| Abstract |
Retinal ischemia leads to neovascularization and excitotoxicity (cell death) both of
which can lead to the development of very severe retinopathies, such as diabetic retinopathy
and age related macular degeneration (Lipton and Rosenberg, 1994; Campochiaro, 2000;
Osborne et al., 2004; Grant et al., 2005), that often result in poor visual acuity and blindness.
Surgical and new pharmacological approaches are available to target the neovascular
component of ischemia induced retinopathies but not the neurodegenerative component. In
order to preserve vision both components must be treated. Therefore, it is mandatory to
establish new pharmacological targets for the treatment of the neurodegenerative component.
The main aim of the present study was to investigate whether the endocannabinoid
system may serve as an efficacious therapeutic target for the treatment of neurodegenerative
retinal disease. Specifically, the neuroprotective actions of the endocannabinoids AEA (Narachidonoylethanolamine,
anandamide) and 2-AG (2-arachidonoylglycerol) and synthetic
cannabinoids MethAEA (Methanandamide) and HU-210 were studied, as well as the
neuroprotective actions of the inhibitors of the enzymes responsible for the metabolism of the
endocannabinoids. In addition, the involvement of the cannabinoid CB1 and CB2 receptors and
the TRPV1 vallinoid receptor in the neuroprotective actions of endo- and synthetic
cannabinoids, as well as the downstream signaling pathways leading to their putative
neuroprotective effects were also investigated.
We employed the in vivo AMPA model of retinal excitotoxicity, previously developed
in our laboratory (Kiagiadaki and Thermos, 2008) and we investigated the neuroprotective
actions of the synthetic HU-210 and MethAEA and the endogenous cannabinoids AEA and 2-
AG. Cannabinoids were intravitreally injected with AMPA (42nmoles/eye) and 24 hours post
injection, the animals were euthanized and their retinas were prepared for
immunohistochemical studies or western blot analysis.
AMPA caused a reduction of bNOS (brain nitric oxide synthase) and ChAT (choline
acetyltransferase) immunoreactive neurons compared to the control tissues and the
cannabinoids afforded neuroprotection against AMPA excitotoxicity, increasing the number of
bNOS and ChAT immunoreactive cells compared to the AMPA treated tissues. The toxic
effects of AMPA and the neuroprotective actions of cannabinoids were also substantiated by
the TUNEL assay. Additional studies revealed that the cell death observed in the presence of
AMPA does not involve activation of the apoptosis mediator caspase-3.
To investigate the involvement of CB1 and CB2 receptors in the neuroprotection, we
performed a series of studies, namely the intravitreal co-injection of cannabinoid receptor
antagonists with AMPA and cannabinoids, radioligand binding studies, study of the
neuroprotective actions of the CB2 selective agonist JWH015, RT-PCR and studies in CB1-/-
andCB2-/- mice, which revealed that the CB1 but not the CB2 receptor is involved in the
neuroprotective actions of cannabinoids. We also performed pharmacological studies to assess
the involvement of the TRPV1 vallinoid receptor in the cannabinoids’ neuroprotective actions.
The data obtained from these studies revealed that the TRPV1 receptor, is not involved in the
neuroprotective actions of cannabinoids in the vivo retinal model of AMPA excitotoxicity.
Having assessed the neuroprotective properties of the cannabinoids in the AMPA model
of excitotoxicity in the retina and the involvement of the CB1 cannabinoid receptor in their
actions, we subsequently investigated the signaling pathways that lead to the neuroprotection.
We performed western blot analysis against phosphorylated and total forms of the PI3K/Akt
and MEK/ERK1/2 kinases, functional studies using the PI3K/Akt inhibitor wortmannin and
neuroprotection studies in Akt2-/- mice. The data obtained suggested the involvement of
PI3K/Akt and/or MEK/ERK1/2 signaling pathways in the neuroprotection afforded by the
cannabinoids. Specifically, the PI3K/Akt signaling pathway seems to be involved in the
Abstract 124
neuroprotective actions of ΑΕΑ, 2-AG and HU-210, while the MEK/ERK1/2 pathway is
involved in the neuroprotective actions of ΑΕΑ and 2-AG, but not those of HU-210.
Besides the direct activation of cannabinoid receptors with specific agonists,
cannabinergic signaling can also be modulated through inhibition of endocannabinoid
metabolism. Therefore, we examined the putative neuroprotective actions of the inhibitors of
the two metabolic enzymes of AEA and 2-AG (Fatty Acid Amide Hydrolase, FAAH and
Monoacylglycerol lipase, MGL for AEA and 2-AG, respectively), to substantiate whether these
inhibitors could provide a new therapeutic target for the treatment of retinal disease. We
performed experiments employing a FAAH inhibitor (AM6642), which inhibits the degradation
of AEA and a dual FAAH/MGL inhibitor (AM9928), which inhibits the degradation of both
AEA and 2-AG. These compounds afforded neuroprotection when co-injected with AMPA,
with AM9928 affording greater neuroprotection than AM6642.The neuroprotective actions of
the two inhibitors were reversed in the presence of the CB1 receptor antagonist AM251.
Subsequently, we co-injected the above mentioned enzyme inhibitors with AMPA plus
endogenous and synthetic cannabinoids to examine if the neuroprotective effects could be
increased due to the elevation of endocannabinoid levels in the synapse and the exogenously
added agents. Both the FAAH and the FAAH/MGL inhibitors failed to afford neuroprotection
in the presence of exogenously applied cannabinoids. These results suggest that blockade of
endocannabinoids’ degradation protects the retina from the AMPA insults. However, the coadministration
of the enzyme inhibitors and exogenously administered endocannabinoids did
not have an additive effect, probably due to desensitization and downregulation of CB1 receptor
(Schlosburget al., 2010). This tenet must be investigated further. MethAEA, a metabolically
stable analogue of AEA that is metabolized by pathways independent of FAAH, did not alter
the neuroprotective action of AM6642 (Abadji et al., 1994). The second synthetic cannabinoid
employed (HU-210) reversed the neuroprotective actions of both AM6642 and AM9928. Since
there are no reports in the literature regarding the metabolic pathways of HU-210, one cannot
make any hypotheses regarding HU-210’s actions.
The data obtained from the present study suggest that endogenous and synthetic
cannabinoids protect the retina from AMPA excitotoxicity, via activation of the CB1, but not
CB2 or TRPV1 receptors, with the involvement of the PI3K/Akt and/or MEK/ERK1/2
signaling pathways. Inhibition of the enzymes that metabolize the endocannabinoids also
afforded neuroprotection, yet the co-administration of exogenous endocannabinoids leads to its
attenuation.
In conclusion, the present study revealed an important role for the endocannabinoid
system in the physiology and pathophysiology of the retina. This system could provide an
important therapeutic target for the development of more efficacious therapeutics for the
treatment of retinopathies whose pathophysiology involves excitotoxic insults.
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