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Ameliorative effects of Fingolimod (FTY720) on microglial activation and psychosis-related behavior in short term cuprizone exposed mice

Ameliorative effects of Fingolimod (FTY720) on microglial activation and psychosis-related... Schizophrenia is a psychiatric disorder that affects around 1% of the population in widespread populations, with severe cases leading to long-term hospitalization and necessitation of lifelong treatment. Recent stud- ies on schizophrenia have highlighted the involvement of inflammatory and immunoregulatory mechanisms with the onset of symptoms, and the usage of anti-inflammatory treatments are being tested against periods of rapid psychosis. In the central nervous system, microglia are the innate immune population which are activated in response to a wide range of physical and psychological stress factors and produce proinflammatory mediators such as cytokines. Microglial activation and neuroinflammation has been associated to numerous psychiatric disor - ders including schizophrenia, especially during psychotic episodes. Thus, novel treatments which dampen microglial activation may be of great relevance in the treatment of psychiatric disorders. Fingolimod (FTY720) is a drug used as an immunosuppressive treatment to multiple sclerosis. Recent clinical trials have focused on FTY720 as a treatment for the behavioral symptoms in schizophrenia. However, the mechanisms of Fingolimod in treating the symptoms of schizophrenia are not clear. In this study we use a recently developed neuroinflammatory psychosis model in mice: cuprizone short-term exposure, to investigate the effects of FTY720 administration. FTY720 administration was able to completely alleviate methamphetamine hypersensitivity caused by cuprizone exposure. Moreover, administration of FTY720 improved multiple measures of neuroinflammation (microglial activation, cytokine production, and leuco - cyte infiltration). In conclusion, our results highlight the future use of FTY720 as a direct anti-inflammatory treatment against microglial activation and psychosis. Keywords Short-term cuprizone exposure, Psychosis, Fingolimod, Microglial activation, Neuroinflammation *Correspondence: Department of Functional Anatomy and Neuroscience, Asahikawa Koki Sakurai Medical University, Hokkaido, Japan ksakurai@protein.osaka-u.ac.jp Department of Neuropsychiatry, Graduate School of Medical Sciences, Takahiro A. Kato Kyushu University, Fukuoka, Japan kato.takahiro.015@m.kyushu-u.ac.jp Takatoshi Hikida hikida@protein.osaka-u.ac.jp Laboratory for Advanced Brain Functions, Institute for Protein Research, Osaka University, Suita, Osaka, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Present Address: Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, Osaka, Japan © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. Molecular Brain (2023) 16:59 Page 2 of 14 expressed in most neuronal lineages and resident CNS Introduction cells, especially neural-derived glia and neurons [23, Schizophrenia is a psychiatric disorder that affects 24]. Moreover, Fingolimod has been reported to interact around 1% of the population in widespread populations, with other targets expressed in the CNS such as Tran- with severe cases leading to long-term hospitalization sient Receptor Potential Cation Channel Subfamily M and necessitation of lifelong treatment [1–4]. Although Member 7 (TRMP7), Histone Deacetylase, and Protein the etiology of schizophrenia has not yet been fully elu- phosphatase 2A (PP2A), functioning as a potent inhibi- cidated yet, genetic and environmental evidence has tor of microglial activation in culture [25]. In clinical pointed to dopaminergic disfunction and glutamater- trials, the efficacy of Fingolimod against patients with gic hypofunction as dominant hypotheses underlying schizophrenia reemphasized the importance of treat- symptoms [5]. Furthermore, recent studies have high- ing dysregulated inflammatory states during acute psy - lighted the involvement of inflammatory and immu - chotic exacerbations [8]. However, the mechanisms of noregulatory mechanisms in the onset of schizophrenia Fingolimod in treating psychosis-related behavioral and related psychotic events [6, 7]. Based on hypotheses symptoms, especially in relation to neuroinflammation that psychotic episodes result from acute exacerbation of is not understood and calls for investigation using ani- neuroinflammatory status, usage of anti-inflammatory mal models to elucidate the pharmacological pathways treatments against periods of rapid psychosis are being targeted by FTY720. investigated [8]. Such use of anti-inflammatory treat - Cuprizone short-term exposure is a recently devel- ment has been supported by further evidence of anti- oped neuroinflammatory psychosis model in mice which inflammatory drugs alleviating symptoms in patients utilizes acute administration of cuprizone (Oxalic acid with early-stage schizophrenia [9]. Despite such clinical bis(cyclohexylidenehydrazide)), a copper depleting agent evidence demonstrating the effectiveness of anti-inflam - that induces oligodendrocytic damage through mito- matory treatments, the causality and mechanism of chondrial dysfunction [26, 27]. Short-term exposure to neuroinflammatory events underlying psychosis-related cuprizone has been shown to induce behavioral abnor- behavioral deficits are yet to be fully uncovered. malities including hypersensitivity to psychostimulants Microglia are the primary innate immune population in (methamphetamine and phencyclidine) and deficits in the central nervous system (CNS), with known implica- short-term memory related tasks [28], most likely asso- tions in brain development, neural network maintenance, ciated with increased expression of proinflammatory and neural injury repair [10, 11]. Microglia are activated cytokine interleukin 6 (IL-6) in the hippocampus [29]. In in response to a wide range of physical and psychologi- this study, we utilized this neuroinflammatory psychosis cal stress factors, which trigger the production of proin- model mouse to investigate the effects of FTY720 admin - flammatory cytokines and reactive oxygen species [12], istration on  behavioral abnormalities, microglial activa- and lead to neuronal degeneration and/or white matter tion status, and proinflammatory induction. abnormality in the long term [13, 14]. In humans, micro- glial activation has been associated to various psychiatric Results and neurological disorders such as schizophrenia [13], Sample groups Alzheimer’s disease (AD) [15], Parkinson’s disease (PD) To investigate the effects of FTY720 on short-term cupri - [16], Amyotrophic lateral sclerosis (ALS) [17], and Mul- zone treated mice, 7  week old C57BL/6NJcl mice were tiple sclerosis (MS) [18]. As an investigative treatment to fed either control (Cont) or cuprizone (CUP) chow for psychiatric disorders, minocycline, a widely used antibi- 1 week and administered either Vehicle (Veh) or FTY720 otic, is known to inhibit microglial activation and have an (FTY) (4 Groups: Cont-Veh, Cont-FTY, CUP-Veh, CUP- ameliorative effect on the clinical performance in schizo - FTY; Additional file 4: Fig. S1). phrenic patients [19–21]. Thus, novel treatments which dampen microglial activation may be of great relevance FTY720 inhibited methamphetamine induced in the treatment of psychiatric disorders [12, 14]. hyperactivity in short term cuprizone treated mice A particular treatment of recent interest regarding The psychiatric symptoms of chronic methamphetamine neuroinflammation is Fingolimod (FTY720). Known addiction closely resemble the positive symptoms of as an immunosuppressive treatment to MS, the active, schizophrenia, and schizophrenic patients are more prone phosphorylated form of Fingolimod depletes circula- to the effects of methamphetamine [30]. Similarly, short- tory lymphocytes after administration by agonizing term cuprizone exposure induces behavioral deficits in lymphocytic sphingosine 1-phosphate (S P) receptors, mice that are in line with human behavioral symptoms, resulting in reduced infiltration of autoreactive lym - including hypersensitivity to acute methamphetamine phocytes into the brain parenchyma [22–24]. Other administration [29]. We investigated the effects of FTY720 than in lymphocytic populations, S P receptors are 1 Li  et al. Molecular Brain (2023) 16:59 Page 3 of 14 Fig. 1 Eec ff ts of FTY720 on behavioral changes in short-term cuprizone-exposed mice. A, B Eec ff ts of FTY720 on locomotor activity in mice after methamphetamine administration. Mice were placed in a circular open field for 30 min to measure baseline locomotion, whereafter an intraperitoneal injection of methamphetamine (2 mg/kg) was administered to the mice and locomotion was measured for 60 min. A The locomotion (ambulatory counts) for 5-min bin within the total 90 min session (− 30 to 0 min before and 0 to 60 min after methamphetamine injection). Arrows indicate methamphetamine injection time. B Total locomotion (cumulative horizontal ambulatory counts) after injection of methamphetamine (0-60 min). n = 10–11 per group. C Rotarod measurement of basal locomotor function. The latency to fall from the rotarod (s) with maximum latency set at 120 s (n = 5 per group). All values are Mean ± SEM. Tukey test *p < 0.05, ***p < 0.001. A, C 3way RM ANOVA. B 2way # ### #### ANOVA p < 0.05, p < 0.001, p < 0.0001 Li et al. Molecular Brain (2023) 16:59 Page 4 of 14 on such increases in methamphetamine sensitivity in a were not significantly affected by FTY720 administration methamphetamine-induced locomotion test. (Fig. 2C, E, G). A significant interaction between Cuprizone exposure To assess microglial activation in further detail, micro- and FTY720 treatment was observed on locomotion glial morphology in the HIP was analyzed in three after methamphetamine administration, with short- dimensions using high magnification, z-stacked micros - term cuprizone-exposed mice (CUP-Veh) showing sig- copy (Fig.  2H, I). A significant effect of both Cuprizone nificantly higher levels of locomotion compared to the administration and FTY720 was observed on microglial control chow group (Cont-Veh) (Fig. 1A, B). Moreover, a ramification ratios, a measure of microglial activation significant effect of FTY720 on locomotion was observed, state (Fig.  2I). In the cuprizone exposed groups (CUP- where the significant increase in locomotor activity in the Veh, CUP-FTY), there was a lower ramification ratio CUP-Veh group was suppressed in the FTY720 treated compared to groups with control chow (Cont-Veh, Cont- Cuprizone group (CUP-FTY), to levels similar to the con- FTY), indicating ameboid-like morphological charac- trol chow group (Cont-Veh) (Fig.  1A, B). There was no teristics of activated microglia. FTY720 was effective in significant effect of FTY administration between groups significantly increasing ramification ratio compared to that were not exposed to cuprizone (Cont-Veh and Cont- Veh treated groups, indicating morphological character- FTY) (Fig.  1A, B). Examination of the basal motor func- istics of resting state microglia. Increased ramification tions of the 4 groups of mice in the rotarod test showed ratio as a result of FTY720 administration was effective no significant effect of either Cuprizone or FTY on motor in restoring decreased ramification ratio in the CUP- ability between groups (Fig.  1C). Evaluation of spatial Veh group to levels similar to untreated Cont-Veh group memory using the Y-maze test showed that short-term (Fig. 2I). cuprizone exposure does not affect total locomotion and arm entry. Although there was no significant difference FTY720 has anti‑inflammatory effects in the hippocampus in correct alternation amongst groups, a downward trend Inflammatory activation in the brain induces the pro - was seen in cuprizone exposed groups (Additional file  4: duction and release of proinflammatory cytokines, Fig. S2). mainly by glial cell populations, and cytokine alteration has been reported in schizophrenia patients [6, 7]. In FTY720 inhibits microglial activation in the hippocampus addition, IL-6 mRNA levels are known to be increased in Glial activation induces the expression and release of the HIP and STR of short-term cuprizone-exposed mice cytokines, and postmortem brain studies of schizo- [29]. We examined the expression levels of proinflam - phrenic patients have reported hyperactivation of micro- matory cytokines interleukin 1 beta (IL-1β), IL-6, Tumor glia [31, 32]. In addition, increased expression levels necrosis factor alpha (TNF-α), along with the expres- of  the microglial gene ionized calcium binding adapter sion level of anti-inflammatory cytokine interleukin 4 molecule 1 (Iba1), indicative of microglial activation, (IL-4) to evaluate changes in neuroinflammatory status have been demonstrated in short-term cuprizone- induced by short-term cuprizone exposure and FTY720 exposed mice [29]. Therefore, we measured microglial treatment. activation by immunohistochemical staining for Iba1 In the HIP, a significant effect of Cuprizone treatment protein (Fig.  2A, Additional file  4: Fig. S3) and assessed was seen in mRNA expression levels of proinflammatory the effects of cuprizone and/or FTY720 treatment. In cytokines IL-6 and TNF-α, with marked increases in the addition, we assessed for Iba1 mRNA gene expression in short-term cuprizone-exposed (CUP-Veh) group com- Hippocampus (HIP), Corpus Callosum (CC), and Stria- pared to other groups (Fig.  3A, C). Moreover, a signifi - tum (STR) using RT-qPCR. cant effect of FTY720 along with a significant interaction Significant effects of Cuprizone and FTY adminis - between Cuprizone exposure and FTY720 administra- tration was observed on Iba1 signal levels in the HIP, tion was observed on IL-6 mRNA in the HIP, with FTY CC, and STR, with Cuprizone administration causing treatment significantly decreasing IL6 expression in marked increases of Iba1 signal, indicating increased the CUP-FTY group compared to the CUP-Veh group microglial activation in the exposed groups (CUP- (Fig.  3A). In contrast, no significant effects of CUP and Veh and CUP-FTY) (Fig.  2B, D, F). In the HIP, FTY720 FTY was observed on expression levels of proinflamma - administration significantly decreased Iba1 levels in both tory cytokines in the CC and STR (Fig. 3E–L). In both the Cont and CUP groups (Fig.  2B). In comparison, in the HIP and CC, a significant effect of FTY720 administra - CC and STR, FTY720 was only effective in decreasing tion was observed on the mRNA expression level of the Iba1 levels in the Cont group (Fig. 2D, F). The expression anti-inflammatory cytokine IL-4, where FTY720 admin - levels of Iba-1 mRNA showed significant increases in istration caused increased IL4 expression (Fig. 3D, H). HIP, CC, and STR as a result of cuprizone exposure but Li  et al. Molecular Brain (2023) 16:59 Page 5 of 14 Fig. 2 Eec ff t of FTY720 on microglial activation in short-term cuprizone-exposed mice. A–C HIP. A Representative image of Iba1 immunostaining in the HIP. Blue: DAPI nuclear stain, Green: Alexa488 (Iba1). B Fluorescence intensity measurement of Iba1 signal in the HIP (n = 3 per group). C mRNA expression levels of Iba1 in the HIP. βActin was used for internal control, and samples were normalized to Cont-Veh. (n = 6 per group). D, E CC. D Fluorescence intensity measurement of Iba1 signal in the CC (n = 3 per group). E mRNA expression levels of Iba1 in the CC. F, G STR. F Fluorescence intensity measurement of Iba1 signal in the STR (n = 3 per group). G mRNA expression levels of Iba1 in the STR. βActin was used for internal control, and samples were normalized to Cont-Veh. (n = 6 per group). H, I Three-dimensional morphology analysis. H Representative image of z stack # ## #### imaging. I Microglial ramification ratio. B–G Mean ± SEM. Tukey test *p<0.05, **p < 0.01. 2way ANOVA p < 0.05, p < 0.01, p < 0.0001. I Violin Plot #### Min-Max values. Dotted lines indicate first, second, and third quartiles. 2way ANOVA p < 0.0001 Li et al. Molecular Brain (2023) 16:59 Page 6 of 14 Fig. 3 Eec ff ts of FTY720 on cytokine changes in short-term cuprizone-exposed mice. A–D mRNA expression levels of A IL-6, B TNF-a, C IL-1β, D IL-4 in the HIP. βActin was used for internal control, and samples were normalized to Cont-Veh. (n = 6 per group) E–H mRNA expression levels of E IL-6, F TNF-a, G IL-1β, H IL-4 in the CC. I–L mRNA expression levels of I IL-6, J TNF-a, K IL-1β, L IL-4 in the STR. βActin was used for internal control, # ## and samples were normalized to Cont-Veh. (n = 6 per group). All values are Mean ± SEM. Tukey test *p < 0.05. 2way ANOVA p < 0.05, p < 0.01 High FTY720 Short‑term administration did not up‑regulate CD45 cells [34] were observed in the CC of the short- leukocyte infiltration and myelin basic protein (MBP) term cuprizone-exposed mice, while no significant differ - expression ence was observed in the HIP (Fig. 4A, B). Neuroinflammation is known to cause disruption of the Furthermore, multiple studies have also reported BBB, resulting in the infiltration of circulatory leukocytes oligodendrocyte and myelin abnormalities and associ- into the brain. Such infiltration has been reported in ated gene dysregulation in the brains of patients with patients with schizophrenia [33]. A significant increase in schizophrenia [35, 36]. FTY720 treatment in rodent Li  et al. Molecular Brain (2023) 16:59 Page 7 of 14 high Fig. 4 Eec ff ts of FTY720 on CD45 cell population and MBP variability in short-term cuprizone-exposed mice. A Representative image of CD45 high high immunostaining in the HIP. Blue: DAPI nuclear stain, Green: Alexa488 (CD45). Arrows indicate CD45 cells. B Number of CD45 cells per area (µm ) in the CC and HIP. C Representative image of MBP immunostaining in the CC. Green: Alexa488 (MBP). D Fluorescence intensity of MBP # ## #### immunostaining in the dotted area within the CC. (n = 3 per group). All values are Mean ± SEM. 2way ANOVA p < 0.05, p < 0.01, p < 0.0001 demyelination models can dampen symptoms by methamphetamine hypersensitivity, microglial activa- enhancing myelin repair and upregulate expression tion, increased proinflammatory cytokine production, levels of MBP, a major component of myelin [37, 38]. leucocyte entry into the parenchyma, and decreased Therefore, we confirmed changes in MBP protein levels myelin protein expression in mice. We revealed that a in the corpus callosum after short-term administration short, 3  day treatment with FTY720 completely allevi- of FTY720 by immunohistochemistry. A significant ates methamphetamine hypersensitivity and improves effect of Cuprizone exposure was seen on MBP staining measures of neuroinflammation (microglial activa - intensities with a marked decrease seen in cuprizone tion, cytokine production, and leucocyte infiltration). exposed groups, but no significant effect of FTY720 These results emphasize the usage of FTY720 as an administration was observed (Fig. 4C, D). anti-inflammatory treatment of psychosis and calls for further investigation of the neuroinflammatory and Discussion immunoregulatory mechanisms in the CNS related to In this study, we examined the effects of short-term psychosis and other psychiatric symptoms. treatment of FTY720 in an inflammatory mouse FTY720 was especially effective in reducing psycho - model of psychosis, demonstrating for the first time stimulant hypersensitivity, reducing levels of locomotion that FTY720, an immunosuppressant which functions after methamphetamine treatment to levels similar to as a potent inhibitor of microglial activation in cul- non-cuprizone-treated controls. Methamphetamine is a ture [25], is effective in alleviating the schizophrenia- powerful and highly addictive stimulant with effects such related behavioral symptoms and neuroinflammation in as excitement, elimination of fatigue, mood enhance- inflammatory psychosis model mice. In agreement with ment, and euphoria, which functions by increasing syn- past studies, short-term cuprizone exposure induced aptic dopamine concentration [38]. In human patients Li et al. Molecular Brain (2023) 16:59 Page 8 of 14 with schizophrenia, hypersensitivity to psychostimu- with such observations, increased signals of Iba1 pro- lants including methamphetamine have been reported tein were observed in the HIP, CC and STR of short- [30, 39], suggesting overlap between methamphetamine term cuprizone exposed mice, suggesting activated states targeted regions and ones responsible for the symptoms of microglia. In particular, a strong increase in staining of schizophrenia. In this study, we chose minimal doses was observed in the HIP. On the other hand, the Cup- of methamphetamine (2  mg/kg) which do not induce FTY group showed decreased Iba1 signal staining in the marked increases in locomotion above basal levels in the HIP, suggesting that FTY720 is effective in attenuating untreated group (Cont-Veh). In contrast to controls and microglial activation in the HIP. A decrease in Iba1 sig- similar to human patients, short-term cuprizone-exposed nal as a result of FTY720 treatment was also observed mice were hypersensitive to methamphetamine treat- in both the HIP and CC of the Cont-FTY group, which ment and exhibited marked increases in locomotor activ- was not exposed to cuprizone, indicating that FTY720 ity. In the short-term cuprizone-exposed mice treated can decrease basal levels of microglial activation in the with FTY720, hyperlocomotion was markedly decreased absence of inflammatory stimulation. Interestingly, acti - and reduced to similar levels as Cont-Veh. vation of microglia in the CC of cuprizone exposed mice Methamphetamine administration has been known to could not be suppressed by FTY720. Cuprizone is known be closely associated to STR dopamine and glutamate to affect different regions of the brain differentially, with levels [40]. Moreover, inflammatory conditions within the CC being a region especially targeted by cuprizone the STR may alter synaptic connectivity and disrupt demyelination [46]. Similar to long-term exposure mod- STR related circuitry [41]. Recent studies have provided els of demyelination, regions surrounding the CC may be evidence that methamphetamine administration is also the primary region affected by acute cuprizone toxicity, relevant to HIP functions, where methamphetamine thus having higher levels of microglial activation which abuse has been reported to correlate with decreased make it difficult to attenuate with drug administration. HIP volume and white matter hypertrophy [42], as well Moreover, recent evidence suggests that there are mul- as alterations of glial cell populations and expression of tiple different subsets of microglia with differential gene inflammatory factors in the HIP [43]. As shown in Figs.  2 expression profiles [47], and population specific accu - and 3, our results are consistent with reports that short- mulation in the CC may have decreased expression of term cuprizone exposure is associated with microglial FTY720 targets. It is possible that the different distribu - activation and increased inflammatory cytokines in HIP tion of microglial subtypes specific to the hippocampus and STR [29]. These findings suggest that short-term and corpus callosum may be related to the ameliorative exposure to cuprizone may have caused some dam- effects against microglial activation. Further studies on age to the STR and HIP, resulting in behavioral abnor- the specific microglial subtypes and regions affected by malities. Furthermore, suppressing HIP inflammation cuprizone exposure may provide insight onto the popula- may improve behavioral abnormalities in response to tions targeted by FTY720. methamphetamine. In the present study, a marked increase in mRNA levels Interestingly, in the Cont-FTY group, which was not of IL-6 was observed in the HIP of the cuprizone exposed exposed to cuprizone but treated with FTY720, an group. Moreover, trends towards increase of TNF-α and increasing trend in methamphetamine induced loco- IL-1β was observed, though the changes were not statis- motor activity was observed. This suggests that at doses tically significant. In contrast, proinflammatory cytokines used in this study, FTY720 most likely does not suppress were not markedly increased in the CC and STR, with locomotor activity, as well as locomotor-function related changes in all measured proinflammatory cytokines (IL- dopaminergic signaling, and counteracts the cuprizone- 6, TNF-α, IL-1β) not being statistically significant. Such induced increase in locomotion through other mecha- results may suggest region-specific differences in the nisms such as altering glial functions related to synaptic surface receptor expression in residential cells [48] and maintenance [23, 44, 45]. consequent differences in susceptibility to neuroinflam - Microglia are the innate immune cells of the CNS mation. Treatment with FTY720 significantly suppressed and are activated in response to various inflammatory/ the elevation of IL-6 in the HIP. Changes in IL-6 as a psychological stimuli such as tissue injury, infection, result of cuprizone exposure and FTY720 administration cytokine signaling and psychological stress. Microglial coincided with observed changes in Iba1, suggesting that activation leads to upregulation of Iba1 as well as the the anti-inflammatory effects of FTY720 are mediated release of response factors including proinflammatory through suppression of microglial activity. In our experi- mediators (ROS, cytokines, etc.) [12], and clinical stud- ments, we saw significant increases of IL-4 expression as ies have reported microglial hyperactivation in the brains a result of FTY720 administration in both the HIP and of schizophrenic and suicidal patients [31, 32]. In line CC. As IL-4 is a cytokine with potent anti-inflammatory Li  et al. Molecular Brain (2023) 16:59 Page 9 of 14 effects [49, 50], the suppression of microglial activation anti-inflammatory and cannot directly rescue cuprizone- and proinflammatory IL-6 expression may be due to induced oligodendrocytic damage/loss, which is the pri- increased IL-4 production, although the effects seem to mary cause of inflammation in this model. differ between the HIP and CC. In summary, this study utilized behavioral, immuno- Microglial activation and subsequent production of histochemical, and transcriptional analyses to evalu- pro-inflammatory cytokines can disrupt the BBB [18], ate the effects of short-term FTY720 administration causing proinflammatory cell populations such as cir - on an inflammatory short-term cuprizone exposure culatory leukocytes to enter into the brain parenchyma model of psychosis. As a result, short-term administra- [51]. Observation of cell populations with high expres- tion of FTY720 improved disorder-related behavior and High sion of CD45 (CD45 ) in the brain can visualize infil - reduced inflammation by suppressing microglial acti - trating leucocytic populations (mainly monocytes) [34]. vation in the HIP, highlighting its future use as a direct High In this study, a marked increase in CD45 popula- anti-inflammatory treatment against microglial activa - tions was observed in the CC of the cuprizone exposed tion and psychosis. These findings are consistent with the groups, suggesting that short-term cuprizone exposure microglial hypothesis in psychosis such as schizophrenia destabilizes the BBB, and allows for increased infiltration and suggests that the suppression of microglial activation of circulatory leukocyte populations. Short-term treat- could alleviate psychosis pathology [13, 14]. It has been ment with FTY720 did not significantly alter the num - reported that neuroinflammation and/or the inflam - High bers of C D45 populations in the HIP or CC. As an masome pathway is associated with many neurological oral therapy for multiple sclerosis, the immunosuppres- diseases [15–18, 57, 58].  New methods such as induced sive effects of FTY720 restrict lymphocytes to lymphatic microglia-like (iMG) cells differentiated from human nodes, which is known to be mediated through the tran- peripheral blood monocytes which exhibit microglia-like sient downregulation and degradation of the S P receptor gene expression, branching morphogenesis, and phago- on circulatory lymphocytic populations [52]. It has been cytic activity with various cytokine releases [59–61] have reported that FTY720 treatment lowers circulating leu- been developed to allow for evaluation in human tissue kocyte count and leukocyte recruitment in the CNS [53]. derived cells. iMG cells differentiated from schizophre - However, our results show that a short-term, three-day nia patients may be used effectively to investigate the treatment does not significantly decrease brain parenchy - inhibitory effects of FTY720 on microglial activation in High mal leucocytic (C D45 ) populations. The neuroinflam - humans. matory state induced by short-term cuprizone exposure Short-term cuprizone exposure causes oligodendrocyte is significantly different from the reported LPS inflamma - dysfunction, which includes disruption of mitochondrial tion model, and it is possible that the effect of FTY720 and metabolic function, as well as ROS production [62]. administration is affected by differences in the degree Cuprizone exposure also causes amino acid depletion of leukocyte infiltration [53]. Additionally, FTY720 has [62]. As a result, it has been reported that the synthesis been shown to pass through the BBB and accumulate in of myelin-related proteins such as MBP may decrease the brain [23, 54] and can target microglial S P receptors [62]. Following oligodendrocyte injury, other glial cells expressed in CNS resident cells. Moreover, there is evi- including microglia are activated, and start to contribute dence of FTY720 preventing BBB disruption by altering to innate immune responses [62] by releasing proinflam - tight junction proteins that form the BBB [55]. There - matory cytokines. Such proinflammatory responses can fore, the anti-neuroinflammatory effects of FTY720 seen cause neural inflammation, resulting in disease-related in this study can most likely reflects the direct effects of symptoms. It has been reported that increased micro- FTY720 on CNS resident cells or BBB forming cells. glial activation causes neuroinflammation and impairs Previous studies on schizophrenia have focused on hippocampal neurogenesis, neuronal synaptic morphol- myelin-related genes as a genetic risk factor of schizo- ogy, and synaptic plasticity [63, 64]. Moreover, it has phrenia [37], and white matter abnormalities correlate been reported that FTY720 attenuates microglial activ- with the stages of schizophrenia [56]. As FTY720 admin- ity and downregulates the production of proinflamma - istration has been reported to have protective effects on tory cytokines by activated microglia [65]. Such insights models of inflammatory demyelination [38], we stained suggest the possibility of FTY720 suppressing neural cell for MBP in the CC, a major white matter structure, to dysfunction (inflammation, synaptic dysfunction, etc.) investigate if FTY720 administration can directly pre- caused by short-term cuprizone exposure to alleviate vent oligodendrocytic damage. We observed a decrease pathological behavior. In addition to the microglial activ- in MBP staining in the cuprizone exposed groups, which ity examined in this study, FTY720 is known to regulate FTY720 administration was not able to rescue. This sug - immunoinflammatory responses through various mecha - gests that the ameliorative effects of FTY720 are mainly nisms (Additional file 4: Fig. S4) [25, 66]. Li et al. Molecular Brain (2023) 16:59 Page 10 of 14 Behavioral analysis The full picture of human psychosis pathology still Rotarod remains largely elusive and continues to be difficult to An accelerating rotarod (ROTA ROD; UGO BASILE) was capture. Elucidation of the causes and development of used, with the initial speed set at 4  rpm, acceleration at therapeutic strategies likely requires further contribu- 20 rpm/min, and maximum speed at 40 rpm. The mouse tions from various fields including animal models, clini - was placed on the rotating rod in the direction opposite cal studies, and human genetics. Short-term cuprizone to the direction of rotation. The test was started after exposure in mice, the model used in this study, can be the mouse was able to stand and move upright on the an effective model of the neuroinflammatory symptoms rotarod. The duration of time till falling to the platform of psychosis, and can be further utilized to study the below was recorded, with the maximum test duration set connections between psychiatric symptoms and inflam - to 120 s after the start of acceleration. Mice were subject matory phenomena due to their relevance in behavio- to three trials in one day, performed 60 min apart. ral characteristics and state of neuroinflammation [32, 33]. Further information into the microglial populations Methamphetamine‑induced locomotion test altered by cuprizone and/or FTY720 and the cellular Methamphetamine (Sumitomo Pharma) was dissolved in mechanisms of underlying the phenomena observed physiological saline and administered intraperitoneally in this study may provide valuable insight on the con- at a final concentration of 2 mg/kg. Mice were left in the nections between neuroinflammatory phenomena and experimental room for over an hour before being trans- behavioral alteration. ferred to a circular open field (diameter 40  cm, height 27  cm, grey plexiglass). After 30  min of baseline loco- Methods motor activity measurement, mice were given intraperi- Animals toneal injections of methamphetamine and locomotion All animal experiments were performed in accordance were measured for 60  min after injection. Mouse move- with protocols of the Animal Experimentation Commit- ment was tracked, and distance traveled (cm/5 min) was tee of the Institute for Protein Research, Osaka Univer- measured using EthoVision XT (Noldus). sity. Male C57BL/6NJcl mice were obtained from CLEA Japan. Mice were maintained in a quiet environment Y‑maze test with a controlled temperature of 24 ± 2  °C. During a The Y-maze test was administered according to Sakurai 12-h light/dark cycle, mice had constant access to either et al. [67], with minor modifications. In brief, the Y-maze a standard solid chow (AIN-93M, ORIENTAL YEAST consisted of 3 arms (42  cm long, 16  cm high and 12  cm CO., LTD.) or identical chow containing 2% w/w cupri- wide) configured 120 degrees apart. Mice were placed in zone (AIN-93M (99.8%) + cuprizone (0.2%), ORIENTAL the center of the arm and allowed to move freely in the YEAST CO.,LTD.) and water. After arriving at 6  weeks, maze for 10  min. Mouse movements and arm entries mice were habituated to the standard chow for seven were tracked, recorded, and counted using EthoVision days, then subsequently divided into four groups (Cont- XT (Noldus), and calculation of successful alternation Veh, Cont-FTY, CUP-Veh, CUP-FTY) and fed either the rate was calculated using an R script (Additional file  1). standard diet (Cont-Veh, Cont-FTY) or the cuprizone- Successful alternation was defined as entering all three formulated diet (CUP-Veh, CUP-FTY) for seven days arms consecutively. (Additional file 4: Fig. S1). Immunohistochemistry Drug administration Immunohistochemistry was performed according to FTY720 {2-amino-2-[2-(4-octylphenyl) ethyl]-1,3-pro- Aomine et  al. [68], with minor modifications. Ani - panediol} was obtained from Cayman Chemical. mals were deeply anesthetized with isoflurane prior Powdered FTY720 was dissolved in dimethylsulfoxide- to experimentation and perfused transcardially with hydrochloride (DMSO-HCl) to make a 0.2  g/ml stock phosphate-buffered saline (PBS, pH 7.4) followed by 4% and stored at − 20 °C. At the time of use, the stock solu- Paraformaldehyde/PBS, and post fixed overnight in 4% tion was diluted with physiological saline to produce a Paraformaldehyde/PBS at 4 ℃. For cryoprotection, brains 0.2 mg/ml solution. Identical DMSO-HCl solution with- were placed in 15% sucrose/PBS for one day, after being out FTY720 was dissolved in saline for control. Mice moved to 30% sucrose/PBS for ~ 36 h at 4 ℃. Brains were were treated for three days with daily intraperitoneal frozen and embedded in embedding agent (TissueTek injections of FTY720 1 mg/kg starting four days after the O.C.T compound, Sakura Finetek Japan Co., Ltd., Tokyo, start of cuprizone exposure (no injections on experimen- Japan) and was sectioned at 40  μm using a cryostat tal day) (Additional file 4: Fig. S1). Li  et al. Molecular Brain (2023) 16:59 Page 11 of 14 3D cell morphology analysis of microglia microtome (Leica CM1860, Leica Biosystems). To avoid Animals were deeply anesthetized with isoflurane prior deformation, the sections were free-float-processed with to experimentation and were perfused transcardially with extreme caution. phosphate-buffered saline (PBS, pH 7.4), followed by a Sections were washed three times with PBS-T (0.3% fixative (a mixture of 4.0% paraformaldehyde and 0.05% Triton-X) and incubated overnight (4  ℃) in an anti- glutaraldehyde in 0.1 M PBS). The brains were left in situ body solution (0.3% Triton X PBS W/ 10% Normal Goat for 2  h at room temperature and were then removed Serum (Thermo Fisher) + primary antibody). Primary from the skull. After cryoprotection with 30% sucrose, antibodies used were Anti Iba1 (1:1000; Rabbit, FUJI- brain blocks were cut into 40-μm-thick sections on a FILM Wako, 019-19741), Anti CD45 (1:100; Mouse, cryostat (Leica CM1950, Wetzlar, Germany). To avoid BIO-RAD, MCA87) and Anti Myelin Basic Protein deformation, the sections were free-float-processed with (1:500; Rabbit, Abcam, ab133620). After primary anti- extreme caution. Sections were blocked for 30  min with body staining, sections were washed three times with 1% bovine serum albumin (BSA) in PBS containing 0.3% PBS and incubated in a secondary antibody solution Triton X-100 and 0.1% sodium azide at 4 °C. After block- (0.3% Triton X PBS W/0.5% Normal Goat Serum + sec- ing, the sections were incubated with Anti Iba1 (1:20000; ondary antibody) for 2 h (RT). Secondary antibodies used Rabbit, FUJIFILM Wako, 019-19741) for 2  days at 4  °C. were Anti Rabbit IgG, Alexa Fluor 488 (1:500; goat, After washing with PBS, the sections were incubated ThermoFisher, A-11034), Anti Mouse IgG, Alexa Fluor with a mixture of Alexa488-conjugated donkey anti-rab- Plus 647 (1:100; Goat, ThermoFisher, A-32728). The sec - bit IgG antibody (1: 300; Jackson ImmunoResearch Lab- tions were washed three times with PBS, mounted on a oratories, West Grove, PA, USA) overnight at 4  °C. The glass slide with Mounting Medium w/DAPI (Abcam, sections were counterstained with DAPI and mounted ab104139), coverslipped, and imaged with a Keyence BZ with Vectashield (Vector Laboratories, Burlingame, CA). X810 microscope. Fluorescent images were acquired with a confocal laser scanning microscope (FV-1000-D; Olympus, Japan). Sec- Fluorescence intensity analysis tions were randomly sampled and processed for immu- HALO analysis nohistochemical staining. The 3D hippocampal microglia Fluorescence intensity was analyzed using HALO images were created with a × 60 objective on an FV- (Indicalab). Three individual images at 40× magnifica - 1000-D at 0.5-μm intervals along the z-axis. Following tion were obtained per region per one mouse were. Care acquisition, images were compiled and transformed into was taken to select the same regions for each individual. 3D microglial renderings using Imaris software (Bitplane, Thresholds were set to automatically detect DAPI posi - Zurich, Switzerland). The morphological microglial tive nuclei and Iba-1 positive signal and were kept the parameters (cell surface area and cell volume) were meas- same for all measurements. Iba-1 fluorescence inten - ured using the Imaris MeasurementPro function. The sity was measured for all Iba-1 positive nuclei and was microglia ramification ratio was calculated as the ratio of average Iba-1 fluorescence intensity was calculated per cell surface area to cell volume [69]. The morphological image analyzed by HALO. The average values of three parameters from 545 to 631 microglia in each group were measurements per individual was used for further calculated from ten sections per mouse (n = 3 mice each). analysis. Quantitative real‑time PCR ImageJ analysis Eight-week-old mice were deeply anesthetized with Sections were stained by the method described above, isoflurane, decapitated, and the entire brain  was then observed and photographed with a Keyence BZ removed  rapidly from the skull. The whole brain was X810 microscope. The captured images were imported sliced into 1  mm sections using a brain matrix (Brain- into Image J, and the fluorescence intensity per area science Idea, Osaka, Japan), and the striatum, hippocam- (300  µm*120  µm; indicated by dotted line) of the cor- pus, and corpus callosum were removed from sections pus callosum region of 3 sections for each individual using a razor blade. Removed sections were placed in was measured. Prior to statistical analysis, mean values RNAlater, kept at 4 ℃ overnight, and then frozen at were calculated for each group (n = 3 mice each) based − 80 ℃. on the measured values, and relative values were cal- RNA was extracted from tissue using RNeasyMini- culated using the mean value of control mice as 100%. Kit (Qiagen) and reverse transcribed into cDNA with Fluorescence intensity statistics results are shown as the ReverTra Ace qPCR RT Master Mix (Toyobo). mean ± SEM. Li et al. Molecular Brain (2023) 16:59 Page 12 of 14 Quantitative real-time PCR was performed using were sacrificed on day 7. All experiments were performed in four groups: GeneAce SYBR qPCR Mix a Low Rox (NIPPON GENE) Cont-Veh, Cont-FTY720, CUP-Veh, CUP-FTY. Fig S2. Eec ff ts of FTY720 on short-term spatial memory in cuprizone-exposed mice. Y-maze test for reagent and QuantStudio 6 pro (Thermo Fisher). Quan - short-term spatial memory was used to assess the effects of cuprizone tification was done using a relative standard curve, and exposure and the effects of FTY720 administration. Total distance. (B) Total βActin was used as an internal control. qPCR run method arm entries. (C) Successful alternation rate. Fig S3. Representative image of immunostaining in hippocampus and corpus callosum. (A) Representa- and Primer information is shown in Additional file 2. tive image of Iba1 immunostaining in the HIP. (B) Representative image of Prior to statistical analysis of quantitative real-time Iba1 immunostaining in the CC. Blue: DAPI nuclear stain, Green: Alexa488 PCR data, the cuprizone group mRNA expression lev- (Iba1). An enlarged view of the top row is shown in the bottom row. Fig S4. Hypothesis of FTY720 mechanism of action in psychosis model mice. els were normalized to the control group for each brain Short-term cuprizone exposure does not cause prominent demyelination, region. Fluorescence intensity and quantitative real-time but specifically causes damage to oligodendrocytes. Such damage results PCR statistical results are shown as mean ± SEM. in activation of glial populations including microglia, triggering the release of proinflammatory cytokines. Extracellular release of proinflammatory cytokines induces neuronal dysfunction and contributes to behavioral Statistical analysis disorders such as psychosis. FTY720 administration inhibits microglial acti- All statistical analysis was performed using Prism8 vation and regulates pro-inflammatory cytokine release, reduces neuronal damage, and improves psychosis-like behavior. (GraphPad Software), except for Fig.  2G which was analyzed using the “rstatix” library in R. The number of individual samples used for each set of experiments is Acknowledgements We would like to thank all Hikida lab members for their valuable comments indicated in the results section and figure legends. Details and support. of ANOVA and post-hoc  analyses are provided in Addi- tional file 3. Author contributions TAK, and TH conceived the study. KS, MO, TAK, and TH designed experiments. SL, KS, and MO performed the experiments. SL, KS, and MO analyzed and interpreted data. SL, KS, and MO wrote the manuscript. All authors read and Abbreviations approved the final manuscript. IL-6 Interleukin 6 TNF-α Tumor necrosis factor-α Funding CNS Central nervous system This study was supported by JSPS KAKENHI, Grants Numbers JP22H03000 to AD Alzheimer’s disease MO, JP22H00494 to TAK, JP21H05694 and JP22H02944 to TH, AMED under PD Parkinson’s disease Grant Numbers JP22dk0207065 to MO, JP21wm0425010 and JP21gm1510006 ALS Amyotrophic lateral sclerosis to TH, JST SPRING under Grant Number JPMJSP2138 to SL and KS, SENSHIN MS Multiple sclerosis Medical Research Foundation to TH, Taiju Life Social Welfare Foundation to PET Positron emission tomography TH, and the Collaborative Research Program of Institute for Protein Research, FTY720 Fingolimod Osaka University, ICR-22-03. SP Sphingosine 1-phosphate TRMP7 Transient receptor potential cation channel subfamily M member Availability of data and materials All data needed to evaluate the conclusions in the paper are present in the PP2A Protein phosphatase 2A paper. The datasets used and/or analyzed during the current study are avail- HIP Hippocampus able from the corresponding author on reasonable request. CC Corpus callosum Iba1 Ioniz ed calcium binding adapter molecule 1 IL1-β Interleukin-1beta Declarations IL-4 Interleukin 4 MBP Myelin basic protein Ethics approval and consent to participate BBB Blood–brain barrier All animal procedures and experiments were performed by the Institute for STR Striatum Protein Research, Osaka University animal committee regulations. Consent for publication Supplementary Information Not applicable. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13041- 023- 01047-5. Competing interests The authors have declared that no conflict of interest exists. Additional file 1:  Y-maze R script. R script to calculate succesful alterna- tion rate for Y-maze. Received: 31 December 2022 Accepted: 21 June 2023 Additional file 2: RT-qPCR Details. qPCR run method and primer information. Additional file 3: Statistics Details. F and P values for ANOVAs, and P values for post-hoc tests. References Additional file 4: Fig S1. Experimental Timeline for Short-Term Cupri- 1. Galderisi S, Davidson M, Kahn RS, Mucci A, Boter H, Gheorghe MD, zone Exposure. 6 weeks old mice were habituated to the control diet Rybakowski JK, Libiger J, Dollfus S, López-Ibor JJ, Peuskens J, Hranov (w/o cuprizone) for 7 days (− 7 to 0 days) before administration of the LG, Fleischhacker WW, EUFEST group. Correlates of cognitive impair- cuprizone-containing diet. FTY720 was administered intraperitoneally 4, 5 ment in first episode schizophrenia: the EUFEST study. Schizophr Res. and 6 days after administration of the cuprizone containing diet. All mice 2009;115(2–3):104–14. Li  et al. Molecular Brain (2023) 16:59 Page 13 of 14 2. Harvey PD, Bowie CR, Friedman JI. 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Ameliorative effects of Fingolimod (FTY720) on microglial activation and psychosis-related behavior in short term cuprizone exposed mice

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Abstract

Schizophrenia is a psychiatric disorder that affects around 1% of the population in widespread populations, with severe cases leading to long-term hospitalization and necessitation of lifelong treatment. Recent stud- ies on schizophrenia have highlighted the involvement of inflammatory and immunoregulatory mechanisms with the onset of symptoms, and the usage of anti-inflammatory treatments are being tested against periods of rapid psychosis. In the central nervous system, microglia are the innate immune population which are activated in response to a wide range of physical and psychological stress factors and produce proinflammatory mediators such as cytokines. Microglial activation and neuroinflammation has been associated to numerous psychiatric disor - ders including schizophrenia, especially during psychotic episodes. Thus, novel treatments which dampen microglial activation may be of great relevance in the treatment of psychiatric disorders. Fingolimod (FTY720) is a drug used as an immunosuppressive treatment to multiple sclerosis. Recent clinical trials have focused on FTY720 as a treatment for the behavioral symptoms in schizophrenia. However, the mechanisms of Fingolimod in treating the symptoms of schizophrenia are not clear. In this study we use a recently developed neuroinflammatory psychosis model in mice: cuprizone short-term exposure, to investigate the effects of FTY720 administration. FTY720 administration was able to completely alleviate methamphetamine hypersensitivity caused by cuprizone exposure. Moreover, administration of FTY720 improved multiple measures of neuroinflammation (microglial activation, cytokine production, and leuco - cyte infiltration). In conclusion, our results highlight the future use of FTY720 as a direct anti-inflammatory treatment against microglial activation and psychosis. Keywords Short-term cuprizone exposure, Psychosis, Fingolimod, Microglial activation, Neuroinflammation *Correspondence: Department of Functional Anatomy and Neuroscience, Asahikawa Koki Sakurai Medical University, Hokkaido, Japan ksakurai@protein.osaka-u.ac.jp Department of Neuropsychiatry, Graduate School of Medical Sciences, Takahiro A. Kato Kyushu University, Fukuoka, Japan kato.takahiro.015@m.kyushu-u.ac.jp Takatoshi Hikida hikida@protein.osaka-u.ac.jp Laboratory for Advanced Brain Functions, Institute for Protein Research, Osaka University, Suita, Osaka, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Present Address: Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, Osaka, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. Molecular Brain (2023) 16:59 Page 2 of 14 expressed in most neuronal lineages and resident CNS Introduction cells, especially neural-derived glia and neurons [23, Schizophrenia is a psychiatric disorder that affects 24]. Moreover, Fingolimod has been reported to interact around 1% of the population in widespread populations, with other targets expressed in the CNS such as Tran- with severe cases leading to long-term hospitalization sient Receptor Potential Cation Channel Subfamily M and necessitation of lifelong treatment [1–4]. Although Member 7 (TRMP7), Histone Deacetylase, and Protein the etiology of schizophrenia has not yet been fully elu- phosphatase 2A (PP2A), functioning as a potent inhibi- cidated yet, genetic and environmental evidence has tor of microglial activation in culture [25]. In clinical pointed to dopaminergic disfunction and glutamater- trials, the efficacy of Fingolimod against patients with gic hypofunction as dominant hypotheses underlying schizophrenia reemphasized the importance of treat- symptoms [5]. Furthermore, recent studies have high- ing dysregulated inflammatory states during acute psy - lighted the involvement of inflammatory and immu - chotic exacerbations [8]. However, the mechanisms of noregulatory mechanisms in the onset of schizophrenia Fingolimod in treating psychosis-related behavioral and related psychotic events [6, 7]. Based on hypotheses symptoms, especially in relation to neuroinflammation that psychotic episodes result from acute exacerbation of is not understood and calls for investigation using ani- neuroinflammatory status, usage of anti-inflammatory mal models to elucidate the pharmacological pathways treatments against periods of rapid psychosis are being targeted by FTY720. investigated [8]. Such use of anti-inflammatory treat - Cuprizone short-term exposure is a recently devel- ment has been supported by further evidence of anti- oped neuroinflammatory psychosis model in mice which inflammatory drugs alleviating symptoms in patients utilizes acute administration of cuprizone (Oxalic acid with early-stage schizophrenia [9]. Despite such clinical bis(cyclohexylidenehydrazide)), a copper depleting agent evidence demonstrating the effectiveness of anti-inflam - that induces oligodendrocytic damage through mito- matory treatments, the causality and mechanism of chondrial dysfunction [26, 27]. Short-term exposure to neuroinflammatory events underlying psychosis-related cuprizone has been shown to induce behavioral abnor- behavioral deficits are yet to be fully uncovered. malities including hypersensitivity to psychostimulants Microglia are the primary innate immune population in (methamphetamine and phencyclidine) and deficits in the central nervous system (CNS), with known implica- short-term memory related tasks [28], most likely asso- tions in brain development, neural network maintenance, ciated with increased expression of proinflammatory and neural injury repair [10, 11]. Microglia are activated cytokine interleukin 6 (IL-6) in the hippocampus [29]. In in response to a wide range of physical and psychologi- this study, we utilized this neuroinflammatory psychosis cal stress factors, which trigger the production of proin- model mouse to investigate the effects of FTY720 admin - flammatory cytokines and reactive oxygen species [12], istration on  behavioral abnormalities, microglial activa- and lead to neuronal degeneration and/or white matter tion status, and proinflammatory induction. abnormality in the long term [13, 14]. In humans, micro- glial activation has been associated to various psychiatric Results and neurological disorders such as schizophrenia [13], Sample groups Alzheimer’s disease (AD) [15], Parkinson’s disease (PD) To investigate the effects of FTY720 on short-term cupri - [16], Amyotrophic lateral sclerosis (ALS) [17], and Mul- zone treated mice, 7  week old C57BL/6NJcl mice were tiple sclerosis (MS) [18]. As an investigative treatment to fed either control (Cont) or cuprizone (CUP) chow for psychiatric disorders, minocycline, a widely used antibi- 1 week and administered either Vehicle (Veh) or FTY720 otic, is known to inhibit microglial activation and have an (FTY) (4 Groups: Cont-Veh, Cont-FTY, CUP-Veh, CUP- ameliorative effect on the clinical performance in schizo - FTY; Additional file 4: Fig. S1). phrenic patients [19–21]. Thus, novel treatments which dampen microglial activation may be of great relevance FTY720 inhibited methamphetamine induced in the treatment of psychiatric disorders [12, 14]. hyperactivity in short term cuprizone treated mice A particular treatment of recent interest regarding The psychiatric symptoms of chronic methamphetamine neuroinflammation is Fingolimod (FTY720). Known addiction closely resemble the positive symptoms of as an immunosuppressive treatment to MS, the active, schizophrenia, and schizophrenic patients are more prone phosphorylated form of Fingolimod depletes circula- to the effects of methamphetamine [30]. Similarly, short- tory lymphocytes after administration by agonizing term cuprizone exposure induces behavioral deficits in lymphocytic sphingosine 1-phosphate (S P) receptors, mice that are in line with human behavioral symptoms, resulting in reduced infiltration of autoreactive lym - including hypersensitivity to acute methamphetamine phocytes into the brain parenchyma [22–24]. Other administration [29]. We investigated the effects of FTY720 than in lymphocytic populations, S P receptors are 1 Li  et al. Molecular Brain (2023) 16:59 Page 3 of 14 Fig. 1 Eec ff ts of FTY720 on behavioral changes in short-term cuprizone-exposed mice. A, B Eec ff ts of FTY720 on locomotor activity in mice after methamphetamine administration. Mice were placed in a circular open field for 30 min to measure baseline locomotion, whereafter an intraperitoneal injection of methamphetamine (2 mg/kg) was administered to the mice and locomotion was measured for 60 min. A The locomotion (ambulatory counts) for 5-min bin within the total 90 min session (− 30 to 0 min before and 0 to 60 min after methamphetamine injection). Arrows indicate methamphetamine injection time. B Total locomotion (cumulative horizontal ambulatory counts) after injection of methamphetamine (0-60 min). n = 10–11 per group. C Rotarod measurement of basal locomotor function. The latency to fall from the rotarod (s) with maximum latency set at 120 s (n = 5 per group). All values are Mean ± SEM. Tukey test *p < 0.05, ***p < 0.001. A, C 3way RM ANOVA. B 2way # ### #### ANOVA p < 0.05, p < 0.001, p < 0.0001 Li et al. Molecular Brain (2023) 16:59 Page 4 of 14 on such increases in methamphetamine sensitivity in a were not significantly affected by FTY720 administration methamphetamine-induced locomotion test. (Fig. 2C, E, G). A significant interaction between Cuprizone exposure To assess microglial activation in further detail, micro- and FTY720 treatment was observed on locomotion glial morphology in the HIP was analyzed in three after methamphetamine administration, with short- dimensions using high magnification, z-stacked micros - term cuprizone-exposed mice (CUP-Veh) showing sig- copy (Fig.  2H, I). A significant effect of both Cuprizone nificantly higher levels of locomotion compared to the administration and FTY720 was observed on microglial control chow group (Cont-Veh) (Fig. 1A, B). Moreover, a ramification ratios, a measure of microglial activation significant effect of FTY720 on locomotion was observed, state (Fig.  2I). In the cuprizone exposed groups (CUP- where the significant increase in locomotor activity in the Veh, CUP-FTY), there was a lower ramification ratio CUP-Veh group was suppressed in the FTY720 treated compared to groups with control chow (Cont-Veh, Cont- Cuprizone group (CUP-FTY), to levels similar to the con- FTY), indicating ameboid-like morphological charac- trol chow group (Cont-Veh) (Fig.  1A, B). There was no teristics of activated microglia. FTY720 was effective in significant effect of FTY administration between groups significantly increasing ramification ratio compared to that were not exposed to cuprizone (Cont-Veh and Cont- Veh treated groups, indicating morphological character- FTY) (Fig.  1A, B). Examination of the basal motor func- istics of resting state microglia. Increased ramification tions of the 4 groups of mice in the rotarod test showed ratio as a result of FTY720 administration was effective no significant effect of either Cuprizone or FTY on motor in restoring decreased ramification ratio in the CUP- ability between groups (Fig.  1C). Evaluation of spatial Veh group to levels similar to untreated Cont-Veh group memory using the Y-maze test showed that short-term (Fig. 2I). cuprizone exposure does not affect total locomotion and arm entry. Although there was no significant difference FTY720 has anti‑inflammatory effects in the hippocampus in correct alternation amongst groups, a downward trend Inflammatory activation in the brain induces the pro - was seen in cuprizone exposed groups (Additional file  4: duction and release of proinflammatory cytokines, Fig. S2). mainly by glial cell populations, and cytokine alteration has been reported in schizophrenia patients [6, 7]. In FTY720 inhibits microglial activation in the hippocampus addition, IL-6 mRNA levels are known to be increased in Glial activation induces the expression and release of the HIP and STR of short-term cuprizone-exposed mice cytokines, and postmortem brain studies of schizo- [29]. We examined the expression levels of proinflam - phrenic patients have reported hyperactivation of micro- matory cytokines interleukin 1 beta (IL-1β), IL-6, Tumor glia [31, 32]. In addition, increased expression levels necrosis factor alpha (TNF-α), along with the expres- of  the microglial gene ionized calcium binding adapter sion level of anti-inflammatory cytokine interleukin 4 molecule 1 (Iba1), indicative of microglial activation, (IL-4) to evaluate changes in neuroinflammatory status have been demonstrated in short-term cuprizone- induced by short-term cuprizone exposure and FTY720 exposed mice [29]. Therefore, we measured microglial treatment. activation by immunohistochemical staining for Iba1 In the HIP, a significant effect of Cuprizone treatment protein (Fig.  2A, Additional file  4: Fig. S3) and assessed was seen in mRNA expression levels of proinflammatory the effects of cuprizone and/or FTY720 treatment. In cytokines IL-6 and TNF-α, with marked increases in the addition, we assessed for Iba1 mRNA gene expression in short-term cuprizone-exposed (CUP-Veh) group com- Hippocampus (HIP), Corpus Callosum (CC), and Stria- pared to other groups (Fig.  3A, C). Moreover, a signifi - tum (STR) using RT-qPCR. cant effect of FTY720 along with a significant interaction Significant effects of Cuprizone and FTY adminis - between Cuprizone exposure and FTY720 administra- tration was observed on Iba1 signal levels in the HIP, tion was observed on IL-6 mRNA in the HIP, with FTY CC, and STR, with Cuprizone administration causing treatment significantly decreasing IL6 expression in marked increases of Iba1 signal, indicating increased the CUP-FTY group compared to the CUP-Veh group microglial activation in the exposed groups (CUP- (Fig.  3A). In contrast, no significant effects of CUP and Veh and CUP-FTY) (Fig.  2B, D, F). In the HIP, FTY720 FTY was observed on expression levels of proinflamma - administration significantly decreased Iba1 levels in both tory cytokines in the CC and STR (Fig. 3E–L). In both the Cont and CUP groups (Fig.  2B). In comparison, in the HIP and CC, a significant effect of FTY720 administra - CC and STR, FTY720 was only effective in decreasing tion was observed on the mRNA expression level of the Iba1 levels in the Cont group (Fig. 2D, F). The expression anti-inflammatory cytokine IL-4, where FTY720 admin - levels of Iba-1 mRNA showed significant increases in istration caused increased IL4 expression (Fig. 3D, H). HIP, CC, and STR as a result of cuprizone exposure but Li  et al. Molecular Brain (2023) 16:59 Page 5 of 14 Fig. 2 Eec ff t of FTY720 on microglial activation in short-term cuprizone-exposed mice. A–C HIP. A Representative image of Iba1 immunostaining in the HIP. Blue: DAPI nuclear stain, Green: Alexa488 (Iba1). B Fluorescence intensity measurement of Iba1 signal in the HIP (n = 3 per group). C mRNA expression levels of Iba1 in the HIP. βActin was used for internal control, and samples were normalized to Cont-Veh. (n = 6 per group). D, E CC. D Fluorescence intensity measurement of Iba1 signal in the CC (n = 3 per group). E mRNA expression levels of Iba1 in the CC. F, G STR. F Fluorescence intensity measurement of Iba1 signal in the STR (n = 3 per group). G mRNA expression levels of Iba1 in the STR. βActin was used for internal control, and samples were normalized to Cont-Veh. (n = 6 per group). H, I Three-dimensional morphology analysis. H Representative image of z stack # ## #### imaging. I Microglial ramification ratio. B–G Mean ± SEM. Tukey test *p<0.05, **p < 0.01. 2way ANOVA p < 0.05, p < 0.01, p < 0.0001. I Violin Plot #### Min-Max values. Dotted lines indicate first, second, and third quartiles. 2way ANOVA p < 0.0001 Li et al. Molecular Brain (2023) 16:59 Page 6 of 14 Fig. 3 Eec ff ts of FTY720 on cytokine changes in short-term cuprizone-exposed mice. A–D mRNA expression levels of A IL-6, B TNF-a, C IL-1β, D IL-4 in the HIP. βActin was used for internal control, and samples were normalized to Cont-Veh. (n = 6 per group) E–H mRNA expression levels of E IL-6, F TNF-a, G IL-1β, H IL-4 in the CC. I–L mRNA expression levels of I IL-6, J TNF-a, K IL-1β, L IL-4 in the STR. βActin was used for internal control, # ## and samples were normalized to Cont-Veh. (n = 6 per group). All values are Mean ± SEM. Tukey test *p < 0.05. 2way ANOVA p < 0.05, p < 0.01 High FTY720 Short‑term administration did not up‑regulate CD45 cells [34] were observed in the CC of the short- leukocyte infiltration and myelin basic protein (MBP) term cuprizone-exposed mice, while no significant differ - expression ence was observed in the HIP (Fig. 4A, B). Neuroinflammation is known to cause disruption of the Furthermore, multiple studies have also reported BBB, resulting in the infiltration of circulatory leukocytes oligodendrocyte and myelin abnormalities and associ- into the brain. Such infiltration has been reported in ated gene dysregulation in the brains of patients with patients with schizophrenia [33]. A significant increase in schizophrenia [35, 36]. FTY720 treatment in rodent Li  et al. Molecular Brain (2023) 16:59 Page 7 of 14 high Fig. 4 Eec ff ts of FTY720 on CD45 cell population and MBP variability in short-term cuprizone-exposed mice. A Representative image of CD45 high high immunostaining in the HIP. Blue: DAPI nuclear stain, Green: Alexa488 (CD45). Arrows indicate CD45 cells. B Number of CD45 cells per area (µm ) in the CC and HIP. C Representative image of MBP immunostaining in the CC. Green: Alexa488 (MBP). D Fluorescence intensity of MBP # ## #### immunostaining in the dotted area within the CC. (n = 3 per group). All values are Mean ± SEM. 2way ANOVA p < 0.05, p < 0.01, p < 0.0001 demyelination models can dampen symptoms by methamphetamine hypersensitivity, microglial activa- enhancing myelin repair and upregulate expression tion, increased proinflammatory cytokine production, levels of MBP, a major component of myelin [37, 38]. leucocyte entry into the parenchyma, and decreased Therefore, we confirmed changes in MBP protein levels myelin protein expression in mice. We revealed that a in the corpus callosum after short-term administration short, 3  day treatment with FTY720 completely allevi- of FTY720 by immunohistochemistry. A significant ates methamphetamine hypersensitivity and improves effect of Cuprizone exposure was seen on MBP staining measures of neuroinflammation (microglial activa - intensities with a marked decrease seen in cuprizone tion, cytokine production, and leucocyte infiltration). exposed groups, but no significant effect of FTY720 These results emphasize the usage of FTY720 as an administration was observed (Fig. 4C, D). anti-inflammatory treatment of psychosis and calls for further investigation of the neuroinflammatory and Discussion immunoregulatory mechanisms in the CNS related to In this study, we examined the effects of short-term psychosis and other psychiatric symptoms. treatment of FTY720 in an inflammatory mouse FTY720 was especially effective in reducing psycho - model of psychosis, demonstrating for the first time stimulant hypersensitivity, reducing levels of locomotion that FTY720, an immunosuppressant which functions after methamphetamine treatment to levels similar to as a potent inhibitor of microglial activation in cul- non-cuprizone-treated controls. Methamphetamine is a ture [25], is effective in alleviating the schizophrenia- powerful and highly addictive stimulant with effects such related behavioral symptoms and neuroinflammation in as excitement, elimination of fatigue, mood enhance- inflammatory psychosis model mice. In agreement with ment, and euphoria, which functions by increasing syn- past studies, short-term cuprizone exposure induced aptic dopamine concentration [38]. In human patients Li et al. Molecular Brain (2023) 16:59 Page 8 of 14 with schizophrenia, hypersensitivity to psychostimu- with such observations, increased signals of Iba1 pro- lants including methamphetamine have been reported tein were observed in the HIP, CC and STR of short- [30, 39], suggesting overlap between methamphetamine term cuprizone exposed mice, suggesting activated states targeted regions and ones responsible for the symptoms of microglia. In particular, a strong increase in staining of schizophrenia. In this study, we chose minimal doses was observed in the HIP. On the other hand, the Cup- of methamphetamine (2  mg/kg) which do not induce FTY group showed decreased Iba1 signal staining in the marked increases in locomotion above basal levels in the HIP, suggesting that FTY720 is effective in attenuating untreated group (Cont-Veh). In contrast to controls and microglial activation in the HIP. A decrease in Iba1 sig- similar to human patients, short-term cuprizone-exposed nal as a result of FTY720 treatment was also observed mice were hypersensitive to methamphetamine treat- in both the HIP and CC of the Cont-FTY group, which ment and exhibited marked increases in locomotor activ- was not exposed to cuprizone, indicating that FTY720 ity. In the short-term cuprizone-exposed mice treated can decrease basal levels of microglial activation in the with FTY720, hyperlocomotion was markedly decreased absence of inflammatory stimulation. Interestingly, acti - and reduced to similar levels as Cont-Veh. vation of microglia in the CC of cuprizone exposed mice Methamphetamine administration has been known to could not be suppressed by FTY720. Cuprizone is known be closely associated to STR dopamine and glutamate to affect different regions of the brain differentially, with levels [40]. Moreover, inflammatory conditions within the CC being a region especially targeted by cuprizone the STR may alter synaptic connectivity and disrupt demyelination [46]. Similar to long-term exposure mod- STR related circuitry [41]. Recent studies have provided els of demyelination, regions surrounding the CC may be evidence that methamphetamine administration is also the primary region affected by acute cuprizone toxicity, relevant to HIP functions, where methamphetamine thus having higher levels of microglial activation which abuse has been reported to correlate with decreased make it difficult to attenuate with drug administration. HIP volume and white matter hypertrophy [42], as well Moreover, recent evidence suggests that there are mul- as alterations of glial cell populations and expression of tiple different subsets of microglia with differential gene inflammatory factors in the HIP [43]. As shown in Figs.  2 expression profiles [47], and population specific accu - and 3, our results are consistent with reports that short- mulation in the CC may have decreased expression of term cuprizone exposure is associated with microglial FTY720 targets. It is possible that the different distribu - activation and increased inflammatory cytokines in HIP tion of microglial subtypes specific to the hippocampus and STR [29]. These findings suggest that short-term and corpus callosum may be related to the ameliorative exposure to cuprizone may have caused some dam- effects against microglial activation. Further studies on age to the STR and HIP, resulting in behavioral abnor- the specific microglial subtypes and regions affected by malities. Furthermore, suppressing HIP inflammation cuprizone exposure may provide insight onto the popula- may improve behavioral abnormalities in response to tions targeted by FTY720. methamphetamine. In the present study, a marked increase in mRNA levels Interestingly, in the Cont-FTY group, which was not of IL-6 was observed in the HIP of the cuprizone exposed exposed to cuprizone but treated with FTY720, an group. Moreover, trends towards increase of TNF-α and increasing trend in methamphetamine induced loco- IL-1β was observed, though the changes were not statis- motor activity was observed. This suggests that at doses tically significant. In contrast, proinflammatory cytokines used in this study, FTY720 most likely does not suppress were not markedly increased in the CC and STR, with locomotor activity, as well as locomotor-function related changes in all measured proinflammatory cytokines (IL- dopaminergic signaling, and counteracts the cuprizone- 6, TNF-α, IL-1β) not being statistically significant. Such induced increase in locomotion through other mecha- results may suggest region-specific differences in the nisms such as altering glial functions related to synaptic surface receptor expression in residential cells [48] and maintenance [23, 44, 45]. consequent differences in susceptibility to neuroinflam - Microglia are the innate immune cells of the CNS mation. Treatment with FTY720 significantly suppressed and are activated in response to various inflammatory/ the elevation of IL-6 in the HIP. Changes in IL-6 as a psychological stimuli such as tissue injury, infection, result of cuprizone exposure and FTY720 administration cytokine signaling and psychological stress. Microglial coincided with observed changes in Iba1, suggesting that activation leads to upregulation of Iba1 as well as the the anti-inflammatory effects of FTY720 are mediated release of response factors including proinflammatory through suppression of microglial activity. In our experi- mediators (ROS, cytokines, etc.) [12], and clinical stud- ments, we saw significant increases of IL-4 expression as ies have reported microglial hyperactivation in the brains a result of FTY720 administration in both the HIP and of schizophrenic and suicidal patients [31, 32]. In line CC. As IL-4 is a cytokine with potent anti-inflammatory Li  et al. Molecular Brain (2023) 16:59 Page 9 of 14 effects [49, 50], the suppression of microglial activation anti-inflammatory and cannot directly rescue cuprizone- and proinflammatory IL-6 expression may be due to induced oligodendrocytic damage/loss, which is the pri- increased IL-4 production, although the effects seem to mary cause of inflammation in this model. differ between the HIP and CC. In summary, this study utilized behavioral, immuno- Microglial activation and subsequent production of histochemical, and transcriptional analyses to evalu- pro-inflammatory cytokines can disrupt the BBB [18], ate the effects of short-term FTY720 administration causing proinflammatory cell populations such as cir - on an inflammatory short-term cuprizone exposure culatory leukocytes to enter into the brain parenchyma model of psychosis. As a result, short-term administra- [51]. Observation of cell populations with high expres- tion of FTY720 improved disorder-related behavior and High sion of CD45 (CD45 ) in the brain can visualize infil - reduced inflammation by suppressing microglial acti - trating leucocytic populations (mainly monocytes) [34]. vation in the HIP, highlighting its future use as a direct High In this study, a marked increase in CD45 popula- anti-inflammatory treatment against microglial activa - tions was observed in the CC of the cuprizone exposed tion and psychosis. These findings are consistent with the groups, suggesting that short-term cuprizone exposure microglial hypothesis in psychosis such as schizophrenia destabilizes the BBB, and allows for increased infiltration and suggests that the suppression of microglial activation of circulatory leukocyte populations. Short-term treat- could alleviate psychosis pathology [13, 14]. It has been ment with FTY720 did not significantly alter the num - reported that neuroinflammation and/or the inflam - High bers of C D45 populations in the HIP or CC. As an masome pathway is associated with many neurological oral therapy for multiple sclerosis, the immunosuppres- diseases [15–18, 57, 58].  New methods such as induced sive effects of FTY720 restrict lymphocytes to lymphatic microglia-like (iMG) cells differentiated from human nodes, which is known to be mediated through the tran- peripheral blood monocytes which exhibit microglia-like sient downregulation and degradation of the S P receptor gene expression, branching morphogenesis, and phago- on circulatory lymphocytic populations [52]. It has been cytic activity with various cytokine releases [59–61] have reported that FTY720 treatment lowers circulating leu- been developed to allow for evaluation in human tissue kocyte count and leukocyte recruitment in the CNS [53]. derived cells. iMG cells differentiated from schizophre - However, our results show that a short-term, three-day nia patients may be used effectively to investigate the treatment does not significantly decrease brain parenchy - inhibitory effects of FTY720 on microglial activation in High mal leucocytic (C D45 ) populations. The neuroinflam - humans. matory state induced by short-term cuprizone exposure Short-term cuprizone exposure causes oligodendrocyte is significantly different from the reported LPS inflamma - dysfunction, which includes disruption of mitochondrial tion model, and it is possible that the effect of FTY720 and metabolic function, as well as ROS production [62]. administration is affected by differences in the degree Cuprizone exposure also causes amino acid depletion of leukocyte infiltration [53]. Additionally, FTY720 has [62]. As a result, it has been reported that the synthesis been shown to pass through the BBB and accumulate in of myelin-related proteins such as MBP may decrease the brain [23, 54] and can target microglial S P receptors [62]. Following oligodendrocyte injury, other glial cells expressed in CNS resident cells. Moreover, there is evi- including microglia are activated, and start to contribute dence of FTY720 preventing BBB disruption by altering to innate immune responses [62] by releasing proinflam - tight junction proteins that form the BBB [55]. There - matory cytokines. Such proinflammatory responses can fore, the anti-neuroinflammatory effects of FTY720 seen cause neural inflammation, resulting in disease-related in this study can most likely reflects the direct effects of symptoms. It has been reported that increased micro- FTY720 on CNS resident cells or BBB forming cells. glial activation causes neuroinflammation and impairs Previous studies on schizophrenia have focused on hippocampal neurogenesis, neuronal synaptic morphol- myelin-related genes as a genetic risk factor of schizo- ogy, and synaptic plasticity [63, 64]. Moreover, it has phrenia [37], and white matter abnormalities correlate been reported that FTY720 attenuates microglial activ- with the stages of schizophrenia [56]. As FTY720 admin- ity and downregulates the production of proinflamma - istration has been reported to have protective effects on tory cytokines by activated microglia [65]. Such insights models of inflammatory demyelination [38], we stained suggest the possibility of FTY720 suppressing neural cell for MBP in the CC, a major white matter structure, to dysfunction (inflammation, synaptic dysfunction, etc.) investigate if FTY720 administration can directly pre- caused by short-term cuprizone exposure to alleviate vent oligodendrocytic damage. We observed a decrease pathological behavior. In addition to the microglial activ- in MBP staining in the cuprizone exposed groups, which ity examined in this study, FTY720 is known to regulate FTY720 administration was not able to rescue. This sug - immunoinflammatory responses through various mecha - gests that the ameliorative effects of FTY720 are mainly nisms (Additional file 4: Fig. S4) [25, 66]. Li et al. Molecular Brain (2023) 16:59 Page 10 of 14 Behavioral analysis The full picture of human psychosis pathology still Rotarod remains largely elusive and continues to be difficult to An accelerating rotarod (ROTA ROD; UGO BASILE) was capture. Elucidation of the causes and development of used, with the initial speed set at 4  rpm, acceleration at therapeutic strategies likely requires further contribu- 20 rpm/min, and maximum speed at 40 rpm. The mouse tions from various fields including animal models, clini - was placed on the rotating rod in the direction opposite cal studies, and human genetics. Short-term cuprizone to the direction of rotation. The test was started after exposure in mice, the model used in this study, can be the mouse was able to stand and move upright on the an effective model of the neuroinflammatory symptoms rotarod. The duration of time till falling to the platform of psychosis, and can be further utilized to study the below was recorded, with the maximum test duration set connections between psychiatric symptoms and inflam - to 120 s after the start of acceleration. Mice were subject matory phenomena due to their relevance in behavio- to three trials in one day, performed 60 min apart. ral characteristics and state of neuroinflammation [32, 33]. Further information into the microglial populations Methamphetamine‑induced locomotion test altered by cuprizone and/or FTY720 and the cellular Methamphetamine (Sumitomo Pharma) was dissolved in mechanisms of underlying the phenomena observed physiological saline and administered intraperitoneally in this study may provide valuable insight on the con- at a final concentration of 2 mg/kg. Mice were left in the nections between neuroinflammatory phenomena and experimental room for over an hour before being trans- behavioral alteration. ferred to a circular open field (diameter 40  cm, height 27  cm, grey plexiglass). After 30  min of baseline loco- Methods motor activity measurement, mice were given intraperi- Animals toneal injections of methamphetamine and locomotion All animal experiments were performed in accordance were measured for 60  min after injection. Mouse move- with protocols of the Animal Experimentation Commit- ment was tracked, and distance traveled (cm/5 min) was tee of the Institute for Protein Research, Osaka Univer- measured using EthoVision XT (Noldus). sity. Male C57BL/6NJcl mice were obtained from CLEA Japan. Mice were maintained in a quiet environment Y‑maze test with a controlled temperature of 24 ± 2  °C. During a The Y-maze test was administered according to Sakurai 12-h light/dark cycle, mice had constant access to either et al. [67], with minor modifications. In brief, the Y-maze a standard solid chow (AIN-93M, ORIENTAL YEAST consisted of 3 arms (42  cm long, 16  cm high and 12  cm CO., LTD.) or identical chow containing 2% w/w cupri- wide) configured 120 degrees apart. Mice were placed in zone (AIN-93M (99.8%) + cuprizone (0.2%), ORIENTAL the center of the arm and allowed to move freely in the YEAST CO.,LTD.) and water. After arriving at 6  weeks, maze for 10  min. Mouse movements and arm entries mice were habituated to the standard chow for seven were tracked, recorded, and counted using EthoVision days, then subsequently divided into four groups (Cont- XT (Noldus), and calculation of successful alternation Veh, Cont-FTY, CUP-Veh, CUP-FTY) and fed either the rate was calculated using an R script (Additional file  1). standard diet (Cont-Veh, Cont-FTY) or the cuprizone- Successful alternation was defined as entering all three formulated diet (CUP-Veh, CUP-FTY) for seven days arms consecutively. (Additional file 4: Fig. S1). Immunohistochemistry Drug administration Immunohistochemistry was performed according to FTY720 {2-amino-2-[2-(4-octylphenyl) ethyl]-1,3-pro- Aomine et  al. [68], with minor modifications. Ani - panediol} was obtained from Cayman Chemical. mals were deeply anesthetized with isoflurane prior Powdered FTY720 was dissolved in dimethylsulfoxide- to experimentation and perfused transcardially with hydrochloride (DMSO-HCl) to make a 0.2  g/ml stock phosphate-buffered saline (PBS, pH 7.4) followed by 4% and stored at − 20 °C. At the time of use, the stock solu- Paraformaldehyde/PBS, and post fixed overnight in 4% tion was diluted with physiological saline to produce a Paraformaldehyde/PBS at 4 ℃. For cryoprotection, brains 0.2 mg/ml solution. Identical DMSO-HCl solution with- were placed in 15% sucrose/PBS for one day, after being out FTY720 was dissolved in saline for control. Mice moved to 30% sucrose/PBS for ~ 36 h at 4 ℃. Brains were were treated for three days with daily intraperitoneal frozen and embedded in embedding agent (TissueTek injections of FTY720 1 mg/kg starting four days after the O.C.T compound, Sakura Finetek Japan Co., Ltd., Tokyo, start of cuprizone exposure (no injections on experimen- Japan) and was sectioned at 40  μm using a cryostat tal day) (Additional file 4: Fig. S1). Li  et al. Molecular Brain (2023) 16:59 Page 11 of 14 3D cell morphology analysis of microglia microtome (Leica CM1860, Leica Biosystems). To avoid Animals were deeply anesthetized with isoflurane prior deformation, the sections were free-float-processed with to experimentation and were perfused transcardially with extreme caution. phosphate-buffered saline (PBS, pH 7.4), followed by a Sections were washed three times with PBS-T (0.3% fixative (a mixture of 4.0% paraformaldehyde and 0.05% Triton-X) and incubated overnight (4  ℃) in an anti- glutaraldehyde in 0.1 M PBS). The brains were left in situ body solution (0.3% Triton X PBS W/ 10% Normal Goat for 2  h at room temperature and were then removed Serum (Thermo Fisher) + primary antibody). Primary from the skull. After cryoprotection with 30% sucrose, antibodies used were Anti Iba1 (1:1000; Rabbit, FUJI- brain blocks were cut into 40-μm-thick sections on a FILM Wako, 019-19741), Anti CD45 (1:100; Mouse, cryostat (Leica CM1950, Wetzlar, Germany). To avoid BIO-RAD, MCA87) and Anti Myelin Basic Protein deformation, the sections were free-float-processed with (1:500; Rabbit, Abcam, ab133620). After primary anti- extreme caution. Sections were blocked for 30  min with body staining, sections were washed three times with 1% bovine serum albumin (BSA) in PBS containing 0.3% PBS and incubated in a secondary antibody solution Triton X-100 and 0.1% sodium azide at 4 °C. After block- (0.3% Triton X PBS W/0.5% Normal Goat Serum + sec- ing, the sections were incubated with Anti Iba1 (1:20000; ondary antibody) for 2 h (RT). Secondary antibodies used Rabbit, FUJIFILM Wako, 019-19741) for 2  days at 4  °C. were Anti Rabbit IgG, Alexa Fluor 488 (1:500; goat, After washing with PBS, the sections were incubated ThermoFisher, A-11034), Anti Mouse IgG, Alexa Fluor with a mixture of Alexa488-conjugated donkey anti-rab- Plus 647 (1:100; Goat, ThermoFisher, A-32728). The sec - bit IgG antibody (1: 300; Jackson ImmunoResearch Lab- tions were washed three times with PBS, mounted on a oratories, West Grove, PA, USA) overnight at 4  °C. The glass slide with Mounting Medium w/DAPI (Abcam, sections were counterstained with DAPI and mounted ab104139), coverslipped, and imaged with a Keyence BZ with Vectashield (Vector Laboratories, Burlingame, CA). X810 microscope. Fluorescent images were acquired with a confocal laser scanning microscope (FV-1000-D; Olympus, Japan). Sec- Fluorescence intensity analysis tions were randomly sampled and processed for immu- HALO analysis nohistochemical staining. The 3D hippocampal microglia Fluorescence intensity was analyzed using HALO images were created with a × 60 objective on an FV- (Indicalab). Three individual images at 40× magnifica - 1000-D at 0.5-μm intervals along the z-axis. Following tion were obtained per region per one mouse were. Care acquisition, images were compiled and transformed into was taken to select the same regions for each individual. 3D microglial renderings using Imaris software (Bitplane, Thresholds were set to automatically detect DAPI posi - Zurich, Switzerland). The morphological microglial tive nuclei and Iba-1 positive signal and were kept the parameters (cell surface area and cell volume) were meas- same for all measurements. Iba-1 fluorescence inten - ured using the Imaris MeasurementPro function. The sity was measured for all Iba-1 positive nuclei and was microglia ramification ratio was calculated as the ratio of average Iba-1 fluorescence intensity was calculated per cell surface area to cell volume [69]. The morphological image analyzed by HALO. The average values of three parameters from 545 to 631 microglia in each group were measurements per individual was used for further calculated from ten sections per mouse (n = 3 mice each). analysis. Quantitative real‑time PCR ImageJ analysis Eight-week-old mice were deeply anesthetized with Sections were stained by the method described above, isoflurane, decapitated, and the entire brain  was then observed and photographed with a Keyence BZ removed  rapidly from the skull. The whole brain was X810 microscope. The captured images were imported sliced into 1  mm sections using a brain matrix (Brain- into Image J, and the fluorescence intensity per area science Idea, Osaka, Japan), and the striatum, hippocam- (300  µm*120  µm; indicated by dotted line) of the cor- pus, and corpus callosum were removed from sections pus callosum region of 3 sections for each individual using a razor blade. Removed sections were placed in was measured. Prior to statistical analysis, mean values RNAlater, kept at 4 ℃ overnight, and then frozen at were calculated for each group (n = 3 mice each) based − 80 ℃. on the measured values, and relative values were cal- RNA was extracted from tissue using RNeasyMini- culated using the mean value of control mice as 100%. Kit (Qiagen) and reverse transcribed into cDNA with Fluorescence intensity statistics results are shown as the ReverTra Ace qPCR RT Master Mix (Toyobo). mean ± SEM. Li et al. Molecular Brain (2023) 16:59 Page 12 of 14 Quantitative real-time PCR was performed using were sacrificed on day 7. All experiments were performed in four groups: GeneAce SYBR qPCR Mix a Low Rox (NIPPON GENE) Cont-Veh, Cont-FTY720, CUP-Veh, CUP-FTY. Fig S2. Eec ff ts of FTY720 on short-term spatial memory in cuprizone-exposed mice. Y-maze test for reagent and QuantStudio 6 pro (Thermo Fisher). Quan - short-term spatial memory was used to assess the effects of cuprizone tification was done using a relative standard curve, and exposure and the effects of FTY720 administration. Total distance. (B) Total βActin was used as an internal control. qPCR run method arm entries. (C) Successful alternation rate. Fig S3. Representative image of immunostaining in hippocampus and corpus callosum. (A) Representa- and Primer information is shown in Additional file 2. tive image of Iba1 immunostaining in the HIP. (B) Representative image of Prior to statistical analysis of quantitative real-time Iba1 immunostaining in the CC. Blue: DAPI nuclear stain, Green: Alexa488 PCR data, the cuprizone group mRNA expression lev- (Iba1). An enlarged view of the top row is shown in the bottom row. Fig S4. Hypothesis of FTY720 mechanism of action in psychosis model mice. els were normalized to the control group for each brain Short-term cuprizone exposure does not cause prominent demyelination, region. Fluorescence intensity and quantitative real-time but specifically causes damage to oligodendrocytes. Such damage results PCR statistical results are shown as mean ± SEM. in activation of glial populations including microglia, triggering the release of proinflammatory cytokines. Extracellular release of proinflammatory cytokines induces neuronal dysfunction and contributes to behavioral Statistical analysis disorders such as psychosis. FTY720 administration inhibits microglial acti- All statistical analysis was performed using Prism8 vation and regulates pro-inflammatory cytokine release, reduces neuronal damage, and improves psychosis-like behavior. (GraphPad Software), except for Fig.  2G which was analyzed using the “rstatix” library in R. The number of individual samples used for each set of experiments is Acknowledgements We would like to thank all Hikida lab members for their valuable comments indicated in the results section and figure legends. Details and support. of ANOVA and post-hoc  analyses are provided in Addi- tional file 3. Author contributions TAK, and TH conceived the study. KS, MO, TAK, and TH designed experiments. SL, KS, and MO performed the experiments. SL, KS, and MO analyzed and interpreted data. SL, KS, and MO wrote the manuscript. All authors read and Abbreviations approved the final manuscript. IL-6 Interleukin 6 TNF-α Tumor necrosis factor-α Funding CNS Central nervous system This study was supported by JSPS KAKENHI, Grants Numbers JP22H03000 to AD Alzheimer’s disease MO, JP22H00494 to TAK, JP21H05694 and JP22H02944 to TH, AMED under PD Parkinson’s disease Grant Numbers JP22dk0207065 to MO, JP21wm0425010 and JP21gm1510006 ALS Amyotrophic lateral sclerosis to TH, JST SPRING under Grant Number JPMJSP2138 to SL and KS, SENSHIN MS Multiple sclerosis Medical Research Foundation to TH, Taiju Life Social Welfare Foundation to PET Positron emission tomography TH, and the Collaborative Research Program of Institute for Protein Research, FTY720 Fingolimod Osaka University, ICR-22-03. SP Sphingosine 1-phosphate TRMP7 Transient receptor potential cation channel subfamily M member Availability of data and materials All data needed to evaluate the conclusions in the paper are present in the PP2A Protein phosphatase 2A paper. The datasets used and/or analyzed during the current study are avail- HIP Hippocampus able from the corresponding author on reasonable request. CC Corpus callosum Iba1 Ioniz ed calcium binding adapter molecule 1 IL1-β Interleukin-1beta Declarations IL-4 Interleukin 4 MBP Myelin basic protein Ethics approval and consent to participate BBB Blood–brain barrier All animal procedures and experiments were performed by the Institute for STR Striatum Protein Research, Osaka University animal committee regulations. Consent for publication Supplementary Information Not applicable. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13041- 023- 01047-5. Competing interests The authors have declared that no conflict of interest exists. Additional file 1:  Y-maze R script. R script to calculate succesful alterna- tion rate for Y-maze. 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Journal

Molecular Brain – Springer Journals

Published: Jul 12, 2023

Keywords: Short-term cuprizone exposure; Psychosis; Fingolimod; Microglial activation; Neuroinflammation

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