Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Neuroscience

Supervisor

Dr. Stephen Pasternak

2nd Supervisor

Dr. Robert Bartha

Joint Supervisor

Abstract

Currently, there is no way to assess apoptotic cell death in living organisms. We have developed a novel contrast agent targeted toward the detection of caspase-3 activity, the key enzymatic mediator of apoptosis. Our contrast agent consists of a dual magnetic resonance imaging/fluorescent probe coupled to a cell penetrating peptide (CPP) sequence by a peptide backbone containing a caspase-3 cleavage site. The CPP allows the agent to cross cell membranes and the blood brain barrier. In cells undergoing apoptosis, activated caspase-3 will cleave the agent removing the CPP and trapping the imaging probes inside the cell.

The purpose of this study was to test the ability of our contrast agent to label apoptotic cells in cultured neurons and to explore its potential to detect apoptosis in vivo. Using multiple methods, we demonstrated that our contrast agent selectively labeled apoptotic but not healthy or necrotic neurons in culture. Furthermore, using a caspase-3 inhibitor we demonstrated that uptake and retention of the contrast agent was dependent on apoptosis and caspase-3 activation.

To test our contrast agent in vivo, we examined the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease to induce apoptosis in the dopaminergic neurons of the substantia nigra. At the time the mice were sacrificed, there was little evidence of apoptosis in the substantia nigra and we were not able to identify any cells with significant retention of the agent. Nonetheless, this data demonstrates that our agent effectively detects apoptosis in cultured neurons and reinforces its potential to image apoptosis in vivo.


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