Novel mechanism of hypoxic neuronal injury mediated by non‐excitatory amino acids and astroglial swelling

Abstract

In ischemic stroke and post-traumatic brain injury (TBI), blood-brain barrier disruption leads to leaking plasma amino acids (AA) into cerebral parenchyma. Bleeding in hemorrhagic stroke and TBI also release plasma AA. Although excitotoxic AA were extensively studied, little is known about non-excitatory AA during hypoxic injury.Hypoxia-induced synaptic depression in hippocampal slices becomes irreversible with non-excitatory AA, alongside their intracellular accumulation and increased tissue electrical resistance. Four non-excitatory AA (L-alanine, glycine, L-glutamine, L-serine: AGQS) at plasmatic concentrations were applied to slices from mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Two-photon imaging, light transmittance (LT) changes, and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. radiatum were used to monitor synaptic function concurrently with cellular swelling and injury. During normoxia, AGQSinduced increase in LT was due to astroglial but not neuronal swelling. LT raise during hypoxia and AGQS manifested astroglial and neuronal swelling accompanied by a permanent loss of synaptic transmission and irreversible dendritic beading, signifying acute damage. Neuronal injury was not triggered by spreading depolarization which did not occur in our experiments. Hypoxia without AGQS did not cause cell swelling, leaving dendrites intact. Inhibition of NMDA receptors prevented neuronal damage and irreversible loss of synaptic function. Deleterious effects of AGQS during hypoxia were prevented by alanine-serine-cysteine transporters (ASCT2) and volumeregulated anion channels (VRAC) blockers. Our findings suggest that astroglial swelling induced by accumulation of non-excitatory AA and release of excitotoxins through antiporters and VRAC may exacerbate the hypoxia-induced neuronal injury.

Promising Molecular Targets in Pharmacological Therapy for Neuronal Damage in Brain Injury

“…One of the first consequences of cerebral ischemia is the loss of synaptic transmission, which is usually reversible in the twilight zone [26]. In fact, in our laboratory, we have shown for the first time that the disappearance of synaptic transmission caused by a period of hypoxia becomes irreversible if the lack of oxygen is accompanied by the presence of certain non-excitatory amino acids [27][28][29].…”

Spreading depolarization causes reversible neuronal mitochondria fragmentation and swelling in healthy, normally perfused neocortex

“…Focal swelling (beading) of dendrites, which resembles beads on a string, was identified by rounded regions extending beyond the initial diameter of the dendrite. The progression of dendritic beading (swelling) and fragmentation of the mitochondrial network was assessed manually by quantifying the amount of dendritic beading or mitochondrial fragmentation in an imaging field following published protocols 25, 28, 40 . Because the resolution and signal-to-noise ratio always present a challenge for analyses of small structures such as mitochondria, images were not randomized to keep track of individual mitochondria in the image sequences.…”

Non-Excitatory Amino Acids, Melatonin, and Free Radicals: Examining the Role in Stroke and Aging

“…We also found that the presence of glutamine in this mixture was necessary but not sufficient (i.e., neither the application of alanine, glycine plus serine without glutamine, nor the individual application of glutamine during hypoxia induced permanent synaptic silencing). These results are remarkable given that L-glutamine is the most concentrated AA in the mixture and it is a substrate for the synthesis of glutamate and GABA [21]. Nevertheless, equimolar substitution of glutamine by histidine or threonine (a mixture of alanine, glycine and serine, plus histidine or threonine at 733 µM concentration) also produced detrimental effects of synaptic transmission [165].…”

“…In contrast, the presence of the seven-AA mixture during the hypoxia period made both the synaptic silencing [20] and the loss of membrane potential irreversible [165] (Figure 3), indicating that neurons have suffered irreversible damage. Lately, we showed that all the seven amino acids were not required to induce this detrimental effect, because with a mixture of just four AA (alanine, glutamine, glycine, and serine) the hypoxia effect was irreversible [21,165]. We also found that the presence of glutamine in this mixture was necessary but not sufficient (i.e., neither the application of alanine, glycine plus serine without glutamine, nor the individual application of glutamine during hypoxia induced permanent synaptic silencing).…”

“…It can attenuate oxidative stress, modulate calcium homeostasis, and inhibit apoptosis, thereby safeguarding neurons against damage induced by stroke and aging processes [19]. Furthermore, the intracellular accumulation of certain non-excitatory amino acids, which were previously not considered excitotoxic, could promote harmful effects during hypoxia-ischemia episodes through the activation of NMDAR [20,21]. Therefore, given that NMDAR antagonists have been shown to be ineffective in clinical trials of ischemic stroke [22], blocking the amino acid transporters involved in the process could be an alternative therapeutic strategy to reduce ischemic damage.…”