Lithium increases synaptic GluA2 in hippocampal neurons by elevating the δ-catenin protein

Abstract

Lithium (Li⁺) is a drug widely employed for treating bipolar disorder, however the mechanism of action is not known. Here we study the effects of Li⁺ in cultured hippocampal neurons on a synaptic complex consisting of δ-catenin, a protein associated with cadherins whose mutation is linked to autism, and GRIP, an AMPA receptor (AMPAR) scaffolding protein, and the AMPAR subunit, GluA2. We show that Li⁺ elevates the level of δ-catenin in cultured neurons. δ-catenin binds to the ABP and GRIP proteins, which are synaptic scaffolds for GluA2. We show that Li⁺ increases the levels of GRIP and GluA2, consistent with Li⁺-induced elevation of δ-catenin. Using GluA2 mutants, we show that the increase in surface level of GluA2 requires GluA2 interaction with GRIP. The amplitude but not the frequency of mEPSCs was also increased by Li⁺ in cultured hippocampal neurons, confirming a functional effect and consistent with AMPAR stabilization at synapses. Furthermore, animals fed with Li⁺ show elevated synaptic levels of δ-catenin, GRIP, and GluA2 in the hippocampus, also consistent with the findings in cultured neurons. This work supports a model in which Li⁺ stabilizes δ-catenin, thus elevating a complex consisting of δ-catenin, GRIP and AMPARs in synapses of hippocampal neurons. Thus, the work suggests a mechanism by which Li⁺ can alter brain synaptic function that may be relevant to its pharmacologic action in treatment of neurological disease.

Keywords: AMPA receptor; GRIP; GluA2; Lithium; PICK1; δ-Catenin.

Figure 1. Li⁺ increases synaptic GluA2 and its associated scaffolding protein in cultured hippocampal neurons

a) Model for the regulation of synaptic GluA2 by Li⁺. The GluA2 AMPA receptor subunit is anchored at the synaptic membrane by an interaction of its C-terminus with the fifth PDZ domain of the scaffolding protein, GRIP, and the related ABP protein. The second PDZ domain of GRIP associates with the C-terminus of δ-catenin. δ-catenin binds to the juxtamembrane region of cadherin, which is a synaptic cell adhesion protein. Phosphorylation of δ-catenin by GSK3 leads to the degradation of δ-catenin, which disrupts the δ-catenin-GRIP scaffold at the synapse and lowers synaptic levels of GluA2. Inhibition of GSK3 by Li⁺ stabilizes δ-catenin, which elevates GRIP and GluA2 at the synapse. See Silverman et al., (2007) and the text for details. Representative immunoblots and quantitative analysis of b) P2 membrane fraction and c) synaptosome fraction from cultured hippocampal neurons treated with NaCl (control) or LiCl showing that LiCl treatment increased GluA2/3, GRIP, and δ-catenin levels (n=5 and 10 experiments in b) and c), respectively, *p<0.05 and ^**p<0.01, unpaired two-tailed Student’s t tests). GSK3β inhibition by LiCl was identified by an increase in its phosphorylation. d) Representative immunoblots of surface biotinylation and a summary graph for cultured hippocampal neurons treated with NaCl or LiCl showing that LiCl treatment increased surface GluA2/3 levels (n=4 experiments, *p<0.05, unpaired two-tailed Student’s t tests).

Figure 2. GRIP is required for a Li⁺ treatment-induced increase in surface GluA2

Schematic diagrams show the binding properties of MycGluA2 and its mutants (Greger et al, 2002) a) GluA2-SVKI (WT) binds to PICK1 and GRIP; b) GluA2-SVKE is unable to bind to either PICK1 or GRIP, and c) GluA2-AVKI is able to bind to PICK1 but not GRIP. Also given are representative confocal images of cultured hippocampal neurons expressing the various mutants of MycGluA2 from Sindbis virus vectors and treated with NaCl or LiCl showing surface GluA2 (green) and total GluA2 (red). Scale bar indicates 10μm. Higher magnification images of highlighted dendrites are shown below. Summary graphs show that: a) LiCl treatment was able to increase surface GluA2-SVKI (WT) levels in cultured hippocampal neurons (n=12 neurons, *p<0.05, unpaired two-tailed Student’s t tests), but b–c) LiCl treatment was unable to alter surface levels of mutant GluA2 (n=30 neurons in b) and 50 neurons in c)), suggesting that the ability of MycGluA2 to bind to GRIP is required for a Li⁺ treatment-induced increase in surface GluA2 in cultured hippocampal neurons.

Figure 3. PICK1 is not necessary for a Li⁺ treatment-induced increase in surface GluA2

a) Representative immunoblots of cell extracts from PICK1 knockdown (KD) cultured hippocampal neurons showing that PICK1 shRNA significantly reduced PICK1 protein levels in these neurons. b) Representative confocal images of hippocampal neurons treated with NaCl or LiCl in the presence of absence of PICK1 shRNA showing surface GluA2 (green) and total GluA2 (red). Scale bar indicates 10μm. Higher magnification images of highlighted dendrites are shown below. Summary graphs show that reduction of PICK1 levels had no effect of LiCl treatment on surface GluA2 levels (n=4 neurons in each condition, *p<0.05 and ^***p<0.001, one-way ANOVA with Fisher’s LSD test).

Figure 4. Li⁺ increases synaptic GluA2 and its associated scaffolding protein in vivo

a) Representative immunoblots and b) quantitative analysis of hippocampal PSD from mice fed with normal mouse chow and Li⁺ chow for 7 days showing that Li⁺ increased GluA2/3, GRIP, and δ-catenin levels (n=8 animals, *p<0.05, unpaired two-tailed Student’s t tests). In vivo GSK3β inhibition by Li⁺ was confirmed by an increase in its phosphorylation and increased β -catenin levels.

Fig. 5. Li⁺ increased the peak amplitude but not the frequency of miniature ESPCs of hippocampal neurons in culture

a) Representative mEPSC traces from cultured hippocampal neurons treated overnight with NaCl (control) or LiCl. Graphs are shown of: b) the peak amplitude and c) frequency of mEPSCs. (n= 13 and 18 cells with Na⁺ and Li⁺ treatment from 3 different cultures, respectively, *p<0.05)