A critical and cell-autonomous role for MeCP2 in synaptic scaling up

Abstract

Rett syndrome (Rett) is the leading genetic cause of mental retardation in females. Most cases of Rett are caused by loss-of-function mutations in the gene coding for the transcriptional regulator methyl-CpG binding protein 2 (MeCP2), but despite much effort, it remains unclear how a loss of MeCP2 function generates the neurological deficits of Rett. Here we show that MeCP2 plays an essential and cell-autonomous role in homeostatic synaptic scaling up in response to reduced firing or reduced sensory drive in rat visual cortical pyramidal neurons. We found that acute RNAi knockdown of MeCP2 blocked synaptic scaling within targeted neocortical pyramidal neurons. Furthermore, MeCP2 knockdown decreased excitatory synapse number without affecting basal mEPSC amplitude or AMPAR accumulation at spared synapses, demonstrating that MeCP2 acts cell-autonomously to maintain both excitatory synapse number and synaptic scaling in individual neocortical neurons. Finally, we used a mouse model of Rett to show that MeCP2 loss prevents homeostatic synaptic scaling up in response to visual deprivation in vivo, demonstrating for the first time that MeCP2 loss disrupts homeostatic plasticity within the intact developing neocortex. Our results establish MeCP2 as a critical mediator of synaptic scaling and raise the possibility that some of the neurological defects of Rett arise from a disruption of homeostatic plasticity.

Figure 1.

MeCP2 KD does not affect basal quantal amplitude. A, Pyramidal neurons transfected for 3 d with ev or MeCP2 shRNA1 (hp1) and untransfected control neurons (unt), fixed, and stained with antibodies against endogenous MeCP2 (red) and NeuN (orange). The ev and hp1 constructs also express soluble EGFP (green). White arrowheads show the position of the same neuron's cell body. Yellow arrowheads indicate a nearby untransfected neuron. Scale bar, 20 μm. B, Quantification of the nuclear MeCP2 fluorescence intensity for unt and ev- and hp1-expressing neurons (n = 164, 48, and 39, respectively). A.U., Arbitrary units. C, Left, Example raw traces of mEPSCs recorded from ev- and hp1-expressing neurons. Right, Average mEPSC waveforms for the same conditions. D, Cumulative histograms of mEPSC amplitudes for ev- and hp1-expressing neurons. Inset, Average mEPSC amplitude for ev-expressing (n = 22) and hp1-expressing (n = 20) neurons. E, Peak scaled average mEPSC waveform for the same conditions. **p < 0.001, different from control.

Figure 2.

MeCP2 KD reduces excitatory synapse number. A, Example dendrites from ev- or hp1-expressing neurons, stained against endogenous surface GluA1 (red) and the excitatory synapse marker VGLUT-1 (green); EGFP is shown in pseudo color blue. Scale bar, 2 μm. B, Length density for total GluA1 and GluA2 puncta in ev- and hp1-expressing cells: ev (n = 5) and hp1 (n = 7) for GluA1; ev (n = 12) and hp1 (n = 6) for GluA2. C, Synaptic length density for GluA1 and GluA2 puncta in ev- and hp1-expressing cells. D, Colocalization of GluA1 and GluA2 with VGLUT-1 in ev- and hp1-expressing cells. E, Synaptic GluA1 and GluA2 surface receptor fluorescent intensity in ev- and hp1-expressing cells. *p < 0.05, different from ev.

Figure 3.

MeCP2 KD blocks or attenuates synaptic scaling up. A, Top, Example raw traces of mEPSCs recorded from ev-expressing (left) and hp1-expressing (right) neurons in untreated control (ctl; top) and DNQX-treated (bottom) conditions. Bottom, Average mEPSC waveform for each condition. B, Cumulative histograms of mEPSC amplitudes from ev-expressing neurons in ctl and DNQX-treated conditions. C, Cumulative histograms of mEPSC amplitudes from hp1-expressing neurons in ctl and DNQX-treated conditions. D, Average amplitude for ctl and DNQX-treated cultures for neurons expressing ev1 (n = 22 and 10, respectively), hp1 (n = 20 and 10, respectively), ev2 (n = 13 and 6, respectively), and hp2 (n = 15 and 7, respectively). E, Plot comparing the percentage change in mEPSC amplitude during scaling (DNQX as a percentage of control) versus the percentage of MeCP2 remaining after KD for unt, ev1, hp1, and hp2 neurons. *p < 0.05, different from ctl; **p < 0.005, different from ctl.

Figure 4.

MeCP2 KD prevents the inactivity-induced accumulation of AMPAR at synapses. A, Examples of staining for endogenous GluA1 (red) and VGLUT-1 (green) for ev-expressing (top) and hp1-expressing (bottom) neurons treated with DNQX for 24 h. Scale bar, 2 μm. B, Fluorescence intensity of synaptic GluA1 puncta for ev- and hp1-expressing neurons treated with DNQX. Here and below, values are from DNQX-treated neurons expressed as a percentage of untreated control neurons. ev, n = 5 and 8, respectively; hp1, n = 7 and 9, respectively. C, Colocalization of GluA1 with VGLUT-1 for ev- and hp1-expressing neurons. D, Length density for synaptic GluA1 for ev- and hp1-expressing neurons. **p < 0.005, different from ev.

Figure 5.

MeCP2 knock-out blocks homeostatic synaptic scaling in vivo. A, Average waveform for mEPSCs recorded from WT (left) and KO (right) neurons from DE and normal 12 h light/dark cycle Ctl mice: WT_Ctl (n = 5 animals), WT_DE (n = 4 animals), KO_Ctl (n = 2 animals), and KO_DE (n = 2 animals). B, Cumulative histogram of mEPSC amplitudes recorded from Ctl (n = 23) and DE (n = 32) WT mice. Inset, Average mEPSC amplitudes for the same conditions. C, Cumulative histogram of mEPSC amplitudes recorded from Ctl (n = 24) and DE (n = 35) KO mice. Inset, Average mEPSC amplitudes for the same conditions. D, Cumulative histogram of mEPSC amplitudes for WT and KO neurons from Ctl mice. Inset, Average mEPSC amplitude for the same conditions. E, Average frequency (left) and input resistance (right) for WT and KO neurons from ctl mice. *p < 0.05, different from WT_Ctl. Cumul. Prob., Cumulative probability.