Malinow

Malinow

Molecular Methods

Quantum Dots

Choquet

AMPAR

Experiment Ideas

experimental notes and highlighted findings

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Issues to consider Overall, we should focus on the key differences in GluR1 vs GluR2 expression in terms of how they are trafficked and their unique roles in potentiation. We know that GluR1 is Ca²⁺ permeable and GluR2 isn't, likewise GluR1 is inward rectifying, GluR2 is not. It should be safe to assume these differences are very meaningful in LTP. Aside from those ion channel differences, GluR1 has a longer C-tail producing differences in trafficking, mobility, and intracellular protein association. Other key LTP-related pathways and mechanisms include: NMDAR-associated calcium influx activates CaMKII, which is rapidly translocated to the active synaptic terminal. Once situated, CaMKII can phosphorylate AMPAR PDZ sites resulting in diffusional trapping at synaptic slots; at active synapses CaMKII can also complex with NMDARs, resulting in a sustained increase in activity of both proteins.

So that is the story, but really to what extent do we know that NMDAR-mediated calcium influx plays dominant role in CaMKII activation. GluR1 homomerics allow calcium to enter the cell too. What evidence is there that GluR1 can't activate CaMKII? Not all synapses even have NMDARs.

• What happens when the PDZ binding site is mutated

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Zac Email
Here are the different labeling techniques that might be applicable with recombinant expression of AMPARs. Roughly ranked from most to least likely to succeed, separated by large vs small AMPAR N-terminal additions. The references in parentheses are for background on the technique.

Large AMPAR N-terminal addition

• Halotag (Promega): AMPAR-enzyme, QD-Halotag substrate
• AMPAR-streptavidin, QD-biotin

Small AMPAR N-terminal addition

• AMPAR-FLAG, QD-anti-FLAG
• AMPAR-biotin ligase recognition peptide, QD-biotin + biotin ligase (Lu Ting 2013 PLOSONE)
• AMPAR-peptide A, QD-peptide B, which binds peptide A (Zhang Kodadek 2000 NatBiotech)
• AMPAR-unnatural amino acid azide, QD-propargyl (Chaterjee Schultz 2013 PNAS)

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Choquet 2010 CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin

• NMDAR activation promotes rapid translocation of aCaMKII::GFP to synapses, causing AMPAR trapping at 1 min (only synapses with CaMKII translocation)
• tCaMKII (active prion) promotes immobilization of endogenous GluR1 (containing) AMPARs (both synaptic and extrasynaptic), and to a much lesser extent GluA2 (containing) AMPARs.
• CaMKII direct phosphorylation of AMPARs unnecessary for synaptic trapping
• GluA1 - SAP97 interaction unnecessary for CaMKII-dependent synaptic trapping
• Stargazin increased tCaMKII-mediated trapping of recombinant GluA1 (homomeric), but tCaMKII had no effect on mobility of recombinant GluA2 (homomeric)
• Stargazin phosphorylation (by tCaMKII) is necessary for GluA1 trapping; blocking phosphorylation caused AMPAR mobility to significantly increase.
• intriguing finding: GluA1 subunit-specific effect of CaMKII, where it immobilizes recombinant GluA1 but not GluA2 homomeric AMPARs.
• findings consistent with specific role of GluA1 in activity-dependent trafficking - but Stargazin can bind all subunits??
• findings raise possibility that during LTP, CaMKII activation triggers both classical LTP and PPD. Interesting that LTP is frequently accompanied by PPD (opposite of PPF: paired-pulse facilitation)

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Experiments

Hayashi, Shi, Esteban, Piccini, Poncer, Malinow • 2000 • Science - PDF

Driving AMPA Receptors into Synapses by LTP and CaMKII: Requirement for GluR1 and PDZ Domain Interaction

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ABSTRACT

• AMPARs with an electrophysiological tag were expressed in rat hippocampal neurons.
• Long-term potentiation (LTP) or increased activity of CaMKII induced delivery of tagged AMPARs into synapses.
• mutating GluR1's CaMKII p-site had no effects on synaptic delivery (Choquet 2010 confirms this)
• mutating GluR1's PDZ domain blocked delivery (NB: Choquet 2010 found opposite {{#info:In their 2010 study Choquet coexpressed tCaMKII with HA:GluA1 lacking the PDZ-binding domain (HA-GluA1D7) and found that HA-GluA1D7 mobility at synapses was strongly reduced by tCaMKII - their measure was diffusion rate and synaptic trapping, which to me is a weaker measure than Malinow's methods which directly test the functional electrophysiological properties conferred to the synapse by GluR1}})
• results show LTP and CaMKII activity drive GluR1 to synapses by mechanism requiring GluR1 and PDZ proteins

FINDINGS

Does tCaMKII-GFP enhanced transmission?

• To examine effect of elevated CaMKII activity, we used tCaMKII-GFP.
  • Genes delivered to neurons in organotypically cultured hippocampal slices, using Sindbis virus
  • * Neurons expressing trans-genes identified by GFP and whole-cell recordings.
• expression of construct increased tCaMKII activity in BHK cells.
• In neurons expressing construct GFP was detected in dendritic arbors and spines.
• we measured synaptic responses in two nearby neurons, one with tCaMKII-GFP one WT.
• tCaMKII-GFP enhanced synaptic transmission but with no effect on rectification

Does tCaMKII enhance transmission via GluR1?

• We used ephys assay to examine if increase in AMPAR-mediated transmission was due to delivery of receptors to synapses.
  • The current-voltage (I-V) relationship of AMPARs is determined by GluR2 subunit (GluR2 has linear I-V relations; GluR1 homomerics are rectified at 140 mV). Most AMPARs in hippocampal pyramidal cells contain the GluR2 subunit.
• We overexpressed GluR1::GFP subunit in hippocampal slice neurons. Most resulting recombinant AMPARs lacked GluR2. Recombinants were functional and showed complete inward rectification in HEK293 cells. Thus, incorporation of these recombinant receptors into synapses would be expected to increase rectification of synaptic responses.

Can CaMKII activity drive recombinant GluR1-GFP into synapses?

• GluR1-GFP is widely distributed throughout dendritic arbors, but little is incorporated into synapses in the absence of activity.
  • In agreement with this, expression of GluR1-GFP had no effect on amplitude or rectification.
• To determine if CaMKII activity could drive recombinant GluR1-GFP into synapses,
  • we coexpressed GluR1-GFP and tCaMKII using an internal ribosomal entry site (IRES) construct.
  • BHK cells expressing this construct showed increased constitutive tCaMKII activity, and slices expressing this construct showed GluR1-GFP expression.
• Pairwise recordings comparing infected and noninfected cells showed infected cells had enhanced transmission, due to increase of tCaMKII activity.
• Notably, infected cell transmission showed increased rectification, indicating a contribution of the homomeric GluR1-GFP to transmission.
• This effect on rectification was due to coexpression of the two proteins, because transmission onto cells expressing either tCaMKII or GluR1-GFP alone had rectification comparable to that in uninfected cells.
• These results show that tCaMKII activity induces the insertion of homomeric GluR1-GFP into the synapse.

Is GluR1 phosphorylation by CaMKII sufficient to drive LTP?

• GluR1 is phosphorylated by CaMKII at Ser831 during LTP.
• To examine if direct phosphorylation of the receptor at this site is required for delivery, we substituted Ser831 with Ala, thus creating GluR1(S831A)-GFP.
• This mutation did not block delivery.
• Expression of this construct alone changed neither amplitude nor rectification, and coexpression with tCaMKII produced potentiated transmission that showed the same increase in rectification as that seen with GluR1-GFP-IREStCaMKII

Is the TGL (PDZ consensus) sequence of GluR1 important for its synaptic trafficking/rectification when tCaMKII is overexpressed

• The subcellular localization of many membrane proteins is controlled by associations with a class of proteins containing PDZ domains.
• In particular, the cytosolic C-terminus of such surface proteins has a PDZ sequence that when mutated prevents associations. GluR1 has this C-terminus TGL consensus sequence.
• We mutated the GluR1 sequence from TGL to AGL, creating GluR1(T887A)-GFP
• GluR1(T887A)-GFP expressed in HEK293 cells formed functional AMPARs with normal rectification.
• GluR1(T887A)-GFP expressed in hippocampal neurons was detected in dendrites, and showed no effect on transmission when expressed alone.
• Coexpression of GluR1(T887A)-GFP and tCaMKII in hippocampal slice neurons completely blocked synaptic response amplitude and rectification, and in fact depressed transmission

Does evoked LTP do the same thing as tCaMKII overexpression, in mediating GluR1 synaptic trafficking/rectification?

• To determine if LTP delivers AMPARs to synapses through a similar mechanism, we examined LTP in cells expressing GluR1-GFP.
• Whole-cell recordings were obtained from cells expressing or not expressing GluR1-GFP.
• LTP was induced with a pairing protocol.
• We added APV to the bath 30 min after potentiated transmission was measured, in order to isolate pure AMPAR–mediated responses.
• The holding membrane potential was then switched to measure rectification of the AMPAR–mediated responses.
• Similar to the effect of coexpressed CaMKII and GluR1-GFP, rectification was increased after LTP in cells expressing GluR1-GFP compared to cells not expressing GluR1-GFP.

Is the PDZ GluR1 binding domain important during LTP?

• We next examined the effects of GluR1(T887A) on LTP.
• As shown above, GluR1(T887A) has a mutated PDZ-interaction site that completely blocks the potentiation produced by tCaMKII.
• we recorded synaptic responses from cells expressing either GluR1-GFP or GluR1(T887A)-GFP.
• After LTP pairing, GluR1-GFP control cells displayed stable potentiation lasting at least 50 min.
• GluR1(T887A)-GFP cells displayed a very different response after LTP pairing: these cells had short-lasting potentiation that decayed over 20 min and after 45 min the responses were significantly depressed from baseline.
• In 4 of the 21 experiments with GluR1(T887A), a control (nonpaired) pathway was monitored, which did not show depression

SUMMARY

• Here, we generated electrophysiologically tagged receptors to monitor their synaptic delivery during LTP and increased tCaMKII. In the absence of plasticity-inducing stimuli, we saw no evidence for their contribution to transmission (consistent with previous results indicating that in the absence of evoked activity, GluR1 is retained within the dendrite).
• Upon coexpression with constitutively active tCaMKII or following LTP induction, we see that tagged receptors contribute to transmission, indicating their delivery to synapses.
• Previous studies indicate that LTP induction increases the CaMKII-dependent phosphorylation of GluR1 at Ser831. Although such phosphorylaton may enhance the function of synaptic receptors, this phosphorylation does not seem to be required for receptor delivery: tCaMKII can deliver GluR1(S831A)-GFP to the synapse. Our results indicate that some protein(s) other than GluR1 must be substrate(s) of CaMKII (Choquet: Stargazin) and participate in the regulated synaptic delivery of AMPARs.
• The most surprising of our results relate to the effects of GluR1(T887A). This protein forms functional receptors and has no detectable effects on basal synaptic transmission. However, this mutant receptor can block the effects of tCaMKII and LTP (24). This has several implications:

1. It reinforces the view that CaMKII and LTP act through similar mechanisms.
2. It indicates that both CaMKII-potentiation and LTP exert their effects through GluR1
3. It indicates that an interaction between GluR1 and a protein with a PDZ domain plays a key intermediate in these forms of plasticity
4. GluR1(T887A) depresses transmission, but only after increased CaMKII or LTP.

• This last finding suggests that activity enables the mutant protein to interrupt a constitutive delivery of endogenous AMPA-Rs (25).

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These results demonstrate that incorporation of GluR1-containing AMPA-Rs into synapses is a major mechanism underlying the plasticity produced by activation of CaMKII and LTP. This process requires phosphorylation of protein(s) other than GluR1. Furthermore, this delivery requires interactions between the COOH-terminus of GluR1 and PDZ domain proteins.

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Kessels, Kopec, Klein, Malinow • 2009 • Nat Neurosci. - PDF

Roles of stargazin and phosphorylation in the control of AMPA receptor subcellular distribution

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Abstract

• Understanding how the subcellular fate of newly synthesized AMPA receptors (AMPARs) is controlled is important for elucidating the mechanisms of neuronal function. We examined the effect of increased synthesis of AMPAR subunits on their subcellular distribution in rat hippocampal neurons. Virally expressed AMPAR subunits (GluR1 or GluR2) accumulated in cell bodies and replaced endogenous dendritic AMPAR with little effect on total dendritic amounts and caused no change in synaptic transmission. Coexpressing stargazin (STG) or mimicking GluR1 phosphorylation enhanced dendritic GluR1 levels by protecting GluR1 from lysosomal degradation. However, STG interaction or GluR1 phosphorylation did not increase surface or synaptic GluR1 levels. Unlike GluR1, STG did not protect GluR2 from lysosomal degradation or increase dendritic GluR2 levels. In general, AMPAR surface levels, and not intracellular amounts, correlated strongly with synaptic levels. Our results suggest that AMPAR surface expression, but not its intracellular production or accumulation, is critical for regulating synaptic transmission.

Kopec, Real, Kessels, Malinow • 2007 • J Neuro - PDF

GluR1 links structural and functional plasticity at excitatory synapses

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Abstract

• Long-term potentiation (LTP), a cellular model of learning and memory, produces both an enhancement of synaptic function and an increase in the size of the associated dendritic spine. Synaptic insertion of AMPA receptors is known to play an important role in mediating the increase in synaptic strength during LTP, whereas the role of AMPA receptor trafficking in structural changes remains unexplored. Here, we examine how the cell maintains the correlation between spine size and synapse strength during LTP. We found that cells exploit an elegant solution by linking both processes to a single molecule: the AMPA-type glutamate receptor subunit 1 (GluR1). Synaptic insertion of GluR1 is required to permit a stable increase in spine size, both in hippocampal slice cultures and in vivo. Synaptic insertion of GluR1 is not sufficient to drive structural plasticity. Although crucial to the expression of LTP, the ion channel function of GluR1 is not required for the LTP-driven spine size enhancement. Remarkably, a recombinant cytosolic C-terminal fragment (C-tail) of GluR1 is driven to the postsynaptic density after an LTP stimulus, and the synaptic incorporation of this isolated GluR1 C-tail is sufficient to permit spine enlargement even when postsynaptic exocytosis of endogenous GluR1 is blocked. We conclude that during plasticity, synaptic insertion of GluR1 has two functions: the established role of increasing synaptic strength via its ligand-gated ion channel, and a novel role through the structurally stabilizing effect of its C terminus that permits an increase in spine size.

Kopec, Real, Kessels, Malinow • 2007 • J Neuro - PDF

GluR1 links structural and functional plasticity at excitatory synapses

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Abstract

• Long-term potentiation (LTP), a cellular model of learning and memory, produces both an enhancement of synaptic function and an increase in the size of the associated dendritic spine. Synaptic insertion of AMPA receptors is known to play an important role in mediating the increase in synaptic strength during LTP, whereas the role of AMPA receptor trafficking in structural changes remains unexplored. Here, we examine how the cell maintains the correlation between spine size and synapse strength during LTP. We found that cells exploit an elegant solution by linking both processes to a single molecule: the AMPA-type glutamate receptor subunit 1 (GluR1). Synaptic insertion of GluR1 is required to permit a stable increase in spine size, both in hippocampal slice cultures and in vivo. Synaptic insertion of GluR1 is not sufficient to drive structural plasticity. Although crucial to the expression of LTP, the ion channel function of GluR1 is not required for the LTP-driven spine size enhancement. Remarkably, a recombinant cytosolic C-terminal fragment (C-tail) of GluR1 is driven to the postsynaptic density after an LTP stimulus, and the synaptic incorporation of this isolated GluR1 C-tail is sufficient to permit spine enlargement even when postsynaptic exocytosis of endogenous GluR1 is blocked. We conclude that during plasticity, synaptic insertion of GluR1 has two functions: the established role of increasing synaptic strength via its ligand-gated ion channel, and a novel role through the structurally stabilizing effect of its C terminus that permits an increase in spine size.

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RANDOM NOTES

Proteins that interact with AMPARs

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Song and Huganir 2002. Use of yeast two-hybrid method to identify AMPA-receptor-interacting proteins was crucial for the rapid progress in this field and has helped to identify a large complex of such proteins. AMPA-receptor-associated protein complex. AMPA receptors are associated with a large protein network. The GluR2 subunit specifically binds to several proteins, including glutamatereceptor- interacting protein (GRIP) 1, GRIP2, protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive factor (NSF). GRIP1, GRIP2 and PICK1 in turn bind to other proteins, including GRIP-associated proteins (GRASPs), EphB receptor tyrosine kinases, ephrins, kinesin 5 (KIF5) and protein kinase Cα (PKCα). The GluR1 subunit binds to synapse-associated protein 97 (SAP97, also known as hDLG) and protein 4.1 (4.1 N). All four AMPA-receptor subunits bind to neuronal activity regulated pentraxin (NARP) and stargazin. Stargazin binds, in turn, to the synaptic scaffolding protein postsynaptic density 95 (PSD95). These associated proteins appear to play an important role in the membrane trafficking of the receptors by escorting the receptor from the cell body to the synapse. In addition, this large complex might regulate novel downstream signal transduction pathways that emanate from the AMPA receptor. Abbreviations: CC, coiled-coil domain; GK, guanylate kinase domain; PDZ, PSD95/Dlg/ZO1 domain; SH3, SRC homology 3 domain.

Qdots

• Quantum Dots

• Qdot 655 Goat F(ab')2 Anti-Rabbit IgG Conjugate
• Qdot Methods Handbook
• Qdot Color Visualizer

Getting a Qdot into the cell

1. Conjugate Qdot with secondary antibody fab
2. Incubate tissue with primary antibodies for AMPAR and PSD95
3. Puff Qdots onto cell body, these will bind the primary at AMPAR N-terminus
4. When AMPARs internalize the Qdot will be dragged into cell
5. Cleave N-terminus of AMPAR to liberate Qdot
6. Qdot can then bind the primary ligated to PSD95

Notes

• Molecular Methods
• FLASH technology
• Bredt
• minisog - gfp
• Acidic basic polypeptide recognition sequences
• Talk with nanotech group about various ways to conj. Qdots
• Nichol and England - couple Qdot to AMPAR agonist
• Have simulation be a competitive model where AMPARs are competing during LTP
• Quantitative review on synaptic numbers (Sheng)

PALM STORM

There are two major groups of methods for functional super-resolution microscopy:

1. Deterministic super-resolution: The most commonly used emitters in biological microscopy, fluorophores, show a nonlinear response to excitation, and this nonlinear response can be exploited to enhance resolution. These methods include STED, GSD, RESOLFT and SSIM.

2. Stochastical super-resolution PALM STORM: The chemical complexity of many molecular light sources gives them a complex temporal behaviour, which can be used to make several close-by fluorophores emit light at separate times and thereby become resolvable in time. These methods include SOFI and all single-molecule localization methods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM and dSTORM.

NRSA

• Dominant negative PSD95