Epilepsy is a neurological disorder characterized by repeated seizures. Nowadays drugs and other approaches to reduce them are available but, unfortunately, around 30% of patients do not respond to medical therapies. In the last decade, optogenetics has emerged as a tool to both explore neuronal networks dynamics and to treat neurological conditions such as epilepsy. The optogenetics strategy involves the expression, in precise brain areas, of light sensitive proteins called opsins that are able to change the membrane potential upon wavelength-specific illumination. This last aspect is, usually, achieved using LED-based hardware. Despite the many advantages of this technique, it still faces practical and translational challenges because of the difficulties of illuminating multiple and deep areas of the brain. In this scenario, the search of alternative light sources is a goal to achieve. Luciferases are enzymes able to emit light upon addiction of their own substrate coelenterazine, and can be used to deliver light to opsins and modulate their action. In this work, the probe called pHIL (pH sensitive inhibitory luminopsin) was developed with the purpose to modulate the epileptic phenotype. pHIL is composed by a bioluminescent protein, RLuc8, coupled to the inhibitory opsin eNpHR3.0. Moreover, the control of seizures will occur only under the intracellular acidic conditions observed in epileptic neurons. The pH sensitivity of the probe is given by the presence of a pH sensor, a pH-sensitive variant of EGFP, called E2GFP. The functioning of the probe is based on the BRET mechanism. The UV light that comes from the luciferase is transferred to the E2GFP that under acidic conditions will emit light and activate the eNpHR3.0, promoting membrane hyperpolarization of epileptic neurons. pHIL is expressed and localizes at the plasma membrane in both HEK293T cells and primary hippocampal neurons. Moreover, pHIL hyperpolarizes HEK293T under acidic conditions and upon addiction of CTZ 400a, the specific luciferase substrate able to induce the UV light emission. On the basis of our data, therefore, we propose pHIL as a potential therapeutic tool to counteract neuronal hyperexcitability.

A novel chemo-optogenetic nanomachine sensitive to intracellular pH shifts

MEROLLA, ASSUNTA
2020-03-13

Abstract

Epilepsy is a neurological disorder characterized by repeated seizures. Nowadays drugs and other approaches to reduce them are available but, unfortunately, around 30% of patients do not respond to medical therapies. In the last decade, optogenetics has emerged as a tool to both explore neuronal networks dynamics and to treat neurological conditions such as epilepsy. The optogenetics strategy involves the expression, in precise brain areas, of light sensitive proteins called opsins that are able to change the membrane potential upon wavelength-specific illumination. This last aspect is, usually, achieved using LED-based hardware. Despite the many advantages of this technique, it still faces practical and translational challenges because of the difficulties of illuminating multiple and deep areas of the brain. In this scenario, the search of alternative light sources is a goal to achieve. Luciferases are enzymes able to emit light upon addiction of their own substrate coelenterazine, and can be used to deliver light to opsins and modulate their action. In this work, the probe called pHIL (pH sensitive inhibitory luminopsin) was developed with the purpose to modulate the epileptic phenotype. pHIL is composed by a bioluminescent protein, RLuc8, coupled to the inhibitory opsin eNpHR3.0. Moreover, the control of seizures will occur only under the intracellular acidic conditions observed in epileptic neurons. The pH sensitivity of the probe is given by the presence of a pH sensor, a pH-sensitive variant of EGFP, called E2GFP. The functioning of the probe is based on the BRET mechanism. The UV light that comes from the luciferase is transferred to the E2GFP that under acidic conditions will emit light and activate the eNpHR3.0, promoting membrane hyperpolarization of epileptic neurons. pHIL is expressed and localizes at the plasma membrane in both HEK293T cells and primary hippocampal neurons. Moreover, pHIL hyperpolarizes HEK293T under acidic conditions and upon addiction of CTZ 400a, the specific luciferase substrate able to induce the UV light emission. On the basis of our data, therefore, we propose pHIL as a potential therapeutic tool to counteract neuronal hyperexcitability.
13-mar-2020
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/996692
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