Neuronal activity regulates PICK1 protein level in cultured neurons
It is well established that chronic changes in synaptic activity result in global remodelling and reorganization of postsynaptic proteins (Ehlers, 2003
). However, the effect of chronic changes in neuronal activity on PICK1 expression has not been examined. Bidirectional homeostatic scaling can be modelled in primary cultured neurons wherein the synaptic strength is scaled up by prolonged suppression of neuronal activity, or scaled down by elevated neuronal activity (O’Brien et al., 1998
; Turrigiano et al., 1998
). We found that 48 hours of TTX treatment, which blocks all evoked neuronal activity, significantly reduced PICK1 expression (73.1 ± 6.8% of control, ). On the other hand, chronic bicuculline treatment, which elevates neuronal firing, had no effect of PICK1 expression (93.2 ± 5.4% of control, ). The reduction of PICK1 protein level was apparent 24 hours after TTX incubation and persisted for as long as 72 hours. Interestingly, short-term treatment with TTX (up to 6 hours) did not have any significant effect on PICK1 protein level (). The time scale by which PICK1 protein level is down-regulated by chronic TTX treatment coincides with the accumulation of surface AMPAR in response to decreased synaptic activity (O’Brien et al., 1998
; Turrigiano et al., 1998
; Sutton et al., 2006
), suggesting a potential role for PICK1 in regulating the trafficking of AMPARs during synaptic scaling.
Synaptic inactivity decreases PICK1 protein level
To gain insight into the mechanism behind reduced PICK1 level following chronic synaptic inactivity, cultured cortical neurons were incubated with TTX for 48 hours in the presence or absence of pharmacological agents that block the proteosomal and the lysosomal degradation pathways. As expected, cortical neurons treated with TTX in the presence of 0.1% DMSO (vehicle) led to a significant decrease in PICK1 protein level (53.9 ± 5.9% of control, ). This effect was largely prevented by co-incubation with the lysosomal inhibitor leupeptin (50 μg/mL, 85.6 ± 2.9% of control, ), but not by the proteosomal inhibitor lactacystin (0.5 μM, 59.4 ± 7.0% of control, ). These data suggest that the degradation of PICK1 during activity deprivation is mediated by the lysosomal degradation pathway.
TTX-induced synaptic scaling is occluded in PICK1 knockout neurons
To examine the role of PICK1 in synaptic scaling, we treated cultured cortical neurons derived from PICK1 knockout and WT littermates with TTX or bicuculine for 48 hours prior to mEPSC recording. WT neurons exhibited normal synaptic scaling up and scaling down of mEPSC amplitudes upon TTX and bicuculline treatment, respectively (Ctrl = 14.45 ± 0.82 pA, TTX = 23.96 ± 2.57 pA, Bic = 9.89 ± 0.69 pA, ). Strikingly, PICK1 knockout neurons showed significantly higher basal mEPSC amplitudes and thus, TTX-induced synaptic scaling appeared to be occluded (Ctrl = 20.13 ± 1.43 pA, TTX = 21.39 ± 2.25 pA, ). In contrast, PICK1 knockout neurons showed a robust synaptic scaling down of mEPSC amplitudes following chronic bicuculline treatment (Ctrl = 20.13 ± 1.43 pA, Bic = 10.84 ± 1.12 pA, ). As expected, neither TTX nor bicuculline treatment changed mEPSC frequency in PICK1 knockout or WT neurons (WT: Ctrl = 2.20 ± 0.42 Hz, TTX = 3.00 ± 0.88 Hz, Bic = 2.29 ± 0.50 Hz; KO: Ctrl = 2.66 ± 0.75 Hz, TTX = 4.22 ± 0.88 Hz, Bic = 3.48 ± 1.18 Hz, ). In addition, we did not observe any change in mEPSC decay kinetics in any condition (WT: Ctrl = 5.18 ± 0.35 ms, TTX = 4.78 ± 0.62 ms, Bic = 6.38 ± 0.73 ms; KO: Ctrl = 5.32 ± 0.60 ms, TTX = 4.74 ± 0.49 ms, Bic = 5.73 ± 0.45 ms), indicating that bidirectional synaptic scaling involves the accumulation and removal of GluA2-containing AMPARs on synapses. Together, these data suggest that PICK is involved in the unidirectional scaling up of AMPAR-mediated mEPSC upon chronic synaptic inactivity.
Synaptic scaling during decreased network activity is occluded in PICK1 knockout neurons
PICK1 knockdown occludes TTX-induced synaptic scaling
To ascertain that the effects we observed in PICK1 knockout neurons were not due to the loss of PICK1 function during neuronal development or the disruption of PICK1 presynaptic function, which may lead to abnormal network activity, we transiently transfected PICK1 shRNA construct to acutely knockdown PICK1 expression in a sparse population of cortical neurons. The PICK1 shRNA#1 efficiently reduced myc-PICK1 expression in heterologous cells and endogenous PICK1 expression in neurons after 3 days of expression (7 days in the case of lentiviral-mediated knockdown, ). We further characterized PICK1 shRNA#1 using two independent assays. Knockdown of PICK1 in cultured hippocampal neurons accelerated GluA2 recycling following NMDA receptor activation, resembling the phenotype seen in PICK1 knockout neurons (Lin and Huganir, 2007
) (Supplemental Fig. 1
). In addition, PICK1 knockdown in young cortical neurons resulted in significantly reduced number of distal dendritic processes, consistent with result from a previous study (Rocca et al., 2008
) (Supplemental Fig. 2
PICK1 knockdown occludes synaptic scaling during decreased network activity
To directly investigate the cell autonomous effect of PICK1 knockdown in synaptic scaling, we treated cultured cortical neurons that had been transfected with either pSuper-Venus or pSuper-Venus-PICK1 shRNA#1 for 24 hours with TTX or bicuculine for 48 hours prior to mEPSC recording. Neurons transfected with pSuper-Venus exhibited normal synaptic scaling up and scaling down of mEPSC amplitudes upon TTX and bicuculline treatment, respectively (Ctrl = 12.57 ± 1.05 pA, TTX = 16.58 ± 1.10 pA, Bic = 8.54 ± 0.41 pA, ). Consistent with our findings in PICK1 knockout neurons, PICK1 knockdown also resulted in significantly higher basal mEPSC amplitudes and occluded TTX-induced synaptic scaling (Ctrl = 16.13 ± 0.80 pA, TTX = 15.99 ± 1.00 pA, ). In addition, PICK1 knockdown neurons showed a robust synaptic scaling down of mEPSC amplitudes following chronic bicuculline treatment (Ctrl = 16.13 ± 0.80 pA, Bic = 9.17 ± 0.52 pA, ). Again, we did not observe any significant changes in mEPSC frequency following TTX and bicuculline treatment in pSuper or PICK1 sh#1 transfected neurons (pSuper: Ctrl = 1.85 ± 0.42 Hz, TTX = 1.62 ± 0.30 Hz, Bic = 0.99 ± 0.27 Hz; sh#1: Ctrl = 2.19 ± 0.46 Hz, TTX = 1.35 ± 0.36 Hz, Bic = 1.08 ± 0.25 Hz, ). Moreover, we did not observe any change in mEPSC decay kinetics in any condition (pSuper: Ctrl = 4.51 ± 0.33 ms, TTX = 4.46 ± 0.23 ms, Bic = 5.52 ± 0.52 ms; sh#1: Ctrl = 4.21 ± 0.23 ms, TTX = 4.36 ± 0.22 ms, Bic = 4.67 ± 0.33 ms), again indicating that bidirectional synaptic scaling involves the accumulation and removal of GluA2-containing AMPARs at synapses. Together, the lack of TTX-induced synaptic scaling in both the PICK1 knockdown and PICK1 knockout neurons provides important complementary results establishing the involvement of PICK1 cell autonomously in postsynaptic neurons in unidirectional scaling up of AMPAR-mediated mEPSC.
Altered surface AMPAR subunit abundance in PICK1 knockout neurons
To assess the contribution of PICK1 to the trafficking of AMPARs during synaptic scaling, we compared the surface expression of GluA1, GluA2 and GluA3 in PICK1 knockout neurons using a biotinylation assay. Under basal conditions, we observed a change in surface AMPAR levels in PICK1 knockout neurons. The levels of surface GluA2 and surface GluA3 in PICK1 knockout neurons were significantly higher than in neurons derived from WT littermates (surface/total ratio; GluA2 = 203 ± 31% of WT, GluA3 = 171 ± 24% of WT ). In addition, we also observed a slight but significant decrease in the level of surface GluA1 expression (surface/total ratio: 74 ± 11% WT, ). The decrease in surface GluA1 may be due to a compensatory mechanism since a reciprocal increase in surface GluA1 expression has also been observed in neurons overexpressing GluA2 siRNA (Gainey et al., 2009
). These altered surface AMPAR levels are most likely due to impaired receptor trafficking events rather than protein synthesis or degradation defects as there was no significant difference in the total expression levels of GluA1, GluA2 and GluA3 subunits in PICK1 knockout neurons (GluA1 = 94 ± 7% of WT, GluA2 = 109 ± 14% of WT, GluA3 = 85 ± 7% of WT, ). These data indicate that the increase in surface GluA2 and GluA3 levels may account for the increase in basal synaptic transmission observed in PICK1 knockout neurons ().
Surface AMPAR protein levels are altered in PICK1 knockout neurons
PICK1 knockout neurons lack an increase in surface GluA2 subunit following TTX treatment
Next, we examined the surface expression of GluA1, GluA2 and GluA3 subunits following TTX and bicuculline treatment. Consistent with electrophysiological recordings, WT neurons exhibited robust increase and decrease in both surface GluA1 and GluA2 expression after TTX and bicuculline treatment, respectively (TTX: GluA1 = 156 ± 13% of control, GluA2 = 133 ± 9% of control; Bic: GluA1 = 64 ± 5% of control, GluA2 = 65 ± 8% of control, ). Strikingly, adaptation of surface GluA2 expression, but not GluA1, was impaired in PICK1 knockout neurons in response to TTX treatment (GluA1 = 146 ± 10% of control, GluA2 = 96 ± 8% of control, ). The inability of GluA2 subunit to scale up is likely due to an occlusion effect since basal surface GluA2 expression was already elevated in PICK1 knockout neurons (). The normal increase in surface GluA1 expression upon TTX treatment indicates a crucial role of PICK1 in selectively controlling GluA2 trafficking. This is consistent with a recent finding that demonstrates a requirement of AMPAR GluA2 subunit for TTX-induced synaptic scaling (Gainey et al., 2009
). In agreement with our electrophysiological recording, no defect of AMPAR trafficking was observed in PICK1 knockout neurons in response to bicuculline treatment (GluA1 = 57 ± 6% of control, GluA2=67 ± 6% of control, ).
The increase in surface GluA2 AMPAR subunit following chronic synaptic inactivity is impaired in PICK1 knockout neurons
To determine whether the lack of an increase in GluA2 surface expression was due to changes in total protein levels, we next examined the total expression of AMPAR subunits following TTX and bicuculline treatment. In WT neurons, chronic TTX treatment selectively increased the total GluA1 level, but not the total expression of GluA2 subunit (GluA1 = 139 ± 13% of control, GluA2 = 91 ± 8% of control, ). Conversely, bicuculline treatment led to significant reduction in total AMPAR expression (GluA1 = 67 ± 6% of control, GluA2 = 70 ± 8% of control, ). We found no significant difference between PICK1 knockout and WT neurons in changes in the total expression of AMPAR subunits upon TTX and bicuculline treatment (PICK1 KO, TTX: GluA1 = 118 ± 5% of control, GluA2 = 91 ± 6% of control; Bic: GluA1 = 63 ± 6% of control, GluA2 = 78 ± 5% of control, ). Moreover, surface to total ratio analyses revealed a distinct mechanism of regulation for GluA1 and GluA2 during TTX-induced synaptic scaling. In both PICK1 knockout and WT neurons, GluA1 total and surface levels change at the similar rate in response to TTX treatment (WT = 121 ± 9% of control, KO = 126 ± 10% of control, ). In contrast, the surface to total GluA2 ratio becomes significantly higher upon TTX treatment in WT but not in PICK1 KO neurons (WT = 156 ± 9% of control, KO = 115 ± 13% of control, ). Altogether, these data suggest that loss of PICK1 function occludes TTX-induced synaptic scaling by specifically impairing the trafficking of GluA2-containing AMPARs.
Chronic activity blockade causes aberrant trafficking of GluA2-containing AMPARs in PICK1 knockout neurons
Unlike surface GluA2, the increase in surface GluA1 induced by TTX treatment is not impaired in PICK1 knockout neurons. Surprisingly, however, mEPSC amplitudes failed to scale up in PICK1 knockout neurons. This result along with a lack of change in mEPSC decay kinetics combined with the lack of surface GluA2 increases suggests that these newly inserted GluA1-containing receptors are most likely extrasynaptic Ca2+
-permeable GluA1 homomers. To test this hypothesis, we combined surface biotinylation and GluA2/3 immunodepletion assays from neuronal lysates and examined the fraction of total and surface homomeric GluA1 AMPARs remaining. Two-rounds of immunoprecipitation effectively pulled down more than 98% of GluA2 and GluA3 leaving an approximately 15% of total GluA1 in the unbound fraction in all conditions (). As expected, we did not observe any significant changes in the amount of surface GluA1 homomers following TTX treatment in WT neurons (Ctrl = 3.9 ± 0.4%, TTX = 3.4 ± 0.6%, ), suggesting that synaptic inactivity drives incorporation of GluA1/2 heteromers to the plasma membrane. Under basal condition, the levels of surface GluA1 homomers were significantly lower in the PICK1 knockout neurons (WT = 3.9 ± 0.4%, KO = 2.7 ± 0.3%, ), consistent with our previous study (Clem et al., 2010
). Surprisingly, the level of surface GluA1 homomers was not altered following activity deprivation in PICK1 knockout neurons (Ctrl = 2.7 ± 0.3%, TTX = 2.0 ± 0.4%, ). This suggests that the TTX-induced increase in surface GluA1 in PICK1 knockout neurons represents an increase in surface GluA1/2 heteromers.
Synaptic inactivity does not induce accumulation of homomeric GluA1 AMPARs on to plasma membrane
How can we account for the lack of change in surface GluA2 subunit in the PICK1 knockout neurons following chronic activity deprivation? Since PICK1 knockout neurons have increased levels of surface GluA2/3 AMPAR complexes, we hypothesize that during chronic activity deprivation, the increase in surface GluA1/2 heteromers is counterbalanced by the removal of heteromeric GluA2/3 receptors from the plasma membrane. Since nearly all of GluA2 subunit forms heteromeric complexes with either GluA1 and GluA3 subunits (Wenthold et al., 1996
; Lu et al., 2009
), we performed GluA1 immunodepletion assays from neuronal lysates and examined the fraction of total and surface heteromeric GluA2/3 AMPARs remaining following chronic activity blockade in both PICK1 WT and knockout neurons. Two-rounds of immunoprecipitation effectively depleted more than 98% of total GluA1 from neuronal lysates in all conditions (). Interestingly, under basal condition total GluA2 and GluA3 in the unbound fraction following GluA1 immunodepletion were significantly higher in the PICK1 knockout neurons compared to the neurons prepared from the WT littermates (WT: GluA2 = 47.4 ± 3.0%, GluA3 = 56.3 ± 3.0%; KO: GluA2 = 59.6 ± 1.4%, GluA3 = 70.8 ± 2.1%, ), suggesting a possible role of PICK1 in AMPAR assembly or stability. Consistent with our previous finding, levels of surface GluA2 and GluA3 subunits were also significantly higher in the PICK1 knockout neurons (WT: GluA2 = 3.9 ± 0.5%, GluA3 = 16.1 ± 2.2%; KO: GluA2 = 6.4 ± 0.4%, GluA3 = 34.9 ± 5.0%, ). We found no significant differences in surface GluA2 or GluA3 levels following activity deprivation in WT neurons, indicating the absence of heteromeric GluA2/3 AMPARs insertion to the plasma membrane (Ctrl: GluA2 = 3.9 ± 0.5%, GluA3 = 16.1 ± 2.2%; TTX: GluA2 = 4.9 ± 0.6%, GluA3 = 21.6 ± 1.9%, ). In PICK1 knockout neurons, however, the level of surface GluA2/3 heteromers decreased significantly, suggesting internalization of these receptors following synaptic inactivity (Ctrl: GluA2 = 6.4 ± 0.4%, GluA3 = 34.9 ± 5.0%; TTX: GluA2 = 3.9 ± 1.0%, GluA3 = 19.6 ± 4.7%, ). Altogether, our data suggest that loss of PICK1 function leads to a complex disruption of proper trafficking of heteromeric GluA2/3 AMPARs, which underlies the occlusion of TTX-induced synaptic scaling in cultured neurons.
Synaptic inactivity causes aberrant trafficking of GluA2-containing AMPARs in PICK1 knockout neurons