The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory
- Remco V Klaassen
- Marta Ruiperez-Alonso
- Azra Elia Zamri
- Jasper Stroeder
- Priyanka Rao-Ruiz
- Johannes C Lodder
- Rolinka J van der Loo
- Huib D Mansvelder
- August B Smit
- Sabine Spijker
- VU University, The Netherlands
- Sylics (Synaptologics BV), The Netherlands
- Vrije Universiteit Amsterdam, The Netherlands
- Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, France
Figures
Figure 1 with 3 supplements
Shisa7 is a type-I transmembrane protein interacting with AMPA-type receptors.
(a) Shisa7 is closely related to the AMPAR auxiliary subunit Shisa6 and Shisa9, bearing a signal peptide (SP; 22 amino acids), an extracellular domain with conserved cysteine-rich motif, a single …
-
Figure 1—source data 1
Sequence of DNA primers.
Primers were used for PCR experiments to detect the presence or absence of exon 4 in Shisa7, as well as for real-time PCR. The forward and reverse primers, as well as the size of the amplicon generated, are indicated.
- https://doi.org/10.7554/eLife.24192.006
Figure 1—figure supplement 1
Shisa7 gene expression.
(a) Quantitative PCR shows that the Shisa7 gene expression is specifically enriched within the brain (note the log2-scale), and is virtually absent in the pancreas (pooled from three adult mice). …
Figure 1—figure supplement 2
Generation of Shisa7 KO mice and antibody testing.
(a) Representation of Shisa7-null mouse generation: The Shisa7 locus around exon 1, encoding the N-terminal part of the Shisa7 protein including the start-site (ATG), with essential restriction …
Figure 1—figure supplement 3
Whole immunoblot compilation.
Whole immunoblots are presented from which sections are included in Figure 1b–e, and Figure 1—figure supplement 2e. Numbers represent apparent molecular weights in kDa. Black dots indicate the …
Figure 2 with 1 supplement
Shisa7 effects on AMPAR desensitization rate and recovery from desensitization.
(a) Peak-scaled example trace of whole-cell recordings from HEK293 cells expressing a homomeric GluA1-containing AMPAR channel in the absence (black) or presence (blue) of Shisa7. Currents were …
Figure 2—figure supplement 1
Shisa7 does not alter AMPAR kinetics in vitro or alter membrane properties in vivo.
(a) Peak-scaled example traces of whole-cell recording from HEK293 cells expressing homomeric GluA1-containing AMPAR channels in the absence (black) or presence (blue) of Shisa7. Currents were …
Figure 3 with 1 supplement
Shisa7 prolongs synaptic AMPAR currents.
(a) Example traces of mEPSC recordings from CA1 pyramidal cells of Shisa7 KO animals and WT littermates. (b,c) Superimposed spontaneous synaptic currents (b), and average synaptic currents (c) of Shi…
Figure 3—figure supplement 1
Shisa7 does not alter membrane properties in vivo.
(a,b) Recorded pyramidal cells in the CA1 hippocampal region showed comparable membrane properties for Shisa7 KO and WT mice; membrane resistance (a) and resting membrane potential (b).
Figure 4 with 2 supplements
Deletion of Shisa7 maintains glutamatergic synapses under basal conditions.
(a) Immunoblots of hippocampal synaptic membrane fractions from WT and Shisa7 KO mice (n = 4–5 each) do not reveal differences in abundance of AMPAR (MWU tests, GluA1, p=0.421; GluA2, p=1.000), …
Figure 4—figure supplement 1
Data as presented in Figure 4, but now with individual data points for WT (gray) and Shisa7 KO (red).
(a) Immunoblots of hippocampal synaptic membrane fractions from WT and Shisa7 KO mice (n = 4–5 each) do not reveal differences in abundance of AMPAR, NMDAR, PSD-95, TARP γ−8, Shisa9 or Shisa6, when …
Figure 4—figure supplement 2
For immunoblots presented in Figure 4, we performed normalization of loading differences based on trichloroethanol-assisted total protein staining of the gel.
The representative protein bands and corresponding loading controls are shown.
Figure 5 with 1 supplement
Shisa7 has no effect on short-term plasticity.
(a) Superimposed example traces of whole-cell recordings voltage clamped at –70 mV from CA1 pyramidal neurons of Shisa7 KO animals (red) and WT littermates (grey) in response to 50 Hz stimulation of …
Figure 5—figure supplement 1
Similar AMPAR and NMDAR amplitudes in Shisa7 KO mice.
(a,b) Correlation analysis of stimulus current injected vs. EPSP amplitude (Ln-transformed data) for AMPAR (a; nA) and NMDAR (b; pA) obtained showed no difference for genotype (a, WT n = 56 data …
Figure 6 with 2 supplements
Deletion of Shisa7 slows down initiation and decreases maintenance of LTP.
(a) Normalized EPSP amplitude over the time course of the LTP experiments shows a clear genotype effect (WT, n = 5 slices, n = 5 mice; Shisa7 KO, n = 6 slices, n = 6 mice). The arrow indicates the …
Figure 6—figure supplement 1
Deletion of Shisa7 slows down initiation and decreases maintenance of LTP.
(a) Normalized EPSP slope over the time course of the LTP experiments shows a clear genotype effect, with a reduction in Shisa7 KO, similar as observed for amplitude (cf. Figure 5)(WT, n = 5 slices, …
Figure 6—figure supplement 2
Deletion of Shisa7 slows down initiation and decreases maintenance of LTP by affecting AMPAR recruitment.
(a) Normalized EPSP amplitude (WT, n = 5 slices, n = 5 mice; Shisa7 KO, n = 6 slices, n = 6 mice mind that the last data point contains n = 5) binned during 5 minute intervals early after LTP …
Figure 7 with 2 supplements
Deletion of Shisa7 specifically affects contextual fear memory.
(a,c) Experimental set-up of measuring contextual fear conditioning memory (a,c), in which mice received a foot shock (US) in a specific environment (CS), and freezing was assessed upon re-exposure …
Figure 7—figure supplement 1
Deletion of Shisa7 specifically affects contextual fear memory.
In addition to the set-ups (a,c,e) and average freezing data of Figure 7, the individual data are shown for short term fear memory (nWT = 8, nKO = 7; b), long term fear memory (nWT = 9 nKO = 10; d), …
Figure 7—figure supplement 2
Shisa7 KO mice display no abnormalities in shock sensation, locomotor activity or anxiety-related behavior.
(a) Mean velocity in the 3 minutes prior to shock (left), or during the 2 s 0.7 mA shock (right) was not different between Shisa7 KO (n = 10) and WT littermates (n = 9), indicating no effect on …
Figure 8
Schematic representation of effects on AMPA type glutamate receptors by Shisa family members and TARP γ−8.
The effect is depicted for different AMPAR kinetic parameters measured in heterologous expression systems (HEK293 cells or oocytes; open ovals), or ex vivo in hippocampal slices (filled ovals). …
Tables
Table 1
Shisa7 is a native interactor of hippocampal AMPARs.
Shisa7 complexes were immunoprecipitated from the hippocampi of Shisa7 WT and KO animals (DDM-extracted crude synaptic membranes; n = 3 IPs per genotype) and subjected to mass spectrometric …
| Gene name | Uniprot recommendedprotein name(s) | Uniprot ID | PDZ-domains | Number of unique peptides | LFQ intensity | Average KO LFQ intensity | Average WT LFQ intensity | Average KO/WT LFQ intensity | Average WT/KO LFQ intensity | T-test significant FDR0.01 = ++ DR0.05 = + | T-test q-value | Percent cover-age | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| KO1 | KO2 | KO3 | WT1 | WT2 | WT3 | KO1 | KO2 | KO3 | WT1 | WT2 | WT3 | |||||||||||
| Shisa7 | Protein Shisa-7 | Q8C3Q5 | 0 | 1 | 1 | 2 | 17 | 18 | 16 | 865 | 873 | 1750 | 319820 | 305190 | 346730 | 1163 | 323913 | 0.4% | 278.58 | ++ | 0.0000 | 39.2 |
| Protein Shisa-7:Exon4-specificpeptideNLYNTMKPSNLDNLHYNVNSPK | - | - | 0 | 0 | 0 | 1 | 0 | 0 | - | - | - | - | - | - | - | - | - | - | - | - | - | |
| Gria1 | Glutamatereceptor 1 | P23818 | 0 | 0 | 0 | 0 | 10 | 12 | 12 | 5921 | 0 | 3217 | 17828 | 52304 | 54212 | 4569 | 41448 | 11.0% | 9.07 | ++ | 0.0000 | 22.8 |
| Gria2 | Glutamatereceptor 2 | P23819 | 0 | 2 | 1 | 0 | 17 | 17 | 17 | 2743 | 0 | 0 | 104860 | 101430 | 93097 | 2743 | 99796 | 2.7% | 36.39 | ++ | 0.0000 | 36.4 |
| Gria3 | Glutamatereceptor 3 | Q9Z2W9 | 0 | 0 | 0 | 0 | 4 | 4 | 5 | 0 | 0 | 0 | 6529 | 7068 | 10102 | 0 | 7900 | 0.0% | NaN | ++ | 0.0000 | 17.7 |
| Cacng8 | Voltage-dependentcalciumchannelgamma-8subunit;TARPgamma-8 | Q8VHW2 | 0 | 2 | 2 | 1 | 2 | 1 | 5 | 4023 | 0 | 0 | 0 | 0 | 3962 | 4023 | 3962 | 101.5% | 0.98 | - | N/A | 30.5 |
| Olfm1 | Noelin | O88998 | 0 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1076 | 0 | 0 | 1004 | 779 | 1076 | 892 | 120.6% | 0.83 | - | N/A | 7.0 |
| Prrt1 | Proline-richtransmem-braneprotein 1;SynDIG4 | O35449 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 969 | 0 | 0 | 0 | 969 | 0.0% | NaN | - | N/A | 7.2 |
| Prrt2 | Proline-richtransmem-braneprotein 2 | E9PUL5 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 333 | 0 | 318 | 0 | 202 | 333 | 260 | 127.9% | 0.78 | - | N/A | 3.8 |
| Rap2b | Ras-relatedproteinRap-2b | P61226 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 2588 | 0 | 0 | 0 | 2588 | 0 | NaN | 0.00 | - | N/A | 12.0 |
| Shisa6 | Proteinshisa-6homolog | Q3UH99 | 0 | 1 | 0 | 0 | 3 | 3 | 1 | 0 | 0 | 0 | 2666 | 3580 | 2136 | 0 | 2794 | 0.0% | NaN | + | 0.0306 | 9.9 |
| Dlg1 | Disks largehomolog 1;SAP97 | Q811D0 | 3 | 0 | 1 | 1 | 3 | 0 | 0 | 0 | 0 | 0 | 2126 | 0 | 0 | 0 | 2126 | 0.0% | NaN | - | N/A | 4.4 |
| Dlg3 | Disks largehomolog 3;SAP102 | P70175 | 3 | 0 | 0 | 0 | 0 | 2 | 2 | 0 | 0 | 0 | 0 | 2441 | 0 | 0 | 2441 | 0.0% | NaN | - | N/A | 4.8 |
| Dlg4 | Disks largehomolog 4;PSD95 | Q62108 | 3 | 0 | 0 | 0 | 9 | 8 | 7 | 0 | 0 | 0 | 11835 | 13410 | 9284 | 0 | 11510 | 0.0% | NaN | ++ | 0.0000 | 21.7 |
| Magi2 | Membrane-associatedguanylatekinase,WW and PDZdomain-containingprotein 2 | Q9WVQ1 | 6 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1558 | 0 | 0 | 1558 | 0.0% | NaN | - | N/A | 1.6 |
-
Table 1—source data 1
Maxquant analysis of hippocampal Shisa7 immunoprecipitation experiments, as detailed in the Materials and methods section.
The Maxquant ‘proteinGroups.txt’ output file was supplemented with ‘LFQ intensity _ Average KO’, ‘LFQ intensity _ Average WT’, and the ‘KO/WT’ and ‘WT/KO’ ratios thereof.
- https://doi.org/10.7554/eLife.24192.008
-
Table 1—source data 2
Statistical analysis of hippocampal Shisa7 immunoprecipitation data, as detailed in the Materials and methods section.
Tab 1: In summary, the Maxquant ‘proteinGroups.txt’ output file was imported into Perseus, and processed in the following manner: (1) Removal of ‘Reverse’, ‘Potential contaminant’, and ‘Only identified by site’ protein groups; (2) Log(2) transformation of all LFQ intensity values; (3) Removal of protein groups with less than three valid ‘Log(2) LFQ intensity’ values in either the WT or KO groups; (4) Imputation of missing values (8.6% of the population) from a normal distribution (width 0.3, down shift 1.8, whole matrix); (5) Performing a Student's t-test followed by permutation-based FDR analysis (S0 = 1, FDR = 0.01, 2500 permutations). Tab 2: Visualization of the data by means of Histogram and Vulcanoplot is presented in the additional sheets. Tab 3: Distribution of LFQ intensities after replacing missing values from a normal distribution. The imputed value distribution is depicted in red.
- https://doi.org/10.7554/eLife.24192.009
Additional files
-
Supplementary file 1
The functional contribution of Shisa family members (Shisa9, Shisa6, Shisa7) and TARP γ−8 on AMPAR complexes are listed for the respective in vitro, ex/in vivo methods used.
In vitro data on TARP γ−8 are from Milstein et al. (2007). The Shisa9 and TARP γ−8 ex/in vivo data originate from Khodosevich et al. (2014), in which experiments were performed using WT, KO and overexpression conditions in the DG, which is regionally more appropriate for Shisa9 than overexpression in CA1 (von Engelhardt et al., 2010), and from the CA1 for TARP γ−8 KO (Rouach et al., 2005). The Shisa6 ex/in vivo data stem from WT vs. KO comparisons in CA1 pyramidal cells activated by (electrically-evoked) CA3 input (Schaffer collaterals (Klaassen et al., 2016)), similar as used for Shisa7 (bold). NA: Not applicable, not measured.
- https://doi.org/10.7554/eLife.24192.026
-
Transparent reporting form
- https://doi.org/10.7554/eLife.24192.027
