Chang Group - Neuroscience: Oxidation Biology

The brain is the body's most oxidatively active organ, consuming over 20% of the oxygen we breathe in every day. On the other hand, many diseases associated with aging and the brain, including cancer and neurodegenerative diseases such as Alzheimer's and Parkinson's, have a strong oxidative stress component stemming from cellular oxygen mismanagement. Oxidative stress is the result of unregulated production of reactive oxygen species, and accumulation of oxidative damage over time leads to the functional decline of organ systems. The biology of reactive oxygen species is much more complex, however, as emerging evidence shows that small oxygen metabolites, such as hydrogen peroxide, can mediate beneficial cellular signal transduction cascades when produced in the right place at the right time at appropriate levels. We are developing and applying new fluorescent, luminescent, and magnetic resonance imaging (MRI) probes for reactive oxygen species, redox status, and enzyme activity to study molecular mechanisms of oxidative signaling and stress pathways in living cells, tissue, and organisms.

Hydrogen Peroxide Signaling and Stress Pathways. Hydrogen peroxide is a major reactive oxygen species in living systems and a common small-molecule marker for oxidative stress. However, the chemical biology of peroxide is much more rich and complex, as recent studies suggest that this oxygen metabolite, in certain situations, can also serve as a messenger in signal transduction by reacting with redox-active sulfur. This signal/stress dichotomy is reminiscent of nitric oxide and offers a brand new view on the potential roles of peroxide in biology. To study the chemistry and chemical biology of hydrogen peroxide in living systems, we are creating new fluorescent, luminescent, and MRI probes to map its generation, translocation, and function. A key challenge for probe design is achieving specificity for hydrogen peroxide over a host of very similar reactive oxygen species, including superoxide, nitric oxide, and alkyl peroxides. We have discovered a new tactic for selective peroxide detection through the peroxide-mediated deprotection of boronic esters to phenols.

First-generation Peroxyresofurin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) can respond to oxidative stress levels of hydrogen peroxide by increases in red, green, or blue fluorescence, respectively. All three probes are cell-permeable, non-toxic, and can be used to image changes in the levels and distributions of hydrogen peroxide in living cells, including primary hippocampal neurons. Near-IR versions are being developed to image peroxide bursts in thicker biological specimens.

Next-generation Peroxy Green-1 (PG1) and Peroxy Crimson-1 (PC1) probes with attenuated sensitivity are the first chemoselective peroxide probes that are capable of visualizing bursts of hydrogen peroxide generated for normal cell signaling. PG1 has been employed to map molecular pathways of peroxide produced through PI3 kinase (PI3K) and NADPH oxidase (Nox) protein complexes in living brain neurons. Current efforts are directed at using these and improved probes for elucidating signal transduction cascades in various cell types.

We are also exploring methods for quantifying peroxide bursts in localized cellular regions. To this end, we are devising new chemical tools for molecular imaging of hydrogen peroxide that can be targeted to specific subcellular locations, as well as optical probes possessing an internal standard that allow quantitation of peroxide levels by ratiometric sensing. Representative indicators are Mito Peroxy Yellow-1 (MitoPY1), Ratio Peroxyfluor-1 (RPF1), and Peroxy Lucifer-1 (PL1). We are also exploring new organic reaction mechanisms for detecting various other reactive oxygen species. Particular species of interest are superoxide, alkyl peroxides, hydroxy radical, hypochlorite, ozone, and singlet oxygen. Optical sensors of redox status are also being pursued. Finally, in addition to small-molecule, newer projects are geared toward protein- and nanoparticle-based strategies for detecting redox function.

Redox Enzyme Activity. In addition to fluorophores for oxygen metabolites, we are also developing activity-based probes for studying enzymes in living cells. Because these reporters are small in size and do not require transfection of additional genes into cells or tissue, they offer an attractive alternative to fluorescent protein and quantum dot approaches to dynamic imaging of enzyme activity. We are broadly interested in devising tools to study proteins involved in neurotransmitter regulation, redox status, and oxygen metabolism in their natural environments. As an example, Monoamine Oxidase Reporters 1 and 2 (MR1/MR2) are two probes that can be used to measure activity levels of monoamine oxidases A and B (MAO A and B) in living neuronal cells.