CREB3L4 upregulates SIRT4 expression to increase GABA/glutamate ratio and mitochondrial homeostasis for epilepsy...

cAMP response element-binding protein 3-like transcription factor 4 (CREB3L4) serves as an important transcriptional regulatory factor and plays a crucial role in neurological diseases, however, its specific mechanism in epilepsy pathogenesis remains unclear. This study aims to investigate the role of the CREB3L4-Sirtuin 4 (SIRT4) regulatory axis in epilepsy pathogenesis and its effects on γ-aminobutyric acid GABA/glutamate balance and mitochondrial function.

A classical epilepsy animal model was established, and quantitative real-time PCR, Western blot, and immunofluorescence staining techniques were employed to detect the expression changes of CREB3L4 and SIRT4 in the epilepsy model. CREB3L4 expression levels were modulated through lentivirus-mediated gene overexpression and RNA interference techniques to observe their effects on epileptic behavioral manifestations. ELISA was used to detect GABA and glutamate contents in brain tissues, and the GABA/glutamate ratio was calculated.

CREB3L4 expression was downregulated 3.2-fold in the epilepsy model (1.026 ± 0.051-0.325 ± 0.028, p < 0.001), with 1.8-fold decreased GABA/glutamate ratio (1.065 ± 0.063-0.581 ± 0.046, p < 0.001). CREB3L4 overexpression upregulated SIRT4 mRNA 3.5-fold (0.185 ± 0.051-0.643 ± 0.039, p < 0.01) and reduced seizure frequency 2.0-fold (67.6 ± 3.1-34.4 ± 2.4 seizures, p < 0.001) and severity 2.2-fold (4.3 ± 0.3-2.0 ± 0.3 grade, p < 0.001). Conversely, CREB3L4 silencing exacerbated epilepsy progression, increasing seizure frequency 1.4-fold (58.7 ± 2.7-79.3 ± 4.2 seizures, p < 0.001) and severity 1.3-fold (3.7 ± 0.3-4.7 ± 0.2 grade, p < 0.001), with 2.2-fold decreased GABA/glutamate ratio (0.639 ± 0.051-0.288 ± 0.041, p < 0.001). Mechanistic studies demonstrated that CREB3L4 overexpression improved mitochondrial function through SIRT4 upregulation, thereby maintaining mitochondrial homeostasis. Meanwhile, activation of the CREB3L4-SIRT4 axis effectively increased brain tissue GABA content, improved the GABA/glutamate ratio, and restored neurotransmitter balance. Rescue experiments demonstrated that SIRT4 silencing (3.1-fold knockdown) significantly attenuated CREB3L4's neuroprotective effects, increasing seizure severity 1.6-fold (3.0 ± 0.5-4.8 ± 0.2 grade, p < 0.01), frequency 1.7-fold (43.2 ± 3.1-74.7 ± 4.2 seizures, p < 0.001), and reducing GABA/glutamate ratio 1.6-fold (1.81 ± 0.05-1.12 ± 0.05, p < 0.001), confirming SIRT4 as the key downstream mediator.

This study elucidates the important role of the CREB3L4-SIRT4 regulatory axis in epilepsy pathogenesis, revealing the molecular mechanism by which CREB3L4 transcriptionally activates SIRT4, subsequently regulates GABA/glutamate balance and maintains mitochondrial homeostasis, ultimately suppressing seizures and associated pathophysiological damage.