Target-derived neurotrophic factors ensure the correct number of neurons survive during development. Purified nerve growth factor promotes neuronal survival and prevents cell death. Major pathways of programmed cell death include the intrinsic mitochondrial pathway activated by loss of trophic factors, and the extrinsic pathway mediated by death receptors. Retrograde transport allows neurotrophins produced distant from the cell body to influence gene expression and survival.

1. Neuronal survival and
programmed cell death
Nitish kumar
M.Sc neurotechnology
JIPMER

2. CONTENTS
 Introduction
 Factors regulating neuronal survival
 NGF Signaling mechanisms and neurotrophic receptors
 Programmed cell death during neural development
 Summary
 References

3. Introduction
 Once a nerve fiber reaches its final target region, the target tissue plays an
important role in determining whether that neuron will survive
 Many target tissue (particularly those in PNS) secrete survival-promoting (trophic)
proteins that help regulate the final number of innervating neurons.
 The release of limited amounts of these proteins at the time of neural innervation
helps ensure that only the correct number of neurons survive in a particular
neuronal population .This is termed as neurotrophic hypothesis.

4.  The neurons that do not obtain the target-derived proteins die off, thus preventing
survival of any excess neurons.
 Another means by which neuronal numbers are regulated in the developing
nervous system is through programmed cell death (PCD).

5. Factors regulating neuronal survival
 From the late nineteenth through to the mid-twentieth century,
neuroembryologists debated what role, if any, target tissues served in regulating
aspects of neural development.
 By the 1950s, the view that target-derived growth factor proteins were important in
promoting neuronal survival and preventing neuronal death during embryonic
development was firmly established.

6.  Target tissues support the required number of innervating neurons.
 (A) The cells in a target tissue secrete limited amounts of survival-promoting
proteins at the time of neuronal innervation.
 (B) Those neurons that receive sufficient quantities of the target-derived protein
survive. The remaining neurons die due to lack of sufficient target derived
protein. This process helps ensure that only the required number of neurons
survive.

7.  Wilhelm Roux, writing in 1881, suggested that neuronal death was a normal part of
embryogenesis, Cajal and others felt that, even if neuronal death occurred for some
developing neurons, “the immense majority of the neuroblasts survive to term and
succeed in collaborating with the normal structures of the adult nervous system.”

8.  , Márian Shorey reported that removing the limb buds from chick or amphibian
embryos resulted in hypoplasia of the motor and dorsal root ganglion (DRG)
neurons innervating the limb bud.
 This hypoplasia resulted from a decrease in the size of the motor neuron pools and
dorsal root ganglia, as well as the individual neurons within these populations.
 Others noted that grafting extra limb tissue resulted in hyperplasia, an increase in
the size of the innervating neuronal populations and individual neurons.

9.  Target tissue size regulates the number of surviving neurons.

10.  When target tissues were removed, the nuclear material in many of the innervating
neurons became pyknotic (irreversibly condensed), indicating that the cells were
dying in the absence of target tissue.
 Using these salivary glands as raw material, by 1956 Stanley Cohen was able to
purify a protein that they called the nerve-growth-stimulating factor, now called
nerve growth factor (NGF).
 Like the tumor extracts and snake venom, purified NGF promoted the outgrowth
of dorsal root and sympathetic ganglia in vitro as well as in vivo
 The discovery of NGF was highlighted in 1986 when Rita Levi-Montalcini and
Stanley Cohen received the Nobel Prize in Physiology or Medicine for their work

11.  NGF was found to affect not only the neurons in dorsal root and sympathetic
ganglia, but also the neurons in most neural-crest-derived ganglia in the PNS and
the basal forebrain cholinergic neurons in the CNS.
 The entire family of NGF-related proteins is now known as the neurotrophins.

12.  In 1982, more than 25 years after the purification of NGF, Barde, Edgar, and
Thoenen reported the discovery of a new growth factor called brain-derived
neurotrophic factor (BDNF).

13.  A. BDNF promotes neurite outgrowth from a dorsal root ganglion explant.
 B. BDNF-induced outgrowth is not inhibited when antibodies specific to NGF are added, indicating that the neurite-
outgrowth-promoting properties of BDNF are distinct from those of NGF.
 C. BDNF also promotes outgrowth of nodose ganglia, a neuronal population that is unresponsive to NGF
 D. nodose ganglia unresponsive to NGF.

14. NGF Signaling mechanisms and
neurotrophic receptors
 Because neuronal cell bodies are often located at a distance from target tissues, the
mechanism by which growth factors reach the cell body to promote cell survival
was initially unclear.
 For many years, scientists suspected that the nerve fibers must express receptors
that take up the growth factor and transport it to the cell body.

15. NGF undergoes retrograde transport from
nerve terminal to cell body
 Proteins similar in size to NGF were not transported to the cell bodies, nor were NGF proteins
whose structure was modified, indicating that the observed retrograde transport was specific for
NGF

16.  When radiolabeled NGF was injected in the region of nerve terminals, scientists
observed that the NGF was transported back to the cell body (arrows).
 It demonstrated that NGF did not need to be expressed near the cell body to exert
its effects.
 Instead, NGF produced at sites distant from the cell body could be carried back to
the cell body via retrograde transport to influence signal transduction pathways
and gene expression

17.  (A) The p75 neurotrophin receptor, initially called the low-affinity NGF receptor (LNGFR), was the
first receptor identified that could bind to NGF. It was later found that NGF, BDNF, NT-3, and NT-
4/5 all bound to this receptor with equal, low affinity. Thus, the name was changed to the p75
neurotrophin receptor (p75NTR).
 (B) Each neurotrophin binds to a specific Trk receptor with high affinity (solid arrows). NGF binds
with high affinity to TrkA, the first Trk receptor discovered. BDNF and NT-4/5 bind with high affinity
to TrkB, while NT-3 binds to TrkC with high affinity. When NT-3 is present at sufficiently high
concentrations, it can also bind TrkA and TrkB (dashed arrows).

18.  Trk A is the high-affinity NGF receptor that is expressed on restricted neuronal
populations as well as on PC12 cells.
 The tropomyosin sequence was located in place of the extracellular binding
domain and this finding led to the name of the receptor—the tropomyosin-
receptor kinase (Trk).
 Other members of Trk family were subsequently discovered and had different
binding specificities were observed for each neurotrophin as shown in above
diagram.

19.  Neurotrophins exist as dimers that bind to Trk monomer subunits. The binding of the neurotrophin
ligand brings the receptor subunits into close contact, so they cross-phosphorylate each other’s
intracellular kinase domains, thus initiating signal transduction cascades in the receptor-bearing
cell.

20.  When a neurotrophin binds to its corresponding Trk receptor, the ligand–receptor complex is internalized
and transported along the axon to the cell body in a transport vesicle called an endosome.
 The ligand remains inside the endosome, while the receptor kinase domains extend outward from the
vesicle surface.
 Because the ligand is still bound to the receptor, the receptor remains active during transport and
recruits additional signaling molecules to the endosome surface.
 Once the endosome complex reaches the cell body, it is able to initiate signal transduction cascades
associated with cell

21. Programmed cell death during neural
development
 PCD is defined as a loss of a large number of cells in a distinct spatial and temporal
pattern within a given species. It refers to the loss of neurons that occurs normally
during neural development.
 PCD is not a result of an abnormality in the cell itself or from extrinsic damage that
leads to cellular injury and necrotic cell death. Instead, PCD is an active cellular
process that is regulated by intracellular signaling pathways activated by either
external signals or intrinsic genetic mechanisms.

23. Difference between necrotic cell death and
apoptotic cell death.
 When a normal cell dies, the stages of cell death differ depending on whether the cell
undergoes necrosis or apoptosis.
 At the onset of necrotic death, the cell begins to swell, and vacuoles form in the
cytoplasm and organelles.
 The cell continues to enlarge as the nuclei and organelles disintegrate, until the cell
membrane ultimately ruptures. The release of cell contents provokes an inflammatory
response.
 As the cell initiates apoptotic death, its cytoplasm and nuclear chromatin begin to
condense.
 At first, the organelles and cell membrane remain intact, even as the cell body and
nucleus begin to shrink. However, the cell surface soon becomes convoluted, and the
nucleus and organelles break off into discrete, membranebound fragments called
apoptotic bodies. These apoptotic bodies are degraded and ultimately phagocytized by
nearby macrophages or glial cells

24.  . Separation of DNA by gel electrophoresis distinguishes apoptotic and non-
apoptotic cell death. During apoptosis, DNA is broken into fragments of 180 base
pairs (bp) or multiples of 180 bp.
 When cell lysates from apoptotic cells are run on an agarose gel, DNA fragments
are separated by size, forming what looks like a ladder. Lysates from non-apoptotic
cells do not form such a pattern

25.  Cell death pathways are shared across species. The cells that are removed by PCD
are always the same cells, and death always occurs at specific times in
development.
 The first genes found to regulate cell death in C. elegans are known as ced (cell
death abnormality) genes.
 The mammalian homolog of ced-4 is Apaf-1 (apoptotic protease activating factor-
1). Apaf-1 and other proteins form a complex called an apoptosome, a critical
component of the cell death pathway.

26. Pathways
1. The mitochondrial pathway or the intrinsic pathway. This pathway can be
activated by cellular stress, DNA damage, or the loss of trophic factor, such as
that observed following the withdrawal of NGF from sympathetic neurons in vitro.
2. The removal of growth factor also leads to the activation of BH3-only proteins
such as Bim and Puma. These pro-apoptotic proteins either activate Bax directly
or inhibit the pro-survival functions of Bcl-2 and Bcl-xL
3. Trophic factors can increase neuronal survival by inhibiting the apoptotic pathway
at various points.

28.  (1) Activation of the pro-apoptotic protein Bax leads to the release of cytochrome c
from mitochondria. Cytochrome c then interacts with Apaf-1, which in the presence of
ATP undergoes a conformational change that allows it to interact with pro-caspase-9, a
pro-apoptotic enzyme.
 Apaf-1, cytochrome c, and pro-caspase-9 form a structure called an apoptosome. Pro-
caspase-9 is cleaved within the apoptosome, and the resulting caspase-9 cleaves pro-
caspase-3. The activated caspase-3 then causes cell death.
 (2) However, if trophic factors are present, they can activate the antiapoptotic protein
Bcl-2, which in turn inhibits the activity of Bax, thereby preventing the release of
cytochrome c from mitochondria and blocking the apoptotic pathway.
 (3) Other proapoptotic proteins such as Bim and Puma are activated upon removal of
growth factors and can promote apoptosis either by activating Bax or inhibiting Bcl-2.

29.  The final element of cell death pathways to consider is the activation of an
extrinsic pathway through “death receptors.”
 Scientists discovered that p75NTR is related to the tumor necrosis family (TNF) of
receptors, a group of receptors that are often called death receptors.
 Adaptor proteins are then recruited to the intracellular portion of the receptor
complex to initiate the signaling cascades that lead to cell death. The exact
mechanism by which this occurs is still under investigation, but in many instances
the c-jun N-terminal kinase (JNK) pathway is involved

31.  When an unprocessed pro-neurotrophin molecule binds to the extracellular region
of a p75NTR–sortilin complex, adaptor proteins are recruited to the intracellular,
non-kinase region of p75NTR that initiate signaling cascades such as the c-jun N-
terminal kinase (JNK) pathway.
 JNK activation can induce cell death via multiple mechanisms. JNK can activate the
transcription factor c-jun, which then translocates to the nucleus (dashed arrow) to
activate proapoptotic genes.
 The JNK pathway can also activate p53, which in turn enhances the activity of Bax.
The pro-apoptotic Bax causes cytochrome c to be released from the mitochondria,
thus allowing it to form an apoptosome and activate the caspase cascade (double
arrows) that leads to cell death.

32. Other types of cell death
mechanisms(induced)
 1. NECROSIS: Degradative changes to cells and tissues as a result of pathology.

33. Types of necrosis:
Necroptosi
s
Necrotic cell death dependent on kinase activity of receptor interacting
(RIP1), RIP3 and their expression
Parthanato
s
A type of regulated necrosis that is dependent on the activity of poly(ADP-Ribose)
polymerase
Ferroptosis An iron dependent form of regulated necrosis that may also be triggered by
exitotoxic stress.
Cell death by
mitochondrial
permeability
transition
Mpt defines a large increase in the permeability of inner mitochondrial membrane
in response to elevated concentration of calcium, usually resulting in uncoupling in
oxidative phosphorylation, cellular energy depletion and necrotic cell death
Lysosomal cell
death
(autolysis)
Mediated by release of lysozyme(protease) in the cytosol.
Pyroptosis Type of regulated necrosis mediated by caspase 1

36. Summary
 The release of limited amounts of these proteins at the time of neural innervation
helps ensure that only the correct number of neurons survive in a particular
neuronal population .This is termed as neurotrophic hypothesis.
 even if neuronal death occurred for some developing neurons, “the immense
majority of the neuroblasts survive to term and succeed in collaborating with the
normal structures of the adult nervous system.
 Purified NGF promoted the outgrowth of dorsal root and sympathetic ganglia in
vitro as well as in vivo

37. Summary
 The entire family of NGF-related proteins is now known as the neurotrophins.
 NGF produced at sites distant from the cell body could be carried back to the cell
body via retrograde transport to influence signal transduction pathways and gene
expression
 Major pathway for programmed cell death includes:-
 1. Intrinsic pathway: activated by cellular stress, DNA damage, or the loss of
trophic factor, such as that observed following the withdrawal of NGF from
sympathetic neurons in vitro
 2. Extrinsic pathway: mediated through TNF Receptors or death receptors.

38. References
 Developmental Neurobiology , Lynne M. Bianchi
 NEURONAL CELL DEATH Michael Fricker et al, Brown Hunter Medical Research
Institute, University of Newcastle, Callaghan, New South Wales, Australia. Physiol
Rev 98: 813–880, 2018