During the last decade, robust preclinical evidence has accumulated demonstrating the neuroprotective potential of compounds that affect the activity of the endocannabinoid system, including both plant-derived cannabinoids and certain signalling lipids (reviewed in Fernández-Ruiz et al., 2010; 2011). This potential has been demonstrated for both acute and chronic brain damage and, in some cases (e.g. Huntington's disease), it is already being investigated at the clinical level (de Yébenes, 2010) based on preclinical results obtained with specific cannabinoid-based drugs (Sagredo et al., 2011; Valdeolivas et al., 2012). The evidence that cannabinoids are neuroprotective compounds is mostly derived from the pharmacological correction of alterations in the endocannabinoid communicating system observed in neurodegenerative disorders (reviewed in Fernández-Ruiz et al., 2007; 2010), alterations that several authors have related to the pathogenesis of these disorders (reviewed in Centonze et al., 2007; Fernández-Ruiz et al., 2010). These alterations are usually deficits, but increases in specific elements of this communicating system (i.e. ligands, receptors, enzymes) have also been found even in early and presymptomatic phases of these disorders, and have been characterized in experimental models and, to a lesser extent, in human subjects (reviewed in Centonze et al., 2007; Fernández-Ruiz et al., 2010). These changes could play a pivotal role in the pathogenesis of these conditions, presumably by aggravating the neuronal injury; although, in some cases, these alterations have been interpreted as being part of an endogenous protectant response against brain damage (reviewed in Fernández-Ruiz et al., 2010; Pacher and Mechoulam, 2011). For example, several studies have described an increased accumulation of arachidonoyl-ethanolamide, 2-arachidonoyl-glycerol or both in specific brain structures in experimental models of different neurodegenerative disorders including multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, ischaemia and brain trauma (Baker et al., 2001; Panikashvili et al., 2001; Witting et al., 2004). Data from patients are also available (Pisani et al., 2005). Experimental brain injury has also been found to be associated with an up-regulation of cannabinoid receptors, including cannabinoid receptor type 1 (CB1) receptors (Jin et al., 2000; Hansen et al., 2001), although the most pertinant data were obtained with the cannabinoid receptor type 2 (CB2) receptor. The CB2 receptor is mostly absent in the brain of healthy individuals but has been reported to be significantly up-regulated in many degenerative pathologies. These results were obtained mainly in animal models of disease but also from a few studies in human samples (reviewed in Fernández-Ruiz et al., 2007; Benito et al., 2008). This response occurs preferentially in activated astrocytes and, in particular, reactive microglial cells (note that resident microglial cells do not express CB2 receptors), which indicates that CB2 receptors may play a role in regulating the trophic and/or the cytotoxic influences of these cells on neurons (Fernández-Ruiz et al., 2007; Benito et al., 2008). The induction/up-regulation of CB2 receptors has also been observed in structures undergoing neuronal damage in patients and animal models of stroke (Ashton et al., 2007), Alzheimer's disease (Benito et al., 2003), Huntington's disease (Palazuelos et al., 2009; Sagredo et al., 2009), Parkinson's disease (Price et al., 2009; García et al., 2011), multiple sclerosis (Maresz et al., 2005; Benito et al., 2007), amyotrophic lateral sclerosis (Yiangou et al., 2006), simian immunodeficiency virus encephalitis (Benito et al., 2005), Down's syndrome (Núñez et al., 2008) and neuropathic pain (Zhang et al., 2003), but not in cases of spinocerebellar ataxias (SCAs), the pathology under investigation in the present study. Importantly, preclinical evaluations of different selective agonists of this receptor have generally demonstrated that these drugs delay the progression of brain damage in most of these disorders, thus indicating their potential to act as neuroprotectants (see below and some reviews in Fernández-Ruiz et al., 2007; Benito et al., 2008).
Autosomal-dominant SCAs are a group of inherited neurodegenerative disorders. The most prevalent cases belong to the family of polyglutaminopathies, which also includes Huntington's disease, and are primarily caused by excessive CAG repeats leading to the expansion of a polyglutamine tract in different recipient proteins (i.e. huntingtin in Huntington's disease, frequently ataxins but also other proteins in SCAs) (Klockgether, 2011). Despite its ubiquitous distribution, the mutant protein usually only affects specific structures within the CNS, the cerebellum being the key structure affected in SCAs, which explains the specificity of the neurological symptoms, that is motor incoordination and ataxia (Matilla-Dueñas et al., 2012). The age of onset of the clinical symptoms is typically between 30 and 50 years of age, although early onset in childhood, as well as cases in which the pathology initiates after 60 years, have been reported for specific SCA subtypes, frequently related to longer or shorter polyglutamine expansions (Durr, 2010). Like other polyglutamine diseases, SCAs are characterized by protein misfolding, failed or incomplete proteolysis and the deposition and formation of intracellular protein aggregates, which represents the key neuropathological characteristic of these disorders (Orr, 2012). Although there is some controversy as regards the role of these aggregates in neurotoxicity, they appear to play a key role in eliciting transcription dysregulation, mitochondrial failure, excitotoxicity, alterations in calcium homeostasis, oxidative stress and local inflammation, which ultimately lead to cell death in specific subpopulations of cerebellar neurons (Fratkin and Vig, 2012). Hence cannabinoid agonists that have been demonstrated to be effective in reducing excitotoxicity (e.g. selective CB1 agonists), oxidative injury (e.g. antioxidant cannabinoids) or inflammation (e.g. selective CB2 agonists) in various chronic neurodegenerative disorders (reviewed in Fernández-Ruiz et al., 2010) may also have a therapeutic effect in SCAs. However, it is also possible that cannabinoids could enhance autophagy, a cellular process responsible, among others, for eliminating the accumulation of toxic proteins, and this has been proposed as a possible therapeutic strategy for SCAs and other polyglutamine disorders, given that mutant proteins are autophagy substrates due to the failure of primary proteolytic processes (Williams et al., 2006). Interestingly, certain cannabinoid agonists have been demonstrated to stimulate cell autophagy in human glioma cells (Salazar et al., 2009), which may also have a therapeutic effect in polyglutamine disorders such as SCAs.
Whether cannabinoids can provide neuroprotection in SCAs as they do in preclinical models of other neurodegenerative disorders also needs to be investigated. However, before this can be done, the type of changes that the development of cerebellar degeneration in the different SCAs produces in particular elements of the endocannabinoid signalling system, specifically alterations in endocannabinoid receptors, needs to be clarified. Hence, we used immunohistochemical procedures to identify and quantify the major endocannabinoid receptors, that is CB1 and CB2 receptors, in the post-mortem cerebellum of SCA patients and control subjects.