ucsd.edu. introduction importance of synapses -brain development -crucial for brain function
TRANSCRIPT
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UCSD.EDU
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Introduction
Importance of Synapses
-brain development
-crucial for brain function
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THE GLUTAMATERGIC SYNAPSE
Presynaptic
Postsynaptic
Andrea Draper
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Why Neuroligin1 vs. Other Proteins?
WADSWORTH CENTER
• Synaptogenic
• Interacts with key post synaptic protein (PSD95)
• Knock-out mice reveal necessary for survival
• Tentative link to neuronal developmental disorders
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Hypothesis
NLG1 may recruit the scaffolding proteins PSD95, SAP102, and S-SCAM to the post synaptic density via different intracellular domains.
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Experimental Strategies
If I get rid of the PDZ domain of NLG1, will that have an effect on the localization of post synaptic proteins?
Answer:
• Over express NLG1 and NLG1-d55 DNA in neurons
• Stain neurons
• Quantify and compare normal neurons to DNA over expressed neurons
Question?
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Goal
Observe the effect of manipulating the NLG1 structure
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Neurons are extracted from hippocampus and cultured
1)
Neurons are either stained or transfected and then stained
2)
Experimental Procedures
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IMMUNOCYTOCHEMISTRY
SAP102 PSD95
CO-LOCALIZATION
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Transfected Neuron Control
Co-localization of pre and post synaptic proteins
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Experiment:Manipulating NLG1
HA
PDZHA-NLG1
HA-NLG1-d55
vs.
HA PDZ
HAPDZ
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Antibody Staining of Transfected Neurons HA-NLG1
Co-localization of pre and post synaptic proteins
HA-NLG1 PSD95
V-GLUT
PSD95
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Antibody Staining of Transfected Neurons HA-NLG1-d55
Co-localization of pre and post synaptic proteins
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ResultsControl
NLG1-d55 TransfectionNLG1 Transfection
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Results
My lab has found previously that by manipulating the PDZ domain of NLG1, it is found that this does have an effect on the co-localization at a synapse.
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Hypothetical Results
HA AChE PDZ Increased # synapse
HA AChE Decreased # synapse
HA-NLG1
HA-NLG1-d55
Untransfected Neuron vs. NLG1 and d55
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Observe effect of NLG1 manipulation on the localization of post synaptic proteins
Goal
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Future Directions
-Test NLG1 difference in younger vs. older neurons
-Effects of NLG1 on localization of other post synaptic proteins
-NLG1 linked to neuronal developmental disorder?
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Acknowledgements
-Jennifer Hoy
-Phil Washbourne and the lab
-SPUR program
-SPUR students
-University of Oregon
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