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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 regulate 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. 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
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 cells. 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
Reports
of our recent studies with CHEC-9 can be found here
and here.
This and other 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. 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 and systemic inflammation,
with peptide inhibitors of sPLA2. Recent experiments also suggest CHEC-9
might be effective for reducing sPLA2 activity for humans with these diseases.
Click here for an example.
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Two
of the bioactive CHEC peptides
models
by VEGA zz
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Protecting
the nervous system from systemic inflammation
The vulnerability of
the nervous system to inflammation may extend to inflammatory diseases like
asthma, arthritis, and sepsis, diseases that begin outside the nervous system
and produce major symptoms in other organs.This problem is discussed in
a recent review by Hugh Perry of the University of Southampton (ref).
For most of the last century, the brain was considered shielded by the blood
brain barrier from the effects of these non-neural diseases. It is now clear
from the work of Perry and others (like Serge Rivest of Laval University,
Quebec, and William Hickey of Dartmouth), that peripheral inflammation signals
are readily transmitted to the brain resulting in activation of the resident
immune/inflammatory cell, the microglia. It is also clear from work on animal
models, and limited clinical data, that the net effect of an increased "peripheral
inflammatory load" is to make the CNS disorders worse. Behaviorally,
that usually means a more rapid deterioration of motor and cognitive skills.
Since our work suggested that sPLA2 inhibition inhibits microglial activation,
we considered the possibility that sPLA2 or one of its metabolites was involved
in transmitting peripheral inflammatory signals to the brain. In fact, sPLA2
inhibition by CHEC-9 treatment inhibited microglia activation in the cerebral
cortex following introduction of bacterial endotoxin (lipopolysaccharide)
into the circulation.
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Inhibition
of systemic sPLA2 activity attenuates microglial response to endotoxin
exposure. Twenty-four hours after exposure to E. Coli lipopolysaccharide,
microglia in the frontal cerebral cortex are activated (examples at
arrows). Activated cells are enlarged with processes. They are labeled
by both the IB4 lectin and antibody ED-1 (double labeling appears red).
Processes of activated cells appear to cover cortical blood vessels
which otherwise stain black. Treatment with sPLA2 inhibitor CHEC-9,
30-60minutes after the exposure, greatly attenuates the microglia response,
compared treatment with vehicle only. Activated microglia are associated
with increased neuron death in most neurodegenerative diseases. |
Further Information
on clinical applications: Application of CHEC peptides to human
disorders, especially those involving the nervous system, is strongly indicated
by the accumulated data with these peptides. For more information concerning
our progress toward developing these compounds for treatment of human diseases
contact Tim Cunningham or the
office of Entrepreneurship
& Technology Commercialization at Drexel University.
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.
Selected Publications:
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.
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