Stargazin Regulates Synaptic Targeting of AMPA Receptors

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Stargazin Regulates Synaptic Targeting of AMPA
Receptors
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A Presentation ByGROUP 6

• Jennifer Miller
• Stewart McIlvena
• Udai Mody
• Ozzie Murray
• Lou Murdoch
• Joshua Michael
• Marissa Miyao
• Bethany Morgan

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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
• 1) Lack of functional AMPARs
• 2) Mutated stargazin
• 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.

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• 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
• 2) How stargazin interacts with AMPAR subunits
  to make it functional.

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Figuring out AMPARs were not Functional in the
Mutant Granule Cells

• Normal granule cells exhibited spontaneous inward
  current that was abolished by CNQX.
• 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
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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
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• 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.

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Figuring out AMPARs were not Functional in the
Mutant Granule Cells

• Immunogold-labeling experiments labeled AMPAR
  subunits.
• - In mutant mice, little labeling was found in
  PSD.
• -In normal mice, abundant labeling was found in
  PSD.

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What If the Mutant Granule Cells Just Couldnt
Produce AMPARs?

• - 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.

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• 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.

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What Could Stargazins Role Be in Granule Cells?

• 1) A calcium channel subunit
• Stargazin has a weak homology to a
  ?-1 Ca channel subunit
• OR
• 2) Regulate AMPARs in the synapse
• When stargazin is mutated, there is a lack of
  functional AMPARs in granule cells
• Which is It???

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Ca2 currents are normal in mutant granule cells

• Because the stargazin protein was similar in
  sequence to the ?-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.

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Ca2 currents are normal in mutant granule cells
Activation of whole-cell calcium currents
Steady-state inactivation of calcium currents
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Ca2 currents are normal in mutant granule cells

• 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.

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• 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 Ca2 channel
  subunit
• Thus, the next reasonable step was to perform
  an experiment to see if stargazin interacted with
  AMPAR subunits.

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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 Stargazins
  PDZ binding site
• When binding site is deleted (known now as
  stargazin?C), AMPAR subunits can still bind, but
  PSD-95 cannot.

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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.

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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.
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Stargazin Rescues AMPAR Responses in Mutant Cells

• 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?C was transfected in mutant cells,
  the spontaneous currents do not increase in
  amplitude or frequency.

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Stargazin?C Cannot Rescue AMPAR EPSCs

• Transfecting stargazin?C in mutant cells cannot
  rescue AMPAR EPSCs
• Transfecting stargazin in mutant cells restores
  AMPAR EPSCs

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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.
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Conclusions

• 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
• The association of the PSD-95 anchor protein with
  stargazin allows the stargazin-AMPAR complex to
  be anchored in the synaptic membrane.