N-glycans Profiling in Pilocarpine Induced Status Epilepticus in Immature Rats

Status epilepticus (SE) is a critical neurological condition that may lead to persistent seizures in newborns due to the immature brain's proclivity for seizure activity (Farmania et al., 2020). The most common aetiology of SE in neonates is hypoxic-ischemic encephalopathy (HIE), which accounts for around 60% of seizures during the neonatal period (Lawrence and Inder, 2010). SE is linked to brain damage and elevates risk of developing epilepsy, as well as cognitive and functional impairments (Kubová et al., 2001). Due to the significant long-term consequences, it is a socioeconomic burden (Allers et al., 2015; Penberthy et al., 2005). There is an urgent need for attention on neonatal SE in research due to the obviously limited diagnostic and therapeutic options available in modern neonatology (Farmania et al., 2020). Glycosylation is the enzymatic attachment of oligosaccharides to proteins and lipids. Aberrant glycosylation is found in a variety of pathophysiological diseases, including cancer, inflammation, autoimmune disease, and aging (Ohtsubo and Marth, 2006). Glycosylation plays an important role in brain development, physiology, and functioning, such as modulating neuronal transmission and regulating synaptic processes. As a result, dysregulated glycosylation can lead to a variety of neurological disorders (Hasan et al., 2021). N-glycosylation impacts glycoproteins in brain development and also has modulatory effects on neural transmission and neural circuit excitability (Scott and Panin, 2014). An N-glycan profile of rat serum is appropriate for pathological and pharmacological studies (Clerc et al., 2016; Gao et al., 2015). The aim of the present study was to assess the overall N-glycans composition from the glycoprofiles in the pilocarpine induced SE model in immature rats.

In this study, immature male Wistar rat pups (weighing 18 g) were used (N=11). The animals were divided into two groups at random: control (n=5) and Li-Pilo (n=6).

Alterations in N-glycans were evaluated in pilocarpine induced SE model in new-born's rat sera. The cluster of 21 N-glycans was appointed and sorted with special emphasis on their structural type (Table 1).

The significant changes between experimental groups (Li-Pilo vs Control) were observed in relative intensities of sialylation in N-glycans, as well as some remodelation trends in variations of relative intensities within N-glycan clusters according to their glycan type.

In the Li-Pilo group, we found significantly lower portion of relative intensities of non-sialylated N-glycans (m/z: 1579.8; 1783.5; 1836.1; 1988.1; 2040.2; 2081.2; 2191.2; 2396.4) compared to the Control group (Figure 1a). There were no statistically significant differences within the clusters of mono- (m/z: 2227.4; 2390.4; 2431.5; 2605.7), di- (m/z: 2736.8; 2792.7; 2966.9), and tetra-sialylated (m/z: 3964.3; 4138.4) N-glycans. However, we detected significantly higher level of relative intensities within the tri-sialylated (m/z: 3154.1; 3328.1; 3603.2; 3777.4;) N-glycan cluster in Li-Pilo group compared to the Control group. The overall relative intensities in tri-sial clusters of N-glycans in Control (29.4%) were augmented by approximately one fifth in Li-Pilo (36.4 %).

Figure 1

There were no statistically significant changes in N-glycans relative intensities based on their glycan types between Li-Pilo and Control groups, even though there are noticeable remodelation trends in high-mannose, complex-tri-fucose, and hybrid N-glycan types in Li-Pilo compared to the Control (Figure 1b).

The purpose of this study was to examine N-glycans changes as the result of metabolic alterations in rodents after epileptogenic insults, such as Li-Pilo-induced SE. N-glycans are crucial entities for brain functions such as neurogenesis, neurite outgrowth, synapse function, and memory formation (Gaunitz et al., 2021). Severely aberrant glycosylation during early development can lead to protein misfolding, which may play a role in the development of neurological deficits (Coss et al., 2014). Moreover, different neuro-pathological symptoms such as mental retardation, seizures, and epilepsy can be induced by N-glycan alterations in the nervous system (Samal et al., 2020).

Our data indicated that blood sera from rats with Li-Pilo-induced SE revealed a significantly lower portion of relative intensities of non-sialylated N-glycans, and higher level of tri-sial N-glycans in compared to the Control groups. N-glycan clusters based on their different glycan types also revealed some remodeling variations within high-mannose, complex-tri-fucose, and hybrid N-glycan types. This research established the framework for using “brain-type” glycans like sialylation (mono-, di-, and tetra-) as biomarkers or therapeutic targets, as well as the potential for modulating their levels within neurodegenerative disorders (Samal et al., 2020).

Moreover, former preclinical studies suggest that the MALDI-TOF-based glycoprofiling method has previously been used to identify and quantify N-glycan structures in glycomic studies, as well as to detect specific N-glycan biomarkers in plasma or serum of patients with various types of disorders (Fogli et al., 2012).

N-linked glycosylated proteins play a variety of roles in the brain, including electrical gradients and neurotransmission. How alterations in N-linked glycosylation affect and may even induce attributes of neurological disorders in not well understood yet (Conroy et al., 2021). To date, very little is known about the precise neurobiological functions of individual N-glycans (Klarić and Lauc, 2022). N-glycosylation has a multifaceted role at the synapses, as sialylated N-glycan structures are ranked among the most powerful modulators of cell excitability, affecting voltage-gated Na⁺ and K⁺ channels particularly (Scott and Panin, 2014). According to Yu et al., (2017), they revealed that improperly expressed glycans were associated with mitochondrial dysfunction in ischemic brain injury. Boll et al. (2020), found that sialylation is a key dynamic modification for synaptic depolarization-dependent processes which are involved in synaptic plasticity and memory formation. In another study, Rebelo et al. (2021) showed that mice lacking sialyltransferases lead to motor and cognitive impairments as well as increased dysmyelination, demonstrating the relevance of sialylation.

In conclusion, in our study, we have demonstrated the significant changes in terms of N-glycans sialylation in Li-Pilo compared to the Control. We also observed some other remodelation trends in different portions of relative intenstities of N-glycan clusters according to their glycan type. Our preliminary findings have laid the foundation for additional investigation into glycosylation alterations in SE in immature rats.