Dystroglycan Mediates Homeostatic Synaptic Plasticity At-Books Pdf

Dystroglycan mediates homeostatic synaptic plasticity at
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Results A C surface, DG GABA A R 3 DG 3 MAP2 GABA A R 2. Characterization of the DG Complex at Hippocampal GABAergic. Synapses We used immunohistochemical staining with mAb. IIH6 antibody which recognizes functionally glycosylated DG. 24 and antibodies to GABAAR 1 to reveal that virtually all. GABAAR 1 clusters colocalize with DG clusters in the CA3. stratum pyramidale of WT mice Fig S1 B and C However in. Mdx mice which do not express the full length isoform of. dystrophin 5 that anchors DG at the cell surface 25 we. Bicuculline 24 hrs, Bicuculline 24 hrs, observed substantially reduced expression of DG and fewer. GABAAR 1 clusters in the CA3 region Fig S1D Similarly. Myd mice which have hypoglycosylated DG due to a muta. tion in the glycosyltransferase LARGE 26 also displayed. reduced DG and 1 clusters in CA3 hippocampus Fig S1E. We further examined the localization of DG using mature. hippocampal cultures and found that DG colocalizes with DG Surface. and with GABAAR clusters apposed to GAD65 puncta but that B DG GABA A R 3 D GABA A R 2. DG does not colocalize with AMPA receptor AMPAR clus. Total Cluster Area Ctrl, Total Cluster Area Ctrl, Total Cluster Area Ctrl. 250 200 200, ters Fig S2A We also observed a high degree of colocalization 200. between DG and several proteins previously identified as part 150 150. of the DGC at neuromuscular synapses including agrin ezrin 100 100. nNOS and utrophin 15 27 Fig S2B Furthermore using im. munoprecipitation from brain lysates we found that DG forms 50. a supramolecular complex not only with DG but also with 0. GABAAR subunits 1 and 2 3 as well as with the inhibitory. Surface Total, synapse scaffolding protein gephyrin Fig S2C Taken together E Ctrl Bic F Ctrl.
these findings suggest that the neuronal DGC is part of the. postsynaptic scaffold at mature inhibitory synapses and may sta DG 150. bilize and or regulate the abundance of GABAARs n s. Activity Dependent Homeostatic Regulation of DG and GABAARs GABA A R. At neuromuscular synapses DG is required for the stabiliza. tion of acetylcholine receptors in the postsynaptic density 15 DG 50 50. In hippocampal neurons however previous work has reported. that DG is not necessary for GABAergic synapse formation and GABA A R. is recruited after the appearance of GABAAR clusters 17 tubulin. GABA A R GABA A R, suggesting that at least in vitro DG is not required for the initial. clustering of GABAARs This suggests that DG may function. Fig 1 Activity dependent homeostatic regulation of DG and GABAARs A. subsequent to inhibitory synapse formation much as it does and C Representative images of permeabilized immunostaining for glyco. at neuromuscular synapses 15 by contributing to activity sylated DG mAb IIH6 green GABAAR 3 red MAP2 blue and cell. dependent regulation of the abundance of synaptic GABAARs surface immunostaining for GABAAR 2 under control conditions and fol. To investigate this possibility we used a model of homeostatic lowing 24 h of bicuculline 10 M treatment Scale bars low magnification. synaptic plasticity in which chronic elevation of neuronal firing 10 m high magnification 2 5 m B and D Quantification of total cluster. rate either by blockade of inhibition with the GABAAR an area corresponding to experiments represented in A and C showing an in. tagonist bicuculline Bic or by elevated extracellular K crease in DG GABAAR 3 and surface GABAAR 2 cluster areas following. results in a multiplicative scaling up of GABAergic synaptic bicuculline Bic treatment n 40 41 images in each condition for DG and. 3 n 20 images in each condition for surface 2 two tailed Student t test. strength 22 28 This process occurs to a large extent through. P 0 001 P 0 01 E Representative Western blots from cell surface. NEUROSCIENCE, an increase in synaptic GABAARs and contributes to the biotinylation assays probing control Ctrl and bicuculline treated 10 M. renormalization of neuronal firing rate 22 23 Following 24 h cultures for surface and total expression of glycosylated DG mAb. bicuculline treatment 10 M 24 h we observed not only an IIH6 DG and GABAAR 3 F Quantification of experiments represented. expected homeostatic increase in the total cluster area of in E showing an increase in surface DG DG and GABAAR 3 following. GABAAR 3 subunits and an increase in the surface level of bicuculline treatment and an increase in the total whole lysate expression. clustered GABAAR 2 subunits but also an increase in the of DG and DG but not GABAAR 3 n 7 10 for surface n 18 21 for. total cluster area of functionally glycosylated DG DG total Wilcoxon signed rank test P 0 01 P 0 05 n s not significant. 209 5 19 9 of control Ctrl GABAAR 3 142 5 10 4, of Ctrl surface GABAAR 2 147 3 9 7 of Ctrl Fig 1 A D In. addition using cell surface biotinylation assays we determined that plasticity However little is known regarding the role of protein. surface expression of glycosylated DG DG and GABAAR 3 synthesis in homeostatic scaling up at GABAergic synapses We. are significantly up regulated following bicuculline treatment found that following bicuculline treatment 10 M 24 h the. DG 125 5 5 2 of Ctrl DG 125 9 7 4 of Ctrl total protein expression levels of glycosylated DG and DG. GABAAR 3 143 6 17 7 of Ctrl Fig 1 E and F These results were significantly increased DG 146 8 13 6 of Ctrl. reveal a parallel homeostatic up regulation of GABAARs and DG 126 8 10 3 of Ctrl Fig 1 E and F However con. DG consistent with a function for DG in homeostatic plasticity sistent with previous reports 22 we did not observe a signifi. at GABAergic synapses cant change in the total expression of GABAAR 3 in response. to chronic activity elevation 100 9 5 5 of Ctrl Fig 1 E and. Downloaded by guest on September 19 2020, Protein Synthesis Is Necessary for Inhibitory Homeostatic Plasticity F Given the colocalization and biochemical association of DG. Protein synthesis plays a crucial role in many forms of synaptic with synaptic GABAARs Fig S2 A and C these data suggest. plasticity 29 including some forms of homeostatic synaptic that the homeostatic increase in DG and GABAAR 3 clustering. Pribiag et al PNAS May 6 2014 vol 111 no 18 6811, and surface expression Fig 1 A F may be coordinated by an Ctrl.
Non Targeting, activity dependent up regulation of DG expression. To determine whether protein synthesis is necessary for ac Bic. tivity dependent homeostatic regulation of DG and GABAAR. 3 clusters we preincubated cultures with the protein synthesis 24 hrs. inhibitor cycloheximide CHX 100 M and then treated with 5 pA. bicuculline 10 M 6 h CHX blocked the homeostatic increase. in total area of both DG and GABAAR 3 clusters DG Bic. 163 0 14 0 of DMSO alone CHX alone 112 4 8 6 of, DG Knockdown. DMSO alone and CHX plus Bic 99 1 5 7 of DMSO alone Bic. 3 Bic 187 9 9 6 of DMSO alone CHX alone 118 0 7 2 50 pA. of DMSO alone and CHX plus Bic 108 3 5 6 of DMSO 2 5 sec. alone Fig 2 A and B By monitoring miniature inhibitory 24 hrs. postsynaptic currents mIPSCs we examined the role of protein. synthesis in homeostatic scaling up at inhibitory synapses and. found that preincubation with CHX blocked this form of plas B Ctrl. ticity DMSO alone 34 1 2 4 pA Bic 49 7 4 4 pA CHX alone Bic n s. 38 7 3 3 pA and CHX plus Bic 33 8 5 2 pA Fig S3 To. mIPSC frequency Hz, mIPSC amplitude pA, gether these findings indicate that this form of plasticity requires n s 4. de novo protein synthesis and are consistent with the proposition 45. that one of the necessary proteins synthesized is DG 3. DG Is Necessary for Homeostatic Scaling Up of GABAergic Synaptic. Strength To determine whether DG is an essential component in 15 1. the recruitment of additional GABAARs to inhibitory synapses. during homeostatic scaling up we recorded mIPSCs from bicu 0 0. culline treated 10 M 24 h neurons several days after trans NT DG KD NT DG KD. fection with a DG knockdown KD miRNA construct that. coexpresses mRFP for visualization of transfected cells Fig S4. C D NT Ctrl, 250 DG KD Ctrl, Treatment mIPSC amplitude pA. and Fig 3A DG KD had no effect on baseline mIPSC ampli 100 DG KD Bic. tude or frequency suggesting that DG loss at least in the short NT Bic Scaled. Cumulative probability, term is not required for GABAergic synapse maintenance 75.
However DG KD abrogated scaling up of mIPSC amplitude 150. induced by bicuculline treatment nontargeting NT miRNA 50 NT Ctrl. Ctrl 32 6 2 8 pA and Bic 50 5 3 9 pA DG KD miRNA Ctrl NT Bic 100. DG KD Ctrl, 33 8 3 3 pA and Bic 37 7 3 3 pA scaling factors vs NT Ctrl 25 DG KD Bic. 1 74 0 01 for NT Bic 1 08 0 01 for DG KD Ctrl 1 20 0 01 NT Bic scaled 50. 0 50 100 150 200 0 50 100 150, mIPSC amplitude pA Control mIPSC amplitude pA. DG GABA A R 3 DG 3, Ctrl Fig 3 Cell autonomous expression of DG is required for homeostatic. Bic scaling up of inhibitory synaptic strength A Representative traces of mIPSC. recordings from control Ctrl and bicuculline treated Bic 10 M 24 h. Ctrl neurons transfected with either a nontargeting miRNA mRFP construct or. a miRNA mRFP construct targeting DG for knockdown Corresponding av. Bic erage mIPSC traces from representative neurons are shown Right B. Group data of average mIPSC amplitude Left and frequency Right cor. responding to experiments shown in A mIPSC amplitude was significantly. GABA A R 3, increased following bicuculline treatment in neurons expressing the non. 200 250 targeting miRNA mRFP construct NT this increase was absent in DG. Total Cluster Area Ctrl, Total Cluster Area Ctrl, knockdown neurons DG KD n 9 14 cells in each condition two way.
150 n s ANOVA Tukey s post hoc test P 0 01 n s not significant C and D. n s Cumulative distribution and linear regression plots of mIPSC amplitudes. 100 showing that multiplicative scaling induced by bicuculline is blocked by DG. 100 knockdown C P 0 001 for NT Bic vs NT Ctrl P 0 99 for DG KD Ctrl vs. 50 50 NT Ctrl P 0 38 for DG KD Bic vs DG KD Ctrl and P 0 72 for NT Bic scaled. vs NT Ctrl Kolmogorov Smirnov test D r2 0 99 for all fitted lines. DMSO CHX DMSO CHX, Fig 2 Protein synthesis is required for homeostatic plasticity at inhibitory for DG KD Bic and 1 07 0 01 for NT Bic scaled Fig 3 A D. synapses A Representative images of dendritic regions immunostained for Because we used conditions where only 1 3 of the cells were. glycosylated DG mAb IIH6 and GABAAR 3 from control Ctrl and bicu transfected the recorded cells were isolated from nontransfected. culline treated Bic 10 M 6 h neurons preincubated either with DMSO cells suggesting that DG requirement is cell autonomous Con. 0 1 or with the protein synthesis inhibitor cycloheximide CHX 100 M versely a trend for increased mIPSC frequency following bicu. Scale bar 2 5 m B Quantification corresponding to experiments repre. sented in A showing that cycloheximide blocks the increase in total cluster. culline treatment was maintained in DG KD neurons NT miRNA. Downloaded by guest on September 19 2020, area of DG and GABAAR 3 induced by bicuculline treatment n 79 80 Ctrl 2 51 0 52 Hz and Bic 3 93 0 63 Hz DG KD miRNA Ctrl. and 70 80 cells in each condition for DG and 3 respectively two way 2 14 0 35 Hz and Bic 3 68 0 57 Hz Fig 3 A and B suggesting. ANOVA Tukey s post hoc test P 0 001 n s not significant that the mIPSC frequency increase observed in this form of. 6812 www pnas org cgi doi 10 1073 pnas 1321774111 Pribiag et al. plasticity is due to DG independent mechanisms acting to in. crease neurotransmitter release probability and or functional. A Ctrl Ctrl, Non Targeting, synapse number In summary DG is required for the recruit Bic. ment of additional GABAARs during homeostatic scaling up of. GABAergic synapses although not for the maintenance of base. line levels of synaptic GABAARs Bic, DG Is Not Required for Homeostatic Scaling Down of Excitatory or. Inhibitory Synapses We investigated the possibility that DG may. influence other aspects of homeostatic synaptic plasticity such as Ctrl. LARGE Knockdown, scaling down at glutamatergic synapses in response to elevated neu Ctrl.
ronal activity and scaling down at GABAergic synapses in response to. reduced neuronal activity 21 Consistent with the absence of DG at. excitatory synapses Fig S2A we observed no effect of DG KD on 50 ms. either baseline excitatory synaptic strength or on scaling down in Bic. response to bicuculline treatment 10 M 24 h NT miRNA Ctrl. 12 3 1 2 pA and Bic 9 02 0 4 pA DG KD miRNA 12 1 0 7 pA. and Bic 9 0 0 5 pA Fig S5 Furthermore strengthening the notion. that baseline inhibitory synaptic strength is set independently of DG B Ctrl. we observed no effect of DG KD on scaling down of mIPSC am 50 Bic 4 n s. mIPSC amplitude pA, plitude in response to prolonged blockade of neuronal activity with. mIPSC frequency Hz, tetrodotoxin TTX 1 M 48 h NT miRNA Ctrl 43 3 3 5 pA 3. and TTX 29 7 2 2 pA DG KD miRNA Ctrl 40 5 3 4 pA and 30. TTX 27 1 1 5 pA Fig S6 Therefore DG appears to function 2. selectively in potentiating inhibitory synaptic strength during pro 20. longed periods of high neuronal activity 1, Glycosylated DG Is Necessary for Homeostatic Scaling Up at. GABAergic Synapses Carbohydrate side chains are necessary for NT LARGE KD NT LARGE KD. DG to bind to agrin and other ligands such as laminin and. neurexin 6 9 24 LARGE is a glycosyltransferase required for Fig 4 The DG glycosylating enzyme LARGE is required for homeostatic. O linked glycosylation of DG 30 31 and is able to com scaling up of inhibitory synaptic strength A Representative traces of mIPSC. static synaptic plasticity maintains this balance though the molecular underpinnings of this plasticity necessary to explain brain dysfunction and define therapies are not well under stood Here we have described regulation of inhibitory syn aptic plasticity by dystroglycan a cell adhesion molecule that

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