Download Understanding the Interactions and Effects of

Protease activated receptor-1 (PAR-1) is part of a family of receptors that use thrombin as the
signaling protein. PAR-1 has already been shown to lead to apoptosis of motor neurons found in the spinal
cord, although the specific mechanism of cell death is not completely understood. While motor neurons do
possess PAR-1, so do oligodendrocytes, the cells that myelinate axons in the
central nervous system [4]. Thus, activation of PAR-1 may affect motor
neurons and oligodendrocytes separately.
If PAR-1 activation on
oligodendrocytes leads to their death or decreases their ability to properly
synthesize myelin, then motor neurons could be affected indirectly. The
myelin that surrounds axons is essential for rapid transduction of signals from
Fig. A: Oligodendrocyte myelinating axons of motor
the motor neurons to their connecting cells (most often muscle cells).
neurons in the CNS
Decreased myelination of motor neurons could lead to reduced speed of signaling between motor neurons
and muscle cells. Motor neurons signal muscle cells to contract and upon contraction, muscle cells release
growth factors that are taken up by motor neurons, which are essential for their survival [7]. Decreased
myelination may cause this relationship between motor neurons and muscles to diminish, which in turn can
cause the amount of growth factors released by muscles to diminish. Unhealthy motor neurons are then
subject to undergo apoptosis.
The purpose of Anna’s project is to determine if the decreased myelination of motor neurons is due to
oligodendrocytes not being able to produce sufficient proteins needed for the
myelination process following PAR-1 activation.
Since Anna is using a
developmental model, spinal cords are isolated from chick embryos at various stages
of development. Spinal cords isolated on the fifth day of development have been
shown to have the largest number of cells in the spinal cord, but by the tenth day of
development approximately one-half of the cells have been lost. This is a naturally
occurring process that happens during the embryonic development of all vertebrates.
Past research has shown that PAR-1 is present during this period [5]. To examine how PAR-1 activation
Fig. B: Protein
Electrophoresis Apparatus affects myelination, developing chick embryos were treated with the amino acid sequence
SFLLRNP, a ligand that causes activation of PAR-1 or phosphate buffered saline (control).
On the specified days, the cords were moved, crushed, and the proteins were removed. The
proteins were separated using a gel and then tested for any differences in levels of PLP1 and
MPZ, two myelin associated proteins, between the control and experimental embryos.
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cyte.gif
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Motor neurons and oligodendrocytes have an
important relationship with one another in the central
nervous system. Oligodendrocytes provide the axons of
motor neurons with a fatty insulation layer called myelin.
This myelin layer helps to relay transmissions more quickly
and effectively throughout the body. It has already been
shown that there is a relationship between motor neurons and
muscle cells that involves healthy motor neurons stimulating
muscle cells and muscle cells releasing growth factors, such
as nerve growth factor (NGF), in return [7]. The lack of
myelin insulation on a motor neuron can decrease its ability
to stimulate muscle cells and result in a lack of NGF being
received by the connecting neuron leading to neuron cell
death. The relationships among various cells are vital for
communication and the maintaining of homeostasis within Fig. C: The interrelationship between motor neurons and muscles. Notice
the unhealthy motor neuron to the right is connected to an atrophied muscle.
the body.
Jordan’s research is focusing on the specific relationship between oligodendrocytes and motor
neurons when PAR-1 is activated. In order to begin this investigation, she must
first establish a model to co-culture the two cells types and to identify each cell
type. After she has determined this model, she will be culturing oligos alone,
oligos plated with motor neurons (each with a different color cell stain), and motor
neurons alone to observe the effects of PAR-1 activation on cell mortality. One
culture set of cells will be the experimental and one culture set of cells will be the
control. Microscopic techniques will be used to make observations and perform
cell counts over time to see if oligodendrocyte mortality has an effect of the number of motor neurons that
undergo apoptosis. Comparisons will be looked at against the control groups to verify any differences in
survivorship.
I would like to thank the lab researchers (Anna, Jordan, Jane, & Kate), Dr. Turgeon, Furman University, and the Howard Hughes
Medical Institute for making this wonderful educational experience possible!
Protease activated receptor-1 is a cell surface receptor found on various cells of the nervous system
and upon activation turns on a host of intracellular signals. PAR-1 can be activated by thrombin cutting
between the arginine and serine amino acids on the extracellular part of the receptor. This cut causes a piece
of the extracellular terminus to be removed and exposes a new amino acid
sequence, SFLLRNP. After its exposure, SFLLRNP can then bind to the second
loop transmembrane portion of the receptor, thus activating the receptor [6]. Once
D: Representation of how Thrombin
the receptor is activated, a specific signaling pathway is turned on depending on the Fig.
activates the PAR-1 receptor [6].
cell type. In motor neurons, PAR-1 activation has been shown to lead to apoptosis, a specific form of cell
death found during development and in neurodegeneration [4].
The purpose of Jane’s project is to determine the steps involved in the apoptosis signaling pathway
after SFLLRNP binds to the transmembrane portion of the PAR-1. To study this, Jane
isolates spinal motor neurons from chick embryos and grows them in culture dishes. This
technique allows her to be certain that she is
studying the effects of PAR-1 activation on one cell
type without any interference from other cell types.
Once the motor neurons have been isolated in
culture, she allows them to develop in either the
presence of absence of SFLLRNP. Other research
has shown that PAR-1 is a G-protein linked receptor, but given that
there are multiple subtypes, more research is needed. G-protein linked
receptors involve a guanine-binding protein (the G-protein), which
binds either GTP or GDP. Activated PAR-1 is bound to GTP [5].
Currently, her studies point to an inositol triposphate (IP3) based
2+
pathway with increases in intracellar Ca and other secondary
Fig. E: The alleged signaling pathways involved with
messengers along that pathway. The next step in her research is for
Thrombin activation of the PAR-1 receptor [5].
her to verify any differences in the amount of calmodulin, a protein that binds Ca2+ binds. This new
2+
Ca /calmodulin complex can then be used to activate other signaling messengers further down the pathway
such as myosin-light chain kinase. Detection of the signals involved in PAR-1 activation involves the use of
specific assay kits and protein isolation. Commercially available detection kits were used to determine the
2+
levels of IP3 and Ca . Since calmodulin is a protein, Jane had to isolate proteins from cultured cells,
separate those proteins using gel electrophoresis and then use specific antibodies to calmodulin for detection.
Since Jane’s previous research shows that Ca2+ is increased following PAR-1 activation, she hypothesizes
2+
that free calmodulin levels will decrease given that Ca binds to calmodulin. If this is the pathway, then the
Ca2+/calmodulin complex should bind to myosin light chain kinase, the next step in the apoptosis pathway.
Neuropsin is a serine protease shown to be expressed by oligodendrocytes found only in the central
nervous system. It has been shown that neuropsin levels can increase following an injury to the
spinal cord. Past research about the role of neuropsin is confounding. Some research suggests
that neuropsin could be involved with degeneration of neurons, while other research suggests that
is aids in repairing damaged neurons following spinal cord injury [8]. The specific role of
Fig. F: Structural
representation of the neuropsin with regards to apoptosis has yet to be understood by scientists.
protein
Kate’s research is focused on trying to gain more of an understanding of the
Neuropsin.
relationship between oligodendrocytes and neuropsin. Oligodendrocytes myelinate
axons of CNS motor neurons. Kate is first examining the various levels of
neuropsin found in developing chick embryo spinal cords at the two different stages
of embryonic development – embryonic day 5 and embryonic day 10. Spinal cords
will be removed at the specified days and proteins will be extracted. Once the
proteins are separated using gel electrophoresis she will identify and quantify the
amounts of neuropsin at an early and late stage of development. Kate will also be
examining the effects of adding neuropsin to isolated oligodendrocyte cultures.
Using a various concentrations of neuropsin, Kate will see if neuropsin affects the survival of the
oligodendrocytes in culture. To examine oligodendrocyte survival, she will use an MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. MTT is a yellow-colored chemical that can
only be taken into the cytoplasm of living cells. Once it is taken into living cells it is incorporated into the
mitochondria where it is converted to a formazol (blue-colored product). Therefore, the blue color indicates
cell viability, which is determined spectrophotometrically. The more blue produced, the larger the number
of viable cells. Since oligodendrocytes are responsible for myelin production in the CNS, the last step in this
investigation will be to determine if neuropsin alters an oligodendrocytes ability to produce myelin.
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neuropsin.gif
Spinal cord injuries are very serious and very traumatic; however, the severity of the injury can range
from short term swelling to actual severing of the neurons themselves. Spinal cord injury that also results in
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lcord1.jpg
injury to the surrounding blood vessels cause the spinal cord to be exposed to proteins
found in the blood. Although several of the blood proteins are also found at various
times in the nervous system, the levels of these proteins become much higher than
normal following injury. One of these substances, thrombin, has been shown to lead to
apoptosis of spinal cord cells. In the blood, thrombin causes clotting by activating a
cascade that cuts several proteins in the extracellular matrix into proteins that cross-link
into a meshwork forming a scab. Thrombin can also work on individual cells by
activating a receptor (PAR-1) located on the plasma membrane of certain cells.
Thrombin is a member of the serine protease family, which works by cutting proteins
between the serine and arginine amino acids of proteins [4]. In addition to thrombin, other
serine proteases such as neuropsin exist in the nervous system and may mediate survival of
spinal cord cells. There are two approaches to studying cell death in the spinal cord. One
method is to induce an injury and examine the changes that take place following the
injury. The other is to use a developmental model. Since spinal cord cells go through a
pattern of cell death during embryonic development that mimics that of injury, using chick
embryos allows researchers to study cell death both in development and neurodegeneration
simultaneously.
Once a more complete knowledge of how and why spinal cord cells undergo cell death is discovered,
better and more effective procedures can eventually be put in place to decrease or even fix any
neurodegeneration within the spinal cord.
The Nervous System has a major responsibility of maintaining homeostasis through coordinating information and
eliciting responses throughout the entire human body. The Nervous System is made up of various cell types and can
be divided into two main divisions, the peripheral and the central nervous systems. The central nervous system
consists of the brain and spinal cord and the peripheral nervous system contains all other nerves that connect to the
brain and spinal cord. The various cells seen throughout the nervous system include;
 Sensory Neurons-carry information gathered from sensory receptors (a stimulus) to the brain or spinal cord.
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 Interneurons-neurons within the brain and spinal cord that carry
information between sensory neurons and motor neurons.
 Motor Neurons-carry information (a response) from the brain or
spinal cord to muscles or glands.
 Glial cells-cells of the nervous system that provide structural and
physical support to the neurons, helping to keep them healthy [2].
 Oligodendrocytes= myelinate axons in the central
nervous system
G: Visual representation of the
 Schwann Cells= myelinate axons in the peripheral nervous system Fig.
interactions of glial cells with a CNS motor
 Astroglia= help to clean up and remove old neurons and waste and neuron.
 provide nutrients to neurons [3]
All of these cells have an important job to do and, in many ways, they depend on one another to function properly. If
one part of the “system” is faulty or broken, traumatic effects can be seen. One example is Amyotrophic Lateral
Sclerosis (ALS), where motor neurons degenerate and eventual die. Since motor neurons are connected to muscles,
the loss of motor neurons removes the signals that cause muscle cells to contract. Without contraction muscle cells
begin to atrophy and can lose functionality or die. Loss of smooth muscle cells can lead to problems with breathing
and/or swallowing, two essential methods used by our bodies to get materials that our cells require (oxygen, food, and
water). Loss of skeletal muscle cells leads to the loss of overall body movements [1]. In addition to ALS, other
problems that involve the nervous system include Multiple Sclerosis (MS), Alzheimer’s Disease, Parkinson’s Disease,
and spinal cord injuries.
[1]
[2]
[3]
[4]
http://www.alsa.org/ accessed 7/13/10
http://health.howstuffworks.com/human-body/systems/nervous-system/nerve.htm accessed 7/13/10
http://faculty.washington.edu/chudler/glia.html accessed 7/13/10
Wang, Hong and Georg Reiser. 2003. Thrombin Signaling in the Brain: The Role of Protease-Activated Receptors. Biological Chemistry, Vol. 384, pp. 193202.
[5] Flynn, A.N. and A.G. Buret. 2004. Proteinase-activated receptor 1 (PAR-1) and cell apoptosis. Apoptosis, Vol. 9, No. 6, pp. 729-737.
[6] Coughlin, Shaun R. 1999. How the protease thrombin talks to cells. Proceedings of the National Academy of Sciences, USA, Vol. 96, pp. 11023-11027.
[7] Murphy, R.A., Singer, R.H., Saide, J.D., Pantazis, N.J., Blanchard, M.H., Byron, K.S., Arnason, B.G.W., & Young, M. 1977. Synthesis and secretion of a high
molecular weight form of nerve growth factor by skeletal muscle cells in culture. Proceedings of the National Academy of Sciences, USA, Vol. 74, No. 10, pp.
4496-4500.
[8] Terayama, R., Bando, Y., Takahashi, T., & Yoshida, S. 2004. Differential Expression of Neuropsin and Protease M/Neurosin in Oligodendrocytes After Injury to
the Spinal Cord. Glia, Vol. 48, pp. 91-101.