UCSD.EDU. Introduction Importance of Synapses -brain development -crucial for brain function.

UCSD.EDU

Introduction

Importance of Synapses

-brain development

-crucial for brain function

THE GLUTAMATERGIC SYNAPSE

Presynaptic

Postsynaptic

Andrea Draper

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

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?

Goal

Observe the effect of manipulating the NLG1 structure

Neurons are extracted from hippocampus and cultured

1)

Neurons are either stained or transfected and then stained

2)

Experimental Procedures

IMMUNOCYTOCHEMISTRY

SAP102 PSD95

CO-LOCALIZATION

Transfected Neuron Control

Co-localization of pre and post synaptic proteins

Experiment:Manipulating NLG1

HA

PDZHA-NLG1

HA-NLG1-d55

vs.

HA PDZ

HAPDZ

Antibody Staining of Transfected Neurons HA-NLG1

Co-localization of pre and post synaptic proteins

HA-NLG1 PSD95

V-GLUT

PSD95

Antibody Staining of Transfected Neurons HA-NLG1-d55

Co-localization of pre and post synaptic proteins

ResultsControl

NLG1-d55 TransfectionNLG1 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

HA AChE PDZ Increased # synapse

HA AChE Decreased # synapse

HA-NLG1

HA-NLG1-d55

Untransfected Neuron vs. NLG1 and d55

Observe effect of NLG1 manipulation on the localization of post synaptic proteins

Goal

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