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Opioids are small signaling molecules which bind to opioid receptors on the surface of cells. The kappa opioid receptor (KOR) is one of three major types of opioid receptors found in human neurons. When an opioid binds to a KOR, a variety of biochemical signaling pathways are activated inside the cell. Each of these pathways are associated with different physiological effects of KOR activation. The production of a small signaling molecule, cyclic adenosine monophosphate (cAMP), is known to be inhibited during KOR activation of the analgesic (pain-killing) signaling pathway. The ability to interrogate the individual responses of KOR signaling pathways in a living mammal would greatly improve our understanding of how opioids work in the brain. To this end, we have developed a biosensor functioning via bioluminescent resonance energy transfer (BRET) as a tool for both fluorescent and luminescent ratiometric quantification of cAMP. We couple two fluorescent proteins, emitting at different wavelengths, to a luciferase which provides chemiluminescent excitation energy for the complex. The intensity of the two emitted wavelengths vary inversely to each other in response to the presence of cAMP. Calculating the ratio of the two emission intensities creates a metric for cAMP concentration that is normalized to the concentration of our sensor, allowing quantitative comparison across trials. The application of our sensor for dual-color live-cell microscopy was demonstrated in mammalian cells using fluorescence and bioluminescence microscopy. Further proof-of-principle studies in KOR-expressing mammalian cells demonstrates the viability of our sensor for live-cell KOR signaling.

Parkinson’s Disease (PD) is a common neurodegenerative disease with over 200,000 new cases each year. In general, the cause of the disease is unknown, but oxidative stress inside of neurons has been associated with the disease’s pathology for some time. Currently, techniques to study the onset of PD inside of neurons are limited. This makes treatments and causes difficult to discover. One solution to this has been fluorescent protein biosensors. In short, these proteins can be engineered to glow when a certain state is achieved inside a cell. The present research discusses the engineering of a genetically-encoded fluorescent protein (FP) sensor able to detect reactive oxygen species (peroxide, hydroxyl, superoxide, etc.) inside of neurons, giving one the ability to enhance their understanding of the role these species play in the onset of the disease. This sensor relies on Förster Resonance Energy Transfer (FRET) between a green fluorescent protein and a red fluorescent protein to facilitate red-shifting of the sensor’s emission spectrum. Linked via a short polypeptide chain, the energy transfer efficiency of these combined FPs can vary greatly. Various linker lengths and FP combinations were experimentally tested to draw conclusions about their performance. The current trajectory of the research currently implies that those combinations with the shortest linker lengths will yield the highest-performing sensors. This sensor is another vital piece in the library of tools which can be used to help us begin answering the many questions we have about PD and its pathology.

Purinergic signaling is a type of extracellular communication that occurs between cells, mediated by adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine. In Parkinson’s Disease, purinergic signaling is disrupted, which contributes to neurodegeneration. In order to monitor this change in cell-to-cell signaling, there is a need for the development of a fluorescent protein (FP) biosensor to study the changes in the concentration of the signaling molecule ATP and its decomposition bioproduct ADP. This summer a genetically encoded ADP sensor that measures changes in ADP concentration was developed. This sensor utilizes Forster Resonance Energy Transfer (FRET) which is a sensing technique that is based on the energy transfer from a donor FP to an acceptor FP. Since this transfer is distance dependent, a change in the sensing domain allows for detection of ADP concentration through changes in fluorescence emission. To develop this FRET based sensor, we are utilizing a cyan-yellow FP pair, as well as a non-fluorescent protein that binds to ADP. Using traditional cloning methods, a small library of ADP sensors from five different versions of both the cyan and yellow proteins was created. This library was screened in E. coli cultures using a method developed to optimize an ATP-sensor. The cloning for this sensor has been confirmed and the library is being tested for sensors responsive to changing concentrations of ADP. With confirmation of a responsive sensor, this sensor design will be validated, allowing for further optimization of this biosensor for the study of purinergic signaling and neurodegeneration.

Parkinson’s disease is a neurodegenerative disease involving the death of neurons in the substantia nigra and loss of the neurotransmitter, dopamine. The disease leads to progressive loss of motor control. Exact causes and mechanisms by which Parkinson’s disease proceeds are unknown, however, previous experiments determine oxidative stress in mitochondria as a factor that results in cell death. Strategies have been implemented to generate fluorescent biosensors to monitor reactive oxygen species (ROS) concentrations while simultaneously measuring the spatiotemporal distribution and correlation between the ROS, cellular function and organelle. Orp1, an enzyme found in yeast, is a sensitive oxidizing species and when coupled with fluorescent protein, roGFP2, the pair acts as a fluorescent biosensor for the ROS, hydrogen peroxide. In this study, Orp1-roGFP2 protein was expressed and purified from bacterial cell cultures and hydrogen peroxide oxidation assays were conducted to compare performance against characteristics reported in the literature.Orp1-roGFP2 is a fluorescence excitation ratiometric probe and the biosensor signal is obtained by the ratio of fluorescent intensities measured with 390 nm and 480 nm excitation. Sigmoidal kinetics were observed for biosensor oxidation by hydrogen peroxide. We also observed the Orp1-roGFP2 is highly susceptible to air oxidation. Finally the mitochondrial targeting mito-Orp1-roGFP2 gene was subcloned into a GW1 plasmid vector for mammalian expression. Future work will entail transfection of mitochondrially-targeted Orp1-roGFP2 into cultured mouse midbrain neurons to enable live-cell imaging of mitochondrial oxidative stress in cellular models of Parkinson’s disease.