Self-inflicted facial lesions of SPRED2 KO mice are indicative of obsessive-compulsive grooming

Starting at ~4 months of age, SPRED2 KO mice developed apparent skin lesions on head, neck, and snout regions. These lesions occurred uni- and bilaterally and progressed to ulcerations with hemorrhage over time (Figure 1a). The penetrance of this phenotype increased with age, and 80% of the KOs were affected at the age of 12 months. We did not detect any lesions in WT littermates, even when they were housed in the same cage with SPRED2 KO mice from birth. This indicated that the lesions in KO mice were not a result of aggressive encounters between cage mates. However, SPRED2 KO mice were often seen engaged in self-grooming regardless of whether they were housed alone or with littermates (Supplementary Video). Thus, we hypothesized that the phenotype of SPRED2 KOs could be the result of excessive and injurious self-grooming, indicating an OCD-like behavior caused by SPRED2 deficiency in brain regions relevant for the onset of OCD.

In SPRED2 KO mice, the Spred2 gene was disrupted by insertion of a gene trap vector between exons 4 and 5 of Spred2 (Figure 1b). The gene trap vector comprised a β-geo reporter gene, which is expressed under control of the endogenous Spred2 promoter. Therefore, in vivo monitoring of Spred2 expression by X-Gal staining is possible. Indicated by the blue color after X-Gal staining of coronal brain sections from heterozygous mice, SPRED2 is expressed in various regions of the brain, including cortex and hippocampus. High promoter activity was especially detected in amygdala and striatum, both brain regions associated with the development of OCD-like behaviors (Figure 1c). Western blot analysis using amygdala lysates from SPRED2 KO mice and WT controls demonstrated SPRED2 expression in WT amygdala but the complete loss of full-length SPRED2 protein in KO amygdala. The deficiency of functional SPRED2 was not compensated by increased expression of homologous SPRED1, demonstrated by unaltered SPRED1 expression after normalization to GAPDH (Figure 1d).

Obsessive-compulsive grooming is associated with changes in anxiety-like behavior in SPRED2 KO mice

Excessive grooming or other OCD-related conditions are often associated with additional behavioral phenotypes in mice. To assess anxiety-like behavior in SPRED2 KO mice, we performed OF, EPM and LDB tests. We used male mice aged 7–10 months, displaying excessive grooming and facial lesions. In the OF an elevated number of grooming events was recorded in SPRED2 KO mice (Figure 2a), supporting our hypothesis of an obsessive-compulsive behavior as cause of the self-inflicted skin lesions. The total distance traveled was reduced in SPRED2 KO mice, indicating diminished locomotor and exploratory activity as a consequence of obsessive grooming (Figure 2a). In comparison to WTs, SPRED2 KO mice tended to spend more time in the center of the OF (Supplementary Table 1), which was indicative of less anxiety. In the EPM, SPRED2 KOs in fact spent a longer time span in the open arms (Figure 2b), traveled a longer distance in, and payed more visits to the open area as compared with control mice; the parameters in the guarded area were accordingly decreased (Supplementary Table 1). Normally, mice avoid exploration of the potentially dangerous open arms, which pointed to reduced anxiety in SPRED2 KO mice. Similar to the OF test, the total distance traveled during the EPM test was decreased (Figure 2b), again suggesting a basically impaired locomotion. Species-conform behaviors, for example, rearing and digging were also generally reduced in favor of compulsive grooming (Supplementary Table 1 and Supplementary Figure 1). In the LDB test, SPRED2 KO mice again preferred the stressful environment and spent more time in the brightly lit chamber. The latency to cross to the save and dark compartment was prolonged, again indicating a less anxious phenotype in SPRED2 KOs (Figure 2c). Like in the OF test, the number of grooming bouts was higher in SPRED2 KO mice compared with WTs (Supplementary Table 1). We also examined the anxiety behavior in a group of younger male mice aged 2–4 months, which did not show the grooming phenotype or skin lesions. However, we could not detect any behavioral changes of these younger SPRED2 KO mice in the above described tests compared with WT controls (Supplementary Table 2). Therefore, we conclude that reduced anxiety of SPRED2 KO mice correlates with the occurrence of obsessive-compulsive grooming.

Figure 2 Obsessive-compulsive grooming is accompanied by altered anxiety-like behavior and can be alleviated by fluoxetine in SPRED2 KOs. (a) In the open field, the number of grooming bouts was elevated in SPRED2 KO mice (n=9), whereas the total distance traveled by SPRED KOs was reduced compared with WT controls (n=7). (b) In the elevated plus maze, SPRED2 KOs (n=9) spent more time in the open arms and the distance traveled was again reduced in comparison to WT littermates (n=7). (c) In the light/dark box, SPRED2 KO mice (n=9) spent more time in the brightly lit chamber than the WT controls (n=7) and took longer to cross from the lit to the dark compartment. (d) Compared with WTs (n=5), SPRED2 KO mice (n=7) showed neither differences in withdrawal thresholds of the hindpaw upon mechanical stimulation with von Frey filaments nor in hindpaw withdrawal latencies upon thermal stimulation with radiant heat. (e) Photo documentations of mice within the placebo group revealed an unaltered or even worsened state of facial lesions in SPRED2 KO mice (n=7) but no occurence of wounds in WT controls (n=6) after 2 weeks. (f) Photo documentations of mice treated with fluoxetine for 2 weeks demonstrated a clear recovery of occurrence and severity of self-inflicted lesions due to reduced hemorrhages and ulcerations in SPRED2 KOs (n=11) but no visible effects of treatment in WT mice (n=7). (g-h) Videotaping of single mice before and after 2 weeks of fluoxetine treatment revealed an increased duration of grooming events in SPRED2 KO mice (n=11) as compared with WT (n=7) at baseline. (g) Duration of grooming events was not affected by placebo treatment in WT mice but seemed slightly increased in SPRED2 KOs (P=0.073) after 2 weeks. (h) Fluoxetine treatment had no effect on grooming time in WT mice but decreased it in SPRED2 KOs after 2 weeks of treatment. Data are mean±s.e.m; *P<0.05, **P<0.01, ***P<0.001. KO, knockout; n.s., not significant; WT, wildtype. PowerPoint slide Full size image

SPRED2 KO mice did not stop grooming even when they already had apparent lesions. Therefore, we investigated the sensitivity to thermal and mechanical stimuli in SPRED2 KOs and WT controls. We used 7–10 months old mice without apparent lesions and only showing first signs of OCD-like behavior. SPRED2 KOs displayed similar heat and mechanical paw withdrawal latencies and thresholds compared with WT littermates, suggesting intact skin sensitivity (Figure 2d). Since nociceptive testing is normally conducted in younger mice and not well suited for mice permanently engaged in self-grooming, assays were also performed with mice aged 2-3 months. Again, we observed no differences in nociception between young WT and SPRED2 KOs (Supplementary Table 2).

Fluoxetine treatment reduces obsessive-compulsive grooming in SPRED2 KO mice

We next evaluated whether drugs used to treat OCD in humans would be effective in reducing the abnormal grooming in SPRED2 KO mice. Because SSRIs are a first-line treatment for OCD, we treated SPRED2 KO mice with apparent OCD-like phenotype and WT controls aged 7–10 months either with fluoxetine or placebo for 2 weeks. We monitored treatment effects on behavior and the occurrence of skin lesions by photo and video documentations. Photo monitoring revealed no occurrence of lesions in placebo-treated WT mice. In contrast, in placebo-treated SPRED2 KO mice facial lesions resulting from overgrooming remained or ulcerations and hemorrhages even worsened (Figure 2e). In the fluoxetine group, no treatment effects were visible in WT mice, while in SPRED2 KOs the occurrence and severity of self-inflicted lesions were diminished (Figure 2f).

These findings were confirmed by video recordings of single mice at baseline and after 2 weeks of placebo vs fluoxetine treatment. Here, ANOVA revealed a significant time × treatment × genotype interaction (P=0.008) and a highly significant main effect of genotype (P<0.001) for the time mice were engaged in grooming. Overall, SPRED2 KO mice did not only display a higher number of grooming events (Figure 2a) but also a longer grooming time compared with WT mice. Neither placebo nor fluoxetine treatment affected the duration of grooming events in WT mice (Figure 2g and h). In placebo-treated KO mice, however, grooming time seemed slightly increased after 2 weeks compared with baseline (Figure 2g), which is in line with aggravation of facial lesions over time. Interestingly, fluoxetine-treated SPRED2 KO mice spent less time grooming after 2 weeks of treatment compared with baseline (Figure 2h). This confirms that grooming can be interpreted as an OCD-like behavior in SPRED2 KOs, which can be treated with fluoxetine.

Digging is a species-typical behavior in mice but responsive to SSRI treatment.44 Therefore, it can also be regarded as OCD-like and is commonly used as control parameter for fluoxetine effects. Digging was reduced by fluoxetine treatment in addition to excessive grooming, confirming the efficiency of fluoxetine treatment and the OCD-like nature of the self-grooming behavior in SPRED2 KOs (Supplementary Figure 1).

Changed synaptic transmission at cortico-striatal and thalamo-amygdala synapses in SPRED2 KO mice

The pathogenesis of OCD is associated with dysregulation in CSTC circuits. SPRED2 is highly expressed in cortex, striatum, and thalamus but also in other parts of the central nervous system. To detect possible defects in striatal neurotransmission caused by SPRED2 deficiency, we performed whole cell patch clamp measurements on acute brain slices of SPRED2 KO mice with apparent OCD phenotype and WT controls aged 6–8 months. The stimulation electrode was placed in fibers of the corpus callosum and the recording electrode in single neurons of the putamen, which is part of the striatum (Figure 3a). We stimulated putamen-innervating corpus callosum fibers with two consecutive pulses of 1 mA and with an interstimulus interval of 50 ms. This presynaptic fiber stimulation elicited two elevated excitatory postsynaptic currents (EPSCs) in the putamen of SPRED2 KO mice, indicating an increased synaptic transmission at cortico-striatal synapses compared with WT controls (Figure 3b and d). According to that, the stimulation threshold tended to be reduced in putamen neurons of SPRED2 KOs (Figure 3c). Since both EPSC1 and EPSC2 were elevated to a similar extent in SPRED2 KO mice (Figure 3d), the paired pulse ratio in KOs was comparable to WTs (Figure 3e), indicating no relevant changes in the presynaptic vesicle release probability.

The effects seen in cortico-striatal neurotransmission, however, were even more pronounced in thalamo-amygdala circuits. Accordingly, we performed another set of whole cell patch clamp measurements by placing the stimulation electrode in thalamic fibers and the recording electrode in single neurons of the lateral amygdala (Figure 3f). Stimulation of thalamic afferents with two consecutive pulses of 1 mA and with an interstimulus interval of 50 ms elicited an elevated EPSC1 in lateral amygdala neurons of SPRED2 KO mice (Figure 3g and i). This indicates an increased transmission also at thalamo-amygdala synapses and is in line with the reduced stimulation threshold in lateral amygdala neurons of SPRED2 KOs (Figure 3h). The provoked EPSC2 was comparable in WTs and SPRED2 KOs (Figure 3g and i). This leads to a reduced paired pulse ratio in SPRED2 KO mice and reflects changes in the presynaptic vesicle release probability at these amygdaloid synapses (Figure 3j). Therefore, we additionally measured response parameters of spontaneously released vesicles by recording mEPSCs (Figure 3k) in lateral amygdala neurons. The frequency of mEPSCs recorded within 1 min was reduced in SPRED2 KO mice, which again indicates presynaptic alterations in vesicle release probability (Figure 3l). mEPSC magnitudes were also decreased in SPRED2 KOs, which might either be caused by changes in postsynaptic sensitivity or in vesicle transmitter load (Figure 3m).

Altered morphology in lateral amygdala neurons is accompanied by dysregulated transcription and expression of synaptic genes

Changes in synaptic input are correlated with morphological alterations in the respective neurons. Hence, we reconstructed pyramidal neurons from the lateral amygdala of SPRED2 KO mice showing apparent OCD-like behavior and of WT controls aged 9–12 months. We detected a higher total spine number on dendritic branches of branch orders 1–4 in SPRED2 KOs (Figure 4a). Spine density per 10 μm of dendritic length was also elevated across these branch orders compared with WTs (Figure 4b).

Figure 4 Differences in lateral amygdala neuron morphology are associated with dysregulated gene transcription and protein expression. (a) Morphological analysis of reconstructed neurons from lateral amygdala of WT (n=26) and SPRED2 KO mice (n=22) revealed a higher total spine number on dendritic branches of branch orders 1–4. (b) Spine density per 10 μm of dendritic length was also higher within these branch orders. (c) Exemplary Western blot analyses showed changed expression levels of different pre- and postsynaptic proteins in the amygdala of SPRED2 KO mice in comparison to WT controls. Protein levels of PSD95, mGluR5, mGluR2, and ERC1 were increased, those of bassoon were decreased. Protein expression of Rab3A, Rab6, α-tubulin, β-tubulin and GAPDH was unaltered; GAPDH was used as loading control. (d) Quantified signals of investigated synaptic proteins after normalization to GAPDH confirmed changed expression levels in the amygdala of SPRED2 KOs (n=11) compared with WTs (n=11). (e) Quantitative RT-PCRs with mRNA isolated from amygdala of SPRED2 KO mice (n=11) and WT controls (n=11) using gene-specific primers confirmed the increased expression of PSD95, mGluR5, mGluR2, and ERC1 on mRNA level. Data are mean±s.e.m; *P<0.05, **P<0.01, ***P<0.001. KO, knockout; n.s., not significant; WT, wildtype. PowerPoint slide Full size image

Since SPREDs are suppressors of Ras/ERK-MAPK signaling and thus critical regulators of cell proliferation and gene expression, we further examined whether changed synaptic excitability and neuron morphology was associated with altered expression of synaptic proteins in the amygdala. Western blot analyses of pre- and postsynaptic proteins demonstrated different expression levels in 10–12 months old SPRED2 KO mice compared with WT controls (Figure 4c). The expression of PSD95, an important anchor of various synaptic proteins at the postsynapse, was upregulated in SPRED2 KOs compared with WT littermates. Protein levels of metabotropic glutamate receptor 5 (mGluR5), which is primarily located at the periphery of postsynaptic densities, were also elevated in SPRED2 KO mice as well as levels of mGluR2, which is primarily distributed at presynaptic axon terminals but may also be expressed at postsynaptic sites. ERC1 (ELKS/RAB6-interacting/CAST family member 1), a structural and functional determinant of the presynaptic active zone, was also upregulated, while presynaptic bassoon, a direct interaction partner of ERC1 in the active zone, was downregulated. The expression of the small GTPases Rab3A and Rab6, which are involved in the regulation of synaptic vesicle transport along microtubules and exocytosis at the presynapse, was not altered. Furthermore, levels of α- and β-tubulin were unchanged (Figure 4c). Quantification of protein amounts by normalization to GAPDH confirmed the dysregulated expression of various pre- and postsynaptic proteins in the amygdala of SPRED2 KOs (Figure 4d).

Differences in protein expression are mainly caused either by altered gene transcription or by protein turnover rate. To address a possible dysregulation at transcriptional level, we performed quantitative RT-PCRs using RNA from the amygdala and gene-specific primers for PSD95, mGluR2, mGluR5 and ERC1. We detected that the dysregulated protein expression observed in Western blots was accompanied by altered expression levels of the selected genes (Figure 4e). Because mRNA and protein levels were changed in the same direction, transcriptional dysregulation is most likely causative for alterations in synaptic protein expression.

SPRED2 deficiency leads to increased TrkB/ERK-MAPK signaling and induces OCD-like grooming in SPRED2 KO mice

Given the dysregulated synaptic gene expression in the amygdala, we focused on the upstream regulatory mechanism that might contribute to the molecular and physiological changes at amygdaloid synapses. An essential regulator of neuronal gene transcription, proliferation and differentiation but also of synaptic transmission and potentiation is the BDNF/TrkB signaling pathway. Upon binding of the neurotrophin BDNF to its preferred receptor tyrosine kinase TrkB, downstream signals are mediated by the Ras/ERK-MAPK cascade. Because SPRED2 is a critical inhibitor of Ras/ERK-MAPK signaling, we investigated if SPRED2 deficiency impacts BDNF/TrkB/ERK-MAPK signaling in SPRED2 KO mice. We analyzed the expression and phosphorylation of ERK1/2 in amygdala of 10–12 months old SPRED2 KO mice and WT littermates by Western blot. The expression of unphosphorylated ERK was not altered, however, P-ERK was increased in SPRED2 KO mice. The 2.5-fold elevated P-ERK/ERK ratio clearly reflected pathway overactivation due to the loss of SPRED2-mediated inhibition (Figure 5a). To unravel whether the increase in Ras/ERK-MAPK signaling is also involved in the development of the OCD-like behavior in SPRED2 KOs, we specifically blocked the Ras/ERK-MAPK in vivo using the MEK1/2 inhibitor selumetinib. In this prospective study, we treated SPRED2 KO mice aged 8 months with selumetinib for one week. Photo documentation revealed reduced hemorrhage and ulceration of self-inflicted wounds in SPRED2 KOs after one week of treatment (Figure 5b). This indicated that SPRED2, as an endogenous suppressor of Ras/ERK-MAPK signaling, is required to ensure normal behavior. Investigation of factors further upstream in this pathway revealed that also Ras was activated 1.9-fold in the amygdala of SPRED2 KO mice compared with WTs (Figure 5c). To test whether pathway activation is elicited by elevated BDNF expression, we estimated BDNF levels in the amygdala of 6–12 months old SPRED2 KO mice and WT controls and found no differences between genotypes (Figure 5d). SPRED2 can be phosphorylated at various confirmed tyrosine residues and might therefore be a direct target of TrkB. To address this, we immunoprecipitated tyrosine-phosphorylated proteins from BDNF-stimulated murine hypothalamic cells and analyzed SPRED2 and TrkB by Western blot. The input controls demonstrated constant expression of both TrkB and SPRED2 in mHypoE44 cells after different times of BDNF stimulation. In the IP samples, BDNF stimulation resulted in increasing TrkB phosphorylation over time. After 60 min of BDNF stimulation, BDNF-mediated TrkB activation provoked phosphorylation of SPRED2 (Figure 5e). Consequently, SPRED2 is a target of TrkB itself or of a kinase downstream of TrkB. Given the unaltered BDNF levels in the amygdala and the interaction between SPRED2 and TrkB, we further investigated whether the augmented activity of Ras and ERK in SPRED2 KO mice might be a result of specifically increased TrkB activation. We used a phospho-RTK array to identify active phosphorylated RTKs in amygdala of 10 months old SPRED2 KO and WT mice. Independently of the genotype, only PDGF-Rα was markedly phosphorylated among the 39 different murine RTKs included in the array. In the amygdala lysates of SPRED2 KOs, we detected highest phosphorylation levels in EGFR, ErbB2 and TrkB (Figure 5f). This demonstrated activation of the TrkB receptor in response to loss of SPRED2; however, a parallel phosphorylation of EGFR and ErbB2 might contribute to induction of downstream pathways. To support our hypothesis that TrkB is responsible for activation of the Ras/ERK-MAPK pathway, we examined possible alterations in TrkB phosphorylation and expression quantitatively. TrkB expression levels were 1.4-fold higher in SPRED2 KO amygdala after normalization to GAPDH in comparison to WT controls (Figure 5g). In addition to TrkB receptor overexpression, phosphorylation of Y515, the tyrosine residue indicative for Ras/ERK-MAPK pathway activation in mouse TrkB, was 1.3-fold elevated in amygdala of SPRED2 KO mice. In contrast, Y817 phosphorylation level, which is relevant for phospholipase C activation, was not altered, indicating that the downstream actions of activated TrkB are specifically mediated by the Ras/ERK MAPK pathway (Figure 5g). Although a contribution of activated EGFR and ErbB2 cannot be excluded, TrkB seems to be a crucial modulator of upregulated ERK-MAPK pathway in SPRED2 KO mice as demonstrated by TrkB overexpression, activation, and association with phosphorylation of SPRED2. Missing inhibition of BDNF/TrkB/ERK-MAPK signaling resulted in OCD-like behavior in SPRED2 KO mice whereas SPRED2-mediated pathway downregulation seems to be necessary for coordinated neuronal protein expression, synaptic function and behavior in vivo.