Krüppel-like factors: potential roles in blood-brain barrier dysfunction and epileptogenesis

Abstract

Epilepsy is a chronic and debilitating neurological disorder, known for the occurrence of spontaneous and recurrent seizures. Despite the availability of antiseizure drugs, 30% of people with epilepsy experience uncontrolled seizures and drug resistance, evidencing that new therapeutic options are required. The process of epileptogenesis involves the development and expansion of tissue capable of generating spontaneous recurrent seizures, during which numerous events take place, namely blood-brain barrier (BBB) dysfunction, and neuroinflammation. The consequent cerebrovascular dysfunction results in a lower seizure threshold, seizure recurrence, and chronic epilepsy. This suggests that improving cerebrovascular health may interrupt the pathological cycle responsible for disease development and progression. Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors, encountered in brain endothelial cells, glial cells, and neurons. KLFs are known to regulate vascular function and changes in their expression are associated with neuroinflammation and human diseases, including epilepsy. Hence, KLFs have demonstrated various roles in cerebrovascular dysfunction and epileptogenesis. This review critically discusses the purpose of KLFs in epileptogenic mechanisms and BBB dysfunction, as well as the potential of their pharmacological modulation as therapeutic approach for epilepsy treatment.

Keywords: blood-brain barrier; cerebrovascular dysfunction; epigenetics; epilepsy; krüppel-like factors; neuroinflammation.

Fig. 1. Main events of epileptogenesis.

An epileptogenic insult (red) prompts a series of events of variable duration (green and blue) that convert healthy brain networks into tissue capable of generating spontaneous recurrent seizures, leading to the establishment of chronic epilepsy (purple) [6, 7]. It is thought to have three phases: the initial epileptogenic event (1); the latent period (2); and the chronic epilepsy phase (3) [3]. BBB blood-brain barrier, SE status epilepticus. Created with BioRender^®.

Fig. 2. Healthy blood-brain barrier (BBB) versus damaged BBB in epilepsy.

a In an intact BBB, brain endothelial cells (BECs) are united by tight junctions (TJs) and adherens junctions (AJs), and encircled by pericytes (1). Astrocytic endfeet cover the basement membrane, while neighboring microglia perform immune vigilance (2). The glycocalyx is also part of the neurovascular unit (NVU) (3) [23, 145]. b In epilepsy, activated astrocytes and microglia are a source of pro-inflammatory molecules (1), including matrix metalloproteinases (MMPs), that prompt the loss of TJs (2), namely claudin-5, occludin and zonula-occludens (ZO)-1, and glycocalyx damage (3). This results in the entry of albumin into the brain (4) and exposure of leukocyte adhesion molecules (LAMs) with consequent infiltration of peripheral leukocytes (5), aggravation of neuroinflammation, and lower seizure threshold [23, 26, 27, 31, 51, 146]. ICAM-1 intercellular adhesion molecule-1, JAM junctional adhesion molecule, VCAM-1 vascular cell adhesion molecule-1. Created with BioRender^®.

Fig. 3. Krüppel-like factors (KLFs) structure and involvement in inflammation through transcriptional activation.

a The N-terminal is the variable region that comprises different combinations of activation and repression domains, responsible for the transcriptional activity [60, 65]. The C-terminal region is the DNA binding domain, consisting of three Cys2/His2 zinc-fingers [64]; b Phylogenetic tree of human KLF family, based on their functional and structural properties: the KLF family is divided into three different groups. In group 1, KLF 3, 8, 12 act as transcriptional repressors; in group 2, KLF 1, 2, 4, 5, 6 and 7 are transcriptional activators; in group 3, KLF 9, 10, 11, 13, 14 and 16 have repressor activity [62]. The activities of KLF15, KLF17 and KLF18 are not yet clear [58, 67]; c Transcriptional activation of nuclear factor kappa-B (NF-κB) promoted by KLFs: the transcriptional activation by the KLF family involves binding to coactivator histone acetyltransferases, such as CREB binding protein (CBP) and p300 [67]. P300/CBP-associated factor (PCAF) is recruited with p300/CBP to specific promoters, forming a complex, which promotes a targeted acetylation of chromatin domains and gene transcription [68]. Adapted from [69]. Created with BioRender®.

Fig. 4. Repressor element-1 silencing transcription factor (REST) structure and function as transcriptional repressor.

a REST is a large KLF with nine zinc-fingers. It has a N-terminal repressor domain (NRD), a DNA binding domain (DBD) and a C-terminal repressor domain (CRD). There is a nuclear localization signal in zinc-finger 5; b Transcriptional repression mediated by REST and co-repressors. REST forms a multimeric complex by recruiting Sin3A/B through the NRD, and REST corepressor 1 (CoREST) through zinc-finger 9 of the CRD. In turn, Sin3A/B interacts with histone deacetylases (HDAC1/2 and 4/5). CoREST recruits HDAC 1/2 and other repressors such as histone demethylase (LSD1) or histone lysine methyltransferase G9a, through chromodomain Y-like (CDYL). Adapted from [71, 72]. Created with BioRender^®.

Fig. 5. Krüppel-like factor (KLF) modulation as potential therapeutic approach in epilepsy.

Excessive glutamate levels trigger microglia activation after seizure onset (1), which gradually enhance astrocyte activation through the release of pro-inflammatory mediators (2). These events aggravate and perpetuate neuroinflammation, leading to the loss of tight junctions (TJs) between brain endothelial cells and overall BBB disruption. This enables the entry of albumin into the brain (3), which is internalized into astrocytes and disturbs K⁺ reuptake, thereby increasing neuronal excitability. The activation of transforming growth factor-β (TGF-β) signaling also contributes to this phenomenon, together with interleukin-1β (IL-1β) (4). Ultimately, seizure threshold decreases, resulting in spontaneous recurrent seizures (5). KLFs inhibit (green) or activate (red) these events, suggesting that their modulation may be a feasible approach to prevent disease progression. IL-6 interleukin-6, JAM-A junctional adhesion molecule A, MMPs matrix metalloproteinases, SE status epilepticus, TNF-α tumor necrosis factor, ZO-1 zonula-occludens 1. Created with BioRender^®.