Excitotoxicity is defined by the excessive buildup of glutamate or other excitatory amino acids in the extracellular space, leading to the heightened activation of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) subtype, in the adult central nervous system (CNS) [1, 2]. This excessive activation causes a sustained influx of calcium (Ca2+) through receptor channels, leading to neuronal cell death. Elevated levels of glutamate in the CNS increase intracellular Ca2+ concentrations, triggering apoptotic stimuli within sensitive organelles like mitochondria and the endoplasmic reticulum (ER). This results in the production of toxic radicals, disruption of cellular energy production, and ultimately, cell death through acute necrosis and/or delayed apoptosis [3]. Additionally, glutamate exposure can activate extrasynaptic NMDA Receptors (NMDARs), halt cAMP response element-binding protein (CREB) activity, cause mitochondrial membrane potential loss, and lead to cell death [4].
Apelin, an endogenous neuropeptide widely distributed throughout various physiological systems, particularly prevalent within the nervous system, undergoes intricate post-translational modifications, resulting in the generation of diverse mature apelin active peptides, with apelin-13 exhibiting the most robust biological activity [5]. Apelin is a natural ligand for the G protein-coupled apelin receptor (APJ) [5]. The apelin/APJ system has been shown to possess neuroprotective properties, including anti-inflammatory, anti-oxidative stress, anti-apoptotic effects, autophagy regulation, and inhibition of excitotoxicity, suggesting its potential as a therapeutic target for neurological disorders [6, 7, 8].
Glutamate excitotoxicity, neuroinflammation, and oxidative stress collectively manifest as a pathogenic “triad” in various brain disorders, leading to cellular demise and perturbations in network dynamics [9]. Neuroinflammation is characterized by the activation of neuronal cells, leading to dysregulation of anti-inflammatory/proinflammatory cytokine ratios due to the excessive secretion of proinflammatory cytokines. These cytokines influence glutamate homeostasis by upregulating glutamate receptors, enhancing glutamatergic neurotransmission, and exacerbating excitotoxicity [10].
Oxidative stress, resulting from an imbalance in the redox state of cells due to excessive production of reactive oxygen species (ROS), can lead to oxidative damage to cellular macromolecules and contribute to various neurodegenerative diseases [11]. Imbalances in the oxidant/antioxidant relationship and excessive ROS production increase the levels of molecules such as advanced glycation end products (AGE), advanced oxidation protein products (AOPP), and kynurenine (KYN), dityrosine (DT), inducing DNA methylation and creating toxic effects on neurons [12, 13]. High ROS levels activate inflammatory pathways such as nuclear factor-kappa beta (NF-κB) and mitogen-activated protein kinase (MAPK), causing cellular damage and decreasing Brain-Derived Neurotrophic Factor (BDNF) expression in neurons [14]. Apelin-13 increases intracellular cyclic Adenosin Monophosphate (cAMP) levels, activating the protein kinase A (PKA) pathway, which suppresses inflammatory signaling pathways and increases BDNF synthesis [15–17]. Additionally, Apelin-13 binds to the APJ receptor, initiating MAPK activation through G-protein coupled receptor (GPCR) signaling pathways, triggering phospholipase C (PLC) activation [18]. PLC stimulates the production of inositol triphosphate (IP3) and diacylglycerol (DAG), leading to intracellular Ca+ 2 release and protein kinase C (PKC) activation. These signals activate the Extracellular signal-Regulated Kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 MAPK pathways, regulating BDNF gene expression [15–17]. Total thiol levels reduce oxidative stress by enhancing cellular antioxidant defense capacity [19]. However, the increase in ROS levels due to mitochondrial dysfunction and the direct interaction of ROS with thiol groups (-SH) leads to the oxidation of free thiol groups [20, 21]. The increase in disulfide bonds (R-S-S-R) in thiol groups causes protein structural degradation and amino acid side chain oxidation, leading to carbonylation, decreased -SH reactivity, and peroxidation [21]. As a result of this process, total thiol levels have been reported to decrease in neurodegenerative diseases [20, 21].
Interleukin-1β (IL-1β) and Tumor necrosis Factor-α (TNF-α) have been shown to play a role in glutamate-mediated excitotoxicity in several studies [22, 23]. These cytokines are secreted by Human Neuroblastoma cell line (SH-SY5Y cell line) in response to NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-induced excitotoxicity, triggering inflammatory responses that lead to cellular damage, neurodegeneration, apoptosis, and further inflammation [24, 25]. Apelin-13, known for its anti-inflammatory effects on neurons, can reduce inflammation and neuronal damage by suppressing the production of IL-1β and TNF-α [18, 26]. Apelin-13 binds to the APJ receptor, enhancing adenylate cyclase activity and cAMP production, which leads to PKA activation and suppression of inflammatory pathways such as NF-κB [27].
Anti-inflammatory cytokines play a crucial role in regulating inflammatory responses and protecting cells from the damaging effects of inflammation. Transforming growth factor β (TGF-β) is a prominent anti-inflammatory cytokine, existing in three isoforms: TGF-β1, TGF-β2, and TGF-β3 [28]. Among these, TGF-β1 is the most abundant subtype in mammals, with significant anti-inflammatory and neuroprotective effects [29]. TGF-β1 has been shown to stimulate the regrowth of damaged neurons after axonal injury [30]. Although its role in neural plasticity and memory in humans is not well understood, TGF-β1’s contribution to learning and memory mechanisms has been demonstrated in rodents [31–33]. Evidence suggests that TGF-β1 may have a protective role in neurodegenerative and neuroinflammatory diseases such as Alzheimer’s and multiple sclerosis [34, 35].
Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), serves as an immunomodulator in the anti-inflammatory process [36]. IL-10 possesses numerous immunoregulatory effects crucial for resolving inflammation and can inhibit the production of various inflammatory cytokines such as TNF-α, IL-1β, Interleukin-6 (IL-6), and Interferon-gamma (IFN-γ) [37]. It is produced by nearly all leukocytes, including T cell subsets, monocytes, macrophages, neutrophils, eosinophils, mast cells, dendritic cells (DCs), B cells, and natural killer (NK) cells, as well as by keratinocytes, epithelial cells, and Nestin+ neuroblasts [36, 38]. In the central nervous system, IL-10 receptors (IL-10R) are expressed by microglia, astrocytes, oligodendrocytes, and even neurons under both physiological and pathological conditions, initiating the Janus kinase (Jak)/tyrosine kinase (Tyk) signaling pathway in neurons [39–41].
BDNF is a neurotrophic factor critical for maintaining neuronal health and enhancing synaptic plasticity in the central nervous system. BDNF supports the growth, differentiation, and survival of neurons while also contributing to the strengthening of synaptic connections [42, 43]. Environmental factors such as stress and toxicity can affect BDNF levels; some studies report that BDNF levels increase with stress, while others indicate a decrease [44, 45]. A reduction in BDNF levels can negatively impact neuronal health and function, and is associated with neurodegenerative diseases [46]. High doses of glutamate increase intracellular Ca2+ levels through the overactivation of NMDA and AMPA receptors, leading to mitochondrial dysfunction and increased ROS production. This process exacerbates neuronal stress and toxicity, suppressing BDNF production and negatively affecting neuronal health [47, 48].
This study uses D-glutamic acid instead of the more commonly studied L-glutamic acid. D-amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are stereospecific enzymes that metabolize D-amino acids (D-AAs). DDO specifically acts on acidic D-AAs like D-aspartate, D-glutamate, and NMDA, converting them into imino acids while reducing flavin adenine dinucleotide (FAD) [49]. Subsequently, FAD is reoxidized in the presence of oxygen, producing hydrogen peroxide (H2O2). High levels of H2O2 cause oxidative stress and trigger events such as mitochondrial dysfunction, pro-inflammatory cytokine activation, and apoptotic and autophagic cell death [50]. Additionally, the accumulation of D-AAs has been associated with immune activation, and cellular exposure to D-AAs is suggested to induce inflammation and cell death through H2O2 and NF-κB activation [51].
Aim
The aim of this in vitro study is to evaluate the protective effect of apelin-13 in a glutamic acid-induced excitotoxicity model in SH-SY5Y cells and to determine the underlying protective mechanisms. Specifically, the study aims to assess the changes in levels of BDNF, IL-10, IL-1β, TNF-α, TGF-β1, and ROS as well as KYN, DT, AGEs, AOPP, and T-SH following apelin-13 treatment. The findings of this study may shed light on the neuroprotective effects of apelin-13, elucidate its mechanisms of action, and identify potential novel targets for the treatment of neurodegenerative diseases.