LRRK2: la proteina che curerà il Parkinson? · LRRK2: la proteina che curerà il Parkinson? Elisa...
Transcript of 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….
Espay et al., Nature Reviews Neurology, 2017
A complex scenario…
Espay et al., Nature Reviews Neurology, 2017
Personalized cocktail therapy
Espay et al., Nature Reviews Neurology, 2017
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
Hypothesis
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
Civiero et al., Frontiers Mol Neurosci, 2017
Active PAK6 rescues LRRK2 neurite outgrowth defects via 14-3-3g phosphorylation
Civiero et al., Frontiers Mol Neurosci, 2017
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
Civiero et al., Frontiers Mol Neurosci, 2017
PAK6 phosphorylates a subset of 14-3-3s
Kinase assays
Phospho-peptide
enrichment
LC-MS/MS