Neurotrophic Factors

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Neurotrophic Factors Programmed Cell Death
November 2, 2007 Scott Zeitlin
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The neurotrophic hypothesis
Targets of innervation secrete limiting amounts
of survival factors to generate a balance
between the size of the target organ and the
number of innervating neurons.
Note This hypothesis is mainly concerned with
peripheral neurons and peripheral target tissues.
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Effect of Removing or Augmenting Neural Targets
on the Survival of Related Neurons

• PN23091.JPG

1934 Victor Hamburger discovered that removal of
a limb bud resulted in reduced numbers of sensory
and motor neurons in the spinal cord.
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Effect of Removing or Augmenting Neural Targets
on the Survival of Related Neurons

• PN23092.JPG

1939 Victor Hamburger showed that
transplantation of a supernumerary limb resulted
in increased numbers of sensory and motor neurons
in the spinal cord.
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Based on his limb-bud experiments, V. Hamburger
hypothesized that the targets of innervating
neurons provide signals that recruit
undifferentiated cells to develop into sensory or
motor neurons.
(he was wrong)
In 1942, Levi-Montalcini and Levi proposed that
target derived signals maintain survival of
differentiating neurons. In 1949, Hamburger and
Levi-Montalcini repeated the limb bud
experiments and found that their results
supported the neurotrophic hypothesis. Hamburger,
V. and Levi-Montalcini, R. (1949) J. Exp. Zool.
111 457-502.
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1954 neurite outgrowth assay
1960 NGF purified
1969 NGF purified to homogeneity
extract
Stanley Cohen
Rita Levi-Montalcini
1986 Levi-Montalcini and Cohen split the Nobel
prize for Physiology or Medicine for their
discovery of growth factors
extract
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NGF sympathetic neurons and some sensory
neurons
(most CNS neurons do not require NGF for survival)
BDNF NGF-related factor purified in 1982 from
pig brain (shares 50 homolog with NGF)
NT-3 and NT-4/5 were obtained by PCR cloning
All these factors are synthesized as 250 aa
precursors that are processed into 120 aa proteins
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Neurotrophin Evolution
Neurotrophins have only been isolated from
chordates
Hallbook (1999) Curr Opin Neurobiol 9 616-21
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Structure of NGF bound to its receptor
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The Trk Family of Receptor Tyrosine Kinases for
the Neurotrophins

• PN23160.JPG

p75NTR purified and cloned 1st, homology to TNFR
Trk tropomyosin-related kinase, originally known
as orphan receptors
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Alternative splicing generates many Trk receptor
isoforms
NGF KD for TrkA10-11M
Roux and Barker (2002) Prog Neurobiol 67203-233
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p75NTR structure
NGF KD10-9M (all neurotrophins can bind p75NTR)
Roux and Barker (2002) Prog Neurobiol 67203-233
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Models for Trk and p75NTR interaction
Chao and Bothwell (2002) Neuron 339-12
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p75NTR is required for developmental myelination
Cosgaya et al. (2002) Science 2981245-1248
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(Found only in fish)
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The effect of NT/NTR knockouts on neurons in the
DRG
Fariñas et al. (2002) Brain Res Bull 57809-816
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Trk receptor signaling
When a neurotrophin binds to a trk receptor, the
kinase domain is activated resulting in
autophosphorylation.
Autophoshorylation results in further activation
of the kinase domain, leading to activation of
three potential signaling cascades
MAPK
PI3K
PLC-g
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Our axons can be gt1 m in length---how does the
neurotrophin/receptor complex signal to the
neuronal cell body?
Miller and Kaplan (2001) Neuron 32767-770
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Campenot, RB (1977) Local control of neurite
development by nerve growth factor. Proc Natl
Acad Sci U S A. 74(10)4516-9.
(A method that can be used to study how NTs added
to distal axons signal retrogradely)
NGF
K252a
Miller and Kaplan (2001) Neuron 32767-770
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Activated Trk can signal locally and retrogradely
using different signalling pathways
Slow (2-20 mm/hr)
Miller and Kaplan (2001) Neuron 32767-770
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Differential control of TrkA trafficking and
signaling may also be the basis for the different
functions of NGF and NT-3
Kuruvilla et al. (2004) Cell 118 243-255
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In vitro assays have shown that NTs enhance both
axonal and dendritic growth In vivo, the
situation is more difficult to study
Why? In standard knockouts, it is difficult to
separate the survival effects of NTs from their
effects on the morphology of neurons. This
problem has begun to be addressed by using
conditional knockouts, or by crossing NT
knockouts with mouse mutants lacking
pro-apoptotic genes. Recent evidence from these
kinds of experiments suggests that long distance
peripheral sensory axon growth in vivo is
NT-dependent.
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NTs roles in neuronal development and function

• NTs are expressed in regions of the developing
  embryo that are traversed by sensory axons en
  route to their targets.
• NTs affect the proliferation and
  differentiation of CNS neuroepithelial
  progenitors, neural crest cells, and progenitors
  of enteric neurons in vitro (and in some cases
  also confirmed in vivo).
• In the CNS, BDNF/TrkB signaling is implicated in
  the development and maintenance of cortical
  circuits.

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NTs in the CNS
The highest levels of NTs are found in the
hippocampus
Lindsay et al. (1995) Trends Neurosci 17182-190.
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Trophic support for peripheral and central
nervous system neurons
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• BDNF can be secreted by the presynaptic neuron in
  an activity-dependent fashion
• Indirect Evidence
• BDNF is found in synaptic vesicle preparations
• TrkB receptors are found in dendrites
• Axotomy of axons from BDNF-expressing neurons
  results in a depletion of BDNF in their target
  area
• Direct Evidence
• Kohara, K. et al. (2001) Activity-dependent
  transfer of brain-derived neurotrophic factor to
  postsynaptic neurons Science 291 2419-2423.

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Experimental Strategy employed by Kohara et al.
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Time-lapse fluorescent microscopy demonstrates
anterograde and retrograde transport of GFP-BDNF
Kohara et al. (2001) Science 291 2419-2423
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Transfer of GFP-BDNF from a presynaptic to
postsynaptic neuron
axon
DsRed
GFP-BDNF
DsRedGFP-BDNF
Kohara et al. (2001) Science 291 2419-2423
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Transfer of GFP-BDNF requires synaptic activity
Kohara et al. (2001) Science 291 2419-2423
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Recently, a similar technique was used to examine
the role of NTs in dendritic remodeling Horch
and Katz (2002) Nature Neuroscience 5 1177-1184
eGFP
Effect on dendrites?
RFP BNDF-myc
Visual cortex pyramidal neurons in a slice
culture
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Result local release of BDNF alters nearby
dendritic structure
Horch and Katz (2002) Nature Neuroscience 5
1177-1184
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Selection of CNS synapses via BDNF
Strong presynaptic activity results in release of
more BDNF. The postsynaptic site responds by
elevating the amount of AMPA receptors and nNOS.
This mechanism could contribute to selective
facilitation (e.g. maintenance of LTP).
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BDNF is also involved in regulating energy
balance/feeding behavior
BDNF/TrkB signaling is downstream of the
melanocortin-4 receptor
Xu et al. (2003) Nature Neuroscience 6 736-742
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Something to contemplate
In rodents, a diet that is high in saturated fat
and refined sugar results in reduced hippocampal
BDNF expression
Molteni et al. (2002) Neuroscience 112 803-814
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Cytokines and Growth Factors in the Nervous System
CNTF, LIF (oncostatin M, cardiotrophin-1)
neuropoietic cytokines. These factors may be
important in neuronal response to injury. GDNF,
neurturin, artemin, persephin exhibit distant
homology with the TGF-b family. They signal
through a receptor complex composed of the Ret
tyrosine kinase and a GPI-linked binding subunit
(GFRa family GFRa1, a2, a3, and a4). These
factors are potent axon-promoting growth factors
in vivo for developing sympathetic and
parasympathetic neurons.
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Neurotrophins can protect striatal neurons from
excitotoxic injury
Albrech et al. (2002) Brain Res Bull 57817-822.
In the striatum, TrkB receptors are the most
abundant, followed by TrkC
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What happens to neurons in the absence of
neurotrophic factors?
apoptosis Pronunciation "a-pp-'tO-ss,
-p-'tO- Function noun Inflected Form(s) plural
apoptoses /-"sEz /Etymology New Latin, from
Greek apoptOsis a falling off, from apopiptein to
fall off, from apo- piptein to fall --
more first use in Kerr et al. (1972)
Apoptosis a basic biological phenomenon with
wide-ranging implications in tissue kinetics.
Br. J. Cancer 26 239-257
Apoptosis is a mechanism contributing to
programmed cell death (PCD)
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Morphological types of cell death Apoptosis
originally defined according to a set of
characteristic ultrastructural features that
include nuclear and cytoplasmic condensation,
cell fragmentation and phagocytosis. Necrosis
cell death as the result of injury, disease, or
pathological state (usually involves large
numbers of cells and is associated with
inflammation). Chromatin condenses in multiple
small clumps and at later stages, cell membranes
and organelles disintegrate. Autophagy (from
the Greek, self-eating) Cytoplasm is destroyed by
lysosomal enzymes before any nuclear changes
become visible. A characteristic feature is the
appearance of large autophagic vacuoles in the
cytoplasm. At later stages, chromatin condenses,
DNA laddering is evident and phagocytosis occurs.
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Necrosis
PCD
apoptotic
autophagic
Morphology of cell death
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Ultrastructure of cell death
apoptosis
necrosis
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Apoptosis the movie
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Stages during neuronal development where PCD
occurs
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PCD also occurs in the glial lineage
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Regulation of PCD in mitotic neural progenitors
depends, in part, on asymmetric distribution of
PAR-4 and elevated ceramide
Ceramide (a lipid that can act as an
intracellular 2nd messenger)
Bieberich et al. (2003) J. Cell Biol. 162
469-579.
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Genes involved in PCD are highly conserved
C. elegans
Mammalian homologs of the C. elegans PCD genes
Ced-3 caspases (cysteine aspartate proteases)
Ced-4 Apaf-1
Pro-death Bax, Bak Bim, Brd, Dp5/Hrk,
Bad Pro-life Bcl-2, Bcl-XL, IAPs
Ced-9 Bcl-2 family
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Death signaling pathways
Extrinsic
Intrinsic
FasL
Bim
Bcl-2
FasR
Bax
PTP
FADD
Bid
Cyt C
pro- casp9
APAF
pro-casp8
casp8
casp9
Apoptosome
APOPTOSIS
casp3
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Intrinisic apoptotic pathway movie
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Discussion paper