A novel chemo-optogenetic nanomachine sensitive to intracellular pH shifts

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

Engineering REST-Specific Synthetic PUF Proteins to Control Neuronal Gene Expression: A Combined Experimental and Computational Study

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA Binding Proteins (RBPs) able to specifically target and dock to REST mRNA. Amongst the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8- and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8- and 16-repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression

Stability Studies of New Caged bis ‐deoxy‐coelenterazine Derivatives and Their Potential Use as Cellular pH Probes

The synthesis of new bis-deoxy-coelenterazine (1) derivatives bearing ester protective groups (acetate, propionate and butyrate esters) was accomplished. Moreover, their hydrolytic stability at room temperature was evaluated in dimethylsulfoxide (DMSO) as solvent, using the nuclear magnetic resonance (NMR) spectra of the key products at different time intervals. The results showed an increasing hydrolysis rate according to longest aliphatic chain, with a half-life of 24 days of the more stable acetate derivative (4a). Furthermore, the analysis of the experimental data revealed the greater stability of the enol tautomer in this aprotic polar solvent. This result was confirmed by theoretical calculations using the density functional theory (DFT) approach, which gave us the opportunity to propose a detailed decomposition mechanism. Additionally, the derivatives obtained were tested by bioluminescence luciferase assays to evaluate their potential use as extracellular pH-sensitive reporter substrates of luciferase. The biological data support the idea that further structural modifications of these molecules may open promising perspectives in this field of research. © 2020 American Society for Photobiolog

Activation of autophagy, observed in liver tissues from patients with Wilson disease and from Atp7b-deficient animals, protects hepatocytes from copper-induced apoptosis

Wilson disease is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in liver and brain. It is caused by mutations in the ATPase copper transporting beta gene (ATP7B), which encodes a protein that transports copper out of heaptocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to reduce copper toxicity in patients with Wilson disease