Download Antibody Staining of Transfected Neurons HA-NLG1-d55

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Introduction
Importance of Synapses
-brain development
-crucial for brain function
Andrea Draper
Presynaptic
Postsynaptic
THE GLUTAMATERGIC
SYNAPSE
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
Hypothesis
NLG1 may recruit the scaffolding
proteins PSD95, SAP102, and S-SCAM
to the post synaptic density via different
intracellular domains.
Experimental Strategies
Question?
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
Goal
Observe the effect of manipulating the
NLG1 structure
Experimental Procedures
1) Neurons are extracted from
hippocampus and cultured
2) Neurons are either stained or
transfected and then stained
IMMUNOCYTOCHEMISTRY
SAP102
CO-LOCALIZATION
PSD95
Transfected Neuron Control
Co-localization of pre and
post synaptic proteins
Experiment:
Manipulating NLG1
HA-NLG1
HA
PDZ
PDZ
vs.
HA
HA-NLG1-d55
HA
PDZ
Antibody Staining of Transfected Neurons
HA-NLG1
HA-NLG1
V-GLUT
PSD95
Co-localization of
pre and post synaptic
proteins
Antibody Staining of Transfected Neurons
HA-NLG1-d55
Co-localization of pre and
post synaptic proteins
Results
Control
NLG1 Transfection
NLG1-d55 Transfection
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.
Hypothetical Results
Untransfected Neuron vs. NLG1 and d55
HA-NLG1
HA
AChE
PDZ
Increased # synapse
HA-NLG1-d55
HA
AChE
Decreased # synapse
Goal
Observe effect of NLG1 manipulation on
the localization of post synaptic proteins
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?
Acknowledgements
-Jennifer Hoy
-Phil Washbourne and the lab
-SPUR program
-SPUR students
-University of Oregon