Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression

“…specific molecular model in which mGluR activation due to parallel fiber stimulation combined with calcium influx due to climbing fiber activation results in activation of PKCa and its targeting to synapses via the protein PICK1 (Jorntell and Hansel, 2006; Perez et al, 2001; Steinberg et al, 2006).”

“Similarly, whereas several forms of LTD depend on PICK1 (which binds to GluA2-containing AMPAR) (Steinberg et al. 2006; Terashima et al. 2008; Volk et al. 2010), scaling down is unaffected by loss of PICK1 (Anggono et al.”

“Anti-Hebbian LTD at cerebellar parallel fiberPurkinje cell synapses involves postsynaptic mGluRs, VSCCs, IP3Rs, and presynaptic CB1R activation but is expressed postsynaptically by AMPAR internalization (Safo and Regehr, 2005; Steinberg et al., 2006).”

“TNF-α is secreted by glia and also acts to increase AMPAR insertion. mice has solidified the idea that the phosphorylation of AMPARs is critical for synaptic plasticity and memory (Lee et al. 2003, Steinberg et al. 2006).”

“In addition, knockout of PICK1 or GRIP1 and 2 eliminated LTD expression (Steinberg et al., 2006; Takamiya et al., 2008).”

“persistent phosphorylation of AMPA receptors at a particular serine residue, which disrupts AMPA receptor clusters and favours receptor endocytosis (Launey et al., 2004; Steinberg et al., 2006).”

“Expression of dominant-negative dynamin 2 inhibits AMPAR endocytosis (Carroll et al., 1999), and interfering dynamin peptides block cerebellar LTD (Wang and Linden, 2000).”

“It is well established that PICK1–GluA2 interaction is required for both hippocampal and cerebellar LTD [40,60 ,61 ,62 ,63].”

“Altogether, these observations point to a role of PICK1 in regulating the intracellular pool of AMPARs after endocytosis, which in turn, can indirectly impact AMPAR internalization.”

“To date, impaired LTD has only been shown in young PICK1 KO, GluR2D7 KI, and GluR2K882A KI mutants (Steinberg et al., 2006), whereas all the behavioral experiments described herein have been performed in adults.”

“These distinct binding properties of PICK1 and GRIP/ ABP are important for AMPA receptor removal during long-term depression (LTD) (Xia et al., 2000; Kim et al., 2001; Chung et al., 2003; Steinberg et al., 2006) and for rapid plasticity Newpher and Ehlers Page 12”

“Mutations in genes encoding BAR domain proteins have been linked to many diseases [9–11], and inactivation of these proteins in cells and animals is often characterized by severe phenotypes resulting from altered membrane dynamics [12–14].”

“Interestingly, both the PDZ and the BAR domains were required for LTD (Steinberg et al, 2006).”

“The GluR2 C-tail is involved in some forms of long-term depression (LTD) induction (Xia et al., 2000; Kim et al., 2001; Chung et al., 2003; Steinberg et al., 2006), raising the possibility that scaling up of synaptic strengths during TTX treatment could be because of block of ongoing LTD in our cultures.”

“Inhibition of Arp2/3-mediated actin polymerization by PICK1 is required for NMDA-induced AMPAR endocytosis As PICK1 is crucial for regulated AMPAR endocytosi”

“Thus, GluA2 phosphorylation is required for AMPAR internalization and its dephosphorylation is important in synaptic retention.(55)”

“PICK1’s role in AMPA receptor trafficking and synaptic plasticity has been further supported by data from Pick1 -knockout mice, which were found to have defects in AMPA receptor trafficking and synaptic plasticity (15, 16).”

“…density-95/Discs large/zona occludens-1 (PDZ)domain containing proteins, glutamate receptor interacting protein 1/2 (GRIP1/2) and PICK1, and modulates the membrane trafficking of GluR2 and the expression of LTD (Xia et al., 2000; Chung et al., 2003; Seidenman et al., 2003; Steinberg et al., 2006).”

“One of the best-defined roles for PICK1 is in mGluR-dependent LTD at parallel fiberPurkinje cell synapses in the cerebellum, expression of which requires GluR2-PICK1 interactions for PKC-dependent AMPAR internalization (Chung et al., 2003; Steinberg et al., 2006; Xia et al., 2000).”

“PDZ domain proteins are heavily implicated in many aspects of neuronal regulation, but are especially known to control the targeting and trafficking of glutamate receptors (Kornau et al., 1995; Dong et al., 1997; Srivastava et al., 1998; Steinberg et al., 2006; Daw et al., 2000; Osten et al., 2000; Wyszinski et al., 2…”

“In addition, targeted in vivo mutations of AMPAR subunits demonstrate that the protein interactions and phosphorylations at the c-tail control their synaptic targeting (Lee et al. 2003; Steinberg et al. 2006).”

“It has been shown that PICK1 interacts with lipids via the BAR domain and the PDZ domain, independently (Jin et al., 2006; Pan et al., 2007; Steinberg et al., 2006).”

“In the case of hippocampal and parallel fiber–Purkinje cell synapses, PICK1 is required for the removal of phosphorylated GluR2 receptors [77,78,81].”

“Male and female GluR2K882A KI mice are viable and fertile without any observable developmental defects (Steinberg et al., 2006).”

“It is possible that AMPARs may scale differentially depending on the patterns of phosphorylation that occurred during earlier LTP or LTD events.”

“Several forms of LTD are dependent on GluA2 C-tail interactions (Chung et al., 2003; Malenka and Bear, 2004; Steinberg et al., 2006; Yoon et al., 2009), but the dependence of L2/3 plasticity on GluA2 has not been assessed.”

“For LTD in the presence of AP5, AP5 was included throughout the experiment.”

“Specific positively charged lysine residues in the concave face of the BAR domain have been identified as mediating binding to negatively charged headgroups of phosphoinositides (Lu and Ziff, 2005; Jin et al., 2006; Steinberg et al., 2006).”

“…(TARP) auxiliary subunits at CA1 synapses (Lee et al., 2000; Tomita et al., 2005), or phosphorylation of GluA2 at Ser880 (Chung et al., 2003; Steinberg et al., 2006) at climbing fiber Purkinje cell synapses, leads to increased AMPAR endocytosis and reduced surface AMPAR expression, probably…”

“Interactions between GluA2 and PICK1 seem important for LTD (Steinberg et al., 2006), but the involvement of PICK1 in LTP and LTD is controversial (Terashima et al.”

“The physiological correlate of enhanced PICK1 binding is an increase in GluR2 endocytosis rate and a reduction in steady-state level of synaptic AMPAR (Kim et al., 2001; Steinberg et al., 2006).”

“…the cause of such slow development of LTD. Specifically, phosphorylation of PKC substrates, which presumably include AMPAR (Chung et al., 2003; Steinberg et al., 2006), during this time leads to internalization of AMPAR via clathrin-dependent endocytosis (Wang and Linden, 2000; Tanaka et al.,…”

“Instead, we favor a role for PICK1 in stabilizing internalized GluA2-containing AMPARs in intracellular pools, which may explain the LTD deficit in PICK1 knock-out mouse (Steinberg et al., 2006; Terashima et al., 2008; Volk et al., 2010).”

“Related to Figure 2, PCs lacking GluA1 or GluA3 have comparable spine density and show comparable levels of LTD induction compared to those in wild type (WT) PCs. (A) Representative confocal optical planes (0.5 µm thick) of PC distal dendrites used to quantify spine densities of WT, GluA1-KO or GluA3-KO PCs. (B) Densi…”

“To test the importance of the BAR domain, we mutated five BAR domain lysines to glutamic acids to disrupt binding to negatively charged head groups in membrane phosphoinositides (Lu and Ziff, 2005; Jin et al., 2006; Steinberg et al., 2006).”

“We next assayed for alterations in AMPA receptor phosphorylation utilizing antibodies directed to sites known to affect GluR1 or GluR2 function [18,19,20].”

“MHCI also binds to a variety of natural killer cell receptors, which are expressed on neurons, including the paired immunoglobulin-like receptor B (PirB) (Syken et al., 2006), Ly49 (Steinberg et al., 2006), and the killer cell-immunoglobulin receptor (KIR) (Bryceson et al., 2005).”

“2142-S1">Supplementary Methods ; Pick1 −/− mouse”

“For example, a well defined role for PICK1 in GluR2-containing AMPAR internalization exists at cerebellar parallel fiber–Purkinje neuron synapses (Xia et al., 2000; Steinberg et al., 2006), whereas stellate cells require PICK1 for synaptic delivery of GluR2-containing AMPARs (Gardner et al.”

“Jack Mellor, in whose lab the electrophysiology was carried out, is confident that he can address all of the referee's comments regarding the electrophysiology, which we will do in the formal rebuttal.”

“LTD and hippocampal spatial learning have all been shown to require AMPAR phosphorylation (8, 18, 35).”

“These findings strongly indicate the presence of a self-regulating mechanism by which Cp-AMPAR-mediated calcium flux triggers recruitment of normal GluA2-containing AMPARs to synapses.”

“Malenka RC, Bear MF (2004) LTP and LTD: An embarrassment of riches.”

“Furthermore, inactivation of PICK1 by gene ablation [28,30,46] or by pharmacological intervention [29] impairs LTD in the cerebellum as well as LTP and LTD in the CA1 region of the hippocampus.”

“PICK1-mediated GluA2 trafficking is also a crucial mechanism in cerebellar LTD (Steinberg et al., 2006; Xia et al., 2000), indicating the central importance of PICK1 in synaptic plasticity.”

“This combination of PDZ domain and BAR domain enables PICK1 to link its membrane cargos to trafficking vesicles, a feature found to be critical for AMPA receptor trafficking and synaptic plasticity [17,18].”

“A role for PICK1 in synaptic plasticity has been established most clearly in the cerebellum, where LTD at multiple synapses requires intact PICK1 function (18, 22, 23).”

“For example, it has been shown that PICK1 is a key regulator of postsynaptic GluR2 AMPA receptor endocytosis in cerebellar Purkinje cells (Steinberg et al., 2006; Xia et al., 2000) and in hippocampus (Kim et al.”

“Expression of a PICK1 BAR-domain mutant that is deficient in lipid binding impairs both hippocampal (15) and cerebellar LTD (8), suggesting a role for the PICK1 BAR domain in mediating the internalization, recycling, and/or intracellular retention of GluA2-containing AMPARs (1, 6).”

“Evidence from PICK1knockout mice, which had deficiencies in AMPA receptor trafficking and synaptic plasticity, further supported PICK1’s indispensable role in these two processes (Gardner et al, 2005; Steinberg et al, 2006).”

“Whereas wild-type PICK1 rescued the cerebellar LTD deficiency in PICK1 knock-out mice, lipid-binding-deficient PICK1 was unable to do so (Steinberg et al., 2006).”

“Finally, LAMP1-Rap1(ca)-IRES-GFP- but not CD8-Rap2(ca)-IRES-GFP-stimulated depression of AMPA transmission was prevented inGluA2(K882A) transgenic CA1 neurons (Figures 6G and 6J; Table S9), in which GluA2-mediated synaptic depression is impaired (Steinberg et al., 2006).”

“…cerebellar LTD did not show any significant morphological abnormalities of PC spines (Aiba et al., 1994; De Zeeuw et al., 1998; Feil et al., 2003; Hansel et al., 2006; Steinberg et al., 2006; van Woerden et al., 2009), suggesting that PF–PC LTD does not trigger morphological changes in PC spines.”

“Indeed, there is considerable evidence for a role of PICK1 in mGluR-LTD in a variety of brain regions, including the cerebellum [37,63,64], VTA [65] and perirhinal cortex [54].”

“The LTD induction signal triggers signaling cascades that involve the activation of enzymes such as PKC (protein kinase C), PP1 (protein phosphatase 1), and calcineurin (Winder and Sweatt, 2001; Song and Huganir, 2002; Steinberg et al., 2006).”

“Disruption of dynamin activity or mutations in the PICK1 gene prevent LTD expression (Wang and Linden 2000; Lin-den 2012; Steinberg et al. 2006).”

“In addition, mutant mice lacking the PKC isoform (Leitges et al., 2004), or PICK1 (Steinberg et al., 2006), do not express LTD.”

“However, GRIP1b palmitoylation is dispensable for cerebellar LTD as GRIP1bwt and nonpalmitoylatable (C11S) GRIP1b both rescue cerebellar LTD in GRIP1/GRIP2 double knock-out (DKO) PNs (DJL, GMT, RLH: our unpublished observations).”

“This interaction between PICK1 and GluR2 is important for cerebellar long-term depression (Steinberg et al. 2006; Yawata et al. 2006).”

“Because the NSF–GluR2 interaction may help maintain AMPARs at synapses (12, 34) and because interactions with GRIP/ABP and PICK1 may have diverse roles in stabilizing AMPARs at synapses or in intracellular compartments (1–6), we examined the cycling of AMPARs lacking GluR2/3 back to the plasma membrane after internali…”

“, 2003) because LTD is impaired by either inhibition of PKC activity or by genetic knock-out of GluR2 in mice (Chung et al., 2003; Gao et al., 2003; Steinberg et al., 2006).”

“Previous studies have shown that PICK1 has important roles in regulating the cytoskeleton and neuron morphology, and PICK1 has been implicated in several diseases (Steinberg et al, 2006; Nakamura et al, 2011; Rocca et al, 2013; Rocca and Hanley, 2015; Kunicka et al, 2016).”

“Changes in the expression, Ser-880 phosphorylation, and clustering of the Ca2+-impermeable GluA2 subunit of AMPA receptors, have been associated with the induction and expression of cerebellar LTD [32-37].”