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160902 Progress Report 進捗報告
- 1. Zebrafish Mecp2 is required for proper axonal elongation of motor neurons and synapse formation Ohshima Laboratory (M1) LIN Yanbin
- 2. Background RTT (Rett Syndrome) • A severe neurological disorder only found in female. • Mainly induced by pathogenic mutation of MECP2 located in X chromosome (Xq28). • The mechanism of Rett Syndrome remains unknown. http://www.rsrt.org/
- 3. MECP2 (methyl-CpG-binding protein 2) R. M. Zachariah, M. Rastegar, Linking epigenetics to human disease and Rett syndrome: the emerging novel and challenging concepts in MeCP2 research. Neural Plasticity. Article ID 415825 (2012).
- 4. BDNF (brain-derived neurotrophic factor) C. Susan, Y. Z. Wu. Active DNA demethylation: many roads lead to Rome. Nature Reviews Molecular Cell Biology 11, 607–20 (2010). BDNF is said to be one of those genes repressed by MECP2.
- 5. • Responsible for neuronal growth & survival • It is reported that the expression of BDNF is reduced in those patients with Alzheimer's, Parkinson's, and Huntington's disease. BDNF (brain-derived neurotrophic factor) J. Budni, et al. The involvement of BDNF, NGF and GDNF in aging and Alzheimer's disease. Aging and Disease 6, 331-41 (2015).
- 6. 72 hpf (hours post fertilization) midline spinal cord dorsal ventral muscle notochord motor neuron (CaP) 1 somite CaP = caudal primary motoneuron
- 7. Plazas et al. Activity-dependent competition regulates motor neuron axon pathfinding via PlexinA3. Proc. Natl. Acad. Sci. USA 110, 1524-9 (2013). Temporal Pattern of CaP CaP MiP RoP
- 8. Temporal Pattern of CaP cue molecule ? ? prepatterning of nAChRs motor axons elongation J. A. Panzer, Y. Q. Song, and R. J. Balice-Gordon. In Vivo Imaging of Preferential Motor Axon Outgrowth to and Synaptogenesis at Prepatterned Acetylcholine Receptor Clusters in Embryonic Zebrafish Skeletal Muscle. The Journal of Neuroscience 26, 934-47 (2006).
- 9. Method (Microinjection) embryo of zebrafish (1~4 cells) • mRNA - overexpression AMO (antisense morpholino oligonucleotide) - knockdown • mRNA coding fluorescent protein (GFP, mCherry) vegetal pole animal pole • overexpression (mRNA) or knockdown (AMO) • fluorescent tag mRNA coding GFP or mCherry • visible marker (Phenol Red)
- 10. 72 hpf (hours post fertilization) (motor neuron) presynapse (muscle) postsynapse vesicle SV2 synaptic vesicle protein 2 nAChR nicotinic acetylcholine receptor midline spinal cord dorsal ventral muscle notochord motor neuron (CaP) 1 somite Neuromuscular Junction (NMJ)
- 11. Method (Immunostaining – SV2, BTX) fluorophore conjugated α-bungarotoxin mouse anti-SV2 antibody goat anti-mouse IgG • SV2 (synaptic vesicle protein 2) staining synaptic vesicle protein 2 mouse anti-SV2 A/b goat anti-mouse A/b • BTX (bungarotoxin) staining α-bungarotoxin can irreversibly bind to nicotinic acetylcholine receptor (nAChR) located on the postsynapse of neuromuscular junction (motor neuron) presynapse (muscle) postsynapse vesicle nAChR nicotinic acetylcholine receptor SV2 synaptic vesicle protein 2
- 12. Analysis of Staining Area Take photos for several layers using a confocal microscope. Layer 1, 2, … Merge all layers. Use ImageJ to quantify the ratio of staining area. Ratio = Staining Area (white dots) / Total Area (circled in red)
- 13. Outline ? Model Animal: Zebrafish mecp2 Temporal Axis Fertilization 24 hpf 48 hpf 72 hpf NMJ Immunostaining ? SV2 BTX [mRNA expression pattern] Whole-mount in situ hybridization [mRNA level] RT-PCR Injection AMO, mRNA Observation bdnf 24 hpf 72 hpf
- 14. Result 1 mecp2 KD 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 1 2 bdnfmRNAlevel uninjected mecp2 AMO uninjected mecp2 AMO SV2 mecp2 AMOuninjected SV2stainedarea/totalarea(%) * 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 * mecp2 bdnf d(pre) ↓ ↑ ↑
- 15. Result 2 Rescue Exp. (mecp2 & bdnf KD) Presynapse density in mecp2 knockdown fish is possible to be recovered by knocking down bdnf. SV2 uninjected mecp2 AMO mecp2 AMO + bdnf AMO 0.0 3.0 6.0 9.0 12.0 15.0 * ** *** mecp2 AMO mecp2 AMO + bdnf AMO uninjected SV2stainedarea/totalarea(%) mecp2 bdnf d(pre) ↓ recovered ↓and
- 16. Result (Previous Study) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 SV2 BDNF mRNAmecp2 AMOuninjected uninjected BDNF mRNAmecp2 AMO * * SV2stainedarea/totalarea mecp2 bdnf d(pre) ↓ ↑ ↑or d(post) ?
- 17. Result 3 bdnf OX / mecp2 KD + BTX Staining 0 0.2 0.4 0.6 0.8 1 1.2 BTX uninjected mecp2 AMO n.s. n.s. BDNF mRNA uninjected BDNF mRNAmecp2 AMO BTXstainedarea/totalarea mecp2 bdnf d(pre) ↓ ↑ ↑or d(post) — n.s.
- 18. uninjected mecp2 AMO (KD) BDNF mRNA (OE) SV2(pre)BTX(post)mergeResult 4 Double Staining scale bar: 50μm
- 19. Discussion • mecp2 KD leads to defected motor activity. (Previous behavioral experiments) • In both mecp2 KD and bdnf OE samples, 1. presynapse density: ↑ 2. postsynapse density: ― 3. altered spatial distributions of both pre- & post- synapse • Knocking down both bdnf and mecp2 could recover presynapse density comparing to mecp2 morphant.
- 20. Future Plan • mecp2 KD leads to defected motor activity. (Previous behavioral experiments) • In both mecp2 KD and bdnf OE samples, 1. presynapse density: ↑ 2. postsynapse density: ― 3. altered spatial distributions of both pre- & post- synapse • Knocking down both bdnf and mecp2 could recover presynapse density comparing to mecp2 morphant.
- 21. Future Plan 0.0 3.0 6.0 9.0 12.0 15.0 * ** *** mecp2 AMO mecp2 AMO + bdnf AMO uninjected SV2stainedarea/totalarea(%) • Result of double knockdown = additive effect? • Dose-dependent manner? bdnf AMO ???
- 22. Thank you for listening!
Notes de l'éditeur
- There is a severe neurological disorder called rett syndrome. This disease is only found in female and mainly caused by the mutation of mecp2 located in x chromosome. We still know less about the mechanism of rett syndrome.
- As it's named, mecp2 can bind to methylated CpG region and repress the expression of the downstream gene. Recently mecp2 is also found to be able to realize some other functions.
- It is discovered that bdnf is one of those genes repressed by mecp2.
- Bdnf (brain derived neurotrophic factor) is responsible for neuronal growth and surivival. Mature bdnf functions mainly by reacting with the high affinity membrane protein called TrkB. This reaction will lead to cell survival. However, immature bdnf can bind to low affinity p75 receptor and cause apoptosis. It is known that the expression of bdnf is reduced in patients with these neurological disorders.
- We use zebrafish embryos to carry out our research. This is a 72 hours post fertilization view of a zebrafish. Here is one somite. We can see spinal cord, notochord here, and muscle in both sides of midline. There is a kind of motor neuron sending its axons toward ventral direction. We call it a caudal primary motoneuron and CaP for short.
- Here is the temporal pattern of CaP. We know they start sending axons from 16 hours post fertilization. Axons will go over the midline at 20 hours post fertilization.
- To form a connection between motor neuron and muscle, a cue molecule will lead to the prepatterning of nicotinic acetylchroline receptors. The cue may also be responsible for directing axons. We know that prepatterning of nicotinic acetylchroline receptors (stained in red) has been done before axons reach their destination.
- We do microinjection of mRNA or AMO to embryos in order to overexpress or knock down genes. We also coinject GFP or mCherry mRNA to help us know if things are absorbed and work normally.
- On the surface of muscle, there are many neuromuscular junction structures. It can be divided into presynapse and postsynapse. We have SV2 proteins on the surface of presynapse vesicle. SV2 is a kind of calcium sensor. We also have nicotinic acetylcholine receptors on the membrane of postsynapse.
- We use these two structures to label pre and post synapse. For presynapse, we add anti-SV2 to label it. For postsynapse, we use bungarotoxin.
- How we quantify the synapse density is that, we take a lot of photos from the surface to the inside of a fish. Then we merge the layers, use imageJ to calculate the ratio of stained area.
- This is the outline of this research. We do injection to change the expression level of mecp2 and bdnf after fertilization. RT-PCR and whole mount in situ hybridization are used to detect the mRNA level and expression pattern. And we do immunostaining to see if there are any changes of presynapse and postsynapse.
- We found that if we knock down mecp2, we will get higher bdnf level and higher presynapse density. I wonder what will happen if we knock down both mecp2 and bdnf.
- So in this result, I knocked down both mecp2 and bdnf. Then I found a lower presynapse density. Maybe if we adjust the ratio of mecp2 and bdnf AMO, it is possible to recover the presynapse density to the equal level as uninjected samples.
- In the previous study, we also know that both overexpression of bdnf and knocking down mecp2 will cause an increase of presynapse density. So how about the postsynapse?
- In fact I found no significant difference in the postsynapse density among three groups. If this is true, it may mean that mecp2 or bdnf won’t influence the postsynapse density.
- Then we did a double staining to check if there is any mismatch between pre and postsynapse. The result is that we found axon branching defect in experimental groups. Staining dots accumulate on the prepatterning position. However in uninjected group, we can see presynapse and postsynapse spread to everywhere of the muscle.
- To summarize, in previous behavioral experiments, we know that mecp2 knockdown may lead to defected motor activity. In both mecp2 knockdown and bdnf overexpression samples, we saw higher presynapse density without the change of postsynapse density. And we saw the spatial distributions of both pre and post synapse changed. If we knock down both bdnf and mecp2, it is possible that presynapse density can be recovered comparing to mecp2 morphant.
- I think it is very interesting to find out why the alterations of mecp2 and bdnf levels influence presynapse and postsynapse density differently. And how they change the spatial distribution of neuromuscular junctions.
- Besides, we can make sure if the result of double knockdown is just a simply additive effect or not, and if it follows a dose-dependent manner. I will inject bdnf AMO and test the staining density.
- Mecp2 is universally expressed everywhere, especially in retina, pineal, whole brain, and cerebellum.
- By the way, bdnf strongly expresses in smooth muscle.
- Zebrafish also has other kinds of motor neurons, RoP (rostral primary motoneuron) and MiP (middle primary motoneuron). But in the ventral side of midline, only CaP exists. It is easy to observe. So I focus on the development of CaP.
- This picture shows several developmental stages of zebrafish embryos. Prepatterning of nAChRs occurs around 14 to 20 hpf. Primary motor axons branch from 16 to 26 hpf. CaPs pass through midline at 20 hpf. After 21 hpf, secondary motor axons will extend.
- We also use whole-mount in situ hybridization technique to visualize the expression pattern of specific mRNA.
- About the axon branching defect, my explanation is that Because the cue molecule is intact, so prepatterning of nAChRs and extending of primary motor neuron axons occur normally. When it comes to the secondary branching, I think it may be high level of immature bdnf that leads to the branching defect. I think this theory needs to be further proved.
- The problem is how to explain the presynapse density increase. In fact, I am not sure which signaling pathway is responsible for it. TrkB or p75. And I still don’t know if the branching of motor neuron axons and secondary patterning of nAChRs are dominated by muscle or neuron.
- We did an experiment to check if injection of AMO itself affects the presynapse or postsynapse density. mecp2 5mis AMO is what carries 5 mispair nucleotides compared with mecp2 AMO. Theoretically it won’tinfluence mecp2 level. The result is that we did not find significant difference. So injection of AMO may not change the density.
- This is the result of in situ hybridization. I found that the expression pattern of bdnf mRNA changed in the head of zebrafish. We can see the size of lens and the head become smaller. And we can only see one staining area among the midbrain comparing to a wild type zebrafish. In fact, obvious difference in the body can not be simply observed in other samples. So I am not sure if there is any change in the body part.