Bob Freeman
Dept. of Pharmacology & Physiology
Med Center, Rm 4-6718
Mechanisms of Programmed Cell Death
and Neurotrophin Signaling
Sources of neurotrophic support
The Neurotrophic Factor Hypothesis
neurotrophic factor
receptor
1.
Target tissues synthesize and release limited quantities of neurotrophic factors that
bind and activate specific receptors on the termini of innervating neurons.
2.
The neurotrophic factor and activated receptor are internalized and transported
retrogradely back to the cell body where they exert trophic effects, including cell
survival.
Nerve Growth Factor: the prototype target-derived
neurotrophic factor
Nobel Prize in Physiology
or Medicine - 1986
Stanley Cohen
Dorsal root ganglion
neurite
outgrowth
w/o NGF
+ NGF
control Ab
cell survival
anti-NGF Ab
anti-NGF Ab
control Ab
Sympathetic ganglia
Rita LeviMontalcini
Neurotrophic factor deprivation-induced cell death
occurs by apoptosis
Chromatin
Fragmented DNA
+ NGF
- NGF
TUNEL = terminal deoxynucleotidyl transferase incorporation of dUTP by nick end-labeling
Neurotrophic factor deprivation-induced death is a metabolically
active process that requires de novo gene expression
+ NGF
Withdraw NGF
+
Inhibitor of protein
or RNA synthesis
Martin et al., 1988
The genetics of programmed cell death was first described in the
nematode C. elegans
The genetics of programmed cell death was first
described in the nematode C. elegans
1090 cells
959 cells
generated during
development of
the hermaphrodite
remain in the adult
131 cells die
Phenotypes
Extra cell deaths
Mutagen
screen
progeny
or
Fewer cell deaths
Functional relationship of cell death genes in C. elegans
Three key cell death genes:
ced-9
ced-4
ced-3
Cell death
Tissue-specific
commitment
signals
ced-9
Gene
mutated
Effect of
mutation
Cell death
phenotype
ced-9
ced-9
ced-4
ced-4
ced-3
ced-3
ced-9 / ced-4
ced-4 / ced-3
lof
gof
lof
gof
lof
gof
lof / lof
gof / lof
extra deaths
no deaths
no deaths
extra deaths
no deaths
extra deaths
no deaths
no deaths
lof = loss of function
gof = gain of function
ced-4
ced-3
The Nobel Prize in Physiology or Medicine 2002
The Nobel Prize in Physiology or Medicine 2002
Cell death
Clearance and
degradation
Sydney
Brenner
Sydney
Brenner
H. Robert
Horvitz
H. Robert
Horvitz
John
E.
Sulston
John E.
Sulston
Cell death mechanisms have been conserved through evolution
BH3-only
BAX
Members of the
‘BCL2 family’ of
proteins
General Scheme for Apoptosis
Neurotrophic factor deprivation
Widespread
proteolysis
Growth factor/cytokine
deprivation
Activation of death receptors
Physiologic, toxic,
or pathologic cell
death stimulus
DNA fragmentation
BAX
+
-
Activation
of
caspases
Cell death
Dismantling of the
cytoskeleton
BCL2
Cytopathic viruses
Oxidative stress
Membrane blebbing
Radiation
Chemotherapy drugs
apoptotic
stimulus
signal
transduction
execution
destruction
Caspases are essential for normal brain development
wild type
caspase-9
knockout
Caspases: Pro-apoptotic cysteine proteases
Activation requires cleavage of inactive ‘procaspases’ into large and
small subunits that then self-assemble into active enzyme.
Procaspase
(inactive precursor)
Active caspase
sites of cleavage
catalytic
sites
‘pro’ domain
C. elegans
CED-3
large
small
Conversion of procaspases to active caspases
requires caspase activity
Initiator caspase
Effector caspase
Initiator activation
Inactive
monomer
Forced
dimerization and
self-cleavage
Apoptotic stimulus
Effector activation
Active initiator
caspase
Inactive effector
caspases
Active effector
caspase
The intrinsic apoptosis pathway: Cytoplasmic cytochrome c
stimulates the recruitment and activation of initiator caspases
NGF withdrawal
Mitochondrion
Mitochondrial
outer membrane
permeabilization
Cytochrome c
APAF-1
Procaspase-9
‘Apoptosome’
+
-
BCL2 family
proteins
BCL2 family proteins include pro-survival and pro-apoptotic members
Pro-survival
BCL2 family
BCL2,
BCLX,
BCLW,
MCL1
Pro-apoptosis
BAX family
BAX,
BAK, BOK
BH3-only
family
BID
BIM, BAD,
BMF, HRK
NOXA,
PUMA
Pro-apoptotic BCL2 family proteins promote mitochondrial outer
membrane permeabilization (MOMP) and release of cytochrome c
Mitochondria
outer membrane
IMS = Mitochondria intra-membrane space
Dual function of BH3-only proteins: (1) bind and inhibit pro-survival
BCL2 proteins; (2) bind and activate pro-apoptotic BCL2 proteins
(BAD, HRK, NOXA)
Sensitization
BCL2
etc.
BH3-only
(BIM, BID, PUMA)
Pro-survival
Activation
BAX,
BAK
Pro-apoptosis
Interactions between the different types of BCL2 family
proteins regulate the intrinsic cell death pathway
BH3-only
Neurotrophin
deprivation
cytochrome c
BH3-only
Neurotrophin
addition
Neurotrophins:
A family of neurotrophic factors characterized by
similar amino acid sequence and tertiary structure and derived from a
common ancestral gene.
NGF
BDNF
NT-3
NT-4
-
nerve growth factor
brain-derived neurotrophic factor
neurotrophin-3
neurotrophin-4
N-Glyc
SIGNAL
SEQUENCE
K/R C
18-24 AA
60-120 AA
proneurotrophin
neurotrophin
SS S
SS
C C C
CC
PRO-SEQUENCE
preproneurotrophin
S
MATURE PROTEIN
neurotrophin homodimer
~ 120 AA
50% AMINO ACID IDENTITY
pI 9-10
Neurotrophin Receptors
Two classes of neurotrophin receptors: low affinity (Kd = 10-9 M) & high affinity (Kd = 10-11 M)
TRK
Transmembrane tyrosine kinase receptor. Ligand binding stimulates the
intrinsic protein kinase activity of its intracellular domain.
Three TRK genes (TRKA, TRKB, TRKC) encode closely related receptors
that bind to distinct neurotrophins.
Required, but not sufficient, for high affinity binding of neurotrophins.
p75NTR
Short intracellular domain contains a protein interaction domain. Binding to
neurotrophin alters the conformation of the receptor in a way that creates new
binding sites for several cytoplasmic proteins.
Binds all mature neurotrophins with similar affinities. Binds to proneurotrophins with even greater affinity. Also binds several other proteins.
Activation can promote cell survival, growth cone repulsion, or even
apoptosis depending on the specific ligand and co-receptor present.
Required, but not sufficient, for high affinity binding.
Neurotrophin Specificity
NGF
BDNF,
NT4
NT3
NGF, BDNF
NT3, NT4
Tyrosine
kinase
domain
TRKA
TRKB
TRKC
p75NTR
High affinity
neurotrophin receptor
complex
Basic NGF
signaling
pathways
Synaptic
plasticity
Survival
Differentiation,
Neurite growth,
Plasticity
Survival
Apoptosis
Neurotrophins, acting through Trk/p75 receptors, inhibit cell
death via effects on BCL2 family proteins
caspase
activation and
cell death
cytochrome c
release
Mitochondrion
Neurotrophins
(1)
Increase the
expression of prosurvival BCL2
family genes
(2)
Bcl-2
Inhibit
transcription
of BH3-only
genes
(4)
Block translocation
of pro-apoptotic
BCL2 proteins to the
mitochondria
BH3
only
(3)
Promote
degradation
of BH3-only
proteins
NGF + TrkA/p75NTR
RAS
PI3K
active
P
BAD
BIM
BAX
BAD
BCL2
AKT
inactive
MAPK
BAX
active
P
P
BIM
P
BCL2
P
BIM
mitochondrion
FOXO
p53
JUN
NFκB
CREB
MYC
AAAAAA
AAAAAA
BH3-only mRNA
expression
AAAAAA
BCL2 mRNA expression