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Timothy J. Cunningham, Ph.D.

Professor

Neuron Survival and Death

Neuroinflammation

Central Nervous System Repair

e-maill: tcunning@drexelmed.edu

Our research program has evolved out of a basic interest in the biology of cell survival and death. In most regions of CNS, neurons that die because of trauma or nutritional losses are not replaced, which limits the healing required for recovery of lost behavioral function. But like all cells, neurons have natural defense mechanisms that allow them to survive when exposed to potentially life threatening situations, for example, after a head injury, interruption of the blood supply, autoimmune disease, or the attack of inflammatory cells that accompanies all these conditions. The nerve cells that escape death in such situations are usually proficient at reorganizing their processes and the circuits they form with other nerve cells. In fact, it is this ability of surviving neurons to reorganize that is the basis for much of the long-term recovery observed following damage to the brain or spinal cord. We have attempted to identify some of the factors that promote the protective responses of neurons following lesions to the nervous system. The idea is to augment the cell's natural defensives so that more survive after the lesions, and as a result, there is more functional recovery. It is hoped that new drugs can be developed from these studies (see below for an example), and used to treat both acute CNS injuries and the deterioration of nervous tissue that occurs in the course of progressive neurodegenerative disorders (like Alzheimer's disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis)..

Increasing neuron survival with new anti-inflammatory proteins and peptides DSEP

Interestingly, molecules that inhibit the immune systems natural response to tissue damage are often neuroprotective, so we are very interested in the activity of inflammatory cells responding to the neurons as the latter degenerate. Part of the mechanism stressed cells use to stay alive may include production of factors that thwart the inflammatory cells. As part of these studies, we stressed a human neural cell line in tissue culture with hydrogen peroxide, assuming such self-protective agents would be produced by the cells [Since this cell line was essentially cancerous, it was presumed to have many such protective mechanisms]. We isolated and identified the amino acid sequence of a novel human polypeptide called DSEP (for Diffusible Survival Evasion Peptide) from the culture medium of the ce?lls.When the DNA coding for this molecule was transfected into mouse neural cells (causing them to produce excessive quantities of DSEP), they became very hard to kill in tissue culture. They also survived when transplanted in the brain, whereas nontransfected cells died. As expected, these transfected cells appeared to inhibit the activities of monocytes and macrophages, and microglia in the nervous system. These are all immune cells that originate in the blood and participate in the inflammatory and immune responses accompanying trauma and nervous system disease. The activity of inflammatory cells is in fact a major cause of the neuron death that is found in nervous system disorders.

We also discovered a small peptide fragment of this new polypeptide could be used like a drug, in that it protected nerve cells after it was injected into animal models of neurodegenerative disorders. This small peptide, which we call CHEC-9, was determined to be a broad spectrum uncompetitive inhibitor of secreted Phospholipases A2 (sPLA2) - enzymes traditionally thought to be part of the acute or early response to inflammation.The fact that CHEC-9 was an uncompetitive inhibitor made it ideal for application to inflammation and for use in vivo. We are now using this inhibitor, along with a bioactive modifications to study the contribution of sPLA2 enzymes to the neuron death that occurs after trauma or during nervous system diseases.

CHEC peptides and sPLA2 enzymes: Applications to human disease

The ongoing research in the lab, as well as the work of others, now suggests that secreted phospholipase A2 (sPLA2) enzymes are directly involved in the pathology of several neurological diseases (Links to recent publications can be found below). Currently our work includes examining sPLA2 activity in human patients with Multiple Sclerosis and Amyotrophic Lateral Sclerosis, and treatment of animal models of MS, ALS, spinal cord injury, traumatic brain injury, systemic inflammation, and psychogenic stress. In all cases we have found that the CHEC peptides reduce the sPLA2-directed inflammatory response. Since we have shown that these peptides are active in human plasma ex vivo, it is suggested that the CHECs will be applicable to a wide variety of human diseases.

Pep models

Simple Monitoring of Systemic sPLA2 Activity

The discovery of the CHECs has led to a close investigation of the target enzyme sPLA2. This enzyme is well known indicator and instigator of inflammatory disease activity and cell death. sPLA2s in healthy individuals prototypehelp to kill and dispose of foreign organisms and unwanted cells. However, when it is recruited during inflammatory diseases or after tissue damage, it often does more harm that good. For example, our work with Multiple Sclerosis patients and the animal model of MS, suggests that elevatedsystemic sPLA2 activity accompanies, and may even trigger relapses, a characteristic of the most common form of MS. The same may be true of several other episodic disorders (i.e., characterized by "flare-ups") like Rheumatoid Arthritis and Asthma. Therefore a monitor of sPLA2 activity would warn of disease activity in thesedisorders and then lead to more efficient and timely treatments. In the case of MS, this efficient recognition of relapse would expedite treatments that could significantly slow long term disability, an unfortunate inevitability in a majority of MS ?patients. Bolstered by the discovery that humans excrete active sPLA2 enzyme in their urine (a real surprise!), we are developing a simple paper strip assay that warns of an unusually high levels of systemic sPLA2 activity. It is based on the affinity of particulate gold for thiols generated after the sPLA2 in urine reacts with a substrate that produces the thiols. The more enzyme activity the more thiols generated. The gold particles react with the thiols, producing an aggregate and a color shift depending on the size of the aggregate. A diagram illustrating a prototype strip and its proposed mechanism is shown on the right.

Biography:

Timothy J. Cunningham received an A.B. in Chemistry in 1968 from Whitman College, and Ph.D. in 1972 from the Department of Biological Structure, University of Washington School of Medicine. He was a Fellow at Vanderbilt University from 1972 to 1974. He joined the faculty of the Medical College of Pennsylvania (later Drexel University College of Medicine) in 1975, and became Professor of Neurobiology and Anatomy in 1989. He served as a regular member of Neurology B2 Advisory Panel for the National Institutes of Health from 1989-1994. Dr. Cunningham is the Chief Scientific Officer of Pultis Biotechnology.

Selected Publications:

Chen, S Yao, L, and Cunningham, T.J. (2012) Secreted Phospholipase A2 Involvement in Neurodegeneration: Differential Testing of Prosurvival and Anti-Inflammatory Effects of Enzyme Inhibition PLoS ONE 7(6): e39257. Full text

Cunningham, TJ, Yao ,L, Lucena, A, Greenstein JI (2009)Uncompetitive Phospholipase A2 Inhibition by CHEC Sequences including Oral Treatment of Experimental Autoimmune Myeloencephalitis, The Open Enzyme Inhibition Journal, in press. Full text

Cunningham, TJ, Yao, L, Lucena, A (2008) Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholine's unexpected therapeutic properties, Journal of Inflammation 5:17 Full text

Cunningham, TJ Maciejewski, J and Yao, L (2006) Inhibition of secreted phospholipase A2 by neuron survival and anti-inflammatory peptide CHEC-9 J. Neuroinflammation, 3:25 Full Text

Cunningham TJ, Yao L, Oetinger M, Cort L, Blankenhorn EP, Greenstein JI (2006) Secreted phospholipase A2 activity in experimental autoimmune encephalomyelitis and multiple sclerosis. J Neuroinflammation, 3:26 Full Text

Cunningham TJ, Souayah, N, Jameson B, Mitchel, J, Yao, L (2004) Systemic Treatment of Cerebral Cortex Lesions In Rats With A New Secreted Phospholipase A2 Inhibitor, J. Neurotrauma, 21:1683-1691

Cunningham TJ, Jing H, Akerblom I, Morgan R, Fisher TS, Neveu M (2002). Identification of the human cDNA for new survival/evasion peptide (DSEP) Studies in vitro and in vivo of overexpression by neural cells. Exp. Neurol. 177, 32-39.

Cunningham, TJ, Jing H, Wang Y, Hodge L (2000) Calreticulin binding and other biological activities of survival peptide Y-P30 including effects of systemic treatment of rats. Exp. Neurol. 163:457-468.

Cunningham, TJ, Hodge l, Speicher D, Reim D, Tyler-Polz C, Levitt P, Eagleson, K, Kennedy S, Wang Y (1998) Identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin. J. Neurosci. 18, 7047-7060.

Milligan CE, Webster L., Piros ET , Evans CJ, Cunningham TJ, Levitt P (1995). Induction of opiod receptor-mediated macrophage chemotactic activity following neonatal brain injury. J Immunol 154:6571-81.

Haun F, Cunningham TJ (1992). Recovery of Frontal Cortex mediated visual behaviors following neurotrophic rescue of axotomized neurons in medial frontal cortex. J Neurosci 113:614-622 .

Milligan CE, Levitt P, Cunningham TJ. (1991) Brain macrophages and microglia respond differently to lesions of the developing and adult visual system. J. Comp. Neurol. 314:136-146.

Eagleson, KL, Cunningham TJ, Haun F. (1992). Rescue of both rapidly and slowly degenerating neurons in the dorsal lateral geniculate nucleus of adult rats by a cortically derived neuron survival factor. Exp. Neurol. 116:156-162.

Cunningham TJ. Naturally occurring neuron death and its regulation by developing neural pathways. In: International Review of Cytology, Vol 74. G.F. Bourne and J.F. Daniell (eds.) Academic Press, New York:163-186, 1982.