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Abstract
Proper synaptic development is fundamental to normal brain function and
requires the appropriate induction of both excitatory and inhibitory
connections. Failure to properly form a network of these different synaptic
types can lead to a myriad of disorders such as autism, schizophrenia and
epilepsy. Our lab recently found two postsynaptically derived fibroblast
growth factors (FGFs), FGF22 and FGF7, which differentially induce the
organization of excitatory and inhibitory presynaptic terminals, respectively.
Interestingly, these FGF’s also affect neurogenesis in an antagonistic
manner, with FGF22 increasing and FGF7 decreasing neurogenesis.
However, the molecular mechanisms through which FGFs affect the
induction of excitatory and inhibitory synapses and neurogenesis are not
identified. A candidate molecule has recently been identified, Insulin-like
Growth Factor II (IGF-2), and my research focus is to elucidate the role of
this molecule in the mechanism of synaptic development and neurogenesis
in the hippocampus.
I hypothesize that FGF22 induces IGF2, which in turn causes neurogenesis
in the SGZ of the hippocampus. The rationale of this hypothesis is (1) in the
hippocampus of FGF22KO mice, IGF2 expression is reduced and (2) IGF2
has been implicated as having a role in neurogenesis.
Ki67
DAPI
Fig. 1 In addition to the synapse
development phenotype observed in
FGF7KO and FGF22KO mice, a
neurogenesis phenotype was seen in
the DG of these mice. FGF22KO
mice have less immature neurons
(DCX), while conversely, FGF7KO
mice seem to have more immature
neurons.
SYNAPTIC DEVELOPMENT
The development of synapses is the result of the following process:
1.  Axon extension and targeting
2.  Initial contact between the axon and its target
3.  Presynaptic and postsynaptic differentiation
4.  Synaptic maturation
5.  Synaptic pruning, and
6.  Maintenance
Unpublished data, Clara Lee
IGF-2
Fig 2. To identify candidate genes
induced by FGF22, microarray
analysis was carried out on cells
of the DG from WT and
FGF22KO mice at P14. One of
the genes found to be down
regulated in FGF22KO mice was
IGF2. We confirmed this
microarray data by qPCR
analysis.
Fig. 3 Between P7 and P14, corresponding to the initiation and peak of synaptogenesis, differences in
proliferation exist between FGF22KO, FGF7KO and WT mice. These differences persist.
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The dentate gyrus is one of only two areas in the brain capable of adult
neurogenesis. Neurogenesis is strictly confined to this area of the
hippocampus.
IGF-2
M.C. Rhodes, Pharmacology. 2003
•  IGF-2 is a secreted molecule that is known to be important for growth
and development, but is not well studied in the brain. It is highly
expressed in the hippocampus throughout development.
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WT
FGF22KO
NeuN
DAPI
Hypothesis
Different responses to FGFs are suggested to be mainly the result of
differential gene expression. I hypothesize that FGF22 and FGF7 induce
distinct sets of genes in excitatory and inhibitory neurons, respectively.
FGF22 induces genes, such as IGF2, which affect excitatory synapse
formation and increase neurogenesis. In contrast, FGF7 controls genes that
play a role in inhibitory synapse formation and the inhibition of
neurogenesis.
I hypothesize that FGF7 induces genes that are involved in the
development of inhibitory synapses and the restraint of neurogenesis in the
SGZ of the DG. I will utilize mice expressing GFP only in inhibitory
neurons via the Gad67 promoter (Gad67:GFP) to select for inhibitory
neurons exclusively and assess any differential gene expression.
Ramon y Cajal
expression by adding FGF22 to an IGF2-/- culture. If the effects of FGF22 are
dependent on IGF2 expression, I would expect to see no rescue.
References
Chen, D.Y. et al., 2011. A critical role for IGF-II in memory consolidation
and enhancement. Nature, 469(7331), pp.491–497.
Fig. 4 The observed proliferation differences between P7 and P14 in FGF22KO and FGF7KO,
correspond to a difference in mature neurons (NeuN).
To address my hypothesis I will perform a rescue experiment in which
persistent, local administration of IGF2 or saline will be applied to the
hippocampus of FGF22KO or WT mice. I will administer IGF2 by
implantation of a resin (Elvax) containing IGF2 or saline on the
hippocampus. This will allow for controlled, sustained release of IGF2 in a
localized area.
I will evaluate the rescue by immunohistochemisty with staining for new
(DCX) and mature neurons (NeuN).
•  This molecule binds to a cell-surface tyrosine kinase receptor, activating
the PI3K/Akt pathway, a signaling cascade that induces gene expression.
Any genes of interest will be validated by qPCR and in situ.
2. in vivo assay
To assess the effect of IGF2 on the synaptic vesicle defects seen in
FGF22KO mice by persistent, local administration of IGF2 or PBS to the
hippocampus of FGF22KO or WT mice. At the induction (P7), peak (P14),
and end (P21) of synaptogenesis, electron microscopy (EM) will be
performed to assess the size, accumulation and docking of vesicles in the
active zone of excitatory neurons in the CA3 region.
I expect to see a rescue of size, accumulation and docking of vesicles in the
active zone of excitatory synapses in IGF2 treated FGF22KO mice and
cultures. In the future I will assess the dependence of FGF22 effects on IGF2
Identify FGF7-induced presynaptic genes involved in the
development and maturation of inhibitory interneurons and
neurogenesis in the hippocampus.
•  IGF-2 is typically viewed as a neurotrophic or neuroprotective protein. It
has been shown to contribute to the proliferation, development and
survival of neuronal and glial cells.
•  Slices taken from Igf2−/− mice fail to exhibit KA-induced spontaneous
epileptiform activity analogous to the seizure resistance observed in
FGF22KO mice.
A. Terauchi, Nature. 2010
I have begun to elucidate the time point at which neurogenesis in the DG is
affected in both FGF22KO and FGF7KO mice (Figs. 3 and 4). It will be
important to also show if this corresponds with a difference in IGF2
expression. I will assess the level of IGF2 mRNA expression by in situ in
each genotype at P7 and P14.
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Unpublished data, Ania Debrowski
•  The hippocampus is composed of three regions, the dentate gyrus (DG),
cornu Ammonis 1 and 3 (CA1 and CA3).These regions form a neuronal
circuit between each other and with the entorhinal cortex.
I have observed less synaptic vesicle clustering in FGF22KO’s compared to
WT (Fig 5). I hypothesize that IGF2, induced by FGF22, stimulates specific
genes involved in the recruitment of synaptic vesicles to the synapses of
excitatory neurons in the hippocampus. The rationale of this hypothesis is
that IGF2 induces the expression of gene products that localize to the
synapse and play a role in synaptic strength. I plan to test this hypothesis in
two ways:
1. in vitro assay
Hippocampal cultures will be prepared from P0 FGF22KO and WT mice,
treated with IGF2 and assessed and DIV14.
A. Terauchi, Nature. 2010
•  The hippocampus is a part of the limbic system and plays a role in
memory formation and learning. It is also plays a major role in
depression, Alzheimer’s disease and Epilepsy.
Determine the role of IGF2 in the FGF22-dependent
neurogenesis in the hippocampus.
Fig 5 Primary neuron cultures from
FGF22KO mice have less synaptic
vesicle clustering than WT at DIV7
Background
THE HIPPOCAMPUS
Determine the role of IGF2 in the FGF22-dependent
synapse formation in the hippocampus.
Preliminary Data
Johnson-Venkatesh, E.M. & Umemori, H., 2010. Secreted factors as
synaptic organizers. The European journal of neuroscience, 32(2), pp.181–
190.
Ming, G., 2005. Adult neurogenesis in the mammalian central nervous
system. Annu Rev Neurosci., 28:223–50.
Russo, V.C. et al., 2005. The insulin-like growth factor system and its
pleiotropic functions in brain. Endocrine reviews, 26(7), pp.916–943.
Terauchi, A. et al., 2010. Distinct FGFs promote differentiation of
excitatory and inhibitory synapses. Nature, 465(7299), pp.783–787.
Acknowledgements
Special thanks to Akiko Terauchi, Ania Debrowski and Masa Yusada for
their helpful insight.
Supported by the NIH Cellular and Molecular Biology Training Grant T32GM007315.