Methods and Compositions for Measuring ATP in Subcellular Compartments

Dysfunction of synaptic metabolism has been linked to neurological and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. However, several basic features of synaptic metabolism remain unclear due to the lack of approaches to quantitatively measure adenosine-tri-phosphate (ATP) levels in subcellular locales.

Firefly luciferase is an efficient optical reporter of ATP. However, its slow catalytic rate and lack of a suitable calibration hamper its use for quantitative subcellular imaging. Dr. Timothy Ryan at Weill Cornell Medicine has developed a novel optical reporter of ATP to overcome these barriers. This reporter consists of a plasmid encoding luciferase fused to a gene for a protein that targets a subcellular locale and a gene for a fluorescent protein, such as mCherry. This reporter allows ratiometric luminescence to fluorescence readouts for determining ATP concentrations at the location of interest. Point mutations incorporated into the luciferase component have increased its thermal stability and catalytic rate.

Using this reporter of ATP targeted to nerve terminals with synaptophysin, Dr. Timothy Ryan's group found that: (1) electrical activity places significant demands on presynaptic ATP levels; (2) at synapses, ATP demand is met by local ATP synthesis triggered by electrical activity; (3) the synaptic vesicle cycle is the major consumer of ATP; (4) compromising local routes of ATP synthesis severely impairs synapse function.

This technology provides a way to examine ATP dynamics, regulation and consumption at nerve terminals in diseased and normal states and to examine energy homeostasis in living tissues. It can also be used as a screening tool to look for molecules that perturb or enhance synaptic metabolism. This technology can also be used to measure ATP concentrations at any other cellular locale that can be specifically targeted.

Potential Applications

• A research tool to examine ATP dynamics, regulation and consumption at nerve terminals as well as other subcellular compartments. Dr. Timothy Ryan's laboratory is completely set up for this research and can partner with industry.
• This method also provides a means to obtain robust quantitative measures of intracellular ATP in intact cells in general and could be applied to examine ATP dynamics in a number of perturbed metabolic states such as those associated with a number of cancers and associated metabolic diseases.

Advantages

• The fluorescent protein component provides a ratiometric way to normalize luminescence signals
• Provide much higher signal-to-noise imaging than cytosolic luciferase
• The luciferase enzyme used exhibits better thermal stability and catalysis