TGFβ1 may be the dominant factor in SCCM to
promote synaptogenesis after trophic inhibition
Writer : 911631
侯思羽
Advisor: Feng Z.H. 南加州大學研究生
Execution: No
Abstract
Previous studies have shown that neurotrophins (BDNF, GDNF, NT3, NT4) and
cAMP can promote neuron outgrowth and survival. But while they make neurons
growth better, they inhibit neurons progressing from growth state to synaptogenesis.
SCCM derived from glia cells have been found to be able to reverse the situation.
They can promote synapse formation after trophic inhibition. However, which factor
in SCCM dominants the work and the mechanism are still unknown. Since TGFβ1 is
presumed to play the dominant role, I have designed experiments to prove it by using
the cell culture model and the techniques of DNA recombination and Immunostaining.
Introduction
The regulation mechanism of synapse formation has become an interesting topic
for research. Besides presynaptic nerve terminal and postsynaptic muscle cell, the
perisynaptic Schwann cell (PSC) is now known to be an important element of synapse
formation as well (Balice-Gordon, 1996). Specific ablation of PSCs in vivo caused
nerve terminal retraction and synapse transmission reduction 1 week after ablation
(Reddy et al., 2003). It shows that PSCs play a vital role in maintaining the structure
and function of synapses.
During synaptogenesis, neurotransmitter receptors and ion channels of muscle
cell cluster at neuromuscular contact site (Mary et al., 2003). One major group of
neurotransmitter receptors of muscle cell is acetylcholine receptors (AchRs).
Researchers observe synapse formation by seeing cluster of AchRs. We have known
that agrin released from neurons and Schwann cells (isoform B8, B11, and B19)
induce the aggregation of acetylcholine receptors (McMahan et al., 1992; Daggett et
al., 1996; Yang et al., 2001). Previous studies have shown that the neurotrophins
cocktail (BDNF, NT3, NT4, glia-derived neurotrophic factor, forskolin1 and IBMX2)
can promote neuron outgrowth and survival (extending life-span from 2days to 5 or 6
days). However, the neurotrophins cocktail will diminish synaptogenesis at
neuromuscular contact site by inhibiting agrin expression of motoneuron (Peng et al.,
2003).
To see whether the factors released from Schwann cells affect synaptogenesis or
not, Schwann cell-conditioned medium (SCCM) has been used. SCCM is selected
from the medium that is used to culture Schwann cells for a period of time, and it
contains proteins released from Schwann cells. Recent studies have shown that SCCM
can reverse the trophic inhibition of synapse formation and promote neurons to shift
from growth state to synaptogenesis state (Peng et al., 2003).
Which factors in SCCM dominate synaptogenesis after trophic stimulation and
how the mechanism works are still not clear. We already known that Schwann cells
release agrin themselves (Yang et al., 2001), but that isn’t the main cause for them to
promote synapse formation after trophic inhibition. That’s because the amount of
agrin released from Schwann cells themselves are less than the amount of agrin
increased after the treatment of SCCM to neuron. So we thought SCCM may
stimulate agrin expression of motoneurons or somehow inhibit neurotrophins’
stimulation to neurons.
We have already known some components of SCCM, including TGFβ1.
Previous studies have shown that TGFβ1 produced long-term facilitation of the
synaptic connections in Aplysia (Fan et al., 1997). TGFβ1 can positively regulate the
release of neurotransmitters and the formation of synapses (Sanyal et al., 2004). We
presume that TGFβ1 may be the dominant factor to recover synaptogenesis after
trophic stimulation. And since normal NMJ in vivo, signals transmit between
presynaptic and postsynaptic region, we use NMJ (neuromuscular junction) culture of
Xenopus as model to study synapses.
1 forskolin－adenylate
cyclase activator, 2 IBMX－phosphodiesterase inhibitor, both elevate
the quantity of cAMP, which can promote survival of mammalian spinal neuron.
Specific Aims
The purpose is to figure out whether TGFβ1 is the factor in Schwann
cell-conditioned medium(SCCM) to reverse trophic inhibition from synaptogenesis.
In order to prove our presumption that TGFβ1 plays the dominant role, I’ll compare
the ability of TGFβ1 in promoting synapse formation with the same ability of SCCM.
Further more, I hope to find out whether TGFβ1 works by it own or by cooperating
with other factors in SCCM.
Materials and Methods
Flow Chart：
Species: Xenopus (African Clawed Frog)
Egg
Testis
Fertilization
Grow to embryonic stage 22 and cut dorsal part
Make NMJ culture
Add treatments to NMJ culture (See Table 1＊)
Table 1)
Fix and Immunostaining
Take pictures and Calculation
＊
Table 1.
Day 1
Day 3
Culture medium only
Fixation and Staining
Add NTs & cAMP to Culture medium
No further addition
Add NTs & cAMP to Culture medium
Add NTs & cAMP to Culture medium
Add further SCCM
Add further TGFβ1
Add NTs & cAMP to Culture medium
Add further SCCM－TGFβ1
◎Neurotrophins
:BDNF, NT3, NT4, glia-derived neurotrophic factor
Factors elevate the quantity of cAMP: forskolin and IBMX
Day 5
Fixation
and
Staining
Fertilization and neuromuscular junction (NMJ) culture
Testis got from male Xenopus is grinded by grinder, and sperms are put onto eggs
collected from female Xenopus. After fertilization, spinal neurons and surrounding
muscle cells were isolated from stage 22 embryos by cutting the dorsal part of
embryos and then dissociating with Ca+, Mg+ free Steinberg solution. Then the cells
are cultured with Xenopus culture medium. After 1 d of culture, neurons and muscles
and their contacts can be detected by microscope.
Preparation of Schwann cell-conditioned medium (SCCM)
Get sciatic nerves from male Xenopus and cut them into small pieces and use
collegenase and trypsin for dissociation. Culture the cells with Xenopus culture
medium. The cells we gain from the procedure are Schwann cells, axons (without cell
bodies), and other stuff. But only Schwann cells will survive because they own
nucleuses. Collect the medium every week for later usage and add fresh medium to
the cells.
Checking TGFβ1 of SCCM (Western blotting)
Samples from SCCM andβ-actin(a loading control) lyse in SDS gel loading buffer
and denatured at
100 °C for 5 minutes. Samples are fractionated by SDS-PAGE and
then transfered to a PVDF membrane (Sambrook et al., 1989). Before probed with
rabbit anti-TGFβ1 or mouse anti-β-actin antibodies, the membrane is blocked by
skim milk. Then the membrane is incubated with anti-rabbit IgG or anti-mouse IgG.
Finally the membrane is visualized with enhanced chemiluminescence (ECL)
(Amersham Pharmacia Biotech.).
Gel images are captured on film. Using a series of known amounts of TGFβ1 for
calibration can quantify the TGFβ1 in normal SCCM. And from which I can have an
idea of the amount of TGFβ1 I should add in the normal physiological range.
Rid of TGFβ1 from SCCM (Block TGFβ1 receptors of neurons)
TGFβ1 mimic are made by inserting short fragment of oligonucleotides into TGFβ1
gene. The recombinant gene is placed downstream of T7 promoter of vector and over
expressed by transformed into E. coli BL21(DE3). I do not actually remove TGFβ1
from SCCM but block TGFβ1 receptors on neurons by TGFβ1 mimic. Before
SCCM are treated to NMJ cultures, TGFβ1 mimic are added to compete the binding
site of TGFβ1 on neurons to block them. (Gel affinity assay should be used to prove
the similar efficiency of TGFβ1 and TGFβ1 mimic.) (Chow et al., 2001)
To make sure that TGFβ1 mimic can really block TGFβ1 receptors, higher
concentration of TGFβ1 mimic(much higher than TGFβ1 to be added) are used.
Blocking can be checked by labeling TGFβ1 before use. And after TGFβ1 are
added to the culture for a while, wash the medium and use liquid scintillation
counting to measure the amounts of labeled TGFβ1 in the medium.
Staining
Fix with 4% paraformaldehyde
wash
Block with goat serum + 0.1% triton
Primary Antibody (rabbit anti Xenopus Synapsin)
(FITC-anti rabbit) + Primary antibody (BTX-Texas Red)
Secondary antibody
Add PPDA to prevent
bleaching. Observe with fluorescence microscope.
Calculation
Calculate the amounts of neuromuscular contacts and the numbers of synapses in the
field of vision under microscope. Totalize the data and gain the proportion of synapses
formation by the equation:
The proportion of synapses formation(%)＝
the numbers of synapses
the amounts of neuromuscular contacts
10μm
m
Anticipated result
I expect that TGFβ1 may promote the synapse formation after trophic inhibition, and
that TGFβ1 may dominant the work without the help of other factors.
The following diagram indicates my hypothesis that TGFβ1 can recruit synapse
formation of almost the same amounts with the amounts SCCM can recruit. And
SCCM without TGFβ1 makes no increase in synapse formation.
The yellow bars are my anticipated results, while the first three bars are what have
been done and published. (Peng et al., 2003)
Discussion
Here are my interpretations to possible alternative results:
TGFβ1
SCCM－TGFβ1
○
○
There are still other factors work besides TGFβ1
○
X
TGFβ1 may be the dominant factor
X
○
TGFβ1 may not be the dominant factor
X
X
TGFβ1 has to coordinate with other factors to work
Indication
○ means to have significant recruit of synapse formation.
There is one thing about TGFβ1 mimics and blocking that I have to improve. Since I
cannot prove that my way of blocking TGFβ1 receptors will not affect other signaling
pathways (which might be important for synaptogenesis), I need further control
experiments as foundation. And if my experiments turn out positive results, I should
try experiments in vivo not merely in culture dishes.
Until now, the mechanism of how TGFβ1 can affect and regulate synapses is not clear.
It is truly an interesting field and worth exploring.
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