Monday, January 16, 2012

Parts 1 and 2










Part One
Determining Which Lactones Antagonize Cocaine













 
Part One
Determining Which Lactones Antagonize Cocaine













Introduction



Testing response in planarians when exposed to cocaine and various gamma lactone structures

Determining the simplest structure that antagonizes cocaine

qrtPCR assays to detect and quantify significant changes in transcription and/or expression rates for various genes in the dopaminergic pathways.

Testing other neuronally active compounds in order to establish anything found to antagonize cocaine does so with specificity.





















Methods and Materials

Animals and chemicals:  Brown planarian worms (Dugesia tigrina) were purchased from Ward’s (Rochester, NY).  General laboratory materials and supplies were obtained from Fisher Scientific (Suwanee, GA) or Sigma-Aldrich (St. Louis, MO); (-) Cocaine hydrochloride was purchased from Sigma-Aldrich (St. Louis, Mo).  The tested Alkyl gamma-lactones were purchased from Chromadex (Irvine, CA).







General Procedure

 The first molecules to be examined were the five sided ringed structure known as gamma lactones. Gamma lactone structures were systematically tested starting with the simplest structure and progressing to sequentially longer hydrocarbon chains.

The simplest gamma lactone tested was gamma Valerolactone. C5H8O2.  The lactone with the longest hydrocarbon chain was gamma Dodecalactone C12H22O2


Figure 10: Gamma Lactones Used in Study





The concentration of each lactone tested was determined by testing planarians in solution with decreasing concentration of lactones until the activity level of planarians in solution demonstrated no significant variation from the control. 

For every trial, a set of controls was also conducted and the activity of the planarians subjected to various lactones with or without cocaine was compared to their corresponding control.

Each planarian was tested once and euthanized with 0.2 M HCl.

Figure 11: Planaria Being Observed for Motility. Photo courtesy One Pagan, PhD.
The concentration of cocaine used was determined by conducting an observation of activity at decreasing concentrations. It was determined that large (>2cm) individuals needed higher concentrations of cocaine in order to exhibit typical cocaine-induced behavioral changes.


Planarian movement was tested by placing an individual planarian in a 6cm diameter petri dish with the specified solution containing the specified concentration added. The petri dish was pre-rinsed in APW (Artificial Pond Water composed of NaCl, CaCl2, NaHCl3 and Distilled H2O) and covered. Each individual was pre-incubated in solution for 10 minutes and the petri dish was set on a grid whose lines formed 1cm squares. When timing commenced, planarians were perturbed by swirling the solution in the dish. Notations were made when the planarians crossed the 1cm hash mark. Movement was documented every minute for a total of five minutes for every observation.   



Gamma Lactones Used in this Observation

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Concentration
γ-Valerolactone
203 µM
γ-Hexalactone
195 µM
γ-Heptalactone
100 µM
γ-Octalactone
100 µM
γ-Nonalactone
51 µM
γ-Decalactone
10 µM
γ-Dodecalactone
10 µM





Cocaine was tested at a concentration of 200 µm.

0.1% DMSO was included in all solutions including controls.

Data were analyzed using Prism Graph Pad software and the data were subjected to regression analysis using a two-tailed t-test.





Surface Area to Volume Ratio



The intensity of responses that planarians demonstrate upon exposure to cocaine is influenced by the surface area to volume ratio of the individual planarian.  Motility in very small and very large individual planarians can be different from individuals that are within one standard deviation of the mean.  This potential problem is resolved by selecting planarians by size. Planarians also need to be starved for at least one week in order to eliminate possible interactions with nutrients and/or metabolites associated with digestion.  After testing responses in planarians that were either extremely small (<1cm) or extremely large (>2cm,) the small individuals responded more vigorously to something that elicited a neurological response and the large individuals generally needed a higher concentration to exhibit a response.   This correlates to the surface area to volume ratio. Because of this phenomenon, planarians were selected for observation based upon their size. Individuals between 1 and 2 cm were selected for observation.


Figure 12: Difference in Motility Comparing Extremely Small and Extremely Lare Planarians



Results

The Effect of γ-Lactones on Planarian Motility

Figure 13 shows a series of concentration-response curves of planarian motility. Motility decreases as a function of γ-lactone concentration. The three smaller lactones (Valerolactone, Hexalactone, and Heptalactone) did not inhibit planarian motility at concentrations below 500 µM.

The larger lactone structures (Octalactone, Nonalactone, Decalactone and Dodecalactone) decreased planarian motility in a concentration-dependent manner. The least potent in the latter group of compounds was Octalactone which had an IC₅₀ of approximately 426 µM while the most active compound, Decalactone displays an IC₅₀ 43µM.



















Motility as a Function of γ-Lactone Concentration


Figure 13: Motility as a Function of Concentration



Appendix Table 2 illustrates the results of parallel experiments using 200 μM cocaine in the absence and in the presence of a single γ-lactone concentration at which the lactone did not induce motility decrease by itself. Cocaine at a concentration of 200 μM decreased planarian motility by about 50 % (Figure 14), which is consistent with previously reported results (Pagán, et al., 2008).  





The only compound capable of antagonizing  cocaine effectively was γ-nonalactone, which, at a concentration of about 50μM, significantly alleviated the 200μM cocaine-induced motility decrease from about 51% (cocaine alone) to about 12% (cocaine + γ-nonalactone, )  See Appendix Table 2.  Figure 14 demonstrates γ-nonalactone’s effect on cocaine-induced motility decrease as concentration-dependent and becomes synergistic with cocaine at γ-nonalactone concentrations higher than 75μM.





     Results for γ-Nonalactone


Figure 14: Results for Gamma Nonalactone expressed at a fraction of the control.




This work has established the γ-lactone moiety associated to a 5-carbon methyl tail attached to position 4 in the lactone ring (γ-nonalactone, Figure 15.) to be the minimum structure necessary to reverse cocaine-induced mobility inhibition in planarians. This is consistent with previous work which indicated that the lactone ring in this class of compounds is essential for their cocaine-antagonist effect in this experimental system (Pagán, et al., 2008).  The results, however, indicate that the γ-lactone moiety is not sufficient to antagonize cocaine effects because none of the other lactones that were tested demonstrated any significant alleviation of cocaine-related symptoms in planarians.  We also determined that the γ-nonalactone effect on cocaine was concentration-dependent, suggesting that γ-nonalactone and cocaine compete for a specific binding site in planarians, presumably a protein receptor. Additional evidence in favor of a common or overlapping binding site for cocaine and the γ-nonalactone can be deduced by the observation that γ-lactones with alkyl chains longer than 5 carbons decrease motility by themselves yet they are inactive antagonists against cocaine.

 This phenomenon is somewhat reminiscent of the cutoff effect observed in some types of general anesthetic molecules. The cutoff effect is the increase in anesthetic potency of a homologous series of compounds, for example, n-alkanes or n-alkanols among others, up to a point where a decrease (or even total loss) of the anesthetic effect is observed in higher molecular weight compounds (Eckenhoff et al., 1999). This effect is frequently used to estimate the molecular dimensions of protein targets (Eckenhoff , et al., 1999; Franks and Lieb, 1985), but other interpretations, including the interaction of the anesthetic compounds with membranes, as opposed to proteins, has also been proposed (Mohr, 2005). It is possible that we are observing a mechanism similar to the cutoff effect in these γ-lactones/cocaine experiments.

 Interestingly, the biggest lactone tested, Dodecalactone, is very similar to Parthenolide in terms of its molecular weight, yet Dodecalactone was inactive against cocaine. This indicates that molecular size must not be the only property that influences parthenolide's (or the γ-lactones) anti-cocaine properties. 

Another consideration that is being explored is the ratio of optic R to S enantiomers found in the various lactones as well as in Parthenolide.  Preliminary findings suggest Octalactone as well as Decalactone favor the R configuration while both parthenolide and nonalactone are either a racemic mixture, have a slight S leaning R to S ratio or favor the S confirmation (get data and facts.)





Similar Results in Mammals

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 (Schwartz, et al., 2011).





Part Two: Determining how the Transcription Rate of Dopamine Receptors is Affected by Parthenolide and Gamma Nonalactone























Introduction



Our lab has recently had a paper published in which we have documented the alkyl γ -lactone structure with a four carbon hydrocarbon chain as the least substituted structure necessary to antagonize the effect cocaine has on the nervous system of planarian worms (Baker, et al., 2011).  The mechanism of action is not known but cocaine is known to obstruct the dopamine transporter protein (Mateo, et al., 2004).  It is also known to decrease the number of post-synaptic dopamine receptors in humans that abuse cocaine (Volkow, 1997). As a result, understanding the changes in transcription in RNA that is translated into the Dopamine transporter protein and associated membrane proteins may be illuminating. 



















Methods



RNA is isolated from planarians

Planarians of a uniform size are separated into four discrete groups according to their treatment. The first group was the control that had been exposed to artificially formulated pond water (APW) and Dimethyl Sulfoxide (DMSO) at 0.1%.  Another group has been exposed to cocaine at a concentration of 200 µM.  A third group will be exposed to a combination of cocaine at 200 µM and gamma nonalactone at a concentration of 50 µM.  The fourth group will be a combination of cocaine at 200 µM and Parthenolide at 50 µM. All four groups will be placed in solution with DMSO at a 0.1% concentration.

The planarians will be decapitated and their heads will be harvested and homogenized.  mRNA will be isolated and the transcription rate for genes of interest will be evaluated by running each group through a qrtPCR procedure.  The results will be compared in such a way that any significant differences between the control and the other variable being tested will be analyzed.













The procedure is as follows:

RNA isolation

Approximately ten planarians will be allocated to each of the four treatments and each group was pre-incubated in its respective solution for ten minutes after which time, they were euthanized, decapitated, and placed in 0.5mL Trizol.  Care was taken to avoid dilution of Trizol when transferring planarians to the container of Trizol. After the planarians were in Trizol, they were homogenized using a Power Gen 125 homogenizer. At this point, the cells have been lysed and organelles are able to be fractionated.

100µL. of chloroform was then added in a microfuge tube and the mixture the mixture was then gently shaken for 15 seconds and was allowed to incubate at room temperature (approximately 20 degrees, C.) for 5 minutes.

 The mixture was then centrifuged at 12,000 rpm/ref. for a total time of ten minutes.

Up until this point, the rationale for this procedure is as follows: Trizol is used to lyse membranes and allow the contents of the cell to become available. Trizol aids in the homogenation process.  Chloroform has an affinity for RNA and helps it go into solution while the Trizol and other organic material precipitate out of solution.

RNA is isolated with the top layer and then precipitated out of solution with the addition of isoproponol. 



The Pellet was washed in cold 75% ETOH and then centrifuged at 12,000 (rpm/ref) for an additional 7 minutes.

The supernatant will be discarded and the pellet will be allowed to dry for five minutes.

The pellet should be re-suspended in 50 µl of DEPC water. If the pellet is having trouble going back into suspension, the solution might need to be gently heated.

After the pellet had gone into suspension, the RNA should be placed on ice.

The concentration of RNA was determined using the Nanodrop and calculations will be conducted in order to ensure that 2µg of RNA will be found at the beginning of each amplification.

RNA may be frozen at this time at -80C degrees.



Reverse Transcription



A reaction for converting RNA into cDNA is as follows:

Determine the volume of RNA solution needed to make 2.0µL. of RNA, add 10.0µL of Master Mix, 3.0µl of oligo (dt) primer, and add enough RNase-free water for 29.0 µL total.

Centrifuge and incubate at 70C degrees for 5 minutes. After 5 minutes, transfer immediately to ice. Cool for 2 minutes and then add 1.0µL Superscript III reverse transcriptase (total volume is 30.0 µL).

Incubate at 42C degrees for 30 minutes.

Centrifuge and incubate at 90C degrees for 5 minutes.

Actin-B or 22Sribosomal subunit will be employed as a positive control.

Thaw Brilliant SYBR Green qftPCR mastermix and take precautions to protect from light.

Use 0.5mL centrifuge tubes.



Quantitative Reverse Transcription Polymerase Chain Reaction

qrtPCR

Set up the following reaction:

12.5µL qrtPCR mastermix

1.8 µL forward primer

2.0 µL reverse primer

5.7µL sterile RNase-free water

1.0 µL cDNA

This should add up to 23.0 µL.

Transfer each reaction to a 96 well plate and program the QPCR machine  (which will need to be programmed with the appropriate data).



Analysis of Results



Determining any potential change in the transcription rate of mRNA for this gene using these variables will help to provide an understanding of the mechanism of action being employed by the lactone that was observed to antagonize cocaine in planarians.  A significant change; either an increase or decrease in transcription may prove illuminating since dopamine receptors’ pharmacokinetics are challenging to predict (Suhara, et al., 2010). 



Genes associated with the CNS are highly conserved across species.  This is beneficial for two reasons; the primers have a good chance of being similar enough for the cDNA to produce similar results in other organisms. More significantly, high conservation of the basic genetic framework lends support for the possibility that understanding the relatively simple planarian system will eventually lead to an enhanced understanding of the more complex mammalian system that is exploited by drugs of addiction (Schwartz, et al., 2011).

Normal controls are being employed to increase confidence in the results. A negative control,No RT cDNA has identified contaminated RNA and those tests have been repeated.  Positive controls namely No Primers and No Template were also incorporated in the procedure to elevate the confidence in this experimental design.



Expected Outcomes/Alternative Approaches

Expected outcomes



The expected outcome of this effort is to quantify the change in the transcription/expression rate of one of the receptors and/or transporters in the dopaminergic pathway that is exploited by cocaine.  The receptors seem to be down regulated and, because cocaine blocks the dopamine transporter (DAT).  The synapse is flooded with excessive dopamine triggering a down regulation of the receptor. The logical expectation is to observe a lower transcription rate for the dopamine receptor in planarians exposed to cocaine and a higher transcription rate for planarians exposed to cocaine and γ-Nonalactone or the cocaine-Parthenolide combination compared with cocaine alone.

Actual results after the first round of qrtPCR: primers for the dopamine receptor did not arrive and the original primer sequence was not available. The β-Actin used as a positive control displayed multiple dissociations that suggest a lack of specificity for that particular locus. 

New primer sequences for the β-Actin and the dopamine receptor were found.

Β-Actin primers have been graciously provided by Dr. Guangwen Chen.

Forward: ACACCGTACCAATCTATG

Reverse: GAGAAACTGTAACCTCGT



Dopamine Receptor primers have been provided by Dr.H. Agata.

Forward: CGAATTGGCGATCGACTTAAATCTGCAC

Reverse: TCCTATAATCGGGATTTTGTGAGCTTTCCA

Dopamine 1Receptor primers (provided by Dr. Agata.)

CGAATTGGCGATCGACTTAAATCTGCAC

TCCTATAATCGGGATTTTGTGAGCTT

























Table 1 Primer Sequences to be Used in qrtPCR

Gene
Source
Forward Primer
Reverse Primer
Just Exon?
β-Actin
Dr. Guangqwn Chen
ACACCGTACCAATCTATG
GAGAAACTGTAACCTCGT
NO
Dopamine Receptor
Dr. Agata
TCCTATAATCGGATTTTGTGAGCTTTCCA
YES
 Dopamine Receptor




 Dopamine Transporter  DAT
 Jayanthi
 5′-TAACCGCATTCTATGTGGATTTC-3′, exon 2) 
 5′-GTTGCACAATTGATGAATGATGTG-3′, exon 7)
 I think
yes
 Alternative DAT

 (RB452: 5′-CAAATCTTCAGACGATCCCGACGAA-3′)
 (RB453: 5′-CTAGGATAATGAAAGTGGAAGACAC-3′)
 I think yes










Results

The results of this observation have been hindered because the genome of D. tigrina seems unstable and the S. mediterranea that the lab has received have not been viable and have certainly not been able to propagate colonies sufficient to supply enough tissue to isolate RNA.  A survey of peer-reviewed literature reveals very little information about the normal pattern of a gel of rRNA for D. tigrina and the gels I have been able to find were not configured in the normal large and smaller band way that is the expected pattern for eukaryotes. 



Discussion

Discussion will vary depending upon future direction.  As of this writing, a colony of S. mediterranea has not been secured sufficiently to isolate enough RNA to carry on a qrtPCR as detailed in Part Two.



Alternative Approaches



Eventually, RNAi techniques will be applied in order to determine the role of various components of the planarian dopaminergic system play in cocaine toxicity and its alleviation when alkyl lactones were tested.

The immediate issue that has prevented the genetic analysis to be conducted efficiently is the questionable stability of the D. tigrigna’s genome.  The double bands of 22s  ribosomal  gel ladder was not evident, even after the procedure was observed by Dr. Gestl in order to detect human error.  Two separate donations of S. mediterranea were not able to survive under the care of members of the Pagan Lab.  Eventually, these problems will be resolved and S. mediterranea will replace D. tigrina. S. mediterranea has a stable genome which opens up the world of microarrays, Blotting, and RNAi.

Another assay that has potential is a spectral analysis to determine the molecular structures that may provide information that will contribute to the general understanding of what is going on at the molecular level at the receptor site.



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