In Vivo Imaging of Brain Proteolysis after Cerebral Ischemia

Zezong Gu, M.D., Ph.D.

University of Missouri

Funded in June, 2008: $200000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY .

LAY SUMMARY

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Monitoring Brain Enzymes that Contribute to Brain Cell Death Following Stroke

Investigators will use a mouse stroke model to develop a new molecular imaging technique for monitoring the activity of enzymes that contribute to blood vessel leakage and nerve cell death following stroke.

The devastating effects of stroke on the brain are not  limited only to oxygen deprivation, but also include the breakdown of the “extracellular matrix.”  This matrix is a mixture of sticky proteins and associated sugars that serves as a cellular glue:  it helps bind cells together and maintain tissue integrity.  Following a stroke, there is aberrant activation of enzymes that break down these extracellular proteins, subsequently causing the disintegration of blood vessel walls, hemorrhage, and additional brain cell death.

Chief among the culprit enzymes in this cascade are the “matrix metalloproteinases” (MMPs).  Currently, the only way to monitor MMP activity after a stroke is by performing test tube assays of enzyme levels in the blood or cerebrospinal fluid, sites that are distant in time and space from the initial brain insult.  Now, however, University of Missouri scientists have developed an approach with a molecular probe that is broken down by MMPs to generate an enhanced signal that can be seen on MRI scans (and is visible immediately right at the site of MMP activity).  This probe will enable researchers to study the dynamics of MMP activation in real time in living brain tissue at the site of a stroke.  They will then determine if, as they hypothesize, measures of MMP activation can be used to predict the extent of hemorrhage and nerve cell death following stroke.

Significance:  Monitoring MMP activity in the brain following a stroke may help predict the extent of tissue damage caused by the stroke, and speed the development of new therapeutics to intervene in the process.

ABSTRACT

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In Vivo Imaging of Brain Proteolysis after Cerebral Ischemia

Ischemic stroke, one of the most devastating of diseases, is caused by blood clotting in the cerebral arteries leading to oxygen deprivation and cerebral infarction. Within three hours (h) of stroke onset, intravenous administration of recombinant tissue plasminogen activator (tPA)—a “clot busting” drug that induces thrombolysis, promotes intravascular fibrinolysis and quickly restores circulation—can improve clinical outcomes. However, because tPA can increase the risk of hemorrhage and induce neurotoxicity, its usage is limited to fewer than 2% of stroke patients. A contributing factor to the deleterious effects of stroke is the pathological activation of matrix metalloproteinases (MMPs), particularly MMP-9, which normally regulate the integrity of cell structures.

Our recently published discoveries indicate that abnormal proteolysis by MMP causes degradation of extracellular matrix components and initiates signaling resulting in neuronal cell death in acute ischemic stroke. Moreover, disruption of neurovascular integrity via MMP signaling can cause blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Current studies of brain proteolysis mediated by MMPs have largely relied on in vitro and/or ex vivo assays by zymography, which do not yield information regarding spatial and temporal enzymatic activity in neurovascular units (comprising neurons, astroglia, and endothelium).

We have implemented a new approach to image brain proteolysis using activatable cell-penetrating peptides (CPP). We hypothesize that proteolysis-activatable CPP conjugated with a clinically-proven safe contrast-enhanced magnetic resonance imaging (MRI) agent super paramagnetic iron oxide (SPIO), will serve as a “target-specific” in vivo MRI contrast agent to evaluate dynamics of brain proteolysis of MMP after stroke. We will test this hypothesis using our well-established cerebral ischemia model in mice, which closely resembles that of acute stroke. We will investigate spatiotemporal activity of brain proteolysis in vivo using a 7-Tesla high-performance microMRI system, which may be adapted for clinical application for early diagnosis of acute stroke and/or to evaluate stroke treatment.

The Specific Aims are to determine: (1) whether the novel strategy using activatable CPPs is effective in the analysis of spatiotemporal proteolysis by MMPs in the ischemic brain and (2) whether analysis of brain proteolysis in vivo using a “target-specific,” biocompatible, contrast-enhanced MRI agent (CPP-SPIO) in conjunction with a high-resolution MRI system, can predict neurotoxicity and hemorrhage after cerebral ischemia in mice. Support from the Dana Foundation will launch this study and enable the junior investigators serving as PI and co-PI to develop an integrative research program in brain-immuno imaging. It will also allow us to generate pilot data crucial to obtain further funding. In summary, we seek to accelerate development of an innovative in vivo imaging strategy to investigate brain abnormal proteolysis after ischemic stroke.

INVESTIGATOR BIOGRAPHIES

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Zezong Gu, M.D., Ph.D.

Zezong Gu earned his Ph.D. in cell biology and neuroscience from the University of Texas Medical Branch at Galveston, and his M.D. from Tianjin Medical University in China.  He received his postdoctoral training at Burnham Institute for Medical Research in San Diego, where he was promoted to Staff Scientist and then Research Assistant Professor before he moved to Columbia, Missouri.  Dr. Gu is currently an Assistant Professor in the Department of Pathology and Anatomical Sciences at the University of Missouri-Columbia, with an adjunct professorship at Burnham Institute.

Research in Gu Laboratory focuses on understanding the molecular mechanisms of redox (reduction-oxidation, a fundamental reaction in chemistry) modulation of proteins and their down-stream signal events that lead to abnormal enzymatic activity, dysfunctional protein degradation, and neuron cell death in neurodegenerative diseases.  Gu Laboratory has established and/or adopted various experimental systems from cell-free protein interactions to disease models in animals.  Multi-disciplinary approaches used in the laboratory include microsurgery, pharmacology, protein biochemistry, cell biology, molecular structure modeling, comprehensive mass spectrometry, immunocytochemistry, confocal/deconvolution fluorescence microscopy, and live brain imaging.

Current research activities in the laboratory are (1) imaging of enzyme activity in live animals in collaboration with molecular imaging authority Dr. Roger Tsien of UC-San Diego; (2) therapeutic potential of mechanism-based proteolytic inhibition in stroke and neurodegeneration in collaboration with renowned medicinal chemist Dr. Shahriar Mobashery of University of Norte Dame; and (3) proteomic analysis of protein posttranslational modifications (PTM) on cysteine residues and molecular modeling for PTM-induced conformational changes in collaboration with an authority in the proteomics research Dr. John Yates of The Scripps Research Institute.  Ultimately, these studies are aimed for the identification of potential therapeutic targets, validation of therapeutic efficacy, and early diagnosis as well.