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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: [email protected]
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