Objectives: Recent studies

described that the expression of

BDNF was decreasing in patients

with depression and severe tinnitus.

However, the relationship between

the incidence of tinnitus and the

role of BDNF still remain unclear in

auditory cells. The aim of this study

is to consider the molecular network

of tinnitus focusing on BDNF signal

and autophagy in auditory cells.

Methods: We used auditory cell line

(HEI-OC1) in this study.

Tunicamycin was used as

Endoplasmic Reticulum (ER) stress.

The viability of HEI-OC1 was

determined by cell viability assays.

Morphological observation was

performed under Transmission

electron microscope (TEM).

Western blot analysis was

performed.

Results: The cell viability after

treatment of Tunicamycin was

decreased in dose- and time-

dependent matter.

Autophagosomes and

autolysosomes were confirmed into

the cytoplasm of HEI-OC1 under

TEM. The expression of LC3-II was

increased in HEI-OC1 after

treatment of Tunicamycin. These

results mean that autophagy was

consistently induced in

Tunicamycin-treated cells. The

expression of CHOP was

decreased after peaking at 12 hours

after the treatment of Tunicamycin.

The expression of BDNF which has

the potential to become a biomarker

of tinnitus and Calcium-dependent

activator protein for secretion 2

(CAPS2) that promotes BDNF

section were decreased in

Tunicamycin-treated cells. The

expression of Bcl-2 that control

apoptosis and Beclin1 that regulate

autophagy were decreased in

Tunicamycin-treated cells.

Conclusion: Our results lead to the

suggestion that the autophagy-

mediated regulation of cell death

and molecular network focusing on

BDNF signal play a major part of

the incidence of tinnitus.

Molecular network of tinnitus focusing on

BDNF and Autophagy in auditory cells

Ken Hayashi1,2, Fumiyuki Goto2, Nana Tsuchihashi2, Yasuyuki Nomura3, Masato Fujioka2, Sho Kanzaki2, Kaoru Ogawa2

1. Department of Otorhinolaryngology, Kamio Memorial Hospital, Japan , 2. Department of Otorhinolaryngology, Keio University, Japan

3. Department of Otorhinolaryngology, Nihon University, Japan

Cell Line: HEI-OC1 (House Ear Research

Institute; now UCLA)

ER stress: Tunicamycin (5μg/ml, 75μg/ml, and

100μg/ml) (Sigma)

Cell viability assay: Cell viabilities were

calculated with Countess® (Invitrogen, Life

Technologies, USA) after stain with trypan blue.

Western blot analysis: Antibody against LC3

was from MBL. Antibody against BDNF, TrkB, Bcl-

2 and Beclin1 were from Santa Cruz BioTec.

Antibody against CHOP and CAPS2 were from

Cell Signaling. Antibody against Math1, Myosin 7a

and Nestin were from Cell Signaling. Antibody

against β-actin was purchased from Bio Legend.

Transmission Electron Microscope: Cells were

fixed for 30 min with ice-cold 3% glutaraldehyde

in 0.1M cacodylate buffer, embedded in Epton,

and processed for transmission electron

microscopy by standard procedure.

Our results lead to the suggestion that the

autophagy-mediated regulation of cell death and

BDNF signaling pathway could play a major part

of the incidence of tinnitus in cell level.

Cells are exposed to not only extracellular stress

such as oxidative stress but also intracellular

stress such as endoplasmic reticulum (ER) stress.

ER stress means intracellular response caused by

the accumulation of unfolded proteins in ER. It has

been reported that ER stress should be involved

in development of degenerative neurological

disorder such as Alzheimer’s disease (1) and

psychiatric disorders such as bipolar disorder （2).

Outer hair cells were reported to be the most

sensitive to ER stress in cochlea (3). This means

that unfold protein response is a phenomenon

exist in inner ear. Emerging evidence points to the

impaired signaling of brain-derived neurotrophic

factor (BDNF) as playing a key role in the

pathophysiology of major depression. Patients

with depression exhibit low circulating levels of

BDNF. The plasma BDNF levels vary with the

severity of tinnitus, suggesting that plasma BDNF

level would be a new biomarker for objective

evaluation of tinnitus (4). However, the molecular

mechanisms of BDNF for development of tinnitus

is unclear at the auditory cellular level under ER

stress. Autophagy is capable of degrading folded

proteins, protein complexes and even entire

organelles. ER stress-induced autophagy is partly

attributed to the AKT/TSC/mTOR pathway (5). We

now discuss molecular mechanisms for

development of tinnitus from the standpoint of the

autophagy-centered regulation of cell death in

auditory cells under ER stress.

INTRODUCTION

METHODS AND MATERIALS

CONCLUSIONS

RESULTS

REFERENCES

ABSTRACT

Name: Ken Hayashi

Organization: Kamio Memorial Hospital

E-mail: ken.hayashi@jcom.home.ne.jp

Website: http://www.kamio.org

CONTACT

Fig.1. The cell viability after treatment of

Tunicamycin in HEI-OC1 cells HEI-OC1 treated with

different concentrations of Tunicamycin for 0-48h

exhibited dose-and time-dependent cell death.

Fig.2. The expression of CHOP after treatment of

Tunicamycin in HEI-OC1 cells The expression of CHOP

was reduced after peaking at12h in Tunicamycin treated

cells.

Fig.3. The induction of LC3-II after treatment of

Tunicamycin in HEI-OC1 cells Tunicamycin treatment

result in time-dependent accumulation of LC3-II. ted

Fig.4. The morphological change after treatment of

Tunicamycin under TEM A small number of

autophagosome (blue arrow) were accumulated at 24 h

in Tunicamycin-treated cells. At the point of 48 h, some

autophagosomes eventually merged with lysosomes to

become autolysosomes (black arrow).

Fig.5. The expression of Bcl-2 and Beclin1 after

treatment of Tunicamycin in HEI-OC1 cells The

expression of Bcl-2 which control apoptosis and Beclin1

that regulate autophagy were decreased in Tunicamycin

treated HEI-OC1 cells.

Fig.6. The expression of BDNF and CAPS2 after

treatment of Tunicamycin

in HEI-OC1 cells The expression of BDNF which has

the potential to become a biomarker of tinnitus and

Calcium-dependent activator protein for secretion 2

(CAPS2) which promotes BDNF section were decreased

in Tunicamycin treated HEI-OC1 cells.

Fig.8. The expression of Inner ear marker in

Tunicamycin-treated HEI-OC1 cells

The expression of Math1, Myosin 7a and Nestin

were reduced in time-dependent manner in

Tunicamycinin-treated HEI-OC1 cells.

Fig.7. The expression of TrkB after treatment of

Tunicamycin in HEI-OC1 cells The expression of

TrkB was reduced after peaking at12h in

Tunicamycin treated cells.

RESULTS

Summary

Intracellular

vesicular

traffic

ER stress

Unfolded protein response

The induction of CHOP

The reduction of CAPS2

The reduction of BDNF

Apoptosis Autophagy

Auditory cell death

CAPS2

BDNF is synthesized as a precursor, pre-proBDNF

protein. Pre-proBDNF has its pre-sequence cleaved

off in ER. The proBDNF moves, via the Golgi

apparatus, into TGN. ProBDNF is packaged in

vesicles, cleaved and secreted as mature BDNF.

Both proBDNF and mBDNF are preferentially

packaged into vesicles of the regulated secretory

pathway. This process is called “intracellular

vesicular traffic”, which won the Nobel prize this

year. Once released, mBDNF binds preferentially to

TrkB receptor. When ER stress was induced in this

process, first unfolded protein response is activated

with the induction of CHOP. Then the expression of

CAPS2 is reduced. Finally, the release of BDNF is

decreased. As a result, apoptosis and autophagy

are induced, the balance between apoptosis and

autophagy determine auditory cell death.

1. Scheper W; Autophagy, 7, 910-911, 2011

2. Kakiuchi C; PLoS One, 4, e4134, 2009

3. Fujinami Y; J Pharmacol Sci, 118, 363-372, 2012

4. Goto F; Neurosci Lett, 510, 73-77, 2012

5. Qin L; Autophagy, 6, 239-247, 2010

PLC-g

CREB

Apoptosis

AKT

PI3K

mTORC1

Autophagy RSK

MAPKK (MEK)

MAPK (ERK)

Raf

Neurogenesis

Neural differentiation

TrkB

BDNF

Nucleus

Cytoplasm

Membrane

IP3

IP3R

Ca2+

regulation

P

P P Ras

BDNF signaling cascade and regulation of cell death

SP356

CAPS2 protein domains