Newborns 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. A ketogenic diet primarily composed of high triglycerides is effective for treating intractable epilepsy, but its application in newborns is challenging. Therefore, studying the role of high triglycerides in seizures can provide new insights for identifying novel therapeutic targets. We chose an HTG ApoC2−/− hamster model and compared it with a control hamster. The HTG hamsters exhibited a reduction in the frequency of acute seizure attacks. Ex vivo experiments 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. However, because the size of hamsters aged P7–P10 is relatively small, we chose P15–P17 to establish an acute seizure model. Hyperlipidaemia 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 showed 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. Pathological staining of the frontal cortex in animals following seizures did not show significant differences in the number or morphology of neurons. This rules out the possibility that hypertriglyceridemia reduces excitability by affecting the structural morphology of the frontal cortex. Thus, 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, there have been no studies investigating the effect of hypertriglyceridemia on acute seizures, particularly on neuronal excitability in neonatal seizure models. One of the possible mechanisms underlying the anti-epileptic effect of a ketogenic diet is that it provides medium-chain fatty acids and ketone bodies as alternative energy sources for epileptogenic brain regions (Han et al. 2021; Steiner 2019). 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 explore the mechanism, we collected microdialysis samples from HTG− and wild-type hamsters before and after seizures and performed lipidomic analysis to examine free fatty acid profiles. We found that both wild-type and HTG hamsters exhibited a decreasing trend in extracellular free palmitic acid levels after induced seizures compared to the baseline state, and after seizures, HTG hamsters showed a significantly lower level of free palmitic acid than wild-type hamsters. Cortical transcriptome and qPCR data revealed upregulation of PAT ZDHHC14 expression in the frontal cortex of HTG hamsters before or after seizures, indicating an upward trend in palmitoylation modification levels in the frontal cortex of HTG hamsters.
Palmitoylation, the covalent attachment of palmitic acid ester to cysteine residues in proteins, is one of the most important lipid modifications. Palmitoyl acyltransferases, which contain a DHHC (Asp-His-His-Cys) zinc finger domain, are responsible for catalysing palmitoylation modification reactions. Therefore, they are also known as the ZDHHC protein family. In mammals, 23 members of this family have been identified. The functions of each member of the ZDHHC family are still being intensively researched. ZDHHC3, ZDHHC5, and ZDHHC8 are involved in substrate recruitment (Gottlieb and Linder 2017), while ZDHHC17 and ZDHHC13 participate in the specific binding of substrates. (Chamberlain and Shipston 2015).
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 (Hayashi 2021). Existing studies have demonstrated that stimulation-dependent endocytosis of the excitatory neurotransmitter receptor AMPA on the synaptic surface is regulated by carboxyl-terminal palmitoylation (Hayashi et al. 2005; Lin et al. 2009). Mice with a defect in carboxyl-terminal palmitoylation of the AMPA receptor subunit GluA1 exhibit cerebral hyperexcitability and elevated seizure susceptibility (Iizumi et al. 2021). Changes in the brain lipid environment are associated with palmitoylation. Spinelli et al. found that mice fed a high-fat diet (HFD) displayed increased expression of ZDHHC3 in the hippocampus, leading to enhanced palmitoylation of the GluA1 subunit, which inhibited its stimulation-dependent translocation to the plasma membrane, thereby affecting synaptic plasticity and memory (Spinelli et al. 2017). Seizure induction involves providing strong stimulation to neurons, and it is reasonable to speculate that such strong stimulation after high-fat diet consumption may alter the palmitoylation of GluA1. Currently, however, there is no research confirming this; further studies are needed.
Our results suggest that ZDHHC14 expression in the frontal cortex of HTG hamsters is upregulated. 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, increasing neuronal excitability (Sanders et al. 2020). To date, there has been no functional research on ZDHHC14 in the frontal cortex. We found that the expression of ZDHHC14 in the frontal cortex was upregulated in the HTG hamster model after PTZ injection. Previous studies have suggested that ZDHHC14 palmitoylates Kv1 channels in hippocampal neurons, an action that can regulate neuronal excitability (Sanders et al. 2020). This suggests that the HTG hamster model shows reduced seizure frequency and decreased EPSP and AP frequencies in cortical neurons through a mechanism relying on the upregulation of ZDHHC14, leading to increased palmitoylation modification in the frontal cortex. This promotes the targeted localization of Kv1 potassium channels at the AIS, increasing outward current and decreasing neuronal excitability. This is an area of ongoing research for us. Additionally, since palmitoylation also affects the trafficking of AMPA and NMDA ionotropic glutamate receptors to the synaptic surface, although there is currently no research suggesting the involvement of ZDHHC14 in this process, we cannot rule it out as another potential mechanism by which ZDHHC14 influences neuronal excitability. This requires further mechanistic investigations.
We discovered a possible association between HTG and palmitoylation, but we currently cannot provide a detailed explanation of this association. Spinelli et al. found that an HFD induced insulin resistance in the hippocampus of mice, leading to upregulated expression of FOXO3a-mediated ZDHHC3 and increased palmitoylation levels in the hippocampus (Spinelli et al. 2017). However, in the ApoC2−/− HTG hamster frontal cortex used in this study, no changes were observed in the expression of the aforementioned genes. The outcomes of neurological experiments depend heavily on the age of the animals, and the hamsters selected in this study were 15–17 days old, which corresponds to the infant stage. In Spinelli's experiments, adult mice aged 10–11 weeks were used. There are differences in blood lipid levels between HFD-fed and genetically engineered HTG animals, and our study focuses on how the lipid environment affects neuronal function during acute seizures. These reasons may contribute to the differences in our results.
In summary, in the HTG hamster model, the frequency of induced seizures and the excitability of cortical neurons decreased during synchronized firing of a large number of neurons. This may be associated with upregulation of ZDHHC14 in the frontal cortex, leading to an increase in palmitoylation modification. This allows us to use more potential mechanisms to achieve higher efficacy, providing new perspectives in the exploration of novel therapeutic targets for neonatal seizures. In future research, we will further verify the impact of ZDHHC14 on cortical excitability and explore the relationship between hypertriglyceridemia and palmitoylation in the cerebrum.