NRSC 500

Shernaz Bamji

Cell Death in Neural Development and

Neurotrophic Factors

I. What is programmed cell death?

II. Mechanisms of cell death

III. The role of cell death in neural development

IV. Role of neurotrophic factors in cell survival and death

BREAK

V. Neurotrophic factor signalling: survival & death pathways

VI. Propagation of neurotrophin signalling (?)

VII. Regulation of neurotrophin secretion and expression (?)

OUTLINE

Programmed cell death (PCD):Death of a cell mediated by an intracellular program – “death by design”

Homeostatic cell death: - occurs throughout all tissues and organs in our body- cells have a finite life – die by PCD – and are replaced by new cells following cell division and differentiation of stem cells

- does not occur in the nervous system

Developmental cell death:First categorized by Ernst (1926) and Glücksmann (1930, 1951) 3 major categories of PCD that subserve distinct functions:

1) morphogenetic (eg. creation of digits by interdigital PCD)2) phylogenetic (eg. the loss of vestigal structures such as the tail)3) histogenetic (eg. PCD during cell development - most neuronal PCD)

I. What is programmed cell death? II. Mechanisms of cell death“Death has a thousand doors to let out life” (poet – Philip Massinger 1624)

Cell shrinks, chromatin condenses(called PYKNOSIS)

“budding”(membrane remains intact)

Inflammation followingRelease of intracellular content

PATHOLOGICAL DEATHCauses include:prolonged exposure to injury, infection, cancer, infarction, poisons, and inflammation.

PHYSIOLOGICAL DEATHProgrammed cell death

Necrosis Apoptosis

Phagocytosis by macrophages ornearby cells

kinder to surrounding cells than necrosis

Mitochondrialswelling, energyfailure, cellblebbing andbursting

Apoptosis in neutrophils:Nissl stain showing pyknotic nuclei (chromatin condensation)

TUNEL Assay: widely used as an assay for apoptosis

Terminal desoxynucleotidylTransferasedUTPNickEndLabelling

The assay relies on the presence of nicks in the DNA(a hallmark of APOPTOSIS) which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker.

III. The role of cell death in neural development

Developmental Cell Death in Invertebrates

I. C. elegans:Individual cells undergo a highly stereotyped sequence of divisions that

determines the number and type of progeny

959 cells total302 neurons in nervous system

2002 Nobel Prize in MedicineSydney Brenner, Robert Horvitz and John Sulston“Cell lineage and PCD in C. elegans”

There are certain fixed “rules” about which cells will die in a lineage (eg. the posterior daughter of of an anterior-posterior cell division is more likely to die)

A neuron requires trophic support from several different sources during its lifetime

In vertebrates, death is not a result of lineage, but a result of competition between nerve cells for trophic factors

Developmental Cell Death in Vertebrates

If the trophic support is insufficient, the neuron will probably die

Developmental cell death occurs in many neuronal systems

Dorsal Root Ganglion (DRG)

Victor Hamburger Rita Levi-Moltacini

A. Removal of a limb bud (target) from chick embryos resulted in a decrease in thenumber of motor neurons and sensory ganglia neurons innervating the limb bud.

Cross-section of the lumbar spinal cord from a 9-day chick embryo 2.5 days after leg primordium was removed. Note: lateral motor column is virtually absent.

9-day chick embryo

Evidence for Competition in Naturally-Occurring Cell Death

I. There is a direct correlation between the number of cells in a nerve centre (eg. ganglia) and the size of the target field

B. Implantation of an extra limb bud in early chick or amphibian embryos resulted in an increase in the number of motor neurons and sensory ganglia neurons innervating the limb bud.

(Victor Hamburger and Rita Levi-Moltacini – 1940s)

Extra legNormal

9-day chick embryo

How do you know it is more death and not less birth?

Showed a relationship between the size of the target and the number of neurons in the innervating population.

Till 1950s many believed that the missing field failed to induce the differentiation or proliferation of the population of innervating neurons.

Assay for cell proliferation

There was an increase in the number of pyknotic cells few days later, but NO change in the amount of tritiated thymadine incorporated into neurons.

Count # cells in DRG at beginning of experiment

Unilateral limb bud removal

Add tritiated thymidine to determine number of dividing cells

In each DRG, a defined number of neurons are bornA period of cell death lasts from E4.5 – E7DRGs that innervate limbs lose less neurons

DRGs for axial muscles lose more neurons

C. Developmental profile of cell numbers in ganglia

1. There is a direct correlation between the number of cells in a nerve centre (eg. ganglia) and the size of the target field.

a) Remove target

b) Add more target

c) Observation – more cells die if innervating a smaller target during development

Evidence for Competition During Nervous System Development

(Due to changes in survival not proilferation)

D ________________________________________________________________________________________________________________________________(Pilar, Landmesser, Burstein J. Neurophys.1980).

Eg. ciliary ganglion neurons of the chick reach their target (the eye) through 3 postganglionic nerves. Cut two nerves lateral to the main blood vessel. Counted the number of cells projecting out of the remaining branch that survived the cell death period (labeled using HRP). Compared to number of surviving cells projecting out the same branch in control ganglia.

II. There is a correlation between the onset of cell death and the timing ofinnervation (ie the timing of cell death is usually quite sharp and occurs atapproximately the same time that axons reach their targets).

Target-dependent cell death is observed in a variety of systems

Extent of cell death is 30-75%

Hamburger, J. Comp. Neurol. 1975

Landmesser and Pilar. J. Phys. 1974

Period of innervation

IV. Role of neurotrophic factors in cell death

The Discovery of Nerve Growth FactorE. Bueker (student of V. Hamburger)

• tested whether implanting neoplastic tissues (tumors) would be an easierway to enhance peripheral nerve survival than implantation of a supernumary limb.

• mouse sarcoma was only one of the 3 they tested that grew

• sensory and sympathetic ganglia in the vicinity of sarcoma implants were enlarged. More growth towards tumour.

♥ this effect was also seen when the tumor was placed on the chorioallantoicmembrane instead of in the embryo proper so concluded that the factor was secreted.

This started the search for the identification of what Montalcini and Hamburger started calling the “nerve growth factor”.

• Levi-Montalcini and Hamburger confirmed what Bueker saw and extended itby showing that this effect was dependent on distance of the tumor fromganglia

Sympathetic chain

Co-culture of mouse sarcoma (not shown) with chick sensory ganglion explant

chick sensory ganglion explant

Tumour extract has similar effects

THE Bioassay…..

• Cohen and Levi-Montalcini wanted to identify the active agent in the tumor extract

Serendipity plays a large role in scientific discovery….

• Knew active fractions contained both nucleic acid and protein

• Used snake venom to digest nucleic acid (source of phosphodiesterase)

Snake venom “control” showed a similar degree of nerve growth stimulation!!

• Looked at mammalian analogue of the snake venom gland – the salivary gland

Salivary gland of male mice makes very large amounts of NGF. Approximately 900 pairs of submaxillary glands from male mice needed to get a pure preparation.

The protein product of NGF includes a signal sequence and a prodomain, followed by the mature neurotrophin sequence (proNGF). The protein must be processed by proteolysis to form a mature protein.

The active neurotrophin consists of 2 identical monomers that form a non-covalently bound dimer with a molecular weight of 25 kD.

NGF

What is Nerve Growth Factor?

1)

2)

3)

4)

5)

The Neurotrophic Factor Hypothesis

Anti-body made in 1960; crystallized in 1971; cloned in 1983

Five arguments that neurons compete for target-derived NGF to survive:

An antibody to NGF applied to the chorioallantois leads to death of sympathetic neurons (Cohen and Levi Montalcini,1960; later repeated in mice; 1972)

Antibody treated

control

�

Antibody treatedcontrol

Sympathetic nerve chain Sympathetic gangliaOnly a subset of neurons are responsive to NGF (eg. neural crest-derived cells & cholinergic neurons of the CNS), which pointed to the existence of other related molecules. Identified a whole family of neurotrophins >>>> Nerve Growth Factor Family of Neurotrophins

Peptide Sequence:Early 1970sCloned in 1983

NGF NT-6

Neurotrophin 6only in teleosts

BDNF

Brain-derivedneurotrophicfactor:Cloned in198950% homology to NGF

Neurotrophin 3Cloned in 19917 groups!!

NT-3 NT-4/5

Neurotrophin 4/5

V. Neurotrophic factor signalling : Survival

& death pathways

How Does NGF Achieve its Effects?

Nerve growth factor (NGF) interacts with two different receptors – the TrkA

receptor tyrosine kinase (binds to NGF with high affinity), and p75NTR (binds to NGF

with low affinity).

NGFdimers

P75 NTR

cysteine rich repeats

Intracellular signalingdomain

PY490

PY674PY675

PY785

TK TK

NGF NGF

TrkA receptor tyrosine kinase

Tyrosinekinase domain

Subsequently, a whole family of Trk receptors were identified that bind to the neurotrophins

Pro-BDNFPro-NT-3Pro-NT4/5

NGF binding to p75NTR not essential for survival.

(For some time the role of p75 remained elusive – now known that p75NTR is a death receptor)

The TrkA receptor tyrosine kinase was shown to be the principle receptor for NGF, and responsible for NGF-mediated survival

PC12 is a cancer cell line derived from a pheochromocytoma of the rat adrenal medulla. PC12 cells stop dividing and terminally differentiate (with a neuronal phenotype) when treated with NGF. Needs NGF for its continued survival.

Early work on NGF-mediated cell survival done using PC12 cell model

SHC SHC

TrkA

PY490

PY674PY675

PY785

Y490

Y674Y675

Y785

P

PP

P

TK TKGRB

SOSRas

MEK

MAPK

Raf

RhoA

FRS2

Growth promoting genesPro-survival genes

PI3K

AKT

PLCγ

DAG IP3

PKCCa2+

Synaptic plasticity/Neurite

outgrowth(local)

NGF NGF

CREB P

Delineation of downstream effectors of NGF stimulation

ERK Pathway first identified as a major pathway in PC12 cell survival

-Neuronal Death (after NGF withdrawal) requires de novo protein synthesis

-Deprive sympathetic neurons of NGF > death within 2 days

-Add cyclohexamide > death response reduced

Neuronal Death is an Active process (not just failure to survive)

Observed in vitro…..

Death Survival

And in vivo:-Identify peak time of naturally occurring cell death

-Block translation

-Block transcription

-reduce # “pyknotic” neurons

N.B. This is an activedeath by apoptosis

Death promoting genes

Rac/cdc42

Ask-1

JNKK

JNK

C-JunP

C-JunP

P75 isNOT a GEF

?

Kinases(add phosphate

group to substrates)

p75 NTR Most work done on p75 activation done using biologically “active”, 25kD NGF. BUT more recently, shown that p75 binds better to proNGFand that proNGF binding to p75NTR increases DEATH.

The prognosis for survival or death of a cell depends on the ratio of Trk to p75NTR receptor and the type of neurotrophin a cell is exposed to.

TrkA

NGF

TrkAp75NTR

BDNF

p75NTR

survivalpathway

deathpathway

deathpathway

CED Genes: The first pro-apoptotic genes identified

Monitor for cells that survive that should have died at defined stages

Identify genes mutated

CED = Cell Death Defective

CED-3, CED-4, CED-9: Have lead to a biochemical understanding of cell death mechanisms

A C. elegans mutagenesis screen for aberrant survival

From worm to mammal: common effectors

proapoptotic

antiapoptotic

More cells survive naturally-occuring cell death in the sympathetic ganglia of Bax-/- mice

More motor neurons survive axotomy-induced death in the facial nucleus of Bax-/- mice

control control

Bax-/- Bax-/-

untreated axotomy

2) The p75 receptorshares homologieswith the Fasreceptor

1) Caspases can be activated upstream of mitochondria (eg. activation of the Fasreceptor)

Anti-apoptotic death effector domain (ADED)-family proteins bind to and prevent activation of initiator DED-containing caspases

Inhibitors of apoptosis (IAP)