以果蝇为模式研究神经发育和

智力低下的分子遗传机制

张永清张永清

中科院遗传与发育生物学研究所

Mental Retardation and MR-related Genes

Ch t i ti f l FMRP tCharacterization of novel FMRP partners

dFMRP interacts with Brat in regulating synapse growthin regulating synapse growth

dFMRP regulates MT formationdFMRP regulates MT formationand interacts with spastin

M t l t d tiMental retardation(Intellectual Disability)(Intellectual Disability)

li d di d h t i da generalized disorder, characterized by significantly impaired cognitive y g y p gfunctioning and deficits in two or more adaptive behaviors with onset beforeadaptive behaviors with onset before the age of 18. 1-3% of general g gpopulation is mentally retarded

from wikipedia.com

>300 MR genes cloned, >1000 entries registered

Inlow and Restifo, Genetics, 2004

Identification of X-linked MR loci

202 201 215202 201

Chiurazzi et al., European Journal of Human Genetics, 2008

82 MR genes on the X-chromon the X-chrom.

Syndromic MR: black yNon-syndromic MR: *greyMR with neuromuscular defects: +grey

Chiurazzi et al., EJHG, 2008

Molecular Functions of MR Genesunknown

4% cell cycle4%

proteinsynthesis

3%

RNAprocessing

ubiquitin cycle3%

4%3%p g3%

DNA 3%

cell adhesion4%

metabolism3%

3 4%metabolism15% signal

transduction2

3

transcription

19%1

35pregulation

22%t k l t

membranecomponent

5

cytoskeleton5%

15%

Chiurazzi et al., EJHG, 2008

Neuronal functions of MR-related genes

1 Neurogenesis (microcephaly)1, Neurogenesis (microcephaly)

2, Neuronal migration (Lissencephaly)

3 Synapse formation and plasticity3, Synapse formation and plasticity (Fragile X syndrome)

Neuronal functions of MR-related genes

1 Neurogenesis (microcephaly)1, Neurogenesis (microcephaly)

2, Neuronal migration (Lissencephaly)

3 Synapse formation and plasticity3, Synapse formation and plasticity (Fragile X syndrome)

Pre-synaptic

Post-synaptic

23 MR Genes

actin

Vaillend et al., Behavioral Brain Research, 2008

Synapse dysgenesis in MR patients

Purpura, Science, 1974Dendritic spines

Modeling of Fragile X Syndrome i F it Fliin Fruit Flies

1, Introduction of Fragile X Syndromeg y

2, Dissecting the Functions of FMRP via a Genetic Approachvia a Genetic Approach

脆性X的临床特征脆性X的临床特征• Occurrence: Most common form of inherited • Occurrence: Most common form of inherited

mental retardation worldwide

• Physical: large ears, enlarged testes

• Behavioral and Cognitive:Mental retardation (IQ < 60)– Mental retardation (IQ < 60)

– Attentional-organizational dysfunction, autismautism

– Speech deficitH ti it i i l i t– Hyperactivity, seizures, social anxiety

脆性X的临床特征• Occurrence: Most common form of inherited

脆性X的临床特征• Occurrence: Most common form of inherited

mental retardation worldwide

• Physical: large ears, enlarged testes

• Behavioral and Cognitive:Mental retardation (IQ < 60)– Mental retardation (IQ < 60)

– Attentional-organizational dysfunction, autismS h d fi it– Speech deficit

– Hyperactivity, seizures, social anxiety

脆性 是 常 遗传病脆性X是一常见遗传病

1 in 4000 males and 1 in 6000 females

1 in 259 women carry Fragile X y g

1 in 800 men carry Fragile X 800 e ca y ag e

1-3% mentally retarded

No cure for mental retardation

1 3% mentally retarded

No cure for mental retardation

Large Ears and Large TestesLarge Ears and Large Testes

Gu et al., Asian Journal of Andrology, 2006

刘备的儿子刘禅 大竖耳 扶不起的阿斗刘备的儿子刘禅、大竖耳，扶不起的阿斗有可能脆性X智力低下

Diagnostic Cytogenetic Marker: a Break at the Tip of X

T i l tid E i DiTrinucleotide Expansion Disease

Coding region5’ 3’CGG

Normal

20-50

g gCGG

50 200

正常

Coding region5’ 3’CGG

Pre-muta.

50-200

CGG

>200

前突变

Coding region5’ 3’CGG

Full muta.全突变

Fragile X全突变

Multi-domain, RNA Binding FMRP Multi domain, RNA Binding FMRP

神经突触发育异常神经突触发育异常

KO mouse

spine

More numerous, structurally structurally abnormal

d d iti idendritic spines

Control mouse

Comery et al., PNAS, 1997 10 μm

212 9

11

823

20

30

21

22

19

31

26

2834 29

1

2

36

54 10

7

14

13 15

1816

17

20

24

21

3125

31

33

32

2730

7 17 24

A Simple System With Complex BehaviorsDrunken Stoned

Sleep

FightingL i dl Learning and

memoryHomosexual

courting adapted from Yi Zhong

Drosophila is an Ideal Model pOrganism to Study Human Disease

• Small size, short life cycle

V d i• Very productive

• Harmless, easy to handle

• Huge collection of mutants

• Well developed and hi ti t d ti t lsophisticated genetic tools

C d • Conserved genome sequence, 75% (1378) disease genes have fly homologueshave fly homologues

The Nobel Prize in Physiology or Medicine 1933

For his discoveries concerning the role played by the chromosome in heredity y

Morgan, T.H. Science, 1910. Sex limited inheritance in Drosophila

The Drosophila model: dfmr1 genep gSingle, Conserved Ortholog

Making Mutantsp[EP]3517

p[EP]3422 1 KB PolyA PolyATransposon Inserts

² 50M² 83M Imprecise

E i i All l ² 113M ² 192N

Revertant

Excision Alleles (null mutants)

A Model Synapse (突触模型)A Model Synapse (突触模型)Neuromuscular Junction (NMJ) SynapsesNeuromuscular Junction (NMJ) Synapses

Motor neuron cell body

axon

NMJ synapses

muscle

Big Simple Accessible

FMRP Has a Conserved Function at Synapsesat Synapses

Comery et al., PNAS, 1997

dfxrNOEWTn

dfxrn

dfxrMOE revertantn

Comery et al., PNAS, 1997

1bn

n

n

n

KO

Control MutantControl MutantWTZhang et al., Cell, 2001

dFMRP Regulates Microtubule Dynamics dFMRP Regulates Microtubule Dynamics in Nervous System and Testes

Nervous system

Futsch /MAP1B

MT stability synaptic structure/function

y

dFMRP/MAP1B structure/function

????? cMT missing immotile sperm????? cMT missing immotile sperm

Spermatogenesis

Zhang et al., Cell 107: 591-603, 2001 Zhang et al., Developmental Biology, 2004

Zhang and Broadie, Trends in Genetics, 2005Pan et al., Current Biology, 2004

FMRP’s Partners Remain Uncharacterized

Zhang and BroadieTIG 21, 2005

Modeling of Fragile X Syndrome i F it Fliin Fruit Flies

1, Introduction of Fragile X Syndromeg y

2, Dissecting the Functions of FMRP via a Genetic Approachvia a Genetic Approach

Forward Genetics–FMRP Suppressors

*

*

突变体中的各种突变位点突变体中的各种突变位点

Point Mutations

Sup29: R47C EMA: E68K (5)Sup10/5b: G80D Sup18: R115C (4), 115HWAug11: A158T, 158V LDec8m: S174F*Point Mutations WSep6Af: L186H LDec8m: G220E ZHNu: G269E (2) G269R XDL: R279C (2)SAug21Df: V354E Sup Nu10/1:Q378P

**

SAug21Df: V354E Sup Nu10/1:Q378P

* *** * *24 missense, 12 AA

**

* **** *

**** **

N CdFMRP

50 aa NLS KH1 NES RGGKH2 *60s

i t tiPhDPPiD

* *** **** * *N CdFMRP

1 113 - 154 224 - 270 290 - 335 426-434 470-499 681

interactionPPiD

32 truncations

点突变对蛋白与蛋白互作的影响点突变对蛋白与蛋白互作的影响

Y t T H b id AMammalian:CYFIP, NUFIP, 82-FIP

Yeast Two-Hybrid Assay

Drosophila:CYFIP NUFIPCYFIP, NUFIP

** *** *** *

17 missenses, 8 AA *

***

** ***

* **N CdFMRP

50 aa NLS KH1 NES RGGKH260s

interactionPhDPPiD

* *1 113 - 154 224 - 270 290 - 335 426-434 470-499 681

N terminal mutations disrupt specific protein-protein interactions

BD-NT218AD-CYFIP1

BDAD

BD AD

CYFIP1 FXR1 82-FIP

R48C (R47C) N N N

E66K (E68K) * N **

R48CA156T

BDAD-CYFIP1

ADBD-NT218

E66K (E68K) * N **

G78D (G80D) N N **

R113C (R115C) ** N **E66K

G78D

A156V

( )

R113H (R115H) * N **

A156T (A158T) N N N

R113C

S172F

L184H

A156V (A158V) N N N

S172F (S174F) ** N N

L184H (L186H) N ** NR113H G217E

L184H (L186H) N ** N

G217E (G220E) N N N

N t i i iti l f it i i f tiN terminus is critical for its in vivo functionReeve et al., J Neurosci, 2008

多途径的突变体筛选多途径的突变体筛选

LOF: EMS化学筛选LOF: EMS化学筛选

染色体缺失筛选染 体缺失筛选

GOF: 共过表达筛选GOF: 共过表达筛选

LOF: loss-of-function; GOF: gain-of-function

To uncover FMRP pathway by characterizing suppressors

Deficiency and Co-overexpression ScreenDeficiency and Co overexpression Screen

GMR promoter Gal4 dfmr1UAS

Gal4

promoter f

Eye specific Gal4

suppress hsuppress enhance

dfmr1 OE dfmr1 OE/Gene Ydfmr1 OE/Gene X

Candidate Modifiers Were Identified 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

XX

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 402L

B tC t 32E2G t2

2R41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Brat37C1-6

Cmet 32E2Cana 32E2

Got222B8

Suppressor Enhancer Weak enhancerWeak suppressor No effect

已得到10个侯选互作基因已得到10个侯选互作基因

基因 染色体 定位 分子功能

Glutamate oxaloacetate 2 22B8 aspartate transaminase activitytransaminase 2

CENP-meta 2 32E2 microtubule motor activity, structural constituent of cytoskeletony

CENP-ana 2 32E2 microtubule motor activity

Brain tumor 2 37C1-C6 protein binding, translation suppressor

Boule 3 66F5 mRNA binding

Rm62 3 83D1-D2 mRNA binding, RNA helicase activity

Adenylate kinase 1 3 69A2 adenylate kinase activityAdenylate kinase-1 3 69A2 adenylate kinase activity

NPFR1 3 83D5-E1 neuropeptide factor receptor 1

CG11843 3 98F6 unknown

CG4484 3 67A3 unknown

brat mutants suppress dfmr1OE rough eyepp f g y

Wild Type dfmr1 OE;Df(2L)pr-A16dfmr1 OE Df(2L)pr-A16

dfmr1 OE;dfmr1 OE; brat11dfmr1 OE; brat1

UAS-brat1Adfmr1 OE; brat11dfmr1 OE; brat1

Tumor Suppressor and Transla. RepressorWT Brat

A polarity protein

B tBrat

A polarity protein as Dlg, Lgl, Scrib etc.

Brat

?Brat

?

Br

at?

BratPumNos

FMRP?

5’ 3’

dMyc

5’ 3’

Hunchback

Asymmetric division Embryo development

Ectopic neuroblast: Misexpression of Hb:Ectopic neuroblast:

brain tumor

Misexpression of Hb:

fewer segment

Brat controls bouton buddingWT dfmr150M brat150/brat192Syt

brat mutants have more numerous, clustered boutons with smaller bouton size

Smaller and clustered boutons in brat mutants SSR

1

SS

AZ2

33

AZ AZ

Wei Xie from Southeast Uni.

Abnormal synapse transmissionWT EJP mEJPA

5mV

brat11/brat192

200ms

500ms

1mV

B

WT brat11/brat192

V) V) z)

WT brat /brat

**C D E F

mpl

itude

(mV

mpl

itude

(m

requ

ency

(H

tal C

onte

nt **

EJP

Am

mEJ

P A

mEJ

P Fr

Qua

nt

brat interacts with dfmr1 genetically at NMJ synapses

FMRP interacts with Brat biochemicallyFMRP interacts with Brat biochemically

In vivo Co IP In vitro pull down Fig 4In vivo Co-IP In vitro pull-down Fig. 4

anti-GFPti dFMRP

ti GFP

anti-dFMRP

anti-GFP

许执恒组

Brat colocalizes with dFMRP

*

Brat and dFMRP interact genetically and biochemicallyBrat and dFMRP interact genetically and biochemically

Wh t i th f th i t ti ?What is the consequence of the interaction?

dFMRP

Futsch/MAP1B

brat and futsch acts antagonistically at synapsesg y y p

Brat suppresses Futsch/MAP1B expressionC

D

Working Model

FMRP

I t t ith MT S i P t i S ti dInteracts with MT Severing Protein Spastin and

Regulates Microtubule Network FormationRegulates Microtubule Network Formation

spastin mutants suppress the dfmr1i h overexpression rough eye

WT dfmr1OE

dfmr1OE;UAS‐spastindfmr1OE;spastin dfmr1OE;spastin‐RNAi

Genetic interaction between dfmr1 and spastin at NMJ

10 μm

Tubulin Poly. MT

Spastin

a tubulin severing protein

Hereditary Spastic ParaplegiaHereditary Spastic Paraplegia遗传性痉挛性截瘫

Microtubule network disrupted in dfmr1 mutants

dfmr1WT dfmr1OE

湖北大学金珊

Disrupted MT network when dfmr1 expression is altered

acetylated tubulin

湖北大学金珊

Tubulin expression is altered in dfmr1 mutants

α-tubulin

Total Soluble Precipitated

α-tubulinactin

WT dfmr1 dfmr1 OE

WT dfmr1 dfmr1 OE

acetylated-tubulin

WT dfmr1 dfmr1 OE

actin

dfmr1 interacts with spastin

dfmr1, spastin mutants have similar synapse phenotypep y p p yp

D bl t t h h d h tDouble mutants have enhanced synapse phenotypes

spastin mutants rescue dfmr1 OE phenotypes

dfmr1 regulates MT network formation

Tubulin Poly MTTubulin Poly. MT

Spastin Futsch/MAP1B

pdFMRP + Brat

Fragile X syndrome

Hereditary Spastic Paraplegia

总结总结

以传统的模式动物果蝇为材料，通过突变

体的筛选和鉴定，利用多学科的试验手段

揭示FMRP在神经突触的功能，从而阐明脆

性X的发病机制,最终为治疗脆性X智力低下

提供理论指导

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

林欣大爱

陈严林欣大

姚爱玉

金珊

东南大学谢维博士；遗传发育所许执恒博士

项目资助:项目资助:

中国科学院（重要方向性项目）

国家基金委（重点、杰青）

科技部(863,973)

美国脆性X研究基金会美 究