Qual Journals - Revision history

Bradley Monk: /* Bianchetta • Lam • Jones • Morabito • 2011 • JNeuro */

2013-06-05T23:23:04Z

Bianchetta • Lam • Jones • Morabito • 2011 • JNeuro

← Older revision		Revision as of 16:23, 5 June 2013
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	===Abstract===		===Abstract===
	The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. [[PSD-95]] is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. [[PSD-95]] is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient [[PSD-95]] ubiquitination has been implicated in NMDA receptor-induced [[AMPA Receptor\|AMPA receptor]] endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with [[PSD-95]] and enhances [[PSD-95]] ubiquitination without affecting [[PSD-95]] protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated [[PSD-95]] at synapses. We show that [[PSD-95]] ubiquitination correlates with increased interaction with β -adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating [[PSD-95]] ubiqutination and its interaction with AP-2 and suggest a mechanism by which [[PSD-95]] may regulate NMDA receptor-induced [[AMPA Receptor\|AMPA receptor]] endocytosis.		The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. [[PSD-95]] is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. [[PSD-95]] is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient [[PSD-95]] ubiquitination has been implicated in NMDA receptor-induced [[AMPA Receptor\|AMPA receptor]] endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with [[PSD-95]] and enhances [[PSD-95]] ubiquitination without affecting [[PSD-95]] protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated [[PSD-95]] at synapses. We show that [[PSD-95]] ubiquitination correlates with increased interaction with β -adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating [[PSD-95]] ubiqutination and its interaction with AP-2 and suggest a mechanism by which [[PSD-95]] may regulate NMDA receptor-induced [[AMPA Receptor\|AMPA receptor]] endocytosis.

			==Intro==
			PSD-95 (SAP90) is a major postsynaptic scaffolding protein of glutamatergic synapses and a substrate of Cdk5. PSD-95 has been implicated in synaptic maturation and regulation of synaptic strength and plasticity. The importance of PSD-95 in synaptic plasticity is underscored by the inhibition of NMDA receptor (NMDAR)-induced AMPA receptor (AMPAR) internalization and the impairment of LTD following PSD-95 knockdown. The rapid and transient ubiquitination of PSD-95 by the Ubiquitin E3 Ligase Mdm2 has been implicated in NMDAR-induced endocytosis of AMPARs, but the mechanisms regulating this posttranslational modification of PSD-95 are still unclear. Since Cdk5 is inactivated by NMDAR stimulation, we investigated whether inactivation of Cdk5 promotes PSD-95 ubiquitination. In this study we report that PSD-95 is ubiquitinated in neurons with reduced Cdk5 activity without affecting PSD-95 protein levels in vivo . We also show that PSD-95 ubiquitination correlates with increased interaction of PSD-95 with β -adaptin, a subunit of the clathrin adaptor protein complex AP-2, and that this interaction is increased under reduced Cdk5 activity or by NMDAR stimulation and is dependent on Mdm2. Together these results suggest a non-proteolytic signaling function for PSD-95 ubiquitination and support a novel function for Cdk5 in the regulation of glutamatergic synapses.

Bradley Monk at 23:20, 5 June 2013

2013-06-05T23:20:04Z

← Older revision		Revision as of 16:20, 5 June 2013
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	;*AMPA auxiliary proteins		;*AMPA auxiliary proteins

	The AMPA auxiliary proteins are another group of molecules important for [[AMPAR]] targeting to the postsynaptic membrane. They include the transmembrane [[AMPAR]] regulatory proteins (TARPs), cornichon-like proteins, neuropilin, and tolloid-like proteins [79 , 80 , 81 , 82 , 83 , 84 ]. TARPs regulate [[AMPAR]] trafficking and channel kinetics and are classified into six isoforms—type 1a (γ -2/stargazin and γ -3), type 1b (γ -4 and γ -8), and type 2 (γ -5 and γ -7)— according to how they control [[AMPAR]] trafficking and channel properties [85 , 86 ]. They stabilize AMPARs at the postsynaptic membrane by direct interaction with scaffolding protein [[PSD95]] and other MAGUKs. TARPs also slow deactivation and desensitization, thereby increasing [[AMPAR]] conductance, and influencing [[AMPAR]] affinity for pharmacological agents [85 , 87 , 88 , 89 , 90 ]. Synaptic TARP phosphorylation is activity regulated and in turn phosphorylation influences stargazin binding to PSD-95 [91 , 92 , 93 ]. CaMKIIdependent phosphorylation of stargazin retains AMPARs at postsynaptic sites by reducing [[AMPAR]] diffusion [94 ]. Interaction of AMPARs with the cornichon-like proteins CNIH2 and CNIH3 was recently reported by Schwenk et al. [95 ], who showed that this interaction is involved in the regulation of [[AMPAR]] expression in the postsynaptic membrane, and also influences [[AMPAR]] channel properties. CNIH2 and CNIH3 bind AMPARs which in turn complex with TARPγ 4 in the [[hippocampus]] [96 ]. In this complex, the cornichon-like proteins control [[AMPAR]] trafficking to the postsynaptic membrane, whereas TARPγ 4 stabilizes the CNIH2/3–[[AMPAR]] interaction in the membrane [97 ].		The AMPA auxiliary proteins are another group of molecules important for [[AMPAR]] targeting to the postsynaptic membrane. They include the transmembrane [[AMPAR]] regulatory proteins (TARPs), cornichon-like proteins, neuropilin, and tolloid-like proteins [79 , 80 , 81 , 82 , 83 , 84 ]. TARPs regulate [[AMPAR]] trafficking and channel kinetics and are classified into six isoforms—type 1a (γ -2/stargazin and γ -3), type 1b (γ -4 and γ -8), and type 2 (γ -5 and γ -7)— according to how they control [[AMPAR]] trafficking and channel properties [85 , 86 ]. They stabilize AMPARs at the postsynaptic membrane by direct interaction with scaffolding protein [[PSD95]] and other MAGUKs. TARPs also slow deactivation and desensitization, thereby increasing [[AMPAR]] conductance, and influencing [[AMPAR]] affinity for pharmacological agents [85 , 87 , 88 , 89 , 90 ]. Synaptic TARP phosphorylation is activity regulated and in turn phosphorylation influences stargazin binding to [[PSD-95]] [91 , 92 , 93 ]. CaMKIIdependent phosphorylation of stargazin retains AMPARs at postsynaptic sites by reducing [[AMPAR]] diffusion [94 ]. Interaction of AMPARs with the cornichon-like proteins CNIH2 and CNIH3 was recently reported by Schwenk et al. [95 ], who showed that this interaction is involved in the regulation of [[AMPAR]] expression in the postsynaptic membrane, and also influences [[AMPAR]] channel properties. CNIH2 and CNIH3 bind AMPARs which in turn complex with TARPγ 4 in the [[hippocampus]] [96 ]. In this complex, the cornichon-like proteins control [[AMPAR]] trafficking to the postsynaptic membrane, whereas TARPγ 4 stabilizes the CNIH2/3–[[AMPAR]] interaction in the membrane [97 ].

	===AMPAR trafficking at nascent versus mature synapses===		===AMPAR trafficking at nascent versus mature synapses===
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	;*Soluble released factors		;*Soluble released factors

	BDNF was the first soluble molecule shown to have a role in homeostatic plasticity [197, 223]. In particular, Rutherford et al. [197] showed that low levels of BDNF trigger synaptic up-scaling in cultured cortical pyramidal neurons. In fact, scavenging endogenous BDNF with a soluble form of its receptor (TrkB-IgG) mimicked activity deprivation and induced up-scaling of excitatory synapses; while exogenous BDNF blocked the synaptic up-scaling that follows chronic activity blockade with TTX. BDNF is also important for the pre-synaptic enhancement that follows chronic activity deprivation. Treatment of cultured hippocampal neurons with the [[AMPAR]] blocker NBQX causes GluA1 accumulation at the synapse, which creates the conditions for retrograde signaling once the block is removed. Factors essential for this signaling include calcium (which enters through GluA1 homotetramers), BDNF, and NO [221]. [[AMPAR]] blockade triggers BDNF synthesis, which drives presynaptic scaling via its presynaptic receptor TrkB. TrkB signaling, together with calcium influx, which enters through P/N/Q presynaptic channels, accelerate synaptic vesicle turnover [224]. TNF-α is a well-known regulator of [[AMPAR]] trafficking [225, 226, 227, 228]. Moreover, TNF-α released from glial cells has been shown to be essential for the up-scaling reaction to TTX—a process that does not occur in TNF-α KO mice [229]. The mechanism by which TNF-α exerts these effects is unclear. β3-integrin could be involved, since acute treatment with TNF-α increases surface levels of β3-integrin: this molecule is known to be required for synaptic scaling [230], and its surface expression correlates with the quantity of [[AMPAR]] at the post-synaptic membrane [230]. Steinmetz and Turrigiano [231] recently proposed that TNF-α’s role in synaptic scaling is not instructive but permissive: it would maintain synapses in a plastic state that allows synaptic compensation to occur. In support of their hypothesis, it has been found that chronic TNF-α signaling blockade alters PSD composition, increasing SAP102 and decreasing PSD-95 expression [231] so that they resemble immature synapses [101], which would in turn have consequences for [[AMPAR]] distribution in the synapse. RA has been identified as a regulator of dendritic protein synthesis in homeostatic plasticity. Following activity blockade with TTX or (2R)-amino-5-phosphonovaleric acid (APV), RA synthesis is enhanced both in cultured hippocampal neurons and [[brain]] slices [217]. In turn, RA enhances the local synthesis of GluA1 (but not GluA2), thus favoring its insertion into the membrane. RA acts through its receptor RARalpha [217] and the fragile X-mental retardation protein FMRP acts downstream of the RA pathway and is necessary for GluA1 synthesis, suggesting that deregulation of homeostatic plasticity might contribute to the pathogenesis of fragile X syndrome [232].		BDNF was the first soluble molecule shown to have a role in homeostatic plasticity [197, 223]. In particular, Rutherford et al. [197] showed that low levels of BDNF trigger synaptic up-scaling in cultured cortical pyramidal neurons. In fact, scavenging endogenous BDNF with a soluble form of its receptor (TrkB-IgG) mimicked activity deprivation and induced up-scaling of excitatory synapses; while exogenous BDNF blocked the synaptic up-scaling that follows chronic activity blockade with TTX. BDNF is also important for the pre-synaptic enhancement that follows chronic activity deprivation. Treatment of cultured hippocampal neurons with the [[AMPAR]] blocker NBQX causes GluA1 accumulation at the synapse, which creates the conditions for retrograde signaling once the block is removed. Factors essential for this signaling include calcium (which enters through GluA1 homotetramers), BDNF, and NO [221]. [[AMPAR]] blockade triggers BDNF synthesis, which drives presynaptic scaling via its presynaptic receptor TrkB. TrkB signaling, together with calcium influx, which enters through P/N/Q presynaptic channels, accelerate synaptic vesicle turnover [224]. TNF-α is a well-known regulator of [[AMPAR]] trafficking [225, 226, 227, 228]. Moreover, TNF-α released from glial cells has been shown to be essential for the up-scaling reaction to TTX—a process that does not occur in TNF-α KO mice [229]. The mechanism by which TNF-α exerts these effects is unclear. β3-integrin could be involved, since acute treatment with TNF-α increases surface levels of β3-integrin: this molecule is known to be required for synaptic scaling [230], and its surface expression correlates with the quantity of [[AMPAR]] at the post-synaptic membrane [230]. Steinmetz and Turrigiano [231] recently proposed that TNF-α’s role in synaptic scaling is not instructive but permissive: it would maintain synapses in a plastic state that allows synaptic compensation to occur. In support of their hypothesis, it has been found that chronic TNF-α signaling blockade alters PSD composition, increasing SAP102 and decreasing [[PSD-95]] expression [231] so that they resemble immature synapses [101], which would in turn have consequences for [[AMPAR]] distribution in the synapse. RA has been identified as a regulator of dendritic protein synthesis in homeostatic plasticity. Following activity blockade with TTX or (2R)-amino-5-phosphonovaleric acid (APV), RA synthesis is enhanced both in cultured hippocampal neurons and [[brain]] slices [217]. In turn, RA enhances the local synthesis of GluA1 (but not GluA2), thus favoring its insertion into the membrane. RA acts through its receptor RARalpha [217] and the fragile X-mental retardation protein FMRP acts downstream of the RA pathway and is necessary for GluA1 synthesis, suggesting that deregulation of homeostatic plasticity might contribute to the pathogenesis of fragile X syndrome [232].

	;*Cell adhesion molecules (CAMS)		;*Cell adhesion molecules (CAMS)
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	;*PSD scaffolding proteins		;*PSD scaffolding proteins

	Scaffolding proteins are crucial for the structural organization of the PSD. They are also involved in the trafficking of receptors to the postsynaptic membrane and their stabilization there [240]. PICK1 and MAGUK scaffolding proteins have recently been shown to be crucially involved in homeostatic plasticity [222, 241]. Anggono et al. [222] showed that PICK1 is specifically required for inactivity-induced (TTX treatment) [[AMPAR]] up-scaling but not for hyperactivity-induced (bicucullin treatment) down-scaling. PICK1 is down-regulated during synaptic up-scaling, enabling GluA2/3 recruitment from the intracellular pool to the membrane. Furthermore, in PICK1 KO neurons, [[AMPAR]] composition and trafficking are impaired and the regulatory mechanisms responsible for homeostatic plasticity are compromised. Sun et al. [241] recently explored the role of the MAGUK scaffolding proteins PSD93 and [[PSD95]] in homeostatic plasticity in neocortical pyramidal neurons. These proteins were found to be essential for assembly of the protein–protein association network, which is required for homeostatic adjustments of [[AMPAR]] abundance in the synapse. PSD-95 was found necessary for down-scaling, whereas PSD-95 and PSD-93 were both involved in synaptic up-scaling [241].		Scaffolding proteins are crucial for the structural organization of the PSD. They are also involved in the trafficking of receptors to the postsynaptic membrane and their stabilization there [240]. PICK1 and MAGUK scaffolding proteins have recently been shown to be crucially involved in homeostatic plasticity [222, 241]. Anggono et al. [222] showed that PICK1 is specifically required for inactivity-induced (TTX treatment) [[AMPAR]] up-scaling but not for hyperactivity-induced (bicucullin treatment) down-scaling. PICK1 is down-regulated during synaptic up-scaling, enabling GluA2/3 recruitment from the intracellular pool to the membrane. Furthermore, in PICK1 KO neurons, [[AMPAR]] composition and trafficking are impaired and the regulatory mechanisms responsible for homeostatic plasticity are compromised. Sun et al. [241] recently explored the role of the MAGUK scaffolding proteins PSD93 and [[PSD95]] in homeostatic plasticity in neocortical pyramidal neurons. These proteins were found to be essential for assembly of the protein–protein association network, which is required for homeostatic adjustments of [[AMPAR]] abundance in the synapse. [[PSD-95]] was found necessary for down-scaling, whereas [[PSD-95]] and PSD-93 were both involved in synaptic up-scaling [241].

	;*Intracellular signaling molecules		;*Intracellular signaling molecules
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	==Bats • Groc • Choquet • 2007 • Cell: Neuron==		==Bats • Groc • Choquet • 2007 • Cell: Neuron==
	{{SlideBox\|The Interaction between Stargazin and PSD-95 Regulates [[AMPA Receptor]] Surface Trafficking\|		{{SlideBox\|The Interaction between Stargazin and [[PSD-95]] Regulates [[AMPA Receptor]] Surface Trafficking\|

	===Abstract===		===Abstract===
	Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an [[AMPAR]] auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate [[AMPAR]] number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of [[AMPAR]] by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 [[AMPAR]] subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases [[AMPAR]] surface diffusion, preventing [[AMPAR]] accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin- PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.		Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an [[AMPAR]] auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as [[PSD-95]]. How [[PSD-95]] and Stargazin regulate [[AMPAR]] number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of [[AMPAR]] by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with [[PSD-95]] and not upon the GluR2 [[AMPAR]] subunit C terminus. Disruption of interactions between Stargazin and [[PSD-95]] strongly increases [[AMPAR]] surface diffusion, preventing [[AMPAR]] accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin- [[PSD-95]] interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.


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	==Bianchetta • Lam • Jones • Morabito • 2011 • JNeuro==		==Bianchetta • Lam • Jones • Morabito • 2011 • JNeuro==
	{{SlideBox\|Cdk5 regulates PSD-95 ubiquitination in neurons\|		{{SlideBox\|Cdk5 regulates [[PSD-95]] ubiquitination in neurons\|

	===Abstract===		===Abstract===
	~~Accumulation of AMPA receptors at synapses~~ is a ~~fundamental feature~~ of glutamatergic synaptic ~~transmission~~. ~~Stargazin, a member of the TARP family, is an~~ [[~~AMPAR~~]] ~~auxiliary subunit allowing interaction of~~ the ~~receptor with scaffold proteins of the postsynaptic density~~, ~~such as~~ PSD-95~~. How PSD~~-~~95 and Stargazin regulate~~ [[~~AMPAR~~]] ~~number in synaptic membranes remains elusive~~. ~~We show, using single quantum dot and FRAP imaging in live hippocampal neurons,~~ that ~~exchange~~ of ~~[[AMPAR]] by lateral diffusion between extrasynaptic and synaptic sites mostly depends on~~ the interaction of ~~Stargazin~~ with PSD-95 and ~~not upon the GluR2~~ [[~~AMPAR~~]] ~~subunit C terminus. Disruption of interactions between Stargazin and~~ PSD-95 ~~strongly increases [[AMPAR~~]] ~~surface diffusion~~, ~~preventing~~ [[~~AMPAR~~]] ~~accumulation~~ at ~~postsynaptic sites~~. ~~Furthermore~~, ~~AMPARs~~ and ~~Stargazin diffuse as complexes in and out synapses~~. ~~These~~ results ~~propose~~ a ~~model~~ in ~~which the Stargazin-~~ PSD-95 interaction ~~plays~~ a ~~key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density~~.		The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. [[PSD-95]] is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. [[PSD-95]] is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient [[PSD-95]] ubiquitination has been implicated in NMDA receptor-induced [[AMPA Receptor\|AMPA receptor]] endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with [[PSD-95]] and enhances [[PSD-95]] ubiquitination without affecting [[PSD-95]] protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated [[PSD-95]] at synapses. We show that [[PSD-95]] ubiquitination correlates with increased interaction with β -adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating [[PSD-95]] ubiqutination and its interaction with AP-2 and suggest a mechanism by which [[PSD-95]] may regulate NMDA receptor-induced [[AMPA Receptor\|AMPA receptor]] endocytosis.

Bradley Monk at 23:16, 5 June 2013

2013-06-05T23:16:05Z

Show changes

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T07:13:11Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

← Older revision		Revision as of 00:13, 29 May 2013
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	;The cell-biological mechanisms underlying synaptic plasticity have been difficult to delineate. In part, this is due to the lack of techniques in intact preparations allowing molecular perturbations with spatial and temporal control, as well as the absence of assays for specific molecular events linked to synaptic plasticity. Here, we generated electrophysiologically tagged receptors to monitor their synaptic delivery during LTP and increased CaMKII. In the absence of plasticityinducing stimuli, we saw no evidence for their contribution to transmission. This is consistent with previous results indicating that in the absence of evoked activity, GluR1 is retained within the dendrite (3). Upon coexpression with constitutively active CaMKII or following LTP induction, we see that tagged receptors contribute to transmission, indicating their delivery to synapses.		;The cell-biological mechanisms underlying synaptic plasticity have been difficult to delineate. In part, this is due to the lack of techniques in intact preparations allowing molecular perturbations with spatial and temporal control, as well as the absence of assays for specific molecular events linked to synaptic plasticity.
			Here, we generated electrophysiologically tagged receptors to monitor their synaptic delivery during LTP and increased CaMKII. In the absence of plasticityinducing stimuli, we saw no evidence for their contribution to transmission. This is consistent with previous results indicating that in the absence of evoked activity, GluR1 is retained within the dendrite (3). Upon coexpression with constitutively active CaMKII or following LTP induction, we see that tagged receptors contribute to transmission, indicating their delivery to synapses.

	;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.		;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.

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T07:12:07Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

← Older revision		Revision as of 00:12, 29 May 2013
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	*Cells expressing GluR1(AGL) showed a short-lasting potentiation that decayed over the next 20 min		*Cells expressing GluR1(AGL) showed a short-lasting potentiation that decayed over the next 20 min
	*45 min after pairing, the responses were significantly depressed from baseline levels		*45 min after pairing, the responses were significantly depressed from baseline levels


			;The cell-biological mechanisms underlying synaptic plasticity have been difficult to delineate. In part, this is due to the lack of techniques in intact preparations allowing molecular perturbations with spatial and temporal control, as well as the absence of assays for specific molecular events linked to synaptic plasticity. Here, we generated electrophysiologically tagged receptors to monitor their synaptic delivery during LTP and increased CaMKII. In the absence of plasticityinducing stimuli, we saw no evidence for their contribution to transmission. This is consistent with previous results indicating that in the absence of evoked activity, GluR1 is retained within the dendrite (3). Upon coexpression with constitutively active CaMKII or following LTP induction, we see that tagged receptors contribute to transmission, indicating their delivery to synapses.

			;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.

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T07:08:21Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

← Older revision	Revision as of 00:08, 29 May 2013
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		*To determine if LTP delivers AMPARs to synapses, we examined LTP in cells expressing GluR1
		*Whole-cell recordings were obtained from cells expressing or not expressing GluR1.
		*LTP was induced with a pairing protocol isolating pure AMPAR–mediated responses
		*The holding potential was then switched to measure rectification of the AMPAR–mediated responses
		*Similar to the effect of coexpressed CaMKII and GluR1, rectification was increased after LTP in cells expressing GluR1 compared to cells not expressing
		*We also examined the effects of GluR1(AGL) on LTP.
		*we recorded synaptic responses from cells expressing either GluR1 or GluR1(AGL)
		*After pairing, cells expressing the control construct displayed stable potentiation lasting at least 50 min
		*Cells expressing GluR1(AGL) showed a short-lasting potentiation that decayed over the next 20 min
		*45 min after pairing, the responses were significantly depressed from baseline levels

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T06:57:57Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

← Older revision		Revision as of 23:57, 28 May 2013
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	;[[Electrophysiology]] assay		;[[Electrophysiology]] assay
	*The current-voltage (I-V ) relationship of ~~AMPA-Rs~~ is determined by the GluR2 subunit:		*The current-voltage (I-V ) relationship of AMPARs is determined by the GluR2 subunit
	*~~AMPA-Rs~~ with GluR2 show linear I-V relations; ~~AMPA-Rs~~ lacking GluR2 show little outward current at +40 mV		*AMPARs with GluR2 show linear I-V relations; AMPARs lacking GluR2 show little outward current at +40 mV
	*Most ~~AMPA-Rs~~ in hippocampal pyramidal cells contain the GluR2 subunit		**GluR1 is [http://en.wikipedia.org/wiki/Inward-rectifier_potassium_ion_channel Inward Rectifying]
	*We overexpressed the AMPA-R GluR1 subunit in hippocampal slice neurons		*Most AMPARs in hippocampal pyramidal cells contain the GluR2 subunit
	*incorporation of these recombinant receptors into synapses would be expected to increase rectification of synaptic responses

			*We overexpressed the AMPAR GluR1 subunit in hippocampal slice neurons
			**incorporation of recombinant receptors into synapses would be expected to increase rectification
			*GluR1-GFP is widely distributed throughout the dendritic arbor, but little is incorporated into synapses in the absence of activity
			*In agreement with this, expression of GluR1-GFP had no effect on either the amplitude or rectification of synaptic transmission

	*GluR1-GFP is widely distributed throughout the dendritic arbor, but little is incorporated into synapses in the absence of activity (3).
	*In agreement with this, expression of GluR1-GFP had no effect on either the amplitude or rectification of synaptic transmission
	*To determine if CaMKII activity could drive GluR1- GFP into synapses, we coexpressed GluR1-GFP and tCaMKII		*To determine if CaMKII activity could drive GluR1- GFP into synapses, we coexpressed GluR1-GFP and tCaMKII
	**used internal ribosomal entry site (IRES) construct		**used internal ribosomal entry site (IRES) construct
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	*Pairwise recordings from infected and noninfected cells showed that transmission was enhanced		*Pairwise recordings from infected and noninfected cells showed that transmission was enhanced
	*transmission showed increased rectification		*transmission showed increased rectification
	**indicating a contribution of ~~the~~ homomeric GluR1-GFP to transmission		**indicating a contribution of homomeric GluR1-GFP to transmission
	*rectification was due to coexpression of the two proteins		*rectification was due to coexpression of the two proteins
	**transmission onto cells expressing either tCaMKII or GluR1-GFP alone had rectification comparable to that in uninfected cells		**transmission onto cells expressing either tCaMKII or GluR1-GFP alone had rectification comparable to that in uninfected cells
	*These results show that CaMKII activity induces the insertion of homomeric GluR1-GFP into the synapse.		;*These results show that CaMKII activity induces the insertion of homomeric GluR1-GFP into the synapse.

			*GluR1 is phosphorylated by CaMKII at Ser831 during LTP.
			*To examine if phosphorylation is required for delivery, we substituted Ser831 with Ala
			**GluR1(S831A)-GFP
			*mutation did not block delivery
			*coexpression with tCaMKII showed the same increase in rectification as that seen previously


			*localization of many membrane proteins is controlled by a class of proteins containing PDZ domains
			*the cytosolic COOH-terminus has a consensus (S/T)X(V/L) (TGL)
			*Serine or threonine at 22 position appears to be crucial because a mutation at this site can prevent associations
			*The last three amino acids of the COOH-terminus of GluR1 are TGL
			*We converted the GluR1 COOH-terminus from TGL to AGL,
			*when expressed in HEK293 cells, formed functional AMPARs that showed the normal rectification
			*When expressed in hippocampal neurons, this protein was detected in dendrites
			*This construct showed no effect on transmission when expressed alone
			*when GluR1(AGL) and tCaMKII were coexpressed in hippocampal the effects of tCaMKII on synaptic response amplitude and rectification were completely blocked

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T06:40:27Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

← Older revision		Revision as of 23:40, 28 May 2013
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	**tCaMKII-GFP enhanced transmission		**tCaMKII-GFP enhanced transmission

	;~~electrophysiological~~ assay		;[[Electrophysiology]] assay
	*The current-voltage (I-V ) relationship of AMPA-Rs is determined by the GluR2 subunit:		*The current-voltage (I-V ) relationship of AMPA-Rs is determined by the GluR2 subunit:
	*AMPA-Rs with GluR2 show linear I-V relations; AMPA-Rs lacking GluR2 show little outward current at +40 mV		*AMPA-Rs with GluR2 show linear I-V relations; AMPA-Rs lacking GluR2 show little outward current at +40 mV

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T05:51:11Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

← Older revision		Revision as of 22:51, 28 May 2013
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	**tCaMKII-GFP enhanced transmission		**tCaMKII-GFP enhanced transmission

			;electrophysiological assay
			*The current-voltage (I-V ) relationship of AMPA-Rs is determined by the GluR2 subunit:
			*AMPA-Rs with GluR2 show linear I-V relations; AMPA-Rs lacking GluR2 show little outward current at +40 mV
			*Most AMPA-Rs in hippocampal pyramidal cells contain the GluR2 subunit
			*We overexpressed the AMPA-R GluR1 subunit in hippocampal slice neurons
			*incorporation of these recombinant receptors into synapses would be expected to increase rectification of synaptic responses


			*GluR1-GFP is widely distributed throughout the dendritic arbor, but little is incorporated into synapses in the absence of activity (3).
			*In agreement with this, expression of GluR1-GFP had no effect on either the amplitude or rectification of synaptic transmission
			*To determine if CaMKII activity could drive GluR1- GFP into synapses, we coexpressed GluR1-GFP and tCaMKII
			**used internal ribosomal entry site (IRES) construct
			*BHK cells expressing this construct showed increased constitutive CaMKII activity
			*Pairwise recordings from infected and noninfected cells showed that transmission was enhanced
			*transmission showed increased rectification
			**indicating a contribution of the homomeric GluR1-GFP to transmission
			*rectification was due to coexpression of the two proteins
			**transmission onto cells expressing either tCaMKII or GluR1-GFP alone had rectification comparable to that in uninfected cells
			*These results show that CaMKII activity induces the insertion of homomeric GluR1-GFP into the synapse.

Bradley Monk: /* Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science */

2013-05-29T04:15:48Z

Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science

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 ==Hayashi · Shi · Esteban · Piccini · Poncer · Malinow • 2000 • Science==
 {{SlideBox|Driving AMPA Receptors into Synapses by LTP and CaMKII: Requirement for GluR1 and PDZ Domain Interaction|
 ===Abstract===
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 }}<!-- END ARTICLE -->
 ==Bats • Groc • Choquet • 2007 • Cell: Neuron==