Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABA_AR density

Treatment of young APP-PS1 mice with artemisinin increases gephyrin and p35 protein levels and results in elevated gephyrin phosphorylation at Ser270

In earlier studies we detected a significant increase of gephyrin and other key inhibitory synapse proteins in the hippocampus of three-month-old APP-PS1 mice (Hollnagel et al. 2019; Kiss et al. 2016, 2020) indicating altered GABAergic inhibition already in the early phase of AD-like pathogenesis.

Based on the recently reported findings showing a direct link between gephyrin and the potentially neuroprotective artemisinins (Qiang et al. 2018) with both positive (Li et al. 2017) and negative (Kasaragod et al. 2019) modulation of GABAergic signaling in vitro, we treated APP-PS1 mice with artemisinin to test its possible modulatory effects on the inhibitory drive in the AD-brain. Six-week-old APP-PS1 mice were fed for six weeks with artemisinin-containing diet (10 and 100 mg/kg) and analyzed for the expression of gephyrin at three months of age, when in the hippocampus amyloid plaques are not yet developed (Kiss et al. 2016; Radde et al. 2006).

We first performed Western blot analysis of hippocampus homogenates prepared in parallel with tissue samples used for immunostaining and IF-microscopic analyses. The genotype of the APP-PS1 mice was demonstrated by the robust expression of hAPP (Figure 1). As shown in Figure 1, the level of gephyrin detected with a pan-gephyrin antibody was increased in the hippocampus homogenates of artemisinin-treated APP-PS1 mice in a concentration-dependent manner, eliciting significant increase with the dose of 100 mg/kg (p < 0.01) (Figure 1B). Interestingly, the signal intensity detected with the phospho-sensitive gephyrin antibody (mAb7a) was significantly elevated with 10 mg/kg artemisinin (Figure 1B, C), suggesting that the phosphorylation of gephyrin at Ser270 may be fostered predominately upon low doses of artemisinin. In earlier studies we have shown that gephyrin is phosphorylated by CDK5/p35 in vitro (Kalbouneh 2013; Kalbouneh et al. 2014; Kuhse et al. 2012) and demonstrated an increased expression of p35 in the hippocampus of young APP-PS1 mice (Kiss et al. 2020). Therefore, we subsequently analyzed the protein levels of p35. Although the quantification of the specific p35 WB bands did not delivered statistically significant differences between groups, p35 protein levels in the hippocampus homogenates of low dose artemisinin-treated mice were constantly increased in comparison to samples from untreated animals (Figure 1).

Figure 1:

Artemisinin fosters gephyrin protein level and gephyrin phosphorylation in hippocampus homogenates of three-month-old APP-PS1 mice.

(A) Representative immunoblots of hippocampus lysates obtained from three-month-old APP-PS1 mice, untreated (APP-PS1) or treated with two doses of artemisinin, 10 mg/kg (A-ARM10) or 100 mg/kg (A-ARM100) and probed with a pan anti-gephyrin (Gephyrin), the phospho-specific anti-gephyrin mAb7a (P-Gephyrin), and an anti-p35 antibody. The antibody against the human transgenic APP (hAPP) was used to verify the genotype, and a mouse anti β-actin antibody was used as a loading control. (B) Quantification of protein band intensities shown in A for gephyrin, phospho-gephyrin and p35; phospho-gephyrin normalized to gephyrin protein is also shown. Note the significant increase of gephyrin protein level with 100 mg/kg artemisinin, whereas gephyrin phosphorylation level was fostered especially in hippocampus extracts from mice treated with 10 mg/kg artemisinin; band intensities were quantified from 3–4 mice/group. Student’s t-test. Means ± SD, **p < 0.01.

Next we performed IF-staining of coronal cryosections cut from fresh-frozen brains using in addition to the phospho-specific anti-gephyrin (mAb7a) and anti-p35 antibodies also an antibody directed against the GABA_AR-γ2 subunits to prove whether a putative change in the gephyrin phosphorylation might coincide with changes of another functionally relevant inhibitory synaptic protein. The labeling with all three antibodies generated a similar distribution in all experimental groups and confirmed our earlier observations in three-month-old APP-PS1 mice (Kiss et al. 2016, 2020): a rather intense gephyrin, GABA_AR-γ2 and p35 immunoreactivity in the somata of neurons of pyramidal (CA1) and granular cells of dentate gyrus (DG) (Figure 2A, C, E). Brightly stained mostly punctate structures were also observed, probably representing the clustered postsynaptic proteins distributed across the dendritic layers of hippocampus (insets) (Figure 2A, C, E). As shown in Figure 2, the analysis of immunolabeled brain sections confirmed the increase of mAb7a and p35 immunoreactivities in the hippocampus of artemisinin-treated mice in comparison to non-treated APP-PS1 mice, and in addition revealed also an increase of GABA_AR-γ2 subunit immunoreactivity. The quantification of mean IF-intensities in CA1 and DG of the hippocampus in contrast to the Western blot analysis of whole hippocampus homogenates detected a significant increase also for p35 in these hippocampal sub-regions of artemisinin-treated APP-PS1 mice (Figure 2B, D, F). This finding in addition to the lack of clear-cut differences between low and high dose induced changes in the sub-regional level of these proteins suggested possible dose- and region-dependent differences in their regulation by artemisinins. However, the detected increase in the IF-intensity of all three proteins was evidently more consistent at the lower dose of 10 mg/kg than at 100 mg/kg, thus showing a consensus with the Western blot data (Figure 1).

Figure 2:

Gephyrin phosphorylation at Ser270, as well as GABA_AR-γ2 and p35 immunreactivity is increased in hippocampal sub-regions of three-month-old APP-PS1 mice after artemisinin-treatment.

Representative IF images demonstrating increased intensity of mAb7a (A), GABA_AR-γ2 (C) and p35 (E) immunoreactivities within the CA1 and dentate gyrus (DG) of the hippocampus of three-month-old APP-PS1 mice treated with two doses of artemisinin, 10 mg/kg (A-ARM10) and 100 mg/kg (A-ARM100). Insets show positive labeled clusters within the stratum radiatum (SR) of the CA1 region. Note the more intense somatic labeling of all three proteins in the neurons of the pyramidal cell layer (PCL) of CA1 and granular cell layer (GCL) of the DG in comparison to probably predominant dendritic segments in SR for all experimental groups. Confocal maximum intensity projection images (four optical sections, 2 μm thick Z-stack). Scale bars: 100 μm; insets: 10 μm. (B) Quantification of mean IF-intensities (see Methods) for mAb7a (D) GABA_AR-γ2 and (F) p35 measured in randomly selected regions of interest in the PLC and SR of CA1 and the GCL of DG from Z-stacks of eight optical section images (one optical section: 500 nm) confirming the significantly higher fluorescence intensity levels in hippocampal sections of APP-PS1 mice after treatment with artemisinin. n = 3–6 animals/group; Means ± SEM; *p < 0.05, **p < 0.01; Student’s t-test.

These results indicate that artemisinin especially at low dose exerts an obvious effect on p35 expression and possibly a subsequent increase of gephyrin phosphorylation at the CDK5-dependent phosphorylation site Ser270 in the hippocampus of young APP-PS1 mice with potential consequences on GABA_A receptor and inhibitory synapse remodeling. Thus, we decided to study the relevance of this CDK-dependent phosphorylation site of gephyrin as a potential player in mediating effects of artemisinin on inhibitory synapses.

Myc-tagged gephyrin is particularly suitable to study the role of CDK5-dependent phosphorylation sites in gephyrin and GABA_A receptor clustering

To investigate, whether the phosphorylation of gephyrin at Ser270 detected by mAb7a might be functionally involved in gephyrin and GABA_A receptor clustering, a lentivirus sh-RNA knockdown approach was established to reduce effectively the endogenous gephyrin expression in cultured hippocampal neurons. We tested two different gephyrin-knockdown shRNA sequences targeting the gephyrin mRNA 3′UTR sequence. The suitability of shRNA1 for this purpose is demonstrated in Supplementary Figure 1. Control hippocampal neurons displayed a high number of somatic and dendritic gephyrin clusters at DIV14, while after infection with shRNA1 expressing lentiviruses (pFSGW-Geph-Kd1) at DIV6, the number, size, and IF-intensities of mAb7a positive immunoreactive puncta was reduced significantly (e.g., the cluster number from 18.68 ± 0.6 to 1.56 ± 0.63) (mean ± standard error of the mean [SEM]) (p < 0.001) (Supplementary Figure 1A, B), attesting that infection of hippocampal neurons with viruses expressing shRNA1 has an efficient “knockdown” effect on gephyrin cluster formation and is suitable for the analysis of the phenotype by gephyrin mutated at specific phosphorylation sites. For this purpose, we used a mEos2-gephyrin fusion construct (Specht et al. 2013), a mutated mEos2-gephyrin^S270A or another N-terminally myc-tagged gephyrin cloned into pFSGW-Geph-Kd1 (see Materials and Methods).

The fluorescence of recombinant mEos2-gephyrin protein in addition to enlarged dendritic puncta revealed larger somatic perinuclear mEos2-gephyrin aggregates in most of the cells (Supplementary Figure 1). Number, size and intensity of clustered mEos2-gephyrin^S270A fluorescence in proximal dendrites of 20 infected neurons from four independent cultures were slightly increased without significant differences (Supplementary Figure 1C–F). The observation of perinuclear mEos2-gephyrin aggregates indicated that the large N-terminal Eos2-fusion protein might interfere with the assembly process of gephyrin that is governed by the trimerization of the N-terminal gephyrin G-domain (Saiyed et al. 2007) possibly hindering an efficient analysis of the phenotype of gephyrin mutants, an observation that might be relevant as a large number of publication used N-terminal GFP-gephyrin fusion proteins for functional analysis in vitro and in vivo.

Therefore, we used for further analysis myc-tagged gephyrin which displayed staining patterns very similar to that of the endogenously expressed gephyrin of control cells (Supplementary Figure 1G, H) and revealed an efficient expression in gephyrin-knockdown cells.

Expression of recombinant myc-gephyrin harboring alanine substitutions at two putative CDK5-dependent phosphorylation sites reduces the synaptic density of gephyrin and GABA_AR-γ2 subunits

Next we tested the functional impact of the phosphorylation at Ser270 by introduction of an alanine codon at this position within the myc-gephyrin reading frame. We analyzed the mutant myc-Gephyrin^S270A expressed in cultured hippocampal neurons by IF microscopy after triple staining using anti-myc, anti-GABA_AR-γ2 and anti-VIAAT antibodies, the latter detecting the presynaptic vesicular inhibitory amino acid transporters. In addition, we included in our analysis another gephyrin mutant with an alanine substitution at Ser200 (myc-Gephyrin^S200A), which is localized within another CDK5-consensus sequence (SPHK) and was shown in earlier studies to be phosphorylated by CDK5 in vitro (Kalbouneh 2013) as well as a gephyrin mutant carrying both substitutions (myc-Gephyrin^S200A,S270A). The number, size and average fluorescence intensity of clustered myc-gephyrin and GABA_AR-γ2-subunits and VIAAT were analyzed. Interestingly, the number and size of gephyrin puncta were not significantly different comparing wild-type and the three different mutant myc-gephyrin proteins (Supplementary Figure 2). Importantly, when measuring the average IF-intensities at the overlapping colocalization areas of the three proteins a significant reduction of gephyrin and GABA_AR-γ2 immunoreactivity was detected with the two single mutants analyzed, whereas no synergistic effect of the double mutation (myc-gephyrin^S200A,S270A) on these parameters could be observed (Figure 3). The VIAAT-IF-intensity in this overlay area was less affected by the gephyrin mutations (Figure 3D), although a tendency to reduced mean values was also observed. Thus, our results suggest that phosphorylation of gephyrin at both CDK5-dependent sites may contribute to the density of gephyrin within the postsynaptic scaffold lattice determining directly or indirectly the density of γ2-subunit harboring GABA_A receptors.

Figure 3:

Alanine substitutions at gephyrin AA S200 or S270 reduce density of postsynaptic immunoreactivities of myc-gephyrin and GABA_AR-γ2 subunit clusters in hippocampal neurons.

(A) Representative IF-microscopy images reveal a large degree of overlapping immunoreactivities specific for transiently expressed recombinant WT myc-gephyrin (left panel) (green), GABA_A receptor γ2 (red) and VIAAT (magenta), or gephyrin mutant myc-gephyrin^S270A (right panel). Scale bar: 20 μm. (B) Quantification of area (a) and IF-intensities of gephyrin (b), GABA_AR-γ2 (c) VIAAT (d) at postsynaptic sites identified as overlapping signals colored in green, red and magenta. Results for gephyrin^Ser270A (green), gephyrin^S200A (blue) and gephyrin^Ser200AS270A (red) compared to wild-type gephyrin (white). Cluster immunoreactivities were quantified at proximal dendritic segments (25 μm length) of 25 cells derived from four independent cultures. Scale bar: 20 μm. The median of values is shown with box-plots with 10th and 90th percentile. One-way ANOVA with Tukey’s post-hoc test. *p < 0.05; **p < 0.001; ***p < 0.0001.

Activity-dependent changes of GABA_AR-γ2 subunits are affected by alanine substitution-mutations of putative CDK5 phosphorylation sites

Next we asked whether the phosphorylation of gephyrin at Ser200 and Ser270 might be important for activity-dependent inhibitory synapse remodeling. For this scope, we used gabazine to block the activity of GABA_A receptors since this dumping of tonic inhibition allows synchronous action potential bursting which is mediated by glutamatergic synaptic transmission (Arnold et al. 2005).

First, we analyzed whether the lentiviral infection causing down-regulation of endogenous gephyrin and its replacement by recombinant myc-gephyrin protein allows the same burst-activation effect of gabazine as in control cultures. We recorded the oscillating burst activity by measuring the changes of fluorescence of the Ca⁺⁺-sensitive protein hSyn:GCaMP6f.NLS.HA, that was co-expressed with the recombinant gephyrin. The quantification of the frequency of nuclear calcium transients (see Material and Methods) revealed that in cell cultures expressing myc-gephyrin, oscillations were efficiently induced with 5 μM gabazine bath application, very similar to the control cultures (Supplementary Figure 3A). More interestingly, in cells expressing myc-gephyrin^S270A, maximal frequency of nuclear calcium transients was induced already with 10 times lower gabazine concentrations (0.5 μM) than in cells expressing wild-type myc-gephyrin, suggesting a decreased inhibitory drive by myc-gephyrin^S270A. Thus, these experiments supported the hypothesis that gephyrin Ser270 is functionally important for GABAergic transmission.

For further analysis, we fixed cells after a bath perfusion of 5 μM gabazine for 5 h, then triple stained them by using anti-myc, anti-GABA_AR-γ2 and anti-VIAAT antibodies and analyzed the average fluorescence intensities at the colocalized and overlapping synaptic areas by confocal fluorescence microscopy. As shown in Figure 4, the inhibition of GABA_A receptor activity with gabazine decreased the density of postsynaptic gephyrin and GABA_AR-γ2 subunits at synapses in cells harboring wild-type gephyrin. However, in neurons expressing gephyrin that lost one of the putative CDK5 phosphorylation sites, the activity-dependent change of synaptic protein density was abolished (see Figure 4C). Surprisingly, the density of synaptic GABA_AR-γ2 subunits in cells expressing the double mutant myc-gephyrin^S200A,S270A was increased upon inhibition of GABA_A receptor activity with gabazine. Thus, the level of phosphorylation on both sites of gephyrin might be important to determine synergistically the receptor density at postsynaptic membrane specializations dependent on the synaptic activity.

Figure 4:

Decreased density of postsynaptic myc-gephyrin and GABA_AR-γ2 subunit cluster-immunoreactivities in hippocampal neurons upon receptor inhibition with 5 μM gabazine.

(A) Typical pattern of immunoreactivities for transiently expressed recombinant myc-gephyrin (green), GABA_AR-γ2 subunit (red), and VIAAT (magenta), and (B) mutated recombinant gephyrin (S200A, S270A) which is also co-localized with GABA_AR-γ2 clusters in hippocampal neurons under steady-state (left side) and gabazine inhibition (GZ) (right side) conditions. Scale bar: 5 μm. (C) The areas of overlaps (AOLs) did not change significantly between gephyrin wild-type and mutants neither under steady-state nor gabazine inhibition conditions (a). Expression of wild-type gephyrin (gray) revealed reduction of average gephyrin (b) and GABA_AR-γ2 (c) IF-intensities in AOLs upon receptor inhibition with gabazine for 5 h (GZ), whereas expression of gephyrin with alanine substitutions at one CDK site (Ser270, green) or (Ser200, blue) prohibited these gabazine induced changes. However, with both sites mutated (gephyrin^Ser200AS270A, red) an increase of average IF-intensity for GABA_AR-γ2 was observed in AOLs upon gabazine inhibition. Average IF-intensity in AOLs for VIAAT (d) did not change significantly comparing steady state and gabazine (GZ) inhibition (5 h) conditions for either WT nor gephyrin mutants. Cluster immunoreactivities were estimated for proximal dendritic segments of 20 μm length from 25–40 cells derived from three independent cultures. The median of values is shown with box-plots with 10th and 90th percentile. Students’ t-test. *p < 0.05.

Construction of expression plasmid

For the expression of gephyrin mutants two different basic constructs were used. In a first set of experiments, a construct harboring the coding sequence for an N-terminal fusion to the switchable fluorescent protein mEos2 was used, basically as described (Specht et al. 2013). A human synapsin promoter (S) in pFSGW was used to drive the expression of the described gephyrin-constructs (Körber et al. 2012).

After assuring that the large N-terminal fusion domain interfered with gephyrin clustering we inserted in the connecting linker region between Eos2 and gephyrin reading frames the coding sequence for the viral peptide P2 (Körber et al. 2012), allowing a cleavage of the N-terminal EOS protein. The design of the inserted sequence encoded also a short myc-epitope peptide allowing a specific detection of the recombinant gephyrin by anti-myc antibodies.

Construction of the gephyrin-knock-down vector

The sequences coding for shRNAs directed toward the 3′UTR of gephyrin were cloned into pFSGW using the human synapsin promoter to drive EGFP expression as described (Körber et al. 2012).

The gephyrin-shRNA1-Geph-kd-2467-sense target sequence was as follows: ttGTCAACATCTTGAACTATATTgtgaagccacagatgAATATAGTTCAAGATGTTGACttttt and the shRNA2-Geph-kd-2546-sense target sequence was: tttGACAACTCTATTCTGGTTATAgtgaagccacagatgTATAACCAGAATAGAGTTGTCtttt. These sequences were first cloned into the BstBI and BbsI restriction sites of the plasmid pCMV-U6 delBbsI, allowing the subsequent recloning of NheI-BstBI restriction fragments containing the described shRNA sequences under the control of the U6 promotor into the NheI and BstBI sites of pFSGW.

Site-directed mutagenesis

Site-directed mutagenesis of Eos2-gephyrin-P1 clone or myc-gephyrin-coding sequence was performed using the QuickChange Lightning mutagenesis kit from Stratagene (Stratagene, La Jolla, USA) following the supplier’s instructions. Mutants were verified by sequence analysis.

Lentivirus preparation

Recombinant lentiviral particles were produced as described previously (Lois et al. 2002). In brief, HEK293LTV cells (Cell Biolabs, San Diego, USA) were transfected with equimolar amounts of pFSGW, pdelta8.9 and pVSVg using polyethyleneimine (Sigma-Aldrich, St. Louis, USA). Virus particles were harvested two days after transfection by collecting the cell culture supernatant and concentrated by ultracentrifugation (90 min at 75,000 g in a SW32Ti rotor, Beckman Coulter, Brea, USA). Virus-containing solution was aliquoted, shock frozen in liquid nitrogen and stored at – 80 °C for further use.

Cell culture

Primary cultures of rat hippocampal neurons were prepared from E19 embryos plated at a density of 40,000 cells/cm² and infected with lentivirus diluted in medium into 24-well plates (Kiss et al. 2020). Infection with lentivirus dilutions was performed two days after plating (days in vitro/DIV2) with lentiviruses expressing either Eos2-gephyrin or rat myc-gephyrin wild-type, or gephyrin mutants with serine residues replaced by alanine by site-directed mutagenesis. Cells were harvested for immunoblotting or fixed for immunocytochemistry at DIV14-15. For details on hippocampal cell cultures and viral infection for Ca⁺⁺-imaging see the online Supplementary Material.

Animals and tissue preparation

The double transgenic mice on a C57BL/6J genetic background (APP-PS1) used in these experiments co-express the KM670/671NL “Swedish” mutated APP and the L166P-mutation carrying human PS1 under the control of a neuron-specific Thy1 promoter element. Based on the elevated production of human Aβ peptide, the APP-PS1 mouse model mimics aspects of cerebral amyloidosis with amyloid plaque deposition in the hippocampus starting at 4–5 months of age (Radde et al. 2006). Cognitive impairment including deficits in the Morris Water maze test and impairments of LTP in the hippocampal sub-region CA1 were reported starting at 7–8 months of age (Gengler et al. 2010).

APP-PS1 mice were obtained from Prof. Dr. M. Jucker (University of Tübingen, Germany) and bred in the animal unit of the University of Kaiserslautern, Germany. All animal procedures were carried out in accordance with the European Communities Council Directive (86/609/EEC) and approved by the responsible regional state authorities (T-65/15 G-72/17 and 23 177-07/G 18-2-041).

Six weeks old male APP-PS1 mice were randomly assigned to three experimental groups: APP-PS1 (A) mice on chow diet, and two artemisinin-treatment groups, artemisinin being mixed in the chow rodent diet at a dose of 10 and 100 mg/kg, respectively (A-ARM10 and A-ARM100) for further six weeks. The doses for artemisinin were chosen after apprehensive literature research (Ho et al. 2014). At three months of age mice (4–6 animals/group) were deeply anesthetized with isoflurane, brains were dissected and hemispheres separated. For immunohistochemistry, one hemisphere of each brain was mounted in OCT embedding compound (VWR Chemicals, Leuven, Belgium) and snap-frozen in an absolute ethanol-dry ice mixture. For biochemical analysis, fresh hippocampus was dissected from the brain hemispheres and immediately frozen in liquid nitrogen. Tissue samples were stored at −80 °C until use (Kiss et al. 2016).

Protein extracts and immunoblot analysis

Protocols were previously described (Kiss et al. 2016, 2020). Primary antibodies used are indicated in Table 1. After exposure to hyper-films (Amersham Bioscience, Amersham, Buckinghamshire, United Kingdom), pixel intensities of the bands of interest were analyzed using ImageJ. Band intensities were averaged from three to five animals (hippocampal tissue/group).

Immunolabeling

For brain sections: The protocol was described in details previously (Kiss et al. 2016). Briefly, coronal cryostat sections (8 μm) cut from fresh-frozen brain hemispheres were fixed with 4% (w/v) PFA (ROTI^®Histofix 4%) (Carl Roth GmbH, Karlsruhe, Germany) for 8 min and incubated overnight at 4 °C with primary antibodies diluted in blocking solution without Triton X-100 (Table 1). Primary antibodies were detected with corresponding secondary antibodies conjugated to fluorophores (Vector Laboratories, Invitrogen, Jackson Immunoresearch Laboratories). Controls omitting the primary antibodies were included. Serial sections from untreated and treated APP-PS1 mice were labeled simultaneously using the same batches of solutions to avoid differences in immunolabeling conditions.

For cultured hippocampal neurons: The protocol was previously described previously (Kiss et al. 2020). Briefly, coverslips containing cultured neurons at DIV15 were washed once with PBS and subsequently fixed with 4% (w/v) PFA for 10 min at RT. After blocking with 5% horse-serum and 1% BSA solution containing 0.1% Triton X-100 (Roth) for 30 min cells were incubated with up to three primary antibodies (Table 1) overnight at 4 °C followed on the next day by incubation with appropriate secondary antibodies conjugated to fluorophores (Vector Laboratories, Invitrogen, Jackson Immunoresearch Laboratories, West Grove, USA) for 30 min at room temperature.

Confocal laser scanning microscopy and quantitative IF-analysis

Images were captured with a Leica TCS SP8 microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) using an HC PL APO CS2 63.0 × 1.40 oil objective or a 63×/1.46 oil objective.

For brain sections: The protocol was described in detail previously (Kiss et al. 2016, 2020). The images were acquired in sequential mode with a frame average of four stacks of eight optical sections (1024 × 1024 pixels) spaced by 500 nm. Laser power and settings were identical for all samples in an experiment. Three randomly chosen fields within CA1 and DG of each hippocampus (n = 3–6 brains/group) were recorded for quantitative analysis. In each field, rectangular areas of 500 × 150 μm within the pyramidal cell layer (PCL) and stratum radiatum (SR) of CA1 and the granular cell layer (GCL) of DG were randomly selected and quantified for mean fluorescence intensities using NIH’s Fiji (pacific.mpi-cbg.de/wiki/index.php/fiji). Mean fluorescence intensity of a region of interest was calculated from maximal intensity projection of eight optical sections. Mean values calculated for each animal were used for final statistics. Mean values of untreated APP-PS1 mice were set to one, and data are given as mean ± SEM.

For hippocampal neurons: The images were acquired using a Zeiss Axio Observer fluorescence microscope (63×/1.46 oil objective) with two-fold electronic zoom in sequential mode with a frame average of two stacks of five optical sections. Laser power and settings were identical for all samples and experiments for each channel. Neurons of similar size and morphology were chosen using the green channel (myc-gephyrin) without knowledge of fluorescence intensities of the red channel (GABA_AR-γ2) and images of proximal dendritic areas (n = 5–10 neurons/experiment) were recorded and maximum intensity projections were used for quantitative analysis. To analyze fluorescence intensities at overlapping sites for myc-gephyrin, GABA_AR-γ2 and VIAAT in the triple-labeled hippocampal neurons an ImageJ/Fiji macro was developed (CellNetworks Math-Clinic Core Facility, University of Heidelberg, Germany) as described (Hollnagel et al. 2019). This first semi-automatically segmented the immunofluorescent puncta using the threshold method and then automatically processed the generated binary masks to find overlapping signals between the three different confocal channels. The number and area of overlapping puncta and corresponding fluorescence intensities for each channel were then counted by the ImageJ/Fiji macro and a customized summary table was created in the output directory for each processed image for further validation and statistical analysis. Mean values calculated for each neuron were used for statistical analysis (25 neurons/group from four independent cultures and 25–40/group from three independent cultures). In addition, single channel images were also analyzed separately with ImageJ/Fiji using the same threshold settings determined manually for all images of one experiment (wt compared to mutants).

Statistical analysis

Statistical evaluation was done in Prism (GraphPad Software Inc). Statistical significance of IF and immunoblot data were determined using unpaired Student’s t-test: *p < 0.05, **p < 0.01 and ***p < 0.001 or ordinary one-way ANOVA with post-hoc test (Tukey’s or Bonferroni multiple comparison-test). Numeric values are given as mean ± SEM or standard deviation (SD) or as medians with box-plots as specified.