Transcript Gaurav Anand - UMKC School of Medicine

Pharmacological Control Of Oxidative Stress-mediated Effects On
Endocannabinoid Signaling Pathways
Gaurav Anand, Christa Montgomery, Ahsan Hussain, Heather Johnson, Peter Koulen
Vision Research Center, Department of Ophthalmology, School of Medicine, University of Missouri-Kansas City, MO
INTRODUCTION
Endocannabinoid signaling is mediated by a group of
receptor proteins that bind endogenous lipid mediators
and exogenous compounds, producing changes in
cellular activity throughout the body1. Although
cannabinoids such as Δ9-tetrahydrocannabinol are
known for their psychoactive effects, they also have
potential therapeutic benefits for treating diseases such
as multiple sclerosis, glaucoma, neurodegenerative
disorders, and anorexia2. Cannabinoids produced
endogenously are upregulated in response to ischemia
and physical trauma, which suggests a neuroprotective
role for these lipid mediators. One endocannabinoid,
palmitoylethanolamide (NAE 16:0), directly protects
neurons against oxidative stress, but may also do so
through competitive inhibition of the enzyme fatty acid
amide hydrolase (FAAH1), resulting in attenuated
hydrolysis and therefore longer availability of
endocannabinoids3. The goal of this study was to
determine effects of NAE 16:0 on expression levels and
subcellular localization of proteins involved in
endocannabinoid signaling.
METHODS
Rat cortical neurons were pretreated with NAE 16:0 and
exposed to the oxidant tert-butyl hydroperoxide to
model neuronal injury secondary to oxidative stress.
Cell viability assays using the fluorescent indicator dye
calcein-AM were conducted to assess the extent of
neuroprotection. Using the immortalized mouse
hippocampal cell line HT-22, immunoblotting and
immunocytochemistry
assays
were
performed
subsequent to NAE 16:0 treatment to analyze changes
in the expression levels and subcellular distribution of
receptors and enzymes involved in endocannabinoid
signaling. Cannabinoid receptor types 1 and 2 (CB1 and
CB2), FAAH1, N-acylethanolamine-hydrolyzing acid
amidase (NAAA), and N-acylphosphatidylethanolaminespecific phospholipase D (NAPE-PLD) were examined.
SUMMARY
RESULTS
Palmitoylethanolamide Protects Neurons Against Oxidative Stress
AA
Figure 1: Chemical structures of select endocannabinoids. Endocannabinoids
are synthesized “on-demand” in response to cellular stress or injury by Nacyl-phosphatidylethanolamine-selective phosphodiesterase (NAPE-PLD)
from phospholipid precursors located in the cell membrane. Following their
release, endocannabinoids can bind to plasma membrane receptors,
cannabinoid receptor 1 (CB1; more abundant in the central nervous system)
and cannabinoid receptor 2 (CB2; more abundant in the immune system).
Endocannabinoids are enzymatically inactivated by fatty acid amide
hydrolase 1 (FAAH1) and N-acylethanolamine-hydrolyzing acid amidase
(NAAA). In the brain, 2-arachidonyl glycerol (top) is more abundant than
arachidonoyl ethanolamide (anandamide; middle). Their congener, palmitoyl
ethanolamide (NAE 16:0; bottom) is also more abundant than anandamide
but does not appreciably interact with CB1 or CB2 receptors. Like other
endocannabinoids, NAE 16:0 potentiates the effect of anandamide by
competing with anandamide for hydrolysis at FAAH1.
BB
CC
Figure 2: Calcein cell viability assay of cortical neurons treated with NAE 16:0 and exposed to oxidative stress. Primary rat cortical neurons were pre-treated for 1-2 hours with media, vehicle, or increasing concentrations of NAE 16:0. Cells were either
exposed to oxidative stress initiated by the addition of 7.5 µM tBHP (insult) or treated with an equal volume of sterile water (mock). After 16-18 hours, fluorimetric calcein-AM viability assays were conducted to measure neuronal viability.4 (A) For each
experimental condition, calcein fluorescence of six replicates was averaged, and three separate experiments were performed using different neuronal cultures. Data was normalized to the vehicle, and statistical significance was assessed using Student’s ttest. (B) One-way ANOVA comparing the viability of cells under mock conditions indicates that pre-treatment with NAE 16:0 does not significantly enhance cell viability when compared to vehicle alone. (C) ANOVA analysis of cells exposed to tBHP indicates
that 100 µM and 10 µM concentrations of NAE 16:0 provide statistically significant neuroprotection when compared to the vehicle alone. Bars represent mean ± SEM. A P-value of < 0.05, < 0.01, and < 0.001 is indicated by *, **, and ***, respectively, as
determined by Dunnett’s post-test.
FAAH Expression Levels are Not Affected by Palmitoylethanolamide
B
A
Cortical neurons treated with NAE 16:0 exhibited an
increase in viability compared to untreated cells.
Treatment of HT-22 cells with NAE 16:0 had no effect on
expression levels of FAAH1. However, NAE 16:0 treatment
increased co-localization of FAAH1 with calnexin, an
endoplasmic reticulum marker protein, indicating
increased localization of FAAH1 to the endoplasmic
reticulum.
CONCLUSION
The endocannabinoid NAE 16:0 exhibited neuroprotective
effects and induced translocation of FAAH1 to the
endoplasmic reticulum. The latter indicates indirect effects
of NAE 16:0 on endocannabinoid signaling that are not
mediated by classical endocannabinoid receptors. Further
research is required to determine the effects of NAE 16:0
on expression levels and localization of CB1, CB2, NAAA,
and NAPE-PLD.
FUTURE DIRECTIONS
Figure 3: Specificity of antibodies targeting endocannabinoid system proteins. Proteins from differentiated HT-22 cells
were detergent solubilized. The protein concentration of the post-nuclear supernatant was determined by BCA assay
using bovine serum albumin as a standard. Proteins were denatured in SDS loading buffer and 10 µg loaded into each
well of a 10% polyacrylamide gel. PageRuler Plus (Thermo Scientific) pre-stained protein ladder was used for molecular
weight estimation. Following protein transfer, nitrocellulose membranes were blocked in 5% non-fat milk. Blots were
incubated in the indicated primary antibody for 48 hours at 4oC. After washing, the membranes were incubated with
horseradish peroxidase-labeled secondary antibodies for 1 hour. Finally, the membranes were washed and incubated
with SuperSignal West Femto (Thermo Scientific) enhanced chemiluminescent substrate and imaged. Left: image of
Western blot. Right: molecular weights of the most prominent band in each lane determined by relative mobility
analysis. The FAAH antibody recognized a single protein of the appropriate molecular weight. Immunoblotting blotting
conditions for the CB1, CB2, NAAA, and NAPE-PLD antibodies need to be further optimized prior to analysis of
expression levels.
Figure 4: Western blot analysis of FAAH1 expression levels after exposure to NAE 16:0. HT-22 cells were grown until reaching 65-75% confluency and then differentiated for 24 hours. The cultures were then treated overnight with
100 µM NAE 16:0, vehicle, or neither (control). Cells were rinsed with ice cold PBS, scraped from the flask, and pelleted by centrifugation. Immunoblotting was performed with anti-FAAH1 antibody as described in Figure 3. For each
condition, three separate replicates were performed using different passages of HT-22 cell cultures. (A) Image of a Western blot showing FAAH1 bands just above the 53 kDa marker. Vinculin (126 kDa) was used as the loading
control. (B) The size and intensity of FAAH1 and vinculin bands were measured using densitometry5. FAAH1 band intensities were normalized to vinculin. Bars represent mean ± SEM. Statistical analysis using a one-way ANOVA
confirms that there is no significant difference in FAAH1 expression among the control, vehicle, or 100 µM NAE 16:0 treated cells.
Conditions for immunoblotting with CB1, CB2, NAAA, and
NAPE-PLD primary antibodies will need to be optimized in
order to investigate other proteins involved in
endocannabinoid signaling.
This will allow us to
determine the effects of NAE 16:0 on expression levels and
localization of these proteins.
Palmitoylethanolamide Induces Translocation of FAAH to the ER
REFERENCES
1Battista,
Figure 5: Examples of methods to qualitatively assess colocalization of fluorescent labels. Left panels demonstrate
the “overlay” method. This representation is produced by acquiring separate images of each label and digitally merging
the images. Areas where pixels from both channels are in close proximity appear yellow. Right panels are intensity
scatterplots of the data presented in the corresponding left panels. Intensity of pixels in channel one are plotted on the X
axis and pixel intensity of channel two are plotted on the Y axis. Linear regression can be applied to determine Pearson’s
coefficient (a quantitative description colocalization). (A) Complete or 100% colocalization produced by duplication. (B)
Complete colocalization as in A but with disparity in channel intensities. (C) Partial colocalization. (D) Exclusion or 0%
colocalization as when one signal is confined to the nucleus and the other to the cytoplasm.
Figure 6: Immunocytochemical colocalization analysis of FAAH and the endoplasmic reticulum protein
Calnexin. HT-22 cells were seeded onto coverslips, incubated overnight, and then differentiated for 24 hours. The
cultures were then treated overnight with 100 µM NAE 16:0, vehicle, or neither (control). Fixed, permeabilized cells
were incubated with primary antibody for 48 hours at 4oC. After washing, the coverslips were incubated with
fluorescently-labeled secondary antibodies for 1 hour. Confocal images were collected at 40x 1 µm above and below
the brightest plane in 0.15 µm slices. Laser intensities and all other parameters were held constant for all images.
Images were analyzed using FIJI image analysis software.5 Noise reduced images were thresholded using the Costes
automated method. Scatterplots were generated using “Cololoc 2” with bisecting linear regression. Pearson’s and
Mander’s coefficients were determined using the “JACoP” plugin. Data is represented as mean ± SEM.
Figure 7: Statistical analysis of FAAH1 colocalization with the endoplasmic reticulum protein
calnexin. Pearson’s and Mander’s coefficients measured in Figure 6 were plotted and statistical
analysis was performed with GraphPad Prism 5.0. Bars represent mean ± SEM. Pearson’s
coefficient describes the extent of overlap between image pairs. A Pearson’s coefficient > 0.5
indicates significant colocalization. Mander’s coefficient describes the contribution of one color
channel to overall colocalization. A Mander’s coefficient > 0.6 indicates colocalization. One-way
ANOVA indicates that there is a significant increase in FAAH1 and calnexin colocalization in HT-22
cells treated with NAE 16:0 when compared to vehicle treated cells. A P-value of < 0.05, < 0.01, and
< 0.001 is indicated by *, **, and ***, respectively, as determined by Dunnett’s post-test.
Natalia, Monia Di Tommaso, Monica Bari, and Mauro Maccarrone. “The
Endocannabinoid System: An Overview.” Frontiers in Behavioral Neuroscience 6
(2012): 1-7. Web.
2Kogan, Natalya M., and Raphael Mechoulam. “Cannabinoids in Health and
Disease.” Dialogues in Clinical Neuroscience 9.4 (2007): 413–430. Web.
3Duncan, R. Scott et al. “Protection of Neurons in the Retinal Ganglion Cell Layer
against Excitotoxicity by the N-Acylethanolamine, N-Linoleoylethanolamine.”
Clinical Ophthalmology (Auckland, N.Z.) 5 (2011): 543–548. PMC. Web.
4Kaja, Simon et al. “Novel Mechanism of Increased Ca2+ Release Following Oxidative
Stress in Neuronal Cells Involves Type 2 Inositol-1,4,5-Trisphosphate Receptors.”
Neuroscience 175 (2011): 281–291. PMC. Web.
5Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al."Fiji: an open-source platform for
biological-image analysis", Nature methods 9.7 (2012): 676-682. PMC. Web.