PP2A and GSK-3beta act antagonistically to regulate active zone development

Abstract

The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. Here, we show that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site.

Figure 1.

Inhibition of PP2A during development impairs synaptic function. Representative traces of spontaneous miniature EJPs (left) and evoked EJPs (right) for wild type (GS Elav Gal 4/+) and dnPP2A (GS Elav Gal 4/UAS dnMts). Spontaneous miniature potentials in dnPP2A animal are as a group lower than those in wild-type animals. Note the dramatic decrease in size of the average evoked potential in the dnPP2A animal. Calibrations: 500 ms, 2 mV for spontaneous miniature potentials and 12.5 ms, 5 mV for evoked potentials.

Figure 2.

Inhibition of PP2A leads to unapposed glutamate receptors. MN 4 1b innervation of muscle 4 in wild type (GS elav Gal4/+) and dnPP2A (GS elav Gal4/UAS dnMts). Larvae are tripled stained with antibodies to the active zone protein Bruchpilot, the glutamate receptor DGluRIII, and the neural membrane marker HRP. Bruchpilot and glutamate receptor staining colocalize at almost all synpases in wild-type animals. In contrast, dnPP2A animals show both colocalized Bruchpilot and receptors (yellow arrowhead) as well as glutamate receptors unapposed to Bruchpilot (white arrows). Scale bar, 10 μm.

Figure 3.

Inhibition of PP2A does not induce synaptic retraction. A terminal branch of MN 4 1b is shown from a dnPP2A (G Selav Gal 4/UAS dnMts) larvae triple labeled with antibodies to the synaptic vesicle protein DVGLUT, Bruchpilot, and DGluRIII. DVGLUT staining is present throughout the nerve terminal, indicating that the presynaptic terminal is not retracted. Note that glutamate receptors apposed to Bruchpilot (yellow arrowheads) and unapposed to Bruchpilot (white arrows) are present in a salt-and-pepper pattern throughout the terminal, which is not consistent with the distal-to-proximal loss of the presynaptic terminal observed with synaptic retraction.

Figure 4.

Unapposed glutamate receptors are most prevalent at the distal NMJ. A, Boutons from third instar larvae with UAS dnMts driven since the first instar stage (GS Elav-Gal 4/UAS-dnMts). Boutons are costained with antibodies to Brp, DGluRIII, and HRP. Upper panels show a terminal NMJ branch, which is the region of the NMJ where new synapses tend to be added. White arrows highlighting several areas of glutamate receptor unapposed to Brp. The lower panel shows proximal boutons, with an asterisk indicating the adjoining nerve. In contrast to distal boutons, the active zones and receptors in the proximal boutons are primarily apposed. Scale bar, 5 μm. B, Boutons from third instar larvae with UAS dnPP2A driven since the second instar stage (GS Elav Gal4/UAS dnMts). Antibody staining and layout of images is as above. In the terminal branch in the top panels, white arrows indicate several sites of glutamate receptors unapposed to Brp. Proximal boutons show uniform apposition of Brp and DGluRIII. Asterisk indicates adjoining nerve. Scale bar, 5 μm. C, Quantification of unapposed glutamate receptors at proximal and distal boutons in wild-type (WT; GS Elav Gal 4/+) and dnPP2A animals driven by GS Elav Gal 4 since first instars. Wild-type animals show negligible unapposed glutamate receptors. The level of unapposed glutamate receptors at proximal boutons in dnPP2A animals, 10%, is not significantly different from wild type (p > 0.4). The level of unapposed glutamate receptors at distal boutons in dnPP2A animals, 48%, differs significantly from distal wild-type boutons (p ≪ 0.001) and proximal dnPP2A boutons (p ≪ 0.001). N = 12 for WT proximal, N = 21 for WT distal, N = 23 for dnPP2A proximal, and N = 47 for dnPP2A distal. D, Quantification of unapposed glutamate receptors at proximal and distal boutons in wild type (GS Elav Gal 4/+) and dnPP2A animals driven by GS Elav Gal 4 since second instars. Wild-type animals show negligible unapposed glutamate receptors. The level of unapposed glutamate receptors at proximal boutons in dnPP2A animals, 6%, is not significantly different from wild type (p > 0.95). The level of unapposed glutamate receptors at distal boutons in dnPP2A animals, 17%, differs significantly from levels in distal wild-type boutons (p < 0.001) and proximal dnPP2A boutons (p < 0.05). N = 12 for WT proximal, N = 25 for WT distal, N = 11 for dnPP2A proximal, and N = 29 for dnPP2A distal.

Figure 5.

Ultrastructural analysis demonstrates decreased active zone density when PP2A is inhibited. TEM of a Type 1 bouton from wild-type (GS elav Gal 4/+) and dnPP2A (GS elav Gal 4/UAS dnMts) animals. Arrows indicate active zones present in these boutons. Both boutons demonstrate examples of active zones with T-bars (asterisks). Scale bar, 500 nm.

Figure 6.

In vivo imaging reveals defects in synapse formation when PP2A is inhibited. A, In vivo imaging of GFP-labeled DGluRIIA at larval NMJs. Shown are two time points of imaging (0 and 6 h) of individual NMJs from (top) a larvae in which PP2A is inhibited (dnPP2A: DGluRIIa-GFP/+;elav-Gal4/UAS-dnMts) and (bottom) a control larvae (control: DGluRIIA-GFP/+;elav-Gal4/+). Regions of formation and loss of synapses within the NMJs are marked. Enlargements of the areas marked in the upper panel by color-coded dots are shown in the lower panel. New synapses are marked with arrows, lost synapses are marked with arrowheads. Scale bar: 5 μm. B, Quantification of synapse formation. Thirteen control junctions (from 5 different larvae) were compared with forty-two mutant junctions (from 13 different larvae). p < 0.05. Error bars equal SEM.

Figure 7.

Inhibition of GSK-3β suppresses synaptic apposition defects due to PP2A inhibition. A, A terminal branch from MN 4 1b innervation is shown costained with antibodies to Bruchpilot and DGluRIII from wild-type (Elav Gal 4/+), dnGSK (UAS dnGSK-3β/+; Elav Gal 4/+), dnPP2A (Elav Gal 4/UAS dnMts), and dnGSK; dnPP2A (Elav Gal 4/UAS dnGSK-3β; UAS dnMts/+) larvae. White arrows illustrate unapposed glutamate receptors in dnMts. For all other genotypes, colocalization of Bruchpilot and receptors appears essentially as in wild-type animals. Scale bar, 7 μm. B, Quantification is shown of the percentage of unapposed glutamate receptors. Levels of unapposed glutamate receptors are not significantly different among wild type, dnGSK-3β, and dnGSK-3β; dnMts (p > 0.9). Unapposed glutamate receptors are increased by >700% relative to wild type in Elav dnMts, *p < 0.01. N = 8 for all genotypes. Error bars equal SEM.

Figure 8.

Inhibition of GSK-3β suppresses cytoskeletal and synaptic terminal morphology defects induced by inhibition of PP2A. A, Representative MN 4 1b NMJs are shown for wild-type (Elav Gal 4/+), dnGSK (UAS dnGSK-3β/+; Elav Gal 4/+), dnPP2A (Elav Gal 4/UAS dnMts), and dnGSK; dnPP2A (UAS dnGSK3-β, Elav Gal 4/UAS dnMts) larvae. NMJs are stained with antibodies to Futsch (left panels) and DVGLUT and HRP (right panels). Note increased Futsch unbundling marked by arrowheads in Elav dnPP2A genotype. Note also that the aberrant synaptic terminal morphology induced by inhibition of PP2A is suppressed by inhibition of GSK-3β. B, Quantification of mean percentage unbundled Futsch at the NMJ. Levels of unbundled Futsch in dnGSK-3β and dnGSK-3β; dnPP2A are not significantly different from wild type p > 0.3, and p > 0.5, respectively. dnPP2A demonstrates an 86% increase in unbundled Futsch relative to wild type, *p < 0.05. Levels of unbundled Futsch in dnGSK-3β, dnPP2A are reduced by 53% from dn PP2A, **p < 0.01. N = 8 for all genotypes. Error bars equal SEM.