Abstract |
ntroduction: The endocannabinoid system is considered as an important therapeutic
target for new treatments of neurodegenerative diseases. The retina, as part of the
CNS, comprises a full and functional endocannabinoid system that consists of the
cannabinoid receptors (CB1 and CB2 receptors), endogenous agonists
(endocannabinoids) and their metabolic enzymes. The endocannabinoids , anandamide
(AEA) and 2-arachidonoyl-glycerol (2-AG), as well as synthetic cannabinoids, like R-
(+)-Methanandamide (MethAEA), have been reported to provide neuroprotection to
retinal neurons in different acute animal models of retinopathies. However, despite
the neuroprotective actions of cannabinoids after acute administration, their
therapeutic potential is limited due to the development of tolerance and dependence
by repeated treatment that correlates with the downregulation of the CB1 receptor.
Aims: Most of the studies suggesting agonist-induced desensitization and
downregulation of CB1R have been performed in brain regions. In the present study,
we focused on the investigation of the effect of endogenous and synthetic
cannabinoids, and the inhibitors of the metabolic enzymes of ΑΕΑ (FAAH;
ΑΜ5206), and 2-AG (MAGL/ABHD6; ΑΜ11920) on the expression of CB1
receptors in normal rat retina, after subchronic or chronic administration, as well as
their potential neuroprotective properties in the experimental in vivo model of AMPA
excitotoxicity. In addition, we examined the involvement of autophagy in the
mechanisms involved in the downregulation of the CB1 receptor.
Methods: We investigated the subchronic and chronic effects of cannabinoids in
normal retina. Sprague-Dawley rats were administered intraperitoneally (i.p) with
AEA, MetAEA, 2-AG or AM5206 and AM11920 daily for 4 or 14 days (subchronic
or chronic administration, respectively). In order to assess the subchronic
neuroprotective properties of these agents, the in vivo AMPA excitotoxicity model
was employed, AMPA (8.4mM) was administered intravitreally and 24 hours later,
rats were administered the above cannabinoids agents i.p daily for 4 days. We also
employed the AMPA model of excitotoxicity in order to assess whether the
administration of 2-AG by a less invasive route, via topical administration as eye
drops (1%, twice/day for 2 days or once/day for 4 or 8 days) will protect the retina.
Ph.D Thesis
Sofia Papadogkonaki
8
The effect of the indirect elevation of endocannabinoid levels, by using inhibitors of
the enzymes that metabolize AEA and 2-AG and subsequently lead to the elevation of
their endogenous levels was also investigated. The FAAH inhibitor AM5206 or the
dual MAGL/ABHD6 inhibitor (metabolic enzymes of AEA and 2-AG, respectively)
were administered i.p for 4 days in control rats, to assess their subchronic effect on
CB1R expression in normal rat retina, or following AMPA administration in order to
study their neuroprotective effects.
Immunohistochemical studies were performed employing antibodies against to CB1R
and markers of retinal neurons to assess CB1R expression and retinal cell
neuroprotection [nitric oxide synthetase, bNOS, marker of amacrine cells), calbindin
(marker of amacrine and horizontal cells), caspase-3 (marker of apoptotic death) or
Iba-1 (marker of microglia) to study the activation of microglia and the potential anti-
inflammatory actions of the endocannabinoid 2-AG. Moreover, western blot analysis
was performed to assess the protein levels of CB1R, and t he phosphorylation of its
downstream signalling proteins, Akt or ERK1/2 kinases.
The mechanism of CB1R’s downregulation was investigated in vivo using
immunoprecipitation (IP) methodology in order to assess the potential interaction of
the CB1R protein with the trafficking marker β-arrestin 2, or endosomal markers
(Rab5, Rab7). In addition, we employed an ex vivo model, where retinas were
incubated with 2-AG in combination with or without the autophagic inhibitor
Bafilomycin A1 (BafA1), in order to examine the involvement of autophagy in the
mechanism of CB1R downregulation.
Results: This study showed that the cannabinoids AEA, MethAEA and 2-AG reduce
CB1R expression in a dose dependent manner, after subchronic or chronic
administration. The reduction of the CB1R’s expression by of MethAEA affected
its downstream signaling (Akt and ERK1/2 phosphorylation). Subchronic treatment
of AEA or 2-AG did not reduce Akt or ERK1/2 phosphorylation. Moreover,
subchronic i.p administration of AEA, MethAEA or 2-AG induced downregulation of
the CB1R in the model of AMPA excitotoxicity and failed to provide protection to
bNOS expressing amacrine cells. However, 2-AG managed to attenuate AMPA-
induced microglial activation, as it reduced the number of reactive microglial cells.
We also assessed the effect of 2-AG, administered topically as eye drops for 2, 4 or 8
days. Our results showed that 2-AG (1%) did not affect CB1R’s expression at any time
Ph.D Thesis
Sofia Papadogkonaki
9
point and protected bNOS expressing amacrine cells after 2 or 4 days, but not after 8 days of
treatment, but reduced the number of caspase-3 positive cells. Also 2-AG (1%) attenuated the
AMPA induced increase in the number of reactive microglia after subchronic (4 days)
administration.
The elevation of the endogenous levels of AEA or 2-AG via the administration of FAAH or
MAGL/ABHD6 inhibitor, respectively, does not induce downregulation of the CB1R at any
of the doses studied (subchronically) in normal rat retina. In the model of AMPA
excitotoxicity, AM5206 and AM11920 provided neuroprotection to bNOS expressing
amacrine cells, after subchronic treatment (i.p) without affecting the expression of CB1R.
Regarding the mechanism of CB1R downregulation in vivo, our findings suggest sthat the
CB1R does not interact with β-arrestin 2 or Rab5 and Rab7 endosomal markers (early and late
endosomes, respectively), however it is possible that the potential interaction could not be
detected due to technical issues related with the use of tissue for IP studies. The data of our ex
vivo studies showed that 2-AG induced downregulation of the CB1R. However, this effect
was reversed when retinas were treated with 2-AG in combination with BafA1, suggesting
that autophagy may be involved in the mechanism of CB1R downregulation.
Conclusions: This study provides important information regarding agonist-induced
CB1R downregulation in rat retina. Endogenous and synthetic cannabinoids may
induce downregulation of the CB1R after subchronic or chronic i.p administration in
normal rat retina, as well as in the experimental model of AMPA excitotoxicity,
where they failed to protect amacrine cells. However, the topical eye drop
administration of the endocannabinoid 2-AG provides neuroprotection to retina up to
8 days of treatment and does not affect the expression of CB1R. The inhibition of the
metabolic enzymes of AEA and 2-AG, by FAAH and MAGL/ABHD6 inhibitors,
respectively, led to the elevation of the endogenous levels of these cannabinoids and
did not alter the levels of CB1R expression in normal retina or in the AMPA
excitotoxicity model. Moreover, the subchronic i.p administration of these inhibitors
was shown to exert neuroprotective actions to the retina, suggesting that the
pharmacological inhibition of the endocannabinoids’ metabolism may be a more
effective therapeutic approach compared to the exogenous systemic cannabinoid
administration. The studies regarding the potential interaction of CB1R with
trafficking markers did not provide satisfying results, however our ex vivo studies
showed that inhibition of autophagy prevents the reduction of CB1R expression,
implying that autophagy may play a role in the mechanism of downregulation of the
receptor.
Ph.D Thesis
Sofia Papadogkonaki
10
In closing, this study provides novel information regarding the effect of
repeated administration of endogenous and synthetic cannabinoids or inhibitors of the
endocannabinoid metabolism in the expression of the CB1R in rat retina. Our results
suggest that inhibitors of the metabolic enzymes of endocannabinoids may be
effective in chronic treatment for retinal disease. Furthermore , we report new
information regarding the trafficking of CB1R that may contribute to the elucidation
of the still unknown mechanism via which the CB1R is downregulated in retina. The
results obtained from this study will serve to evaluate the chronic use of cannabinoids
as neuroprotectants in retinal disease
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