CHAPTER 3 Neurobeachin regulates transmitter receptor ... 3 Neurobeachin regulates...early in the biosynthetic pathway, and mature NMDA, kainate and GABA A-receptors do not reach the

CHAPTER 3

Neurobeachin regulates transmitter receptor trafficking tosynapses

Juliane Lauks1, Ramya Nair2, SangYong Jung2, Nancy E. Cooke 3, Heidi de Wit1, NilsBrose2, Manfred W. Kilimann4, Matthijs Verhage1 and JeongSeop Rhee2

1 Departments of Functional Genomics and Clinical Genetics, Center for Neurogenomics andCognitive Research, Neuroscience Campus Amsterdam, Vrije Universiteit (VU) andVU Medical Center, 1081 HV Amsterdam, Netherlands

2 Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine,37075 Goettingen, Germany

3 Department of Genetics and Medicine, University of Pennsylvania, Perelman School of Medicine,Philadelphia, PA 19104, USA

4 Department of Neuroscience, Uppsala University, 75124 Uppsala, Sweden

J. Lauks and R. Nair contributed equally to this work

Results of this chapter have been accepted for publication in the Journal of Cell Biology

Chapter3

Abstract

At synapses, the surface density of clustered neurotransmitter receptors is a key de-terminant of synaptic efficacy. Synaptic receptor accumulation is regulated by thetransport, postsynaptic anchoring, and turnover of receptors. These processes arecontrolled by multiple trafficking, sorting, motor, and scaffold proteins. We foundthat neurons lacking the BEACH domain protein Neurobeachin have strongly reducedsynaptic responses due to a reduction in surface levels of glutamate and GABAA-receptors. In the absence of Neurobeachin, immature AMPA-receptors accumulateearly in the biosynthetic pathway, and mature NMDA, kainate and GABAA-receptorsdo not reach the synapse, while maturation and surface expression of other membraneproteins, synapse formation, and presynaptic function are unaffected. These datashow that Neurobeachin regulates synaptic targeting of neurotransmitter receptorsunder basal conditions and thereby exerts a major influence on synaptic transmis-sion.

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Nbea regulates transmitter receptor trafficking

Introduction

Strength and plasticity of nerve cell synapses are controlled by modifications of thenumber and functionality of neurotransmitter receptors in the postsynaptic density.The number of functional synaptic receptors is regulated by a complex interplay be-tween mobilization and immobilization, by internalization and local recycling, and bydesensitization. While many features of the activity-dependent local receptor traffick-ing mechanisms have been characterized in detail, the mechanisms that orchestratedelivery of new receptors - from their biosynthesis and heteromeric assembly in theendoplasmic reticulum (ER) via their maturation in the Golgi apparatus to their fi-nal insertion into the target membrane - are poorly understood. These mechanismshave both shared and unique features for different receptor types. Already in the ER,the assembly of oligomeric glutamate and GABAA receptors, and the combinationof receptor subunits are controlled by different scaffolding, adapter, and traffickingproteins (Greger and Esteban, 2007; Jacob et al., 2008; Prybylowski and Wenthold,2004). Likewise, the molecular determinants of the motor-based intracellular trans-port of different receptor types are different (Hirokawa and Takemura, 2004).

Defined organizing processes are likely to control the assembly and targeting ofspecific multiprotein complexes to neuronal synapses. In the present study, we iden-tified the putative neuronal trafficking protein Neurobeachin (Nbea) as a moleculewith such a function for several major ionotropic excitatory and inhibitory receptorsin nerve cells. Nbea is a large multidomain protein (327 kDa) and selectively ex-pressed in neurons and endocrine cells (Wang et al., 2000). Its C-terminus containsa highly conserved BEACH (beige-Chediak/Higashi) domain, a pleckstrin homology-like (PH) domain and WD40 repeats (Gebauer et al., 2004; Wang et al., 2002, 2000),an arrangement that is shared within a family of eight BEACH domain proteins. Mu-tations in the BEACH domain of LYST cause Chediak-Higashi Syndrome in humansand the beige syndrome in mice, which are characterized by enlarged lysosomes andrelated organelles (Burkhardt et al., 1993; Introne et al., 1999; Nagle et al., 1996;Zhao et al., 1994). Mutations in the BEACH protein NBEAL2 cause Gray Plateletsyndrome with perturbations in the biogenesis of secretory α-granules and other en-domembrane compartments (Albers et al., 2011; Gunay-Aygun et al., 2011; Kahret al., 2011). Human mutations in LRBA cause a syndrome dominated by immuno-logical abnormalities (Lopez-Herrera et al., 2012). Heterozygous rearrangements inthe human NBEA gene were detected in patients with autism (Castermans et al.,2003) or with multiple myeloma (O’Neal et al., 2009), and reduced Nbea expressioncauses overweight in mice and possibly also in humans (Olszewski et al., 2012).

In neurons, Nbea is recruited to tubulovesicular endomembranes in a GTPγSstimulated manner (Wang et al., 2000), which led to the notion that Nbea might reg-ulate post-Golgi protein trafficking. Accordingly, Nbea associates with the traffickingprotein Vacuolar Protein Sorting 35 in a complex with glycine receptor β-subunits(del Pino et al., 2011). Two knock-out (KO) alleles were reported for murine Nbea,and consequent defects in synapse morphology, enrichment of synaptic molecules andsynaptic transmission were described (Medrihan et al., 2009; Niesmann et al., 2011;

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Su et al., 2004). Here, we demonstrate that profound defects in the synaptic localiza-tion of ionotropic receptors for the key excitatory and inhibitory neurotransmittersare a major cause of these synaptic defects, and that in the absence of Nbea, thesereceptors accumulate in the biosynthetic pathway. Our observations identify Nbea asa general organizer of synaptic receptor targeting with a major role in the regulationof synaptic transmission.

Materials and Methods

Laboratory animals

Two independent Nbea KO mouse lines were used in this study (Medrihan et al., 2009;Su et al., 2004). Central aspects of the synaptic phenotype, i.e.glutamate responsive-ness, were tested and confirmed in both lines. The ultrastructural analyses, analysesof Nbea levels in rescued neurons, analyses of glutamate responses in high densitycultures, analyses of connectivity in Nbea KO and WT neurons, were performed inthe laboratory of M.V. using the mouse line generated by the laboratory of N. E.C. (Su et al., 2004). Electrophysiological recordings on autapses and heterologousneuronal pairs, as well as the FM1-43, biotinylation and deglycosylation experimentswere performed in the laboratory of J.S.R. using the mouse line generated by thelaboratory of M.W.K. (Medrihan et al., 2009). Mouse embryos were obtained at em-bryonic day 18 (E18) by caesarian section of pregnant females from timed matingof Nbea heterozygous animals (C57/Bl6 background). Heterozygous Nbea KO micedisplay no obvious phenotypic changes in their viability or fertility. However, Nbea-deficient mice can be easily distinguished from wild-type or heterozygous littermates- they exhibit hunched posture and an omphalocele, i.e. an abdominal defect wherethe intestine is not withdrawn into the abdominal cavity during embryonic develop-ment. In addition, they don’t show any response to tactile stimuli and die perinatally(Medrihan et al., 2009; Su et al., 2004), most likely due to defective neuromuscularsynaptic transmission and concomitant breathing failure (Su et al., 2004). NewbornP0-P1 pups from pregnant female Wistar rat (Harlan or Charles River) were used forrat glia preparations. All animals were housed and bred according to the institutionaland Dutch governmental guidelines for animal welfare.

Plasmids and antibodies

The full-length Nbea cDNA clone was generated from fragments of a yeast-two-hybrid cDNA library (Clontech CAT# ML408AH) and a partial image clone (KazusamKIAA1544). First, the N-terminal part of Nbea was obtained from the yeast-two-hybrid cDNA library and subcloned in pCR-Script (Stratagene Cat# 211190) usingthe following primers: rz62 5’TGCACAGCTCCTCAGCAGCG3’; rz63r 5’GCTGGGT-GTTCTGACATTAGAGCC3’and rz64 5’CAGCTCATATTAAAGGATCGAGG’3;rz65r 5’GGATGAGG-GATAGATGGTATGACC3’. The resulting subclones were lig-ated at PstI and ScaI sites. Then, the C-terminal part from the Kazusa image clone

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was connected to the N-terminal part using NotI and SpeI resulting in a full-lengthNbea in a pCR-Script backbone. For creating the Nbea-IRES2EGFP construct thefull-length Nbea script clone was digested with Ndel and SalI. After modifying theNdel site with Klenow polymerase (New England Biolabs CAT# M0210S) the Nbea-containing fragment was ligated into the pIRES2EGFP (Clontech PT3267-5), whichwas digested with SmaI and SalI. A fusion of EYFP and Nbea was made by di-gesting the Nbea full-length pCR-Script with SalI & KspI and ligating this into thepEYFP-C1, digested with the same enzymes. pEGFP was purchased from Clontech(CAT#PT2039-5). For the analysis of the number of synapses and connectivity inboth genotypes specific primary antibodies were used against MAP2 (mouse mono-clonal AP20, Chemicon, 1:1000), Nbea (rabbit polyclonal, Synaptic Systems, 1:1000;for details see Chapter 2) and VAMP2 (mouse monoclonal 69.1, Synaptic Systems,1:1000). Suitable secondary antibodies were obtained fromMolecular Probes (1:1000).Protein levels in mouse E18 brain homogenate and lysates prepared from cultured neu-rons were assessed using the following primary antibodies against: actin (mouse mon-oclonal, Sigma, 1:4000), GABAAα1 (rabbit polyclonal, Chemicon, 1:1000), GABAAα5(rabbit polyclonal (Brunig et al., 2002)1:2000), GABAAγ2 (rabbit polyclonal, Abcam,1:1000), GluA1 (rabbit polyclonal, Chemicon, 1:1000), GluA2/3 (rabbit polyclonal,Chemicon, 1:1000), GluK2/3 (rabbit polyclonal, Chemicon, 1:1000), GluN1 (mousemonoclonal M68, Synaptic Systems, 1:1000), GluN2A (rabbit polyclonal, Chemicon,1:1000), munc13-1 ((Rhee et al., 2002); 1:1000 ), NL1 (mouse monoclonal 4C12,Synaptic Systems 1:5000), PSD95 (mouse monoclonal 6G6-1C9, Abcam, 1:2000),synapsin1 (rabbit polyclonal, Synaptic Systems, 1:2000), synaptophysin (mouse mon-oclonal, Synaptic System 1:400000), synaptotagmin1 (mouse monoclonal, SynapticSystems, 1:2500), transferrin receptor (mouse monoclonal H68,4, Zymed, 1:2000),tubulin (mouse monoclonal, Sigma, 1:30000), VGLUT1 (rabbit polyclonal, SynapticSystems, 1:2000) and VIAAT (Synaptic Systems, 1:1000). The blots were detectedwith peroxidase-labeled secondary antibodies (1:5000, Sigma or DAKO), developedwith ECL/ECF and the protein levels were quantified using ImageJ.

Dissociated neuronal culture and transfection

Microisland and high-density cultures of hippocampal and striatal neurons were pre-pared and cultured as described previously (Jockusch et al., 2007). For electrophys-iological measurements in high density neuron cultures, neurons were plated on glialcells. The cell cultures were transfected at DIV3-4 using the calcium-phosphate pre-cipitation method as described previously (Kohrmann et al., 1999). For heterologouspairs of cells on a single astrocyte microisland, glutamatergic (presynaptic) neuronswere obtained from hippocampus and inhibitory (postsynaptic) cells were obtainedfrom striatum. Cell types in culture were identified based on their morphology andsynaptic characteristics.

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Electrophysiology

For autaptic and paired recordings cells were whole cell voltage clamped at -70 mVwith an axoclamp amplifier under the control of the Clampex program 10.1. Allanalyses were performed using Axograph 4.1, Axograph X or Clampfit 10.2. Thecells were recorded between DIV10-19. EPSCs and IPSCs were evoked by depolar-izing cells from -70 mV to 0 mV. The response to triggered release of the RRP wasmeasured after application of hypertonic sucrose solution (500 mM). mEPSCs andmIPSCs were recorded in the presence of 300 nM TTX. The extracellular solutioncontained (mM): 140 NaCl, 2.4 KCl, 4CaCl2, 4 MgCl2, 10 HEPES, 10 glucose (pH7.3;320 mOsm). The patch-pipette solution for autaptic recordings contained (mM): 136KCL, 17.8 HEPES, 1 EGTA, 4.6 MgCl2, 4 NaATP, 0.3 Na2GTP, 15 creatine phos-phate and 5 U/ml phosphocreatine kinase (315-320 mOsm, pH 7.4). The series re-sistance was compensated by about 60-80 %. All extracellular solutions were appliedwith a custom-built fast flow system consisting of an array of flow pipes controlled by astepper monitor that allows complete and rapid solution exchange with time constantsof approximately 30 ms. In high-density cultures (25k/well), whole-cell voltage-clamp(Vm= -70 mV) recordings were acquired with an Axopatch 200B amplifier, Digidata1440A and pCLAMP 10 software (all from Axon Instruments). Recordings were per-formed at DIV 1418 with borosilicate glass pipettes (2.5 - 4 mOhm) containing in(mM): 125 K+-gluconic acid, 10 NaCl, 4.6 MgCl2, 4 K2-ATP, 15 creatine phosphate,1 EGTA, and 20 U/ml phosphocreatine kinase (pH 7.3, 300mOsm).The external so-lution contained (mM): 140 NaCl, 2.4 KCl, 4CaCl2, 4 MgCl2, 10 HEPES, 10 glucose(pH7.3; 300 mOsm). In addition, 1µM TTX and 20 µM gabazine were added freshlyto the external medium prior to recording. 100 µM glutamate was applied by a 20msec pressure ejection from a glass electrode (∼ 2.8 mOhm). All experiments wereperformed at RT (20 - 23◦C).

Electron microscopy

Hippocampal and cortical high-density cultures of WT and Nbea KO mice (E18)grown on glass coverslips were fixed (DIV14) for 45 min at RT with 2.5% glutaralde-hyde in 0.1M cacodylate buffer (pH 7.4). After fixation, cells were washed three timesfor 5 min with 0.1M cacodylate buffer (pH 7.4), post-fixed for 2 h at RT with 1%OsO4/1% KRu(CN)6 in bidest, washed and stained with 1% uranyl acetate for 40min in the dark. Following dehydration through a series of increasing ethanol con-centrations, cells were embedded in Epon and polymerized for 24 h at 60◦C. Afterpolymerization of the Epon, the coverslip was removed by alternatively dipping itin liquid nitrogen and hot water. Cells of interest were selected by observing theflat Epon embedded cell monolayer under the light microscope, and mounted on pre-polymerized Epon blocks for thin sectioning. Ultrathin (∼ 90 nm) were cut parallelto the cell monolayer and collected on single-slot, formvar-coated copper grids, andstained in uranyl acetate and lead citrate. Hippocampal and cortical synapses wererandomly selected at low magnification using JEOL 1010 electron microscope. Foreach condition, the number of docked synaptic vesicles (SV), total SV number, post-

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synaptic density (PSD) and active zone (AZ) length, and SV cluster surface (nm2)were measured on digital images taken at 100.000-fold magnification using analysissoftware (Soft Imaging System). The observer was blinded for the genotype. Forall morphological analyses we selected only synapses with intact synaptic plasmamembranes with a recognizable pre- and postsynaptic density and clearly defined SVmembranes. SV were defined as docked if there was a distance of 0 nm between the SVmembrane and the active zone membrane. The active zone membrane was recognizedas a specialized part of the pre-synaptic plasma membrane that contained a cleardensity opposed to the PSD and docked SVs. To obtain an estimate of the total SVpool, distances of undocked synaptic membranes to the active zone membrane werealso included in our measurements. The SV cluster size indicates the size (perimeter)occupied by the total pool of SVs (docked and undocked) present in a synapse. Incase of hippocampal neurons, cells were cultured from 6 different WT and 7 differentKO mice (originating from 4 different litters). Cortical neurons were cultured from 4different WT and 4 KO mice (originating from 3 different litters). Approximately 30synapses were analyzed per culture stemming from 1 animal.

Immunofluorescence staining and confocal microscopy

Neurons were fixed in 3.7% formaldehyde (Electron Microscopy Sciences) in Dul-becco’s phosphate buffered saline (D-PBS; Gibco) for 20 min before being rinsedwith D-PBS. Subsequently, they were permeabilized for 5 min in D-PBS containing0.5% Triton X-100, followed by a 30 min incubation in PBS containing 0.1% TritonX-100 and 2% normal goat serum to block aspecific binding. The same solution wasused for diluting antibodies. Neurons were incubated for 2 h in primary antibodiesat room temperature (RT), washed 3 times with D-PBS and incubated in secondaryantibodies for 1 h. After additional 3 washes they were mounted with ProLong R©Gold(Invitrogen) on microscopic slides and imaged with a 63x Plan-Neofluar lens (Numer-ical aperture 1.4, Carl Zeiss b.v. Weesp) on a Zeiss 510 Meta confocal microscope(Carl Zeiss). For fixable FM1-43 labeling, hippocampal neurons were grown for 14days and then stained at 30◦C for 10 s with 20 µM fixable FM1-43 (Molecular Probes)in modified depolarizing external medium containing 86 mM K+ and 83.5 mM Na+,immediately followed by an incubation for 30 s with the same dye concentration instandard external medium (167 mM Na+, 2.4 mN K+, 10 mM HEPES, 10 mM glu-cose, 4 mM Ca2+, 4 mM Mg2+, 330 mOsm, pH 7.3). All further procedures wereperformed at RT. The cells were washed several times with external medium, fixedfor 5 min with 2.5% formaldehyde in external medium, and then incubated for 15min with 5% formaldehyde in PBS. Reactive sites were blocked with 25 mM glycinein PBS for at least 30 min. Then, cell membranes were permeabilized under mildconditions to avoid the formation of dispersive aggregates from membrane contentsand the membrane staining dye. For this purpose, the cells were kept for 20 min in 1mM sodium cholate (Sigma) in an otherwise salt-free 300 mM sucrose solution. Be-fore and after the permeabilization, cells were carefully washed with 300 mM sucroseto remove salt and detergent residues. To identify all synapses independently of their

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exocytic activity, the cultures were stained for α-VGLUT1 and α-MAP2 (Jockuschet al., 2007). For comparing the expression levels of endogenously expressed Nbea,with Nbea re-expressed in KO neurons and Nbea overexpressed in WT neurons, high-density hippocampal neurons were transfected with EGFP and Full-length Nbea oran empty vector at DIV3-4. These neurons were left to grow until DIV14-15, whenthey were fixed and subsequently stained for MAP2 and Nbea (using the α-Nbeaantibody). The latter signal was used for quantification, like the number of Nbeapuncta per µm dendrite etc. For WT and KO analysis a given set was treated inparallel and the images were taken with the same settings.

Biotinylation assay

Live hippocampal neurons in high-density culture were washed with the extracellularsolution and incubated with 0.5 mg/ml of biotin (Pierce) for 30 min at RT. Theunbound biotin was removed with a glycine wash and the cells were lysed using RIPAbuffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100,0.1% SDS, and protease inhibitors 17.4 µg/ml PMSF, 1 µg/ml aprotinin, 0.5 1 µg/mlleupeptin ). The cells were scraped off and centrifuged at 14000 x g for 15 min at4◦C. The supernatant was collected and the protein concentrations were measuredusing BCA (Pierce). Equal amounts of control and Nbea KO proteins were incubatedwith streptavidin beads to capture biotinylated proteins. After washing in extractionbuffer, biotinylated proteins were eluted from streptavidin beads by boiling in SDS-PAGE sample buffer, separated by SDS-PAGE, and immunobloted.

Deglycosylation assay

Neuron culture lysates were collected and treated with glycoprotein denaturing buffer(NEB). The lysates were boiled for 10 min and incubated with Endoglycosidase-Hand PNGase-F (NEB) according to the manufacturer’s instructions. Samples wereanalyzed using 7.5% SDS/PAGE gels, and immuno-blotted. Quantification of ratiosbetween the levels of mature and immature proteins were assessed based on Endo-Hsensitivity. For each brain sample and protein, data were obtained by dividing theintensity of the band representing the Endo-H insensitive protein fraction (i.e. highermolecular weight bands at the level of the bands detected in the respective untreatedControl samples) by the intensity of the band reflecting the Endo-H sensitive proteinfraction (i.e. lower molecular weight bands at the level of the band detected in therespective PNGase samples).

Data analysis and statistics

All data in this study are provided as mean ± standard error of the mean and werealways obtained in at least two independent experiments. For the analysis of Nbea’slevels in the re- and over-expression studies, single plane images of neurons (1024 x1024) were analyzed using SynD software (Schmitz et al., 2011). For the analysisof autaptic data confidence was assessed by two-tailed unpaired Student’s t tests be-

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tween control and KO datasets using Instat software for all statistical analysis.For theanalysis of the electrophysiological data of from high-density cultures (exogenous glu-tamate application) and the corresponding Nbea’s levels, as well as for the analysis ofthe number of synapses and branching in both genotypes, we used MATLAB R2007a(The MathWorks Inc, Natick, Massachusetts). To test our datasets for normalitywith Lilliefors goodness-of-fit and heterogeneity of variance with Bartlett’s test forequality of variance. If data allowed, a two-tailed unpaired Student’s t test was per-formed. Alternatively, the Wilcoxon-Mann-Whitney test was used. When comparingthree groups the Kruskal-Wallis ANOVA was used. n and p values are given in thecorresponding figure legends.

Results

Defective glutamatergic and GABAergic synaptic transmission in NbeaKO neurons

Nbea KO mice die perinatally (Medrihan et al., 2009; Su et al., 2004). Therefore,we used autaptic and high-density cultures of hippocampal and striatal neurons fromE18 embryos to study the functional consequences of Nbea loss. We detected nosignificant morphological or functional differences between wild-type (WT) and het-erozygous Nbea KO neurons (data not shown), and therefore pooled all the dataobtained with these genotypes, designating them as control.

Evoked excitatory postsynaptic current (EPSC) amplitudes in Nbea KO hip-pocampal neurons and inhibitory postsynaptic current (IPSC) amplitudes in NbeaKO striatal neurons were significantly reduced by 64% and 67%, respectively, as com-pared to control cells (Cont; Figure 3.1A-C). Similar changes were observed in analysesof postsynaptic responses triggered by hypertonic sucrose solution, which causes therelease of the readily releasable pool (RRP) of synaptic vesicles (SVs; Jockusch et al.,2007; Rosenmund and Stevens, 1996). We found that glutamatergic and GABAergicNbea KO neurons showed reductions in apparent RRP sizes of 75% and 70%, respec-tively (Figure 3.1A, B and D). The vesicular release probabilities (Pvr) in the twotypes of neurons, calculated by dividing the charge transferred during action poten-tial evoked PSCs by the RRP charge, were slightly reduced upon Nbea KO (Figure3.1E). Amplitudes of miniature EPSCs and IPSCs (mEPSCs/mIPSCs) were reducedby 23% and 16%, respectively. The corresponding mEPSC/mIPSC frequencies werereduced by about 60% (Figure 3.1F-I), i.e. by a similar degree as evoked EPSC/IPSCamplitudes and the corresponding responses to hypertonic sucrose solution (Figure3.1C and D). In analyses of short-term plasticity, we found that EPSC and IPSCamplitudes in hippocampal and striatal Nbea KO neurons depressed progressivelyduring 10 and 40 Hz stimulation trains to the same steady state depression levels ascontrol cells (Figure 3.2A and B). These data show that loss of Nbea causes strongdefects in synaptic transmission, without changes in synaptic short-term plasticity.

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Glutamatergic neuronsA B GABAergic neurons

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Figure 3.1 Reduced evoked and spontaneous synaptic transmission in Nbea KO

neurons. (A) Example traces of depolarization evoked EPSCs (left) and responses afterapplication of hypertonic sucrose solution (right) in glutamatergic neurons. (B) Exampletraces as in panel (A) but for depolarization evoked IPSCs and responses after application ofhypertonic sucrose solution in GABAergic neurons. (C) Mean evoked EPSC amplitudes inhippocampal glutamatergic (left) and striatal GABAergic cells (right, on gray background).(D) Mean charge transfer during the response to hypertonic sucrose solution (apparent RRPsize estimate) in hippocampal glutamatergic (left) and striatal GABAergic cells (right, ongray background). (E) Average vesicular release probability pvr in hippocampal glutamater-gic (left) and striatal GABAergic cells (right, on gray background). The pvr is expressed as% of the RRP and was calculated by dividing the charge transfer during an action potentialevoked response by the charge transfer measured during a response to hypertonic sucrose so-lution (reflecting the apparent RRP). (F) Example mEPSC trace recorded at -70mV holdingpotential in presence of 300 nM TTX in glutamatergic neurons. (G) Example mIPSC tracesrecorded at -70mV holding potential in the presence of 300 nM TTX in GABAergic neurons.(H) Mean mPSC amplitudes and (I) mean mPSC frequencies in hippocampal glutamatergic(left) and striatal GABAergic (right, on gray background) neurons. Responses of Nbea KOneurons are always depicted in gray (traces and bars) and responses of control neurons inblack (traces and bars). Error bars indicate SEM. The number of cells analyzed are givenin/above the histogram bars (*P<0.05, **P<0.01, ***P<0.001 in Student’s t-test).

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Figure 3.2 Unaltered presynaptic plasticity in Nbea KO neurons. (A) EvokedEPSC depression during 10 Hz (left; WT, black, n=94; Nbea KO, gray, n=69) and 40 Hz(right; WT, black, n=35; Nbea KO, gray, n=32) stimulation trains. (B) Evoked IPSCdepression during 10 Hz (left; WT, black n=94; Nbea KO, gray, n=69)and 40 Hz (right;WT, black, n=35; Nbea KO, gray, n=32) stimulation trains. Data were normalized to thefirst response in the train. (C) Left panel: Example of calcimycin triggered (10 µM) EPSCsin glutamatergic Nbea KO (KO, gray) and control neurons (Cont, black). Right panel:Example traces of calcimycin triggered (10 µM) IPSCs in GABAergic Nbea KO (KO, gray)and control neurons (Cont, black). (D) Mean charge transfer during calcimycin triggeredPSCs in hippocampal glutamatergic (left) and striatal GABAergic cells (right) of control(Cont, black) and Nbea KO (KO, gray) mice. The smaller histograms show correspondingdata obtained with stimulation by hypertonic sucrose solution (see Figure 3.1A, B and D).Error bars indicate SEM (*P<0.05, **P<0.01, ***P<0.001 in ttest).

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We next examined the effects of voltage-gated Ca2+ channel independent activa-tion of the presynaptic release machinery by the Ca2+ ionophore calcimycin (Jockuschet al., 2007). Irrespective of the genotype and transmitter type of the tested neurons,elevation of intracellular Ca2+ levels [Ca2+]i by 10 µM calcimycin triggered massiverelease of SVs, leading to a complete depletion of all releasable SVs. Calcimycinresponses in Nbea KO cells were reduced to a similar degree as the responses to hy-pertonic sucrose solution (Figure 3.2C and D). These findings indicate that synaptictransmission is severely affected in Nbea KO neurons and that the dominant defectis downstream of presynaptic Ca2+ influx.

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Figure 3.3 Intact presynaptic activity in Nbea KO neurons. (A) Examples of control(Cont) and Nbea KO neurons (KO) loaded with FM1-43 (green) and costained for VGLUT1(red) and MAP2 (blue). Scale bar = 10 µm.(B) Quantification of VGLUT1/FM1-43 colo-calization in control (Cont, black) and Nbea KO (KO, gray) neurons. (C) Recordings frompairs of neurons of different transmitter type on microislands as indicated in the diagram onthe left. In each of the three genotype configurations tested (i.e. presynaptic glutamater-gic cell control or Nbea KO, and postsynaptic GABAergic cell control or Nbea KO), bothcells were whole-cell patch clamped, the presynaptic cell was stimulated, and responses weremeasured in the presynaptic (autaptic) and the postsynaptic (heterosynaptic) cell. Data formean autaptic responses are shown in the middle histogram panel, and data for the meanheterosynaptic responses are shown in the right histogram panel. Genotype combinations(presynaptic cell/postsynaptic cell) are given below the histogram bars. Error bars indicateSEM (*P<0.05, **P<0.01, ***P<0.001 in t-test).

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In addition we determined the total number of presynaptically active synapses inglutamatergic Nbea KO neurons by combining antibody staining with fixable FM1-43, which stains synapses with exocytosis and endocytosis activity. The proportionof active synapses was similar in Nbea KO and control neurons (Figure 3.3A and B),and the total number of synapses in Nbea KO neurons was not altered (Figure 3.7).These findings indicate that the synaptic transmission deficits in Nbea KO neuronsare not due to changes in the number of active synapses or to a profound presynapticdefect.

To further test if presynaptic defects (e.g. in neurotransmitter loading into SVs)contribute to the reduction in synaptic strength in Nbea KO neurons, we establishedmicroisland cultures of cell pairs on a single astrocyte microisland, consisting of onehippocampal glutamatergic and one striatal GABAergic neuron (Figure 3.3C), whichwere distinguished by their morphology and synaptic features. The combination ofan inhibitory and an excitatory neuron on a single island allowed us to discriminateautaptic and intercellular synaptic glutamate evoked responses, and seeding togetherinhibitory and excitatory neurons from Nbea KO and control genotypes allowed usto determine presynaptic and postsynaptic contributions to the Nbea KO phenotype.The EPSC amplitudes from pairs consisting of two KO neurons were smaller thanthose of two WT neurons. In contrast, the postsynaptic currents recorded in WTneurons receiving synaptic inputs from an Nbea KO neuron were normal (Figure3.3C). Together with the facts that FM-dye loading is unaltered in KO cells (Figure3.3A and B), responses evoked by action potentials, hypertonic sucrose solution, andcalcimycin are similarly reduced in KO neurons (Figure 3.2C and D), and presynapticplasticity is unaffected (Figure 3.2A and B), the paired recordings indicate that thealtered synaptic strength of Nbea KO neurons cannot be explained by presynapticdefects.

Less functional neurotransmitter receptors at Nbea KO synapses

To further investigate the causes of the reduced synaptic transmission in Nbea KOneurons, we measured their responses to exogenous application of 100 µM gluta-mate on high-density hippocampal cultures (Jones and Westbrook, 1996). Comparedto WT cells from DIV14-18, Nbea KO neurons exhibited a significant reduction intheir responses to exogenous application of 100 µM glutamate (Figure 3.4A and D).Additionally, autaptic hippocampal and striatal neurons, exhibited dramatic reduc-tions in their responses to exogenous application of 10 µM kainate, 3 µM GABA, 30µM glutamate and 100 µM NMDA, but only when recorded from mature neuronswith functional synapses (Supplementary material Figure 3.11). The same patternsof reduced glutamatergic and GABAergic responses were seen in glutamatergic andGABAergic NbeaKO cells, with defects of similar extent (approximately 65%). Areduction in response to exogenous application of 10 µM kainate and 3 µM GABAwas also observed in high-density cultures (data not shown). The above data indicatethat the dramatic loss of synaptic transmission in the absence of Nbea is caused bysevere postsynaptic transmitter receptor defects.

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KO

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Figure 3.4 Diminished response to exogenous application of glutamate in Nbea

KO neurons can be rescued by re-expression of the full-length Nbea. (A-C) Rep-resentative traces of glutamate application on WT and Nbea KO neurons. The green KOtrace depicts responses triggered in KO neurons expressing EGFP, the green WT trace rep-resents the responses triggered in WT neurons expressing EGFP, the red Nbea rescue tracerepresents responses triggered in KO neurons expressing full-length Nbea, and the red Nbeaoverexpression trace represents responses triggered in WT neurons overexpressing full-lengthNbea. (D) Mean amplitudes of responses to 100 µM glutamate in hippocampal glutamater-gic neurons of WT (WT, black) and Nbea KO mice (KO, gray). (E) Mean amplitudes ofresponses to 100 µM glutamate in hippocampal glutamatergic neurons with indicated geno-types (WT or KO) after calcium phosphate transfection at DIV3 with EGFP or Full-lengthNbea (rescue experiments). (F) Mean amplitudes of responses to 100 µM glutamate in hip-pocampal glutamatergic neurons of Nbea WT mice, after calcium phosphate transfection atDIV3 with EGFP or Full-length Nbea (overexpression experiments). Responses from neu-rons transfected with EGFP are represented as green bars, while responses from neurons(over-)expressing Full-length Nbea are depicted as red bars. Error bars indicate standarderror of the mean (SEM; *P<0.05 **P<0.01).

It is possible to distinguish synaptic and extrasynaptic NMDA receptors in autap-tic cultures by first measuring the response to exogenous NMDA application, followedby blockade of synaptic NMDA receptors by applying a 0.33 Hz train of 100 stim-uli in the presence of the irreversible open-channel blocker MK-801, and subsequentassessment of the cellular response to a second exogenous NMDA application (Au-gustin et al., 1999). In this regimen, synaptic NMDA receptors become selectivelyblocked by MK-801, and the ratio between the second and the first response to exoge-

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nous NMDA application can be used to assess the ratio of synaptic vs. extrasynapticNMDA receptors.

MK801 @ 0.33Hz

100µM NMDA

3s

0.5 nA

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0.3 nA

3s

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Stimulus number

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lize

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plit

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lative

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Figure 3.5 Reduced density of synaptic neurotransmitter receptors in Nbea KO

neurons. (A) Example traces of responses to exogenous 100 µM NMDA in glutamatergicNbea KO (KO, gray) and control neurons (Cont, black) before (left panel) and after MK801(right panel) stimulation with 100 stimuli at 0.33 Hz in the presence of MK801 to blocksynaptic NMDA receptors. (B) Mean amplitude of responses to exogeous 100 µM NMDAin glutamatergic Nbea KO (KO, gray) and control neurons (Cont, black) before stimulationwith 100 stimuli at 0.33 Hz in the presence of MK801 application [see panel (A)]. (C) Timecourse of depression of synaptic responses in glutamatergic Nbea KO (KO, gray) and controlneurons (Cont, black) upon stimulation with 100 stimuli at 0.33 Hz in the presence of MK801to block synaptic NMDA receptors [see panel (A)]. Amplitudes were normalized to the firstresponse. The inset shows mean NMDA component relative to the mean AMPA componentin the cells tested. The numbers of cells analyzed are given in the histogram bars. (D)Mean amplitude of responses to exogenous 100 µM NMDA in glutamatergic Nbea KO (KO,gray) and control neurons (Cont, black) after stimulation with 100 stimuli at 0.33 Hz in thepresence of MK801, i.e. after blockade of synaptic NMDA receptors [see panel (A)]inducedresponse after MK801 application. Error bars indicate standard error of the mean (SEM;*P<0.05 **P<0.01).

To ensure that the measured NMDA-mediated currents, after the 0.33 Hz/100stimuli protocol, were not affected by the length of the protocol and correspond-ing run-down of synaptic responses, we also employed a 40 Hz/200 stimuli protocol,but the two protocols yielded very similar data, which were therefore pooled (Figure

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3.5B and D). The ratios of responses to exogenously applied NMDA after and beforeMK-801 blockade obtained in control cells indicate that typically about 80% of allfunctional receptors are at synapses (data not shown). In contrast, Nbea KO neuronshave much less synaptic NMDA receptors while the surface density of extrasynapticreceptors is unaffected (Figure 3.5). The synaptic release probability, which we esti-mated on the basis of the decay time course of the synaptic NMDA receptor currentduring the 0.33 Hz train in the presence of MK-801, was similar between Nbea KOand control cells, further indicating that the synaptic release machinery is not affectedby Nbea KO loss (Figure 3.5C).

Nbea re-expression rescues the phenotype of Nbea KO neurons

Calcium phosphate re-expressed full-length Nbea in Nbea KO neurons, displayed animmunoreactivity pattern similar to the one exhibited by endogenous Nbea. It lo-calized throughout the somatic cytoplasm and showed also the prominent dendriticpuncta (Figure 3.6A).

While the Nbea clusters in rescued neurons did not differ from endogenous Nbeaof WT neurons expressing EGFP, either in their size nor number, the intensity ofthe Nbea immunorectivity was significantly increased, both in dendrites and soma(Figure 3.6B-E). These result indicate that rescued neurons express more Nbea thanWT neurons and that the endogenous Nbea does not saturate the system. In linewith this, also the intensity of Nbea’s immunoreactivity measured in WT neuronsover-expressing Nbea, differs from endogenous Nbea levels. As the intensity of Nbea’simmunoreactivity measured in ”rescued” KO neurons re-expressing Nbea, was compa-rable to the intensity measured in WT neurons over-expressing Nbea (Figure 3.6B-E),a ceiling seems to exist that defines the maximal amount of Nbea’s expression levelin the cell.

The electrophysiological responses triggered by external application of 100 µM glu-tamate mirrored this situation (Figure 3.4B, C, E and F), in that the re-expressionof full-length Nbea not only fully rescued the Nbea-null phenotype, but resulted incurrents with an amplitude exceeding the one observed in WT neurons expressingEGFP (Figure 3.4B and E). Similarly, WT neurons over-expressing Nbea also exhibitan increased glutamate induced response compared to WT neurons expressing EGFP(Figure 3.4C and F).

The rather small responses of WT neurons expressing EGFP in the rescue exper-iment (Figure 3.4E) might be a consequence of non-optimal neuronal cultures, sincesuch low responses were not observed in autaptic cultures (Supplementary materialFigure 3.12).

The effects of Nbea loss on responses to other agonists, as well as evoked EPSC/IPSC amplitudes were also fully rescued (Supplementary material Figure 3.12). Thisindicates that the phenotypic effects of the Nbea KO are cell autonomous and thatNbea loss does not lead to complex secondary effects that cannot be reverted by Nbeare-expression.

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0

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.u.)

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Figure 3.6 Expression levels and subcellular localization of endogenous and over-

expressed Nbea in neurons. (A) Immunostaining for MAP2 (top) and Nbea (bottom)in DIV14 Nbea KO neurons (plated on glia cells) expressing full-length Nbea from a rescueplasmid that had been introduced by transfection with a calcium phosphate precipitationprotocol at DIV4.The middle panel shows the enlarged soma, and the right panel shows adendrite. Scale bar = 5 µm. (B) Quantification of the average number of Nbea punctaper µm dendrite in wild type (WT) neurons expressing EGFP (green) or over-expressingfull-length Nbea (red) and Nbea KO (KO) neurons expressing full-length Nbea (red). Errorbars indicate SEM. The number of cells analyzed are given in the histogram bars (*P<0.05in Student’s t-test). (C) Analysis as in (B) but for the average size of dendritic Nbea puncta.(D) Analysis as in (B) but for the average intensity of dendritic Nbea puncta. (E) Analysisas in (B) but for the average intensity of somatic Nbea signals.

Nbea KO neurons show minor presynaptic differences

To study the effects of Nbea loss on synapse formation, i.e. connectivity in high den-sity cultures, we stained high-density hippocampal neurons for the presynaptic markerVAMP2 and subsequently quantified the number of synapses. These were unalteredin Nbea KO neurons (Figure 3.7A-D). Because certain Nbea-related BEACH domainproteins play an important role in the control of membrane dynamics and membrane

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trafficking, thus contributing to cell maturation and development (Wang et al., 2000),we tested whether the reduced synaptic transmission in Nbea KO neurons was due toimpaired cell maturation and dendrite development. Staining high-density hippocam-pal neurons at day 14 in vitro (DIV14) using an α-MAP2 antibody, and analyzingthe dendrite complexity by Sholl analysis (Sholl, 1953), did not reveal any abnormal-ities in Nbea KO neurons (Figure 3.7E). These results further support our conclusionthat the reduced synaptic transmission in Nbea KO neurons is not due to impaireddendrite growth or to a reduced synapse number.

A

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ea

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ap

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.)

Figure 3.7 Nbea KO neurons show normal connectivity. (A) High-density DIV14-15 hippocampal neurons of WT (WT, black) and Nbea KO mice (KO, gray), stained forthe dendritic marker MAP2, the synaptic marker VAMP2 and Nbea. Scale bar = 20 µm.(B) Quantification of the number of synapses per µm dendrite in high-density DIV14-15hippocampal neurons of WT (WT, black) and Nbea KO mice (KO, gray). (C) Mean synapsearea in high-density DIV14-15 hippocampal neurons of WT (WT, black) and Nbea KO mice(KO, gray). (D) Mean synapse intensity in high-density DIV14-15 hippocampal neurons ofWT (WT, black) and Nbea KO mice (KO, gray). (E) Sholl analysis conducted on high-density DIV14-15 hippocampal neurons of WT (WT, black) and Nbea KO mice (KO, gray).Error bars indicate SEM.

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A

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les

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210183 154121 210183 154121

210183 154121 210183 154121

210183 154121

Figure 3.8 Minor perturbations in synaptic ultrastructure in Nbea KO neurons.

(A) Electron micrographs of synapses in hippocampal (top) and cortical (bottom) highdensity neuronal cultures (DIV14-15) from wild type (WT) and Nbea KO (KO) mice. Scalebar = 200 nm. (B) Quantification of the average number of docked SV per synapse. (C)Quantification of the active zone size. (D) Quantification of the average number of dockedSVs per nm of active zone. (E) Quantification of the average total number of SVs persynapse. (F) Quantification of the postsynaptic density size. (F) Quantification of the SVcluster size per synapse. Error bars indicate SEM. The number of synapses analyzed aregiven in the histogram bars. *P<0.05 **P<0.01 in Student’s t-test.

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At the ultrastructural level, synapses of Nbea KO hippocampal and neocorticalneurons showed 12-26% reductions in the number of total and docked SVs and in thesize of the active zone and postsynaptic density (Figure 3.8). However, these subtlepresynaptic morphological defects do not appear to cause detectable functional defectsin the presynaptic compartment of Nbea KO neurons (Figure 3.1 - Figure3.3; see alsofurther discussion below).

Reduced surface expression of transmitter receptor proteins in Nbea KOneurons

A

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xp

ressio

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rote

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urf

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Tubulin

Control samplesBiotinylated

samples

input sup input sup

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55

130

130

95

55

170

43

130

130

55

Cont KO

5 5 4 4 5 2 4 45 5

Figure 3.9 Impaired cell surface trafficking and synaptic targeting of multiple

neurotransmitter receptors in Nbea KO neurons. (A) Biotinylation assays wereperformed on high-density DIV19 hippocampal neuron cultures from control (Cont) andNbea KOmice (KO) to assess the surface expression of proteins indicated. Fractions obtainedin the assay were processed for SDS-PAGE and immuno-blotting for the indicated proteins.Immunolabeled bands were visualized by enhanced chemiluminescence. Input, total proteincontent before removal of the biotinylated fraction; Sup, remaining supernatant after removalof the biotinylated fraction; affinity purified biotinylated samples are shown on the right. (B)Quantification of surface expression of the indicated proteins in Nbea KO neurons. Data arefor from at least four independent experiments, except for GABAAα5. Data are normalizedto control levels. Error bars indicate SEM. *P<0.05 **P<0.01 in Student’s t-test.

We next tested if the reduced surface expression of functional neurotransmitter re-ceptors in Nbea KO neurons is also detectable at the protein level. We first usedsemiquantitative Western blotting to compare total receptor protein expression inNbea KO and control neurons using whole brain homogenates (E18) and DIV19 hip-pocampal cultures (Supplementary material Figure 3.13). Expression of glutamateand GABA receptor subunits, as well as of all other pre- and postsynaptic proteins

90


tested, were similar in Nbea KO and control preparations, partially unlike earlierfindings on Nbea KO brainstem (Medrihan et al., 2009).

Endo HPNgase Control

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re r

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5 44 24

Figure 3.10 Aberrant glycosylation of neurotransmitter receptors in Nbea KO

neurons. (A) Sample of control (Cont) and Nbea KO (KO) hippocampal cultures (DIV19)that had either been left untreated (Control) or had been digested with Endoglycosidase-H(Endo H) or PNGase F (PNgase), and were then processed by SDS-PAGE and immono-blotting for the indicated proteins. (B) Quantification of ratios between the levels of matureand immature proteins in Nbea KO cultures as assessed by Endo-H sensitivity. For eachsample, data were obtained by dividing the intensity of the band representing the Endo-H insensitive protein fraction (i.e. higher molecular weight bands in the Endo-H treatedsample at the level of the bands detected in the respective untreated Control samples)by the intensity of the band reflecting the Endo-H sensitive protein fraction (i.e. lowermolecular weight bands in the ENdo-H treated sample at the level of the band detectedin the respective PNGase samples). Data are from at least four independent experiments(except for GABAAα5) and were normalized to the control levels. Error bars indicate SEM.The number of cultures analyzed are given in the histogram bars (*P<0.05 in Student’st-test).

Next, we performed cell surface biotinylation assays on high-density hippocampalcultures (DIV19) to analyze surface expression of glutamate and GABAA receptorsubunits. Nbea KO cells showed a 60% reduction in the cell surface level of GluA2/3and 40% reduction in the cell surface levels of GluA1, GluN1, GluN2A, GluK2/3,GABAAγ2 and GABAAα1 (Figure 3.9). Surface expression of other cell surface pro-teins, such as neuroligin 1 or transferrin receptor, was unaffected or even slightlyincreased in Nbea KO cells. Also, the cell surface expression of the extrasynapticGABAA receptor subunit GABAAα5 (Brunig et al., 2002) was slightly increased inNbea KO cells, indicating that Nbea acts specifically on the surface expression ofdefined glutamate and GABAA receptors.

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To obtain independent evidence for compromised receptor targeting in Nbea KOneurons and to identify the step in the secretory pathway that may be affected, weanalyzed the levels of mature receptor subunits by assessing their sensitivity to en-doglycosidase H, which can be used as evidence for post-translational modificationin the Golgi apparatus. Nbea KO neurons showed 50% reduced levels of maturelyglycocylated GluA2/3 subunits, but not GluK2/3, GABAAα1, extrasynaptic GABAA

receptors (GABAAα5), or the synaptic adhesion protein neuroligin-1 (Figure 3.10).This indicates that Nbea deletion compromises GluA2/3 subunit trafficking from theER to Golgi apparatus, or early within the Golgi apparatus, while other receptorsubunits can reach the Golgi apparatus, and are probably arrested late within theGolgi apparatus or further downstream, given their reduced surface expression.

Discussion

The present study shows that Nbea is dispensable for cell autonomous programs ofneuronal development and synaptogenesis, but required for normal synaptic trans-mission. Defective postsynaptic trafficking of the major ionotropic receptor typesfor excitatory and inhibitory neurotransmitters is the dominant consequence of Nbealoss, at least in the experimental system studied here. Electrophysiology data (Figure3.3 C, Figure 4 and Figure 5) show that postsynaptic receptors are specifically lost inNbea KO neurons, but the overall expression levels of receptor subunits is not affected(Supplementary material Figure 3.12). Analysis of their maturation showed that theprogress of AMPA, GABAA and kainate receptors through the secretory pathway isdifferentially affected by Nbea loss (Figure 3.10).

Surprisingly in view of previously published data, the present study on centralnervous system neurons yielded primarily evidence for postsynaptic Nbea functions.Some indications for presynaptic defects in Nbea KO neurons were observed at theultrastructural level (Figure 3.8), but several lines of evidence indicate that presynap-tic defects are not the primary cause for the synaptic dysfunction upon Nbea loss.The most important evidence against primary presynaptic functional defects in NbeaKO neurons is the fact that in neuron pairs in culture (Figure 3.3C) the synaptic phe-notype of Nbea deficiency is exclusively observed if the responding neuron is Nbeadeficient, but not if the transmitter releasing neuron lacks Nbea. Second, presynap-tic short-term plasticity is unaffected in Nbea KO cells (Figure 3.2A and B). Thiswould not be expected if aspects of presynaptic function were altered due to Nbealoss. Third, the defects in synaptic transmission in Nbea KO neurons prevailed whencalcimycin was used to trigger transmitter release (Figure 3.2C and D), indicatingthat the functional defects caused by Nbea loss occur downstream of voltage-gatedCa2+ channel signaling, and vesicular and synaptic release probabilities were not, orweakly, perturbed in Nbea KO neurons (Figure 3.1 E, Figure 3.5 C). Fourth, theFM1-43 loading experiments revealed no defects in the SV cycle or in the number ofpresynaptically active synapses (Figure 3.3A and B). Fifth, Nbea immunoreactivitydoes not accumulate in presynaptic compartments (as shown in the previous chapter).

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Sixth, our Western blot analyses did not reveal altered expression of key presynapticproteins in Nbea KOs (Supplementary material Figure 3.12). This is, in part, differ-ent in the brainstem of Nbea KOs, where the expression levels of certain presynapticproteins were found to be reduced (Medrihan et al., 2009). However, mIPSC frequen-cies were also reduced in Nbea KO brainstem neurons (Medrihan et al., 2009). Thisis compatible with a role of Nbea in postsynaptic receptor trafficking as describedin the present study, assuming that some synapses in Nbea KO neurons contain lessreceptors, leading to reduced mPSC amplitudes (Figure 3.1F-H), while others con-tain very few or no receptors, causing an apparent reduction in mPSC frequencies(Figure 3.1F, G and I). Taking all of the arguments above into account, we concludethat Nbea does not have a major cell autonomous presynaptic role in glutamater-gic hippocampal and GABAergic striatal neurons, although we cannot unequivocallyexclude additional minor presynaptic roles of Nbea. The presynaptic changes in theNbea KO mutants that we observed at the ultrastructural level (Figure 3.8) are rathersmall (0-30%) and in this respect different from the strong presynaptic defects thathad previously been observed in Nbea KO neuromuscular junctions (NMJ; Su et al.,2004). Thus, Nbea has different functions in glutamatergic and GABAergic synapsesvs. NMJs. The effects on docked SVs and SV cluster size in Nbea KO cells that weobserved, might then be a secondary effect of the chronically altered synaptic trans-mission in mutant neurons. Such structural changes are known to occur upon chronicchanges in activity and are in accord with other ultarstructural changes that werepreviously observed in the Nbea KO brain (Niesmann et al., 2011).

Several key regulators of synaptic glutamate receptor localization and functionhave been identified, such as CaMKII (Pettit et al., 1994), TARPs (Tomita et al.,2005), Cornichon (Schwenk et al., 2009) or CKAMP44 (von Engelhardt et al., 2010),and their role in synaptic function and plasticity is well established (Bredt and Nicoll,2003; Malinow and Malenka, 2002). Upon synaptic activity AMPA receptor GluA1and GluA2/3 subunits are differentially trafficked to synapses (Hayashi et al., 2000;Shi et al., 2001; Wenthold et al., 1996). Instead, Nbea loss equally affects the synapticlocalization of GluA1 and GluA2/3 at rest (data not shown), indicating that Nbea actsupstream of the activity dependent bifurcation point at which GluA1 and GluA2/3subunits are trafficked differentially. This is consistent with the fact that AMPA re-ceptors accumulate early in the biosynthetic route and are partially immature in theabsence of Nbea (Figure 3.10).

Apart from exocytosis and endocytosis at synapses, transmitter receptors canmove into and out of synapses by lateral diffusion from the extrasynaptic plasmamembrane (Bogdanov et al., 2006; Newpher and Ehlers, 2008). Based on electrophys-iology (Figure 3.5) and surface biotinylation experiments (Figure 3.9), we present twolines of evidence indicating that Nbea deficiency has little effect on extrasynaptic re-ceptors. However, the levels of all receptor subunits present at synapses are reducedin the absence of Nbea (Figure 3.9). Altogether, these data point towards a role ofNbea in targeting several major ionotropic neurotransmitter receptors directly to thesynapse. The regulation of receptor distribution between extrasynaptic and synapticlocations and of their activity dependent redistribution between these locations ap-

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pears to operate downstream of the initial Nbea-dependent targeting step.Nbea deletion affects at least four different types of neurotransmitter receptors,

but different receptor types are affected differentially. AMPA receptors accumulateupstream of or early within the Golgi apparatus in the secretory pathway (e.g. inthe ER), while GABAA and kainate receptors appear to accumulate late within ordownstream of the Golgi apparatus (Figure 3.10). Unfortunately, the deglycosylationexperiment for the NMDA receptor subunits was not conclusive, hence we can notidentify the precise step of the secretory pathway where NMDA receptor traffickingis compromised. The fact that AMPA receptors are not properly glycosylated andmay become trapped at the ER level in Nbea KO neurons, indicates that the earliestaction of Nbea in the biosynthetic pathway is at the level of ER exit, e.g. in a qual-ity control mechanism that ensures that only properly assembled multimeric proteincomplexes leave the ER. The effect of Nbea loss in trafficking of GABAA, kainate andNMDA receptors indicates an additional role of Nbea in the transport of receptorsto the synaptic plasma membrane. This latter role does not seem to involve effectson the synaptic targeting of receptor scaffolding proteins like PSD95 and gephyrin,whose localization is unaffected by Nbea loss (Figure 3.9).

Acknowledgements

We would like to thank F. Benseler, I. Beulshausen, R. Dekker, J. Wortel, A. Galin-ski, K. Hellmann, Y. Thanhaeuser, D. Schwerdtfeger, and the staff of the Trans-genic Animal Facillities in Goettingen and Amsterdam for invaluable support. Wethank M. Hoon for discussions and advice, R. Zalm for cloning the full length NbeacDNA construct, and T.C. Suedhof, J. van Minnen and J.M. Fritschy for kind gifts ofreagents. This work was supported by the Max Planck Society (N.B.), the NeuromicsMarie Curie Early Stage Training grant (MEST-CT-2005-020919, J.L. and M.V.), theDeutsche Forschungsgemeinschaft (Ki 324/15, M.W.K. and N.B.), Vetenskapsradet(VR-M, M.W.K.), Uppsala University (Start-up Grant, M.W.K.), the Eurospin andSynSys Consortia, (FP7-HEALTH-F2-2009-241498, FP7-HEALTH-F2-20009-242167,N.B. and M.V.), and the Dutch Organization for Scientific Research (NWO-ZonMWVeni grant 916-36-043 to H.d.W).

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KO neurons. (A-C) Example traces of repsponses to exogenous 10 µM kainate (A), 30µM glutamate (B), and 3 µM GABA (C) in glutamatergic Nbea KO (KO, gray) and controlneurons (Cont, black). (D) Mean amplitudes of responses to exogenous 10 muM kainate inhippocampal gluatamatergic (left) and striatal GABAergic cells (right, on gray background)of control (Cont, black) and Nbea KO (KO, black) mice. (E) Mean amplitudes of responsesto 30 muM glutamate in hippocampal glutamatergic (left) and striatal GABAergic cells(right, on gray background) of control (Cont, black) and Nbea KO (KO, black) mice. (F)Mean amplitude of responses to exogenous 3 µM GABA in hippocampal glutamatergic (left)and striatal GABAergic cells (right, on gray background) of control (Cont, black) and NbeaKO (KO, black) mice. Error bars indicate SEM. The number of cells analyzed are givenin/above the histogram bars (*P<0.05 **P<0.01 ***P<0.001 in Student’s t-test).

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CHAPTER 3 Neurobeachin regulates transmitter receptor ... 3 Neurobeachin regulates...early in the biosynthetic pathway, and mature NMDA, kainate and GABA A-receptors do not reach the