kennedy’s disease research at the lim lab...vcp valosin-containing protein, also named p97 or...

Janghoo Lim, Ph.D.

Department of Genetics

Department of Neuroscience

Program in Cellular Neuroscience, Neurodegeneration and Repair

Yale University School of Medicine

Kennedy’s Disease Research at the Lim Lab

- Signaling pathway modulating Androgen Receptor

post-translational modifications and its clearance

as well as their contribution to KD

Neurodegenerative Diseases

Basic Neuroscience

gene identification

disease models

pathogenesis studies

successful therapies?

pre-clinical trials

candidate targets for therapeutic intervention

drug discovery

Translational Neuroscience

SBMA

Are there any good candidate targets fortherapeutic intervention for SBMA?

Nemo-Like Kinase (NLK)

Are there any good candidate targets fortherapeutic intervention for SBMA?

Conserved MAPK-like serine/threonine protein kinase


Cargnello et al., 2011


Linked to several cell signaling pathways and processes

(Wnt, TGF-β, Notch, etc.)



Linked to several cell signaling pathways and processes

(Wnt, TGF-β, Notch, etc.)

Interacts with many disease-associated proteins


Nemo-Like Kinase as a potential modifier of

many neurodegenerative diseases

Disease-causing proteins Interacting proteins

HIPK2

ATXN1

ATXN2A2BP1

RBM17

HIP1

NLK NARF

QKI

Htt

PCBP4

HIP2

Puratrophin-1

COIL

PCBP1

NOVA-1

CIC

SCYL1

Akt1 CBPAR

MYB

STAT3

TAK1

APP

Fbxw7

Notch

Nurr1

Lim et al., 2006; unpublished

NLK interacts with both wild-type and polyQ-

expanded mutant AR

Todd et al., 2015

0 0.2

0.6

1

1.4

1.8

HA-ARFLAG-NLK-WT

DHT+ +

++

*

25Q 120Q

IB: α-HA

IB: α-GST

Cell Extract

(input)

GST

Pulldown

100 -

50 -

25 -

(kDa)

HA-AR25Q FL FL N FL FL NGST-NLK ++- ++-

A B C

HA-AR120Q

+pcDNA3.1

HA-AR120Q

+FLAG-NLK-WT

HA-AR120Q

+FLAG-NLK-KN

α-ARα-FLAG

α-ARα-FLAG

α-ARα-FLAG

D G

IB: α-GAPDH

*

AR

co-I

Pe

d

AR

inp

ut

E F

HA-AR25QHA-AR120Q

FLAG-NLK

+ ++

-+ +-

WT KN

# C

ells

with

ag

gre

ga

tes

# C

ells

Cou

nte

d

0

0.2

0.4

0.6

0.8DHTDHT+

-

+- -

- WT KN

- - -

********

n.s.

****n.s.

150 -

100 -

50 -

150 -

100 -

50 -

37 -

(kDa)

*

IB: α-AR

IB: α-AR

IB: α-FLAG

IB: α-FLAG

IB: α-GAPDH

HA-ARFLAG-NLK-WT

DHT

25Q 120Q 25Q 120Q

IP: α-FLAG

Cell

extract

(Input)

-- - - + ++ + + + +

NSC-34 motor neuron cells

S81

S94S256

S308S424

S515S630

NLK influences the phosphorylation status of AR

in cell culture

NSC-34

motor neuron cells

Todd et al., 2015

A potential model for the role of NLK in SBMA pathogenesis

SBMA phenotypes- Aggregation

- Cytotoxicity

- Neuromuscular degeneration

PPP

polyQ-AR

NLK

polyQ-AR

ATP

DHT

?

X

Altering Nlk expression

Modulating NLK activitySBMA pathogenesisor

?

Can NLK modulate SBMA pathogenesis?

KN: Kinase Activity

Defective

GFP (AR)

Α-FLAG (NLK)

DAPI (nuclei)

Todd et al., 2015

NLK promotes polyQ-expanded mutant AR phenotypes

in cell culture system

NLK modulates polyQ-expanded mutant AR toxicity

in a Drosophila model of SBMA

Todd et al., 2015

NLK modulates polyQ-expanded mutant AR toxicity

in a Drosophila model of SBMA

Todd et al., 2015

IB: α-AR (low exposure)

IB: α-AR (high exposure)

IB: α-Tubulin

100 -

100 -

50 -

stacking

gel

(kDa)

AR61QnmoDHT

- - --- - - -

--

adk1

+ +

- + + +- -

+

+adk2 adk1 adk2

*

0

0.4

0.8

*

HM

W A

R/ T

ub

ulin

AR61QnmoDHT - + +

+ + +- -

+

+adk1 adk2

1.2

+-

- + +

- + + +- -

+

+adk1 adk2

+ +

+ +-

+

+adk1 adk2

+--

E

F

Control SBMA SBMA; nmoadk1/+ SBMA; nmoadk2/+

+DHT +DHT +DHT +DHTA B C D

Reduced expression of NLK suppresses the toxicity

induced by a mutant AR fragment

trAR112Q: N-terminal 130 amino acids

contains polyQ region

contains S81 and S94 (purple dots)

AR

130Todd et al., in revision

SBMA mouse

model

Mice with reduced

Nlk expression

Compare the

male progeny

Can loss of Nlk affect SBMA-related phenotypes

in vivo?

A fifty percent reduction in NLK partially rescues

SBMA pathology in mice

Todd et al., 2015

Loss of one copy of Nlk improves the pathogenic

change in motor neuronal soma size in SBMA mice

Todd et al., 2015

Loss of one copy of Nlk significantly extends

overall lifespan of AR-BAC mice

Kaplan Meier, log rank test, p= 0.00107

! " # $%&' " ( )&

* +&, * - ( )& * +&, * - ( )&.# $%&' " ( )&

! "

#! ! "

$! ! ! "

$#! ! "

%! ! ! "

%#! ! "

&! ! ! "

'()

*+"

,-

."/

01

%2"

3

$! %! &!

. 4* "/5 * * 672

! "

%! ! "

8! ! "

9! ! "

: ! ! "

$! ! ! "

3"

3"

;<

=4">

(1*

"/72

$! $8 $:

. 4* "/5 * * 672

! "

# $%&' " ( )&

* +&, * - ( )&

* +&, * - ( )&.# $%&' " ( )&

! "

! ?%"

! ?8"

! ?9"

! ?: "

$"

! " $! ! " %! ! " &! ! " 8! ! " #! ! " 9! ! " @! ! "

-A+B

(B<

C"+<

>*

. 4* "/D<E72

! " #

$

! " # $%&' " ( )&

* +&, * - ( )& * +&, * - ( )&.# $%&' " ( )&

AR-BAC/+

AR-BAC/+; Nlk/+ WT (n=27)

Nlk/+ (n=27)

AR-BAC/+ (n=51)

AR-BAC/+; Nlk/+ (n=37)

Todd et al., 2015

Interim Summary

NLK interacts with, and phosphorylates, polyQ-expanded mutant AR.

Increased NLK expression promotes SBMA phenotypes in cell culture and Drosophila in a kinase activity-dependent manner.

Decreased expression of Nlk partially, but significantly, suppresses SBMA-related phenotypes in Drosophila and mice.

Next questions are …

1. What is the molecular mechanism for the NLK-mediated effects on SBMA?

2. Whether pharmacological inhibition of NLK could suppress SBMA phenotypes?

A potential model for the role of NLK in SBMA pathogenesis


- Cytotoxicity


PPP

polyQ-AR

NLK

polyQ-AR

ATP

DHT

X

Other

modifications

S81

S94S256

S308S424

S515S630

NLK influences the phosphorylation status of AR

in miceAR-BAC mouse muscle

Todd et al., 2015

NLK increases AR-dependent gene transcription in a

kinase activity-dependent manner

Androgen Response

Element (ARE)Luciferase Reporter

DH

T

AR

NSC-34 cells

Todd et al., 2015

Increased NLK elevates AR protein expression

Todd et al., 2015

0 0.2

0.6

1

1.4

1.8

HA-ARFLAG-NLK-WT

DHT+ +

++

*

25Q 120Q

IB: α-HA

IB: α-GST

Cell Extract

(input)

GST

Pulldown

100 -

50 -

25 -

(kDa)

HA-AR25Q FL FL N FL FL NGST-NLK ++- ++-

A B C

HA-AR120Q

+pcDNA3.1

HA-AR120Q

+FLAG-NLK-WT

HA-AR120Q

+FLAG-NLK-KN

α-ARα-FLAG

α-ARα-FLAG

α-ARα-FLAG

D G

IB: α-GAPDH

*

AR

co-I

Pe

d

AR

inp

ut

E F

HA-AR25QHA-AR120Q

FLAG-NLK

+ ++

-+ +-

WT KN

# C

ells

with

ag

gre

ga

tes

# C

ells

Cou

nte

d

0

0.2

0.4

0.6

0.8DHTDHT+

-

+- -

- WT KN

- - -

********

n.s.

****n.s.

150 -

100 -

50 -

150 -

100 -

50 -

37 -

(kDa)

*

IB: α-AR

IB: α-AR

IB: α-FLAG

IB: α-FLAG

IB: α-GAPDH

HA-ARFLAG-NLK-WT

DHT

25Q 120Q 25Q 120Q

IP: α-FLAG

Cell

extract

(Input)

-- - - + ++ + + + +

NSC-34 motor neuron cells

AR is degraded by the autophagy-lysosomal pathway


- Cytotoxicity


PPP

polyQ-AR

NLK

polyQ-AR

ATP

DHT

XLysosome

Can NLK modulate SBMA via regulating the autophagy-lysosomal pathway?


- Cytotoxicity


PPP

polyQ-AR

NLK

polyQ-AR

ATP

DHT

XLysosome

NLK

?

Loss of NLK increases lysosome number/size

N2a cells

In preparation

Loss of Nlk leads to up-regulation of a subset of lysosomal genes

N2a cells

- RNA sequencing from WT and Nlk KO N2a cells

- The location or function of lysosomal genes based on Gene

Ontology, significantly up-regulated in Nlk KO cells

In preparation

NLK decreases expression of TFEB, a master regulator of autophagy-lysosome biogenesis

In preparationNSC- 34 cells

Next questions are …

1. What is the molecular mechanism for the NLK-mediated effects on SBMA?

2. Whether pharmacological inhibition of NLK could suppress SBMA phenotypes?

unpublished

SB23080 can inhibit NLK activity in cell culture

unpublished

SB23080 can inhibit NLK activity in cell culture

P

NLK


- Cytotoxicity


PPP

polyQ-AR

NLK

polyQ-AR

ATP

DHT

XLysosome

NLK

Working model for the role of NLK in SBMA pathogenesis

Summary

NLK promotes SBMA phenotypes in a kinase activity-dependent manner.

Reducing NLK expression is beneficial in SBMA.

NLK interacts with, and phosphorylates, polyQ-expanded mutant AR.

NLK can regulate expression levels of AR proteins by controlling the autophagy-lysosomal pathway.

The role of VCP in Kennedy’s Disease

- Supported by 2016 KDA Research Grant

VCP

Valosin-containing protein, also named p97 or cdc48.

ATPase associated with other activities (AAA+) protein family.

Implicated in multiple cellular processes

- cell cycle regulation, DNA replication, organelle biogenesis, and

protein degradation, etc.

Missense mutations in VCP cause IBMPFD (Inclusion Body Myopathy with Paget Disease of Bone and/or Frontotemportal Dementia).

=> Multisystem degenerative disorder with three variably penetrant

phenotypes.

- Inclusion Body Myopathy (IBM)

- Paget’s Disease of the Bone (PDB)

- Frontotemporal Dementia (FTD)

- other phenotypes: cardiomyopathy, cataracts and neuropathy

unpublished

VCP co-localizes with mutant AR in cultured cells

PC12 cells

+ DHT

unpublished

VCP co-localizes with mutant AR in SBMA mouse

skeletal muscle

unpublished

VCP expression is increased in SBMA mouse

skeletal muscle at symptomatic stages

For 10 weeks, n = 2 vs. 3



**p < 0.005 (t-test).

unpublished

Increased expression of VCP suppresses the

toxicity induced by a mutant AR fragment in

Drosophila

unpublished

Generation of Vcp knockout mice

Vcp

Actin

Spinal Cord

+/+ +/-

Muscle

+/+ +/-

Cortex

+/+ +/-

Cerebellum

+/+ +/-Vcp:

> Backcrossed over 10 generations onto pure C57BL/6J and

129S6/SvEv backgrounds.

Future VCP Questions in SBMA

Whether, and how, VCP modulates the pathogenesis of SBMA?

1. Can altered expression of VCP modulate mutant AR phenotype and toxicity in SBMA?

2. Is increased VCP expression in SBMA mice beneficial or detrimental?

3. What mechanisms underlie the function of VCP in SBMA?

4. Is AAA+ ATPase enzymatic activity in VCP important for modulating SBMA phenotypes?

Neurodegenerative Diseases

Basic Neuroscience

gene identification

disease models

pathogenesis studies

successful therapies?

pre-clinical trials

candidate targets for therapeutic intervention

drug discovery

Translational Neuroscience

Kennedy’s Disease

NLK, VCP & ……

Acknowledgements

Lab members:

- Hiroshi Kokubu

- Terri Driessen

- Tingting Dong

- Ivy Hu

- Cleo Smeets

- Xiaowen Yu

Collaborators:

- Albert La Spada, UC San Diego

- Diane E. Merry, Thomas Jefferson University

- J. Paul Taylor, St. Jude Children’s Research Hospital / HHMI

Funding: NIH, Kennedy’s Disease Association

kennedy’s disease research at the lim lab...vcp valosin-containing protein, also named p97 or...

Documents