Treatment with the Ketone Body D-β-hydroxybutyrate Attenuates Autophagy Activated by NMDA and Reduces Excitotoxic Neuronal Damage in the Rat Striatum In Vivo

Abstract

Background: The ketone bodies (KB), β-hydroxybutyrate (BHB) and acetoacetate, have been proposed for the treatment of acute and chronic neurological disorders, however, the molecular mechanisms involved in KB protection are not well understood. KB can substitute for glucose and support mitochondrial metabolism increasing cell survival. We have reported that the D-isomer of BHB (D-BHB) stimulates autophagic degradation during glucose deprivation in cultured neurons increasing cell viability. Autophagy is a lysosomal degradation process of damaged proteins and organelles activated during nutrient deprivation to obtain building blocks and energy. However, impaired or excessive autophagy can contribute to neuronal death.

Objective: The aim of the present study was to test whether D-BHB can preserve autophagic function in an in vivo model of excitotoxic damage induced by the administration of the glutamate receptor agonist, N-methyl-Daspartate (NMDA), in the rat striatum.

Methods: D-BHB was administered through an intravenous injection followed by either an intraperitoneal injection (i.v+i.p) or a continuous epidural infusion (i.v+pump), or through a continuous infusion of D-BHB alone. Changes in the autophagy proteins ATG7, ATG5, BECLIN 1 (BECN1), LC3, Sequestrosome1/p62 (SQSTM1/ p62) and the lysosomal membrane protein LAMP2, were evaluated by immunoblot. The lesion volume was measured in cresyl violet-stained brain sections.

Results: Autophagy is activated early after NMDA injection but autophagic degradation is impaired due to the cleavage of LAMP2. Twenty-four h after NMDA intrastriatal injection, the autophagic flux is re-established, but LAMP2 cleavage is still observed. The administration of D-BHB through the i.v+pump protocol reduced the content of autophagic proteins and the cleavage of LAMP2, suggesting decreased autophagosome formation and lysosomal membrane preservation, improving autophagic degradation. D-BHB also reduced brain injury. The i.v+i.p administration protocol and the infusion of D-BHB alone showed no effect on autophagy activation or degradation.

Keywords: Autophagic flux; excitotoxicity; ketone bodies; lysosomal degradation; neuronal death; striatum..