Transcript 559(2015)x - University of Washington

A mitochondrial quality control
pathway revealed from studies of
familial Parkinson’s Disease genes
Leo Pallanck
Department of Genome Sciences
OUTLINE
I. Overview of Parkinson’s disease (PD)
-characteristics
-influence of environment
-influence of genetics
II. Studies of the familial PD genes PINK1 &
Parkin reveal a mitochondrial QC pathway
-Part I: contributions from work in flies
-Part II: contributions from work in cell
culture
Parkinson’s disease (PD)
-Motor dysfunction (rigidity, tremor, bradykinesia, etc.).
-Highly prevalent (~1% over age 65).
-Dopamine neuron degeneration in midbrain.
-Lewy body pathology (cytoplasmic proteinaceous inclusions).
Composed of:
--synuclein
-ubiquitin
-synphilin-1
-neurofillaments
-etc.
What causes Parkinson’s disease?
Environmental Toxins
OR
Genetic Factors
The potential role of environmental factors
•1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
MPTP
MPP+
Dopamine
transporter
The potential role of environmental factors
•1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
MPTP
MPP+
MPP+
Dopamine
transporter
Mitochondrial
Complex I
The potential role of environmental factors
•1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
MPTP
MPP+
Dopamine
are mitochondria?
transporter
MPP+
-What
-What does mitochondrial complex
Mitochondrial
Complex I
I do?
-Why does inhibition of complex I
kill neurons?
CELL DEATH
Oxidative
Stress
Some background information on
mitochondrial biology
Textbook view of
mitochondria
inner membrane
outer membrane
cristae
matrix
Roles:
-ATP synthesis
-Ca2+-buffering
-apoptosis
-metabolite
synthesis
intermembrane
space
The secret lives of mitochondria
HeLa
cell
yeast
cell
mouse
embryonic
fibroblast
Mitochondria undergo continual fission
and fusion events
Video: Lab of David Chan
Fission and fusion are mediated by
evolutionarily conserved factors
FISSION:
Drp1
Fission and fusion are mediated by
evolutionarily conserved factors
FISSION:
Drp1
FUSION:
Mitofusin (Mfn)
Fission and fusion are mediated by
evolutionarily conserved factors
FISSION:
FUSION:
Drp1
Mitofusin (Mfn)
fission and fusion are dynamic processes and
their rates influence mitochondrial morphology
Mfn
Drp1
Fission and fusion are mediated by
evolutionarily conserved factors
FISSION:
For Drp1
example…
Untreated HeLa cells
FUSION:
Mitofusin (Mfn)
fission
or
fusion
fission and fusion are dynamic processes and
fusion
their rates influence mitochondrial
morphology
Legros S, et al., (2003) MBC 13, 4343-54
or
fission
Mfn
Why?
Drp1
Mitochondrial anatomy
outer
membrane
matrix
respiratory
chain
intermembrane
space
intermembrane space
inner
membrane
inner membrane
~1,000 mitochondrial
proteins encoded in the
nucleus and imported
H2O2
O2- H2O2
O
post-translationally
2
O2-
Complex I
Complex III
Complex II
mitochondrial
genome
matrix
Complex IV
(~40 genes)
Complex V
The ‘Vicious Cycle’ theory of aging
Harman D. (1972) Journal of the American Geriatrics
Society 20: 145.
O2
OH O - OH
2
H2O2
H2O2
-
OH
OH
H2O2
O2OH
H2O2
O2-
Damage lipids,
proteins & DNA
H2O2
O2- H2O2
OH
O2-
O2-
OH
Proximity to ROS
makes mitochondria a
particularly
vulnerable target
O2-
The vicious cycle theory: The progressive
accumulation of ROS-induced mitochondrial
damage leads to cell death and aging
The potential role of environmental factors
•1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
MPTP
MPP+
MPP+
Dopamine
transporter
OH O
2
Mitochondrial
Complex I
CELL DEATH
-
H2O2
O2-
OH
O2OH
OH
Additional evidence for environmental
factors in PD
•Rotenone (a pesticide) produces Parkinsonism in
rats
Rotenone is a mitochondrial complex I inhibitor
Additional evidence for environmental
factors in PD
•Rotenone (a pesticide) produces Parkinsonism in
Muftuoglu M, et al.
rats
(2004) Mov Disord 19,
544-8.
Rotenone is a mitochondrial complex I inhibitor
(complex I activity is decreased in many cases of
idiopathic PD)
controls
Parkinson’s
disease
Additional evidence for environmental
factors in PD
•Rotenone (a pesticide) produces Parkinsonism in
rats.
Rotenone is a mitochondrial complex I inhibitor.
(complex I activity is decreased in many cases of
idiopathic PD)
Smoking:
Coffee consumption:
≥40 years
≥28 oz/day
•Epidemiological
studies: 20-24 oz/day
20-39 years
-associations
with altered
risk: farming
0-19
years
12-16 oz/day
(herbicides?
nonsmoker
pesticides?),
welding (Fe?Mg?),
4-8 oz/day
well water
smoking,
0 0.5(toxins?),
1
nondrinkercoffee, etc.
Relative Risk
0
0.5
1
Relative Risk
Evidence for genetic factors in PD
•Twin studies:
Dizygotic twins
(~50% genome in common)
Monozygotic twins
(100% genome in common)
•No difference in concordance rate between monozygotic and dizygotic
twins with onset after age 51
However…
-Positron emission tomography revealed high concordance of preclinical
neuronal dysfunction in monozygotic twins, regardless of age
-Concordance was 2X greater for monozygotic twins in PD individuals
with onset before 51 years-justified search for genes among earlyonset cases
-May provide insight into common forms of PD
-May help distinguish cause and consequence
The identification of rare PD-causing
gene mutations has been a major advance
Gene
Mode of Inheritance
PARK1 (-synuclein)
AD
PARK2 (parkin)
AR
PARK3
AD
PARK5 (UCH-L1?)
AD(?)
PARK6 (PINK1)
AR
PARK7 (DJ-1)
AR
PARK8 (LRRK2)
AD
PARK9 (ATP13A2)
AR
PARK10
?
AD=autosomal dominant
Age of Onset
~45yrs
3-64yrs
~60yrs
~50yrs
~40yrs
~30yrs
~50yrs
teens
>50yrs
AR=autosomal recessive
The identification of rare PD-causing
gene mutations has been a major advance
Gene
Mode of Inheritance
PARK1 (-synuclein)
AD
PARK2 (parkin)
AR
PARK3
AD
PARK5 (UCH-L1?)
AD(?)
PARK6 (PINK1)
AR
PARK7 (DJ-1)
AR
PARK8 (LRRK2)
AD
PARK9 (ATP13A2)
AR
PARK10
?
AD=autosomal dominant
Age of Onset
~45yrs
3-64yrs
~60yrs
~50yrs
~40yrs
~30yrs
~50yrs
teens
>50yrs
AR=autosomal recessive
A little more about -synuclein
-synuclein:
-A major component of Lewy Body inclusions
-Normal cellular function unknown
Parkinson’
s disease
(PD) dominant fashion:
-Phenotype
inherited
in autosomal
Increased
gene
dosage
-Motor
dysfunction
(rigidity,
tremor, bradykinesia, etc.).
Missense alleles that reduce -synuclein turnover
-Highly prevalent (~1% over age 65).
Suggests
that
may be a
-Dopamine
neuron excess
degeneration -synuclein
in midbrain.
pathogenic factor in most cases of
-Lewy body pathology (cytoplasmic proteinaceous inclusions).
Parkinson’sComposed
disease
of:
-a-synuclein
Major questions:
-ubiquitin
-why is -synuclein toxic?
-synphilin-1
-neurofillaments
-how does it become toxic in sporadic
cases?
-etc.
-does it influence mitochondrial integrity?
The identification of rare PD-causing
gene mutations has been a major advance
Gene
Mode of Inheritance
PARK1 (-synuclein)
AD
PARK2 (parkin)
AR
PARK3
AD
PARK5 (UCH-L1?)
AD(?)
PARK6 (PINK1)
AR
PARK7 (DJ-1)
AR
PARK8 (LRRK2)
AD
PARK9 (ATP13A2)
AR
PARK10
?
AD=autosomal dominant
Age of Onset
~45yrs
3-64yrs
~60yrs
~50yrs
~40yrs
~30yrs
~50yrs
teens
>50yrs
AR=autosomal recessive
Background info on Parkin & PINK1
Parkin:
-loss-of-function mutations cause autosomal
recessive Parkinson’s disease
-encodes a cytoplasmic E3 ubiquitin protein ligase
The ubiquitin proteasome degradation pathway
E1
E2
E2
Ubiquitin
E3
E3
X
(parkin)
X
+
X
Degradation
by proteasome
Target
protein
E2
E3
X
Mitochondrial
anatomy
Background info on Parkin
& PINK1
Parkin:
outer
membrane
matrix
-loss-of-function mutations
respiratory cause autosomal
chain
recessive Parkinson’s disease
intermembrane
space
inner
-encodes a cytoplasmic
ubiquitin protein ligase
~1,000E3
mitochondrial
proteins encoded in the
nucleus and imported
post-translationally
PINK1:
membrane
mitochondrial genome
(~40 genes)
-loss-of-function mutations cause autosomal
recessive Parkinson’s disease
-encodes a Ser/Thr protein kinase with a
mitochondrial
targeting sequence
OUTLINE
I. Overview of Parkinson’s disease (PD)
-characteristics
-influence of environment
-influence of genetics
II. Studies of the familial PD genes PINK1 &
Parkin reveal a mitochondrial QC pathway
-Part I: contributions from work in flies
-Part II: contributions from work in cell
culture
Part I: Genetic studies
of PINK1 and Parkin
homologs in fruit flies
parkin & PINK1 mutants exhibit
mitochondrial pathology
wild-type
parkin-
Transverse
sections
from flight
muscle
wild-type
park-/-
PINK1-/-
myofibrils
mitochondria
Mitochondrial defects also seen in other
affected tissues (e.g., germline, neurons)
Data from Park, J et al.
(2006) Nature 441, 1157.
PINK1 and Parkin act in a common pathway
PINK1
WT
PINK1-/-
PINK1-/Parkin
…but, PINK1 over-expression does
not rescue the parkin phenotypes
also, PINK1 parkin double mutants
are indistinguishable from the single
mutants
Parkin
mitochondrial
integrity
?
Park, J. et al. (2006) Nature 441, 1157; Clark, I., et al. (2006) Nature 441, 1162; Yang, Y
et al. (2006) PNAS 103, 10793; Poole, A., et al. (2008) PNAS 105, 1638.
How does the PINK1/Parkin pathway
influence mitochondrial integrity?
A Clue:
Mutations in PINK1 & parkin result in fewer, but larger
mitochondria:
Flight
muscle:
wildtype
Parkin-/-
Male
germline:
Do PINK1 and Parkin regulate mitochondrial morphology?
Parkin & PINK1 inhibit mitochondrial fusion…
Evidence?
=>Promoting fragmentation (e.g., Drp1 overexpression, mitofusin
KO, etc.) suppressed the PINK1 & parkin phenotypes
=>Promoting fusion (e.g., Drp1 KO, Mitofusin overexpression,
etc.) worsened the PINK1 & parkin phenotypes
Poole, A., et al (2008) PNAS 105, 1638
…and they do so(promote
by fusion)
targeting Mitofusin for
A., et al (2010)
Mitofusins
ubiquitin-mediated
degradation Poole,
PLoS ONE 5, e10054
Mitofusins
Mfn
Drp1 (promotes fission)
Ub-Mfn
anti-Mfn WB
Anti-Mfn IP
anti-Ub WB
A hypothesis:
PINK1 and Parkin promote the
fragmentation of damaged
mitochondria, such that they can
be degraded through autophagy
Poole, A., et al (2008) PNAS 105, 1638
Whats autophagy????
Mitochondrial quality control
Three general categories:
I. damage prevention
(e.g., antioxidant enzymes)
II. local repair
(e.g., proteases, UPS, DNA
repair enzymes, etc.)
O2–
Mitochondrial quality control
Three general categories:
I. damage prevention
(e.g., antioxidant enzymes)
II. local repair
(e.g., proteases, UPS, DNA
repair enzymes, etc.)
III. Complete degradation
of damaged mitochondria
Mitochondrial degradation occurs
through autophagy
Image from:
http://www.smallerquestions.org/blog/2012/3/5/brucellais-running-the-show-here.html
-individual steps mediated by conserved autophagy-promoting
factors (Atg factors)
-first recognized >50 years ago
-evidence for selective targeting of damaged mitochondria only
over the past 10-15 years
Evidence for selective autophagic degradation
of damaged mitochondria (mitophagy)
Photodamaged
portion of a
hepatocyte
TMRM: a membrane
potential-dependent
dye
LC3-GFP: an
autophagosome
marker
Kim, et al (2007) Arch Biochem Biophys 462:245.
Mitochondrial turnover occurs
through autophagy
-individual steps mediated by conserved autophagy-promoting
factors (Atg factors)
-first recognized >50 years ago
-evidence for selective targeting of damaged mitochondria only
over the past 10-15 years
Mitochondrial turnover occurs
through autophagy
-individual steps mediated by conserved autophagy-promoting
factors (Atg factors)
-first recognized >50 years ago
-evidence for selective targeting of damaged mitochondria only
over the past 10-15 years
Mitochondrial turnover occurs
through autophagy
-individual steps mediated by conserved autophagy-promoting
factors (Atg factors)
-first recognized >50 years ago
-evidence for selective targeting of damaged mitochondria only
over the past 10-15 years
Part II: Cell biological
studies of PINK1 and
Parkin in vertebrate cell
culture
Parkin is
recruited to
depolarized
mitochondria…
DMSO
…and promotes
their turnover in
an atg5-dependent
manner
CCCP 24h CCCP 24h
CCCP
Parkin
WT
Tom20
Merge
Data from: Narendra D, et al. (2008) J Cell Biol 183:795.
atg5-/-
PINK1 accumulates upon mt
depolarization
CCCP treated cells
PINK1
PINK1 required for Parkin
recruitment to depolarized mt
& their subsequent turnover
DMSO
PINK1 siRNA
CCCP
control
PINK1 siRNA
Parkin-YFP
MTR
PINK1 is selectively
stabilized on depolarized
Tom20
Data from: Narendra et al. (2010)
PLoS Biol 26: e1000298.
merge
->provides a cell biological
explanation for genetic data linking
PINK1 & Parkin in a common pathway
Data from: Vives-Bauza C, et al
(2010) PNAS 107:378
A model of how PINK1 and parkin
target mitochondria for autophagy
PINK1: a
mitochondrially
localized kinase
Parkin: a cytosolic ubiquitinprotein ligase
A model of how PINK1 and parkin
target mitochondria for autophagy
1. PINK1 accumulates on depolarized mitochondria
2. PINK1 recruits Parkin to mitochondria
3. Parkin degrades mitofusin (& other proteins)
A model of how PINK1 and parkin
target mitochondria for autophagy
1. PINK1 accumulates on depolarized mitochondria
2. PINK1 recruits Parkin to mitochondria
3. Parkin degrades mitofusin (& other proteins)
4. Depolarized mitochondria are degraded in the lysosome
Summary
-Environment and genetics contribute to PD
-Mitochondrial dysfunction is strongly implicated in
PD (mitochondrial toxins, decrease complex I
activity in sporadic PD, genetic forms associated
with defects in mitochondrial QC)
-Studies of PINK1 and Parkin indicate that they
promote the fragmentation and degradation of
damaged mitochondria
There is still a lot left for you guys
to do!
-Why are only dopaminergic neurons affected
by mutations in PINK1 and Parkin?
-Does alpha-synuclein impinge on mitochondrial
health?
How much do the various cellular QC pathways
contribute to mito QC?
-Does accumulated mitochondrial damage
contribute to aging?
-Exactly how do PINK1 and Parkin promote
mitophagy?
-Why is the accumulation of damaged
mitochondria bad for the cell?
-etc.
Acknowledgements
Evvie Vincow
Nick Shulman
Ruth Thomas
Jonathon Burman
Michael MacCoss
Department of Genome
Sciences
University of Washington
Former Contributors:
Angela Poole
Cornell University
Alex Whitworth
University of Sheffield
Jessica Greene
Fred Hutchinson Cancer
Research Center
NIH (NINDS, NIGMS)