Characterization of Usp15 constitutive KO and conditional KO mice
To analyze acute effects of Usp15 knockout after status epilepticus (Fig. 1a), we used a constitutive Usp15 KO mouse line (Usp15−/−) and respective WT littermates (Usp15+/+, referred to as WT in the following). To determine a possible therapeutic effect of a Usp15 knockdown in chronic epilepsy (Fig. 4a), we used a newly generated inducible Usp15 KO mouse line which allows the conditional inactivation of Usp15 via the Cre/loxP system (Usp15fl/flCAGCreERT2+). Inducible KO mice will be referred to as Usp15fl/flCre+ before tamoxifen treatment and Usp15Δ/Δ after tamoxifen treatment, respective Cre-negative littermates as Usp15fl/fl.
To confirm the full deletion of USP15 in Usp15−/− mice, we performed Western blot analysis of the brains at the end of the in vivo experiment (Suppl. Figure 1a). To determine if the lack of USP15 triggers a compensatory reaction, we measured protein levels of the closely related DUBs USP4 and USP11. However, protein levels of both were unchanged in Usp15−/− mice compared to WT either with or without ihKA application (Suppl. Figure 1b and c).
As this is the first study reporting the Usp15fl/flCre+ / Usp15Δ/Δ mouse model, we validated the protocols to achieve efficient Usp15 induced deletion. Both Usp15fl/fl and Usp15fl/flCre+ mice were treated with tamoxifen. The weight of mice during, before and after tamoxifen treatment did not differ between genotypes (Suppl. Figure 2). In addition, upon visual inspection, behavioral parameters (feeding, grooming, social interaction) did not appear to change after tamoxifen injection. These results show that deletion of Usp15 in adult mice does not cause overall detrimental effects, which is also an important information regarding safety considerations for potential therapeutic approaches targeting USP15.
The efficiency of Usp15 depletion in Usp15Δ/Δ mice was determined in the brains by Western blot analysis (Suppl. Figure 1d, e). USP15 protein expression levels were reduced on average to 9.3 ± 5.3% in Usp15fl/flCre+ mice after tamoxifen application (i.e., Usp15Δ/Δ), indicating a highly efficient induced deletion. Again, USP4 and USP11 protein levels were unchanged after USP15 induced deletion (Suppl. Figure 1f, g).
To assess potential side effects due to the lack of USP15, we performed microscopic evaluation of tissues derived from multiple organs of both Usp15−/− and Usp15Δ/Δ in comparison with WT and Usp15fl/fl mice, respectively. There was no detectable difference in hematopoiesis between Usp15−/− mice and WT or Usp15Δ/Δ and Usp15fl/fl mice, respectively. Likewise, no pathological abnormalities were observed in the spleen or bone marrow (both from sternum and femur). In the bone marrow, the cellularity was not altered and the erythroid/myeloid ratio was maintained. None of the immune organs (mesenteric lymph node, axillary lymph node, spleen, bone marrow, Peyer’s patches, thymus and bone marrow) showed any alteration. Any minor microscopic findings recorded were either part of the background or were attributable to tamoxifen (direct effect of tamoxifen or the injection procedure; data not shown).
Usp15 −/− mice and WT littermates show comparable severity of behavioral status epilepticus
First, we tested the hypothesis whether constitutive deletion of Usp15 affects hippocampal damage and or inflammatory reaction early (4 days) after status epilepticus. At this time point the initial cell loss following KA injection is nearly complete and the inflammatory reaction has previously been shown to be strong in C57Bl/6 mice [48–50]. Usp15−/− mice and WT littermates were injected with KA or NaCl and the severity of the initial status epilepticus was monitored by video analysis. As expected, ihNaCl control mice (not video-recorded) never showed any behavioral signs resembling status epilepticus. Analysis of status epilepticus in ihKA mice using modified Racine stages (see Methods) revealed that the time course of the mean highest stages aligned to the time point of awakening was mostly overlapping for Usp15−/− and WT mice (Fig. 1b; nUsp15−/−=15, nWT=14). For statistical comparison, we calculated the area under the curve (AUC) normalized to the total observation time (AUC/h) for time courses of individual animals. This did not reveal any difference between WT and Usp15−/− mice (mean AUC/h Usp15−/−=1.84, WT = 1.69, difference = 0.154, 95% CI=[-0.200, 0.508], unpaired Student’s t-test p = 0.380). In addition, neither the time for each mouse to reach its individual highest stage (ihKA injection as t = 0 min; Fig. 1c), nor the time to reach stages 3–6, nor the incidence of these stages differed between Usp15−/− and WT (Suppl. Figure 3). Only mice that showed clear signs of status epilepticus (reaching at least stage 3) were used for further analyses. Mice were then randomly allocated to histological (nUsp15−/−, KA=6, nWT, KA=6) and transcriptome analysis (nUsp15−/−, KA=9, nWT, KA=8, nUsp15−/−, NaCl=7, nWT, NaCl=7).
(a) Schematic depicting the experimental design to induce status epilepticus in Usp15−/− and wildtype (WT) mice. (b) Mean time course of the maximum stage reached within 5 min-long windows aligned to the time point of awakening from anesthesia. WT mice (n = 14) are shown in blue, Usp15−/− mice (KO, n = 15) are shown in red, shadows in corresponding color depict 95% confidence intervals. The time courses are overlapping. (c) Maximum stage reached during status epilepticus (mean, individual values) did not differ between Usp15−/− and WT littermates.
Cell loss, granule cell layer width and acute gliosis 4d after status epilepticus did not differ in Usp15 −/− mice and WT littermates
For histological analysis, we focused on the comparison of Usp15−/− and WT mice, because ihKA-induced changes have already been thoroughly described [48, 51, 52]. We selected sections surrounding the injection site which show the strongest effects following the ihKA injection [50, 53]. Analysis of hippocampal NeuN immunostaining revealed neuronal loss in the hilus and substantial thinning of principal cell layers in CA3 and in CA1 together with a beginning dispersion of the granule cell layer in the ipsilateral hippocampus, but without any salient differences between WT and Usp15−/− mice (Fig. 2a1-a4). Indeed, neither statistical comparison of granule cell layer (GCL) width, nor relative NeuN-positive area in the pyramidal cell layer of CA3 and CA1, which represents an indirect parameter for neurodegeneration, revealed any difference between Usp15−/− and WT mice for the ipsi- or the contralateral hippocampus (unpaired Student’s t-tests assuming equal variances for GCL, CA3, CA1; Fig. 2e-g, Suppl. Table 3).
To analyze the glia activation after KA injection, we performed immunostaining for GFAP, Iba1 and CD68 to label astrocytes, resident and activated microglia, respectively. At 4d after ihKA, GFAP staining was strong throughout both hippocampi in WT and Usp15−/− mice (Fig. 2b1-4) with an accumulation of astroglia in the ipsilateral hilus and CA1 pyramidal cell layer (Fig. 2b2, b4). In the contralateral hippocampus of both genotypes, GFAP-positive cells were evenly distributed (Fig. 2b1, b3). Analysis of integrated density of fluorescence staining in both hippocampi did not reveal any significant differences (Fig. 2h, Suppl. Table 3). Iba1-positive microglia clustered at the sites of cell loss in the hilus, CA3 and CA1 pyramidal cell layer of the ipsilateral hippocampus of WT and Usp15−/− mice (Fig. 2c2, c4). In the contralateral hippocampus of both genotypes, strongest Iba1 staining was visible in dendritic layers of CA1 and in the inner molecular layer - two areas that receive commissural projections from the ipsilateral hippocampus (Fig. 2c1, c3). Quantification of integrated density of Iba1 staining in the whole hippocampus did not reveal any difference between WT and Usp15−/− mice (Fig. 2i, Suppl. Table 3). Finally, the density of CD68-positive activated microglia was strongly elevated throughout the ipsilateral hippocampus following ihKA but appeared similar in WT and Usp15−/− mice (Fig. 2d2, d4), whereas the contralateral hippocampus was mostly devoid of any CD68 staining (Fig. 2d1, d3). Again, integrated density measurement did not show any difference between WT and Usp15−/− mice (Fig. 2j; Suppl. Table 3).
Together these results indicate that despite strong alterations between the contralateral and the KA-injected side in both genotypes, USP15 deficiency does not affect the pattern of granule cell dispersion, cell loss or glia activation shortly after status epilepticus.
(a1-4) NeuN immunostaining in the ipsi- (a1) and contralateral hippocampus (a2) of wildtype (WT) and (A3, A4) of Usp15−/− (KO) mice at 4d after ihKA. Note cell loss in the hilus and CA1 (white arrows) in the ipsilateral hippocampus in both genotypes. (b1-4) GFAP immunocytochemistry in WT and Usp15−/− mice. The density of astrocytes and local distribution appears comparable in both genotypes. (c1-4) Iba1 immunostaining. Microglia accumulates at the sites of strong cell loss in WT and Usp15−/− mice. (d1-4) CD68 immunostaining for activated microglia does not reveal a salient difference between WT and Usp15−/− mice. (e) Granule cell layer (GCL) width does not differ between genotypes for the ipsi- or contralateral hippocampus, respectively. (f) CA3 NeuN density was measured in a 300*200 µm region of interest (ROI, indicated in a1) placed above the pyramidal cell layer and pixels exceeding a threshold of mean grey value + 1SD (measured in the whole hippocampus) were detected. Area > threshold / ROI area is given. WT and Usp15−/− mice do not differ in the ipsi- or contralateral hippocampus, respectively. (g) Same for CA1 but with a 400*200 µm ROI (see a1). (h) Integrated density measurement of GFAP immunostaining does not reveal differences between WT and
Usp15−/− mice (unpaired Student’s t-test after log-transformation to induce symmetry and equal variance; results were back-transformed to the original scale; geometric means are displayed). (i) Same for integrated density measurement of Iba1 immunostaining. (j) Same for integrated density measurement of CD68 immunostaining. Mean and individual values are displayed. Scalebar 100µm. GCL granule cell layer, CA cornu ammonis.
Lack of USP15 does not significantly affect the global gene expression profile in ihKA mice
By reanalyzing a previous dataset from the pilocarpine mTLE model [35], we identified a set of seven gene modules (modules 5.o, 12.o, 16.o, 18.o, 20.o, 22.o, 24.o). Remarkably, all were co-regulated in epileptic hippocampi and predicted to be regulated by the USP15-associated IFN-α/β, TGF-β and NRF2 pathways.
In the current study, the ihKA injection had a strong impact on gene expression four days after administration: 4791 and 2691 genes responded to KA (compared to NaCl) in the ipsi- and contralateral hippocampus of WT mice, respectively (false discovery rate (FDR) p ≤ 0.05). In addition, we found that the co-regulation patterns within six of the seven gene modules were conserved after ihKA-induced status epilepticus (Suppl. Table 4) and that five of these modules were significantly upregulated in response to ihKA (Fig. 3a). In conclusion, while Usp15 mRNA expression was not altered itself, gene modules expected to be under upstream USP15 control were conserved in the ihKA model. These data validate the use of the ihKA model to test our mechanistic hypothesis of inhibiting USP15 as a therapeutic approach to modulate pathways (NRF2, IFN-α/β and TGF-β) that play an important role in epilepsy.
Next, we assessed the impact of USP15 deficiency on the transcriptome. Overall, the effect of the lack of USP15 only affected expression levels of three genes which were significantly differentially expressed between WT and Usp15−/− ihNaCl mice (FDR p < 0.05): besides the downregulation of Usp15, which validated the experimental approach, we found a moderate downregulation of Rnf41 (E3 ubiquitin protein ligase) and an upregulation of Gm9770. Functional analysis (i.e., gene set enrichment analysis, see Methods) detected only mild effects of Usp15 deletion on USP15-regulated co-regulation modules. Gene modules 16.o and 18.o were significantly underexpressed in Usp15−/− compared to WT mice after ihNaCl, but with a minimal magnitude compared to the effect caused by ihKA (data not shown).
In the Usp15−/− mice 4543 genes responded in the ipsilateral and 787 in the contralateral hippocampus after ihKA compared to ihNaCl (FDR p ≤ 0.05). On both, the gene-level and for functional analysis, the same conclusions could be drawn for ihKA Usp15−/− as for WT mice Fig. 3b). Only two genes were significantly (FDR p < 0.05) differentially expressed between WT and Usp15−/− mice after ihKA injection: Usp15 was downregulated and Gm9770 was upregulated. Moreover, gene modules 16.o and 18.o were again expressed at significantly lower levels in Usp15−/− compared to WT mice, but with a minimal effect compared to the ihKA effect itself and far from any normalization of the changes brought by ihKA injection (Fig. 3c). Finally, the effect of ihKA in WT and Usp15−/− mice was highly correlated (r = 0.72 in the ipsilateral hippocampus, r = 0.73 in the contralateral hippocampus, Pearson correlation; Suppl. Figure 4). In conclusion, the effect of ihKA on the transcriptome was very similar in Usp15−/− and in WT mice, thus ruling out the hypothesis that a constitutive deletion of Usp15 could rescue transcriptomic changes caused by ihKA-induced status epilepticus.
The distribution of mean log2FC per gene is plotted. Each dot represents the mean log2FC for a gene in the module; the color of the dot indicates if the gene is significantly overexpressed (red), underexpressed (blue), or not significantly differentially expressed (grey). The following comparisons were made (nWT,NaCl=7, nUsp15−/−,NaCl=6, nWT, KA=8, nUsp15−/−, KA=8): (a) the effect of ihKA in WT mice (ihKA WT vs NaCl WT; M5.o FDR < 10− 4, M12.o FDR > 0.05, M16.o FDR < 10− 4, M18.o FDR < 10− 4, M20.o FDR < 0.05, M22.o FDR < 10− 4, M24.o FDR < 10− 4); (b) the effect of ihKA in Usp15−/− mice (ihKA Usp15−/− vs ihNaCl Usp15−/−; M5.o FDR < 10− 4, M12.o FDR > 0.05, M16.o FDR < 10− 4, M18.o FDR < 10− 4, M20.o FDR < 0.05, M22.o FDR < 10− 4, M24.o FDR < 10− 4); (c) comparison of ihKA effect in Usp15−/− and WT mice (ihKA Usp15−/− vs ihKA WT; M5.o FDR > 0.05, M12.o FDR < 0.05, M16.o FDR < 10− 2, M18.o FDR < 10− 3, M20.o FDR > 0.05, M22.o FDR < 10− 3, M24.o FDR > 0.05).
Induced deletion of Usp15 after epilepsy onset does not alter epileptic activity in the ihKA model of MTLE
The constitutive deletion of Usp15 had no effect on strength and duration of status epilepticus and downstream pathophysiological processes as it might have been compensated during development. In contrast, the induced deletion of Usp15 in chronic epilepsy resembles potential therapeutic intervention and allows monitoring direct consequences. Therefore, we next tested how an induced deletion of Usp15 after epilepsy onset would alter spontaneous recurrent seizure events, histological hallmarks and the transcriptome in a long-term experiment (Fig. 4a).
Starting from two weeks after ihKA injection, Usp15fl/flCre+ mice and Usp15fl/fl littermates underwent local field potential (LFP) recordings for five consecutive days followed by a two-day break before tamoxifen injection and two (nΔ/Δ=2, nfl/fl=2) or three weeks (nΔ/Δ=11, nfl/fl=10) of recordings after tamoxifen (Usp15fl/flCre+ mice will be termed Usp15Δ/Δ after the tamoxifen-induced deletion of Usp15). NaCl-injected control mice (nΔ/Δ=12, nfl/fl=11) underwent the same protocol but were only occasionally recorded to confirm that these mice did not show any epileptic activity in the LFP.
Upon visual inspection, there was some inter-individual variability in epileptic activity but no salient difference between the genotypes, neither before nor after tamoxifen injection (Fig. 4b, c). After automated detection of epileptic activity [42], we determined the mean EA ratio as the cumulative duration of any kind of epileptic activity per total recording time for each week (Fig. 4d). Before tamoxifen injection, EA ratio was comparable between both genotypes, which agrees with the full expression of USP15 in both groups at this time point (Fig. 4d, Suppl. Table 5). During week 1 and week 2 after tamoxifen injection there was a small (~ 4%) but significant increase in EA ratio in Usp15Δ/Δ compared to Usp15fl/fl littermates, whereas in week 3 after tamoxifen the EA ratio of both groups converged again.
Next, we compared the cumulative distribution of epileptic burst duration using binning with a 10s-wide window (occasional events longer than 90s are included in the 90s group) before and after tamoxifen injection. Individual values were fitted with logistic regression curves (Fig. 4e-h). The week before tamoxifen, regression curves were overlapping (Fig. 4e). In contrast, after tamoxifen injection, the curves for Usp15Δ/Δ mice were shifted to the right for weeks 1–3, respectively, indicating longer bursts in Usp15Δ/Δ mice with an odds ratio for a longer duration being ~ 1.35-fold greater than in Usp15fl/fl mice for each week (Fig. 4f-h). Finally, we performed classification of epileptic bursts into different degrees of severity (severe = high spike load, moderate = medium spike load, mild = low spike load [41]). We compared the average weekly number of each class in Usp15Δ/Δ and Usp15fl/fl mice in week 1, 2 and 3 after tamoxifen using the average number before tamoxifen injection as covariate (analysis of co-variance (ANCOVA; Suppl. Figure 5). This did not reveal any difference between the genotypes for severe events. For moderate and mild events, lower average numbers for Usp15Δ/Δ mice were only found during week 3 and week 2, respectively (Suppl. Figure 5).
Together, our LFP data indicate that there is a transient shift to a higher EA ratio, which is reflected in longer durations of severe events but not in their number. The numbers of moderate or mild events were partly reduced, followed by convergence in the third week after induced deletion of Usp15. The induced deletion of Usp15 did therefore not reduce the susceptibility for epileptic activity.
After the recording phase, mice were randomly distributed to a transcriptome analysis group (KA: nΔ/Δ=9, nfl/fl=8, NaCl: nΔ/Δ=9, nfl/fl=8, one KA-injected Usp15fl/fl mouse died between EEG and preparation) and a histology group (KA: nΔ/Δ=4, nfl/fl=3 WT, NaCl: nΔ/Δ=3, nfl/fl=3).
(a) Scheme depicting the experimental time line for the long-term experiment with Usp15fl/fl and Usp15fl/flCre+ mice, termed Usp15Δ/Δ after tamoxifen-induced Usp15 deletion. (b) Representative examples of 200s-long cutouts from LFP recordings in a Usp15fl/fl mouse. Recordings show epileptic bursts consisting of high amplitude population spikes and intermittent fast oscillations, which can be distinguished from low amplitude baseline. Selected traces are from the same mouse at the end of the week before tamoxifen injection (pre), and the end of weeks 1–3 (post 1–3) after tamoxifen injection and show no major changes. (c) Same as in (b) but for USP15Δ/Δ mice before (pre) and after tamoxifen-induced knockdown of Usp15. There is no salient difference between genotypes in the raw data, nor does the induced deletion of Usp15 lead to any visible changes in the raw data after tamoxifen. (d) Average weekly EA ratio, determined as the time spent in epileptic bursts divided by the total recording time for Usp15fl/fl (blue) and Usp15Δ/Δ (red; means and individual values). During week 1 and 2 after tamoxifen injection the EA ratio is significantly increased in Usp15Δ/Δ mice (*p < 0.05, **p < 0.01, analysis of covariance with pre as the covariate). (e) Cumulative frequency of duration of epileptic bursts in 10s bins (< 10s, 10-20s, etc. 80–90 s bin also contains rare events > 90s). Individual values for each mouse are displayed, curves represent a logistic regression analysis assuming proportional odds. (f, g, h) Same as (e), but for weeks 1, 2, 3 after tamoxifen, respectively. Note the convergence of regression for both genotypes at the late stage. Scalebars in B and C 2mV, 10s.
Histological analysis does not show differences between Usp15∆/∆ and USP15fl/fl mice in chronic epilepsy
To determine whether the induced deletion of Usp15 affected structural alterations after ihKA injection, we performed immunostaining for NeuN after the EEG experiment, i.e., 6 weeks after ihKA/ihNacl (3 weeks after tamoxifen) (Fig. 5a1-a6). The ipsilateral hippocampus displayed strong granule cell dispersion, whereas in the contralateral hippocampus and in NaCl-injected hippocampi the granule cell layer width appeared normal (Fig. 5a1-a6). Quantitative analysis revealed a significant increase of granule cell layer width after ihKA compared to ihNaCl injection in USP15Δ/Δ mice as well as in USP15fl/fl littermates (Fig. 5e, Suppl. Table 6), but no difference between genotypes. In the contralateral hippocampus a slight increase in granule cell layer width after ihKA injection was observed in Usp15Δ/Δ mice only (Fig. 5e, Suppl. Table 6).
Neuronal degeneration was indirectly determined by NeuN immunocytochemistry in CA3 and CA1. IhNaCl-treated hippocampi (Fig. 5a1, a2) and contralateral CA3 and CA1 regions after ihKA injection did not show any difference for genotype or treatment (Fig. 5a3, a5, f, g, Suppl. Table 6). In contrast, the ipsilateral CA1 pyramidal cell layer was nearly gone after KA injection in both genotypes and NeuN-positive cells in CA3 were reduced in density (Fig. 5a4, a6). The quantification of NeuN expression confirmed significantly reduced density in ipsilateral CA3 and CA1 in both genotypes in ihKA compared to ihNaCl mice (Fig. 5f, g, Suppl. Table 6). The comparison of genotypes, however, did not reveal any differences in CA3 or in CA1 after ihKA, but, surprisingly, NeuN density was slightly lower in Usp15Δ/Δ after ihNaCl injection (Fig. 5g, Suppl. Table 6).
GFAP expression was weak throughout the NaCl-injected hippocampus, except for some accumulation of astrocytes along the electrode tracks (Fig. 5b1, b2). After ihKA, GFAP staining in the contralateral hippocampus was slightly increased in CA1, but a stronger increase was visible in the subgranular zone (Fig. 5b3, b5), reflecting increased contralateral progenitor proliferation and neurogenesis after KA injection [53]. In contrast, GFAP staining was strongly enhanced throughout the ipsilateral hippocampus after ihKA, which was also highly significant in the quantification (Fig. 5h, Suppl. Table 6), but again, GFAP density after ihKA injection did not differ between genotypes (Fig. 5h, Suppl. Table 6). In the contralateral hippocampus a slight elevation of GFAP density after ihKA was observed in Usp15Δ/Δ mice, but all other comparisons did not reveal any differences for genotype or treatment. Iba1 immunostaining was weak in NaCl-injected hippocampi as well as in contralateral hippocampi of both, Usp15Δ/Δ and Usp15fl/fl mice (Fig. 5c1-3, c5). In contrast, the ipsilateral hippocampus of both genotypes showed increased Iba1 expression in the dentate gyrus and pronounced scarring in CA1 (Fig. 5c4, c6). Quantification of integrated density revealed high variability across mice and a significant difference occurred only between ipsilateral hippocampi of KA- and NaCl-injected Usp15fl/fl mice (Fig. 5i, Suppl. Table 6). Finally, CD68-positive activated microglia were much less prominent than shortly after status epilepticus. A weak accumulation of CD68-positive cells in CA1 of the ipsilateral hippocampus after ihKA resulted in significantly higher density in Usp15fl/fl and Usp15Δ/Δ mice but without a difference between genotypes (Fig. 5d1-6, j, Suppl. Table 6). In summary, the treatment with KA compared to NaCl injection and the presence of epileptic activity has a major effect on cell death and glia reaction in both genotypes, while the genotype alone plays a subordinate or no role.
(a1-6) Representative images of NeuN immunostaining at 6 weeks after ihKA/ihNaCl (3 weeks after tamoxifen injection) in mice that were LFP recorded. (a1, a2) NaCl-injected ipsilateral hippocampus of an Usp15fl/fl and an Usp15Δ/Δ mouse. (a3, a4) Contra- (A3) and ipsilateral hippocampus (a4) of a KA-injected Usp15fl/fl mouse. Note the loss of pyramidal cells in CA3 and CA1 and the dispersion of the granule cell layer. (a5,6) Same, but for an Usp15Δ/Δ mouse. (b1-6) GFAP immunostaining. In b2 the implantation track of an electrode is visible. After KA injection, GFAP-positive cells accumulate in the contralateral subgranular layer (b3, b5) and in the entire ipsilateral hippocampus (b4, b6), irrespective of the genotype. (c1-6) Iba1 immunostaining. Iba1-positive microglia accumulate at the sites of cell death in CA3 and CA1 (c4, c6), irrespective of the genotype. (d1-6) CD68 immunostaining. No activated microglia is visible in control mice (d1,2) nor in the contralateral hippocampus (d3,5). (d4,6) In the ipsilateral hippocampus, CD68-positive microglia accumulated mainly in CA1. (e) Quantification of granule cell layer width for the ipsi- and contralateral hippocampus (mean, individual values). (f) NeuN density in CA3, area > threshold / ROI area is given. (g) Same for CA1 indicating an almost complete loss of CA1 neurons after ihKA in both genotypes. (h) Quantification of integrated density of GFAP immunostaining in the whole hippocampus. (i) Same for Iba1 immunostaining. (j) Same for CD68 immunostaining. Two-way factorial analysis of variance was made on the log-transformed data for the different comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar 100µm.
Induced deletion of Usp15 after epilepsy onset does not affect the hippocampal global gene expression profile in the ihKA model of MTLE
We investigated gene expression profiles in the Usp15Δ/Δ mice at the end of the experiment. After ihKA injection 3600 genes were differentially expressed in the ipsilateral hippocampus of Usp15fl/fl mice compared to ihNaCl (1583 over-/2017 underexpressed), whereas only three genes were overexpressed in the contralateral hippocampus. In Usp15Δ/Δ mice a similar number of genes was differentially expressed after ihKA (1918 over-/2308 underexpressed ipsilateral; 1 overexpressed contralateral). However, when comparing genotypes, we found only three genes that were significantly differentially expressed in Usp15fl/fl and Usp15Δ/Δ ihNaCl-injected mice: As expected, Usp15 was underexpressed in Usp15Δ/Δ mice in both hippocampi. In addition, Esr1 (estrogen receptor α, part of the inducible KO system) was found to be overexpressed in both hippocampi of Usp15Δ/Δ mice, whereas Dihydropyrimidine Dehydogenase (Dpyd) was slightly downregulated in the contralateral hippocampus only. After ihKA, only Usp15 and Esr1 were significantly differentially regulated in both hippocampi. As shown above, the effect of ihKA in Usp15Δ/Δ mice was highly correlated to the effect of ihKA in Usp15fl/fl (Suppl. Figure 4b), indicating that the ihKA response is not affected by the genotype and Usp15Δ/Δ does not impact gene expression after ihKA.
Usp15 knockdown does not impact microglial functions in vitro
Previous studies in experimental cerebral malaria and autoimmune encephalomyelitis models have implicated USP15 as a regulator of type 1 IFN responses in innate immune and T cells. It remained unclear whether the reported attenuation of CNS inflammation in USP15 KO mice [23] is a consequence of direct action on microglia or attenuation of the peripheral innate and adaptive immune response in brain infiltrating cells. Furthermore, there is a lack of understanding of the role of USP15 in microglial essential functions. In parallel to the in vivo target validation studies, we fill this gap by exploring the impact of USP15 knockdown on microglial immune response and clearance functions.
An immortalized murine microglial cell line (BV2) was used for this purpose. Prior to functional assays efficient Usp15 knockdown using an Usp15 siRNA was assessed against a scrambled control siRNA. Western blot data showed > 85% decrease in protein levels at 48h post-siRNA exposure in unstimulated BV2 cells (Fig. 6a). Next, we assessed the impact of Usp15 knockdown in downstream pathway genes and cytokine release in BV2 cells challenged with 100 ng/mL lipopolysaccharide (LPS) for 24h. LPS stimulation induced significant increases in Irf7 and Oasl2 transcript levels suggesting LPS to be a relevant stimulus for the exploration of USP15 function in microglia (Fig. 6c). Interestingly, unlike reported previously for innate immune cells such as macrophages and neutrophils in in vivo models of cerebral malaria and autoimmune encephalomyelitis [23], Usp15 knockdown did not affect expression levels of its downstream nodes (Irf7 and Oasl2; Fig. 6a, c and suppl. Figure 6a). Next, we investigated the consequences of Usp15 knockdown in microglial inflammatory responses, more specifically cytokine release. LPS exposure of BV2 cells induced a significant release of pro-inflammatory cytokines (TNF-α, IL1-β, IL6, IL1), however, Usp15 knockdown did not attenuate cytokine levels in LPS-stimulated microglia (Fig. 6d). To determine the effect of the Usp15 knockdown on microglial clearance functions we measured lysosomal acidification (LysoTracker™ and LysoSensor™; Suppl. Figure 6b, c) and phagocytic uptake of pHrodoTM-conjugated zymosan particles (Fig. 6e). At 48h following Usp15 knockdown BV2 cells were exposed to LysoSensor™ dye or pHrodoTM-labeled zymosan particles for 2h. Longitudinal time lapse imaging revealed that USP15 knockdown did neither affect phagocytosis nor lysosome acidification in BV2 cells (Fig. 6e and Suppl. Figure 6b, c).