Role of Lactate and TREK1 Channels in Neuroprotection during Cerebral Ischemia – in Vitro Study in Rat Hippocampus

Abstract

Cerebral ischemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Insufficient blood supply to the brain leads to cerebral ischemia and increase in extracellular lactate concentrations. Rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischemia. Lactate is a neuroprotective metabolite whose concentrations increase to 15-30 mM during ischemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischemia, however the connecting link between the two is missing. We hypothesized that lactate might interact with TREK1 channels and mediate neuroprotection. The aim of this study was to investigate the effect of lactate on activity and expression of TREK1 channels and evaluate the role of lactate-TREK1 interaction in conferring neuroprotection in the ischemia-prone hippocampus

Ischemic concentrations (15-30 mM) of lactate at pH 7.4 increased whole cell TREK1 current in CA1 stratum radiator astrocytes and caused membrane hyperpolarization. We confirmed the intracellular action of lactate on TREK1 in hippocampal slices using mono carboxylate transporter blockers. The intracellular effect of lactate on TREK1 channels is specific since other mono carboxylates such as pyruvate at pH 7.4 failed to increase TREK1 current. We used immunostaining, western blot and electrophysiology to show that 15-30 mM of lactate increased functional TREK1 protein expression by 1.5-3 fold in hippocampal astrocytes. Next, we performed quantitative PCR to investigate if the increase in TREK1 protein expression was due to increased transcription and found that lactate stimulated TREK1 mRNA transcription to increase TREK1 protein. Lactate mediated increase in TREK1 expression was dependent on protein kinase A as inhibitors of protein kinase A abolished the increase in TREK1 mRNA and protein.

The role of lactate-TREK1 interaction in neuroprotection was subsequently investigated using an in vitro oxygen glucose deprivation model of ischemia. Addition of 30 mM lactate to oxygen glucose deprived slices reduced neuronal death in the hippocampal CA1 pyramidal layer. However, 30 mM lactate failed to reduce cell death in rat hippocampal slices treated with TREK1 channel blockers signifying the requirement of active TREK1 channels for lactate mediated neuroprotection. However, lactate in the presence of protein kinase inhibitor failed to reduce cell death. This might be related to the role of protein kinase A in upregulation of TREK1 channels.

We also estimated CA1 pyramidal neuronal TREK1 channel expression and found both lactate and oxygen glucose deprivation to decrease TREK1 channel expression that was surprisingly opposite to the effects on astrocytes. As TREK1 channel activation and upregulation decreases neuronal excitability, a decrease in neuronal TREK1 channel expression in response to lactate is expected to cause higher neuronal death and fails to explain lactate mediated neuroprotection. Since, lactate upregulated TREK1 channel expression and functional activity in CA1 stratum radiate astrocytes, we reasoned that the lactate mediated neuroprotection might be via astrocytic TREK1 channels requiring viable functional astrocytes. This was tested by disrupting astrocyte function using gliotoxin, and estimating cell death in oxygen glucose deprived hippocampal slices. Lactate failed to reduce cell death in presence of gliotoxin signifying the importance of viable astrocytes for lactate mediated neuroprotection.

The above effects were specific to lactate as pyruvate failed to increase TREK1 expression and reduce cell death. TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression and activity in astrocytes during ischemia. This pathway serves as an alternate mechanism of neuroprotection.