Inhibition of Vesicular Glutamate Transporters (VGLUTs) with Chicago Sky Blue 6B Before Focal Cerebral Ischemia Offers Neuroprotection.

Abstract

Brain ischemia is one of the leading causes of death and long-term disability in the world. Interruption of the blood supply to the brain is a direct stimulus for many pathological events. The massive vesicular release of glutamate (Glu) after ischemia onset induces excitotoxicity, which is a potent stress on neurons. Loading of presynaptic vesicles with Glu is the first step of glutamatergic neurotransmission. Vesicular glutamate transporters 1, 2, and 3 (VGLUT1, 2 and 3) are the main players involved in filling presynaptic vesicles with Glu. VGLUT1 and VGLUT2 are expressed mainly in glutamatergic neurons. Therefore, the possibility of pharmacological modulation to prevent ischemia-related brain damage is attractive. In this study, we aimed to determine the effect of focal cerebral ischemia on the spatiotemporal expression of VGLUT1 and VGLUT2 in rats. Next, we investigated the influence of VGLUT inhibition with Chicago Sky Blue 6B (CSB6B) on Glu release and stroke outcome. The effect of CSB6B pretreatment on infarct volume and neurological deficit was compared with a reference model of ischemic preconditioning. The results of this study indicate that ischemia upregulated the expression of VGLUT1 in the cerebral cortex and in the dorsal striatum 3 days after ischemia onset. The expression of VGLUT2 was elevated in the dorsal striatum and in the cerebral cortex 24 h and 3 days after ischemia, respectively. Microdialysis revealed that pretreatment with CSB6B significantly reduced the extracellular Glu concentration. Altogether, this study shows that inhibition of VGLUTs might be a promising therapeutic strategy for the future.

Neuroprotective Effects of VGLUT1 Inhibition in HT22 Cells Overexpressing VGLUT1 Under Oxygen Glucose Deprivation Conditions

“…VGLUT1 and VGLUT2 have been shown to in uence the extent of Glu release and subsequent excitotoxic damage (Takamori et al, 2000). Therefore, targeting VGLUTs activity could provide therapeutic bene ts in mitigating the effects of stroke and other neurological diseases (Pomierny et al, 2023;Yan et al, 2020). In our previous studies we showed in in vivo model of brain ischemia, that unspeci c inhibition of VGLUTs, reduced neurological de cit, infarct volume as well as reduced excitotoxicity and neuroin ammation in rats (Pomierny et al, 2023(Pomierny et al, , 2024.…”

“…Therefore, targeting VGLUTs activity could provide therapeutic bene ts in mitigating the effects of stroke and other neurological diseases (Pomierny et al, 2023;Yan et al, 2020). In our previous studies we showed in in vivo model of brain ischemia, that unspeci c inhibition of VGLUTs, reduced neurological de cit, infarct volume as well as reduced excitotoxicity and neuroin ammation in rats (Pomierny et al, 2023(Pomierny et al, , 2024. Unfortunately, due to high homology of VGLUTs, there are no speci c and selective inhibitors of VGLUT1, which is known as a main vesicular transporter in the CNS (Thompson et al, 2005).…”

“…In our previous studies using a rat model of focal cerebral ischemia we showed that stroke upregulates VGLUT1 and inhibition of this transporter using an azo-dye Chicago Sky Blue 6B (CSB6B) resulted in reduced Glu e ux, smaller infarct size and improved neurological condition. Treatment with CSB6B revealed also a profound anti-in ammatory effect, what may suggest a broad, multifactorial mode of action (Pomierny et al, 2023(Pomierny et al, , 2024.…”