A role of parkin in stress response pathways [Elektronische Ressource] / Lena Bouman

Lena Bouman

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 26 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München










A Role of Parkin in Stress Response Pathways








Lena Bouman

aus

Heidelberg



2010

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 4 der Promotionsordnung vom
29. Januar 1998 von Frau Priv.-Doz. Dr. Konstanze Winklhofer betreut.





Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.







München, am 15. 03. 2010


_______________________






Dissertation eingereicht am 22.01.2010
1. Gutachter: Priv.-Doz. Dr. Konstanze F. Winklhofer
2. Gutachter: Prof. Dr. F.-Ulrich Hartl
Mündliche Prüfung am 08.03.2010



Acknowledgements

I
first
and
foremostt
hank
my
supervisor,
Priv.‐Doz.
Dr.
Konstanze
F.
Winklhofer,
for
her

guidance
throughout
my
thesis.
I
thank
her
for
her
constant
advice
and
help
and
for

always
having
an
open
door
for
me.

I
would
like
to
thank
Prof.
F.
Ulrich
Hartl
for
reviewing
my
thesias
s
second
referee
as
well

as
for
the
good
working
environment
I
had
during
the
time
when
our
group
was
at
his

department
of
Cellular
Biochemistry
at
the
Max
Planck
Institute
of
Biochemistry.

I
thank
my
thesis
committee
Professor
Martin
Biel,
Professor
Jörg
Tatzelt,
Professor
Dietmar

Martin
and
Professor
Christian
Wahl ‐Schott
for
taking
the
time
to
review
my
work.

I
specially
want
to
thank
Professor
Jörg
Tatzelt
that
he
always
kept
an
eye
on
my
work

and
was
always
available
when
I
needed
his
advice
and
support.

I
am
truly
grateful
to
Professor
Christian
Haass
for
his
fantastic
support,
his
inspiring

enthusiasm
and
the
excellent
working
conditions
I
had
in
his
laboratory
at
the
Adol‐f
Butenandt‐Institute
at
the
Ludwig‐Maximilians ‐University.

I
am
veryt
hankful
for
the
financial
support
that
I
received
from
the
Hans
and
Ilse
Breuer

Stiftung.


I
 also
 thank
 all
 past
 and
 present
 members
 of
 the
 Winklhofer/Tatzelt
 Lab
 for
 being

excellent
colleagues
and
for
keeping
up
a
good
lab
spirit.
Specially,
I
want
to
thank
Ir
is,
Geli,
Julia,
Margit,
Zenzi,
Anita,
Mareike,
Veronika,
Kathrin,
Anna,
Vignesh,
Uli,
Natalie,

Vincenza,
and
Alex
for
many
happy
lunches,
celebrations
and
hours
shared
at
the
bench
.

I
thank
my
colleagues
Anita,
Kathrin,
Iris,
Vincenza,
Christian,
and
Kenr
sftoir
their
great

help
with
experiments.

Some
parts
of
this
work
were
only
possible
by
successful
collaborations.
Therefore,
I

thank
 Professor
 Michael
 S.
 Kilberg,
 PhD
 Jinxiu
 Shan,
 Professor
 Carsten
 Culmsee,

Professor
 Gerald
 Thiel,
 Priv.‐Doz.
 Dr.
 Daniel
 Krappmann
 and
 Professor
 Thomas

Herdegen.

I
am
grateful
to
all
past
and
present
members
of
the
Haass
lab
for
the
good
atmosphere

on
the
floor
and
in
the
kitchen
and
their
willingness
to
share
reagents.
Specially,
I
want
to

thank
my
lab
colleagues
Nicole,
Bettina,
Ulf
and
Doro
for
being
always
helpful
and
for

sharing
many
nice
moments
in
and
outside
of
the
lab,
Michael
for
his
willingness
to
be

part
of
my
thesis
committee
and
for
his
support,
and
Sabine
for
running
the
daily
lab

business
and
all
her
help
with
ordering.

Thanks
to
Kathryn,
Ulf,
Kathrin
and
Richard
for
proofreading
some
parts
of
my
thesis
.
Last
but
not
least,
I
thank
my
parents
for
their
great
support
and
constant
encouragement

and
Konstantin
for
being
there
for
me
every
step
of
the
way.

Table of content
Table of content
1 SUMMARY ....................................................................................................1
2 INTRODUCTION...........................2
2.1 Parkison’s Disease............................................................................................................2
2.1.1 History...........................2
2.1.2 Neuropathological characteristics.................3
2.1.3 Symptoms and therapeutic approaches .......................................................................5
2.1.4 Etiology .........................................................................................8
2.2 Familial forms of PD and their genetics..........9
2.2.1 Autosomal-dominant genes........................................................11
2.2.2 Autosomal-recessive genes................................12
2.3 Pathogenesis of PD and its associated cellular processes ........................................15
2.3.1 Mitochondrial dysfunction and oxidative stress...........................................................15
2.3.2 Mitochondria-induced apoptosis .................................................17
2.3.3 Protein aggregation and dysfunction of the ubiquitin proteasome system..................19
2.3.4 Endoplasmic Reticulum (ER) stress...........................................22
2.3.5 Interaction of ER and mitochondria.............................................26
2.3.6 Pathways implicated in neuronal survival and cell death............27
2.4 Parkin-associated Parkinson’s Disease........................................31
2.4.1 Clinical and neuropathological characteristics of parkin-associated PD.....................31
2.4.2 Molecular genetics and cell biology of parkin .............................................................31
2.4.3 Parkin mutations .........................................................................33
2.4.4 Putative parkin substrates...........................34
2.4.5 Parkin-deficient animal models ...................................................37
2.4.6 Parkin has a neuroprotective potential........................................38
3 RESULTS ....................................................................41
3.1 Parkin is a target of the unfolded protein response and protects cells from ER
stress-induced mitochondrial damage...................................................41
3.1.1 Parkin is up-regulated in response to ER stress.........................41
3.1.2 Parkin as target of the unfolded protein response (UPR)...........42
3.1.3 c-Jun represses the ER stress-induced transcriptional activation of parkin................49
3.1.4 Parkin protects cells from ER stress-induced cell death.............................................54
3.1.5 Parkin has no direct effect on ER stress.....................................58
3.1.6 Involvement of parkin in protective signaling pathways..............65
3.1.7 Parkin maintains mitochondrial integrity after ER stress.............................................73
3.2 Regulation of PD-associated genes in response to pathophysiological stress
conditions..................................................................................................................................76
3.2.1 Parkin gene expression is stress-regulated................................76
Table of content
3.2.2 PACRG is up-regulated in response to ER stress ......................................................79
3.2.3 α-synuclein, DJ-1, PINK1, LRRK2 and HtrA2/omi are up-regulated in response to ER
stress ....................................................................................................................................80
3.2.4 The expression of α-synuclein, DJ-1, PINK1, LRRK2 and HtrA2/omi is not changed in
response to mitochondrial stress, excitotoxicity and dopamine treatment..............................82
3.2.5 PINK1 and parkin expression are not transcriptionally linked to each other...............84
3.2.6 Parkin point mutations lead to misfolding ...................................................................87
4 DISCUSSION...............................................................89
4.1 Parkin is a target of the unfolded protein response and protects cells from ER
stress-induced mitochondrial damage ...................................................................................89
4.2 Regulation of PD-associated genes in response to pathophysiological stress
condition....................................................................................................................................95
5 METHODS .................................................................100
5.1 Cell culture methods .....................................................................100
5.1.1 Cell culture................100
5.1.2 Preparation and cultivation of mouse embryonic fibroblasts.....100
5.1.3 Preparation and cultivation of mouse and rat primary cortical neurons ....................101
5.1.4 Transient transfection ...............................................................................................101
5.1.5 RNA interference......................................101
5.1.6 Starvation of cells......102
5.2 Nucleic acid biochemistry ............................................................................................102
5.2.1 Polymerase chain reaction (PCR).............102
5.2.2 Agarose-gelelectrophoresis......................103
5.2.3 Isolation and purification of DNA fragments from agarose gels................................103