New perspectives in neuroprotection and anti-inflammatory therapy of this Parkinson's neurodegenerative disease

 

ABSTRACT

Cannabinoid type-1 receptors (CB1Rs) modulate synaptic neurotransmission by participating in retrograde signaling in the adult brain. Increasing evidence suggests that cannabinoids through CB1Rs play an important role in the regulation of motor activities in the striatum. In the present study, we used human brain samples to examine the relationship between CB1R and dopamine receptor density in case of Parkinson’s disease (PD).

Post mortem putamen, nucleus caudatus and medial frontal gyrus samples obtained from PD patients were used for CB1R and dopamine D2/D3 receptor autoradiography. [125I]SD7015, a novel selective CB1R inverse agonist, developed by a number of the present co-authors, and [3H]raclopride, a dopamine D2/D3antagonist, were used as radioligands. Our results demonstrate unchanged CB1R density in the putamen and nucleus caudatus of deceased PD patients, treated with levodopa (l-DOPA). At the same time dopamine D2/D3 receptors displayed significantly decreased density levels in case of PD putamen (control: 47.97 ± 10.00 fmol/g, PD: 3.73 ± 0.07 fmol/g (mean ± SEM), p < 0.05) and nucleus caudatus (control: 30.26 ± 2.48 fmol/g, PD: 12.84 ± 5.49 fmol/g, p < 0.0005) samples. In contrast to the putamen and the nucleus caudatus, in the medial frontal gyrus neither receptor densities were affected.

Our data suggest the presence of an unaltered CB1R population even in late stages of levodopa treated PD. This further supports the presence of an intact CB1R population which, in line with the conclusion of earlier publications, may be utilized as a pharmacological target in the treatment of PD. Furthermore we found discrepancy between a maintained CB1R population and a decreased dopamine D2/D3 receptor population in PD striatum. The precise explanation of this conundrum requires further studies with simultaneous examination of the central cannabinoid and dopaminergic systems in PD using higher sample size.

Keywords: Parkinson’s disease, Endocannabinoid CB1 receptor, Dopamine D2/D3 receptor, Molecular imaging biomarker, Human brain autoradiography, Striatum

1. Introduction

The endocannabinoid (EC) system is commonly described as a neuromodulatory system that interacts with and regulates the functions of many neurotransmitter systems, including cholinergic (Ach), dopaminergic (DA), serotoninergic, adrenergic, opiate, glutamatergic and GABAergic systems [,,]. The main contribution of ECs to the control of synaptic neurotransmission is to act as retrograde messengers through type 1 cannabinoid receptors (CB1R) [,]. Presynaptic CB1Rs are abundant in the adult mammalian brain []. CB1Rs are coupled to Gi/o proteins and, under specific conditions to Gs proteins [,,]. CB1Rs regulate the activity of various plasma membrane proteins and signal transduction pathways, including ion channels, context-dependent recruitment of second messengers (Erks, STATs, etc.) and various kinases. In addition, CB1Rs activate G protein-independent pathways, as well [,,]. Among various other functions, endocannabinoids have neuromodulatory functions, as well, [] and play an important role in long term potentiation (e.g. []).

Multiple levels of evidence suggest that ECs have a potential to protect neurons under chronic degenerative conditions via CB1R-dependent and -independent mechanisms [,,,,]. An increasing number of studies have demonstrated that CB1R density and binding is altered in the extrapyramidal system of humans in e.g., Huntington disease and PD [,,,,,,,].

However, the observed alterations in CB1R’s in various neurodegenerative diseases, such as HD or PD, may be of diverse origins. The GABAergic spiny neurons (MSNs) are the most populous neuronal cell type of the striatum (90–95% in rats and over 85% in humans), along with several small populations of interneurons [,]. CB1Rs are primarily expressed my MSNs. The HD brain is characterized by loss of the MSNs of the striatum, which results in robust down-regulation of CB1Rs. A severe loss of CB1R’s in the striatum has, consequently, been described as a landmark of HD [,]. On the other hand, the alteration of striatal CB1R population in PD is full of controversies and the most important striatal cells expressing the CB1R are affected in a lesser extent compared to HD and the EC systems shows a strong tendency for reorganization [,].

It is well known that a progressive degeneration of the dopaminergic system, especially the dopaminergic neurons of the substantia nigra pars compacta (SNc) [,], underlies the pathogenesis and clinical manifestations of PD. The decrease in striatal dopamine (DA) alters the regulation of synaptic dopamine levels, and dopamine receptor density and functional state [,,,,,,,,,]. Alterations in basal ganglia CB1R density or EC levels have been described in rat models of PD on the basis of which a strong functional connection between the striatal dopamine and endocannabinoid systems has been hypothesized [,]. However, experimental models in small animals are inconclusive regarding the direction of changes of CB1R density in Parkinson models: whereas there is evidence for the decrease of CB1Rs in the striatum, as a consequence of 6-hydroxydopamine-induced nigrostriatal terminal lesion in rats []; studies using postmortem human PD brain samples, 6-hydroxydopamine (6-OHDA) or reserpine-treated rat models of PD, MPTP-lesioned marmoset and mouse mutant models of PD indicate an up-regulation [,,,,,], no change [,,,] or down-regulation [,] of CB1Rs in Parkinson’s disease.

In order to investigate changes in CB1R: D2/D3 balance in PD in the human basal ganglia, we explore correlative alterations in dopamine D2/D3 receptors, key players in the disease process [,,], and the alteration in CB1R using selective radioligands – [3H]raclopride [,], [125I]SD7015 [] – in brain tissues obtained from PD and age-matched control subjects.

Discussion

We investigated the relationship of CB1R and D2/D3 receptor densities in PD human brains by means of receptor autoradiography. [125I]SD7015, a novel CB1R agonist, [] and [3H]raclopride, a dopamine D2/D3 receptor antagonist [], were applied as radioligands.

CB1R densities in putamen, nucleus caudatus and frontal cortex samples seem to be unchanged in PD while in contrast, dopamine D2/D3 receptor density in PD putamen and nucleus caudatus decreases. The latter is in line with previous findings, namely this decrease of D2/D3 receptor density in PD putamen and nucleus caudatus could be the consequence of longterm antiparkinsonian treatment. It is generally agreed that dopaminergic denervation leads to striatal D2 dopamine receptor up-regulation as postsynaptic compensatory mechanism in response to deficiencies in synaptic dopamine signaling [,,,,]. Treatment of PD patients with dopaminergic drugs returns the striatal dopamine D2 receptor expression to near normal levels [,,]. Frontal cortex samples presented no difference between the subject cohorts.

The CB system in experimental PD models and PD patients has been extensively studied, yet with contradictory conclusions. In rat Parkinson models (reserpine treatment or 6-OHDA-lesion models) an increase in endogenous endocannabinoid levels was observed in the striatum [,,,] as it was also observed in the CNS of 16 untreated PD patients []. Other authors found significantly altered CB1R mRNA expression in animal models of PD or in postmortem human PD brain specimens. [,,,]. Finally, changes in CB1R binding sites [,,] and activation of GTP-binding proteins in the basal ganglia of PD patients and of MPTP-treated marmosets were also reported []. On the other hand, using the 6-OHDA rat model Romero et al. [] did not find significant changes in CB1 receptor binding, measured by [3H]WIN-55,212,2 autoradiography, or in the activation of signal transduction mechanisms, measured by WIN-55,212,2-stimulated [35S]GTPgammaS binding autoradiography, between the lesioned and non-lesioned sides at the level of the lateral caudate-putamen, globus pallidus and substantia nigra.

In the present study we did not find changes in CB1R densities in the striatum and frontal cortex of PD subjects. One of the explanations for the unaltered CB1R density found by us could be the unchanged density of high affinity CB1Rs in the investigated PD brain regions [,]. This may be possible due to the presence of the large reserve of CB1Rs and their likely inter-conversion between low and high affinity states [,]. Due to low sample size results are only suggestive in the aspect of an intact CB1R density. This statement requires further justification in the future by studies using more specimens and performing the detailed investigation of this problem.

On the other hand, functional relationship have been reported between CB1Rs and both dopamine D1 and D2 receptors [,,,,,,,,, ]. For instance, Giuffrida et al. [] proved that striatal administration of D2 agonist results in release of endocannabinoids. Furthermore, activation of CB1 or dopamine D2 receptors alone resulted in inhibition of cAMP accumulation whereas simultaneous activation of both receptors increased cAMP levels []. Kreitzer and Malenka [] reported in animal models of Parkinson’s disease that DA depletion blocked the generation of endocannabinoid-mediated long-term depression (eCB-LTD) in indirect striatal pathway but administration of dopamine D2 receptor agonist together with inhibitors of endocannabinoid degradation rescued indirect-pathway eCB-LTDs and in vivo reduced parkinsonian motor deficits. Dopamine receptor antagonists inhibited cannabinoid induced striatal mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activation [].

Administration of CB1R agonists increased dopamine turnover and release [,,,], excited dopaminergic neurons in the ventral tegmental area and substantia nigra [], decreased the tremor associated with overactivity of the subthalamic nucleus and improved motor impairment [,,], at the same time a CB1 antagonist (rimonabant) alleviated hypokinesia in animal models of Parkinson’s disease [,], probably effecting on the lateral globus pallidus. It has been demonstrated that glutamatergic and GABAergic terminals strongly express CB1Rs and CB1R agonists significantly inhibited glutamate and GABA release, however, cannabinoids in vitro, directly did not affect the release of dopamine [,,]. Yin et al. [] observed that presynaptic reduction in glutamate release was the consequence of a retrograde signal through eCBs; this eCB synthesis and release from the postsynaptic cell results from cooperating, convergent glutamate and dopamine inputs. A potential indirect dopamine–CB1R interaction through the cannabinoid induced regulation of the upper mentioned neurotransmitters (GABA, glutamate) of striatal neuronal pathways could be the basis of cannabinoid effect on motor activity. However, the findings of [] that dopaminergic cells also express CB1R as well as observations about functional interactions between CB1Rs and both dopamine D1 and D2 receptors [,,,,,,,,,]. could contribute to the upper mentioned effect as well. Due to the complexity of this cannabinoid–dopamine receptor conundrum further researches are required with well designed study protocols. Although our results base on relatively small sample size, they refer to the presence of an apparently intact CB1 receptor population may be usable in PD therapy, even in advanced PD.

On the other hand, it is accepted that classical neuroinflammatory diseases such as multiple sclerosis present aspects of neurodegeneration, while classical degenerative disorders such as Alzheimer’s disease, Parkinson’s disease are demonstrably affected by inflammation []. In CNS CB1 receptors exist in all types of neural cells, in astrocytes [,], microglia [,], and oligodendrocytes [] whereas CB2receptors are expressed on cells of immune system and microglia []. Studies report neuroprotective [,,] and anti-inflammatory effects through CB receptors [,,,,,]; CBs protected against dopaminergic cell death, as well []. Thus CB1 and CB2 receptors could provide substrate for neuroprotective and anti-inflammatory actions of cannabinoids in neurodegenerative diseases, however, the effects through CB1Rs are more relevant to neuroprotection, whereas CB2Rs modulate the immune response primarily, although a potential overlap as well as non CB1/CB2-mediated mechanisms may exist [].

In this study we used PD putamen, nucleus caudatus, medial frontal gyrus samples in order to correlate CB1 receptor density with dopamine D2/D3 receptor density. Our results refer to an unchanged CB1R and decreased dopamine D2/D3 receptor density in nucleus caudatus and putamen of PD patients whereas medial frontal gyrus sections did not show any alteration. Our data suggest that in case of long-term l-DOPA treatment and long disease progression CB1R density does not fall under control levels, although, dopamine D2/D3 receptor density is significantly decreased. Various explanations could exist: (1) neurodegeneration induced affinity or sensitivity increase of ‘reserve’ CB1Rs could compensate CB1R density changes, (2) reactive changes of CB system could go along with PD progression, until more effective compensatory mechanisms come into action, (3) despite the decreased density, dopamine D2receptor signal transduction seems functionally intact []) and possible physiological interactions with CB1 receptors could be maintained even in later stages of PD, which could result in unchanged CB1R density; however, functional receptor crosstalks between these receptor types are, yet, unequivocally unproven. Nevertheless the combination up to various degree of the aforementioned or other, yet, unknown mechanisms is the most probable phenomenon.

We concluded that an intact CB1R population could represent alternative target for treatment of PD; additionally it could open new perspectives in neuroprotection and anti-inflammatory therapy of this neurodegenerative disease. Better understanding and further exploration of central cannabinoid system and related questions need further in vivo and in vitro detailed designed studies, emphasizing study population/sample group homogeneity regarding to data about PD neuropathological stage, disease duration and l-DOPA substitution/dopamine agonist therapy. This is suggested since one of the limitation of this study is the low sample number and consequently the questionable reliability of statistical calculations on these data. By the parallel investigation of CB1 and DA D2/3 receptors in PD striatum we wished to base and pioneer future researches aiming this field of neuroscience.