PICK1 loss of function occludes homeostatic synaptic scaling

Abstract

Homeostatic synaptic scaling calibrates neuronal excitability by adjusting synaptic strengths during prolonged changes in synaptic activity. The molecular mechanisms that regulate the trafficking of AMPA receptors (AMPARs) during synaptic scaling are largely unknown. Here, we show that chronic activity blockade reduces PICK1 protein level on a time scale that coincides with the accumulation of surface AMPARs. PICK1 loss of function alters the subunit composition and the abundance of GluA2-containing AMPARs. Due to aberrant trafficking of these receptors, the increase in synaptic strength in response to synaptic inactivity is occluded in neurons generated from PICK1 knock-out mouse. In agreement with electrophysiological recordings, no defect of AMPAR trafficking is observed in PICK1 knock-out neurons in response to elevated neuronal activity. Overall, our data reveal an important role of PICK1 in inactivity-induced synaptic scaling by regulating the subunit composition, abundance, and trafficking of GluA2-containing AMPARs.

Figure 1.

Synaptic inactivity decreases PICK1 protein level. A, Western blot analysis of PICK1 from total protein extracts of mouse cortical neurons treated for 48 h with control solution (Ctrl), TTX (2 μm), or bicuculline (Bic, 40 μm). B, Quantification of PICK1 protein levels after normalizing to α-tubulin. Data represent mean ± SEM of band intensities normalized to control values of untreated neurons (ANOVA, *p < 0.05; **p < 0.01; n = 15). C, D, Western blot analysis (C) and quantification (D) of TTX-dependent decrease of PICK1 protein over time. Data represent mean ± SEM of band intensities normalized to control values of neurons at 0 h time point (Mann–Whitney test, **p < 0.01; n = 6). E, Western blot analysis of PICK1 from lysates of cortical neurons treated for 48 h with TTX in the presence of either the vehicle control DMSO (0.1%), the proteosome inhibitor lactacystin (Lac; 0.5 μm) or the lysosomal inhibitor leupeptin (Leu; 50 μg/ml). F, Quantification of PICK1 protein levels after normalizing to β-actin. Data represent mean ± SEM of band intensities normalized to control values of untreated neurons (ANOVA, **p < 0.01; ***p < 0.001; n = 4).

Figure 2.

Synaptic scaling during decreased network activity is occluded in PICK1 knock-out neurons. A, B, Representative whole-cell recording sample traces of mEPSC events (A) and cumulative distribution plots of mEPSC amplitude (B) from cultured cortical neurons derived from PICK1 knock-out and WT littermates after treatment with control solution (Ctrl), TTX (2 μm), or bicuculline (Bic, 40 μm) for 48 h. C, D, Quantification of mean mEPSC amplitude (C) and frequency (D) for each population. Data represent mean ± SEM (ANOVA, *p < 0.05; ***p < 0.001; n = 8 neurons per group).

Figure 3.

PICK1 knockdown occludes synaptic scaling during decreased network activity. A, PICK1 shRNAs decrease expression of Myc-PICK1 in HEK293 cells. B, Lysates from cultured cortical neurons either uninfected or infected with GFP (FuGW) or PICK1-shRNA#1::GFP (sh#1) lentivirus were subjected to immunoblot analysis with anti-PICK1, anti-GluA1, and anti-α-tubulin antibodies. C, Cortical neurons (DIV 8) expressing Venus or a bicistronic Venus::PICK1-shRNA#1 vector for 3 d were fixed and stained with anti-PICK1 antibody. The neuron expressing PICK1-shRNA showed a significant decrease in endogenous PICK1 staining (bottom panel) compared with the vector control-transfected neuron (top panel). Scale bar, 50 μm. D, E, Representative whole-cell recording sample traces of mEPSC events (D) and cumulative distribution plots of mEPSC amplitude (E) from cultured cortical neurons transfected with either pSuper-Venus (pSuper) or pSuper-Venus-PICK1 shRNA (sh#1) after treatment with control solution (Ctrl), TTX (2 μm), or bicuculline (Bic, 40 μm) for 48 h. F, G, Quantification of mean mEPSC amplitude (F) and frequency (G) for each population. Data represent mean ± SEM (ANOVA, **p < 0.01; n = 7–10 neurons per group). n.s., Not significant.

Figure 4.

Surface AMPAR protein levels are altered in PICK1 knock-out neurons. A, Cultured cortical neurons from PICK1 knock-out or WT littermates were subjected to surface biotinylation assay. The relative amount of surface and total AMPARs was assessed by Western blot using specific antibodies against GluA1, GluA2, and GluA3. PICK1 blot confirmed the genotype of the cultured neurons. B, C, Quantification of the surface:total ratio (B) and total (C) GluA1, GluA2, and GluA3 after normalizing against α-tubulin. Data represent mean ± SEM of band intensities normalized to control values of WT neurons (Mann–Whitney test, *p < 0.05; n = 12).

Figure 5.

The increase in surface GluA2 AMPAR subunit following chronic synaptic inactivity is impaired in PICK1 knock-out neurons. A, Cultured cortical neurons from PICK1 knock-out or WT littermates were treated with control solution (Ctrl), TTX (2 μm), or bicuculline (Bic, 40 μm) and were subjected to surface biotinylation assay 48 h later. The relative amount of surface and total AMPARs was assessed by Western blot analyses using specific antibodies against GluA1 and GluA2 subunits after normalizing against α-tubulin. B–D, Quantification of the surface (B), total (C), and surface:total ratio (D) of GluA1 and GluR2 AMPAR subunits in PICK1 WT and knock-out neurons. Data represent mean ± SEM of band intensities normalized to control values of untreated neurons (ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001; n = 18–24).

Figure 6.

Synaptic inactivity does not induce accumulation of homomeric GluA1 AMPARs on to plasma membrane. A, Cultured cortical neurons from PICK1 knock-out or WT littermates were treated with control solution (Ctrl) or TTX (2 μm) and were subjected to surface biotinylation assay 48 h later. Neuronal lysates were then subjected to two-rounds of GluA2/3 immunodepletion before Neutravidin chromatography steps. I, Input; T, total unbound fraction; S, surface unbound fraction following GluA2/3 immunodepletion. Surface fraction was loaded at 17× input concentration. B, C, Quantification of the surface (B) and total (C) GluA1, GluA2, and GluA3 AMPAR subunits in PICK1 WT and knock-out neurons. Data represent mean ± SEM of band intensities normalized to control values of untreated neurons (Student's t test, *p < 0.05; n = 6).

Figure 7.

Synaptic inactivity causes aberrant trafficking of GluA2-containing AMPARs in PICK1 knock-out neurons. A, Cultured cortical neurons from PICK1 knock-out or WT littermates were treated with control solution (Ctrl) or TTX (2 μm) and were subjected to surface biotinylation assay 48 h later. Neuronal lysates were then subjected to two-rounds of GluA1 immunodepletion before Neutravidin chromatography steps. I, Input; T, total unbound fraction; S, surface unbound fraction following GluA1 immunodepletion. Surface fraction was loaded at 13× input concentration. B, C, Quantification of the surface (B) and total (C) GluA1, GluA2 and GluA3 AMPAR subunits in PICK1 WT and knock-out neurons. Data represent mean ± SEM of band intensities normalized to control values of untreated neurons (Student's t test, *p < 0.05; **p < 0.01; n = 7).

Figure 8.

Model of PICK1 regulation of GluA2-containing AMPARs during synaptic scaling. A, Under basal conditions, PICK1 maintains pools of GluA2-containing AMPARs intracellularly. Chronic synaptic inactivity results in graded reduction in total PICK1 protein level, relieving the intracellular retention of GluA2-containing AMPARs in the form of GluA1/2 heteromers, which can then be incorporated into the plasma membrane. By lateral diffusion, these receptors are then inserted into and accumulate at the synaptic plasma membrane, presumably through the interaction between GluA2 and NSF. B, In the PICK1 knock-out neurons, there is a change in AMPAR subunit composition resulting in increased levels of total and surface GluA2/3 heteromers and elevated basal AMPAR-mediated mEPSC amplitudes. In the absence of PICK1, activity deprivation induces aberrant removal of GluA2/3 heteromers in exchange for the newly inserted GluA1/2 heteromers on the plasma membrane.