Using Drosophila to Study the PINK1-Parkin Mitochondrial

Quality Control PathwayLeo Pallanck

University of Washington

MitofusinsDrp1

The PINK1-Parkin PathwayMitofusins

ParkinPINK1

Lysosome

MitofusinsDrp1

The PINK1-Parkin PathwayMitofusins

ParkinPINK1

Contributions of fly genetics to this model:=>PINK1 & Parkin mutants accumulate enlarged defective mitochondria=>PINK1 & Parkin act in a common pathway=>Parkin ubiquitinates mitofusin to promote mt fragmentation

The PINK1-Parkin Pathway

Lysosome

MitofusinsDrp1

Mitofusins

ParkinPINK1

Some important remaining questions:

=>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons?

=>Do PINK1 & Parkin promote mitochondrial turnover in vivo?

=>What other factors act in the PINK1-Parkin pathway?

The PINK1-Parkin Pathway

Lysosome

MitofusinsDrp1

Mitofusins

ParkinPINK1

Our approach: stable isotope labeling and mass spectrometry

(collaboration with the laboratory of Mike MacCoss)

D3D3

D3 D3D3

time

trypsin digest total

proteins 5 & 10 days

CD3

D3-leucine (mass:

+3)

% o

f leu

cine

in p

eptid

es

D3-leucine

unlabeled leucine

Do PINK1 & Parkin promote mitochondrial turnover in vivo?

Technical challenges with this approach:

Solution: TOPOGRAPH; an algorithm that we helped developed in collaboration with the MacCoss laboratory

2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover

1. Quantifying the amount of D3-leucine incorporation into protein

actual amount of isotope

% 0 D3-leucines% 1 D3-leucine

isotope (additional mass units)

Frac

tion

al

Abun

danc

e

predicted

1. Quantifying the amount of D3-leucine incorporation into proteinTopograph finds the “best fit” (natural isotope distribution + n D3-leucines)

2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover

1. Quantifying the amount of D3-leucine incorporation into protein

Technical challenges with this approach:

Solution: TOPOGRAPH; an algorithm that we developed in collaboration with the MacCoss laboratory

D3-leucine content after

X hours of labeling: 25%

e.g., AIGLPEDLIQK (2 leucines)

Requires multiple Leu containing

peptides

precursor pool100% D3-leucine

precursor pool 50% D3-

leucine

25% turnover

2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover

50% turnover

“Old”

“New”

“Old”

“New”

TOPOGRAPH determines

this distribution

= 50% turnover

and uses probability-based calculations to estimate the D3-leucine content of the precursor pool

enables an estimate of turnover

“New”:

“Old”:

2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover

control

time

% D

3-le

ucin

e

Mitochondrial proteins

PINK1 or parkin mutants

Predictions of the PINK1-Parkin mitochondrial QC

model:

PINK1 or Parkin overexpression

control

time

% D

3-le

ucin

e

Non-mitochondrial

proteins

PINK1 or parkin mutants

PINK1 or Parkin overexpression

mitochondrial protein turnover is decreased in parkin mutants

0

200

400

600

800

1000

1200

1400

controlparkin–/–

half-

life

(hou

rs)

cont

rol

parki

n–/–

0

100

200

300

400

500

600

700

800

900

mea

n ha

lf-lif

e (h

ours

)

*p<0.0001

mean decrease in half-life: ~31%

nonmitochondrial protein turnover is similarly decreased in parkin

mutants

0

2

4

6

8

10

12

controlparkin–/–

half-

life

(hou

rs)

cont

rol

parki

n–/–

0

50

100

150

200

250

300

350

400

450

500

mea

n ha

lf-lif

e (h

ours

) *p<0.02

Why are nonmitochondrial proteins affected?

• Parkin regulates protein turnover more broadly than anticipated?

• The accumulation of defective mitochondria has a general effect on mitochondrial turnover?

0

100

200

300

400

500

600

700

800

controlPINK1 OE

half-

life

(hou

rs)

cont

rol

PINK1 O

E0

100

200

300

400

500

600

mea

n ha

lf-lif

e (h

ours

)

mean decrease in half-life: ~10%

*p<0.0001

PINK1 overexpression increases mitochondrial protein turnover

Importantly, mitochondrial proteins are selectively affected by PINK1

overexpression

0

2

4

6

8

10

12

mea

n %

cha

nge

(PIN

K1

OE

vs. c

ontr

ol)

mitochondrial nonmitochon-drial

*p < 0.0001

Important remaining questions:

=>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons?

=>Do PINK1 & Parkin promote mitochondrial turnover in vivo?

=>What other factors act in the PINK1-Parkin pathway?

The PINK1-Parkin Pathway

Lysosome

MitofusinsDrp1

Mitofusins

ParkinPINK1

Do PINK1 & Parkin influence mitochondrial QC in dopamine

neurons?First needed to develop a simple method to purify specific dopaminergic neurons from the fly brain:

-Uses the UAS/GAL4 system to mark the neurons of interest

(Brand and Perrimon, 1993)

GFP

ExamplesDopaminergic neuronsCholinergic neuronsEtc.

-Uses FACS to collect the marked neurons following mechanical and enzymatic brain dissociation

Non-transgenic flies

DAP

I

FITC (GFP)

TH-GAL4; UAS-GFP

FITC (GFP)

FACS purification of dopaminergic neurons from the adult Drosophila brain:

DAP

I

2% of events 0.02% of events

FACS-purified dopaminergic neurons also express appropriate markers (e.g., TH, VMAT, etc.)

Predictions:-mt membrane potential should correlate with PINK1-Parkin activity-accumulation of enlarged mt in parkin mutants.

+CCCP

-CCCP

Mitotracker fluorescence

# of

cel

ls#

of c

ells 78%22%

0%100%

55%45%

DA neurons DA neuronspark+/-

park-/-

38%62%

Rela

tive

MM

P

WT Park-/- PINK1OE

1.20.80.4

0

P < 0.01

P < 0.05

% fu

sed

mt

WT Park-/-

10080

40

0

P < 0.01

Mitotracker fluorescence

20

60

Do manipulations that promote mt fragmentation and turnover influence the

parkin neuronal phenotypes?

WT

0.40

0.8

park-/-

TgDRP1park-/-

Mfn Rnai

park-/-

TgAtg8a

1.2

park-/-

P < 0.01

Lysosome

MitofusinsDrp1

Mitofusins

PINK1Re

lativ

e M

MP

WT

0.40

0.8

park-/-

TgDRP1park-/-

Mfn Rnai

park-/-

TgAtg8a

1.2

park-/-

P < 0.001

Rela

tive

#PP

L1 n

euro

ns

-An enhanced sensitivity of mitochondria?-An increased sensitivity of cell survival in response to a general mitochondrial defect?

What explains the selective sensitivity of dopaminergic neurons to mutations in PINK1

and parkin?Re

lativ

e M

MP

WT DA neuronsPark-/-

1.20.80.4

0

P < 0.01

P < 0.05

% fu

sed

mt

WT Park-/-

10080

40

0

P < 0.01

20

60

=>Mitochondria in dopaminergic neurons are more sensitive to perturbations of the PINK1-Parkin pathway

Cholinergic neurons

Park-/-

Important remaining questions:

=>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons?

=>Do PINK1 & Parkin promote mitochondrial turnover in vivo?

=>What other factors act in the PINK1-Parkin pathway?

The PINK1-Parkin Pathway

Lysosome

MitofusinsDrp1

Mitofusins

ParkinPINK1

UAS-PINK1ey-GAL4

Cy

Del

Sb

++

++

; ;X

UAS-PINK1ey-GAL4

+;

Del

+suppression or enhancement

?

A screen for PINK1 overexpression modifiers:

WT

PINK1

PINK1park-/-

A deletion bearing chromosome

Will this work???

Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway?-mutations of activators (like parkin) should suppress-mutations of inhibitors (like mitofusin) should enhance

A screen for PINK1 overexpression modifiers:

WT

PINK1

PINK1park-/-

Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway?-mutations of activators (like parkin) should suppress-mutations of inhibitors (like mitofusin) should enhance

Will this work???

Eye severity

score

WT PINK1OE

24

0

6

PINK1OE

Park-/-PINK1OE

Park+/-PINK1OE

Mfn+/-

A screen for PINK1 overexpression modifiers:

WT

PINK1

PINK1park-/-

Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway?-mutations of activators (like parkin) should suppress-mutations of inhibitors (like mitofusin) should enhance

Will this work???

Eye severity

score

WT PINK1OE

24

0

6

PINK1OE

Park-/-PINK1OE

Park+/-PINK1OE

Mfn+/-

Suppressor

Enhancer

A screen for PINK1 overexpression modifiers:

-We have thus far screened deletions covering >95% of the genes residing on two of the three major chromosomes in Drosophila31 suppressors53 enhancersSome suppressors: Parkin, p62Some enhancers: mitofusin, Afg3L2Ongoing efforts:

Some are stronger modifiers than Parkin and Mitofusin

-Testing the specificity of modifiers

-mapping the modifier gene

Summary-The PINK1-Parkin pathway promotes selective mitochondrial turnover in vivo

-PINK1 & Parkin influence mitochondrial QC in dopaminergic neurons

-Mitochondria in dopaminergic neurons are selectively sensitive to the loss of the PINK1-Parkin pathway (a toxic effect of dopamine?)

-We’ve identified a large collection of candidate PINK1-Parkin pathway components

AcknowledgementsEvvie VincowJonathon BurmanRuth ThomasMichael MacCossNick ShulmanDepartment of Genome SciencesUniversity of Washington

Former Contributors:Angela PooleCornell UniversityAlex WhitworthUniversity of SheffieldJessica GreeneFred Hutchinson Cancer Research Center

National Institutes of HealthUMDF

How accurately does Topograph estimate the

precursor pool?-A validation experiment using yeast

1. Uniformly label yeast proteins for many generations in media containing a defined D3-leucine content

2. Subject the yeast proteins to mass spectrometry and use Topograph to measure the precursor poolAmount of D3-

Leucine in media33%67%

Topograph’s estimate of D3-Leucine precursor

pool33%68%

Pretty Accurately!!!