저작자표시-비영리 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. l 이차적 저작물을 작성할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다.
Early onset of age related hearing loss in
LDHB knock out mouse
By
SAIHALI
Major in Molecular Medicine
Department of Physiology
Early onset of age related hearing loss in
LDHB knock out mouse
By
SAIHALI
A Dissertation Submitted to The Graduate School of
Ajou University in Partial Fulfillment of the Requirements
for the Degree of
Master of Biomedical Sciences
Supervised by
Chan Bae Park, Ph.D
Major in Molecular Medicine
Department of Physiology
The Graduate School, Ajou University
July, 2014
This certifies that the dissertation of
SAIHALI is approved.
SUPERVISORY COMMITTEE
Chan Bae Park
Yup Kang
Gye Soon Yoon
The Graduate School, Ajou University
July, 4th, 2014
i
Abstract
Age related hearing loss (AHL) is a main feature of mammalian aging and is t he most common sensory dysfunction in the elderly. AHL is associated with an age depended loss of sensory hair cell, spiral ganglion neurons, and stria vascularis cell i n the inner ear(Gates and Mills,2005 and Yamasoba et al., 2007). The progressive los s of these cells finally results in hearing loss because hair cells and cochlear neuron cells do not regenerate. Mitochondria are key of energy supply, cellular redox balance, signaling and regulation of intrinsic apoptosis in inner ear, especially in the high en ergy demanding cells, such as hair cells and neuron cells (Hengchao, Chen et al., 201 4).
Lactate dehydrogenates B is important enzyme in maintaining high level of pyruvate which in the presence of oxygen will be further metabolized in the TCA cycle to produce NADH and FADH2 for oxidative phosphorylation in the mitochondria. This study aimed to
observe the metabolism study of Age-related hearing loss in LDHB knockout mice.
In response to deletion of LDHB gene in mouse caused early onset of age re lated hearing loss, as increase of high frequency threshold, hair cell death, which are the typical features of Age-related hearing loss. We investigate the molecular mechani sm of hearing loss caused by LDHB deficiency by using in vitro hair cell UB-OC1. We found that differentiated UB-OC1 showed LDHB and increase of the mitochondri al function: mitochondrial respiratory subunits and MMP enhanced, increase of ATP a nd NAD+/NADH ratio were accompanied. LDHB knock down decreased mitochondria
ii
l function in differentiated UB-OC1 cell, the decrease of NAD+ and increase of HIF1a
which probably cause mitochondrial dysfunction. LDHB knock down sensitized hair c ell to antibiotics which support important clinical information.
iii
TABLE OF CONTENTS
ABSTRACT --- i
TABLE OF CONTENTS --- iii
LIST OF FIGURES --- v
I.INTRODUCTION ---1
II.MATERIALS AND METHOD A. Creation of LDHB knockout mice--- 4
B. Auditory brainstem response (ABR)--- 4
C. Cell Culture --- 5
D. siRNA Transfection --- 5
E. Cell viability assay --- 5
F. ATP level measurement --- 6
G. Mitochondrial membrane potential--- 6
H. Reactive oxygen species (ROS) --- 7
I. NAD+/NADH ratio measurement --- 7
J. LDH activity--- 7
K. Lactate production assay --- 8
L. LDH isozyme pattern assay --- 8
M. Western blot assay --- 9
III.RESULTS A. Generation of whole body LDHB knock out mouse ---11
iv
B. LDHB knock out mouse grows normally---13
C. LDHB knock out mouse show early onset of age related hearing loss---15
D. Differentiated hair cell express LDHB and show increased mitochondrial function --- 17
E. LDHB knockdown decreased the mitochondrial function in differentiated UB-OC1---21
F. LDHB knockdown differentiated UB-OC1 cells stabilized the Hif1a expression -23 G. LDHB knockdown differentiated UB-OC1cells are sensitive to mitochondrial toxins ---24
IV.DISCUSSION ---27
V. CONCLUSION ---30
v
LIST OF FIGURES
Fig.1.Generation of LDHB knockout mouse---12 Fig.2. LDHB knock out mouse grows normally---14 Fig.3.LDHB knock out mouse show early onset of age related hearing ---16 Fig.4.Increased LDHB expression and mitochondrial function observed in differentiated UB-OC1 cell---19 Fig.5.Decreased mitochondrial function in LDHB knockdown differentiated UB-OC1 cell---22 Fig.6.LDHB knockdown differentiated UB-OC1 cells stabilized the Hif1a expression----23 Fig.7.LDHB knockdown increases sensitivity to mitochondrial toxins in differentiated UB-OC1 cell ---26
1
I. INTRODUCTION
Decreased mitochondrial function with age has been documented in multiple mammalian species. Studies on isolated mitochondria from human muscle biopsies or rodent muscles support the existence of an intrinsic, aging-dependent mitochondrial defect associated with ATP production (Short et al., 2005). Age related hearing loss (AHL), the hearing loss in response to aging, is a universal disorder in current society. The progressive of hearing loss is related with the degeneration of cochlear neuron cells. AHL is characterized by an age associated defect of hearing function which beginning with hearing threshold increase in high frequency region, spread towards the low frequency region, this is accompanied by the loss of hair cells and neurons in the cochlea of the inner ear with age. The process of AHL depends on many molecular, physiological and biochemical changes (Fischel-Ghodsian et al., 1997).
Mitochondria are the main source of adenosine triphosphate (ATP), and contribute to variety of cellular functions, such as ROS generation, calcium signaling. The mitochondrial dysfunction is thought to play a central role in age related hearing loss. The mitochondrial DNA damage, ROS production and ATP deficiency contributes to age related degeneration of cochlear in inner ear (Fischel-Ghodsian., 2003)
Pyruvate is described as the substrate of the mitochondrial ATP production, which is the product of the final step of glycolysis, in the presence of oxygen will be furth er metabolized in the citric acid cycle to produce NADH and FADH2 for oxidative p
2
Lactate dehydrogenase (LDH) catalyzes the inter convertion of pyruvate and lac tate using NAD+ and NADH as a cofactor in the last step of glycolysis. LDH is a h
omo or hetero tetrameric enzyme composed of two subunits. M and H encoded by t he LDHA and LDHB genes respectively. Form five different isozymes regarding to t he ratio of the M and H subunits. The major isozymes of skeletal muscle and liver have four muscle (M)-LDHA subunits called LDH5 mainly contribute to generate the lactate from pyruvate, while the major isozymes for heart muscles contains four heart (H)-LDHB subunits known as LDH1 work for produce pyruvate from lactate. The isozyme contain LDHA subunit more, the reaction prefer to produce lactate from pyruvate more, the isozyme contain more LDHB, the reaction produce more pyruvate from lactate. Th e other variants contain both type of subunits (M3H1:LDH4, M2H2:LDH3, M1H4: L DH3) (Di Zhao, 2013). Organ of corti in cochlear of inner ear as a high energy de manding tissue mainly contains the LDH1 (LDHB) rather than LDH5 (LDHA) (Ren t ian ying, 1989).
The conversion from lactate to pyruvate depends on LDHB containing isozyme, which is important in maintaining high level of pyruvate and highly expressed in high energy d emanding tissues. If the conversion from lactate to pyruvate is destroyed, the substrate of mitochondrial ATP production will be decreased, therefore the ATP production will be disturbed cause mitochondrial dysfunction as consequence.
Age-related mitochondrial dysfunction is thought to contribute to mammalian aging. A recent study (Gomes et al., 2013) provided that decreased level of nicotinamide adenine dinucleotide (NAD+) contributes to the mitochondrial defects related with skeletal muscle
3
aging and that sirtuin 1 (SIRT1) modulates this process.
In this study we showed the LDHB deficiency decreases the mitochondrial dysfunction cause age-related hearing loss as consequence in the mouse in the way of high frequency region hearing threshold increase, hair cell loss, neuron cell and lateral wall degradation, which are the typical features of Age related hearing loss.
LDHB deficiency in UB-OC1 decreased NAD+ and stabilized HIF1a which prob
ably cause mitochondrial dysfunction. LDHB knock down sensitized hair cell to antib iotics which support important clinical information.
4
II. MATERIALS AND METHOD
A.Creation of LDHB knockout mice
A targeting vector was generated which contains Loxp 2 sites flanked LDHB and
Frt-PGK-NEO-Frt cassette (Frt-site-flanked neomycin gene expressed from the
phosphoglycerate kinase promoter). The targeting vector was transfected to the embryonic stem cells (ES cells). The targeting vector replaced the ES cells endogenous DNA by homologous recombination. The Frt-PGK-neo-Frt gene was excised by mating LDHB-neo-Loxp mice with transgenic Flp-recombinase expressing mice. The LDHB knockout mice were generated by crossing Loxp 2 sites flanked LDHB mice with protamine-cre
recombinase carrying mice.
B. Auditory brainstem response (ABR)
Hearing ability was assessed by a Biosig 32 ABR system (Tucker-Davis Technologies, Gainesville, FL) as previous study (Tian Chunjie et al., 2013). Each mouse was anesthetized with an injection of Zoletil 50 (Virbac Laboratories, Carros, France) and Rompun 2% (Bayer Korea, Ansan, Korea). Before placement of earphone, researcher examined all tympanic membranes an otoscope. The mouse was placed into a sound –shield booth, which keep outside noise from disturbing the hearing measurement. ABRs were transcutaneously recorded with sterile electrode needles. The active electrode was placed at vertex; the reference electrode at the contra lateral mastoid process: and the ground electrode on the
5
ipsilateral mastoid. Sound was presented through an earphone, which was placed directly in the ear canal. By selecting the hearing frequency, pre calibrated stimuli of 8 kHz, 16 kHz or 32 kHz were presented from 75 dB down to 10 dB in five steps. An average of 1,000 stimuli at each frequency was collected, and every result was recorded and saved.
C. Cell culture
Immortalized organ of Corti cells derived from the mouse (Rivolta MN et al 1998), UB/OC-1 cells were cultured in High Glucose DMEM (Hyclone) with 10% FBS (GIBCO) and 100kU/L penicillin at 33°C in an incubator with 10% CO2 or at 37°C in 5% CO2.
D. siRNA Transfection:
Thermo Scientific DharmaFECT1 transfection reagent, Bioneer negative control siRNA and Bioneer LDHB siRNA (sequence: 5’-3’GAAAUGUCAACGUGUUCAA), Opti-MEM Reduced Serum Medium (GIBCO) were applied for all transfections according to manufacturer’s instructions. Briefly, the siRNA was mixed with DharmaFECT1 in the certain ratio (according to cell number) and incubated with Reduced Serum Medium for 20 min at room temperature. The siRNA mixture was directly added to cells and incubated for 48h-96h then processed for further study.
E
. Cell viability assay
Cell viability was determined by MTT assay, the cells were treated with various concentrations of oxidative drug and mitochondrial toxins for 24 hours, then the MTT assay
6
was estimated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dipheny-2H-tetrazolium bromide - MTT with DMEM were added per cultured cell, the optical density (OD) of culture well was measured by a microplate reader at 595 nm.
F
. ATP level measurement
ATP was monitored by detection of light caused by the reaction of ATP with added luciferase and D-luciferin. According to the manufacture’s instruction ATPlite™ Luminescence Assay System, 1000 Assay Kit PerkinElmer, cells were lysed with ATP Lit e lysis buffer (ATPLlite: NaOH, 100Mm Triton X100 4ml/L), with shaking and sonicatio n, centrifuge the cell lyste at 13,000rpm, 4oC, 5min, the supernatant was the samp
le measured. In 96 well micro plate, 65ul of dilution buffer (PBS:lysis buffer = 2:0) w ith 10ul of sample and 15 ul of substrate solution were mixed, kept in dark condition for 10min, then measured the luminoscence intensity. The ATP level was normalized by protein concentration.
G. Mitochondrial Membrane potential
The MMP of intact cell was determined by flow cytometry with the TMRM whic h is a potentiometric, cell permeable fluorescent indicator that accumulates highly neg atively charged interior of mitochondria. The culture medium was removed from the adherent UB-OC1 cells, rinsed with PBS and resuspended in 500ul DMEM with 5ul of 1Mm TMRM(10uM final concentration), incubated at 37oC for 30mins. The cells
7
tified by flow cytometry (FACS Vantage, Becton Dickinson).
H. Reactive Oxygen species (ROS)
Intracellular ROS level was monitored by flow cytometry with 2’, 7’-dichlorodihyd rofluorescein (CM-H2DCFDA) fluorescent probe. The cells were rinsed with PBS prio r to pellet the cells, resuspended in 500ul PBS with 5uM CM-H2DCFDA, incubated at 37oC for 30mins. Fluorescence was quantified by flow cytometry (FACS Vantage,
Becton Dickinson).
I. NAD
+/NADH ratio measurement
Whole cell NAD+ and NADH were measured using Elite Fluorimetric NAD/NADH
ratio assay kit (Red Fluorescence) according to the manufacture’s instruction. Briefly Cells were lysed with NAD+ /NADH lysis buffer, with freeze thaw cycle and sonication, the lysate
was centrifuged at 13,000rpm, 4oC for 15min, the supernatant was collected as the sample. NAD+, NADH, NAD+/NADH ratio measurements were based on an enzymatic cycling
reaction, using a fluorescent plate reader(Ex/Em=540/590) . The NADH level was normalized by protein concentration.
J. LDH activity
The enzyme activity was determined by measuring the change in absorbance at 340nm as previously described (Krieg AF et al., 1967). Briefly, we lysed the cells usi ng lysis buffer (100mMK2HPO4, 30mMKF, 1mM EDTA, Protease inhibitor cock tail)
8
with homogenization. The lysate was centrifuged at 13,000rpm, 4oC for 15 min, the su
pernatant was collected as the sample. The spectrophotometric assays of Pyruvate to l actate (P to L) were performed at 25oC, pH7.4 (0.1M potassium phosphate buffer), fi
nal concentration of NADH 0.25mM, 0.5mM Sodium pyruvate. Assay of Lactate to Pyruvate(L to P) LDH activity were performed at 25oC, pH8.8( 50mM sodium pyrop
hosphate buffer), final concentration of 5.25mM NAD, final concentration of 5mM La ctate. In all assays, absorbance at 340 nm was measured at per min intervals for 10 min with Flex station. We used the Vmax as the main parameter of LDH activity, n ormalized the with protein concentration.
K. Lactate production assay
The lactate level in cultured media was analyzed by spectrophotometric assay as previous study (Lundholml et al. 1963). Briefly, 100ul of cultured medium was mixed with 200ul of perchloric acid and centrifuged at 3000g for 15min, the supernatant wa s neutralized with KOH (3M) at the ratio of (400:17), then incubated in ice for 15m in, then centrifuged at 3000g for 15min. The supernatant was applied for spectrometri c lactate assay using Thermo Max micro plate reader (Molecular Devices Co., Sunny vale, CA, USA). Lactate level was assessed from standard lactate calibration curve pr epared at the same condition and expressed as lactate (ug) released from 1ug of cell lysate.
9
LDH isozymes pattern was identified by non-denaturing Tris-glycine PAGE gel.
Briefly, the proteins were lysed with lysis buffer (100mMK2HPO4, 30MmKF, 1mM ED
TA, Protease inhibitor cock tail) with homogenization, the lysate was centrifuged at 1 3,000 rpm, 4oC for 15min, the supernatant was used as sample. Samples were kept in
ice all the time. The loading samples were prepared by adding the 40% sucrose whic h had same volume as sample, colored by bromophenol blue, the samples should not be boiled. 10ug of samples in each well were separated by SDS free Tris-glycine ele ctrophoresis buffer. Then the gel was stained with developer solution: Lactate (3.24m g/ml), NAD+ (β-nicotinamide adenine dinucleotide 0.3mg/ml), NBT(Nitroblue Tetrazoliu
m 0.8mg/ml), and PMS (Phenazine methosulfate 0.167mg/ml) dissolved in 10mM Tris -Hcl (pH8.5) buffer, incubated at 37oC for 30min, or longer time until saw the expec
ted bands, stoppted the reaction by 5% acetic acid.
M. Western blot assay
Total proteins were extracted with the Rippa buffer. Protein concentration was assessed by Bio-Rad DC protein assay (Bio Rad Laboratories, Hercules.CA). Prot eins were separated by electrophoresis on 8%-12% sodium dodecyl sulfate polyacryla mide gels. Same amount of proteins (ug) were loaded in each lane. After electrophor esis, the proteins were transferred to the polyvinylidene difluoride membrane (PVDF) or nitrocellulose membrane (NC) and subsequently subjected to immunoblottin g analysis using appropriate antibodies. The amount of loading was further detected b y western blotting housekeeping protein (Lamin A, 1:5000 dilution). Using horseadish
10
peroxidase - conjugated secondary antibody. Protein bands on the blots were visualized by ECL western blot detection reagent.
11
Ⅲ
. RESULTS
A. Generation of whole body LDHB knock out mouse
Our lab generated whole body LDHB gene knock out mouse as inactivation of LDHB gene by Cre/Loxp system. The conditional mouse harbors the floxed allele, in which LDHB gene has flanked by two Loxp sites. The LDHB gene can be exc ised and inactivated by Cre recombinase recognizing two Loxp sites.
Two mouse lines were required for conditional gene-LDHB deletion. First, a c onventional transgenic mouse line with Cre targeted to protamine in testis. Secondl y a mouse strain that embodies a target gene–LDHB flanked by two Loxp sites. R ecombination occurs only in those cells expressing Cre recombinase. Thus, the LD HB remains active in all cells and tissues which do not express Cre. We successfu lly generated whole body LDHB knock out mouse, which express the loxP and Cr e. LDHB expression protein level and LDH isozyme in heart tissue (Figure1, A). Th e mouse contains only loxP but not cre was the wild type mouse which we genera ted for control group.
12
Figure1. Generation of the LDHB knockout mouse. Restriction map of the 5’
region of the wild-type LDHB locus (wild-type locus), the targeting vector, the LDHB locus after homologous recombination (Targeted/neo locus), the LDHB locus after Flp-recombinase-mediated excision of PGK-neo gene, and knockout LDHB locus (knock out locus) (A), analysis of control (C) and targeted (T) ES cells. Digestion of ES cell DNA with BamH1 generates a novel fragment of 7.5 kb in (Targeted/neo locus) clones (B), protein level of LDHB expression in LDHB knock out mouse heart tissue(C), LDH isozyme pattern in LDHB knock out mouse heart tissue (D).
13
B. LDHB knock out mouse grows normally
LDHB is important in maintaining high level of pyruvate and highly expressed in hig h energy demanding tissues such as heart and kidney. In response to knock out, the LDH activity from lactate side of reaction (lactate to pyruvate: L-P) showed clear decrease due to deficiency of LDHB in total activity. The LDH activity from pyruvate side of reaction (pyruvate to lactate: P-L) showed decrease of total LDH activity as well, it mainly because in LDHB KO mouse the LDHA level in protein level was not changed, therefore the total LDH was decreased by LDHB deficiency(Fig.2A). To confirm whether loss of LDHB gene aff ects on the growth of mouse, we measured the body weight of LDHB knockout mous e in male and female groups for 48weeks. The LDHB knock out mice in male and female both groups showed normal growth (Fig2B).
14 Fig.2. LDHB knock out mouse grows normally.
LDH activities were decreased in heart and kidney of LDHB knockout mouse (A), L DHB knockout mouse grows normally (B). White bars and circles represent control mice, and black bars and circles represent Knockout mice. *p<0.05,**p<0.01,***p<0.0 01 vs. wild type by Student’s t-test.
15
C. LDHB knock out mouse show early onset of age related he
aring loss
Inner ear as higher energy demanding organ expresses high level of LDHB in organ of corti which contains hair cells and supporting cells, to transduct the hearing s ignal to spiral ganglion cells hair cells need numerous of ATP to serve the potassium ion recycle. To investigate the hearing function in the response of LDHB knockout in mo use, we conducted the brainstem response (ABR), a common electrophysiological test of hearing function. The results showed the age-dependent increase of hearing threshol ds were observed at high (32kHz) frequency region and middle (16kHz) frequency region i n 10 month old wild type mice (Fig.3C). Interestingly, in LDHB knock out group incr ease of hearing thresholds was observed in 5 month old mouse, which more specifically increased at high frequency region (Fig.3B) indicates LDHB KO mouse lost hearing ability earlier than WT. It lost hearing ability in high frequency sound, while low frequency is preserved. At the age of 10 months, the LDHB knockout mouse showed more severe hearing loss than WT. It is widely known that the onset of AHL begins in the high frequency region and spreads toward the low frequency region during agi ng (Keithley et al., 2004). These results suggested that the LDHB KO mice show early onset of age related hearing loss.
16
A B C
Fig.3.LDHB knock out mouse show early onset of age related hearing loss.
Auditory brainstem response (ABR) hearing threshold test with 2 months(A), 5 month s and 10 months old Wild type and LDHB Knockout mice at 8kHz, 16 kHz and 32 kHz three different frequencies. The wild type mice has hearing threshold increase at the age of 10 months(C). Increase of auditory threshold was observed in knock out mice at the age of 5 month(B), at the age of 10 months, both wild type and knock out mice showed hearing loss, the knock out mice showed more severe hearing threshold increase than wild type. *p<0.05,**p<0.01,***p<0.001 vs. wild type by Stud ent’s t-test.
17
D. Differentiated hair cell express LDHB and show increased m
itochondrial function
Hair cell damage with ROS production related mitochondrial dysfunction is thought to be the essential role of age-related hearing loss (Hengchao, Chen et al; 2014). In vivo study we observed the LDHB knockout mouse has the high frequency hearing threshold increase and hair cell loss earlier than wild type group. To investigates the molecular mechanism of hair cell loss in LDHB knock out mouse. UB-OC1 cell as hair cell precursor was app lied for further study. It differentiates to the hair cell at 37oC, 5%CO
2 (Hasson et al.,
1997). UB-OC1 cell up-regulated the cyto-skeletal protein Myosin VIIa as hair cell m arker and down regulated the supporting cell marker Jagged1 under differentiating co ndition(Fig,4A). We determined the expression level of LDHA and LDHB (Fig, 4A). The clear increase of LDHB was observed in differentiated UB-OC1 which mainly contain LDH1 and LDH2, but there was no change in LDHA expression compare wi th proliferative UB-OC1 (Control group). To investigate the affect of LDHB induction on mitochondrial function we investigated two critical indicators of energy status: the i ntracellular ATP and NAD+/NADH levels, differentiated UB-OC1 had higher ATP lev
el (Fig, 4C). Moreover, increased expression of mitochondrial respiratory proteins (Fig, 4D), mitochondria in RNA level increase (Fig, 4F), companied with mitochondrial me mbrane potential increase (Fig, 4F) were observed as well. Indicates the mitochondrial function was increased after differentiation. With increase of mitochondrial respiratory chain activity the more electron donor NADH convert to NAD+ therefore the redox state NAD+/N
18
It is widely known that mitochondria, beside play the critical role in aerobic ATP generation and in different pathways, are the primary source of ROS as well. We found that the intracellular ROS production had increased (Fig, 4H) in differentiated UB-OC1.
To examine whether the mitochondrial function increase was related to LDH isozyme changes, the LDH isozymes of the UB-OC1 cells were analyzed using a native PAGE gel electrophoresis assay. Interestingly, the differentiated UB-OC1 showed a distinct LDH isozyme pattern (Fig, 4B), they retained LDH1 and LDH2, which normally highly ex pressed in energy demanding tissue, whereas proliferative UB-OC1 retains mostly LD H5. Differentiated UB-OC1 had higher LDH activity (Fig, 4I) and, correlatively, relea sed greater amount of lactate production (Fig, 4J) into the media than proliferative UB -OC1. These results suggest that differentiated UB-OC1 express LDHB and there is c lear increase of mitochondrial function.
19
Fig.4. Increased LDHB expression and mitochondrial function observed in differe ntiated UB-OC1 cell.
Hair cell precursor UB-OC1 cell undergoes differentiation at 37oC, 5% CO
2. LDHB e
xpression (A), ATP level (C), mitochondrial respiratory subunits (D), Mitochondrial R NA levels (E), Mitochondria membrane potential (F) NAD+/NADH levels (G),ROS le
vel(H), LDH activity(I), Lactate production in media (J) are increased in differentiated UB-OC1 cells. The isozymes pattern shift to LDH1, LDH2 in differentiated UB-OC1. White bars represent control cells and black bars represent differentiated cells. *p<0.0
20
21
E.
LDHB knockdown decreased the mitochondrial function in
differentiated UB-OC1
To investigate the mechanism of hair cell loss in LDHB deficient condition, we studied mitochondrial function in LDHB knockdown differentiated UB-OC1. In LDHB knockdown UB-OC1 cell, LDHB expression was decreased in the protein level (Fig.5A), without changes in LDHA expression (Fig.5A). It companied with LDH isozymes pattern shift (Fig.5B) toward contains LDH5 mainly. We observed deficiency of LDHB cause the ATP production decrease (Fig.5D), mitochondrial membrane potential (Fig.5F) N AD+/ N ADH level decrease (F ig. 5G). Even though there was no clear change in
mitochondrial respiratory subunits. We concluded deficiency of LDHB causes the mitochon drial dysfunction in differentiated UB-OC1 cell. It companied by the intracellular ROS pr oduction increase (Fig.5H). The LDHB knock down cell showed lower LDH activity (Fig. 5C), the result is consistent with LDH activity in the heart and kidney tissue. But there was no clear change was observed in the release amount of lactate production into the m edia compare with control siRNA group (Fig.5I).Collectively, these results explain that LDHB knock down decreased mitochondrial function in differentiated UB-OC1 cell.
22
Fig.5. Decreased mitochondrial function in LDHB knockdown differentiated UB-O C1 cell.
LDHA and LDHB expression (A), LDH activity (C), ATP level (D), mitochondrial m embrane potential (F), NAD+/NADH ratio (G) are decreased in differentiated UB-OC1
cells. There is no change in LDHA expression (A) and Mitochondrial respiratory sub units (E). ROS level (H) is increased in differentiated UB-OC1. Lactate level in medi a is no significant change in differentiated UB-OC1 (I). White bars represent control cells and black bars represent LDHB knockdown cells. *p<0.05,**p<0.01,***p<0.001 vs. control siRNA by Student’s t-test.
23
F. L D H B k n o c k d o w n differentiated UB-OC1 c e l l s
stabilized the Hif1a expression.
Recent study shows that decreased levels of NAD+ contribute to the mitochondrial
dysfunction related with Hif1a stabilization, skeletal muscle aging and that sirtuin 1 (SIRT1)modulates (Gomes et al., 2013) . In our study deficiency of LDHB in differentiated UB-OC1 showed the decreased level of NAD+/NADH and reduced
mitochondrial function. To investigate the mechanism of LDHB deficiency induced mitochondrial dysfunction, we checked the Hif1a expression in LDHB knockdown differentiated UB-OC1. The Hif1a expression was stabilized in response of LDHB knockdown in differentiated UB-OC1 (Fig6). Thus LDHB knock down decrease NAD
+ and stabilize HIF1a which probably cause mitochondrial dysfunction in
differentiated UB-OC1.
Fig.6. Induced Hif1a expression in LDHB knockdown differentiated UB-OC1 cells.
24
G. LDHB knockdown differentiated UB-OC1 cells are sensitive to
mitochondrial toxins
We observed clear mitochondrial defects in the response of LDHB deficiency in hair cell. The mtDNA mutations/deletions, ROS production related mitochondrial dysfunctions are thought to be an essential component of the age-associated disorder in auditory sensitivity (T. Yamasoba et al., 2007). To investigate the mitochondrial toxin affect on LDHB deficient hair cell. The test on respiratory inhibitors, such as Rotenone (a complex I inhibitor), CCCP (mitochondrial uncoupling agent), Antimycin a (a complex III inhibitor) and Oligomycin (a complex V inhibitor) were studied. We measured the cell viability by MTT assay with LDHB knockdown UB-OC1 cells which treated with the respective doses of inhibitors for 24 hours. LDHB knockdown UB-OC1 group showed less cell viability compare with control group (Fig.7A).Cisplatin is a widely used chemotherapeutic agent to treat variety of cancer. But it shows the high does regimens ototoxicity as side affect. It mainly damages the outer hair cells in the basal turn, spiral ganglion neuron cells and the stria vascularis, resulting in hearing loss. The mechanisms appear to involve theproduction of reactive oxygen species (ROS), which can trigger cell death (Rybak L et al., 2006). Cisplatin accumulates in mitochondria causes a direct and significant impairment of mtDNA and mtRNA thesis (Nuria GARRIDO et al., 2008). Gentamicin is one of most common applied antibiotic treat variety of bacterial infections, particularly those caused by Gram-negative organisms.
25
Gentamicin toxic to the sensory cell in ear, gentamicin -induced ototoxicity is usually bilateral, symmetrical, irreversible and typically affects the higher frequencies (J. Schacht 1998). LDHB deficiency increased sensitivity to cisplatin, gentamicin treatment in hair cell. These results explained the LDHB knockdown induces the sensitivity to the mitochondrial toxin and antibiotics, which is important clinical information for future study.
26
Fig.7.LDHB knockdown increases sensitivity to mitochondrial toxins in differe ntiated UB-OC1 cell.
100uM of H202 ; 2.5uM of Cisplatin; 1mM Gentamicin; 5uM of Rotenone; 10uM
of CCCP; 5uM of Antimycin a; 1uM of Oligomycin a were respectively treated on differentiated UB-OC1 for 24 hours, there are decrease of cell viability in LD HB knock down UB-OC1 group. White bars represent control cells and black bar s represent LDHB knockdown cells. *p<0.05,**p<0.01,***p<0.001 vs. control siR NA by Student’s t-test.
27
Ⅳ
. Discussion
Mitochondrial pathology plays important role in different type of hearing loss (Pickles. J.O et al., 2004). Acquired mitochondrial dysfunction in hearing loss seems to involve increased ROS and decreased energy production, redox imbalance. It is well known that pyruvate as the substrate of the mitochondrial ATP production, is the fina l product of glycolysis. LDHB is important in maintaining high level of pyruvate lev el, LDHB expression could be directly correlated to mitochondrial function and energ y production.
A widely accepted hypothesis of how aging leads to age-related hearing loss is through mitochondrial dysfunction related with reactive oxygen species (ROS), mtDNA mutants and ATP deficiency plays a key role in aging process of cochlear cells (Shinic hi Someya et al,.2010)..
In this study, first we show that the deficiency of LDHB gene causes the early onset of age related hearing loss, even though the growth is normal in mouse. LDHB KO mouse lost hearing ability earlier than WT, even though it lost hearing ability in high frequency sound, while low frequency is preserved, which companied hair cell loss, spiral ganglion neuron cell and lateral wall degradation in high frequency region. Hence, these results suggest that LDHB play a critical role in cochlear aging, AHL in general. In response of knocking out LDHB, the total LDH activity in both side reaction: lactate side of reaction (lactate to pyruvate: L-P) and pyruvate side of reaction (pyruvate to lactate: P-L) were decreased (Fig.2A). Based on previous study (Krieg AF et al., 1967) and our experimental
28
observation the LDHB is highly sensitive to substrate inhibition. In higher concentration of pyruvate, LDHB activity is inhibited by pyruvate. In our experiment we selected the 0.5mM of pyruvate as substrate which avoided the substrate inhibition. In addition LDHB KO mouse the LDHA level in protein level was not changed, therefore the total LDH activity was decreased by LDHB deficiency in pyruvate side of reaction as well, which constant with cell line study result (Fig.5C).
We found that enhance of LDHB mediates the increase of mitochondrial function followed an enhanced NAD+/NADH balance, ATP level, mitochondrial respiratory chain
subunits (Fig4.G,C,D).Mitochondrial respiratory chain catalyzes the NADH oxidation. The increase of mitochondrial respiratory chain activity elevates the NAD+/NADH ratio and
translocates protons across the inner mitochondrial membrane, which ultimately leads to increase of ATP production.
The deficiency of the LDHB causes the mitochondrial dysfunction in hair cell of cochlear, triggered decline in NAD+ /NADH level (Fig5.G), stabilization of the Hif1a
expression (Fig.6). The decreased NAD+ and the accumulation of HIF-1α under normoxic
conditions which probably cause mitochondrial dysfunction in LDHB knock down mouse. Tissues that are composed of postmitotic cells such as the brain and the inner ear are particularly vulnerable to mitochondrial dysfunction because of their high energy requirements, and inability to undergo regeneration.
We observed mitochondrial dysfunction in the response of LDHB deficiency in hair cell. Mitochondrial dysfunctions are thought to be an essential component of the age-associated disorder in auditory sensitivity (Fig.7). With LDHB deficiency, the hair cell becomes more
29
sensitive to the mitochondrial toxin. We postulated that the deletion of LDHB leading the mitochondrial dysfunction and related increase of oxidative stress accelerates the hair cell damage with mitochondrial toxin challenge.
Cisplatin and Gentamicin are most commonly applied drug in clinic, unfortunately both of them show ototoxicity as side affect (Leonard Rybak et al., 2006), induce the oxidative stress and mitochondrial defects in cochlear of inner ear. In our results suggested that LDHB deficiency results the mitochondrial defects which increases the sensitivity to cisplatin, gentamicin treatment in hair cell (Fig.7). These postulated the LDHB knockdown induces the sensitivity to the mitochondrial toxin and antibiotics, which is important clinical information for future study.
30
V. Conclusion
In summary, the deficiency of LDHB gene causes the early onset of age related hearing loss. LDHB knock down decreased NAD+ level and stabilized Hif1a which pro
bably cause mitochondrial dysfunction in hair cell. LDHB knock down sensitized hair cell to antibiotics which support important clinical information.
31
Ⅵ
. References
1. Di Zhao., Shao-Wu Zou, Ying Liu, Xin Zhou, Yan Mo, Ping Wang, Yan-Hui Xu, Bo Dong,Yue Xiong,1 Qun-Ying Lei, and Kun-Liang Guan. 2013. Lysine-5 Acetylation Negatively Regulates Lactate Dehydrogenase A and Is Decreased in Pancreatic Cancer. Cancer Cell. 23:464–476
2. Fischel-Ghodsian.2003. Mitochondrial deafness. Ear Hear., 24 : 303–313 3. Fischel-Ghodsian., Y. Bykhovskaya, K. Taylor, T. Kahen, R. Cantor, K. Ehrenman, R. Smith, E. Keithley. 1997. Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations .Hearing Research.110 : 147– 154
4. Gates and Mills. 2005. Presbycusis. Lancet. 366 : 1111–1120
5. Hasson T and Mooseker MS. 1997. The growing family of myosin motors and their role in neurons and sensory cells.Curr Opin Neurobiol. 7:615-623.
6. Heng chao, Chen and Jian guo,Tang. 2014. The role of mitochondria in age-related hearing loss. Biogerontology. 15:13–19
32
7. J. Schacht. 1998. Aminoglycoside ototoxicity: Prevention in sight? Otolaryngol. Head Neck Surg. 118 :674–677
8. Krieg AF., Rosenblum LJ, Henry JB. 1967. Lactate dehydrogenase isoenzymes a comparison of pyruvate-to-lactate and lactate-to-pyruvate assays. Clin Chem. 13:196-203
9. Lundholml, Mohme-Lundholme and VamosN. 1963. Lactic acid assay with L (plus)lactic acid dehydrogenase from rabbit muscle. Acta Physiol Scand. Jun-Jul. 58:243-9.
10. Leonard.Rybak,CraigA.Whitworth, Debashree Mukherjea,Vickram Ramkumar. 2006. Mechanisms of cisplatin-induced ototoxicity and prevention. heares.09:015
11. Nuria Garrido, Acisclo Perez-martos, Jos´e Manuel .2008.Cisplatin-mediated impairment of mitochondrial DNA metabolism inversely correlates with glutathione levels Biochem. J. 414: 93–102
12. Pickles. J.O. 2004. Mutation in mitochondrial DNA as a cause of presbyacusis. Audiology Neurotologyl. 9 :23–33
33
13. Ren tian ying.1989. Lactate dehydrogenase isozyme patterm in Cochlear. Chinese Journal of Otorhinolaryngology 15(3):196-201
14. Rivolta MN, Grix N, Lawlor P, Ashmore JF, Jagger D andHolley MC.1998. Auditory hair cell precursors immortalized from the mammalian inner ear. Proceedings of the Royal Society of London. 265:1595–1603.
15. Rybak LP, Whitworth CA, Mukheriea D, Ramkumar V. 2007. Mechanisms of cisplatin-induced ototoxicity and prevention. Hear. Res. 226:1-2
16. Shinichi Someya, Wei Yu, William C. Hallows, Jinze Xu, James M. Vann, Christiaan Leeuwenburgh, Masaru Tanokura,John M. Denu, Tomas A. Prolla. 2010. Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction. Cell. 143: 802–812.
17. Tian C, Kim YH, Kim YC, Park KT, Kim SW, Kim YJ, Lim HJ, Choung YH. 2013. Korean red ginseng ameliorates acute 3-nitropropionic acid-induced cochlear damage in mice. Neurotoxicology. 34:42-50.
18. T. Yamasoba, S. Someya, C. Yamada, R. Weindruch, T.A. Prolla, M. Tanokura.2007. Role of mitochondrial dysfunction and mitochondrial DNA mutations in age-related hearing loss Hearing Research. 226:185–193.
