Seizures are the most common clinical manifestation of neonatal neurological dysfunction, with an incidence rate of 1‰ to 5‰ (1). During the neonatal period, significant neurogenesis and synapse formation occur to meet the demands of neurodevelopment. The brain is in a physiologically active state, actively receiving excitatory signals (2, 3). Additionally, the immature nature of the developing brain during this period leads to an excitatory/inhibitory imbalance (4), which can be observed in the following three aspects: (i) Calcium channels that mediate excitatory inward currents develop early, while stabilizing excitatory potassium currents appear later (2). (ii) Excitatory glutamatergic neurons are heavily expressed during development, with the NMDA receptor GluN2B subunit expressed in various brain regions, promoting intracellular calcium influx and prolonging postsynaptic currents. (iii) Activation of GABA receptors in the early stages of development can cause chloride ion efflux, resulting in neuronal depolarization and increased excitability (5). Therefore, due to the unique physiology and vulnerability of the immature brain to damage, the incidence of seizures is higher in this age group than in others. Currently, the first-line treatment drug is phenobarbital, with an effectiveness rate of 50 to 64% in treating neonatal seizures (6). Uncontrolled seizures may lead to various neurological sequelae, including epilepsy, cerebral palsy, developmental delay, and psychomotor disorders (1, 7). Some studies have found that anti-seizure medications themselves can increase cellular apoptosis and potentially affect normal brain development by altering neurogenesis, synaptogenesis, cell proliferation, and migration (8–10). Consequently, there is still an ongoing search for new therapeutic targets in the treatment of neonatal seizures to improve the prognosis of affected infants.

A ketogenic diet has been shown to be significantly effective in the clinical treatment of intractable epilepsy. The characteristics of a ketogenic diet include high fat, low carbohydrates, and adequate protein. One type of ketogenic diet, known as the classic ketogenic diet, consists mainly of long-chain triglycerides, with approximately 80 to 90% of calories coming from fat. In this regimen, cellular metabolism relies on the oxidation of fatty acids as the primary source of energy (11). The mechanisms behind the reduction of seizures through a ketogenic diet are not fully understood. Existing research suggests that these mechanisms may be related to improvements in brain energy metabolism (12), neurotransmitter release (13), ion channels (14), and oxidative stress (15, 16). It is worth noting that whether triglycerides are involved in the neuroprotective effects during seizures remains unclear. Some clinical studies in adults have suggested that triglycerides may exert protective effects against cognitive impairment (17–19). However, research focusing on their role in neuronal excitability and their relevance during the neonatal period remains limited. To investigate this, we used a genetically edited hamster model with extreme hypertriglyceridemia to study the impact of high triglycerides on neuronal excitability and its underlying mechanisms. This research aims to provide new insights for identifying therapeutic targets for neonatal seizures.

This study utilized apolipoprotein C2 (Apoc2) knockout golden Syrian hamster models with hypertriglyceridemia. Apoc2 is a crucial component of chylomicrons, very low-density lipoprotein, and high-density lipoprotein, and it serves as an essential activator of lipoprotein lipase, which is necessary for triglyceride metabolism (20, 21). Consequently, Apoc2^−/− hamsters exhibit extremely high levels of triglycerides and cholesterol. Hamsters possess many lipid metabolic features similar to those of humans, such as a lipoprotein profile dominated by low-density lipoprotein and a high level of endogenous cholesteryl ester transfer protein expression (22), making them an ideal animal model for studying pathophysiological processes related to lipid metabolism. Furthermore, Apoc2 is almost absent in the brain, and the knockout of this gene does not affect neuronal function. Therefore, Apoc2 knockout hamsters can be employed to investigate the role and mechanisms by which triglycerides and their metabolites contribute to neonatal seizures.

This study aims to elucidate the mechanisms through which elevated triglycerides modulate neuronal excitability and confer neuroprotection during seizures. By identifying the underlying pathways, this research seeks to uncover novel therapeutic targets for the treatment of neonatal seizures, offering new strategies to improve outcomes for affected infants.

Neonates have the highest incidence of seizures among all age groups. The effectiveness rate of anti-seizure medication is only approximately half, and if the condition is not well controlled, it can result in residual neurological sequelae. Triglycerides—the primary fat source in the ketogenic diet—have an unclear role. Studying the role of high triglycerides in seizures can provide new insights for identifying novel therapeutic targets. We chose an Apoc2^−/− hypertriglyceridemic hamster model and compared it with a control group. The HTG hamsters exhibited a reduction in the frequency of acute seizure attacks. Ex vivo studies also demonstrated a decrease in neuronal excitability, indicating that HTG indeed has a protective effect when there is excessive synchronous neuronal firing. Furthermore, the reduction in free palmitic acid content and the increased expression of ZDHHC14 suggest that palmitoylation modification may be a mechanism underlying its protective effect.

First, the in vivo results showed significantly fewer tonic–clonic seizures in Apoc2^−/− hamsters than in wild-type hamsters. Given the size of hamsters aged P7–P10 is relatively small, we chose P15–P17 to establish an acute seizure model. Hypertriglyceridemia may also introduce confounding factors in terms of the efficiency of drug absorption via intraperitoneal injection. Therefore, we also conducted ex vivo experiments using brain slices from neonatal hamsters aged P7–P10, inducing neuronal discharges by perfusing brain slices with magnesium-free artificial cerebrospinal fluid. The results indicated that the EPSP frequency of cortical neurons in HTG hamsters was significantly lower than that in wild-type hamsters, and the AP frequency was also significantly lower under various intensities of electrical stimulation. This suggests that cortical neurons in HTG hamsters exhibit lower excitability than those in wild-type hamsters, which is consistent with the results of in vivo experiments. Consequently, we have confirmed at both in vivo and ex vivo levels that hypertriglyceridemia reduces neuronal excitability in the seizure model and has an inhibitory effect on the synchronization of the firing of a large number of neurons.

To our knowledge, no studies have investigated the effect of hypertriglyceridemia on acute seizures, particularly on neuronal excitability in neonatal seizure models. One possible mechanism underlying the anti-seizure effect of a ketogenic diet is that it provides medium-chain fatty acids and ketone bodies as alternative energy sources for epileptogenic brain regions (29, 30). Therefore, the lipid environment of hypertriglyceridemia may provide abundant metabolic substrates for the nervous system, meeting the energy demands of synchronized neuronal firing. To further investigate the mechanisms, we focused on the extracellular fluid of the cortex, which serves as a mediator between the nervous system and the periphery. We collected microdialysates from Apoc2^−/− and wild-type hamsters before and after seizures and conducted fatty acid metabolomics. We found a trend towards a decrease in free palmitic acid in both wild-type and Apoc2^−/− hamsters after seizure induction compared to pre-seizure levels, with a significant reduction in free palmitic acid in Apoc2^−/− hamsters compared to wild-type post-seizure. This finding aligns with prior research by Leitner et al., which demonstrated a reduction in cortical palmitic acid levels in epilepsy patients on a ketogenic diet, suggesting a possible association between reduced free palmitic acid and seizure protection (31). However, the specific physiological role of this reduction in modulating neuronal excitability remains to be elucidated. The neuroprotective roles of fatty acids remain a significant research focus. While our study centers on palmitic acid and no significant differences were observed in the levels of other fatty acids, it is possible that other fatty acids and lipid metabolites, such as caprylic acid, capric acid, oleic acid, linoleic acid, and polyunsaturated fatty acids, may also contribute through mechanisms like α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition, PPAR-γ activation, and anti-inflammatory or antioxidant effects (32–34). Given the age-specific impacts of fatty acids on neuronal protection, systematic analyses of their metabolic profiles and functions in neonatal seizure models could advance the development of personalized therapeutic strategies for neonatal seizures.

Cortical transcriptomics and qPCR showed an upregulation of ZDHHC14 expression in the cortex of Apoc2^−/− hamsters before and after PTZ injection. Since free palmitic acid serves as the substrate for protein palmitoylation, these findings suggest an increased level of palmitoylation in the cortex of Apoc2^−/− HTG hamsters. Palmitoylation, the covalent attachment of a palmitic acid ester to cysteine residues in proteins, is one of the most important lipid modifications. Protein palmitoylation dynamically regulates the transport of proteins between the plasma membrane and intracellular compartments such as the Golgi apparatus, endoplasmic reticulum, and endocytic vesicles (35, 36). In the nervous system, palmitoylation is closely associated with the transport of ion channel receptors and ion channels. Kang et al. published a study in Nature that conducted a palmitoyl-proteomics analysis on rat cortical neurons, identifying 89 palmitoylated proteins, including neurotransmitter receptors, ion channels, and scaffold proteins. Most of these proteins are transmembrane proteins, with palmitoylation contributing to their targeted trafficking to membrane microdomains. Furthermore, after inducing seizures in the animals, Kang et al. re-examined the levels of protein palmitoylation in neurons and found an increase in palmitoylation levels for most proteins (37). Spinelli et al. found that overexpression of ZDHHC3 can lead to enhanced palmitoylation modification of the GluA1 subunit of AMPA receptors, inhibit their stimulus-dependent trafficking to the plasma membrane, and thereby affect synaptic plasticity (38). Mice with palmitoylation-deficient C-terminal GluA1 subunits of AMPA receptors exhibit increased brain hyperexcitability and heightened seizure susceptibility (39). The palmitoylation modification of Kv1.5 can regulate its stable expression on the membrane (40).

Palmitoyl acyltransferases (PATs), which contain a DHHC (Asp-His-His-Cys) zinc finger domain, are responsible for catalyzing palmitoylation modification reactions. Therefore, they are also known as the ZDHHC protein family. In mammals, 23 members of this family have been identified (41, 42). The functions of each member of the ZDHHC family are still being intensively researched. Although existing evidence suggests that protein palmitoylation is crucial for maintaining the normal physiological functions of neuronal ion channels and ion channel receptors (40, 43–45), research on the role of protein palmitoylation in the onset and progression of epilepsy remains insufficient. Kang et al. found that after inducing seizures in rats, the level of neuronal protein palmitoylation was enhanced compared to the non-convulsive group (37), indicating that the regulation of palmitoylation is closely related to neuronal firing activity. It warrants further investigation to uncover novel therapeutic targets.

Our results suggest that ZDHHC14 expression in the cerebral cortex of HTG hamsters is upregulated after seizure induction. Although ZDHHC14 is highly expressed in the brain, there is limited research on its functions in the nervous system. A recent study by Sanders et al. suggests that ZDHHC14 in the hippocampus mediates palmitoylation of postsynaptic density 93 and type I voltage-gated potassium (Kv1) channels, targeting them to the axon initial segment (AIS). Moreover, the loss of ZDHHC14 leads to reduced palmitoylation of Kv1 channels, resulting in a decrease in AIS-targeted potassium channels and a subsequent reduction in voltage-dependent outward currents, thereby, increasing neuronal excitability (28). To date, there has been no functional research on ZDHHC14 in the cortex. Therefore, we further examined the magnitude of voltage-gated potassium currents in the cortical neurons. The results showed that the transient outward potassium current (I_A) was enhanced in the cortical neurons of Apoc2^−/− hamsters. I_A can slow the approach of the membrane potential to the threshold in excited neurons, prolong the inter-spike interval of action potentials, and reduce the firing frequency of action potentials (46, 47). Additionally, I_A can quickly recover from inactivation, facilitating the repolarization of action potentials. This is consistent with our observation of decreased excitatory postsynaptic potentials and evoked action potential frequencies in the frontal cortex neurons of Apoc2^−/− hamsters.

This study revealed the differential expression of ZDHHC14, highlighting its significant role in regulating neuronal excitability and conferring protection against seizures. However, given the complexity of the ZDHHC family and the potential functional redundancy among its members, other ZDHHC proteins may also contribute to this process. For instance, ZDHHC17 has been shown to catalyze the palmitoylation of the voltage-gated potassium channel Kv1.1, thereby regulating its subcellular localization at the axon initial segment (43). Similarly, ZDHHC3 and ZDHHC15 have been demonstrated to influence synaptic transmission and plasticity through their regulatory roles in the trafficking of AMPA receptors, NMDA receptors, and GABA_A receptors (45, 48–50). Moreover, the crystal structures of ZDHHC family members remain insufficiently characterized, which may account for substrate overlap among some members. In this study, while differential expression of ZDHHC14 was observed, the expression levels and functions of other family members have yet to be comprehensively evaluated. Therefore, future studies will employ gene knockout or overexpression approaches to further elucidate the specific role of ZDHHC14 in neuronal excitability and seizure modulation.

In recent years, a few studies have begun to focus on the impact of high triglycerides on palmitoylation. Our research results also suggest a potential association between hypertriglyceridemia and palmitoylation. However, we are currently unable to provide a detailed explanation for this, and further research is needed. Spinelli et al. found that a high-fat diet induced insulin resistance in the hippocampus of mice, leading to upregulated expression of FOXO3a-mediated ZDHHC3 and increased palmitoylation levels in the hippocampus (38). It is evident that the lipid abundance provided by high triglycerides offers substrates for palmitoylation in the nervous system, maintaining active palmitoylation activity and resulting in more pronounced molecular interactions. However, in the Apoc2^−/− HTG hamster cortex used in this study, no changes were observed in the expression of insulin resistance-related genes. The outcomes of neurological experiments depend heavily on the age of the animals. In Spinelli’s experiment, adult mice aged 10–11 weeks were selected, with the research focus on synaptic plasticity of hippocampal neurons rather than excessive abnormal synchronization of numerous cortical neurons, which differs from the focus of this study.

The ketogenic diet, which utilizes fat as the primary energy source, has demonstrated well-established anti-seizure effects. However, its application in the neonatal period is substantially constrained due to the requirement for high-fat intake, which can result in feeding intolerance, hypoglycemia, and severe metabolic acidosis, potentially posing life-threatening risks (51). Elucidating the mechanisms by which triglycerides regulate neuronal excitability, including the identification of specific pathways and molecular targets, holds promise for optimizing ketogenic therapies and developing precision-targeted pharmacological interventions, such as ZDHHC14 modulators, for use in conjunction with lipid-based therapies like the ketogenic diet. Despite the potential of voltage-gated potassium channels as therapeutic targets for anti-seizure drug development, relatively few pharmacological agents specifically target these channels. The role of ZDHHC14-mediated palmitoylation in regulating voltage-gated potassium channels remains incompletely understood. This highlights a significant avenue for clinical research. Investigating the catalytic domains and regulatory regions of ZDHHC14, alongside resolving its three-dimensional crystal structure, could facilitate the development of novel therapeutic strategies for epilepsy management.

This study identified a potential regulatory role of ZDHHC14 in the cortex on transient outward potassium current. However, further research is required to elucidate how ZDHHC14 influences transient outward potassium channels in the cortex, its regulatory effects on the intracellular trafficking of potassium channels, and the specific subtypes of interacting target proteins. These investigations are essential to clarify the functional role of ZDHHC14 in the nervous system and its mechanisms for modulating neuronal excitability.

In summary, this study demonstrates that the Apoc2^−/− hypertriglyceridemic hamster model exhibits a reduced frequency of induced seizures and decreased cortical neuron excitability. These findings confirm, both in vivo and ex vivo, the seizure-protective effects of elevated triglycerides. Mechanistic exploration indicates that the upregulation of ZDHHC14 expression in the cortex, leading to enhanced transient outward potassium currents, may contribute to this protective effect. This study provides novel insights into potential therapeutic targets for neonatal seizures. Future research will focus on further elucidating the role of ZDHHC14 in modulating neuronal excitability in the cortex by knocking down or overexpressing ZDHHC14. Additionally, we will explore the relationship between hypertriglyceridemia and neural palmitoylation.