Pharmacological intervention to reduce the level or content of Glu under the cerebral ischemia effectively alleviate brain injury and produce neuroprotective effects[12–15]. But the significance of regulating Glu-Gln cycle metabolism in the treatment of cerebral ischemia has not been reported so far. In this study, as shown in Fig. 6,we investigated the neuroprotective potential and changes in Glu-Gln cycle between astrocytes and neurons.
As expected, our results verified the regulation of Glu-Gln balance by AMP or CB-839 produced neuroprotective effects in MCAO/R mice. Under ischemia, the activity and expression of GS on the damaged astrocytes were decreased, which inhibited the conversion of Glu to Gln. Meanwhile, the expression of GLS on the damaged neurons was increased, which promoted the hydrolysis of Gln to Glu. The above dual effects made the imbalance of Glu and Gln content in the brain of ischemic mice leading to a large amount of Glu accumulation, which aggravate the injury and neurotoxic reaction of ischemic mice. However, the impact was reversed by pre-treatment with AMP or CB-839, our research highlights the glutamate homeostasis in cerebral ischemia-mediated toxicity
Interestingly, we found that the activity of GLS in neurons of affected brain tissue was dramatically reduced, while it was significantly increased when mice treated with AMP (300 mg/kg) or CB-839 (200 mg/kg). It seems contradict with the inhibitory effect of CB-839 on GLS. It has been shown that in gerbil models of MCAO, GLS1 activity is enhanced, aggravating, and promoting neuronal apoptosis[5]. However, the research in which brain tissues from MCAO rats were partitioned to detect GLS activity found that GLS activity was highest in non-ischemic tissues. In contrast, in the central ischemic regions, GLS activity decreased rapidly, with 85% and 97% of the enzyme inactivated at 6 and 24 h of reperfusion, respectively[5, 16, 17]. The above results indicated that GLS activity is differentially expressed in MCAO rats, the determinants of its activation were complicated and related to the functional condition of neurons, the time of reperfusion, and the state of neural tissues.
It should be noted that the content and fluorescence expression of GLS in the dorsal, ischemia-vulnerable region (CA1) was not compatible with the it’s activity changes in the affected brain tissue. To our surprise, the protein expression levels of GLS were markedly up regulated under ischemia and the fluorescence of GLS was strongly displayed in the injured areas of neurons. While after treated with AMP (300 mg/kg) or CB-839 (200 mg/kg), the protein levels of GLS were decreased, and the fluorescence of GLS was down-regulated in the regions with high expression of NeuN-labeled neurons. Earlier studies have identified that the ischemia-induced neuronal death promoted the release of large amounts of GLS synthesized in mitochondria[18, 19]. GLS overexpression induced microglia activation, which in turn led to neuroinflammation, and application of inhibitors of GLS improved neuroinflammation caused by brain ischemia[20]. In summary, the reason for the inconsistent results of GLS activity and expression may be that the high activity of GLS is related to the functional state of neurons. The GLS is mainly expressed on neurons, which are most sensitive to ischemic injury. The substantial death of neurons after ischemia decreased total GLS activity, while AMP or CB-839 rescued neuronal apoptosis and increased the number of surviving neurons, resulting in an elevation of the sum activity of GLS. In contrast, the amount of GLS expression was related to the degree of neuronal damage. The NeuN-labeled neurons under ischemia showed weak fluorescence expression, obvious nucleus fixation, and significantly reduced in number and sparse distribution, which suggests serious apoptosis and necrosis of neurons. The strong fluorescence expression of GLS on such neurons might be because those neurons are in the apoptotic state with low GLS activity and the compensatory expression raised to maintain the function of GLS or it could be due to a large release of GLS synthesized in mitochondria when neurons died.
Glutamate dyshomeostasis results in considerable accumulation of extracellular Glu and severe deficiency of Gln, which triggers a series of neurotoxic damage such as Glu toxicity[21], memory impairment, oxidative stress, and inflammatory response [22] that leads to disruption of neurotransmitter and energy homeostasis[23]. When the induced excitotoxic Glu is excessively released into the extracellular space by neurons, the balance of Glu mainly depends on the uptake of Glu in astrocytes due to the absence of extracellular enzymes that can directly utilize Glu in the extracellular space[24, 25]. When the presynaptic reuptake of Glu is hindered, the extracellular accumulation of Glu is increased, and NMDAR in neurons is over-activated by Glu, which directly causes excitatory neurotoxic reactions[26, 27]. The upstream and downstream active molecules evoked excitotoxic responses because of the Glu overdose. Our results show in neurons, the GLS-mediated hydrolysis of Gln to Glu is hindered by AMP or CB-839 through reducing the expression contents of GLS, which results in elevated Gln content and downregulated Glu levels under cerebral ischemia. Both AMP and CB-839 enhanced GS-mediated conversion of Glu to Gln on astrocytes by increasing ischemic brain GS activity and reducing Glu accumulation via facilitating GLT-1 expression and promoting Glu uptake. At the same time, the expression of NMDAR was decreased to prevent the excitatory neurotoxicity caused by Glu's excessive activation.
To date, the significance of modulating Glu-Gln balance in treating cerebral ischemia has yet to be reported in the literature. Our results primarily demonstrate the neuroprotective potential and changes in Glu-Gln related pathway influenced by AMP or CB-839. Both AMP (300 mg/kg) and CB-839 (200 mg/kg) through suppressing the expression of GLS, removing the excess Glu in the circulating to maintain the homeostatic balance of Glu-Gln and alleviate the neurological damage of cerebral ischemia. GLS may serve as a key mediator and promising target in cerebral ischemia.