4. DISCUSSION
In the present study, we found that fingolimod, an S1P receptor modulator, attenuated PCP-induced impairments of spatial and fear memory by promoting neurogenesis and attenuating glial activation and IL-6 and IL-1β pro-inflammatory cytokine expressions. The underlying mechanism involves the upregulation of BDNF expression and activation of the ERK signalling pathway. The above results demonstrate that fingolimod is a potential therapeutic drug for treating cognitive impairment in schizophrenia.
Increasing evidence supports the idea that the dysregulated glutamatergic system plays a crucial role in the development of schizophrenia (Dauvermann, Lee et al., 2017), and administration of the N-methyl-D-aspartic acid receptor antagonist PCP, especially chronic use, induces typical symptoms in humans similar to those observed in patients with schizophrenia (J. D. Jentsch & Roth, 1999). This has been the basis for inducing animal models that mimic the symptoms of schizophrenia. Consistent with previous studies, our study showed that chronic PCP (10 mg/kg, 14 days) treatment induced spatial and fear memory impairments that correlated well with those thought to occur in schizophrenia (Cadinu, Grayson et al., 2017).
Since learning and memory processes are closely related to hippocampal neurogenesis (Deng, Aimone, & Gage, 2010) and fingolimod has been shown to facilitate hippocampal neurogenesis through the internalisation of the S1P1 receptor in hippocampal neural stem cells (NSC)s of adult mice (Yili Sun, Hong et al., 2016) (Efstathopoulos, Kourgiantaki et al., 2015), we reasoned that the treatment effect of fingolimod may be, at least in part, due to modulation of neurogenesis. In line with our animal behavioural tests, PCP treatment suppressed the proliferation and differentiation of NSCs, as indicated by the decreased number of BrdU-, NeuN-, and DCX-positive cells in the dentate gyrus. In contrast, fingolimod ameliorated this neurogenic deficit by facilitating adult neurogenesis.
Recently, the potential involvement of the immune response and consequent neuroinflammation in the pathogenesis of schizophrenia has attracted more attention. Similar to MS, schizophrenia is often considered to be associated with the dysregulation of the immune response and chronic inflammation in the CNS (Pape, Tamouza et al., 2019). In contrast, schizophrenia-like symptoms have also been described in some patients with MS (Meier, Ramagopalan et al., 2020). This suggests that the dysregulated immune system and inflammation observed in schizophrenia and MS somewhat overlap. Therefore, the aetiologies and therapeutic implications may be shared between both diseases.
Resident myeloid cells of the brain, microglia, astrocytes, and inflammatory cytokines are involved in mediating neuroinflammation. Similar to macrophages, microglia account for approximately 10% of all cells in the CNS (Soulet & Rivest, 2008). They are thought to originate from myeloid precursor cells during early neonatal development and are the principal resident innate immune cells of the CNS (Santambrogio, Belyanskaya et al., 2001). Under normal circumstances, microglia maintained a downregulated phenotype. When exposed to stimuli such as chronic stress, injury, or inflammation, the microglia can be “activated” or “sensitized,” by which a lower level of the same stimulus, such as cytokine response, can cause them to proliferate and increase the production of pro-inflammatory cytokines (Norbert & Müller, 2018; Rahimian, Wakid et al., 2021). This sensitisation process heightens responsiveness to inflammatory signals, inducing psychopathological symptoms and cognitive deficits by exacerbating or re-exacerbating the inflammatory pathology in the CNS. Postmortem studies have found a significant increase in microglial density in the brains of patients with schizophrenia despite substantial heterogeneity between studies (Kesteren, Gremmels et al., 2017). In line with these findings, our study found significantly more Iba-1-labelled microglia in the hippocampus of PCP-treated rats, which may indicate a vulnerable environment for an inflammatory insult. Astrocytes are known to provide neurotrophic support for neurones, maintain a homeostatic environment, and modulate synapse pruning (Clarke, Laura et al., 2013). However, little is known about their functional changes in response to immune attacks or neuroinflammation (referred to as “reactive astrocytes”). Studies have shown that microglia can cause astrocytes to lose their normal functions and become neurotoxic, contributing to neuronal death (Liddelow, Guttenplan et al., 2017). In our study, a higher expression of GFAP-positive astrocytes was also found in the hippocampus, which suggested a highly hostile environment for neural function. Fingolimod may have an ameliorative effect by suppressing microglial and astroglial activation.
The presence of the BBB creates an “immune privileged” environment for the brain, shielding the CNS from neurotoxic insults from peripheral immune cells, cytokines, and chemokines. However, the loss of BBB integrity appears to be a common pathological finding in patients with schizophrenia (Greene, Hanley, & Campbell, 2020), which often leads to an increase in the infiltration of the brain parenchyma and pro-inflammatory cytokine release. This possibly links increased neuroinflammation to detrimental effects on memory, neural plasticity, and neurogenesis. Among these, IL-6 and IL-1β appear to play essential roles in the pathogenesis of schizophrenia, and their findings are consistent. Both drug-naïve (Upthegrove, Manzanares-Teson et al., 2014) and chronic schizophrenia patients (Goldsmith, Rapaport et al., 2016) demonstrate higher expressions of IL-6 and IL-1β than healthy controls. It has been demonstrated that the overproduction of IL-6 and IL-1β may result in aberrant hippocampal neurogenesis and cognitive impairment (Vallières, Campbell et al., 2002) (M. Wu, 2013). In line with these findings, we found higher levels of IL-6 and IL-1β in the hippocampus of PCP-treated rats, which were negatively correlated with their neurogenic potential. In contrast, fingolimod partially attenuated the IL-1β and IL-6 levels in a dose-dependent manner.
BDNF is one of the neurotrophins that plays a significant role in neurogenesis, synaptic plasticity, and cognitive function (Kowiański, Lietzau, Czuba, Waśkow, Steliga, & Moryś, 2018). It is also involved in an upstream ERK cascade. The essential role of ERK activation in the CNS has been well-described in synaptic plasticity and memory (Iii, 2008). Multiple lines of evidence indicate that peripheral levels of BDNF are reduced in both drug-naïve and medicated patients with schizophrenia compared with healthy controls. (Green, Matheson et al., 2011). Although the interactions between BDNF and inflammatory cytokines require further elucidation, it has been demonstrated that patients with higher levels of inflammatory cytokines have lower BDNF expression in both postmortem and blood sample studies (Fillman, Cloonan et al., 2013; Valeria, Mondelli et al., 2011). A recent study found that aging-related and lipopolysaccharide-induced microglial activation in mice could be reversed by BDNF supplementation (S.-Y. Wu, Pan et al., 2020). In our study, ERK signalling was downregulated in parallel with BDNF expression in PCP-treated rats, which also suggests that PCP can induce cognitive deficits by compromising neurogenesis via the reduction of BDNF signalling. Furthermore, since a previous study showed that fingolimod-induced neurogenesis was dependent on the activation of ERK signalling (Y. Sun, Feng et al., 2016), we reasoned that the overall improvement in learning and memory function may be related to the restoration of BDNF/ERK signalling in PCP-treated rats.
It should be noted that an eight-week, double-blind pilot study was recently conducted to assess the effects of fingolimod in patients with schizophrenia and schizoaffective disorder (Francis, Hummer et al., 2020), which proposed that dysregulated inflammatory processes may contribute to the white matter abnormalities observed in schizophrenia. By analysing the fractional anisotropy values in diffusion tensor imaging, a significant relationship was found between the degree of lymphocyte reduction and improved white matter microstructure in the corpus callosum and the right superior longitudinal fasciculus. Significant improvements in the Brief Assessment of Cognition in Schizophrenia were not demonstrated; this may be partly due to the small sample size (40 patients in total) and the complex medication history of the recruited patients. However, studies involving larger samples and longer treatment durations are required to clarify the potential therapeutic effects of fingolimod in schizophrenia.
Some limitations of this study should be noted. The complex and unclear pathogenesis of schizophrenia is considered to be an outcome of gene–gene and gene–environment interactions; therefore, the current animal model based on N-methyl-D-aspartic acid receptor manipulation is focused on the cognitive deficits of schizophrenia, and cannot completely mirror the complex and heterogeneous aetiology of this disease. Therefore, other animal models of schizophrenia which adequately replicate ”positive-like” symptoms (e.g., hyperactive stimulant sensitivity) and ”negative-like” symptoms (e.g., social withdrawal) are needed to fully assess the treatment effects of fingolimod. In addition, for planning personalised treatment, further study is needed to help discover potential biomarkers of therapeutic relevance, so that we can identify the types of patients for whom fingolimod therapy may be suitable.
In conclusion, our study demonstrated that fingolimod improved PCP-induced learning and memory deficits in a rat model of schizophrenia. We highlight the ability of fingolimod to modulate neurogenesis and neuroinflammatory processes, which are markedly altered in schizophrenia. The study also showed that the BDNF/ERK pathway influenced the treatment effect of fingolimod. To the best of our knowledge, this is the first preclinical evidence supporting fingolimod as a potential therapeutic agent for cognitive deficits in schizophrenia.