Acid sphingomyelinase activity suggests a new antipsychotic pharmaco-treatment strategy for schizophrenia

Association of SMPD1 and SMPD3 with schizophrenia in humans

In order to test whether natural genetic variance in genes of sphingolipid control predisposes to schizophrenia, we performed a human genome-wide association study. For four key sphingolipid regulatory genes involved in cognitive and emotional behavioral regulation [50, 51], the major two related to ceramide synthesis (SMPD1 and SMPD3) and major two related to ceramide metabolism (ASAH1 and ASAH2), we extracted single-nucleotide polymorphisms (SNPs) that cover a gene region (extended to ± 10 KB) and their association with schizophrenia from the latest genome-wide association study (GWAS) summary statistics of wave 3 Psychiatric Genomics Consortium (PGC) GWAS of schizophrenia. It included 74,776 cases and 101,023 controls [25]. Among the four analyzed sphingolipid-genes, we identified three SNPs in SMPD3, coding for neutral sphingomyelinase-2 (NSM), that showed significant association with schizophrenia (Fig. 1a). No associations were found for SMPD1, ASAH1 and ASAH2.

a LocusZoom plot cantered on SMPD3 gene for the Schizophrenia genome-wide association study. In this plot, each point is one variant, with the x– and y-axes representing physical position on the chromosome and −log₁₀(p-value), respectively. The lead SNP (rs11862968; p-value = 4.72 × 10⁻⁹) is represented by the purple diamond symbol. The color coding of all other SNPs indicates LD with the lead SNP (estimated by Phase II HapMap CEU r² values): red, r² ≥ 0.8; gold, 0.6 ≤ r² < 0.8; green, 0.4 ≤ r² < 0.6; cyan, 0.2 ≤ r² < 0.4; blue, r² < 0.2; gray, r² unknown. Recombination rates are estimated from 1000 Genomes phase 3 data. The dashed horizontal line represents the statistical threshold of genome-wide significance (−log₁₀ (P = 5 × 10⁻⁸)). b Comparisons of SMPD1 gene expression values in each of the three-brain region. The normalized (log2) expression values of SMPD1 gene were compared between disease states (CON: Control and SCZ: Schizophrenia) in Associative Striatum (AS): CON = 18 and SCZ = 18, Hippocampus (HIP): CON = 18 and SCZ = 15, and Pre-frontal Cortex (PFC): CON = 19 and SCZ = 15 using the Wilcoxon test. In the box plots, the box represents the interquartile range (IQR), with the upper and lower bounds indicating the 75th percentile (Q3) and 25th percentile (Q1) respectively. The horizontal line inside the box represents the median. The upper and lower horizontal lines outside the box indicate the maximum and minimum values (excluding outliers), which correspond to Q3 + 1.5 × IQR and Q1 − 1.5 × IQR respectively. The whiskers extend from each quartile to the minimum and maximum values.

Next, we compared the normalized expression values of these sphingolipid controlling genes between controls (CON) and schizophrenia (SCZ) patients in three different brain regions: the associate striatum (AS: n_CON = 18 and n_SCZ = 18), hippocampus (HIP: n_CON = 18 and n_SCZ = 15), and the prefrontal cortex (PFC: n_CON = 19 and n_SCZ = 15) [52]. Among four sphingolipid genes, we identified significant expression alterations for SMPD1, coding for ASM, specifically in the PFC (log₂ Fold change = −0.14; P = 0.005, Wilcoxon test; Fig. 1b). Altogether, these findings point towards an association of genes coding for NSM and ASM with schizophrenia, with the PFC as a potential locus of action.

Psychosis-like behavior can be reversed by short-term haloperidol treatment

After principle relevance of sphingolipid genes in schizophrenia was identified, we searched for brain mechanisms of sphingolipid involvement in schizophrenia pathogenesis and in APD therapy. To interrogate the role of sphingolipid regulatory enzymes in schizophrenia, we investigated behavior in a rat model of an amphetamine (AMPH)-induced psychosis [28, 53] after short-term treatment with haloperidol (HAL) [30, 54]. Escalating AMPH-treatment induced psychosis-like behavior in a test of AMPH-induced acute hyperlocomotion (AIH) and in the pre-pulse inhibition of an acoustic startle response (PPI). A seven-day short-term chronic administration of HAL with osmotic mini-pumps (Figs. 2a and S1) improved psychotic-like symptoms of previously AMPH-sensitized rats in the AIH test and PPI test. These two tests are commonly used in preclinical assessment of novel APDs and correspond to agitation behavior and sensorimotor gating deficits of schizophrenic patients. In detail, for the short-term treatment model, two-way ANOVAs showed significant effects for the factor Group in the baseline (F_2,104 = 6.374, p = 0.002) and challenge period (F_2,208 = 29.562, p < 0.001) of the total locomotion level in the AIH test (Fig. 2b). The factor Time yielded a significant effect during baseline (F_3,104 = 40.005, p < 0.001), but only a tendency after the AMPH challenge (F_7,208 = 1.923, p = 0.067). No significant effects of Time×Group interaction were observed (F_6,104 = 1.112, p = 0.361 and F_14,208 = 1.212, p = 0.268), before and after AMPH injection. AMPH-sensitized vehicle-treated animals (AMPH-VEH) displayed a longer distance moved in the open field than a control group (SAL-VEH) at 10-, 15-, 20-, and 25-min time points after the AMPH challenge (p = 0.005, p = 0.002, p = 0.002, p = 0.009, respectively, Fig. 2b). AMPH-sensitized HAL-treated animals (AMPH-HAL) showed significantly lower locomotion then controls at the baseline and after AMPH challenge (p = 0.012 and p = 0.008 at −10 min and 25 min time points). An area under the curve (AUC) analysis with two-way ANOVA with LSD pre-planned comparisons verified the higher locomotion level for the AMPH-VEH group compared to controls (p = 0.002 for the first 20 min after AMPH injection), which was reversed by HAL treatment (Fig. 2c).

Data are presented as means ± SEM. a Study design for the short-term (7 days of treatment) experiment. b Total locomotion of animals in AMPH-induced hyperlocomotion (AIH) test with short-term HAL treatment (7 days of osmotic mini-pumps delivery, 0.5 mg/kg). AMPH-sensitized rats treated with vehicle (AMPH-VEH) showed a psychotic-like agitation state by the dramatically increased locomotion after the AMPH challenge (1.5 mg/kg, i.p.) in comparison with HAL-treated (AMPH-HAL) and control (SAL-VEH) groups. c Area under the curve (AUC)for total locomotion level after short-term HAL treatment (7 days, 0.5 mg/kg). d Time spent in the central zone of the open field in the AIH test after short-term HAL treatment. e AUC for time spent in the central zone of the open field in the AIH test after short-term HAL treatment. f AUC for prepulse inhibition (PPI) of acoustic startle response for three pulse stimuli: 100, 110, and 120 dB after short-term HAL treatment. Rats with psychotic-like symptoms demonstrate lower AUC of PPI and thus higher sensorimotor gating deficit that was restored by short-term treatment with HAL. g Activity of acid sphingomyelinase (ASM) after short-term HAL treatment in three brain regions: prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS). h Neutral sphingomyelinase (NSM) activity in three brain regions: PFC, DS, and VS after short-term HAL treatment. i Acid ceramidase (AC) activity in three brain regions: PFC, DS, and VS after short-term HAL treatment. j Neutral ceramidase (NC) activity in three brain regions: PFC, DS, and VS after short-term HAL treatment. k Sphingomyelin synthase (SMS) activity in three brain regions: PFC, DS, and VS after short-term HAL treatment. (*p < 0.05, ^#p < 0.01, ^$p < 0.001; n = 8–10 animals/group).

The anxiety level of animals was evaluated by the time spent in the center area of the Open field (OF). Animals from both AMPH-sensitized groups displayed higher anxiety levels after the AMPH challenge, indicated by a more prominent decline during the second 20 min of testing (Fig. 2d). There were significant effects of the factors Time (F_2,78 = 11.973, p < 0.001) and Group (F_2,78 = 8.194, p < 0.001), but not for the Interaction (F_4,78 = 1.760, p = 0.145). AUC analysis with pre-planned comparisons showed significant differences between AMPH-VEH (p = 0.001) and AMPH-HAL (p < 0.001) groups vs. controls (Fig. 2e).

We found sensorimotor gating deficit in the PPI test in AMPH-VEH rats (p = 0.014 vs SAL-VEH) for the P100 dB stimulus (Factor: Prepulse stimulus (ppSt), F_2,75 = 39.278, p < 0.001; Factor: Group, F_2,75 = 3.751, p = 0.028; Interaction, F_4,75 = 3.751, p = 0.594, Fig. 2f). For P110 dB, we found an increase of the PPI AUC in the AMPH-HAL group (p < 0.001 vs SAL-VEH; two-way ANOVA, Factor: ppSt, F_2,75 = 89.632, p < 0.001, Factor Group, F_2,75 = 6.686, p = 0.002, Interaction, F_4,75 = 1.657, p = 0.169) (Fig. 2f). These findings replicate the previously established schizophrenia model along the sensomotor-, emotional- and cognitive dimensions of the pathology.

Amphetamine-induced psychotic-like behavior is associated with sphingolipid enzyme activity changes

In order to identify the sphingolipid neurobiology underlying schizophrenia pathogenesis and treatment outcomes, we measured the activity of five sphingolipid-metabolizing enzymes: ASM, NSM, acid- (AC) and neutral (NC) ceramidases, as well as sphingomyelin synthase (SMS) in three different brain regions most related to schizophrenia pathogenesis: the PFC, which is mostly associated with cognitive and negative symptoms, and the dorsal- (DS) and ventral striatum (VS) that are predominantly claimed to be responsible for psychotic symptoms [3]. After psychosis-induction, we observed a significantly enhanced ASM activity in the PFC (F_2,26 = 6.387, p = 0.006; AMPH-VEH vs Sal-VEH: p = 0.002) and VS (F_2,25 = 12.697, p < 0.001, AMPH-VEH vs Sal-VEH: p = 0.002). Also, in the VS, AC activity (F_2,26 = 40.821, p < 0.001; p < 0.001) and SMS activity (F_2,25 = 9.205, p = 0.001; p < 0.001) were significantly enhanced in these animals (Fig. 2g–k). This may suggest a potential pathological mechanism linking psychosis-induction and sphingolipid metabolism. Sphingolipid changes coincide with the observation of psychotic-like behavioral symptoms.

Psychosis-remission is associated with normalization of sphingolipid enzyme activity

If a patho-mechanism is associated with both, psychosis-induction and APD treatment efficacy, it should emerge during psychosis induction, but disappear when APD treatment is effective. Effective short-term treatment with HAL did not impact the psychosis-induced rise in the activity of the enzymes, except ASM in the PFC. ASM activity was restored to the levels of the SAL-VEH group after 7-day administration with HAL, mirroring the efficacy pattern at behavioral level (Fig. 2g). In addition, HAL treatment attenuated NSM activity in the PFC (p p < 0.001). Short-term HAL treatment did not affect the AMPH sensitization induced increase in VS ASM (p < 0.001; Fig. 2g), AC (p < 0.001; Fig. 2i) and SMS activity (p < p < 0.001; Fig. 2k). These findings may suggest a specific role for ASM in the PFC for psychosis induction and reversal.

Brain sphingolipids during psychosis-induction and reversal

Sphingolipid enzymes tightly regulate ceramide and SM levels in a brain-area specific way [16, 26, 50]. In order to detect potential patterns of sphingolipids that may be associated with psychosis induction and reversal, we measured single species and total ceramide and SM levels in the PFC, VS and DS. We found a decline in most of the analyzed ceramide species in the PFC and some species in the VS in AMPH-sensitized animals, regardless of the treatment (Fig. 3a–f). This was reflected in a significant decline in total ceramide levels in the PFC (F_2,27 = 9.862, p < 0.001; AMPH-VEH and AMPH-HAL vs. SAL-VEH: p = 0.005 and p < 0.001), but not in VS or DS (p > 0.05) (Fig. 3g).

Data are presented as means ± SEM. a Levels of ceramide 16:0 (Cer 16:0). b Levels of ceramide 18:0 (Cer 18:0). c Levels of ceramide 20:0 (Cer 20:0). d Levels of ceramide 22:0 (Cer 22:0). e Levels of ceramide 24:0 (Cer 24:0). f Levels of ceramide 24:1 (Cer 24:1). g Levels of total ceramide (Cer Total). h Levels of sphingomyelin 16:0 (SM 16:0). i Levels of sphingomyelin 18:0 (SM 18:0). j Levels of sphingomyelin 20:0 (SM 20:0). k Levels of sphingomyelin 22:0 (SM 22:0). l Levels of sphingomyelin 24:0 (SM 24:0). m Levels of sphingomyelin 24:1 (SM 24:1). n Levels of total sphingomyelin (SM Total). Data were analyzed by one-way ANOVA followed by LSD pre-planned comparisons with Bonferroni’s correction (n = 8–10 animals/group; *p < 0.05, ^#p < 0.01, ^$p < 0.001).

Surprisingly, the remission of psychosis–like behavior with HAL was not accompanied by reversal of ceramide alterations. In fact, the visually detectable decline was now significant in the PFC for Cer 16:0 (p = 0.024), Cer 20:0 (p < 0.001), Cer 22:0 (p = 0.005), and Cer 24:1 (p = 0.009) in the AMPH-HAL group compared to controls. Also the most abundant Cer 18:0 further declined in the PFC (p < 0.001), driving the decline in total ceramide levels (p < 0.001). Furthermore, Cer 16:0, Cer 20:0 and Cer 24:1 levels were found to be significantly lower in the VS of AMPH-HAL rats compared to controls (p = 0.006, p = 0.017, p = 0.008). The treatment had only little effects on SM levels in the PFC, VS or DS (Fig. 3h–n). Only SM20:0 levels were reduced in the PFC after AMPH-HAL treatment (p = 0.018; Figs. 3j and S2). Altogether, these findings suggest PFC and VS specific alterations of Cer levels following psychosis induction. However, they may not be directly involved in the APD treatment response as they do not reverse after HAL short-term chronic treatment.

AMPH-induced psychosis is diminished after 14 days

A major problem of current APD therapy of schizophrenia is the frequently observed failure in the clinic, for which mechanisms have only recently started to emerge [30]. Here we tested for a potential sphingolipid involvement. In a model of APD treatment failure (Fig. 4a), long-term treatment with HAL for 14 days abrogated its therapeutic effects, expressing an increase in locomotion boost not only for AMPH-VEH, but also for AMPH-HAL-treated rats. The long-term treatment abrogated HAL therapeutic effects as no inhibition of the AIH was observed any more (ANOVA, Factor Group, F_2,48 = 8.292, p < 0.001, Factor Time, F_1,48 = 0.903, p = 0.347, Interaction, F_4,72 = 2.566, p = 0.088, Fig. 4b). Over the last 20 min of AIH testing, the HAL-treated group showed a noticeable drop in locomotor activity (p = 0.002, p < 0.001, p < 0.001 for 30, 35 and 40 min, respectively, vs controls, Fig. 4b). However, pre-planned comparisons of % AUC values, as groups differed in locomotion at the end of the baseline, revealed a significant increase in locomotion boost for AMPH-HAL (p < 0.001) and AMPH-VEH (p = 0.044) vs SAL-VEH in the first 20 min of AMPH challenge in AIH test, but not in the second 20 min (p > 0.05; Figs. 4c and S3).

Data are presented as means ± SEM. a Study design for the long-term (14 days of treatment) experiment. b Total locomotion of animals in AMPH-induced hyperlocomotion (AIH) test with long-term HAL treatment (14 days, 0.5 mg/kg). c Locomotion boost between baseline level and after AMPH challenge for two 20-min intervals after long-term HAL treatment (14 days, 0,5 mg/kg). d Time spent in the central zone of the open field in the AIH test after long-term HAL treatment. (p) AUC for time spent in the central zone of the open field in the AIH test after long-term HAL treatment. e The discrimination level between novel and familiar objects in the novel object recognition (NOR) test after long-term HAL treatment. AMPH-sensitized animals display a deficit in short-term memory resulting in a lower discrimination rate. f AUC for prepulse inhibition (PPI) of acoustic startle for three pulse stimuli 100, 110, and 120 dB after long-term HAL treatment. g The activity of acid sphingomyelinase (ASM) after long-term HAL treatment in the PFC, DS, and VS. h NSM activity in three brain regions: PFC, DS, and VS after long-term HAL treatment. i AC activity in three brain regions: PFC, DS, and VS after long-term HAL treatment. j NC activity in three brain regions: PFC, DS, and VS after long-term HAL treatment. k SMS activity in three brain regions: PFC, DS, and VS after long-term HAL treatment (*p < 0.05, ^#p < 0.01, ^$p < 0.001; n = 8–10 animals/group).

AMPH sensitization did not cause altered emotional behavior in the AIH when tested after 14 days. Evaluation of center time spending in OF arena in AIH showed the higher anxiety level of rats treated with HAL 20-40 min after the AMPH challenge, as indicated by an ANOVA (factors: Time, F_2,72 = 29.342, p < 0.001, Group, F_2,72 = 6.229, p = 0.003, Interaction, F_4,72 = 0.648, p = 0.630; Fig. 3d) and AUC analysis (p = 0.012 AMPH-HAL vs. SAL-VEH, Fig. 4e).

AMPH sensitization did not affect PPI significantly after 14 days (Fig. 4f). The AUC analysis of PPI nevertheless revealed a dramatic rise after long-term HAL treatment compared to the control group, although no sensorimotor deficit was observed in untreated rats (P100 dB, pp 74 dB: p < 0.001, AMPH-HAL vs. SAL-VEH, Fig. 4f). An ANOVA demonstrated a significant effects of factor ppSt (F_2,72 = 26.193, p < 0.001) and factor Group (F_2,72 = 8.388, p < 0.001), but not for the Interaction (F_4,72 = 1.548, p = 0.198).

No role of sphingolipids in APD failure

No significant elevation of the sphingolipid enzyme activities was found 14 days after psychosis induction (p > 0.05; Fig. 4g–k), where only a decrease in AC activity in the VS was observed (F_2,25 = 4.518, p = 0.021; AMPH-VEH vs SAL-VEH: p = 0.009, Fig. 4i). When psychosis-induction and APD treatment effects were no longer visible, sphingolipid responses in the brain were also largely diminished (Fig. 5). The AMPH-sensitization alone had no significant effects on Cer or SM levels in the PFC, VS, and DS (p > 0.05). Only after long-term HAL treatment, effects emerged. Cer 20:0 (p = 0.009), Cer 22:0 (p = 0.002), and Cer 24:1 (p = 0.022) levels were significantly decreased in the PFC (Fig. 5c–f). Remarkably, the Cer 22:0 concentration was raised in the VS in the same group of animals (p = 0.023, Fig. 5d). We further report decreased levels of SM 20:0 in the DS after AMPH-HAL long-term treatment (p = 0.009; Figs. 5j and S4). Altogether, sphingolipid changes coincide with the observation of psychotic-like behavioral symptoms, but vanish when induced symptoms are no longer present.

Data are presented as means ± SEM. a Levels of ceramide 16:0 (Cer 16:0). b Levels of ceramide 18:0 (Cer 18:0). c Levels of ceramide 20:0 (Cer 20:0). d Levels of ceramide 22:0 (Cer 22:0). e Levels of ceramide 24:0 (Cer 24:0). f Levels of ceramide 24:1 (Cer 24:1). g Levels of total ceramide (Cer Total). h Levels of sphingomyelin 16:0 (SM 16:0). i Levels of sphingomyelin 18:0 (SM 18:0). j Levels of sphingomyelin 20:0 (SM 20:0). k Levels of sphingomyelin 22:0 (SM 22:0). l Levels of sphingomyelin 24:0 (SM 24:0). m Levels of sphingomyelin 24:1 (SM 24:1). n Levels of total sphingomyelin (SM Total). Data were analyzed by one-way ANOVA followed by LSD pre-planned comparisons with Bonferroni’s correction (n = 8-10 animals/group; *p < 0.05, ^#p < 0.01, ^$p < 0.001).

ASM inhibition with KARI201 reverses psychosis-like behavior

To further investigate the relationship between ASM activity and psychotic symptoms, we used the novel selective ASM inhibitor, KARI201 [33] and compared it with the effects of HAL treatment. Inducing an AMPH-psychosis in a rat model significantly enhanced AIH responses in the rats. This effect could be blocked by short-term chronic treatment with HAL. However, HAL reduced locomotor activity already at baseline level. KARI201 (10 mg/kg) [33] short-term treatment also reduced AIH responses to the level of vehicle controls. Interestingly, KARI201 did this without affecting locomotor baseline levels (Fig. 6b, c). An ANOVA of locomotor activity showed a significant effect of factors Time (F_3,156 = 40.662, p < 0.001) and Group (F_3,156 = 18.060, p < 0.001), but not Time×Group interaction (p > 0.05), during the baseline period. There was a significantly lower level of locomotion in the AMPH-HAL group compared to control rats during baseline after 5 min (p < 0.001) and 10 min (p < 0.001) of testing, which however, normalized thereafter (p > 0.05; Fig. 6b). No significant difference was observed between the AMPH-KARI201 and SAL-VEH groups compared to controls at the baseline (p > 0.05). The AMPH challenge induced a significant increase in locomotion (ANOVA, factor Group: F_{3, 312} = 152.541, p < 0.001). This was most pronounced in the AMPH-VEH group (10, 15, 20, and 25 min after the AMPH injection: p < 0.001, p = 0.002, p = 0.012, and p = 0.008 vs. SAL-VEH). The increase was significantly attenuated in the AMPH-HAL group (all time points after the AMPH injection: p < 0.001 at vs. SAL-VEH). No difference was observed between KARI201-AMPH and the SAL-VEH groups (p > 0.05). These results were confirmed in the AUC analysis (Figs. 6c and S5).

Data are presented as means ± SEM. a Study design for the experiment with HAL and KARI201 treatment. b. Total locomotion of animals in AMPH-induced hyperlocomotion (AIH) test with 10-day treatment (oral gavage, per os). One group was a control (SAL-VEH), and three groups were sensitized with AMPH and treated with either VEH (AMPH-VEH), HAL at 1 mg/kg (AMPH-HAL), or KARI201 at 10 mg/kg (AMPH-KARI). c The area under the curve (AUC) for total locomotion represents the baseline level (Bl), first and second 20 min after the AMPH challenge. Two-way ANOVA of the AUC analysis of locomotion indicated significant effects by factors Time (F_2,117 = 65.427, p < 0.001), Group (F_3,117 = 50.167, p < 0.001), and Time × Group interaction (F_6,117 = 6.778, p < 0.001). The AMPH-VEH group was more active during the first 20 min after AMPH injection compared to SAL-VEH rats (^#p < 0.001), while HAL-treated rats demonstrated a dramatic drop in AUC locomotion in comparison with the control group (p < 0.001). d Visits made in the central zone of the open field (Center visits) in the AIH test after VEH or HAL or KARI treatment. e AUC for visits in the central zone of the open field (Center visits) in the AIH test. AUC analysis revealed a significant effect of factors Time (F_2,117 = 28.716, p < 0.001), Group (F_3,117 = 21.858, p < 0.001), and Time × Group interaction (F_6,117 = 3.465, p = 0.004). The AMPH-HAL group demonstrated lower number of center visits during AMPH challenge period (p < 0.001), while the AMPH-KARI group showed the same drop but only at the second 20-min interval of AMPH challenge period (p < 0.008) vs. controls. f–h Prepulse inhibition (PPI) of acoustic startle for three pulse stimuli: 100, 110, and 120 dB, and for three prepulse stimuli: 74, 80, and 86 dB. The lower the percentage of PPI the higher the sensorimotor gating animals demonstrate. i The discrimination level between novel and familiar objects in the novel object recognition (NOR) test to evaluate short-term memory. j–p Levels of ceramide species (Cer total, Cer 16:0, Cer 18:0, Cer 20:0, Cer 22:0, Cer 24:0, Cer 24:1) in AMPH-sensitized animals treated with VEH (AMPH-VEH) or HAL (AMPH-HAL) or KARI201 (AMPH-KARI) and controls (SAL-VEH). Brain samples were collected from the prefrontal cortex (PFC). q–w Levels of sphingomyelin species (SM total, SM 16:0, SM 18:0, SM 20:0, SM 22:0, SM 24:0, SM 24:1) in AMPH-sensitized animals treated with VEH (AMPH-VEH) or HAL (AMPH-HAL) or KARI201 (AMPH-KARI) and controls (SAL-VEH). Brain samples were collected from the prefrontal cortex (PFC). Data were analyzed by one- or two-way ANOVA followed by LSD pre-planned comparisons with Bonferroni’s correction. (n = 10-12 animals/group; *p < 0.05, ^#p < 0.01, ^$p < 0.001).

HAL treated animals showed an increase in anxiety levels at baseline as indicated by reduced number of center visits. An ANOVA indicated a significant effect of factors Time (F_3,156 = 26.340, p < 0.001) and Group (F_3,156 = 8.796, p < 0.001), but not Time×Group interaction (p > 0.05), during the baseline period. The AMPH-HAL group made less center visits at first 5 min of testing (p < 0.001 vs. SAL-VEH). The AIH challenge increased center visits. This was not affected by KARI201, but significantly reduced by HAL treatment (Fig. 6d, e). After AMPH challenge, there was a significant effect of factor Group (F_3,312 = 51.609, p < 0.001), but not Time or Time×Group interaction (p > 0.05). The AMPH-HAL group differed from SAL-VEH 5 min (p = 0.007), 10 min (p = 0.004), and 15-40 min (p < 0.001) after AMPH injection. We suggest that effect of HAL is explained not by higher anxiety levels of animals but by a prominent sedative action of the drug, which was also observed in general locomotor activity.

In the same animals, the sensorimotor gating deficit, induced by the AMPH sensitization, was rescued by both, HAL and KARI201 (Fig. 6f–h). The PPI for the pp+P pair 74 + 100 dB was significantly decreased in the AMPH-VEH group (p = 0.002 vs SAL-VEH, Fig. 6f). This was reversed by both HAL and KARI201 treatment (p > 0.05 vs. SAL-VEH). No significant differences between the groups in PPI levels was observed for 110 dB pulse intensity (Fig. 6g). While the AMPH sensitization did not yield a significant decline in PPI at 120 dB pulse stimulus, HAL and KARI201 treatment significantly enhanced the PPI at a 80 and 86 pre-pulse stimulus intensity (p = 0.003 and p = 0.015 vs. SAL-VEH; Fig. 6h). These findings suggest that KARI201 has a similar antipsychotic potential as HAL.

While the AMPH sensitization did not affect cognitive function in the NOR test, treatment with HAL caused a significant decline (p < 0.001 vs. SAL-VEH; Fig. 6i). This was not seen in the KARI201-treated animals. This may suggest that KARI201, at a therapeutically active dose, may have less cognitive impairing side effects than HAL [55].

Brain sphingolipids after KARI201 treatment

Given that the key alterations in ASM activity and sphingolipid levels were PFC-specific, we measured the effects of an ASM inhibitor, KARI201, on ceramide and SM levels in the PFC compared to HAL treatment. Similar to the results of our first experiment, a notable reduction in both ceramide and SM levels were revealed for the AMPH-sensitized VEH treated group (AMPH-VEH) that demonstrated psychotic-like symptoms in the behavioral tests. Total ceramides were decreased in AMPH-VEH (p < 0.001) and AMPH-HAL (p < 0.001) groups in comparison with controls (SAL-VEH), showing the significant effect of factor Group (F_3;42 = 10.779, p < 0.001) and suggesting no impact of HAL treatment (Fig. 6j). The most abundant Cer 18:0 species displayed the same aberrations (Group: F_3;42 = 11.334, p < 0.001) in AMPH-VEH (p < 0.001) and AMPH-HAL (p < 0.001) animals (Fig. 6l). Cer 20:0 levels were diminished only in AMPH-VEH rats (p = 0.004), significantly affected by factor Group (F_3;42 = 4.410, p = 0.009) (Fig. 6m). Other ceramide species were impacted neither by AMPH-sensitization nor by treatment (Figs. 6k, n–p and S6).

Levels of total SM and few SM species were found to be remarkably decreased in psychotic-like animals (Fig. 6q–w). An ANOVA revealed a significant effect of factor Group for total SM (F_3;42 = 4.205, p = 0.011) and SM 18:0 (F_3;42 = 4.149, p = 0.012) with a strong decline in its levels for AMPH-VEH group (p = 0.004 and p = 0.005, respectively) (Fig. 6q, s). We also observed a similar drop in SM 20:0 levels in both AMPH-VEH (p < 0.001) and AMPH-HAL (p < 0.001) animals (Group: F_3;42 = 9.101, p < 0.001) (Fig. 6t). Interestingly, that SM 24:0 concentrations were diminished only in AMPH-HAL group compared to SAL-VEH (p = 0.011) (Fig. 6v). We reported no alterations in the levels of SM 16:0, SM 22:0, and SM 24:1.

Altogether, we identified the striking effect of AMPH-sensitization on the sphingolipid balance in the PFC, similarly to the previous findings. The majority of alterations were independent of HAL treatment. However, ASM inhibition through KARI201 administration recovered all ceramides and SMs to the levels of control animals.

Gene expression in the PFC after AMPH-induced psychosis and APD treatment

Membrane sphingolipids regulate classical transmitter signaling through lipid membranes of the brain [17, 56]. As the observed changes in sphingolipid regulation cannot fully explain behavioral alterations after psychosis-like state induction and its reversal with HAL or KARI201, we measured gene expression in the PFC using RNA sequencing [57], after psychosis induction and successful APD treatment. Despite a considerable interindividual variance, results showed that psychosis-induction was accompanied by a significant upregulation of the genes Slc2a5, Pld4, Olig1, Fgfr1, Gpr17, Cxcl14, Phlda3, Gna12, Abca2, Sox1, RGD1566085 and a down-regulation of Dpm2, Ergic2, Rab2a, Vma21 when AMPH-VEH was compared to the SAL-VEH group (Fig. 7a, b). The HAL treatment did not reverse any of these effects, but upregulated expression of Col6a3, Slc22a8, Ndufs5-ps2, Net1, Rasl11b, and downregulated Leo1 and Bmal1 expression in the comparison of the AMPH-HAL vs. AMPH-VEH groups. KARI201 treatment did also not reverse the effects of psychosis-induction. Nor did it share effects with the HAL treatment. Instead, the KARI201 treatment upregulated expression of Tmem238 and Nr2f6 in the comparison of the AMPH-KARI vs. AMPH-VEH groups. These data may suggest that psychosis induction is accompanied by numerous changes in gene expression on the PFC. Successful APD treatment does not work by a reversal of these changes, but by other mechanisms. Despite a shared effect on ASM activity in the PFC, the new ASM targeting antipsychotic drug KARI201 does not share effects on gene expression with HAL.

Antipsychotic (APD) treatment with haloperidol (HAL/H) or KARI201 (KARI/K) does not reverse these effects, but has its own expression profile. Animals were tested at a time of confirmed psychotic-like state and efficacy of the APD treatment (n = 6/group). a, b Differential expression analysis of RNA sequencing data for all genes analyzed. c, d Selective analysis of sphingolipid controlling genes.

A selective analysis of sphingolipid controlling genes did not show different expression after psychosis induction (AMPH-VEH vs. SAL-VEH). However, it showed a selective reduction in the expression of Asah2 and an enhanced expression of Cers2 after HAL treatment (AMPH-HAL vs. AMPH-VEH). KARI201 did not affect sphingolipid controlling genes (AMPH-KARI vs. AMPH-VEH). Interestingly, none of the treatments affected the expression of the ASM coding gene Smpd1 (Figs. 7c, d and S7).