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neurotrophic
regulation of
synapse
development and plasticity
Bai Lu, PhD, Head, Section on Neural
Development and Plasticity Eugene Zaitsev, PhD, Research
Fellow Guhan Naggapan, PhD, Visiting
Fellow Kazuko Sakata, PhD, Visiting
Fellow Mingrui Zhao, PhD, Visiting
Fellow Hyun-Soo Je, BS, Predoctoral
Fellow Petti Pang, BS, Graduate Student |
|
We
study molecular mechanisms underlying synapse development and function, with
particular emphasis on the role of neurotrophic factors. Traditionally, neurotrophic
factors are defined as secretory proteins that regulate neuronal survival and
differentiation. Recent studies indicate that a major function of
neurotrophic factors in the brain is the regulation of synaptic transmission
and plasticity. Our laboratory was among the first to reveal this novel
function. Using rodent models, we demonstrated that brain-derived
neurotrophic factor (BDNF) plays a key role in hippocampal long-term
potentiation (LTP), a cellular model for learning and memory. Using the Xenopus neuromuscular synapse as a
model, we identified two modes of regulation by neurotrophins: acute modulation of synaptic
transmission and plasticity and long-term
alteration of the structure and function of synapses. In the past year, we
discovered the mechanistic link between tissue plasminogen activator (tPA),
an extracellular protease implicated in long-term plasticity and memory, and
BDNF in late-phase LTP (L-LTP) in the hippocampus. We have also made progress
in understanding how BDNF signaling through TrkB receptor is controlled. In
addition, we discovered a dynamin-independent form of synaptic vesicle
endocytosis. Long-term regulation of
synaptic structures and function by BDNF Pang, Zaistev, Woo,
Sakata, Lu; in collaboration with Hempstead, Welker The rodent barrel cortex offers
an excellent model system in which to study activity-dependent
synaptogenesis. Mechanical stimulation in adult whisker induces the
expression of BDNF as well as synapse formation in layer IV of the barrel
cortex. We used serial section electron microscopy to study the
ultrastructural alterations of synaptic connectivity in the barrel cortex,
including measurement of synapse density, spine morphology, and synaptic
vesicle distribution. BDNF heterozygote (+/−) mice, with a reduced
level of BDNF mRNA and protein, had a barrel neuropil indistinguishable from
that of wild-type (+/+) controls. After 24 hours of whisker stimulation,
however, the +/+ mice, but not the +/− mice, exhibited a significant
increase in the number of synapses formed. The balance between excitatory and
inhibitory synapses was modified in the +/+ mice, but not in the +/−
mice. The distribution of synaptic vesicles in excitatory synapses was the
same in +/+ and +/− mice and was not influenced by the stimulation
paradigm. Moreover, the spine volume was significantly more increased by
stimulation in +/− mice than in +/+ mice. These results provide direct
evidence that, in vivo, BDNF plays
a crucial role in the structural rearrangement of cortical circuitry as a
consequence of an enhanced sensory stimulation. Long-term
memory is believed to be mediated by protein synthesis–dependent L-LTP.
Two secretory proteins, tPA and BDNF, have been implicated in L-LTP, but
their relationship is unclear. We demonstrated that, by activating the
extracellular protease plasmin, tPA converts the precursor proBDNF to the
mature BDNF (mBDNF) in the hippocampus and that such conversion is critical
for L-LTP expression. Our electrophysiological studies demonstrated that
mBDNF, but not the cleavage-resistant proBDNF, rescues L-LTP in tPA- and
plasminogen-knockout mice. Biochemical experiments also showed that tPA, by
converting plasminogen to plasmin, cleaves proBDNF to form mBDNF. In
addition, genetic and pharmacological experiments revealed that, in L-LTP
expression, mBDNF is downstream of plasmin, which is downstream of tPA.
Moreover, application of mBDNF converts early-phase LTP (E-LTP) to L-LTP and
rescues L-LTP when protein synthesis is blocked by the inhibitor anisomycin.
These results suggest that mBDNF is a key protein synthesis product
responsible for L-LTP expression. Taken together, the present study has
identified both tPA/plasmin as an endogenous enzyme system that converts
proBDNF to mBDNF in the hippocampus and a physiological role of such
conversion in the brain. Further, the results have provided a mechanistic
link between these two seemingly independent molecule systems in L-LTP
expression. Egan
MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Zaistev E, Bertolino
A, Gold B, Goldman D, Dean M, Lu B, Weinberger DR. The BDNF val66met
polymorphism affects activity-dependent secretion of BDNF and human memory
and hippocampal function. Cell
2003;112:257-269. Genoud
C, Knott GW, Sakata K, Lu B, Welker E. Reduced BDNF expression prevents
synapse formation in the adult somatosensory cortex. J Neurosci 2004;24:2394-2400. Lu
B. Pro-region of neurotrophins: role in synaptic modulation. Neuron 2003;38:735-738. Pang
PT, Lu B. Regulation of late-phase LTP and long-term memory in normal and
aging hippocampus: role of secreted proteins tPA and BDNF. Aging Res Rev 2004;3:407-430. Pang
PT, Teng H, Zaitsev E, Woo KN, Sakata K, Zhen S, Teng K, Yung W-H, Hempstead
B, Lu B. Cleavage of proBDNF by tPA/plasmin is essential for long-term
hippocampal plasticity. Science
2004;306:487-491. Mechanisms controlling
BDNF signaling through the TrkB receptor Sakata, Lu; in
collaboration with Kojima, Zheng Although
BDNF plays a key role in synapse development and plasticity, the underlying
signaling mechanisms remain largely unknown. We found that BDNF rapidly
recruits full-length TrkB (TrkB-FL) receptor into lipid rafts, cholesterol-
and sphingolipid-enriched membrane microdomains thought to be signaling
platforms for extracellular stimuli, from nonraft regions of neuronal plasma
membranes. Truncated TrkB lacking the intracellular kinase domain was not
translocated, and Trk inhibitors blocked the translocation of TrkB-FL,
suggesting that phosphorylation by TrkB tyrosine kinase is required for such
translocation. Disruption of lipid rafts by depleting cholesterol from the
cell surface blocked the ligand-induced translocation. Moreover, disruption
of lipid rafts prevented potentiating effects of BDNF on transmitter release
in cultured neurons and synaptic response to tetanus in hippocampal slices.
In contrast, lipid rafts are not required for BDNF regulation of neuronal
survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent
a signaling mechanism selective for synaptic modulation by BDNF in the
central nervous system. We further tested the role of
lipid rafts in BDNF signaling during axon growth, which requires signal
transduction of extracellular cues for directional motility. We showed that
lipid raft disruption abolished growth cone attraction and repulsion in
gradients of BDNF and netrin-1, respectively, but exerted no effects on
glutamate-induced attraction. Interestingly, local raft disruption on one
side of the growth cone exposed to uniform BDNF or netrin-1 produced opposite
turning responses to that induced by the gradients. Lipid raft manipulation
also blocked semaphorin 3A–induced growth cone repulsion, inhibition,
and collapse. Finally, we found that guidance cue–elicited MAP kinase
activation depended on raft integrity and that specific guidance receptors
were associated with lipid rafts. The results demonstrate an essential role
for lipid rafts in growth cone guidance and suggest that localized signaling
through these dynamic microdomains underlies specific actions of
extracellular cues on developing axons. We
next examined how cAMP controls BDNF/TrkB signaling. While it has been
hypothesized that the synaptic actions of BDNF are “gated” by cAMP,
the underlying molecular mechanisms remain unclear. We showed that cAMP
regulates BDNF function by modulating TrkB receptor signaling and
trafficking. TrkB phosphorylation is controlled by cAMP, with three features
characteristic for cAMP gating: first, inhibitors of cAMP signaling
attenuated BDNF-induced TrkB phosphorylation; second, cAMP analogs
potentiated such phosphorylation; and third, activation of cAMP pathway alone
had no effect on TrkB phosphorylation. cAMP also facilitated trafficking of TrkB
to dendritic spines, possibly by promoting its interaction with synaptic
scaffolding protein PSD-95, and gated BDNF’s long-term modulation of
spine density, but not dendritic branching, in cultured hippocampal neurons.
These results reveal a specific role of cAMP in controlling BDNF actions in
the brain and provide new insights into the molecular mechanism underlying
cAMP gating. Du J, Feng L, Zaitsev E, Je
H-S, Liu X, Lu B. Regulation of TrkB receptor tyrosine kinase and its
internalization by neuronal activity and Ca2+ influx. J Cell Biol 2003;163:385-395. Guirland C, Suzuki S, Kojima
M, Lu B, Zheng JQ. Lipid rafts mediate chemotropic guidance of nerve growth
cones. Neuron 2004;42:51-62. Ji Y, Pang PT, Feng L, Lu B.
Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine
formation in hippocampal neurons. Nat
Neurosci 2004, in press. Lu B, Je H-S. Neurotrophic
regulation of the development and function of the neuromuscular synapses. J Neurocytol 2004;32:931-941. Suzuki S, Numakawa T, Shimazu
K, Koshimizu H, Hara T, Hatanaka H, Mei L, Lu B, Kojima M. BDNF-induced
recruitment of TrkB into lipid rafts: roles in synaptic transmission. J Cell Biol 2004;167:1205-1215. A novel mechanism for
synaptic vesicle endocytosis Lu; in collaboration
with Zheng In
addition to studying neurotrophic regulation, we are interested in the basic
mechanisms for the development and function of synapses. One of the essential
mechanisms is the activity-dependent endocytosis of synaptic vesicles.
Synaptic vesicle endocytosis is believed to require Ca2+ and the
small GTPase dynamin. We have recently identified a novel form of rapid
endocytosis (RE) in dorsal root ganglion (DRG) neurons that, unlike
previously described forms of endocytosis, is completely independent of Ca2+
and the GTPase dynamin. The RE is tightly coupled to a Ca2+-independent
but voltage-dependent secretion (CIVDS). Using FM dye and capacitance
measurements, we showed that membrane depolarization induces RE in the
absence of Ca2+. Inhibition of dynamin function does not affect
RE. Inhibitors of protein kinase A (PKA) suppress RE induced by
high-frequency depolarization while PKA activators enhance RE induced by
low-frequency depolarization. Biochemical experiments demonstrated that
depolarization directly upregulates PKA activity in Ca2+-free
medium. The results reveal a Ca2+- and dynamin-independent form of
endocytosis in DRG neurons, with endocytosis controlled by neuronal activity
and PKA-dependent phosphorylation. Bamji
SX, Shimazu K, Kimes N, Huelsken J, Birchmeier W, Lu B, Reichardt LF. Role of
beta-catenin in synaptic vesicle localization and presynaptic assembly. Neuron 2003;40:719-731. Guhan
N, Lu B. PIKE/Homer complex: a novel link between mGluR1 and PI3 kinase. Trends Neurosci 2004;27:645-648. Luo ZG, Je H-S, Yang F, Xiong
WC, Lu B, Mei L. Implication of geranylgeranyltransferase I in synapse
formation. Neuron 2003;40:703-717. Yang F, He X, Russell J, Lu
B. Ca2+ influx-independent synaptic potentiation mediated by
mitochondrial Na+-Ca2+ exchanger and protein kinase C. J Cell Biol 2003;163:511-523. Zhang C, Xiong W, Zheng H, Wang LC, Lu B, Zhou Z. Activity-dependent but Ca2+-independent endocytosis in dorsal root ganglion neurons. Neuron 2004;42:225-236. COLLABORATORS Barbara Hempstead,
MD, Masami Kojima,
PhD, National Institute of Advanced
Industrial Science and Technology (AIST), Louis F.
Reichardt, PhD, Herbert Boyer Program
in Biological Science, University of California, San Francisco, CA Egbert Welker,
PhD, James Zheng, PhD, The Zhuan Zhou, PhD, For further information, contact lub@mail.nih.gov |