Potential of the cannabinoid CB₂ receptor as a pharmacological target against inflammation in Parkinson's disease

Highlights

• CB₂ receptors are elevated in microglial cells in the post-mortem substantia nigra of PD patients.

• CB₂ receptors are also elevated in the striatum and the substantia nigra in LPS-lesioned mice.

• The genetic inactivation of CB₂ receptors aggravates LPS-induced inflammation.

• The pharmacological activation of CB₂ receptors reduces LPS-induced proinflammatory responses.

Abstract

Inflammation is an important pathogenic factor in Parkinson's disease (PD), so that it can contribute to kill dopaminergic neurons of the substantia nigra and to enhance the dopaminergic denervation of the striatum. The cannabinoid type-2 (CB₂) receptor has been investigated as a potential anti-inflammatory and neuroprotective target in different neurodegenerative disorders, but still limited evidence has been collected in PD. Here, we show for the first time that CB₂ receptors are elevated in microglial cells recruited and activated at lesioned sites in the substantia nigra of PD patients compared to control subjects. Parkinsonian inflammation can be reproduced experimentally in rodents by intrastriatal injections of lipopolysaccharide (LPS) which, through an intense activation of glial elements and peripheral infiltration, provokes a rapid deterioration of the striatum that may extend to the substantia nigra too. Using this experimental model, we recently described a much more intense deterioration of tyrosine hydroxylase (TH)-containing nigral neurons in CB₂ receptor-deficient mice compared to wild-type animals, supporting a potential neuroprotective role for this receptor. In the present study, we further explored this issue. First, we found elevated levels of the CB₂ receptor measured by qRT-PCR in the striatum and substantia nigra of LPS-lesioned mice, as well as an increase in the immunostaining for this receptor in the LPS-lesioned striatum. Second, we found a significant increase in CD68 immunostaining, which serve to identify activated microglia and also infiltrated peripheral macrophages, in these brain structures in response to LPS insult, which was much more intense in CB₂ receptor-deficient mice in the case of the substantia nigra. Next, we observed that the activation of CB₂ receptors with a selective agonist (HU-308) reversed LPS-induced elevation of CD68 immunostaining in the striatum and the parallel reduction in TH immunostaining. Lastly, we found that LPS elevated the gene expression of different pro-inflammatory mediators in both the striatum and the substantia nigra, whereas the selective activation of CB₂ receptors reduced a part of these mediators, e.g. inducible nitric oxide synthase, although exclusively in the striatum. In conclusion, we have provided the first evidence on the up-regulation of CB₂ receptors in glial elements in postmortem tissues of PD patients, which has been confirmed in an inflammatory model of this disease. In addition, we have provided evidence on the benefits derived from their activation in relation with the activation of microglial cells, the infiltration of macrophages and also certain capability of these cells to generate proinflammatory factors.

Introduction

Cannabinoids have important anti-inflammatory properties that may preserve neuronal homeostasis and survival in different neuroinflammatory/neurodegenerative disorders (Chiurchiù et al., in press). These effects are preferentially exerted by the activation of the cannabinoid type-2 (CB₂) receptor (Fernández-Ruiz et al., 2007), although recent studies also situate the cannabinoid type-1 (CB₁) receptor (Chung et al., 2011, de Lago et al., 2012) and even the nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) family (Fidaleo et al., 2014) as additional potential targets for the anti-inflammatory action of certain cannabinoids. The anti-inflammatory properties of compounds activating the CB₂ receptors are facilitated by the presence of these receptors in glial cells, mainly astrocytes and microglial cells, and the up-regulatory response that they experience when these cells become reactive in inflammatory, excitotoxic, oxidative or infectious conditions (Fernández-Ruiz et al., 2007, Fernández-Ruiz et al., 2010). Frequently, glial CB₂ receptors may work alone but they can also work in conjunction with CB₁ receptors, for example in the regulation of astrocyte activity in conditions of brain damage (reviewed by Stella, 2010). Irrespective of the cannabinoid receptor type involved, the benefits that cannabinoids may provide appear to be associated with the trophic role exerted by these glial cells, including improvements in the supply of metabolic substrates to neurons (lactate or ketone bodies: Duarte et al., 2012). They could act: (i) by enhancing the generation of neurotrophins or anti-inflammatory/pro-survival mediators that could potentially rescue damaged neurons, e.g., interleukin-10 (IL-10), IL-1 receptor antagonists, transforming growth factor-β (TGF-β) (Molina-Holgado et al., 2003, Smith et al., 2000), and/or (ii) by inhibiting the production of chemokines by astrocytes, e.g., fractalkine (Sheng et al., 2009). In contrast to astrocytes, the effects of cannabinoids on microglial cells are much more dependent on CB₂ receptor function. Indeed, CB₂ receptors appear to play an important role in the proliferation and migration of these cells at lesion sites (Carrier et al., 2004, Walter et al., 2003). In addition, the activation of CB₂ receptors dampens the generation of a variety of neurotoxic factors by microglial cells (Fernández-Ruiz et al., 2007, Fernández-Ruiz et al., 2010), for example tumor necrosis factor-α (TNF-α), a major contributor to the pathophysiology of brain injury (reviewed by Stella, 2010). Activation of CB₂ receptors apparently inhibits the production of TNF-α by inhibiting NFκB (Oh et al., 2010), a transcription factor that plays a key role in the regulation of pro-inflammatory responses.

Therefore, the presence of CB₂ receptors in reactive microglia and also in activated astrocytes, places these receptors in a promising position for their use as a target for neuroprotection (Fernández-Ruiz et al., 2010). An important advantage of compounds targeting these receptors is that they do not provoke the frequent psychotropic side effects elicited by cannabinoids that activate the CB₁ receptor, indicating that they may be safe and well-tolerated in clinical applications. In addition, such pharmacological manipulations may be the best way to reproduce the endogenous response provoked by these receptors, which, as mentioned above, are up-regulated in activated astrocytes and, in particular, reactive microglia in response to inflammatory, excitotoxic and traumatic insults, such as those that occur in most of neurodegenerative disorders including Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis and others (reviewed in Fernández-Ruiz et al., 2007, Fernández-Ruiz et al., 2010). This response, which had remained elusive for years, has been also identified in Parkinson's disease (PD) in a couple of recent studies. Thus, Price et al. (2009) described an elevation of CB₂ receptors in microglial cells recruited at the lesion sites in mice intoxicated with MPTP, a model with a modest glial response. In addition, these authors found that targeting these receptors reduced the damage of nigrostriatal neurons (Price et al., 2009), although a recent study showed that the inhibition of microglial activation and the preservation of nigrostriatal dopaminergic neurons in MPTP-lesioned mice involved surprisingly the activation of CB₁ receptors (Chung et al., 2011). In our laboratory, we studied this issue in a more inflammatory model of nigrostriatal damage consisting in intrastriatal injection of lipopolysaccharide (LPS) (García et al., 2011). We found elevated levels of CB₂ receptors in the striatum of LPS-lesioned mice, although we did not investigate the cell substrates in which this response is occurring (García et al., 2011). In addition, we found that CB₂ receptor-deficient mice were more vulnerable to LPS lesion than wild-type animals, a difference that was not found in mice lesioned with 6-hydroxydopamine, a model with poor inflammatory responses and in which the death of dopaminergic neurons is directly related to mitochondrial dysfunction and oxidative damage (García et al., 2011). In agreement with this difference, LPS-lesioned mice responded to compounds targeting the CB₂ receptor (García et al., 2011), whereas 6-hydroxydopamine-lesioned mice did not (García-Arencibia et al., 2007), although other authors described that mice overexpressing CB₂ receptors were more protected against 6-hydroxydopamine-induced nigrostriatal damage (Ternianov et al., 2012). All this evidence supports that CB₂ receptor may be also a promising pharmacological target in PD, although more specifically in those conditions having greater glial activation and inflammatory events. In the present study, we wanted to further explore the issue by addressing some unexplored objectives: (i) to elucidate whether such up-regulatory response of CB₂ receptors also occurs in PD patients using postmortem tissues provided by two biobanks; (ii) to quantify the magnitude of such response in LPS-lesioned mice and the differences between wild-type and CB₂ receptor-knockout mice; and (iii) to investigate some molecular substrates presumably controlled by the selective activation of these receptors in LPS-lesioned mice.