a b c 200 - H S1 P1 S2 P2 SYN TIF PSD Shrm4 95 - PSD95 34 - Synaptophysin 120-100 - Wil type GABA B1a -/- GABA B1b -/- -GABA B1a -GABA B1b e f 5 μm 10 μm 20 μm 100 - GABA B1a 600 400 200 Ponceau GABA B Shrm4 0 I A I A I A GABA B1a mean intensity (a.u) 5 μm 10 μm 20 μm I A I A I A Supplementary Figure 1
Supplementary Figure 1. Shrm4 is localize in the pre- an postsynaptic compartments of neurons. (a) (Top) Images an (Bottom) histogram showing quantitative co-localization of enogenous Shrm4 staining with presynaptic (Bassoon) as well as with postsynaptic markers of both excitatory (PSD-95) an inhibitory (GABA A β3) synapses in 18DIV (ays in vitro) culture hippocampal neurons. n= 15, Scale bar: 10 µm (b) Electron micrographs of culture cortical neurons at 20DIV immunolabele with anti-shrm4 an seconary antiboy conjugate with Nanogol particles, showing gol particles in enrites an synaptic boutons. Sv: synaptic vesicles; MT: microtubules; m: mitochonria. Scale bar: 200 nm. The histograms show that in Shrm4-staine neurons, gol particles were significantly enriche (*p <0.0001, see Table 1) in enrites an presynaptic terminals (synaptic boutons) an also tene to accumulate at the postsynaptic compartments. (c) Western blot analyses of postsynaptic ensity (PSD) subcellular fractionation from rat brain. H, total homogenate; S1, P1, Whole-brain postnuclear supernatant an membrane fractions, respectively; S2, cytosolic proteins; P2, crue synaptosomal fraction; SYN, synaptosomes; TIF, Triton X-100 insoluble fraction; PSD, Triton X-100-extracte PSD. () Western blot of wil-type, GABA B1a -/- an GABA B1b -/- mouse brain lysates using anti-gaba B1 for immunoblotting. The blot shows that anti- GABA B1 antiboy recognizes both GABA B1a an GABA B1b isoforms. (e) Full image of irect stochastic optical reconstruction microscopy (STOM) imaging of GABA B -ATTO 488 (shown in re) an Shrm4-Alexa 647 (shown in green) on 14DIV culture hippocampal neurons. Scale bar: 0.4 µm. (f) (Left) GST pull-own experiments using GST-PDZ on lysates of cells overexpressing GABA B1a -GFP with increasing concentrations (5, 10 an 20 µm) of Tat-control (I) or Tat-859-870 (A) peptie. (ight) Histogram showing the GABA B1a mean intensity compare to the Tat-control peptie. Incubation with the Tat 859-870 at 10 an 20 µm showe a reuction in Shrm4/GABA B1 bining compare to the Tat control peptie.
a b c scramble/#2 Surface GABA B p150-cc1 Surface GABA B Control sina Shrm3 sina GABA B GABA B scramble/#2 shna shna Surface GABA B Surface GABA B Surface GABA B p150-cc1 rescue rescue Surface GABA B Surface GABA B Surface GABA B Normalize GABA B1 Surface signal intensity 1.5 1.0 0.5 0 * * * scramble/#2 shna shna#2 p150-cc1 rescue Normalize GABA B1 signal intensity 2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 Normalize spine ensity 2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 control sina shrm3 sina shna#2 Surface GABA B shna#2 Surface GABA B Supplementary Figure 2
Supplementary Figure 2. Valiation of knockown an rescue constructs. (a) Western blots of lysates from transfecte an non-transfecte (NT) HEK293 cells emonstrating the effectiveness of the two knockown constructs (shna an shna#2). HA-tagge Shrm4 (HA-Shrm4) was use to overexpress Shrm4 in these cells. Transfecte constructs were HA-Shrm4, shna with HA-Shrm4 (rescue) or HA-Shrm4 with shna#2. α-tubulin serve as loaing reference an polyclonal anti-ha antiboy was use to reveal Shrm4 expression. The blot shows that HA-Shrm4 is resistant to shna but not to shna#2. (b) (Left) epresentative images of hippocampal neurons transfecte at 8DIV an immunostaine at 18DIV with polyclonal anti-shrm4 antiboy to reveal enogenous Shrm4 expression an monoclonal anti-ha to reveal overexpresse Shrm4. Transfections were scramble, shna, shna#2, shna plus HA-Shrm4, an shna#2 plus HA-Shrm4. Scale bar: 10 µm. (ight) Quantification of enritic Shrm4 expression normalize to scramble transfecte neurons. Both shna (**p < 0.01; one-way ANOVA, n = 10) an shna#2 (**p < 0.01; one-way ANOVA, n = 10) reuces enogenous Shrm4 expression an expression of HA-Shrm4 (resistant) with shna rescues Shrm4 expression whereas it oes not with shna#2. (c) Full representative images an histogram showing surface immunostaining for GABA B in culture hippocampal neurons at 18DIV after transfection with scramble, shna, shna#2, p150-cc1 or rescue constructs at 8DIV. Scale bar: 15 µm. Histograms show mean ± SEM; n = 5-15, *P < 0.05, One-way ANOVA. () epresentative images of hippocampal neurons transfecte at 8DIV with GFP coexpressingcontrol sina or Shrm3 sina an immunostaine at 18DIV for surface GABA B1. No significant changes in GABA B1 intensity or spine ensity were observe from Shrm3-siNA expressing neurons compare to controls. Histograms show mean ± SEM; n = 10.
scramble shna escue EGFP PSD95 merge EGFP PSD95 merge EGFP PSD95 merge EGFP Bassoon merge EGFP Bassoon merge EGFP Bassoon merge EGFP GluA2 merge EGFP GluA2 merge EGFP GluA2 merge EGFP Synapsin merge EGFP Synapsin merge EGFP Synapsin merge Supplementary Figure 3
Supplementary Figure 3. Shrm4 regulates synaptic protein composition. Full representative images of rat hippocampal neurons transfecte at 8DIV with Shrm4 scramble or shna with or without rescue constructs an immunostaine for PSD95, Bassoon, GluA2 an Synapsin post-synaptogenesis at 18DIV. Scale bars: 10 µm.
a b 12 DIV Supplementary Figure 4
Supplementary Figure 4. Shrm4 regulates enritic spine ensity an GABA B transport before synaptogenesis. (a) epresentative images an histograms of enrites () an axons (ax) at 14DIV from hippocampal neurons transfecte before synaptogenesis at 8DIV with control (scramble#2) or knockown (shna#2) shnas. The enritic spine ensity an GABA B expression levels were measure in enrites an axon by co-staining for brevican to localize axons. The histograms compare mean (± SEM) spine ensity (normalize number of spines/20 µm; 20 neurons examine per construct) an polarity inex (PI) of GABA B expression. Spine ensity (***p < 0.001; t-test, n = 15) is significantly lower in Shrm4-silence neurons than scramble controls. In aition, the PI of Shrm4 knockown neurons is significantly lower than scramble (*p < 0.05; t-test, n = 10). Scale bar: 20 µm. (b) epresentative images with histograms comparing spine ensity of rat hippocampal neurons transfecte at 12DIV with knockown shna or scramble shna. Spine ensity oes not iffer between Shrm4- silence an control neurons when transfecte post synaptogenesis at 12DIV (normalize number of spines/ 20 µm) Histogram mean ± SEM, n=20.
a b scramble shna GABA B1 Average F/F 0 0.8 0.6 0.4 0.2 0.0 shna + 1 ΔCT 2 shna 0 300 600 900 1200 Time (ms) Decay (ms) 2000 1500 1000 500 0 Mean ise Time (ms) 300 200 100 0 Supplementary Figure 5
Supplementary Figure 5. (a) Valiation of GABA B1 shna. epresentative images (Left) an histogram (ight) of hippocampal neurons transfecte at 8DIV with scramble (control) or knockown shna that specifically target GABA B1. The neurons were transfecte at 8DIV an analyze by confocal microscopy at 18DIV for GABA B expression. Normalize GABA B fluorescence intensity is significantly lower in shna-transfecte neurons than scramble controls. The histograms compare mean (±SEM); n=20; ***p < 0.001, t-test. Scale bar: 20 µm. (b) Calcium ynamics in neurons expressing 1ΔCT. (Left) Time course of average ΔF/F 0 values an single exponential fits of Ca 2+ signals in control neurons expressing shna an neurons expressing 1b subunits with a eletion of the C-terminus tail (1ΔCT) co-expresse with 2 an shna. e lines show exponential fits of the ecays. (ight) Mean rise times an ecay τ of Ca 2+ signals for control an 1ΔCT. Histograms show mean ±SEM; n = 11-18.
a b c f e 200-120- 100- Input (5%) IgG ip Shrm4 Shrm4 KIF5B 200-120- 100- Input (5%) IgG ip Shrm4 Shrm4 KIF5C 1a Decay τ (min) g I II h III Supplementary Figure 6
Supplementary Figure 6. Shrm4 an ynein/ynactin regulate GABA B transport to enrites. (a) Schematic showing a GABA B heteroimer with an α-bungarotoxin (BgTx) bining site (BBS) in the N-terminus of 1a (1a BBS ). (b) Live cell surface fluorescence monitore over 60 min at 30-32 C, in hippocampal neurons at 14DIV, expressing 1a BBS 2 with BgTx couple to Alexa-fluor 555 (BgTx-AF555), with either Shrm4 knockown (shna) or scramble constructs (scramble). (c) ates of constitutive internalization for BgTx- AF555-tagge GABA B s in Shrm4 knockown ( ) an scramble controls ( ). () Exponential ecay time constants for membrane fluorescence for GABA B s in knockown an control cells. (e) Membrane fluorescence of GABA B s after 1 hr in knockown an scramble control cells. Histograms show mean ±SEM; n = 6-8. Scale bar 10 µm. (f) Blots of Shrm4 immunoprecipitation experiments on rat brain lysates, which emonstrate the absence of interaction with the subunit B an C of KIF5. (g) Supramolecular complex between the Shrm4 PDZ omain, GABA B1b, GABA B2 an the 104-138 filaments of ynein IC2 interacting with imeric ynein LC8. (I) The GABA B1b (green) an GABA B2 (aquamarine) moele heteroimer comprises the seven-helices bunle, the intracellular an extracellular loops, an the C-terminus up to the en of the coil-coil region. The Shrm4 PDZ is violet. The 110-138 filament of ynein IC2C is orange. (II) A sie view of the complex in (I) is shown. In this panel imeric LC8 (gray an yellow protomers) an an aitional filament of IC2C (blue) of ynein are shown as well. The spheres centere on the Cα-atoms of GABA B1b an PDZ mark a selection of salt briges at the interface between the two proteins. (III) A zoom on the GABA B1b /PDZ/IC2/LC8 interfaces is shown. The C-terminal 867-886 amino aci segment of GABA B1b makes contacts with PDZ. The selecte salt briges marke by spheres at the GABA B1b -PDZ interface inclue: 1) E869 from the receptor an both K26 an 70 from β2 an α2 of PDZ, respectively; 2) K875 from the receptor an E41 from the β2-α1 loop of PDZ;
an 3) E886 from the coil-coil helix of the receptor an 53 from the α1-β4 loop of PDZ. The β1 stran of PDZ forms an antiparallel β-sheet with the 128-135 stran of IC2 (orange). The latter in turn forms an antiparallel β-sheet with the yellow protomer of LC8. A secon segment of IC2 (blue) forms an antiparallel β-sheet with the gray protomer of LC8. For both IC2C filaments of ynein the 110-120 segment is not shown. (h) epresentative Western blots of Shrm4, α-tubulin an GFP from hippocampal brain extracts prepare three weeks after injection of AAV5-scramble or AAV5-shNA (knockown) into opposing hippocampi of rats. Hippocampi infecte with AAV5-shNA show robust reuction in Shrm4 expression (Histograms show mean ±SEM; n = 3; **p < 0.01; t-test).
a ax ax GFP GABA B escue GABA B () () (ax) GFP GABA B (ax) escue GABA B ax ax shna GABA B GFP-p150-cc1 c1 GABA B () () (ax) shna GABA B (ax) GFP-p150-cc1 GABA B b Supplementary Figure 7
Supplementary Figure 7. Shrm4 an ynein/ynactin regulate GABA B transport to enrites. (a) Full representative images of immunostaining of enogenous GABA B on enrites () an axons (ax) in 18DIV neurons transfecte at 7DIV with Scramble, shna, escue or p150-cc1 construct. Scale bar 10 µm. (b) epresentative images of enrites () an axons (ax) of 18DIV hippocampal neurons transfecte after synaptogenesis at 12DIV with knockown or scramble controls. Neurons were immunostaine to reveal enogenous GABA B s an brevican to localize axons. Histogram showing that PI of knockown shnaexpressing neurons is lower than that of scramble-expressing neurons (Histograms show mean ±SEM; n = 10; **p < 0.01; t-test,). Scale bar: 20 µm.
GFP () (ax) 1A-myc GFP ax shna () (ax) 1A-myc shna 1A-myc 1A-myc ax GFP-p150-cc1 () (ax) 1A-myc GFP-p150-cc1 1A-myc ax GFP () (ax) 1B-myc GFP 1B-myc ax shna () (ax) 1B-myc shna 1B-myc ax () (ax) 1B-myc GFP-p150-cc1 1B-myc GFP-p150-cc1 ax GABA B1 GABA B2 0 0,5 1 1,5 Correlation coefficient (Pearson r) scramble shna a b Supplementary Figure 8
Supplementary Figure 8. Shrm4 an ynein/ynactin regulate overexpresse GABA B transport to enrites. (a) Full representative immunostaining images of overexpresse myc-tagge GABA B -1a (left) an -1b (right) on enrites () an axons (ax) at 18DIV neurons, transfecte at 7DIV together with GABA B2 an either scramble, shna, escue or p150-cc1 construct (b) epresentative images (top) an histogram (bottom) of 18DIV culture hippocampal neurons transfecte at 8DIV with GABA B1 -myc an GABA B2 -flag with either knockown shna or its scramble control an immunostaine using the anti-tag antiboies. A high egree of co-localization is seen for both knockown an scramble shna-expressing neurons.
a b shna rescue 10 μm baclofen 100pA 1s Current Density (pa/pf) 10 µm Baclofen -2.0-1.5-1.0-0.5 0 * 100 μm baclofen 100pA 1s Current Density (pa/pf) 100 µm Baclofen -3.0 * -2.5-2.0-1.5-1.0-0.5 0 c Supplementary Figure 9
Supplementary Figure 9. Changes in electrophysiological properties in vitro upon Shrm4 silencing. (a) Whole-cell GIK currents, recore in response to GABA B activation by 10 or 100 µm baclofen, from rat hippocampal neurons at 14DIV, transfecte with knockown shna with or without the rescue construct (HA-Shrm4). Peak K + current ensity in Shrm4-silence neurons was significantly lower than in the presence of the rescue construct for both oses of baclofen. Histograms show mean ±SEM; *P<0.05, t-test, n = 7-10. (b) Whole-cell GIK currents recore from culture rat hippocampal neurons at 14DIV in response to glutamate o not iffer between neurons expressing knockown (shna), those expressing scramble, or those expressing the coile-coil omain of ynactin (GFP-p150-cc1). GIK currents were inuce by mglu activation with 10 µm glutamate in the presence of 2 mm kynurenic aci an 20 µm bicuculline. (c) Amplitue, ecay time, area an frequency of mipscs recore from 14DIV hippocampal neurons transfecte (8DIV) with knockown shna or scramble, measure in the presence of kynurenic aci (3 mm) an liocaine (500 µm) Histograms show mean ±SEM; n=12-17.
a b c scramble shna Slope (% of maximum response) 100 80 60 40 20 scramble shna 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Current (ma) fepsp slope (% change) 140 120 100 80 60 40 20 0.5 mv 10 ms AAV5-scramble AAV5-shNA 0 0 10 20 30 40 50 60 Time (min) fepsps slope (last 10 minutes) (% of control) pre LFS post LFS 100 80 60 40 20 0 e scramble shna 100000 PSD95 Mean intensity (a.u) 80000 60000 40000 20000 0 Supplementary Figure 10
Supplementary Figure 10. Changes in electrophysiological properties in vivo upon Shrm4 silencing. (a) Input-output curves measure in vitro for fiel excitatory postsynaptic potential (fepsp) in hippocampal slices from rat injecte with AAV5-shNA compare to AAV5- scramble controls. n = 6 (b) Iniviual traces of fiel excitatory postsynaptic potentials (fepsps) at baseline (grey) an after high frequency stimulation (black) of acute hippocampal slices of ault rats previously injecte with either knockown AAV5-shNA (right hemisphere) or AAV5-scramble (left hemisphere) (n = 9 animals). (Bottom) epresentative traces an bar chart of changes in fepsps. Baseline fepsps were recore from the apical enritic layer of CA1. LTP was inuce by high frequency stimulation (100 stimuli at 250 Hz) of Schaffer collaterals. The traces show no ifferences in LTP inuction or maintenance between Shrm4-silence hippocampal slices an scramble controls. (c) Iniviual representative traces of fepsps at baseline (line) an after low frequency stimulation (otte line) of acute hippocampal slices of ault rats previously injecte with either knockown AAV5-shNA (right hemisphere) or AAV5-scramble (left hemisphere) (n = 6 animals). (Bottom) epresentative traces an bar chart of changes in fepsps. Baseline fepsps were recore from the apical enritic layer of CA1. LTD was inuce by low frequency stimulation (900 stimuli at 1 Hz) of Schaffer collaterals. The traces show no ifferences in LTD inuction or maintenance between Shrm4-silence hippocampal slices an scramble controls. () epresentative images (top) an histogram (bottom) of the mean intensity of PSD-95 positive puncta (excitatory synapses) immunolabele in the CA1 region of the hippocampus of rats previously injecte with either knockown AAV5-shNA (right hemisphere) or AAV5-scramble (left hemisphere). The results show no ifferences in PSD95 mean intensity between Shrm4-silence hippocampal slices an scramble slices (n= 3 animals).
Scale bar: 20 µm (e) (Left) epresentative traces an (ight) histograms of amplitue, ecay time, area an frequency of mipscs recore from DGGCs of ault rats previously injecte with either knockown AAV5-shNA (right hemisphere) or AAV5-scramble (left hemisphere) (n= 3 animals). Amplitue, ecay time, area an frequency of mipscs o not iffer significantly between Shrm4-silence hippocampal slices an scramble controls. mipscs were measure in the presence of kynurenic aci (3 mm) an liocaine (500 µm).
fig.1b Input (5%) IgG ip Shrm4 fig.1 (1) fig.1 (2) fig.1 (3) -PDZ(Mut AA) fig.1e 200- Shrm4 120-110- GABA B1 fig.1f fig.1g NT Input (2%) Tat control Tat 859-870 GST-PDZ mutaa GST-PDZ Input (2%) GST-PDZ mutaa GST-PDZ GABA B1b fig.1i fig.3b fig.3c(top) fig.3c(bottom) Ponceau fig.3 fig.5f fig.3f 80-70 - Input GST GST-PDZ GST-PDZ (Mut AA) His-IC-2C 100- GABA B2 fig.3e 85-70 - Input (10%) GST GST-PDZΔ14 GST-PDZ DIC e Ponceau e Ponceau Supplementary Figure 11
Supplementary figure 11. Full versions of the western blots presente in the stuy. The blots have been labelle with figure panels where they have appeare.
Supplementary Table 1: Distribution of gol nanoparticles in culture rat cortical neurons immunolabele with (Shrm4) or without (Control) primary antiboies against Shrm4 Compartments Χ 2 Values Observe Gol, No Observe Points (P) LD Values (No/P*a p) Expecte Gol, Ne LI Values (No/Ne) Shrm4 Synaptic bouton 15 5 3 2.66 5.64 57.30* Post-synaptic terminal 1 1 1 0.53 1.88 0.41 Thin enrites 102 86 1.19 45.72 2.23 69.27* Other un-etermine 4 70 0.06 37.22 0.11 29.65 structures Cell structure absent 4 75 0.05 39.87 0.10 32.27 Total 126 237 0.53 126 1 188.91$ Control Synaptic bouton 1 1 1 0.60 1.67 0.27 Post-synaptic terminal 0 1 0 0.60 0 0.60 Thin enrites 17 22 0.77 13.14 1.29 1.13 Other un-etermine 18 29 0.62 17.32 1.04 0.03 structures Cell structure absent 10 24 0.42 14.34 0.70 1.31 Total 46 77 0.60 46 1 3.34
Supplementary Table 2: Statistical analysis parameters for Fig. 2 Figure panel Statistical test n Conitions p value F 2c (Left) PSD95 Orinary one-way ANOVA 15, 14, 11 Scramble, shna, rescue p=0,0011 F(2,32)=8,191 2c (Left) Bassoon Orinary one-way ANOVA 10, 10, 15 Scramble, shna, rescue p=0,0002 F(2,32)=11.16 2c (Left) GluA2 Orinary one-way ANOVA 10, 10, 15 Scramble, shna, rescue p<0,0001 F(2,32)=32.72 2c (Left) Synapsin Orinary one-way ANOVA 10, 10, 15 Scramble, shna, rescue p=0,0002 F(2,32)=11.22 2c (ight) PSD95 Orinary one-way ANOVA 15, 14, 11 Scramble, shna, rescue p=0,0011 F(2,37)=2,37 2c (ight) Bassoon Orinary one-way ANOVA 15, 20, 15 Scramble, shna, rescue p=0,0148 F(2,32)=4,615 2c (ight) GluA2 Orinary one-way ANOVA 17, 20, 15 Scramble, shna, rescue p=0,0069 F(2,32)=5,592 2c (ight) Synapsin Orinary one-way ANOVA 10, 10, 15 Scramble, shna, rescue p=0,0174 F(2,32)=5,592 2 Orinary one-way ANOVA 15, 15, 20 Scramble, shna, rescue p=0,0029 F(2,47)=6,641 2f Orinary one-way ANOVA 15, 15, 20 Scramble, shna, rescue p<0,0001 F(2,42)=75.59 2g Orinary one-way ANOVA 15, 15, 20 Scramble, shna, rescue p<0,0001 F(2,47)=28.23