In the present study, we report for the first time that CPZ increased the level of expression of CS2 and SPTLC1, and decreased the level of expression of SMS1 in the mouse Cg of cortex. Interestingly, it also enhanced the level of ROS in mouse cortical tissue. CPZ significantly caused astrocytopathy, microgliosis, oligodendropathy, and neurodegeneration in mouse Cg of cortical tissue. Furthermore, CPZ significantly caused deficits in motor function and muscle strength, and, increased anxiety-like behaviours in the animals. UA and FTY720 significantly attenuated the CPZ-induced altered behavioural, biochemical and molecular parameters in the rodents. Thus, UA could be a potential therapeutic alternative in the management of MS. Further, the ceramide biosynthetic pathway could be a potential therapeutic target in the management of MS.
MS exhibits motor deficits and anxiety-like behavioural characteristics in addition to other behavioural manifestations (Khodanovich et al., 2019). In the present study, CPZ significantly caused motor dysfunction and deficit in muscle strength, and, increased anxiety-like behaviours in the animals as per the previous report (Khodaei et al., 2019; Khodanovich et al., 2019). UA alleviates motor function and anxiety-like behaviour of mice in OFT and, EPM and HBT, which is supported by the study (Yamamoto et al., 2020). Here, we, for the first-time report that UA promotes muscular strength in CPZ-challenged animals in the Kondziela's Inverted Screen Test. FTY720 also exerts a beneficial effect against CPZ-induced motor dysfunction in animals (Mitra et al., 2022; Moradi et al., 2022). Interestingly, FTY720 exerted anxiolytic activity in the CPZ-induced animal model of MS. These observations indicate the fact that UA, in addition to FTY720, could be a potential therapeutic candidate in terms of alleviating the phenotypic characteristics of MS.
It is well established that cortical demyelination is well associated with motor and cognitive disabilities in the pathophysiology of MS (Calabrese et al., 2009). The pathophysiological studies suggest that oxidative stress, astrocytopathy, microgliosis, oligodendropathy, neuroinflammation, and neurodegeneration in the cortical region are the cardinal features of MS (Honarvar et al., 2019; Honarvar et al., 2022; Yamamoto et al., 2020; Zhang et al., 2020). It is also suggested that estriol attenuates oligodendropathy in the cortex and thus reduced demyelination in the experimental model of MS (Meyer et al., 2023). Taurochenodeoxycholic acid attenuated astrocytopathy and demyelination in the cortex of the animals subjected to EAE, a model of MS (Xu et al., 2023). It is also evident that Siponimod attenuates microgliosis and demyelination in experimental animal model of MS (Gentile et al., 2016). Thus, it can be assumed that one or multiple of oxidative stress, astrocytopathy, microgliosis, oligodendropathy, neuroinflammation, and neurodegeneration could be considered in the management of demyelination in cortical region in MS. In the present study, CPZ caused oxidative stress, astrocytopathy, microgliosis, oligodendropathy, neuroinflammation, and neurodegeneration in the mouse cortical tissue. Schröder et al. (2023) suggested that CPZ causes astrocytopathy, neuroinflammation, and neurodegeneration in rodent corpus callosum (Schröder et al., 2023). In another study, it has been documented that CPZ also caused microgliosis in addition to astrocytopathy, neuroinflammation, and neurodegeneration in the corpus callosum of rodents (Mohamadi & Borhani-Haghighi, 2023). Martinović et al. (2023) have reported that CPZ can also cause oligodendropathy in the corpus callosum of a demyelinating mouse model of X-linked adrenoleukodystrophy (Martinović et al., 2023). Similar to our study, Aldhahri et al. (2022) have reported that CPZ can cause behavioural deficits and neurodegeneration in the pre-frontal cortex in animals (Aldhahri et al., 2022). Interestingly, in the present study, UA (100 and 200 mg/kg) and FTY720 significantly attenuated CPZ-induced oxidative stress, astrocytopathy, microgliosis, oligodendropathy, neuroinflammation and neurodegeneration in the mouse cortex. It is interesting to note that all the doses of UA (50, 100 and 200 mg/kg) and FTY720 was able to attenuate the CPZ-induced oligodendropathy in terms of a decrease in the expression level of Olig2 in the mouse cortical tissue. However, UA (50 mg/kg) did not cause any significant change in the CPZ-induced decrease in the expression level of MBP in the cortex. The discrepancy in the dose of UA on CPZ-induced changes in oligodendropathy markers such as MBP and Olig2 is yet to be clarified in future studies.
Ceramide is an important intracellular signalling molecule that regulates cell proliferation, differentiation, and apoptosis in CNS (Levy & Futerman, 2010; Nyonda et al., 2022). It is also documented that the ceramide biosynthetic pathway plays an important role in the physiology of myelination in CNS (Kitatani et al., 2008). In the present study, CPZ significantly altered the ceramide biosynthetic pathway in terms of an increase in the level of expression of CS2 and SPTLC1, and a decrease in the level of expression of SMS1 in mouse cortical tissue. Similar to our study, it has been documented that CPZ can aberrantly upregulate the astroglial ceramide which is responsible to potentiate oligodendrocyte injury in the animal study (Barcelos et al., 2019; Kim et al., 2012). Further, it is also reported that serine palmitoyl transferase, the rate-limiting enzyme for ceramide de novo biosynthesis, was consistently upregulated in reactive astrocytes in the cuprizone mouse model of demyelination (Barcelos et al., 2019; Kim et al., 2012). It has been documented that there is a significant elevation in the level of CS2 and the level of expression of its mRNA in monocytes and neutrophils in animals with characteristic features of demyelination and clinical patients of MS (Tanaka et al., 2022), indicating the fact that ceramide biosynthetic pathway could be considered as one of the potential contributors in the pathophysiology of demyelination which is subject to future clarifications. In our study, UA (100 and 200 mg/kg) and FTY720 significantly attenuated CPZ-induced increase in the level of expression of CS2 and SPTLC1, and a decrease in the level of expression of SMS1 in mouse Cg of cortical tissue. However, UA (50 mg/kg) did not alter the CPZ-induced increase and decrease in the level of expression of CS2 and SPTLC1, and SMS1 in mouse Cg region of cortical tissue, respectively. Similar to our study, FTY720 exhibited neuroprotective activity in terms of ceramide-induced deleterious effects in astrocyte culture derived from postmortem brain tissue of MS patients (van Doorn et al., 2012). In contrast to our result, the liposomal formulation of UA significantly increased the level of ceramide in the human skin cell culture study (Both et al., 2002; Yarosh et al., 2000). The discrepancy in the results could be due to the consideration of different experimental models of study, which needs further clarification in the near future.
In conclusion, CPZ increased the level of expression of CS2 and SPTLC1, and decreased the level of expression of SMS1 in the mouse cortex. It also enhanced the level of ROS in mouse cortical tissue. CPZ significantly caused astrocytopathy, microgliosis, oligodendropathy, and neurodegeneration in mouse cortical tissue. Furthermore, CPZ significantly caused deficits in motor function and muscle strength, and, increased anxiety-like behaviours in the animals. UA and FTY720 significantly attenuated the CPZ-induced altered behavioural, biochemical and molecular parameters in the rodents. Thus, UA could be a potential therapeutic alternative in the management of MS. Further, the biomarkers of ceramide biosynthetic pathway such as CS2, SMS1, and SPTLC1 could be potential therapeutic targets in the management of MS.