lrrk2: la proteina che curerà il parkinson? · lrrk2: la proteina che curerà il parkinson? elisa...

LRRK2: la proteina checurerà il Parkinson?

Elisa Greggio, PhD

Department of Biology

University of Padova

Email: [email protected]

Pavia, 15 marzo 2019

Parkinson’s disease

• No cure• Only symptomatic

therapies

• 230.000 Italians with PD

• ~1% of the population > age 65

• Median age of onset is 60 years, with slight male preponderance

• Disease onset to death is about 15 years

• Cardinal Clinical Features:

Tremor at restRigidityAkinesiaPostural instability

Dauer and Przedborsky, Neuron, 2003

• Degeneration of dopaminergic neurons

• Lewy bodies containing alpha-synuclein

DA cell loss is accelerated in PD

Dop

am

inerg

ic n

eu

ron

s (%

)

Age (years)

100

50

0

0 20 40 60 80

Lewy bodies

Parkinson’s disease: a syndrome rather than a disease?

Tremors BradikinesiaRigidity

Lewy pathology

Single gene mutations

?

Diagnostic tiers as a ‘single disease’

Espay et al., Nature Reviews Neurology, 2017

A complex scenario….


A complex scenario…


Personalized cocktail therapy


Targeting mutant LRRK2

Importance to categorize PD sub-types

Leucine-rich repeat kinase 2

• Strong genetic link• Most common monogenic cause

of PD (1-5% in Europe)• Risk factor

• LRRK2-PD similar to sporadic PD:• Often classical features• But clinical and pathological

variability within the same mutation carriers are common

• Mutations increase kinase activity: therapeutic target?

GWA risk alleles

CV risk alleles

Causal mutationsLRRK2

LRRK2

LRRK2

Singleton & Hardy, Neuron 2017

LRRK2: many domains….

R1441C/G/H**Y1699C**

G2019S*I2020T*

Decrease GTPase*

Prolonged GTP-bound state

Prolonged signaling(e.g PAK6)

Increase kinase activity*

Auto-P

Rabs

LRRK2 subcellular localization

and activity

Pharmacologicl inhibition cause

de-phosphorylation

14-3-3

P-S910P-S935

PdeP

CK1a

IKKs

PP1A

autoP-S1292

…many functions?

Civiero et al., BST, 2018

Increased kinase activity due to pathogenic mutant alters

these processes

• One more relevant than others?

• Distinct pathogenic mechanisms converging or independent?

• Interaction with other genetics and environmental factors?

LRRK2 inhibition: a valuable therapeutic strategy?

Adapted from West 2017

Mutations confer gain of kinase function Kinase inhibition confers neuroprotection in rodent models of synuclein-based neurodegeneration

Daher et al., JBC, 2015

Last generation inhibitors: oral availability, good selectivity, cross BBB

Scott et al., JMC 2017

Lack of a lrrk2-rodent model recapitulating PD manifestations

LRRK2 inhibition: a valuable therapeutic strategy?

Adapted from Denali Therapeutics

LRRK2 inhibition: safety liabilities

Fuji et al., Sci Trans Med, 2015

Lysosome-related lamellar bodies and pulmonary toxicity in non-human primates (but not mice!) treated with LRRK2 inhibitors

Control LRRK2 inhibitor

LRRK2 inhibitor

LRRK2 is expressed in different tissues…

Cogo and Greggio, 2018

Hum

an0

1

2

3

4

5

FP

KM

NeuronsMicroglia/MarcophagesAstrocytes

Identification of brain-specific LRRK2 partners

Protoarrays® ~ 9,000 human recombinant proteins

Laura Civiero

The p21-activated kinase family

Nekrasova et al., Dev Biology, 2008 Furnari et al., PLoSOne, 2013

• Pak6 knock-out mice:viable and fertile

• Pak5/Pak6 doubleknockout: reducednumber of neurites

• PAK6 and PAK5 arepredominantly expressedin the neurons

Huang et al., Mol. Cell. Biol., 2011Kelly and Chernoff, Mol. Cell. Biol., 2011

Group I

Group II

Does PAK6 kinase activity influence neurite morphology in LRRK2 models?

K436M

S531N

kinase-dead (KD)

hyper-active (HA)

Kinase

PRR

S560

(Auto-P)

CRIB

kinase activity (relative to WT)

0 1 2 3

K436M wild-type S531N

AAV

Striatal injection

Striatal injection

LRRK2 and PAK6 cooperate to control neurite growth in vivoW

ild-t

yp

eK

no

ck-o

ut

Civiero et al., J Neurochem, 2015

PAK6 and LIMK1 phosphorylation is reduced in Lrrk2-/-

PAK6 pS560

LIMK1 pT508

Cofilin pS3

F-actin

neurite complexity

Cofilin

G-actin

Less branching

PAK6

LIMK1

WT KO0.0

0.5

1.0

1.5

2.0

P-S

er5

60 / P

AK

6

**

WT KO0.00

0.05

0.10

0.15

0.20

0.25

P-T

508 / L

IMK

1 *

p-PAK6

p-LIMK1

PAK6 pS560

LIMK1 pT508

Cofilin pS3

F-actin

neurite complexity

LRRK2(GTP)

Cofilin

G-actin

Less branching

PAK6

LIMK1

Adapted from Civiero et al., Bioc Soc Trans, 2018 Civiero et al., J Neurochem, 2015

Summary (1)

• LRRK2 and PAK6 interacts in the striatum

• PAK6 kinase activity promotes neurite complexity

• LRRK2 is required for PAK6-dependent neurite outgrowth

• LRRK2 controls PAK6 activity

PAK6 kinase activity has a potent dephosphorylating effect on pSer935 similar to LRRK2 kinase inhibition

Hypothesis

Civiero et al., Frontiers Mol Neurosci, 2017

PAK6 phosphorylates 14-3-3g at Ser59

PAK6 phosphorylates 14-3-3s decreasing their affinity for LRRK2

14-3-3

P

LRRK2

14-3-3g pre-phosphorylated in vitro by PAK6

P

14-3-3

P

LRRK2

IB: 14-3-3

IB: LRRK2

In vitro In cells


Active PAK6 rescues LRRK2 neurite outgrowth defects via 14-3-3g phosphorylation


empty vector

Changes in PAK6 phosphorylation in G2019S brains

Alice Biosa

Midbrain from 12 month-old mice

Is PAK6 activity protective in a synuclein-G2019S mouse model?

rAAV2/7-asyn + rAAV2/7-caPAK6

Histology Biochemistry

BAC-G2019S

Summary (2)

• PAK6 causes LRRK2 dephosphorylation at Ser935 in cells

• PAK6 phosphorylation at Ser59 results in 14-3-3g-LRRK2 complex dissociation

• PAK6 phosphorylation of 14-3-3g rescues the neurite shortening phenotype of BAC-LRRK2 G2019S neurons

• PAK6 less active in aged G2019S mice

• Therapeutic target with more selective cellular specificity than LRRK2?

PAK6 pS560

G-actin

Less branching

LRRK2G2019S

14-3-3g

pS59

F-actin

neurite complexity

LRRK2G2019S

14-3-3g

Acknowledgments

University of Padova

Lab of Molecular Physiology

KULeuven

Evy Lobbestael

Veerle Baekelandt

NIH - LNG-NIH

Mark Cookson

Alice KaganovichSasha Beilina

University of Trento

Giovanni Piccoli

University of Ferrara

Michele Morari

Paolo Pinton

Claudia Morganti

Laura Civiero

Alice Biosa

Susanna Cogo

Jean-Pierre Aubert Research

Center, Lille

Jean-Marc Taymans

Marie-Christine Chartier-Harlin

Giulia Tombesi

Luigi Bubacco

Isabella Tessari

Nicoletta Plotegher


PAK6 phosphorylates a subset of 14-3-3s

Kinase assays

Phospho-peptide

enrichment

LC-MS/MS

lrrk2: la proteina che curerà il parkinson? · lrrk2: la proteina che curerà il parkinson? elisa...

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