Neurotrophins

“Nature seems unaware of our intellectual

need for convenience and unity, and very
often takes delight in complication and
diversity.”
Santiago Ramón y Cajal, Nobel Prize
Lecture, 1906.

http://en.wikipedia.org/wiki/Santiago_Ram%C3%B3n_y_Cajal
 History: Ross G. Harrison
Early 1900s
• Harrison isolated neural tissues from
amphibian embryos and grew them in vitro.
He is credited with developing the method of
tissue culture.
• In 1910 Harrison observed processes
projecting from neural explants which were
grown in the presence of a preparation derived
from clotted lymph.
• Harrison thought that the projections (axons)
might be directed by a physical property of the
substrate or by electrical fields.
• Harrsion also showed that the amphibian
nervous system could innervate limbs and
organs grafted from other species.
http://books.nap.edu/html/biomems/rharrison.pdf
 Viktor Hamburger: 1930s and 1940s
• 1934: Hamburger developed the chick
embryo model for studying innervation of
peripheral tissues; this model came to
replace the amphibian model used by
Harrison and other investigators.
• He found that removal of the wing bud led
to hypoplasia of the innervating motor
and sensory neurons. Subsequent
studies revealed that the hypoplasia
resulted from death of differentiated
neurons, rather than a failure to recruit
neurons from a pool of precursor
neurons.
• Hamburger also used the chick embryo
model to study the innervation of tissue
grafts.

http://www.sdbonline.org/archive/SDBMembership/hamburger-obit.html
 Rita Levi-Montalcini
• She graduated summa cum laude from the University of
Turin Medical School in 1936, and then completed a
degree for specialization in neurology and psychiatry in
1940. Fascist laws prevented Italian Jews from practicing
medicine or working in universities at that time, so Levi-
Montalcini set up laboratory equipment in her bedroom to
continue her research on neurogenesis. When the family
was forced to leave Turin in 1941due to the heavy Allied
bombing of the city, Levi-Montalcini rebuilt her laboratory
in the family's country cottage. When the Germans
invaded Italy in the fall of 1943, the family moved to
Florence where they lived underground until the end of
the war. After Allied armies forced the Germans out of
Florence in August 1944, Levi-Montalcini worked as a
medical doctor in an Italian refugee camp, treating http://en.wikipedia.org/wiki/Rita_Levi-Montalcini
epidemics of infectious diseases and abdominal typhus.
• After the war, her family returned to Turin and Levi-
Montalcini resumed her position as an assistant at the
University of Turin Institute of Anatomy. Two articles that
Levi-Montalcini had published in foreign scientific journals
interested Viktor Hamburger, head of the Zoology
Department of Washington University in St. Louis. In
September 1947 Rita Levi-Montalcini accepted
Hamburger's invitation to collaborate with him as a
research associate. Though she initially planned to stay
at Washington University for less than one year, Levi-
Montalcini stayed for thirty years.
Text from: http://beckerexhibits.wustl.edu/mowihsp/bios/levi_montalcini.htm
http://www.sfn.org/skins/main/pdf/nq/summer_2010.pdf
 Levi-Montalcini and Hamburger:
1948; sarcoma 180
• 1948: Elmer D. Bueker, a former student
of Hamburger, reported that the chick
embryo nervous system could innervate a
grafted mouse tumor (“sarcoma 180”)
isolated from connective tissue. Bueker
observed that sensory nerve fibers from
the dorsal root ganglia adjacent to the
grafted tumor had innervated the tumor,
while the tumor was devoid of innervation
by motor neurons.
• Levi-Montalcini and Hamburger decided
to use this experimental system, rather
than studying grafted limbs and organs.

http://www.unm.edu/~neurohsc/history.htm
 Levi-Montalcini and Hamburger:
1948-1951
• They reproduced the studies of Bueker but also
noted that, in addition to sensory neurons,
sympathetic neurons innervated the tumor.
They noted that the sympathetic ganglia
innervating the tumor were greatly (~6X)
increased in size compared to control ganglia.
• They proposed that the sarcoma 180 tumor was
releasing a soluble, diffusible, growth-promoting
factor that altered the differentiative and growth
properties of the target cells.
 Levi-Montalcini and Hamburger:
1951-1952
• In order to test their proposal, they transplanted a mouse
sarcoma onto the chorioallantonic membrane (an
extraembryonic membrane) of the chick embryo to
prevent direct contact of the tumor and chick tissues.
The circulatory system connects the two tissues. It was
found that the extra-embryonic transplants elicited the
same effects as the intraembryonic grafts, supporting the
proposed diffusible nature of the nerve-growth promoting
agent being produced by the tumor.
• Their attempts to replicate their findings using dried
tumors and by injecting tumor extracts into the embryos
were unsuccessful.
• They reasoned that the use of cell culture techniques
would greatly facilitate the identification of the factor.
 Levi-Montalcini goes to Rio de Janeiro
• Dr. Levi-Montalcini developed a collaboration with
Professors Carlos Chagas and Hertha Meyer at the
University of Brasil in Rio de Janerio. She reports
boarding an airplane (presumably in St. Louis) “carrying
in my handbag two mice bearing transplants of mouse
sarcomas 180 and 37.” (Levi-Montalcini 1986 Nobel
lecture).
• “The tumor had given a first hint of its existence in St.
Louis, but it was in Rio de Janeiro that it revealed itself,
and it did so in a theatrical and grand way, as if spurred
by the bright atmosphere of that explosive and exuberant
manifestation of life that is the Carnival in Rio” [ref. 14 in
1986 Nobel lecture].
 “Halo” effect identified by
Rita Levi-Montalcini (1954)

• Sensory and sympathetic ganglia

were removed from chick embryos
and cultured in a semi-soft
medium in proximity to, but not in
contact with, fragments of mouse
sarcoma tumor 180 or 37. Within
24-hours, Levi-Montalcini and
colleagues noted a halo of nerve
fibers growing out from the
ganglia, with the highest density of
fibers being on the side facing the
tumor.

Figure 3. Eight-day-old sensory ganglia from chick embryos. (a) The ganglion, which faces a
fragment of chick embryonic tissue (ct), shows fibroblasts but few nerve fibers. (b) Ganglion cultured
in the presence of fragments of mouse sarcoma for 24 h. (c) Ganglion cultured in the presence of
fragments of mouse sarcoma for 48 h. In (b) and (c), the ganglia, facing fragments of sarcoma (s),
show the typical ‘halo’ effect elicited by the growth factor released from the sarcoma. In (c), note the
first evidence of a neurotropic effect of the growth factor. Reproduced, with permission, from Ref. [7].
© (1986) The Nobel Foundation.
Aloe 2004
 Effect of NGF

Figure 4. Photomicrographs of sensory ganglia removed from an eight-day-old

chick embryo and cultured for 24 h at 37 8C. Ganglia were cultured (a) in a medium
containing no nerve growth factor (NGF) and (b) in a medium containing 10 ngml
of NGF. Note that only the ganglion that was exposed to NGF displays a dense halo
of nerve fibers. Reprinted, with permission, from Ref. [14]. q (1964) American
Association for the Advancement of Science (http://www.sciencemag.org/).

Aloe 2004
 1954: Stanley Cohen
• Levi-Montalcini returned to St. Louis
(1952-1954?).
• Shortly before her return, Stanley
Cohen, a biochemist, had begun
working with Viktor Hamburger. Cohen
isolated a fraction from the sarcomas
that promoted neuronal growth in vitro.
Cohen, Hamburger, and Levi-Montalcini
proposed the name “Nerve Growth
Stimulating Factor”, which was later
shortened to Nerve Growth Factor
(NGF).
 1956-1964
• 1956-1960: Cohen purified and characterized NGF,
first from snake venom, then from mouse
submandibular salivary glands.
• 1960: Cohen, while purifying NGF from
submandibular glands, began to test for other growth-
promoting activities. He noted that, when fractions
that did not contain NGF-like activity, were injected
into newborn mice, there was a precocious opening of
the eyelids, an early eruption of the incisors, and a
stunting of growth.
• 1962: Cohen isolated the second factor and called it
“tooth-lid” factor
• 1964: Cohen renamed the second factor “epidermal
growth factor” (EGF), based on his observation that in
vitro it had a direct effect on epidermal cell growth.
Identification of other neurotrophins
(post 1964)
• 1982: brain-derived neurotrophic factor (BDNF) was
purified
• 1990: neurotrophin-3 (NT3) was identified by cloning
techniques
• 1991-1992: the gene for neurotrophin 4 (NT4) was
cloned from Xenopus laevis; subsequently, the
mammalian homolog of NT4 was cloned but was given
the name neurotrophin 5 (NT5) because its sequence is
more divergent from the Xenopus counterpart than are
the sequences of other neurotrophin homologs.
Generally, the term NT4/5 is used to refer to this gene
and its product.
 Summary

4 neurotrophin genes:
• NGF
• BDNF
• NT3
• NT4/5
 Neurotrophins are synthesized as
proneurotrophins

The protein product of each neurotrophin gene includes a signal sequence,

which is cleaved in the ER, a prodomain, and the mature neurotrophin
sequence.
Proneurotrophins are cleaved intracellularly by furin and other prohormone
convertases in the trans-Golgi network. Extracellular proBDNF is cleaved by
several matrix metalloproteinases (MMPs) and by plasmin following its
activation (cleavage) by tissue plasminogen activator (tPA).
 Pro- and mature neurotrophins
• Both pro- and mature forms of neurotrophins are
released by cells.
• Both processed (mature) and unprocessed
(proform) neurotrophin proteins form
homodimers in solution.
• NGF is primarily secreted in its mature form,
whereas BDNF is primarily secreted as
proBDNF.
• NGF is released through the constitutive
secretory pathway, while BDNF (proBDNF) is
predominantly secreted through the regulatory
(activity-dependent) pathway.
 Pro- and mature neurotrophins
• Pro-domains play a role in protein folding
and dimerization
• Pro domain appears to play a role in
apoptosis
• In general, the proneurotrophins and the
neurotrophins exert opposing biological
actions
 BDNF gene
• Each neurotrophin is translated from a single coding exon.
• In the case of BDNF, multiple mRNA transcripts are present within a cell.

From Aid et al. 2007 J Neurosci Res. 85:525-535

 Why so many transcripts?
• May allow for cell-type or subcellular
localization differences
• May (probably) have different mechanisms
for controlling expression from differing
promoters. Different signals may generate
different transcripts.
• Transcripts may have differing stability-
may be able to regulate duration of local
BDNF production
 Functions of proneurotrophins
• proNGF: apoptosis
• proBDNF: apoptosis; dendritic remodeling
• proBDNF: may regulate synaptic efficacy
during development
• proBDNF: induction of LTD
 Functions of neurotrophins

• Target-derived survival factor for developing

neurons (original identification)
• Growth cone guidance
• Synaptic modulation
• Roles in learning and memory
• Neurogenesis
 Neurotrophin-receptor interactions
Two types of neurotrophin
receptors have been
characterized:
1. Trk (tropomyosin receptor
kinase) family
2. p75NTR (p75 neurotrophin
receptor), which is a
member of the tumor
necrosis factor receptor
(TNFR) superfamily.
A third neurotrophin
receptor has been identified
but not well characterized:
sortilin.

Reichardt 2006
Complexity of the TrkB gene
Complexity of the TrkB gene
Complexity of TrkB proteins
 Neurotrophin-receptor interactions
Trk receptors display selectivity for
neurotrophins. p75NTR binds all neurotrophins
with similar affinity.
Trk and p75NTR bind neurotrophins with an
equilibrium binding constant of Kd ~ 10-9M when
expressed alone. When they are co-expressed,
the Kd is increased to ~10-11M.
The ligand specificity of the Trk-p75NTR is
determined by the Trk moiety.
Neurotrophins have been proposed to physically
interact solely with the Trk constituent of the Trk-
p75NTR complex. p75NTR is thought to alter the
conformation of the Trk receptor, increasing its
affinity for ligand.

Sortilin binds NGF with relatively low affinity (Kd~10-8M)

and proNGF and proBDNF with realtively high affinity:
10-9M and Kd~10-10M, respectively). Sortilin associates
with p75NTR further increasing it affinity for proNGF
(~10-10M).

Schweigreiter (2006) BioEssays

p75NTR complexes with sortilin and with TrkA

Nykjaer et al (2004) Nature 427: 843-848

 Functions of proNGF binding to the
p75NTR/sortilin complex

• cell death occurring during development

• injury- e.g., acute neuronal loss following spinal
cord injury and in seizure models
• disease progression- e.g., neuronal loss during
aging and in Alzheimer’s Disease patients

Antagonists of proNGF binding to the

p75NTR/sortilin complex may be useful in the
treatment/prevention of disorders resulting from
injury and in the treatment of Alzheimer’s Disease
and aging
Functional duality of neurotrophins and
proneurotrophins in the nervous system

Mature neurotrophins Proneurotrophins and/or

and/or Trk receptor p75NTR/sortilin

Cell fate survival apoptosis

Glial migration & Stimulatory & inhibitory, Inhibitory & stimulatory,

differentiation respectively respectively

Transmitter phenotype noradrenergic cholinergic

Activity-dependent
LTP LTD
synaptic plasticity

Schweigreiter 2006
 Neurotrophin signalling.

Reichardt L F Phil. Trans. R. Soc. B 2006;361:1545-1564

 BDNF and synaptic plasticity
• BDNF produces fast effects on synaptic transmission by post-
translational modification of synaptic proteins in both the
presynaptic and postsynaptic compartments.
• BDNF increases depolarization-dependent glutamate release
(note: a presynaptic event), possibly as the result of the
phosphorylation of components of the exocytotic machinery (e.g.,
synapsin I and II).
• BDNF also exerts effects on postsynaptic events via mechanisms
dependent on protein phosphorylation: BDNF increases NMDA
receptor single channel open probability as the result of NMDA
receptor phosphorylation; BDNF stimulates delivery of GluA1-
containing AMPA receptors as the result of protein
phosphorylation.
• BDNF also regulates synaptic plasticity via effects on gene
trascription and mRNA translation.
 BDNF-dependent regulation of synaptic transmission

Fig. 2. BDNF regulates glutamatergic synaptic transmission by acting at the pre- and post-synaptic level. BDNF
sequestered in secretory vesicles present in the post-synaptic region is released by a Ca2+-dependent mechanism, following
activation of glutamate receptors. BDNF acts on pre-synaptic TrkB receptors, potentiating glutamate release, and exerts short-
and long-term effects in the post-synaptic cell. BDNF induces the translocation of AMPA receptors to the synapse and
increases the activity of NMDA receptors by phosphorylation-dependent mechanisms. Furthermore, BDNF induces local
protein synthesis at the synapse from mRNAs transported along dendrites in RNA granules, by promoting the disassembly of
the granules (1) and activating the translation machinery (2). Additional effects of BDNF include the regulation of RNA
transport along dendrites, which is mediated by kinesin motor proteins, and activation of gene expression (3).
 Local protein synthesis

• The majority of mRNA translation in neurons occurs in the cell body

• Translation of some mRNAs can occur independent of the soma at synaptic
sites in dendrites and growing axons
• Various stimuli induce local protein synthesis at synapses
• Local protein synthesis has been shown to play a role in synaptic plasticity,
neurite growth and development.
• BDNF regulates translation initiation and elongation through mechanisms
dependent on the activation of mTOR, PI3-K and ERK signaling pathways.
BDNF, like
netrin-1,
induces
synthesis of β-
actin in
neuronal growth
cones under
attractive
conditions

Figure 1. The ‘differential translation’ model for local translation in growth cones. (a) A gradient of
attractive guidance cue, such as netrin-1, induces asymmetrical activation of translation and transport
of mRNAs, causing asymmetrical translation of proteins that build up the cytoskeleton, which leads to
attractive turning. (b) A gradient of repulsive guidance cue, such as Slit-2, induces similar asymmetrical
activation of translation but induces transport and translation of different mRNAs, causing asymmetrical
translation of proteins that disassemble the cytoskeleton, which leads to repulsive turning.

Lin & Holt 2008

 Figure 2. Local translation and communication between the axon and cell body. (1) Stimulation of axons
leads to transcription-independent differential localization of mRNAs to the axon through transport on
microtubules, changing the population of mRNAs available for local axonal translation. (2) Newly synthesized
transcription factors can be retrogradely transported on microtubules to the cell body where they influence
transcription.

Lin & Holt 2008

 Why synthesize proteins locally rather than
transport proteins (either “mature” or
“immature”) that were synthesized in the soma?
• increased flexibility for the regulation of localization and
activation: regulatory elements in the 5’UTR and 3’UTR
do not affect function of the protein; in contrast,
regulatory elements in the protein do affect localization
and function
• increased speed of response
• more efficient: possible macromolecular crowding in
axons/synapses may preclude storage of multiple copies
of each of the proteins that may be needed; it is more
efficient to store mRNA and synthesize and degrade
proteins, as needed.
Mitogen-activated protein kinases
(MAP kinases; MAPKs)
 Mitogen-activated protein kinases
(MAPKs)
• proline-directed, serine/threonine kinases- i.e., MAPKs
phosphorylate serine or threonine residues that precede proline
residues: PX(S/T)P
• Three families:
1. extracellular signal-regulated protein kinase (ERK)
2. C-Jun amino-terminal kinase (JNK, also called stress-
activated protein kinases, SAPK)
3. p38 stress-activated protein kinase (p38; also called
RK/CSBP)
• Activated by dual phosphorylation on both tyrosine and threonine
residues in the “activation lip”.
• Regulate several MAPKAPKs (MAPK-activated protein kinases),
transcription factors, and post-translational processes (e.g., mRNA
stability).
 MAPK activation/inactivation

2ATP
MEK
2ADP

MAPK MAPK
T-X-Y
T-X-Y
P P

2Pi

MKP
 MAPK phosphorylation sites

• minimal consensus substrate sequence of

(S/T)-P
• optimal consensus substrate sequence of
P-X-(S/T)-P
• ~90% of all proteins contain an (S/T)-P
sequence, yet, not all of these are MAPK
substrates
 Mechanisms of MAPK interaction
with a substrate
• 2 site model: phospho-acceptor site and docking
site
• 2 types of docking domains:
1. D-domain; Related motifs have been
identified in a number of proteins and have
been given various names, including DEJL
(docking sites for ERK and JNK, LXL) domain,
kinase interaction motif (KIM), MAPK-docking
site, D box, D-site and D-domain.
2. FXFP motif; also called the DEF (docking
site for ERK, FXFP) motif
 D-domains
• Although these domains were identified based on the
ability to bind one or more MAPK, there are differences
in the consensus sequences used to identify each of
them.
• Consensus sequences
D-domain (Kornfeld et al.):
(K/R)-X-(X/K/R)-(K/R)-X(1-4)-(L/I)-X-(L/I)
(K/R)-(K/R)-(K/R)-X(1-5)-(L/I)-X-(L/I)
KIM sequence (McKenzie et al.):
(V/L)X2(R/K)(R/K)X(3-6)L
MAPK-binding site (Bardwell et al.):
(R/K)2X(2-6)(L/I)X(L/I)
 D-domains
• Model of Sharrocks and colleagues

Basic LXL Φ
 The CD domain and docking
groove on MAPKs
• Proteins bind to a region on MAPKs
termed the common docking (CD) domain.
This region is enriched in negatively
charged amino acids.
• Docking of proteins to the CD domain is
mutually exclusive.
• Regions adjacent to the CD domain are
also important for docking; thus, the
concept of a docking groove
 MAPK pathways

MAPKKK

MAPKK

MAPK

http://www.brc.riken.jp/lab/dna/en/GENESETBANK/301MAP_kinase.html
MAPK pathway inhibitor
 References
• Aloe (2004) Trends Cell Biol. 14: 395-399.
• Keshishian (2004) J. Exp. Zoo. 301A:
201-203
• Reichardt (2006) Phil. Trans. R. Soc. B.
361:1545-1564.
• Schweigreiter (2006) BioEssays 28: 583-
594.