IN HEALTH AND DISEASES

DR. HAMSA PUTHALAKATH

LA TROBE UNIVERSITY

Apoptosis and tissue homeostasis

Two distinct but converging pathways to cell death

Bim Bik

Bax/Bak Bcl-2

AdaptorsFADD

initiator

caspases effector

caspases

caspase-8

FasL

Fas

intracellular and other

death stimuli

APOPTOSIS

The mitochondrial pathway simplified!

BIM Bcl2

THE LIFE AND DEATH SWITCH

Bim

Bcl-2/Bim: a life-death switch

Apoptosis is a silent death!

Normal cell Apoptotic cell

PS (“eat me!” signal)

Measuring Apoptosis:

Apoptotic cell blebbing

Macrophages

(Scavenger cells)

Pla

sm

a m

em

bra

ne

Nucleus

Annexin V-FITC

Phosphatidylserine

Propidium Iodide

Live cell Early Apoptosis Late Apoptosis

Measuring Apoptosis:

BIM Bcl2

THE LIFE AND DEATH SWITCH

GENOME ORGANIZATION

Chromosome 14Chromosome 18

Lots and Lots

Of

Antibodies!!!

Bcl-2

Origin of B-Cell cancer

Bcl-2

Bcl-2 Bcl-2

Bcl-2

Bcl-2Bcl-2

- IL3

FDC-P1

FDC-P1 Bcl-2 FDC-P1

Electroporation

Blastocyst Homologous

recombination

Antibiotic selection

Gene knockout in mice:

Foster Mother

Chimeric mouse Normal mouse

Chimeric mouse Normal mouse

Heterozygous

Knockout mouseNormal mice

Gene knockout in mice:

c-myc!"bim chimeric mice!

Bim

“Blue” gene

Normal mouse “Blue” mouse

Gene Targeting

GENE TARGETING IN MICE

Bim

Bcl-2 Bim

Bcl-2

Life and Death in the balance!

Phenotype of bcl-2-/- mice

Veis et al Cell 75 229 1993Nakayama et al PNAS 91 3700 1994Kamada et al Cancer Res 55 354 1995

• Short ears

• Runting

• Polycystic kidneys

• Lymphopenia

• Greying (at the second hair follicle cycle)

• Early death (5-6 wks on C57BL/6 background)

Bim is critical for the development of our immune system

Bim!!

Cardiomyocytes’ apoptosis: Bim on the beat?

Bim in dilated cardiomyopathy?

!s " #

Adenylate

cyclase

ATP

cAMP

PKA

Bim

Cardiomyocyte

apoptosis

Catecholamines, autoantibodies etc

Cardiomyopathy

Heart failure

HYPOTHESIS:

#-AR

#2-AR transgenic hearts develop cardiomyopathy and display

increased apoptosis

WT WT WT

WT

Tg Tg Tg

Tg

#2ARTg,400X #2ARTg+bim peptide,400XWT,400X

WT

WT

Tg1

Tg2

Tg3 M

yoca

rdial

infa

rctio

n

BimEL

HSP70

Cont

Isop

rot

BimEL

HSP70

Bim is induced in #2-AR transgenic heart tissues

WT

bim-/-

Freshly Isolated 24 h in culture 24 h Iso+rolipram

bim deficiency protects cardiomyocytes in culture

Blocking Bim induction could protect against heart failure!

Bim mimetics in therapies

Bim

Bcl-2

Rheumatoid arthritis and various cancers

Bim mimetics in Cancer treatment:

Summary:

• Apoptosis or PCD is a process where cells are programmed to

die during development or to remove autoreactive, cancerous or

infected cells

• Proper apoptosis programme is necessary for cellular

homeostasis. Improper regulation of this process could lead to

various diseases such as autoimmune disorders,

neurodegenerative disorders, cancer, heart failure etc.

• Understanding the molecular mechanism(s) behind this process

could help in designing better therapeutic drug targets in treating

these disorders.