Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

Abstract

Hypoxia is characterized by low oxygen content in the tissues. The central nervous system (CNS) is highly vulnerable to a lack of oxygen. Prolonged hypoxia leads to the death of brain cells, which underlies the development of many pathological conditions. Despite the relevance of the topic, different approaches used to study the molecular mechanisms of hypoxia have many limitations. One promising lead is the use of various genetically encoded tools that allow for the observation of intracellular parameters in living systems. In the first part of this review, we provide the classification of oxygen/hypoxia reporters as well as describe other genetically encoded reporters for various metabolic and redox parameters that could be implemented in hypoxia studies. In the second part, we discuss the advantages and disadvantages of the primary hypoxia model systems and highlight inspiring examples of research in which these experimental settings were combined with genetically encoded reporters.

The Uniqueness of Clear Cell Renal Cell Carcinoma: Summary of the Process and Abnormality of Glucose Metabolism and Lipid Metabolism in ccRCC

“…The products of aerobic glycolysis provide energy to cancer cells, while generating a large number of by-products and lactic acid ( 46 ). Glucose is broken down by glycolysis into pyruvate, which is eventually converted to acetyl-CoA ( 47 ). So far, everything seems to be consistent with what happens in normal cancer cells when a gene mutation occurs: the mutation genes that regulates GLUT causes a lot of glucose to enter the ccRCC cells.…”

A guide to genetically encoded tools for the study of H2O2

“…The explosive growth of fluorescent protein-based technologies has led to the creation of a toolbox of genetically encoded instruments with a wide range of opportunities to study processes in live cells. In particular, the appearance of genetically encoded fluorescent redox biosensors has revolutionized redox biology [49,[72][73][74][75][76][77]. Apart from fluorescent biosensors that allow for passive observation of the redox events in the cell, several genetically engineered fluorescent proteins were found to generate ROS upon illumination.…”

“…Since the introduction of the first H 2 O 2 biosensor HyPer in 2006 [44], more than ten H 2 O 2 ‐sensitive GEFIs have been created. The most frequently used GEFIs for H 2 O 2 imaging can be subdivided into three families according to their fluorescent and H 2 O 2 ‐sensing moieties: (a) GEFIs consisting of circularly permutated fluorescent proteins linked to the highly H 2 O 2 ‐sensitive bacterial transcription factor OxyR [45] (HyPers [44,46–49] and NeonOxIrr [50]); (b) roGFP‐based sensors in which a thiol‐based peroxidase oxidizes roGFP via a process named the thiol‐disulfide exchange, after interaction with H 2 O 2 (roGFP2‐Orp1 [51], roGFP2‐Tsa2 [52], roGFP2‐Prx1 [53], roGFP2‐Tpx1 [54]); and (c) Förster resonance energy transfer (FRET)‐based sensors consisting of a H 2 O 2 ‐sensitive unit that when oxidized brings two fluorescent proteins fused to the unit's edges into close proximity (OxyFRET and PerFRET [55]). These GEFIs provide either intensiometric (HyPerRed) or ratiometric (HyPer family and roGFP‐ and FRET‐based sensors) readouts enabling retrieval of quantitative data regarding changes in H 2 O 2 concentrations.…”

“…To understand how the specificity of H [44], more than ten H 2 O 2 -sensitive GEFIs have been created. The most frequently used GEFIs for H 2 O 2 imaging can be subdivided into three families according to their fluorescent and H 2 O 2sensing moieties: (a) GEFIs consisting of circularly permutated fluorescent proteins linked to the highly H 2 O 2 -sensitive bacterial transcription factor OxyR [45] (HyPers [44,[46][47][48][49] and NeonOxIrr [50]); (b) roGFPbased sensors in which a thiol-based peroxidase oxidizes roGFP via a process named the thiol-disulfide exchange, after interaction with H 2 O 2 (roGFP2-Orp1 [51], roGFP2-Tsa2 [52], roGFP2-Prx1 [53], roGFP2-Tpx1 [54]); and (c) F örster resonance energy transfer (FRET)-based sensors consisting of a H 2 O 2 -sensitive unit that when oxidized brings two fluorescent proteins fused to the unit's edges into close proximity (Oxy-FRET and PerFRET [55]). These GEFIs provide either intensiometric (HyPerRed) or ratiometric (HyPer family and roGFP-and FRET-based sensors) readouts enabling retrieval of quantitative data regarding changes in H 2 O 2 concentrations.…”

Genetically Encoded Fluorescent Redox Indicators for Unveiling Redox Signaling and Oxidative Toxicity

“…An extensive array of methods and assays have been developed to detect redox-active molecules and processes. These methods include electron paramagnetic resonance spectroscopy, chemiluminescence assays, and fluorescence assays based on either small molecule-based synthetic dyes − or genetically encoded redox indicators (GERIs). − GERI-based fluorescence methods have gradually gained popularity for several reasons: First, these indicators can be expressed by cell machinery, providing chances to measure redox signals in the natural cellular contexts while introducing minimal toxicity. Second, using tissue-specific promoters or subcellular localization sequences, GERIs can be readily localized to specific tissues and subcellular compartments, giving superior spatial and temporal resolution over small molecule synthetic dyes.…”