Mitochondrial activity and quality control are essential for neuronal homeostasis as neurons rely on glucose oxidative metabolism. The ketone body, D-β-hydroxybutyrate (D-BHB), is metabolized to acetyl-CoA in brain mitochondria and used as an energy fuel alternative to glucose. We have previously reported that D-BHB sustains ATP production and stimulates the autophagic flux under glucose deprivation in neurons; however, the effects of D-BHB on mitochondrial turnover under physiological conditions are still unknown. Sirtuins (SIRTs) are NAD+-activated protein deacetylases involved in the regulation of mitochondrial biogenesis and mitophagy through the activation of transcription factors FOXO1, FOXO3a, TFEB and PGC1α coactivator. Here, we aimed to investigate the effect of D-BHB on mitochondrial turnover in cultured neurons and the mechanisms involved. Results show that D-BHB increased mitochondrial membrane potential and regulated the NAD+/NADH ratio. D-BHB enhanced FOXO1, FOXO3a and PGC1α nuclear levels in an SIRT2-dependent manner and stimulated autophagy, mitophagy and mitochondrial biogenesis. These effects increased neuronal resistance to energy stress. D-BHB also stimulated the autophagic–lysosomal pathway through AMPK activation and TFEB-mediated lysosomal biogenesis. Upregulation of SIRT2, FOXOs, PGC1α and TFEB was confirmed in the brain of ketogenic diet (KD)-treated mice. Altogether, the results identify SIRT2, for the first time, as a target of D-BHB in neurons, which is involved in the regulation of autophagy/mitophagy and mitochondrial quality control.

Altered protein homeostasis is associated with neurodegenerative diseases and acute brain injury induced under energy depletion conditions such as ischemia. The accumulation of damaged or unfolded proteins triggers the unfolded protein response (UPR), which can act as a homeostatic response or lead to cell death. However, the factors involved in turning and adaptive response into a cell death mechanism are still not well understood. Several mechanisms leading to brain injury induced by severe hypoglycemia have been described but the contribution of the UPR has been poorly studied. Cell responses triggered during both the hypoglycemia and the glucose reinfusion periods can contribute to neuronal death. Therefore, we have investigated the activation dynamics of the PERK and the IRE1α branches of the UPR and their contribution to neuronal death in a model of glucose deprivation (GD) and glucose reintroduction (GR) in cortical neurons. Results show a rapid activation of the PERK/p-eIF2α/ATF4 pathway leading to protein synthesis inhibition during GD, which contributes to neuronal adaptation, however, sustained blockade of protein synthesis during GR promotes neuronal death. On the other hand, IRE1α activation occurs early during GD due to its interaction with BAK/BAX, while ASK1 is recruited to IRE1α activation complex during GR promoting the nuclear translocation of JNK and the upregulation of Chop. Most importantly, results show that IRE1α RNase activity towards its splicing target Xbp1 mRNA occurs late after GR, precluding a homeostatic role. Instead, IRE1α activity during GR drives neuronal death by positively regulating ASK1/JNK activity through the degradation of 14-3-3 θ mRNA, a negative regulator of ASK and an adaptor protein highly expressed in brain, implicated in neuroprotection. Collectively, results describe a novel regulatory mechanism of cell death in neurons, triggered by the downregulation of 14-3-3 θ mRNA induced by the IRE1α branch of the UPR.

Ischemic stroke is a leading cause of disability worldwide. There is no simple treatment to alleviate ischemic brain injury, as thrombolytic therapy is applicable within a narrow time window. During the last years, the ketogenic diet (KD) and the exogenous administration of the ketone body β‐hydroxybutyrate (BHB) have been proposed as therapeutic tools for acute neurological disorders and both can reduce ischemic brain injury. However, the mechanisms involved are not completely clear. We have previously shown that the D enantiomer of BHB stimulates the autophagic flux in cultured neurons exposed to glucose deprivation (GD) and in the brain of hypoglycemic rats. Here, we have investigated the effect of the systemic administration of D‐BHB, followed by its continuous infusion after middle cerebral artery occlusion (MCAO), on the autophagy‐lysosomal pathway and the activation of the unfolded protein response (UPR). Results show for the first time that the protective effect of BHB against MCAO injury is enantiomer selective as only D‐BHB, the physiologic enantiomer of BHB, significantly reduced brain injury. D‐BHB treatment prevented the cleavage of the lysosomal membrane protein LAMP2 and stimulated the autophagic flux in the ischemic core and the penumbra. In addition, D‐BHB notably reduced the activation of the PERK/eIF2α/ATF4 pathway of the UPR and inhibited IRE1α phosphorylation. L‐BHB showed no significant effect relative to ischemic animals. In cortical cultures under GD, D‐BHB prevented LAMP2 cleavage and decreased lysosomal number. It also abated the activation of the PERK/eIF2α/ATF4 pathway, partially sustained protein synthesis, and reduced pIRE1α. In contrast, L‐BHB showed no significant effects. Results suggest that protection elicited by D‐BHB treatment post‐ischemia prevents lysosomal rupture allowing functional autophagy, preventing the loss of proteostasis and UPR activation.

Anti-N-methyl D-aspartic acid receptor (anti-NMDAR) encephalitis is caused by anti-NMDAR antibodies (Abs) that induce neurologic and psychiatric symptoms, explained mainly by NMDAR hypofunction. In the long-term, these Abs decrease surface NMDAR and NMDAR-mediated intracellular Ca2+ ([Ca2+]i) influx. However, there are contradictory findings regarding short-term mechanisms. We investigated NMDAR function in cultured neurons after 60 min treatment with three commercial, rabbit, anti-NMDAR Abs (anti-GluN1 extracellular (EC) domain; anti-GluN2B EC domain; and anti-GluN1 intracellular (IC) domain). The anti-GluN2B and anti-GluN1 IC Abs were previously reported to mimic patients Ab effects in a rodent in vivo model and decreased NMDAR-mediated [Ca2+]i entry after 24 h treatment in our cells. After 60 min incubation with anti-GluN2B or anti-GluN1 IC decreased the NMDAR-mediated [Ca2+]i rise, whereas anti-GluN1 EC slightly increased it. Interestingly, all Abs induced p38 phosphorylation (p-p38). However, surprisingly, it was also elicited by a rabbit Ab directed against a non-NMDAR intracellular epitope, which also reduced NMDAR-mediated [Ca2+]i entry. We further investigated the cellular mechanisms regulated by the anti-GluN2B Ab after 60 min. This Ab did not reduce surface NMDAR and p38 inhibition partially prevented its effect on NMDAR function. This Ab did not elicit per se an [Ca2+]i rise, whereas NMDAR inhibitors 7DCK and MK-801 did not prevent p-p38. Nonetheless, 7DCK prevented NMDAR-mediated [Ca2+]i reduction by the Ab, suggesting a role of GluN1 flux-independent signaling. These data indicate that anti-NMDAR and non-anti-NMDAR Ab modulate NMDAR function distinctly and p38 signaling in the short-term, and a role of a third-party mediator. Finally, our results suggest the involvement of NMDAR flux-independent signaling.