RESUMEN
The strength of inhibitory neurotransmission depends on intracellular neuronal chloride concentration, primarily regulated by the activity of cation-chloride cotransporters NKCC1 (Sodium-Potassium-Chloride Cotransporter 1) and KCC2 (Potassium-Chloride Cotransporter 2). Brain-derived neurotrophic factor (BDNF) influences the functioning of these co-transporters. BDNF is synthesized from precursor proteins (proBDNF), which undergo proteolytic cleavage to yield mature BDNF (mBDNF). While previous studies have indicated the involvement of BDNF signaling in the activity of KCC2, its specific mechanisms are unclear. We investigated the interplay between both forms of BDNF and chloride homeostasis in rat hippocampal neurons and in utero electroporated cortices of rat pups, spanning the behavioral, cellular, and molecular levels. We found that both pro- and mBDNF play a comparable role in immature neurons by inhibiting the capacity of neurons to extrude chloride. Additionally, proBDNF increases the endocytosis of KCC2 while maintaining a depolarizing shift of EGABA in maturing neurons. Behaviorally, proBDNF-electroporated rat pups in the somatosensory cortex exhibit sensory deficits, delayed huddling, and cliff avoidance. These findings emphasize the role of BDNF signaling in regulating chloride transport through the modulation of KCC2. In summary, this study provides valuable insights into the intricate interplay between BDNF, chloride homeostasis, and inhibitory synaptic transmission, shedding light on the underlying cellular mechanisms involved.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Cloruros , Cotransportadores de K Cl , Neuronas , Miembro 2 de la Familia de Transportadores de Soluto 12 , Animales , Femenino , Ratas , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Cloruros/metabolismo , Hipocampo/metabolismo , Homeostasis , Neuronas/metabolismo , Precursores de Proteínas/metabolismo , Simportadores/metabolismoRESUMEN
The potassium-chloride co-transporter 2, KCC2, is a neuron-specific ion transporter that plays a multifunctional role in neuronal development. In mature neurons, KCC2 maintains a low enough intracellular chloride concentration essential for inhibitory neurotransmission. During recent years, pathogenic variants in the KCC2 encoding gene SLC12A5 affecting the functionality or expression of the transporter protein have been described in several patients with epilepsy of infancy with migrating focal seizures (EIMFS), a devastating early-onset developmental and epileptic encephalopathy. In this study, we identified a novel recessively inherited SLC12A5 c.692G>A, p. (R231H) variant in a patient diagnosed with severe and drug-resistant EIMFS and profound intellectual disability. The functionality of the variant was assessed in vitro by means of gramicidin-perforated patch-clamp experiments and ammonium flux assay, both of which indicated a significant reduction in chloride extrusion. Based on surface immunolabeling, the variant showed a reduction in membrane expression. These findings implicate pathogenicity of the SLC12A5 variant that leads to impaired inhibitory neurotransmission, increasing probability for hyperexcitability and epileptogenesis.