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.