. METHODS: The authors performed a retrospective study in 218 senior patients (255 eyes) with cardiovascular disease, which were treated by PSI in the past 5 years. RESULTS: Thirteen cases (14 eyes) did not have PSI due to surgical contraindication or impossible to tolerate the operation or impossible to obtain an improvement of visual acuity after operation. Two hundred and five cases (241 eyes) with cardiovascular disease were treated with PSI. After follow up for 1 - 18 months (mean 6.3 months), 165 cases (193 eyes) had a best corrected visual acuity (BCVA) equal or more than 20/60 (80.5%); 32 cases (39 eyes) had BCVA of 20/400 or less than 20/60 (15.6%); and 8 cases (9 eyes) had BCVA less than 20/400 (3.9%). There were no severe complications during the operation. One case developed inhalation pneumonia postoperatively and was transferred to internal medicine for rescue. CONCLUSIONS: Senile cataract patients with cardiovascular disease must be evaluated before performing PSI. The advantages of general hospital should be utilized to treat patients with cardiovascular disease before, during and after the operation. The use of sedatives, adrenalin and steroids must be individualized; high-risk patients should be monitored by EKG and supplied by constant inhalation of low dose oxygen to minimize the surgical risk. In senile cataract patients with cardiovascular disease and IV grade or more lens nucleus, small incision cataract surgery with intraocular lens implantation is better than phacoemulsification with intraocular lens implantation.
Subject(s)
Cataract Extraction , Cataract/complications , Heart Diseases/complications , Lens Implantation, Intraocular , Phacoemulsification , Aged , Aged, 80 and over , Contraindications , Follow-Up Studies , Humans , Hypertension/complications , Male , Middle Aged , Perioperative Nursing , Preoperative Care , Retrospective Studies , Visual AcuityABSTRACT
Age-associated degeneration in the central auditory system, which is defined as central presbycusis, can impair sound localization and speech perception. Research has shown that oxidative stress plays a central role in the pathological process of central presbycusis. Thioredoxin 2 (Trx2), one member of thioredoxin family, plays a key role in regulating the homeostasis of cellular reactive oxygen species and anti-apoptosis. The purpose of this study was to explore the association between Trx2 and the phenotype of central presbycusis using a mimetic aging animal model induced by long-term exposure to d-galactose (d-Gal). We also explored changes in thioredoxin-interacting protein (TXNIP), apoptosis signal regulating kinase 1 (ASK1) and phosphorylated ASK1 (p-ASK1) expression, as well as the Trx2-TXNIP/Trx2-ASK1 binding complex in the auditory cortex of mimetic aging rats. Our results demonstrate that, compared with control groups, the levels of Trx2 and Trx2-ASK1 binding complex were significantly reduced, whereas TXNIP, ASK1 p-ASK1 expression, and Trx2-TXNIP binding complex were significantly increased in the auditory cortex of the mimetic aging groups. Our results indicated that changes in Trx2 and the TXNIP-Trx2-ASK1 signal pathway may participate in the pathogenesis of central presbycusis.
Subject(s)
Auditory Cortex/metabolism , Carrier Proteins/metabolism , MAP Kinase Kinase Kinase 5/metabolism , Thioredoxins/metabolism , Aging , Animals , Antioxidants/metabolism , Apoptosis/genetics , Auditory Cortex/cytology , Auditory Cortex/physiology , Auditory Cortex/ultrastructure , Carrier Proteins/genetics , Cell Cycle Proteins , Gene Expression Regulation , MAP Kinase Kinase Kinase 5/genetics , Malondialdehyde/metabolism , Mitochondria/metabolism , Oxidative Stress , Rats, Sprague-Dawley , Superoxide Dismutase/metabolism , Thioredoxins/geneticsABSTRACT
Aging is a natural process usually defined as a progressive loss of function with an accumulation of senescent cells. The clinical manifestations of this process include age-related hearing loss (AHL)/presbycusis. Several investigations indicated the association between a mitochondrial common deletion (CD) (mtDNA 4977-bp deletion in humans, corresponding to 4834-bp deletion in rats) and presbycusis. Previous researches have shown that peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a key regulator of mitochondrial biogenesis and energy metabolism. However, the expression of PGC-1α in the inner ear and the possible effect of PGC-1α on presbycusis are not clear. Our data demonstrated the distribution of PGC-1α and its downstream transcription factors nuclear respiratory factor-1 (NRF-1), mitochondrial transcription factor A (Tfam) and nuclear factor κB (NF-κB) in marginal cells (MCs) for the first time. To explore the role of PGC-1α in cellular senescence, we established a model of marginal cell senescence harboring the mtDNA4834 common deletion induced by d-galactose. We also found that PGC-1α and its downstream transcription factors compensatorily increased in our cell senescence model. Furthermore, the overexpression of PGC-1α induced by transfection largely increased the expression levels of NRF-1 and TFAM and significantly decreased the expression level of NF-κB in the cell senescence model. And the levels of CD, senescent cells and apoptotic cells in the cell model decreased after PGC-1α overexpression. These results suggested that PGC-1α might protect MCs in this cell model from senescence through a nuclear-mitochondrial interaction and against apoptosis. Our study may shed light on the pathogenesis of presbycusis and provide a new therapeutic target for presbycusis.
Subject(s)
Cellular Senescence , Cochlea/metabolism , DNA, Mitochondrial/genetics , Presbycusis/metabolism , RNA-Binding Proteins/metabolism , Sequence Deletion , Transcription Factors/metabolism , Adenoviridae/genetics , Animals , Animals, Newborn , Apoptosis , Cells, Cultured , Cellular Senescence/drug effects , Cellular Senescence/genetics , Cochlea/drug effects , Cochlea/pathology , Dose-Response Relationship, Drug , Galactose/pharmacology , Genetic Predisposition to Disease , Genetic Vectors , NF-kappa B/metabolism , Nuclear Respiratory Factor 1/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phenotype , Presbycusis/genetics , Presbycusis/pathology , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-Dawley , Time Factors , Transcription Factors/genetics , Transfection , Up-RegulationABSTRACT
Aging has been associated with mitochondrial DNA damage. P66Shc is an age-related adaptor protein that has a substantial impact on mitochondrial metabolism through regulation of the cellular response to oxidative stress. Our study aimed to establish a D-galactose (D-gal)-induced inner ear aging mouse model and to investigate the potential role of p66Shc and its serine 36-phosphorylated form in the inner ear during aging by using this model. Real-time PCR was performed to detect the mtDNA 3873-bp deletion and the level of p66Shc mRNA in the cochlear lateral wall. Western blot analysis was performed to analyze the total and mitochondrial protein levels of p66Shc and the level of Ser36-P-p66Shc in the cochlear lateral wall. Immunofluoresence was performed to detect the location of the Ser36-P-p66Shc expression in the cochlear lateral wall. The results showed that the accumulation of the mtDNA 3873-bp deletion, total and mitochondrial protein levels of p66Shc and level of Ser36-P-p66Shc were significantly increased in the cochlear lateral wall of the D-gal-treated group when compared to the control group and that Ser36-P-p66Shc was mainly localized in the cytoplasm of the cells in the stria vascularis. During aging, the oxidative stress-related increase of p66Shc and Ser36-P-p66Shc might be associated with the accumulation of the mtDNA 3873-bp deletion in the inner ear.
Subject(s)
Aging/drug effects , DNA, Mitochondrial/metabolism , Ear, Inner/metabolism , Galactose/pharmacology , Shc Signaling Adaptor Proteins/metabolism , Animals , DNA Damage/drug effects , DNA, Mitochondrial/genetics , Male , Mice , Oxidative Stress/drug effects , Shc Signaling Adaptor Proteins/genetics , Src Homology 2 Domain-Containing, Transforming Protein 1ABSTRACT
Oxidative damage to mtDNA is associated with excessive reactive oxygen species production. The mitochondrial common deletion (mtDNA 4977-bp and 4834-bp deletion in humans and rats, respectively) is the most typical and frequent form of mtDNA damage associated with aging and degenerative diseases. The accumulation of the mitochondrial common deletion has been proposed to play a crucial role in age-related hearing loss (presbycusis). However, the mechanisms underlying the formation and accumulation of mtDNA deletions are still obscure. In the present study, a rat mimetic aging model induced by D-Gal was used to explore the origin of deletion mutations and how mtDNA repair systems modulate this process in the inner ear during aging. We found that the mitochondrial common deletion was greatly increased and mitochondrial base excision repair capacity was significantly reduced in the inner ear in D-Gal-treated rats as compared with controls. The overexpression of mitochondrial transcription factor A induced by D-Gal significantly stimulated mtDNA replication, resulting in an increase in mtDNA copy number. In addition, an age-related loss of auditory sensory cells in the inner ear was observed in D-Gal-treated rats. Taken together, our data suggest that mitochondrial base excision repair capacity deficiency and an increase in mtDNA replication resulting from mitochondrial transcription factor A overexpression may contribute to the accumulation of mtDNA deletions in the inner ear during aging. This study also provides new insights into the development of presbycusis.
Subject(s)
Aging/metabolism , DNA Repair , DNA-Binding Proteins/biosynthesis , Ear, Inner/metabolism , Mitochondrial Proteins/biosynthesis , Oxidative Stress , Presbycusis/metabolism , Transcription Factors/biosynthesis , Aging/drug effects , Aging/genetics , Animals , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , DNA Polymerase gamma , DNA Repair/drug effects , DNA Replication/drug effects , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Ear, Inner/drug effects , Ear, Inner/pathology , Galactose/toxicity , Gene Deletion , Gene Dosage/drug effects , Gene Expression Regulation, Developmental/drug effects , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , Male , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxidative Stress/drug effects , Presbycusis/genetics , Presbycusis/pathology , RNA/metabolism , RNA, Mitochondrial , Random Allocation , Rats , Rats, Sprague-Dawley , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
OBJECTIVE: determine the feasibility of manganese superoxide dismutase (MnSOD) gene therapy for protecting the cochlear function against aminoglycoside-induced oxidative stress in aging rats. METHODS: The aging model of SD rats were obtained with 8 weeks daily of D-gal (150 mg/kg per day) hypodermic injection. In the 9th week, amikacin (500 mg/kg per day) were injected intramuscularly into some aging SD rats. The viral particles of recombinant adeno-associated viral vector II/MnSOD (6 microl, 5 x 10(11) vector genomes/ml) were injected into the perilymph through the round window membrane (RWM). The feasibility of MnSOD gene therapy against aminoglycoside-induced oxidative stress in aging rats was evaluated with the methods of caspase-3 protein analysis, apoptosis detection with immunohistochemical, the detection of MnSOD concentration, stretched preparation of basilar membrane and evaluation of hearing threshold with ABR-click. RESULTS: Compared with the control group, the concentration of MnSOD of cochlear tissue was increased (P < 0.05), and the active fragment expression of caspase-3, the numbers of apoptosis bodies and the hearing threshold were decreased (P < 0.05). CONCLUSIONS: MnSOD could play a partly role to treat cochlear aminoglycoside-induced oxidative damage in aging rats.
Subject(s)
Aminoglycosides/adverse effects , Cochlea/pathology , Genetic Therapy , Oxidative Stress , Superoxide Dismutase/genetics , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cochlea/metabolism , Dependovirus/genetics , Ear, Inner/cytology , Ear, Inner/metabolism , Female , Genetic Vectors , Male , Rats , Rats, Sprague-Dawley , TransfectionABSTRACT
OBJECTIVE: To set up the oxidative stress experimental model of rat cochlea with stria vascularis marginal cells injury induced by hydrogen peroxide in vitro. METHODS: Cultured marginal cells of rat were treated by 200, 300, 400, 600 and 800 micromol/L hydrogen peroxide (H(2)O(2)) for 0.5, 1, 2, 4, 16 and 24 hours, respectively. Cell viability was assessed by the CCK-8 assay. The content of the lipid peroxidation production malondialdehyde (MDA) were detected in H(2)O(2) induced marginal cells injury with different concentration H(2)O(2). Apoptosis was assessed by flow cytometry by propidium sodium staining. The expression of the cleaved-caspase-3 was assessed by Western blot. RESULTS: Being exposed to H(2)O(2), marginal cells displayed nuclear pyknosis and margination, cytoplasmic condensation, cell shrinkage and formation of membrane and bounded apoptotic bodies. A time-dependent and dose-dependent decrease of cellular viability was detected with the treatment of H(2)O(2). Cellular maleic dialdehyde was generated in proportion to the concentration of H(2)O(2) at 2 hours and the number of apoptotic cells increased significantly (P < 0. 05). Western blot showed the expression of the cleaved-caspase-3 increased when 200 micromol/L, 300 micromol/L and 400 micromol/L H(2)O(2) treated cultured marginal cells. Thereafter the expression of the cleaved-caspase-3 decreased with 600 micromol/L H(2)O(2) and with 800 micromol/L H(2)O(2) the expression of cleaved-caspase-3 was weak. CONCLUSIONS: The findings indicated that the experimental model can be established successfully using cultured cells exposed to H(2)O(2) and activation of caspase-3 is associated with hydrogen peroxide induced rat marginal cells the oxidative stress injury.
Subject(s)
Hydrogen Peroxide/metabolism , Oxidative Stress , Stria Vascularis/metabolism , Tinnitus/metabolism , Animals , Animals, Newborn , Apoptosis , Caspase 3/metabolism , Cells, Cultured , Disease Models, Animal , Lipid Peroxidation , Rats , Rats, WistarABSTRACT
OBJECTIVE: To research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of kanamycin. METHODS: Fifty wistar rats were randomly divided into four groups: group A (D-galactose group, n = 14) were treated with hypodermic 5% D-galactose (150 mg x kg(-1) x d(-1)) for 8 weeks and then with intraperitoneal saline for 10 days; group B (D-galactose and kanamycin group, n = 14) were given the same dose of D-galactose but kanamycin (500 mg x kg(-1) x d(-1)) instead of saline; group C (kanamycin group, n = 12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n = 10) were given saline only. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and the mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. RESULTS: The incidence of mitochondrial DNA 4834 bp deletion mutation was 100% (28/28) in group A, 92.86% (26/28) in group B and 0% in group C or group D. The activity of GSHPX in group A was (59.07 +/- 8.70)U, (63.29 +/- 12. 40)U in group B, (136.67 +/- 9.53)U in group C and (142.10 +/- 7.02)U in group D. The difference between group A and D was significant (P = 0.000) while the difference between group A and B was not significant (P = 0.307), which was similarly as between group C and group D (P = 0.151). ABR threshold was (5.36 +/- 3.08) dB peSPL in group A, (61.79 +/- 11.20) dB peSPL in group B, (34.17 +/- 4.69) dB peSPL in group C and (6.50 +/- 3.37) dB peSPL in group D. No difference was found between group A and D (P = 0.398) while the difference in shift of ABR threshold between group B and group C (or group D) was significant (P = 0.000). CONCLUSIONS: The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats present high incidence of mtDNA4834 deletion which can greatly enhance the sensitivity of the inner ear to the kanamycin.
Subject(s)
Aging, Premature/chemically induced , Ear, Inner/drug effects , Kanamycin/toxicity , Animals , DNA, Mitochondrial/genetics , Disease Models, Animal , Disease Susceptibility , Galactose/toxicity , Rats , Rats, Wistar , Sequence DeletionABSTRACT
OBJECTIVE: To investigate the balance function of the patients with unilateral vestibular hypofunction (UVH) by timed balance tests and static posturography (SPG). METHODS: Sixty-five subjects with UVH and 92 healthy subjects were taken the timed balance tests under differential stance including (1) standard Romberg test, (2) feet apart stance test, (3) tandem and (4) unilateral standing tests with eyes open and eyes closed. The average timing that subjects kept balance before falling in each standing conditions was recorded by stopwatch as the timed result. The body sway velocity during the test (1) and (2) were also recorded by the SPG. RESULTS: The timed results of the tandem and unilateral standing with eyes open and eyes closed in the UVH group were decreased (P < 0.001) compared with the control group. The body sway velocity of the standard Romberg test and foot apart stance with eyes open was not different between the UVH group and control group (P-value was 0.118 and 0.110 for the two tests respectively), and the difference was significant in the eyes closed condition (P < 0.001). For the two groups, the body sway velocity of foot apart standing was decreased than that of the standard Romberg test with eyes open and eyes closed (P < 0.05 or P < 0.001). Significant correlations were not found between the timed results and sway velocity results in both two groups respectively (P > 0.05). CONCLUSIONS: According to clinical assessment of balance function in UVH, the tandem and unilateral stance test could provide the additional information about the upright stance to the SPG measurement. The effect of foot position on the results of SPG should been considered in clinic.
Subject(s)
Postural Balance , Vestibular Diseases/physiopathology , Adult , Case-Control Studies , Female , Humans , Male , Middle Aged , Neurologic ExaminationABSTRACT
Mutations in mitochondrial DNA (mtDNA) are associated with diverse pathological states in humans, notably sensorineural deafness. In humans, mtDNA4977 deletion, known as common deletion, is thought to play a critical role in presbyacusis. A similar mtDNA deletion occurs in the naturally aging rats is mtDNA4834 deletion. Today, it is still obscure about the effect of common mtDNA deletion on the presbyacusis and hearing loss. We establish a model of rat associated with mtDNA4834 deletion in inner ear by d-galactose. It was found that the malondialdehyde (MDA) increased with superoxide dismutase (SOD) decreasing in the inner ear of the rat treated with d-galactose than of the control. However, there was no significant difference in elevation of ABR threshold between the rat with mtDNA4834 deletion induced by d-galactose and control. After aminoglycoside antibiotic injected, the hearing threshold of the rats carrying mtDNA4834 deletion increased significantly compared with the rats without mtDNA4834 deletion. The results show that resembled accelerated aging in the inner ear of the rat could be induced by injecting d-galactose. Moreover, those suggest that mtDNA4834 deletion can not directly induce the hearing loss, but acting as a predisposing factor which can greatly enhance the sensitivity of the inner ear to the aminoglycoside antibiotic.