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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Cell Neurosci. Author manuscript; available in PMC Nov 1, 2007.
Published in final edited form as:
PMCID: PMC1936977
NIHMSID: NIHMS23137

The ubiquitin proteasome system postsynaptically regulates glutamatergic synaptic function

Abstract

The ubiquitin proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome β2 and β6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors.

Keywords: ubiquitin, proteasome, postsynaptic, glutamate receptor, PDZ scaffold, discs large, neuromuscular junction, Drosophila

Introduction

Recently, the ubiquitin proteasome system (UPS) has been shown to function locally at the neuronal synapse, placing protein degradation alongside protein transport and synthesis as a primary means for regulating the abundance of proteins critical for neurotransmission (DiAntonio and Hicke, 2004; Bingol and Schuman, 2005; Yi and Ehlers, 2005). The UPS entails a sequential enzymatic cascade in which an ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3), catalyze the attachment of ubiquitin to a lysine residue on the target protein. Polyubiquitination, particularly with lysine 48 linked chains of at least four residues, targets proteins to the proteasome for degradation (Thrower et al., 2000; Glickman and Ciechanover, 2002). At the synapse, chronic activity-dependent changes in postsynaptic density composition require UPS-mediated protein degradation (Ehlers, 2003; Pak and Sheng, 2003). Thus, understanding UPS regulation of synaptic function is critical to understanding the physiological changes in synaptic strength that occur during development and plasticity, as well as the pathological changes that occur in inherited neurological diseases.

At glutamatergic synapses, fast excitatory synaptic transmission is mediated by ionotropic glutamate receptors (GluRs); glutamate-gated cation channels formed as tetrameric subunit complexes (Dingledine et al., 1999). The UPS has been shown to regulate GluR synaptic abundance both directly and indirectly. In C. elegans, direct ubiquitination and proteasomal degradation of GluR1 subunits has been demonstrated (Burbea et al., 2002), but the UPS also indirectly regulates GluR1 synaptic abundance through multiple other pathways (Juo and Kaplan, 2004; Dreier et al., 2005; Schaefer and Rongo, 2006). In cultured rat hippocampal neurons, indirect regulation of AMPA receptors has been demonstrated through ubiquitination and degradation of the postsynaptic scaffolding protein PSD-95 (Colledge et al., 2003), and agonist-induced AMPA receptor internalization has been shown to be dependent on UPS-mediated degradation of an undetermined synaptic protein (Patrick et al., 2003). Little is known, however, about the kinetics of this GluR regulation, or whether UPS-mediated regulation of GluR subunits plays a role in the regulation of specific subclasses of synaptic glutamate receptors.

Using the Drosophila NMJ glutamatergic synapse, we have shown previously that the UPS regulates presynaptic release probability via the acute regulation of the dUNC-13 synaptic vesicle priming protein, regulated downstream of G-protein coupled receptor PKA- and PLC-dependent pathways (Aravamudan et al., 1999; Aravamudan and Broadie, 2003; Speese et al., 2003). Postsynaptically, five GluR subunits (GluRIIA-E) share homology to mammalian AMPA/kainate ionotropic GluRs in mammals (Petersen et al., 1997; Marrus et al., 2004). Recent evidence has demonstrated the presence of two spatially and functionally distinct GluR classes, composed of common GluRIII (IIC), IID, and IIE subunits and either GluRIIA (A-class) or GluRIIB (B-class) (Marrus et al., 2004; Qin et al., 2005; Featherstone et al., 2005). These two GluR classes are known to be regulated by separable mechanisms (Davis et al., 1998; Chen and Featherstone, 2005; Chen et al., 2005), but possible differential regulation by the UPS has not been investigated. In the current study, we take advantage of Drosophila genetic techniques to spatially and temporally target dominant negative proteasome mutants to the postsynaptic muscle cell at the Drosophila NMJ, allowing for examination of the specific postsynaptic effects of impaired UPS function at this glutamatergic synapse. We demonstrate that postsynaptic proteasome inhibition, even for periods as short as 6 hours, causes the B-class glutamate receptor and its interacting MAGUK PDZ-domain scaffold DLG to be selectively misregulated, resulting in alterations in synaptic transmission strength and activity-dependent plasticity. Thus, UPS regulation in the postsynaptic cell is glutamate receptor subunit specific and important for the acute regulation of synaptic function.

Materials and Methods

Drosophila Stocks

All stocks were maintained at 25°C on standard cornmeal medium supplemented with dry yeast. The homozygous UAS-DTS5 2B(2), DTS7 1B(3) line (generously provided by Dr. John Belote, Syracuse University (Belote JM and Fortier, 2002)) was crossed with the homozygous muscle-specific myosin heavy chain (MHC)-GAL4 driver line to generate heterozygote MHC-GAL4/UAS-DTS 5,7 larvae containing a single copy of the UAS transgene and a single copy of the GAL4 driver. To generate heterozygote controls, UAS-DTS 5,7 and MHC-GAL4 lines were crossed with the wild-type Oregon-R (+/+) line. For inducible UAS transgene expression, the GeneSwitch system was utilized (Osterwalder et al., 2001). GeneSwitch uses a RU486-dependent GAL4-progesterone receptor fusion protein and the MHC-GeneSwitch GAL4 stock, generously provided by Dr. Haig Keshishian (Yale University). For induction of the UAS transgene, 96 hour third instar larvae were transferred to cornmeal medium containing 15 μg/ml RU486 in 4% EtOH or 4% EtOH alone for 6 hours at 29°C prior to utilization.

Immunohistology

Preparations were fixed and immunohistologically stained as reported previously (Broadie and Bate, 1993; Broadie et al., 1995; Rohrbough et al., 2000). Third instar larvae were dissected along the dorsal midline and secured flat with histoacryl glue in standard saline, fixed for 30–45 min with Bouin’s (Sigma), then washed in PBS-TX (0.1% Triton X-100 in PBS) containing 0.5% BSA. Primary antibodies were applied overnight at 4°C, and antibody concentrations and sources are as follows: GluRIIA (1:50 mouse monoclonal, DSHB, University of Iowa), GluRIIB (1:2500 rabbit polyclonal; a generous gift of Dr. Aaron DiAntonio, Washington University St. Louis), DLG (1:1000 mouse monoclonal, DSHB, University of Iowa), FK1 antibody directed against polyubiquitinated proteins (1:1000 mouse monoclonal, Biomol). Preparations were incubated with a fluorescently-tagged secondary antibody (1:300) or Cy3-conjugated anti-HRP (1:400) for 90 min at room temperature. All antibody dilutions were in PBS-TX. Images were collected with a Zeiss LSM 510 META confocal microscope and are presented here using Adobe Photoshop and Illustrator (Adobe). FK-1 immunostaining intensity was quantified by determining the fluorescence intensity per unit area from matched areas in muscle 6 segments A2 and A3 and in the CNS neuropil, with results normalized to the MHCGAL4/+ values. NMJ immunostaining intensity was quantified at Type I boutons on both the upper and lower nerve branch to muscle 12 segment A2 and A3 on dissected larvae. The intensity per unit area of the protein of interest was normalized to HRP immunostaining intensity for each synapse. In all cases, control and experimental matched pairs were fixed side by side on the same coverslip; processed, imaged and quantified using identical methods.

Electrophysiology

All recordings were made at 18°C from muscle 6 in abdominal segments A3 or A4 of freshly dissected third instar larvae, as described previously (Rohrbough et al., 1999). Briefly, two-electrode voltage-clamp recordings (TEVC) were made at a holding potential of -60 mV with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Dissections and recordings were made in modified standard Drosophila saline composed of (in mM): 128 NaCl, 2 KCl, 4 MgCl2, 70 sucrose, 5 HEPES, and 0.20–1.8 CaCl2; pH-adjusted to 7.2 with NaOH.Intracellular electrodes were filled with a 3:1 mixture of 3 M K+acetate:KCl and had resistances of 15–30 MΩ Segmental nerves were severed near the CNS to eliminate junctional responses originating from CNS activity, and EJCs were evoked by stimulating (0.5 ms) the severed nerve at 0.2–20 Hz frequencies with a suction electrode filled with external saline. mEJCs were recorded at -60 mV in 0.2 m M Ca2+ saline. Currents were filtered (500–1000 Hz), digitized to disk (5 kHz), and analyzed using pClamp9 acquisition and analysis hardware and software (Axon Instruments, Foster City, CA). Exemplar EJC traces were averaged from 10–20 consecutive individual responses. Event averaging and amplitude analysis was performed using pClamp9 and commercial spreadsheet software. Statistical analysis was performed using Instat Graphpad software, with one-way ANOVA or two-tailed T-tests. All data are presented as mean ± SEM unless otherwise indicated.

Immunoprecipitation and Immunoblotting

For immunoprecipitation experiments, larvae were lysed in PLC-LB buffer including protease inhibitors and NEM as described previously (Oldham et al., 2002). DLG was isolated from lysates using the 4F3 ascites antibody (DSHB, University of Iowa). Following immunoprecipitation, precipitates were washed five times with PLC-LB, fractionated by SDS-PAGE and then transferred to Immobilon P Membranes. Membranes were then probed with either anti-4F3 (DLG) or, following guanidine hydrochloride treatment, ubiquitin antibody (Covance) to detect ubiquitination.

Proteomic Analysis

2D difference gel electrophoresis (DIGE) using a mixed sample internal standard, spot identification by mass spectrometry, and database searching were done largely according to Friedman et al., 2004 (reviewed in (Lilley and Friedman, 2004)). For each of four independent replicate experiments, using genotypes MHC-GAL4/+, UAS-DTS/+ and DTS, body wall muscles were dissected from 50 third instar larvae by holding the tail with forceps and applying firm pressure on the cuticle with the flat side of a second pair of forceps starting at the tail and advancing rostrally. The extruded muscle masses were immediately transferred to 100 μL of lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 17 mM DTT) and centrifuged for 5 min at 12000k. The supernatant was collected and precipitated with methanol and chloroform (Wessel and Flugge, 1984) and resuspended in 40 μL labeling buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 5 mM magnesium acetate). One-third of each sample (13 μL, or ca. 16 muscle masses) was removed and combined into a single tube to comprise the pooled-sample internal standard which was then labeled en masse with 1200 pmol Cy2. The remaining two-thirds of each sample were individually labeled with 200 pmol of either Cy3 or Cy5 such that a given genotype was individually labeled twice with each dye to control for any dye-specific labeling artifacts. The labeled samples were combined such that each pair wise Cy3/Cy5-labeled sample was mixed with an equal aliquot of the Cy2-labeled mixed sample; in total, ca. 50 muscle masses (composed of two labeled samples of controls or mutant along with an equal aliquot of the 12-mix) were loaded on each gel. Quantitative measurements were made relative to the signal from the Cy2-labeled internal standard, allowing for simultaneous analysis of all 12 samples regardless of which two samples are co-resolved on a given DIGE gel. Mass spectrometry and database interrogation data are listed in supplementary Table 1. The four sets of tripartite-labeled samples were separated by standard 2D gel electrophoresis using an IPGphor first-dimension isoelectric focusing unit and 24 cm 4–7 immobilized pH gradient (IPG) strips (Amersham Biosciences/GE Healthcare, Piscataway NJ), followed by second-dimension 12% SDS-PAGE using an Ettan DALT 12 unit (Amersham Biosciences/GE Healthcare). The Cy2 (mixed standard), Cy3 (sample n), and Cy5 (sample m) components of each gel were individually imaged using mutually exclusive excitation/emission wavelengths with a Typhoon 9400 fluorescence imager (Amersham Biosciences/GE Healthcare). A Sypro Ruby post-stain (Molecular Probes/Invitrogen) was used to ensure accurate protein excision. DeCyder software (Amersham Biosciences/GE Healthcare) was used for simultaneous comparison of abundance changes across all three sample pairs with statistical confidence and without interference from gel-to-gel variation (Alban et al., 2003; Friedman et al., 2004). Normalized volume ratios for each resolved protein were calculated relative to the internal standard present on every gel and were used to calculate average abundance changes and Student's t test P values for the variance of these ratios for each protein pair across all six independent gels. Proteins of interest were excised and digested in-gel with modified porcine trypsin protease (Promega). Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS/MS) and data-dependant TOF/TOF tandem MS/MS was performed on a Voyager 4700 mass spectrometer (Applied Biosystems, Framingham MA). For each protein, intact peptide masses and associated fragmentation spectra were collectively used to interrogate sequences present in the NCBInr database to generate statistically significant candidate identifications using GPS Explorer software (Applied Biosystems) running the MASCOT search algorithm. Searches were performed without constraining taxonomy or protein molecular weight or isoelectric point, with complete carbamidomethylation of cysteine, partial oxidation of methionine residues, and 1 missed cleavage also allowed in the search parameters. Significant Molecular Weight Search (MOWSE) scores (p<0.05), number of matched ions, number of matching ions with independent MS/MS matches, percent protein sequence coverage, and correlation of gel region with predicted MW and pI were collectively considered for each protein identification.

Results

Constitutive postsynaptic proteasome inhibition alters synaptic function

DTS5 and DTS7 are dominant temperature-sensitive mutants that result from single-amino-acid substitutions in the β6 and β2 proteasome subunits of the 20S proteasome, respectively (Saville and Belote, 1993; Smyth and Belote, 1999). Mutant subunits act in a dominant negative manner to interfere with proteasome function; in multiple previous studies, they have been shown to inhibit UPS-mediated degradation of known proteasome substrates (Schweisguth, 1999; Speese et al., 2003; Neuburger et al., 2006). Transgenic co-expression of both proteasome subunit mutants in the Drosophila eye using the UAS-GAL4 system has a synergistic effect (Belote JM and Fortier, 2002). Previously, it was shown in our laboratory that the proteasome is highly localized at the Drosophila NMJ synapse and that presynaptic inhibition of proteasome function rapidly leads to changes in synaptic transmission that are likely mediated through degradation of the synaptic vessel priming protein DUNC-13 (Aravamudan and Broadie, 2003; Speese et al., 2003). To examine the effect of inhibiting proteasome function specifically in the postsynaptic cell, we crossed the double-mutant UAS-DTS5, DTS7 line with the muscle-specific MHC-GAL4 line to drive expression of the DTS proteasome mutant subunits specifically in muscle (Fig. 1A). The resulting MHC-GAL4 / UAS-DTS5, DTS7 line will hence be referred to as DTS. DTS animals develop normally with no distinguishable morphological phenotypes. In DTS larval muscle, there is a marked accumulation of polyubiquitinated proteins, as assayed with FK-1, an antibody specific for polyubiquitinated proteins (Fig. 1B). FK-1 immunostaining intensity was significantly elevated in DTS larval muscle relative to controls (MHC-GAL4/+ 1.00 ± 0.070, UAS-DTS/+ 0.90 ± 0.077, DTS 1.83 ± 0.112, n=12, p < 0.001, one-way ANOVA and Newman-Keuls post-test), with no significant difference in immunostaining intensity measured in the brain neuropil between DTS and control animals (MHC/+ 1.00 ± 0.048, UAS-DTS/+ 1.02 ± 0.093, DTS 0.88 ± 0.036, n = 12). Thus, this method selectively inhibits proteasome function within the postsynaptic muscle.

Figure 1
DTS mutant animals accumulate polyubiquinated proteins in muscle

When the DTS proteasome mutants were constitutively expressed, the average EJC amplitude recorded in 0.2 mM calcium was more than twice that of both the heterozygote controls (MHC-GAL4/+ 8.29 ± 1.77 nA, n=9, UAS-DTS/+ 8.96 ± 1.39 nA, n=10, DTS 18.1 ± 2.48, n=10; p < 0.01, one-way ANOVA and Newman-Keuls post-test; Fig. 2A,B). A similar but smaller increase in EJC amplitude was recorded in 0.4 mM calcium (MHC-GAL4/+ 71.1 ± 5.14 nA, n=14, UAS-DTS/+ 56.8 ± 5.56 nA, n=7, DTS 96.0 ± 8.57 nA, n= 14; p < 0.05). There was no significant difference in EJC amplitude in high (1.8 mM) extracellular calcium (MHC-GAL4/+ 198.2 ± 10.5 nA, n=8, UAS-DTS/+ 178.5 ± 4.56 nA, n=6, DTS 170.6 ± 4.56 nA, n=5; Fig. 2C). Short-term facilitation was assessed by taking the ratio of the average EJC amplitude after 10 consecutive stimuli at 20 Hz frequency relative to the baseline EJC amplitude assessed at the basal 0.5 Hz stimulation. In DTS animals, short-term facilitation was very significantly reduced in both 0.2 mM calcium (MHC-GAL4/+ 3.56 ± 0.71, n=9, UAS-DTS/+ 3.49 ± 1.53, n=9, DTS 2.05 ± 0.34, n=10; p < 0.01) and 0.4 mM calcium (MHC-GAL4/+ 1.29 ± 0.19, n=11, UAS-DTS/+ 1.19 ± 0.12, n=6, DTS 0.91 ± 0.19, n=9; p < 0.01) (Fig. 3A-C). Elevated EJC amplitude at lower extracellular calcium concentrations combined with impaired short-term facilitation is often considered diagnostic of altered presynaptic properties, yet for these experiments the proteasome inhibition was specifically inhibited postsynaptically.

Figure 2
Postsynaptic proteasome inhibition increases synaptic transmission
Figure 3
Postsynaptic proteasome inhibition impairs short-term facilitation

To further evaluate presynaptic versus postsynaptic mechanisms, miniature EJCs properties were analyzed. This analysis revealed significantly smaller mEJC amplitudes in DTS proteasome mutants when compared to both control conditions (MHC-GAL4/+ 0.86 ± 0.042 nA, n=8, UAS-DTS/+ 0.71 ± 0.028 nA, n=10, DTS 0.62 ± 0.018 nA, n=10; p < 0.05). mEJC decay was analyzed by averaging the first 25 mEJCs for each recording and fitting the decay with a single exponential function. mEJC decay was significantly more rapid in the DTS proteasome mutants relative to both controls (MHC-GAL4/+ 15.6 ± 0.71ms, n=8, UAS-DTS/+ 13.9 ± 0.42 ms, n=10, DTS 12.6 ± 0.39 ms, n=10; p < 0.05). However, there was no change in mEJC frequency between DTS proteasome mutants and controls (MHC-GAL4/+ 2.80 ± 0.25 Hz, n=8, UAS-DTS/+ 2.49 ± 0.28 Hz, n=10, DTS 2.37 ± 0.31 Hz, n=10). The change in mEJC amplitude without a change in mEJC frequency is supportive of a postsynaptic effect. Importantly, smaller mEJC amplitudes and faster mEJC decay times occur when B-Class GluRs are expressed exclusively, or when these receptors are present in greater relative abundance (DiAntonio et al., 1999; Chen and Featherstone, 2005).

Increased B-Class GluRs and DLG scaffold after constitutive proteasome inhibition

To assess the postsynaptic GluR field, animals were immunostained with specific GluRIIA or GluRIIB antibodies. Consistent with the prediction of increased B-class receptors based on the functional characterization, GluRIIB immunofluorescence was clearly qualitatively increased in the postsynaptic domains of DTS proteasome mutants (Fig. 4A). GluRIIB fluorescence intensity was quantified in isolated synaptic boutons per unit area relative to an internal fluorescence control (HRP). In DTS proteasome mutants, there was a higher level of B-class receptors compared to either genetic control (MHC-GAL4/+ 1.00 ± 0.035, n=18, UAS-DTS/+ 1.00 ± 0.058, n=18, DTS 1.19 ± 0.052, n=18; p < 0.05) (Fig. 4B). In contrast, GluRIIA immunofluorescence was unchanged in DTS and genetic controls (Fig. 4B). Thus, inhibition of the proteasome causes a specific up-regulation of B-class GluRs without altering the synaptic abundance of A-class GluRs.

Figure 4
Postsynaptic proteasome inhibition selectively increases GluRIIB

It has been recently demonstrated that synaptic expression of B-class GluRs correlates with expression of DLG, a PDZ domain-containing MAGUK scaffolding protein that is a homologue to mammalian PSD-95 and hDLG/SAP97 proteins (Chen and Featherstone, 2005). DLG regulates the synaptic abundance of B-class GluRs, but has no detectable role in the synaptic localization of A-class GluRs. Consistently, immunostaining with a specific anti-DLG antibody showed a clear qualitative increase in the postsynaptic NMJ domains of DTS proteasome mutants (Fig. 5A). DLG fluorescence intensity was quantified in isolated synaptic boutons, as above, and was increased in DTS proteasome mutants compared to both genetic controls (MHC-GAL4/+ 1.00, n=13, UAS-DTS/+ 1.02 ± 0.069, n=13, DTS 1.19 ± 0.054, n=13; p < 0.05) (Fig. 5B). Note that the quantitative increase in B-class GluRs and DLG was nearly identical, consistent with a regulatory correlation between the DLG scaffold and B-class GluRs.

Figure 5
Postsynaptic proteasome inhibition increases DLG scaffold expression

Acute postsynaptic proteasome inhibition alters synaptic function

Understanding the time course of UPS regulation of synaptic function is critical to understanding its role in acute synaptic plasticity versus chronic synaptic remodeling. To begin to assess this, we used the GeneSwitch system (Osterwalder et al., 2001) to attain temporal control over expression of the DTS proteasome mutant subunits. The GeneSwitch MHC-GAL4 line was crossed with the UAS DTS proteasome mutant line. To determine the best timing interval, RU486 induction of expression of a reporter protein, τ tubulin-GFP, was examined by crossing the GeneSwitch MHC-GAL4 line with a UAS τ-tubulin GFP line. No GFP expression was detected in muscle cells up through the early third instar larval period (96 hours after egg laying), although a low level of GFP expression was observed in muscles of –RU486 control late wandering third instar larvae (data not shown), similar to a previous report (Osterwalder et al., 2001). Thus, early third instar larvae were used in subsequent experiments. Following RU486 feeding, the first reporter expression was detected after several hours, with a high level of expression present at 6 hours (data not shown). Six hours post-induction was therefore selected as the interval for all subsequent assays.

When synaptic function was examined in 6-hour RU486-induced animals, EJC amplitude was nearly tripled compared to the un-induced control (-RU486 4.24 ± 0.58 nA, n=10, +RU486 11.5 ±1.74 nA, n=10; p < 0.001, two-tailed T test) (Fig. 6A,B). As an additional control for RU486 effects, GeneSwitch MHCGAL4/+ larvae fed RU486 for 6 hours were also examined, and EJC amplitudes were similar to those in control animals (3.31 ± 0.56 nA, n=7). As with constitutive proteasome inhibition, the strongly enhanced synaptic function was specific to low external calcium condition, with no change detectable in high (1.8 mM) calcium saline (-RU486 105 ± 4.92 nA, n=11, +RU486 92.2 ± 6.70 nA, n=9). Similarly, short-term facilitation was grossly impaired over stimulus frequencies ranging from 0.5 Hz to 20 Hz (Fig. 6C). Interestingly, both the elevated basal transmission and the depressed facilitation were more pronounced following this acute proteasome inhibition. This difference may be due to more efficient proteasome inhibition with the GeneSwitch system, a lack of time for compensatory mechanisms, or simply an effect of the superior genetic control conditions. Similar to results seen with constitutive DTS expression, mEJC amplitudes were significantly reduced following acute DTS expression (-RU486 0.64 ± 0.028 nA, n=12, +RU486 0.54 ± 0.030 nA, n=12; p < 0.05) (Fig. 6 D,E), and mEJC frequency was unchanged (+RU486 0.95 ± 0.13 Hz, n=12, -RU486 0.98 ± 0.16 Hz, n=12) (Fig. 6F). These results show that altered synaptic function happens quickly, on the time course of hours, following proteasome inhibition.

Figure 6
Acute postsynaptic proteasome inhibition increases synaptic transmission

Presumably, the acutely altered function should correlate with the same changes in postsynaptic composition as reported for constitutive proteasome inhibition. GluRIIB immunostaining confirmed an increased synaptic abundance of B-class GluRs (-RU486 1.00 ± 0.032, n=20, +RU486 1.24 ± 0.056, n=20; p<0.001) but also showed a coincident significant decrease in GluRIIA synaptic expression (-RU486 1.00 ± 0.024, n=13, +RU486 0.900 ± 0.041, n=13; p <0.05) (Fig 7A,B). Interestingly, unlike with constitutive proteasome inhibition, synaptic DLG immunostaining was not significantly changed (Fig 6C). Therefore, as an alternative means to assess DLG abundance and putative ubiquitination, larval lysates were immunoprecipitated with anti-DLG antibody. Strikingly, the amount of immunoprecipitated DLG was increased by more than 5-fold in +RU486 lysates compared to un-induced controls (Fig. 8A). Blotting with anti-ubiquitin antibody revealed a smear of higher molecular weight bands, consistent with DLG ubiquitination (Fig. 8B). These results show that acute inhibition of proteasome function postsynaptically causes a marked accumulation of DLG and a coincident, specific elevation of B-class GluRs in the postsynaptic domain.

Figure 7
Acute postsynaptic proteasome inhibition increases GluRIIB expression
Figure 8
Acute postsynaptic proteasome inhibition causes ubiquitinated DLG to dramatically accumulate in muscle

Proteomic analysis for protein alterations after postsynaptic proteasome inhibition

To identify additional postsynaptic proteins that are regulated by the UPS, we performed proteomic analyses using two-dimensional difference gel electrophoresis (2D-DIGE; Fig. 9). This analysis was used to resolve proteins isolated from body wall muscle dissections in constitutive DTS-expressing and genetic control animals. 2D DIGE is a proteomics technology that overcomes many of the caveats of conventional 2D gel analysis; it employs controls and multiple sampling that allows changes in protein abundance to be detected with statistical confidence (Alban et al., 2003, Friedman et al., 2004). Approximately 1500 protein spots with isoelectric points between pH 4 and 7, and molecular weights between 15 and 150 kDa were resolved on these gels (Fig. 9). Sixteen protein spots representing ten distinct identifiable proteins showed significant changes in DTS proteasome mutants when quantified across the four independent experiments (Fig. 9). Several proteins were present as multiple charged or processed isoforms, some of which were reciprocally up- and down-regulated in the mutant (rather than an overall change in protein abundance).

Figure 9
Proteomic analysis following postsynaptic proteasome inhibition

These results show that postsynaptic proteasome inhibition affected several cellular processes and structures that may be critical regulators of postsynaptic function including: chaperones (lethal(2)essential for life, FK506 binding protein 59), the cytoskeleton (tubulin αchain), glucose/AA metabolism (phosphoglucomutase, phosphoenolpyruvate carboxykinase), oxidative stress response (GST omega), and deubiquitinating enzymes (ubiquitin C-terminal hydrolase). However, the mass spectroscopic identification of randomly selected proteins from multiple DIGE gels, showed a relative absence of proteins specific to the postsynaptic density (data not shown). Possible explanations for this include the relatively lower abundance of these proteins in the muscle, the high molecular weights of many postsynaptic density proteins, and the relative insolubility of membrane associated proteins. Of the proteins identified, the upregulation of the ubiquitin C-terminal hydrolase (Fig. 9) is particularly interesting as this deubiquitinating enzyme is involved in cleaving small ubiquitin chains to regenerate free ubiquitin. This change is therefore suggestive of a compensatory change in response to increased utilization of ubiquitin in polyubiquitin chains during proteasome inhibition.

Discussion

B-Class glutamate receptors are specifically regulated by the UPS

Constitutive and acute proteasome inhibition in the postsynaptic muscle of the Drosophila NMJ increases synaptic current amplitudes in low external calcium, although not in high calcium, and strongly impairs short-term facilitation. This targeted postsynaptic proteasome inhibition also increases the synaptic abundance of B-class glutamate receptors, as demonstrated by both GluRIIB immunostaining and GluRIIB-correlated changes in mEJC properties. Constitutive UPS inhibition shows that this regulation is specific to B-class receptors, with A-class receptors being unaffected. Acute UPS inhibition reveals not only a highly significant increase in B-class glutamate receptors, but also a simultaneous decrease in A-class glutamate receptors. Thus, the synaptic functional changes likely result from this increase in B-class relative to A-class receptors, reflecting differences in A-class and B-class GluR properties. In particular, it has been shown that B-class receptors desensitize much more rapidly than A-class receptors based on analysis of currents in outside-out patches from GluRIIA and GluRIIB knockout animals (DiAntonio et al. 1999). If B-class receptor desensitization is regulated through a calcium-dependent mechanism, this could explain the increased EJC amplitudes in lower calcium with no amplitude changes in higher calcium. Another possibility may reflect the fact that A-class and B-class receptors localize to separate postsynaptic domains (Marrus et al., 2004), with B-class domains potentially more active at lower calcium concentrations. Further characterization of the biophysical properties and synaptic localization of A-class and B-class receptors will be necessary to test these possibilities.

The pattern of synaptic functional change we observed following postsynaptic proteasome inhibition is often seen when presynaptic function is altered (Rohrbough et al., 1999; Rohrbough et al., 2000). Such a presynaptic effect would necessitate that postsynaptic UPS inhibition was triggering a retrograde signaling pathway that modifies presynaptic neurotransmitter release. There is ample evidence for retrograde signaling at the Drosophila NMJ synapse (Peterson et al., 1997; Davis et al., 1998; Haghighi et al., 2003) and this signaling can occur very rapidly (minutes) as a homeostatic response to postsynaptic GluR changes (Peterson et al., Davis et al., Frank et al., 2006). This mechanism has been proposed for the functional changes in GluRIIA subunit knockouts and following acute pharmacological GluR inhibition. The lack of change in mEJC frequency following constitutive or acute postsynaptic proteasome inhibition does not support this, but in other examples where a retrograde signaling mechanism is proposed at this synapse, changes in miniature excitatory postsynaptic potential (mEPSP) frequency were similarly not observed (Peterson et al., 1997; Frank et al., 2006). Thus, since postsynaptic UPS inhibition increased the proportions of B-class GluRs, GluR-mediated modulation of a retrograde signal that alters presynaptic properties is another potential mechanism for the synaptic functional changes we observed.

Experiments using the GeneSwitch system to induce acute postsynaptic proteasome inhibition demonstrate that the increase in B-Class receptor abundance and coincident changes in synaptic function are rapid. Just how rapid is this UPS regulation? With this transgenic system, induced protein expression is not detectable at 2.5 hours, but becomes detectable by ~5 hours post induction (Osterwalder et al., 1999). This delay reflects the time it takes for larvae to ingest and absorb RU486, followed by transcription, translation, and incorporation of the induced protein into its appropriate cellular pathway. In the case studied here, mutant proteasome subunits have to incorporate into the proteasomal complex, and then replace the existing population of proteasomes in muscle. Presumably, these combined events take several hours, at the least showing that the UPS-mediated regulation of B-class receptors is occurring within a few hours. This rapid turnover of B-class receptors stands in contrast to results from a recent study using fluorescently tagged GluRIIA showing that A-class receptors are relatively stable for up to 24 hours after being inserted in postsynaptic membrane (Rasse et al., 2005). A-class GluR abundance has been shown to increase with heightened synaptic activity (Sigrist et al., 2002; Sigrist et al., 2003). Increasing expression of the GluRIIA subunit results in an increase in the amplitude of spontaneous synaptic potentials while over-expression of GluRIIB results in a decrease in the amplitude of spontaneous synaptic potentials (DiAntonio et al., 1999). This suggests that B-class GluRs may be important in fine tuning synaptic responsiveness, but during periods of heightened synaptic activity, they may be rapidly turned over via the UPS and replaced by more stable, slowly desensitizing A-class receptors.

This study expands on previous work showing that A-class and B-class GluRs are differentially regulated. A-class receptor synaptic abundance decreases with protein kinase A application (Davis et al., 1998) and when the actin cytoskeleton interacting Coracle/4.1 protein is absent (Chen et al., 2005), but increases with p21 activated kinase (PAK) pathway activation (Albin and Davis, 2004) and when the anaphase promoting complex (APC) is absent (van Roessel et al., 2004). Similarly, in mammalian CNS glutamatergic synapses, there are many examples of differential regulation of AMPA GluR subtypes occurring in physiological and pathological processes (Ogoshi and Weiss, 2003; Stellwagen et al., 2005; Thiagarajan et al., 2005; Gardner et al., 2005; Brooks-Kayal, 2005). Our results demonstrate that UPS-mediated turnover provides an additional important mechanism for rapid differential regulation of glutamate receptor subclasses.

DLG scaffold is acutely targeted by UPS regulation

The DLG scaffold is a critical determinant of B-class GluR abundance in the postsynaptic domain (Chen and Featherstone, 2005). Thus, increased DLG levels following constitutive proteasome inhibition may explain the increased abundance of B-class receptors. For acute proteasome inhibition, there is direct evidence of increased DLG levels based on quantitative immunoprecipitation. However, this acute effect on DLG is not apparent in the NMJ immunostaining. One explanation for this finding is that ubiquitination of DLG leads to higher levels of membrane-dissociated DLG, which is more readily immunoprecipitated. There are multiple potential explanations for the UPS-mediated regulation of B-class glutamate receptors and DLG after transgenic proteasome inhibition. UPS inhibition may directly regulate DLG leading to an increase in GluRIIB, may independently directly regulate DLG and GluRIIB, or may act on a protein or proteins that regulate DLG and/or GluRIIB. We have been unable to determine whether B-class GluRs are directly ubiquitinated, but are currently working to develop an antibody that works for GluRIIB immunoprecipitation experiments. These possibilities would best be addressed in future studies involving DLG or GluRIIB over-expression or knockdown.

This study provides the first evidence that DLG is ubiquitinated in Drosophila. Previous studies have shown that human DLG/SAP97 (hDLG) becomes a target for proteasomal degradation when bound by the human papilloma virus protein E6 (Thomas et al., 2005; Matsumoto et al., 2006). In cultured rat hippocampal neurons, another mammalian homologue of Drosophila DLG, PSD-95, has been shown to be polyubiquitinated in its N-terminal domain and targeted for proteasomal degradation (Colledge et al., 2003). However, the N-terminal region required for PSD-95 ubiquitination is not present in Drosophila DLG, indicating that it must have a different ubiquitination site. Since the binding of the E6 protein can facilitate hDLG ubiquitination, it is possible that a native postsynaptic protein fulfills this role to facilitate Drosophila DLG ubiquitination. DLG is a critical postsynaptic scaffold that organizes the subsynaptic reticulum postsynaptic (SSR) domain (Budnik et al., 1996), forms a physical complex with integrin receptors (Beumer et al., 2002), and localizes Shaker potassium channels, the cell adhesion molecule fasciclin II, and other PDZ-domain scaffold proteins to the postsynaptic domain (Tejedor et al., 1997; Thomas et al., 1997; Bilder et al., 2000). Thus, UPS regulation of MAGUK family PDZ-domain containing postsynaptic density organizing proteins may a fundamental step in synaptic remodeling.

Conclusion

This study is the first in vivo demonstration of a selective UPS regulation of a specific subclass of postsynaptic glutamate receptors. This study also begins to define the critical temporal dynamics of UPS synaptic regulation, showing that the UPS remodels the postsynaptic apparatus on the time course of hours. Future work must focus on identifying specific ubiquitin ligases and delineating synaptic proteins, in addition to the DLG scaffold, that are directly ubiquitinated by these ligases. Combining temporal and spatial targeting of proteasome inhibition with more focused and specific proteomic approaches will prove especially valuable in identifying synaptic proteins that are ubiquitinated over specified time courses. Several neurological diseases that lead to synaptic dysfunction specifically involve mutations in UPS proteins, including Alzheimer’s disease, Angelman Syndrome, and Parkinson’s Disease (Ciechanover and Brundin, 2003; Jiang and Beaudet, 2004). Understanding the UPS role in regulation of synaptic function offers the hope of identifying new therapeutic targets to treat or prevent these diseases.

Supplementary Material

Acknowledgments

We are particularly grateful to Dr. John Belote for providing the UAS-DTS line and to Dr. Haig Keshishian for providing the Geneswitch MHC-GAL4 line that were instrumental to this study. We are most grateful to Dr. Aaron DiAntonio for providing the critical GluRIIB antibodies. We thank the Iowa Developmental Studies Hybridoma Bank for providing GluRIIA and DLG antibodies used in this study. This work was supported by NIH grant NS048882-01 to K.F.H. and NS41740 to K.B.

Footnotes

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