apoptose cerebro

8/3/2019 apoptose cerebro

1/57

Genetic pathways of cell

death in mammals

Transgenesisto unravel

cell death mechanisms


2/57

studying a death pathway

In knockout orTransgenic mice

(in development)

In cellular systems ofembryonic origin

In surviving individuals


3/57

1. Transgenic technology

2. Knockout technology

4. Conditional knockout technology

6. Conditional transgenesis technology

3. Gene trap

The mutant mouse approach

5. Knockout-knockin technology


4/57

By Knudson


5/57

The apoptoticpathways

The apoptosome


6/57

Control STS

Cyt c

Hsp 60

Nuclei

Merge

Cytochrome cReleasein apoptosis


7/57


8/57

By Knudson


9/57

Bcl- 2- like proteins:

Bad

ANTI-APOPTOTIC

PRO-APOPTOTIC


10/57


The apoptosome


11/57

2. Knockout technology to studycell death in CNS development

E8. 5 E9. 0 E1 1 . 0


12/57

PCD within the proliferative ventricular zones a potential rolein regulating the

size of the

progenitor pool PCD of postmitotic neurons a mechanism to match

the neuron populationto its target fields

PCD specific for early brain regioni.e. the lateral edges of the hindbrainneural fold

essential fornormal neuraltube closure

MULTIPLE FUNCTIONSOF PROGRAMMED CELL DEATH

IN BRAIN DEVELOPMENT


13/57

Several genes implicated in PCDduring brain development

K.O. mouse exhibits an apparently normal forebrain (FB)formation and shows a decreased PCD in specific neuronalsubpopulations

K.O. mouse shows a reduction of PCD in most peripheral

ganglia and in spinal cord. K.O. mouse has more motor neurons than its w.t.

counterpart

Bax:

E 12

From White et al.,1998

From Roth et al., 1999

Bax BclXL Apaf1 Casp9 Casp3

Jnk1/Jnk2

?


14/57


Jnk1 /Jnk2: dK.O. mouse exhibits the lack of closureof the hindbrain neural tube

w.t. Jnk1/J nk2 dKO

E 9.0

E 11 .5

Bax BclXL Apaf1 Casp9 Casp

Jnk1/Jnk2

?

From Kuan et al., 1999


15/57


BclXL: K.O. mouse shows an increased PCD in specific neuronalsubpopulations (postmitotic neurons of the developingbrain, spinal cord and dorsal root ganglia). However it dies at E 13for hematopoietic defects

Preplate (PP) andventricular zone (VZ) of the

developing cortical wall of

E12.5 embryos

From Kuan et al.,

2000


Jnk1/Jnk2

?


16/57

K.O. mouse exhibits forebrain overgrowth, mostly throughthe expansion of the neural progenitor cells population.

It also affects the postmitotic neurons

Apaf1:

E 16.5E 12.5


From Cecconi et al., 1998


Jnk1/Jnk2

?


17/57

Caspase- 9: K.O. embryo exhibits severe brain malformationwith a large overproduction of forebrain progenitors

Several genes implicated in PCDduring brain developmen

E 10.5E 16.5

From Kuida et al.,1998


Jnk1/Jnk2

?


18/57


K.O. mouse exhibits forebrain abnormalities (*) andincreased forebrain surface area. By preventing selected PCD in the early forebrain progenitor

lineage, caspase deletion causes an increase in forebrainfounder cells and leads to a convoluted cerebrum

Caspase- 3:

E 12.5

Lamina terminalis PN 16

From Rothet al.

,1999 From Haydar et al., 1999


Jnk1/Jnk2

?

S l i li t d i PCD


19/57


dKO BclXL/Apaf1, BclXL/Casp9, BclXL/Casp3:compensative effect in the PCD of neuronal postmitotic cells, butthey still show severe malformations of brain. They die at E1 3 as

KO BclXL: epistatic and independent actions in neural PCD

dKO BclXL/Bax:compensative effect in the PCD of neuronal postmitotic cells, they

still die at E13 as KO BclXL: epistatic action in neural PCD

Epistatic and independent apoptotic functions


20/57

Epistatic and independent apoptotic functionsof Apaf1 , Caspase- 3, Bax and BclXL in the

developing nervous system

Apaf1

From Kuanet al., 2000


21/57


Jnk1 /Jnk2: dK.O. mouse exhibits the lack of closureof the hindbrain neural tube

w.t. Jnk1/J nk2

dKOE 9.0

E 11 .5


Jnk1/Jnk2

?

From Kuan et al.,

1999


22/57

3. The Gene Trap Strategy

Example: Apaf1

A t i i d d i A f1 / b


23/57

Apoptosis is reduced in Apaf1-/- embryos

The Apaf1-/- phenotype


24/57

The Apaf1 / phenotype

Onset Organ Description

e11.5 CNS Open brain and/or spina bifida

e13.5 CNS Forebrain overgrowth

e14.5 Eye Retina overgrowth, lens reduction, lens

mispolarization, accumulation of hyaloidendothelial cells

Lack of fusion of palatal shelvesPalate

Skull Absence of skull vault, absence of the

basisphenoid ossification centre

Limb Persistence of interdigital webs

Brain Differentiation into 2 layers of thechoroid plexus,expansion of the neural progenitor cellspopulation,thickening of the hindbrain

Inner ear Malformation of the otic vesicle

Th A f1 / h t


25/57

The Apaf1-/- phenotype

PCD i di l li b d l


26/57

PCD in distal limb development


27/57

Lack of apoptosis physiologiacl Apoptosis

PCD in interdigital webs


28/57

Apaf1-/-

wt

Palate formation


29/57

Retina hyperplasyin Apaf1 -/- embryos


30/57


31/57


The apoptosome

4 The Conditional knockout technology


32/57

4. The Conditional knockout technologyExample: caspase-8 and Lck-Cre

Ph t i th T


33/57

Phenotype in the Tcell lineage:

1. Marked increase in

the number ofperipheral T cell

2. Impaired T cellresponse

Caspase-8 isrequired for

CD95/Fas but notmitochondrial-mediated apoptosisin the T-cell lineage


34/57

Caspase-8 isrequired for T cells

homeostasis

5 Fine tuning of phenotypes: the knockout knockin technology


35/57

Specific knockout-

knockinof Cytochrome cKAform leads to

Specific inhibition ofapoptosome formation inKA/KA mice

5.Fine tuning of phenotypes: the knockout-knockin technology

All l f C h b t i bili bi d A f1


36/57

All roles of Cytochrome c but its capability to bind Apaf1

are preserved

The phenotype of Apaf1 knockout is surprisingly more severe


37/57

The phenotype of Apaf1 knockout is surprisingly more severe

than in KA/KA mice

Additional

roles for

Apaf1 ?


38/57

6. The conditional (over)expression strategy. IExample: Apaf1

*

This mouse expresses Cre Recombinasejust in the developing cortex

cortical

promoter

Cre

Recombinase

pA

This mouse expresses GFP in all tissue

CMV prom.

-act ehn.

loxP

siteGFP pA Apaf1 cDNA IRES LacZ pA

loxP

site


39/57

6. The conditional (over)expression strategy.IIExample: Apaf1

Apaf1Apaf1 ++

This mouse (over)xpresses Apaf-1 and --galgal in the cortex, and GFP in the others tissues

In the cortex, LacZ translation is not dependent from Apaf1 translation because of the

IRES box

Apaf1 cDNA IRES LacZ pA

--galgal

loxP GFP pA loxP Apaf1 cDNA IRES LacZ pA


40/57

REDUNDANCY IN CELL DEATH

1. Among members of the same gene family

2. Among cell death alternative pathways


41/57


42/57


43/57

By Knudson


44/57

By Knudson


45/57

By Knudson


46/57

Apaf1 mutantIW undergo celldeath by a

nonapoptoticpathway


47/57

The cellulardeathmorphotype

mimicksnecrosis

The apoptotic


48/57


The apoptosome

AIF DAPI


49/57

AUTOPHAGY: complex catabolic


50/57

Widely used autophagy pathways

during development: lessons fromyeast to nematodes

A role for autophagy in

stress-induced development

AUTOPHAGY: complex catabolicprogram for lysosomaldegradation of proteins and other

subcellular constituents

CELLDEATH

Beclin-1 (Atg6 homolog) mutant embryos show early


51/57

Beclin 1 (Atg6 homolog) mutant embryos show earlylethality

Bak-/-/Bax-/- dKO cells undergo anyway cell death by


52/57

Bak /Bax dKO cells undergo anyway cell death byautophagy


53/57

1. The fog(Apaf1 hypomorph) mouse exhibits anabnormal brain architecture

2. The main features of the phenotype are:extraventricle, inflammation (gliosis), abnormal

cortical layering, cortical hyperproliferation, enlargedchoroid plexus

3. The fogbrains show fried-eggcells, a typicalhallmark of oligodendrogliomas AND neurocytomas:

autophagy



54/57

1. Transgenic technology

2. Knockout technology

4. Conditional knockout technology

6. Conditional transgenesis technology

3. Gene trap


5. Knockout-knockin technology


55/57

The Gene Disrupters:

R.A. Flavell, T.W. Mak,

R. Hakem, P. Gruss,A. Strasser, J. Penninger,

K.A. Roth, C.B. Thompson,T. Knudson, N. Heintz,

many others and myself


56/57

studying a death pathway

In knockout orTransgenic mice(in development)

In cellular systems ofembryonic origin

In surviving individuals

Laboratory of Molecular Embryology Dulbecco Telethon Institute

D f Bi l U i i f T V R I l


57/57

Department of Biology, University of Tor Vergata, Rome, Italy

Elisabetta Ferraro

Marco CorvaroDaniela De Zio

Luigi Giunta

Giovanni Marchetti

Daniele Soroldoni

Maria TrignettiEmanuele Zaina

Elisa Tino

Francesca Fausti

University of Roma TreSandra Moreno

CollaboratorsKevin A. Roth, Marjo Salminen, Mauro Piacentini,

Date post:	06-Apr-2018
Category:	Documents
Upload:	filipa-branco-filipe
View:	222 times
Download:	0 times

apoptose cerebro

Documents