1. Stargazin Regulates Synaptic Targeting of AMPA Receptors

2. A Presentation By: GROUP 6 Jennifer Miller Jennifer Miller Stewart McIlvena Stewart McIlvena Udai Mody Udai Mody Ozzie Murray Ozzie Murray Lou Murdoch Lou Murdoch Joshua Michael Joshua Michael Marissa Miyao Marissa Miyao Bethany Morgan Bethany Morgan

3. Background In the year 2000, a mouse was found exhibiting epileptic seizures and ataxia (which results in wobbliness). Through a number of experiments, two defects, (among others), were located in the cerebellar granule cells of the mouse : In the year 2000, a mouse was found exhibiting epileptic seizures and ataxia (which results in wobbliness). Through a number of experiments, two defects, (among others), were located in the cerebellar granule cells of the mouse : 1) Lack of functional AMPARs 1) Lack of functional AMPARs 2) Mutated stargazin 2) Mutated stargazin * For an AMPAR to be “functional,” it needs to be localized at a synapse. * For an AMPAR to be “functional,” it needs to be localized at a synapse. * In the rest of the presentation, “mutated granule cells” refers to those that have the defective stargazin protein, and “normal granule cells” refers to those that had the normal stargazin.

4. In the first half of this presentation, I am going to present the experiments performed that proved AMPARs were non-functional in the mutant granule cells….. And in the second half of the presentation, I am going to present the experiments that showed… 1) Why Mutant stargazin caused the AMPARs to be non-functional 1) Why Mutant stargazin caused the AMPARs to be non-functional 2) How stargazin interacts with AMPAR subunits to make it functional. 2) How stargazin interacts with AMPAR subunits to make it functional.

5. Figuring out AMPARs were not Functional in the Mutant Granule Cells Normal granule cells exhibited spontaneous inward current that was abolished by CNQX. Normal granule cells exhibited spontaneous inward current that was abolished by CNQX. Mutant granule cells exhibited essentially no spontaneous current. Mutant granule cells exhibited essentially no spontaneous current. *Spontaneous currents were mediated by the release of glutamate from a presynaptic granule cell and recorded via whole-cell patch clamp recording Normal – 1.7 currents/sec Mutant – 1 current/10 sec

6. Figuring out AMPARs were not Functional in the Mutant Granule Cells To eliminate the possibility that the current was abolished due to defective NMDARs, the granule cell cultures were depolarized in the presence of CNQX. Frequency: Insignificantly different for the two cell types

7. As stated before, AMPARS need to be localized at the synapse to be functional because they need the glutamate that is released there in order to activate. As stated before, AMPARS need to be localized at the synapse to be functional because they need the glutamate that is released there in order to activate. Thus, the next experiment compares the localization of AMPARS in mutant and normal cell cultures via immunogold-labeling. Thus, the next experiment compares the localization of AMPARS in mutant and normal cell cultures via immunogold-labeling.

8. Figuring out AMPARs were not Functional in the Mutant Granule Cells Immunogold-labeling experiments labeled AMPAR subunits. Immunogold-labeling experiments labeled AMPAR subunits. - In mutant mice, little labeling was found in PSD. -In normal mice, abundant labeling was found in PSD.

9. What If the Mutant Granule Cells Just Couldn’t Produce AMPARs? - A Cytoplasmic AMPAR subunit labeling was performed basically the same in both normal and mutant granule cell cultures. - A Cytoplasmic AMPAR subunit labeling was performed basically the same in both normal and mutant granule cell cultures. This indicates the problem was not due to the production of AMPAR subunits, but rather their transport to the synapse.

10. Now, that we have shown that there is a lack of functional AMPARs in the granule cells, we are going to talk about the mutant protein stargazin, and what its role in the cell could possibly be. Now, that we have shown that there is a lack of functional AMPARs in the granule cells, we are going to talk about the mutant protein stargazin, and what its role in the cell could possibly be.

11. What Could Stargazin’s Role Be in Granule Cells? 1) A calcium channel subunit *Stargazin has a weak homology to a *Stargazin has a weak homology to a γ-1 Ca channel subunit OR OR 2) Regulate AMPARs in the synapse *When stargazin is mutated, there is a lack of functional AMPARs in granule cells *When stargazin is mutated, there is a lack of functional AMPARs in granule cells Which is It???

12. Ca 2+ currents are normal in mutant granule cells Because the stargazin protein was similar in sequence to the γ-1 calcium channel subunit, it was possible the primary defect in mutant granule cells was due to altered calcium channel function. Because the stargazin protein was similar in sequence to the γ-1 calcium channel subunit, it was possible the primary defect in mutant granule cells was due to altered calcium channel function. Whole-cell patch clamping showed no difference in calcium currents. Whole-cell patch clamping showed no difference in calcium currents.

13. Ca 2+ currents are normal in mutant granule cells Activation of whole-cell calcium currents Steady-state inactivation of calcium currents

14. Ca 2+ currents are normal in mutant granule cells Result: Result: Even though stargazin shows weak homology to the calcium channel subunit, this data makes it unlikely that the primary defect is altered calcium channel function.

15. As of now, we know the following things: As of now, we know the following things: 1) Mutant granule cells lack functional AMPARs 2) Stargazin is mutant in granule cells 3) Stargazin is probably not a Ca 2+ channel subunit Thus, the next reasonable step was to perform an experiment to see if stargazin interacted with AMPAR subunits. Thus, the next reasonable step was to perform an experiment to see if stargazin interacted with AMPAR subunits.

16. Stargazin interacts with AMPARs and PDZ Proteins Experiment 1 Stargazin, upon being transfected into cells and co- immunoprecipitated, was found to bind with GluR1, 2 and 4. Experiment 2 Stargazin interacts with PSD-95 via Stargazin’s PDZ – binding site * When binding site is deleted (known now as stargazinΔC), AMPAR subunits can still bind, but PSD-95 cannot.

17. Is Stargazin the Connector Between AMPARs and PSD-95? Because PSD-95 can mediate clustering of ion channels, it was hypothesized that maybe this was the connection between stargazin and the clustering of AMPAR subunits at the synapse.

18. Stargazin Interaction with PSD-95 and GluR4 Co-expression of GluR4 with PSD- 95 or with Stargazin results in diffuse distributions of these proteins at the cell surface. However, transfecting the three together causes patch-like clusters at the cell surface.

19. Stargazin Rescues AMPAR Responses in Mutant Cells Transfecting stargazin in mutant cells restores synaptic AMPAR function Transfecting stargazin in mutant cells restores synaptic AMPAR function When stargazin-GFP expressing cells were transfected into mutant cells spontaneous currents increased in amplitude and frequency When stargazin-GFP expressing cells were transfected into mutant cells spontaneous currents increased in amplitude and frequency When stargazinΔC was transfected in mutant cells, the spontaneous currents do not increase in amplitude or frequency. When stargazinΔC was transfected in mutant cells, the spontaneous currents do not increase in amplitude or frequency.

20. StargazinΔC Cannot Rescue AMPAR EPSCs Transfecting stargazinΔC in mutant cells cannot rescue AMPAR EPSCs Transfecting stargazinΔC in mutant cells cannot rescue AMPAR EPSCs Transfecting stargazin in mutant cells restores AMPAR EPSCs Transfecting stargazin in mutant cells restores AMPAR EPSCs

21. Interaction Between AMPAR, Stargazin, and PSD-95 Stargazin interacts with AMPA receptors in an intracellular compartment in the cell and promotes their delivery to the cell surface. The carboxyl terminus of stargazin binds specifically to the anchor protein PSD-95 and mediates recruitment of the stargazin-AMPA receptor complex to postsynaptic sites.

22. Conclusions Mutant cerebellar granule cells lacked AMPAR synaptic currents because they contained defective stargazin proteins Mutant cerebellar granule cells lacked AMPAR synaptic currents because they contained defective stargazin proteins Normal stargazin proteins are necessary to localize AMPAR subunits in the synaptic membrane Normal stargazin proteins are necessary to localize AMPAR subunits in the synaptic membrane The association of the PSD-95 anchor protein with stargazin allows the stargazin-AMPAR complex to be anchored in the synaptic membrane. The association of the PSD-95 anchor protein with stargazin allows the stargazin-AMPAR complex to be anchored in the synaptic membrane.