Abstract
The Planarian as a Model Organism for
Neuropharmacology and Regenerative Biology
By
Debra Baker
Chairperson: Oné Pagán,
Ph.D.
Planarians
have been historically associated with Regenerative
Biology but they have recently emerged as model organisms for Neuroscience and
Neuropharmacology. This is, in part, because they are thought
to be the closest extant species that is descended from the common
ancestor that gave rise to all organisms that evolved a central nervous
system. These simple flatworms possess
every major neurotransmitter that is found in mammalian systems including
humans. Homology across species that have evolved a centralized nervous system
gives reason to envision the possibility that analogous results might be
observed in mammals.
Parthenolide,
a sesquiterpene lactone, has been investigated using planarians and has been
established to alleviate the effect cocaine has upon these organisms. The gamma
lactone structure has previously been found to be an essential substructure in
the sesquiterpene molecule in order for it to alleviate cocaine’s behavioral
responses in planarians. We have
determined the simplest structure than can alleviate cocaine-induced motility
reduction is γ-nonalactone.
Specificity
is a desirable quality because the extent to which a substance interacts with
other binding targets will contribute to the risk of side effects and adverse
reactions that might prevent a potential pharmaceutical from being used
therapeutically. The specificity of
parthenolide was established by testing planarians for the presence of seizure
like movements that normally exist upon exposure to neuronally active substances. After a series of tests using parthenolide
against neuronally active substances, parthenolide was found to alleviate
cocaine-induced seizure-like movements (SLM) but not SLMs induced by any of the
other substances that were tested.
Because
planarians have a remarkable ability to regenerate, several neuronally active
substances were tested in decapitated and regenerating planarians to determine
when recovery was sufficient to allow the response that was found in intact
individuals.
This
series of behavioral observations and analysis has supported the use of
planarians as a model for investigating regeneration from a pharmacological
perspective as well as investigating pharmacodynamics using the behavioral
consequences of regeneration.
The
Planarian as a Model Organism for Neuropharmacology and Regenerative Biology
A
Thesis Presented to the Faculty of the
Department
of Biology
West
Chester University
West
Chester, Pennsylvania
In
Partial Fulfillment of the Requirements
For
the Degree of Master of Science
By
Debra
Baker
2012
©
2012 Debra Baker
All
Rights Reserved
Debra
Baker
Approval
of Thesis
For
Master
of Science Degree
In
Biology
COMMITTEE
MEMBERS Date
Oné
Pagán, Ph.D. Chairperson
Gustave
Mbuy, Ph.D.
Erin
Gestl, Ph.D.
Darla
Spence Coffey, Ph.D., M.S.W.
Associate
Provost and Dean of
Graduate
Studies
“Do not let fear make decisions for you
and what you do, do in love. Let love govern your heart for love is the
currency of Heaven.”
“Fear creates discord; it interferes
with the harmony, the song that resonates throughout the universe. It is hard
to impossible to listen to the sound of the universe and stay in harmony when
all you can hear is the demanding rhythm of your own naked fear. Love, on the
other hand, is the very essence of the song the Universe is singing. When
someone acts out of love, he or she is in harmony with the rhythm of all of
creation. Things have a way of working out in the end in spite of the
circumstances. There is no explanation but this is part of the deep laws that
hold everything together and that unite all of Creation to the other. Against
love there is no law, with love, there are no limitations.”
Acknowledgements
I
thank God for the gift of life and for my unique temperament.
I
thank my parents, Joan Sander and James Sander, for doing the best they could
with what they were given.
I
want to thank my husband, Pat, for supporting me and picking up the slack at
home.
Also
my children, Jessica, Jennifer, Joanna, Jonathan, Jeanette, Joshua, Joseph, and
Julianna, who inspire me with their own unique giftings.
Thanks
to my granddaughters, Keli and Fiona who was born as this was being written,
and to her future siblings and cousins. To my cousin, Adriana for also being my
friend.
Thank
you, Dr. Gestl, for graciously tolerating my endless questions.
Thank
you, Dr. Mbuy, for teaching me how to learn thereby opening my mind to behold
so many possibilities.
To
Dr. Pagán, who showed me how to believe in myself by believing in me first, for
your advice, your compassion and, most of all your friendship, I will always be
in your debt.
Sean
Deats, you met me in the middle of the Biology-Psychology Bridge; thank you for
brainstorming with me. I hope we get to collaborate in the future.
To
all of my lab partners, Sean, Peter, Daniel, Matthew, Erica, Clinita, Dharini,
and Galia; you not only helped me tremendously, you have become dear
friends. Whenever I remember you it will
include laughter
Table
of Contents
Introduction………………………………………………………….……….
….. 8
Evolution
of the Central Nervous System ………………………………………..9
Dopamine
…………………….………..………………………………………...20
Serotonin
and Octopamine.……………..………………………………..………23
Cocaine: not Species Specific. ………………………………………………….27
Planarians………………………………………………………………………...32
Sesquiterpenes……………………………………………………………………34
Parthenolide
and Gamma Lactones……………………..……………………….35
Why
Parthenolide……………………………………………………………..….42
Planarians
as a Model Organism…………………………………………………39
Testing
Sesquiterpenes ……………………….………………………………….33
Why
Parthenolide………….……………………………………………………..43
Chapter Two: Determining the simplest
lactone structure that does what?……………………48
Introduction
…….………………………………………………………………. 48
Methods
………………………………………………………………………….49
Results……………………………………………………………………………58
Discussion………………………………………………..………………………65
Determining
Specificity……………...…………………………………………..67
Chapter Three: Molecular Biology...………………………..……………………
71
Introduction
……………………………………………………………………...71
Methods
and Materials …………………………………………………………..72
Procedure……………………………………………………...…………………73
Reverse
Transcription………………………………...………………………….75
qrtPCR…………………………………………………………………………...76
Results…………...……………………………………………………………….79
Discussion…………………………………...…………………………………..
79
Alternative
Approaches.…………………………………………………………80
Chapter Four: Behavior Mapping………………..…………………………..……83 Introduction……………………………………………………...………………83
Experimental
Design…………………………………………………..…………84
Discussion………………………………………………………...…………..….92
Additional
Data for NMDA……………………………………..……………….93
Additional
Data for Cytisine…………………………………….……………….96
Chapter Five: Perspectives.….……………………………….…………..…………..98
Bibliography ………………………………………………..…………………..……101
Published Papers Based Upon This Work……...………………………………109
List
of Figures
1 Clade………………………………….………………………………………………..13
2Key Genes for the
CNS………………………..……………………………………….14
3 Bilateral Symmetry in
Planarians………………..…………………………………….17
4 Dopamine Molecule……………………………………………………………………18
5 DAT/Da Complex……………………………………………………………………...19
6 DAT…………………………………………...……………………………………….22
7 Octopamine…………………………………………………………………………….25
8 Octopamine Compared
To Dopamine………...……………………………………….26
9 Cocaine Molecule…………………………...…………………………………………31
10 Sesqueterpene………………………………………………………………...………34
11 Parthenolide Molecule……..…………………...…………………………………….38
12 Dimethyl-amino Parthenolide
……………………..…………………………………38
13 Parthenolide and
Euniolide…………………………………………………….……..46
14 Parthenolide and
Gamma Lactones…………………………………………………..47
15 Gamma Lactones Used in
Study……………………………………………………..50
16 Testing
Motility………………………………………………………………...…….52
17 Surface Area to Volume………………………………………………………………57
19 Results…………………………………………………………………………….….63
20 Results for Gamma Nonalactone………………..……………………………………64
21 Substances Tested
………………………………………………….69
22 PSLM/Parthenolide……………………………………….…………………………..69
23Karyotype of D. tigrina………………………………….…………………………….80
24 D. tigrina in APW………………………………………………….…………………82
25 Dashed-line Site of
Decapitation……………………………………………………..85
26 Comparing
Seizure-like Responses in Intact and Decapitated Planarians…………...86
27 Planarian
Seizure-like Movement…………….………………………………………87
28 Comparing Planarian
Seizure-like Movements………………………………………88
29 Seizure-like
Positions in Intact and Decapitated/Regenerating Planarian Controls….90
30Seizure-like
Positions in Intact and Decapitated/Regenerating Planarians upon Exposure to
1mM Cocaine……………………………………………………………….91
31 Seizure-like
Positions in Intact and Decapitated/Regenerating Planarians upon Exposure to
1mM NMDA……………………………………………………………….94
32 NMDA Molecule……………………………………………………………………..95
33 Cytisine Molecule…………………………………………………………………….95
34 Seizure-like
Positions in Intact and Decapitated/Regenerating Planarians upon Exposure to
1mM Cytisine………………………………………………………………97
List
of Tables
1 Gamma Lactones Used
in this Observation……………………………………………50
2 Solubility……………………………………………………………………………….59
3 Data for Gamma Lactones…………………………………………………….……….62
4 Primer
Sequences……….…………………………………….……………………..…78
5 Difference Between
Groups on the Same Day (Cocaine)…………………….……….89
6 Difference Between
Groups on the Same Day (Cytisine)……………………………..98
Chapter
One: General Background
Introduction
Planarian worms are
being used as model organisms in research related to Neuroscience because they
occupy a unique niche in the evolution of the Central Nervous System. They are
believed to be the extant species that is the closest, morphologically, to the
common ancestor shared by all subsequent species that possess a centralized
nervous system (Sarnat and Netsky, 2002).
The planarian nervous system is relatively simple but, paradoxically, it
possesses the same neuronal elements that are found in more evolved
species. Genes of the central nervous
system are very highly conserved (Mineta, et al., 2003). Consequently, there is reason to believe
results obtained by observing planarians have the potential to contribute to a
better understanding of similar processes in mammalian species including
humans. Because the planarian genome has been sequenced and resulting data are
being made available, observing and analyzing behavior in planaria has become
an attractive avenue for investigation.
Another quality that is
expressed in planarians is an extraordinary ability to regenerate. Planarians
have the highest concentration of pluripotent stem cells found in adult
organisms (Agata et al., 2008). The
potential applications using stem cells is just beginning to be explored. Because planarians are both able to respond
to psychoactive substances in a quantifiable and predictable manner and they
are able to regenerate any organ in their system including the cerebral
ganglia, also known as a brain, this organism is a unique model to study
regeneration from both a pharmacological as well as a behavioral perspective.
Evolution
of the CNS
The famous quote given
to us by Theodosius Dobzhansky (1900-1975), “Nothing in Biology Makes Sense Except in the Light of Evolution,”
holds true when studying the Central Nervous System. How the nervous system evolved and how
related one organism is to the others in this respect has, among other things,
revived planarians as a model organism to study the Central Nervous System in
addition to its well-known status as a model organism in the study of
regeneration.
The development of
bilateral symmetry and cephalization were important precursors to the evolution
of the Central Nervous System (CNS).
Bilaterians most likely descended from a common flatworm-like ancestor
during the Cambrian geological period approximately 550-600 million years ago
(Jacobs, et al., 2005).
Until a few years ago, the dominant theory was
that vertebrates, insects, and flatworms diverged from a common ancestor,
appropriately named Urbilateria, and
went on to evolve giving rise to three distinct nervous systems; the ganglion
and ladder-like nerve cord of planarians, the segmented ganglia of arthropods,
and the brain and spinal cord and inverted dorsoventral axis of vertebrate
systems, (Sarnat and Netsky, 1985). New
evidence suggests the Urbilateria had not only developed bilateralism but also
acquired the genes that form the foundation of a CNS (Jacobs, et al. 2005). All
organisms with a defined CNS have origins in this common ancestor. This is reflected in the evidence that many
associated genes are extremely highly conserved across all species that possess
a CNS (Mineta, et al., 2003). Although
significant differences exist in the morphology of individual species, each
possesses corresponding regions that, even when their neurological systems are
morphologically dissimilar, posses, “similar molecular fingerprints,” that
reflects a high degree of conservation (Sarnat and Netsky, 2002).
Planarians possess a
CNS with simple primitive morphology. The, “brain,” is a centralized U-shaped
ganglion with ventral nerve cords. The ganglion has nine branches that form
pairs of connections to symmetrical sensory organs. The cephalic ganglion is
similar to more complex vertebrate CNS.
Some examples of features both share include multi-polarized neurons and
homologs of the otx (orthodenticle) genes that are expressed in the anterior
region of the planarian cephalic ganglion (Ribeiro,
2005).
A large anterior commissure
crosses medially uniting planarian hemispheres (Sarnat and Netsky, 1985). The planarian ganglion can be considered a primitive brain. Their simple
cerebral ganglia demonstrates bilateralism with
regions being dedicated to mechano-sensory, chemosensory, light sensory and
inter-neuronal communication (Agata, et al., 1998). Planarians also possess a peripheral nervous
system that is distinct from the central nervous system. The planarian cephalic
ganglion exhibits many morphological features similar to the vertebrate CNS,
such as multi-polar neurons. The ratio
of brain size to body mass in planarians is similar to rats and young
planarians have a higher ratio compared to adults. This is similar to mammalian
species but is not a feature in other simple animal examples (Sarnat and
Netsky, 1985; Raffa and Rawls, 2004).
A consequence of
evolving a central nervous system is the emergence of special sense organs and
these are represented in simple presumably newly evolved form in the planarian
brain. The eyespots are not eyes as mammals experience eyes but are organs able
to sense light from dark. The word, “auricle,” suggests the ear or hearing and
planarian auricles contribute to the edges of a triangularized head but the
auricles are not ears at all and are actually more akin to olfactory organs
than any other mammalian sensory organ. Some species of planarians have a
statocyst which is a simple vestibular organ (Sarnat and Netsky, 1985).
Taxonomically,
planarians belong to the phylum Platyhelminthes. The taxonomic niches are being
adapted by molecular analyses of the 18s rRNA sequences and, using
this measure, bilateral animals are being classified into three groups:
Deuterostomia, Ecdysozoa, and Lophotrochozoa (Huson and Scornavacca,
2011). Phylogenically, Platyhelminthes
are found near the root of Lophotrochozoa.
Because planarians are
so close to the morphology of the early bilateral ancestor and because
planarians occupy a unique place wherein they possess qualities shared by
vertebrates, they are an ideal species to use in the study of the evolution of
the CNS (Agata, et al., 1998)
Figure
1: Phylogeny of the Central Nervous System
Phylogenic Clade Source; Michael Gregory SUNY
Figure
2: Conservation of Key Genes of the CNS
Key genes for the CNS across species. Source, K.
Agata (put the complete reference here).
Periwinkle is for neurotransmission, pink is for the neural network, yellow is
for brain morphogenesis and neural differentiation, aqua is the sensory system,
and dark purple is for other features.
If this figure is
black and white, the colors are all in the same order, from left to right, they
are periwinkle, pink, yellow, pale blue-green, and purple.
When key genes in the
central nervous system were compared across species, 110 of 116 head genes
found in planarians were shared by all of the bilateral animals that have been
examined (C. elegans, D. melanogaster,
and H. sapiens.) The absent genes, the 6 out of 116 genes that were
analyzed, were absent in C. elegans and/or in D. melanogaster. These three species belong to the three different
groups of bilaterians that have a CNS (Agata, et al., 1998). These genes include FGF signaling molecules,
noggin, and frizzled, among many others.
Because so many functional genes are so highly conserved, one leading
theory suggests that all bilaterians with a CNS share a common ancestor (Sarnat
and Netsky, 1985).
Because the CNS appears
to be so highly conserved and because planarians are taxonomically and
morphologically the closest extant organism to a shared common ancestor,
several research laboratories are observing and documenting normal behavioral
patterns in planarians that have been exposed to various chemicals that act
upon various neurotransmission pathways.
Responses to compounds such as cocaine (Raffa, et al., 2003), caffeine (Pagán,
et al., 2009), nicotine, (Pagán, et al., 2009), and NMDA (Rawls, et al., 2007),
have been observed in planarians. These
observations have established a variety of behaviors that are considered normal
which has, in turn, made it possible to identify behavior that departs from the
standard (Raffa, et al., 2001).
With respect to
cocaine, planarians exhibit predictable behavior patterns upon exposure to
cocaine at sufficient concentrations. (We determined a concentration of 200µM
was ideal, the reasoning behind that decision can be found in Chapter Two). Their
movement is reduced significantly. They also go through seizure-like movements
during which they frequently curl up into a, “C-like position.” This is a spasm-like behavior that is
distinguishable from the more smooth movements that are within the planarians
repertoire. Another behavior that is
associated with cocaine toxicity is the “head bop,” in which they appear to
have the tail pinned to the bottom of the dish while they waggle back and forth
(Raffa, et al., 2001; Rowlands and Pagán, 2008).
Figure 3
Bilateral
Symmetry in D. tigrina
Photograph taken by Dr. Pagán
Figure 4, Dopamine Molecule
|
Dopamine
Molecule Source Dr. Pagán
|
Figure
5: Dopamine/Transporter Complex
|
Source: Beuming, et al 2008: The Dopamine
Transporter Complexed With Dopamine (key amino acids shown).
|
Dopamine (Not
Specific to Planarians)
Dopamine
is a neurotransmitter that can be found in the brain of animals including
planarians but the information in this section is generalized and not
species-specific to planarians unless noted.
Dopamine is synthesized from the essential amino acid Tyrosine. The first modification in this pathway is a
hydroxyl group added by Tyrosine Hydroxylase forming the intermediary, 3,
4-Dihydroxyphenylalanine is formed. This intermediate is commonly called DOPA.
The last modification is the removal of a carboxyl group is by DOPA
decarboxylase to form dopamine (Hyman, 2005). Dopamine is stored in vesicles in
the end of an axon. With the appropriate stimulation, dopamine is secreted into
the synaptic cleft. Dopamine travels
across the synaptic cleft to the receptor on the neuron on the other side of
the cleft (Callier, et al., 2003; Craig and Rice, 2004)
There
are two major types of dopamine receptors and planarians have both types. They
are D1 (including D1 like) and D2 (including D2 like.) The major difference
between these two receptors is that, upon stimulation, D1 receptors are coupled
with the G-protein coupled receptors (GPCRs) that stimulates the release of
adenylate cyclase increasing the concentration of adenosine monophosphate
(cAMP.) Conversely, when D2 receptors
are stimulated, the activity level of cAMP decreases or is unaffected (Venturini,
et al., 1989; Missale, et al., 1998; Ribeiro, et al., 2005).
The
dopamine transporter (DAT) is a transmembrane protein that pumps dopamine out
of the synapse and into the neuron from which it originated (Venturin, et al.,
1989). Dopamine is repackaged into
vesicles to be used again. Until very
recently, cocaine was thought to bind competitively at a site in the DAT that
overlapped the receptor site of dopamine but recent data seem to support the
cocaine binding site as a non-competitive allosteric site on the DAT (Graczyk,
T. 2011 not yet peer-reviewed).
Dopamine
can be modified by Beta-hydroxylase and processed into norepinephrine. Because
it was regarded (correctly) as a precursor of norepinephrine and epinephrine,
Dopamine was not recognized as a neurotransmitter in its own right until
Swedish Biologist, Arvid Carlsson, discovered it in 1957
(Carlsson, 1957). In 2000, Dr. Carlsson
shared the Nobel Prize for his work.
Cocaine
is an addictive drug as a result of the way that it exploits the dopaminergic
neural pathways, and is experienced as a sense of euphoria in human subjects (Hall,
et al., 2004; Gainetdinov and Caron, 2003).
Dopamine is prevented from being cleared from the synapse when cocaine
acts as a competitive reuptake inhibitor. Eventually, more cocaine is needed
for the addict to experience the same intensity of euphoria. Recently, knockout mice without dopamine
transporters have demonstrated addictive “seeking” behaviors which led to the
discovery that cocaine affected serotonin transporters as well as dopamine
transporters (Ribeiro, et al., 2005; Iversen, 2009; and NIH,1998).
Figure 6, Dopamine Transporter
|
|
DAT Source: Kurian, et al, 2009. This is a two-dimensional representation of a
three-dimensional transmembrane protein.
Some of the dopamine binding substrates are represented as are sodium
ion binding and glycosylation site.
Serotonin and Octopamine (Not Species-Specific)
Serotonin
Serotonin
is another neurotransmitter that is related to reward and motivation in mammals
that also exists in planarians (although determining the extent to which
flatworms experience motivation is beyond the scope of this work.)
Because
dopamine and serotonin are both associated with motivation, pleasure, and
reward, cocaine is an extremely addictive substance. The dopaminergic system is
actually altered in chronic addicts (Volkow, et al., 1993). Because cocaine blocks the dopamine
transporter, and excessive dopamine accumulates in the synapse, the number of
postsynaptic receptors is actually decreased in long-term addicts.
Up
until recently, cocaine was thought to bind to an orthosteric site that there
was a bit of overlap between the cocaine binding site on the DAT and the region
of the DAT that is used to hold dopamine while it is transported to the
presynaptic vesicle (Callier, et al,
2003). Recently, one of Dr. Pagán’s
graduate students has produced data that provides strong support for the active
binding site for cocaine being on an allosteric site with no direct overlap of
the binding site that dopamine uses in order to be taken back to the
pre-synaptic vesicles where it can be used over and over again before it is
metabolized.
Additionally,
some physiological effects of cocaine exposure include tachycardia, elevated
body temperature, decreased appetite, and an increase in available energy; the
result of cocaine’s effect upon the availability of dopamine that is
metabolized into norepinephrine (Koehntop, et al., 1977).
Octopamine
Many invertebrates,
including planarians, possess a dopamine-related neurotransmitter called,
“Octopamine.” In insects, Octopamine is
the primary reward pathway. Octopamine has transporters that are similar to
dopamine and serotonin which are also affected by cocaine (in insects
(Nathanson, et al., 1993). The primary subject of most investigations
related to octopamine is insects but octopamine is produced in planarians (Barron,
et al., 2010). Octopamine is configured almost like norepinephrine but
possesses one hydroxyl group, either Para or Meta conformation.
Structurally,
Octopamine looks more like Norepinephrine than dopamine with tyramine
resembling dopamine (figure 8).
Figure
7: Octopamine in the Para and Meta Conformation
Octopamine (Para,
Left and Meta, right)Molecular structures courtesy of Dr. Pagán
Figure
8: Octopamine and Dopamine
Octopamine compared to Dopamine (Barron, et
al., 2010)
Cocaine
Cocaine is a derivative
of the coca plant, Erythroxylum coca,
which thrives in its woodland environment of South America. There are actually
several species of shrub that are in the Erythroxylum
Genus but the species most used and most associated with cocaine is Erythroxylum coca.
Indigenous peoples of
that region have taken advantage of the medicinal properties coca leaf and have
also incorporated it into their religious rituals. Generally, indigenous
peoples struck a balance with their natural environment and used the coca
leaves with respect and moderation (Personal conversation with Sue Mustalisch,
Professor of Health Science at West Chester University). There have been no documented
cases of addiction as the result of using coca leaves
According to
anthropologists, natives of Peru were aware of the anesthetic properties of the
coca plant. They would chew the leaves and drool on the part of the body that
needed to be cut. Herbal treatments derived from coca leaves were applied to
the wounds associated with trepanning procedures that were performed into the
early part of the twentieth century (Musto, 1991).
There are some accounts
of the indigenous use of white powder and herbs that would increase energy and
take away painful sensation. These
accounts were documented by the priests and scribes that accompanied the
Spanish in the fifteenth and sixteenth century (Calatayud, and Gonzalez 2005).
Cocaine was initially
transported to Europe and studied by Friedrich Gaedcke. Starting in 1855, he extracted an “amorphous
substance” from the coca plant and identified it as an alkaloid naming it
Erythroxyline. Another chemist, Dr. Carl
Von Sherzer, transported coca leaves to Europe. Von Scherzer sent some of the
coca leaves to Friedrich Wohler in Gottingen, Germany who then gave the leaves
to his graduate student, Albert Niemann for analysis. In 1859, Niemann isolated
cocaine from the coca leaves, correctly identified the extracted powder as an
alkaloid and named it “Cocaine” by combining the name of the leaves, “coca”
with the Latin suffix, “-ine” for its alkaloid identity. Niemann noticed that, “Its solutions have an alkaline reaction, a bitter taste, promote the
flow of saliva and leave a peculiar numbness, followed by a sense of cold when
applied to the tongue.” His findings can be found in his 1860 Ph.D.
dissertation entitled, “Uber eine neue
organische Base in den Cocablattern” translated by me, “On a new organic base in the coca leaf.”
In 1868, Thomas Moreno y
Maiz, a surgeon in the Peruvian army, wrote a book based on records he kept
while experimenting on animals in his care (Moreno y Maiz, T., 1868). In 1879, Vassili von Anrep noticed that
applying cocaine in solution would numb the skin on the exposed region. He also
dropped the same solution onto the eye of an animal which would numb the
eyeball and dilate the pupil (Vandam, 1987).
It took another five years before cocaine was recognized as a local
anesthetic.
Cocaine was the first
clinically used local anesthetic (Sholz, 2011).
Albert Niemann isolated cocaine from the coca leaves (Koller, 1941) and
Sigmund Freud was the first one to treat a patient with it. Ironically, Freud
used cocaine to wean his patients from morphine. He and his colleague, Karl Koller,
were also the first to demonstrate its numbing effect when applied
occularly. A conundrum exists in this
narrative that may lead the reader to believe that two men were given credit
for discovering cocaine as a local anesthetic.
Although Koller cited Anrep’s previous work that was published, Koller
was credited with the discovery because his work that confirmed Anrep’s
original findings was published in a professional journal that was prominent to
the surgeons of his era (Yentis and Vlassakov, 1999) Dr. William Stewart
Halsted is credited to be the first to describe the effect when cocaine is
injected into the sensory nerve trunk (Osborne, 2007). Unfortunately, no one knew about the
addictive qualities of cocaine. Freud, as well as many other prominent people
of the late Victorian era, became cocaine addicts before laws could be passed
to regulate the substance (Koller, 1941).
The mechanism of action that interferes with
the transmission of sensation is a disturbance in the resting potential in the
sodium gated ion channels. Most theories
involve open/closed, active/inactive channels. Other theories propose a
disturbance in the phospholipid bilayer of the cell membrane. Local anesthetics are generally divided into
amides and esters (Vandam, 1987).
Amylocaine was the
first synthetic local anesthetic. It was synthesized in 1903 and was quickly
overshadowed by the formulation of procaine in 1904. Both Amylocaine and
Procaine (Novacaine) are esters and analogs of cocaine (McLure and Rubin,
2005). The slight change in molecular
conformation was sufficient to lose the addictive properties associated with
cocaine while preserving its effectiveness as a local anesthetic. Soon after
Procaine was first synthesized in 1904, it became widely available in dental
offices and emergency rooms. During the
second half of the twentieth century, Amides took the place of esters and local
anesthetics took effect more rapidly and had a lower incidence of allergic
reactions (Calatayud and Gonzalez, 2005).
From the plant’s
perspective, cocaine evolved as a potent weapon in E.coca’s botanical arsenal. Cocaine affords effective protection
against predation. It is worth mentioning the likelihood that many potential
future pharmaceuticals may be found in the arsenal of hundreds of thousands of
known and yet-to-be discovered plants, animals, and microscopic life forms that
are players in the evolutionary arms race that share habitat with Erythroxylum coca (Perfecto and Vandermeer, 2008). The
cocaine molecule has four chiral centers which allow it to form 16 isomers (McLure
and Rubin, 2005). Out of these 16
isomers, only one is pharmacologically active (Dr. Pagán’s Ph.D. Dissertation).
Figure
9: Cocaine Molecule
Source, Dr.
Pagán
Planarians
Planaria is the common name for several genera
of free-living non-parasitic flatworms. Taxonomically, planarians belong to the
phylum Platyhelminthes, class Turbellaria, order Tricladida (Carranza, et al. 1998).
Planarians make up various family, genera, and species. Although they possess three germ layers,
planarians are acoelomates.
Generally, planarian
biologists commonly use three species of planarians namely Dugesia japonica, Dugesia
tigrina, and Schmidtea mediterranea.
The genome of Schmidtea mediterranea
has been sequenced by Dr. Alejandro
Sánchez Alvarado and his colleagues at the University of Utah (Newmark
and Sanchez-Alvarado, 2002). The
accomplishment of the Sanchez lab opens up access to genetic assays that will
enable biologists to determine transcription and expressing rate while siRNA
will be used to silence genes to determine function (Gentile, et al., 2011;
Sofia, 2007). The use of S. mediterranea in labs that focus on
molecular investigation has become almost ubiquitous because of Sanchez’s
pioneering work (Gentile, et al., 2011).
Planarians occupy an
important position in the evolution of the central nervous system the details
of which can be found on page 9.
Although their system is simple, planarians possess features that
suggest a pivotal place in evolution They are closely related to the first
organism to evolve a brain and they share essential central nervous system
genes with almost every contemporary organism that possess a CNS (Sarnat and
Netsky, 1985).
Planarians process and dispose of nitrogenous
waste using flame cells. Recently, the
physiology of flame cells has been studied and these cells form a prototype of
a nephron (O’Donnell, 1997).
Planarians are triclad
but do not possess a coelom and do not have an alimentary canal. They access food using an appendage called a
proboscis. Solid waste also exits the body the same way it entered (O’Donnell,
1997).
Most species of
planarians (including D. tigrina used
in this project) are hermaphroditic possessing both male and female sex organs
(Chong, et al., 2011).
Figure
10: Sesquiterpenes
Sesquiterpenes Used in the Pagán, et al., 2008
paper. The lactone structure was found to be necessary in order for the effects
of cocaine in planarians to be alleviated. Molecular structures were generated
by Dr. Oné Pagán.
Parthenolide
and Gamma Lactones
Parthenolide, a
Sesquiterpene lactone, has been established to antagonize behavior that is
associated with cocaine exposure in planarians.
Parthenolide is a relatively simple ringed structure. Because of this,
determining the simplest structure that has this capacity offers the potential
to contribute to a better understanding of the mechanisms that work to
significantly reduce the behavioral effects cocaine has on the nervous system.
Recent findings have identified γ-Nonalactone
as the simplest structure needed to effectively antagonize cocaine toxicity
(Baker, et al., 2011). Observations of
quantifiable behavior responses were used by our lab in order to determine the
presence and the intensity of the inhibitory properties of substances that were
being tested.
Eventually, genetic and
immune assays will be used to better understand why these substances are
inhibiting cocaine (Raffa, et al, 2005). Determining any changes in the
transcription rate of key components in the cocaine-sensitive dopaminergic pathway,
for example, has the potential to contribute to a more comprehensive
understanding of the mechanism of action lactones employ as cocaine
antagonists.
In one sense,
parthenolide was a logical choice. A
detailed explanation of the reasoning behind choosing parthenolide can be found
on page 42 of this document.
When several compounds
were introduced to the aqueous environment in which planarians were contained,
the compounds with the four-carbon lactone ring structure were found to
alleviate the withdrawal symptoms in planarians with Parthenolide demonstrating
the greatest effect (Pagán, et al., 2008).
Parthenolide is similar in many ways to compounds such as β-eudesmol,
which shares similarities in molecular structure but had no significant effect on
planarians that were exposed to cocaine. The molecules that antagonized cocaine
always had the gamma lactone structure (a “lactone” is a ringed ester). These ringed structures can also be found in
many traditional herbal medicines that have been used by people for thousands
of years. Feverfew (Tanacetum parthenium)
is a good example. Parthenolide is one of the identified molecules in this
herbal remedy (Erlich, 2008).
Parthenolide
in Mammals
Parthenolide
in Humans
Parthenolide has
demonstrated the ability to selectively destroy leukemic stem cells while
sparing other hematopoietic stem cells as well as other hematopoietic stem
cells in humans (Guzmán, et al. 2007).
It seems to be an effective chemotherapy for leukemia but it has not
been approved by the FDA.
Barriers exist that
impede the ability to get parthenolide from the in vitro environment to the
practical in vivo world of effective pharmacokinetics and chief among these
barriers is parthenolide’s extremely low solubility. One possible solution for making parthenolide
bio-available is to create a soluble parthenolide analog (Guzmán, et al.,
2007).
The researchers who
were investigating the aforementioned anti-leukemia properties of parthenolide
have also formulated an analog of parthenolide that is 70% soluble (Guzmán, et
al., 2007). This is an impressive improvement over parthenolide that is about
5% soluble according to the Materials Safety Data Sheet
Apparently,
parthenolide elicited a cytoprotective response in the leukemic stem cells it
was targeting, but the effort this team made to increase the solubility by
formulating an analog seems to be effective with tolerable side effects in vivo
in humans (Guzmán, 2007.)
Figure
11: Parthenolide
Figure
12: Dimethylamino Parthenolide
Parthenolide
in the Rat Model
Parthenolide has
recently been administered to rats that had been exposed to “acute” injections
of cocaine. Parthenolide has been found to block the inhibitory effect of
cocaine upon the dopamine neurological firing rate in the rats. In mammalian systems, cocaine works on the
ventral tegmental area of the dopaminergic network which is associated with
motivation and reward. These more
sophisticated pathways do not exist in planarian systems but it is significant
that Parthenolide seems to inhibit the activity of cocaine using a pathway in a
mammalian subject that is not found in the planarian brain (Schwarz, et al.,
2011).
Parthenolide
in a Human Cell Line
As indicated previously, very recent
unpublished (as of this date) data suggests that parthenolide (in particular)
inhibits the mechanism of action of a
cocaine analog against the dopamine transporter in human embryonic cell lines
that had been transfected with the gene that codes for the DAT (Graczyk, 2011
unpublished data).
Planarians
as a Model Organism
By the turn of the
twentieth century, Biologists were aware of the regenerative abilities of
planarians. The earliest known reference
was found in a Japanese encyclopedia dating to the seventeenth century (Sanchez
Alvarado Lecture, 2010). Over 600
scholarly papers about planarians were published around the turn of the twentieth
century. Research varied from
light-dark studies to regeneration after exposure to radiation.
The reason planarians
stopped being used in advanced research was a direct result of the intellectual
rivalry between two distinguished developmental and genetic biologists, namely
Charles Manning Child and Thomas Hunt Morgan (Mitman and Sterling, 1992).
Thomas Hunt Morgan
embraced Mendelian Inheritance theories and believed there was a distinction
between somatic and germ line genetics. Since Drosophila melanogaster expresses its germ line earlier in
development, D. melanogaster became a model organism for use in developmental and
genetics research (Nobel Prize Biography).
Child used planarians
in his research and was much more interested in environmental influences and
phenotypes. Child was not quite
convinced that genes were stored on chromosomes. He also rejected the notion that two discrete
sets of molecular instructions exist; the somatic and germ line (Mitman and
Sterling, 1992).
Child and Morgan chose
to ignore one another’s existence and certainly did not acknowledge the other’s
contribution to genetics and developmental biology.
Eventually, Morgan’s
ideas were supported by subsequent discoveries and his work with D. melanogaster
became associated with the emerging discipline while planarian research fell
out of favor (Mitman and Sterling, 1992). Ironically,
some trends have come full circle and the work Morgan pioneered in the field of
genetics has resulted in the sequencing and eventual mapping of the human
genome as well as the genomes of many other species. As a result, the focus of research has
shifted to epigenetics; the study of how environment interacts with the basic
instructions contained within the cell, and stem cell technology that holds the
hope of innovative therapies. Because
they have adult pluripotent stem cells, neoblasts, distributed throughout their
body, planarians are able to allow regeneration of an entire individual from a
tiny fragment of tissue. As a consequence of their remarkable ability to
regenerate and because of their status as the least evolved organism with a
central nervous system, planarians are being rediscovered as model organisms in
labs all around the world (Gentile, 2011).
Advancements in
sequencing DNA have contributed to resurgence in planarian Biology. Dr. Alejandro
Sánchez Alvarado was among the first to recognize the potential benefits
of sequencing the genome of at least one species of planarians. It was
necessary for him to harvest planarians from a fountain in Spain. The genus and
species of his planarians just happened to be Schmidtea mediterranea which is why this particular genus and
species will most likely emerge as the model species for planarian research
into regeneration (Newmark and
Sánchez-Alvarado, 2003).
Planarians are emerging
as model organisms in the areas of regeneration research, particularly using
adult stem cells and in the area of neurological and neuropharmacological
research (Gentile, et al., 2011). Along with
the ability to regenerate and their position on the phylogenetic clade that
places them as the most anciently evolved species with a centralized nervous
system and rudimentary special sensory organs, planarians are able to reproduce
sexually and asexually. S. mediterranea houses its genome on four stable
diploid chromosomes (Lau, et al., 2007).
The lack of a stable
genome within the Dugesia genus will adversely affect the ability to conduct
the tests necessary to discern what is happening to the transcription rate of
the associated RNA and subsequent changes to the expressing level of key
protein products.
Testing
Sesquiterpenes
Various organic
compounds have been tested to determine if any of them was able to antagonize
cocaine’s effect in planarians. Eventually,
a group of ringed organic Sesquiterpenes were suspected to be good candidates
for investigation. After careful observation, a Sesquiterpene with the five
sided gamma lactone, Parthenolide, was found to antagonize the symptoms of
cocaine’s activity in planarians (Pagán, et al., 2008).
The idea of testing Parthenolide against
cocaine in planarians originated in Dr. Oné Pagán’s dissertation. The following
narrative is a direct quote taken from Dr. Pagán’s dissertation defense.
“Why parthenolide? The idea of using parthenolide as a
cocaine-displacing ligand came from its close structural similarity to another
class of natural products, the cembranolides.
Cembranolides are cyclic diterpenoids widely distributed in nature. More than 300 examples have been described
(Wahlberg & Eklund 1992).
The
cembranolides are established ligands of the nicotinic acetylcholine receptor
(nAChR). The nAChR is the best-known
member of the ligand-gated ion channel superfamily. This receptor will not be discussed here, and
the reader is referred elsewhere (Karlin, 2002). Cembranolides bind in a mutually-exclusive
manner with the local anesthetic procaine, on the nAChR (Pagán, 1998; Pagán et
al., 2001).
The
“line of thought” that led me to consider parthenolide as a cocaine antagonist
was:
1. Cembranolides and an example of a local
anesthetic bind to nicotinic acetylcholine receptors (nAChRs) in a
mutually-exclusive manner (Pagán, 1998, Pagán et al., 2001).
2. Cocaine is the prototype of the local
anesthetic class of molecules, with neuronal sodium channels as the target
(Ruetsch et al., 2001; Scholz, 2002).
However, its behavioral effects are due to cocaine’s interaction with
neurotransmitter transporters, mainly the dopamine transporter (Uhl et al.,
2002).
3. Parthenolide shows close structural similarity
with cembranolides.
4. Parthenolide interacts with nAChRs and is
capable of alleviating cocaine inhibition of this receptor in
electrophysiological studies (Pagán and Hess, unpublished results).
Based on the points above, I thought
that parthenolide and cocaine had the potential to interact with the dopamine
transporter” (Pagán, 2005).
Dr. Pagán’s educated
hunch seems to have served him well as yet-to-be published data that is the
Master’s thesis of one of his graduate students strongly supports the
hypothesis articulated in Dr. Pagán’s dissertation. Thomas M. Graczyk, MS, has transfected
dopamine transporter genes (DAT) into embryonic kidney cells that normally do
not possess dopamine transporters. He
then exposed these cell lines to procaine (an analog of cocaine) in the
presence and absence of parthenolide and the parthenolide reverses the effect
procaine has upon the DAT. His experimental design included negative and
positive controls one of which allowed for repeated tests in cloned cell lines
that did not possess DAT. These non-DAT lines did not see the same effects
(Graczyk, 2011, not yet peer-reviewed as of this writing). One of the more intriguing results of this
endeavor includes the discovery that the binding site of parthenolide to the
dopamine transporter is allosteric and non-competative with dopamine or
cocaine. It apparently increases the
affinity for dopamine. Up until this point, the model for the binding site
cocaine occupied to antagonize movement and increase seizure-like movements was
thought to be both orthosteric and competitive with dopamine at the transporter
site (Graczyk, 2011).
Cocaine ester acts as
an anesthetic as well as a dopamine reuptake antagonist (Koehntop, 1977).
Cocaine amide acts as a local anesthetic but does not produce the behavioral
effects that are associated with excessive dopamine levels (Iversen, 2009).
Figure
13: Structural Similarities Between Parthenolide and Euniolide (a
Cembranolide.)
Parthenolide
(left) and Euniolide (right) Shown together to illustrate structural
similarity.
Figure
14: Structural and Molecular Similarities Between Dodecalactone and
Parthenolide
Comparing the structural and molecular similarities
between Decalactone and Parthenolide. Molecules courtesy of Dr. Pagán
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