Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture Samuel J. Jackson, David Baker, M. Louise Cuzner and Lara T. Diemel Department of Neuroinflammation, Institute of Neurology, University College London, 1 Wakefield Street, London WC1N 1PJ, UK Keywords: caspase 3, CB 1 , myelin basic protein, neurofilament, SMI-32 Abstract Multiple sclerosis is increasingly recognized as a neurodegenerative disease which is triggered by inflammation in the central nervous system (CNS). Demyelination-associated axonal or neuronal damage is a primary cause of disability and has thus far not been successfully targeted by available drug therapies. The neuroprotective properties of both endogenous and administered cannabinoids have been shown in in vivo and in vitro models of CNS damage following excitotoxic, oxidative, traumatic and ischaemic insults, with a predominantly apoptotic effector mechanism. In this study a foetal mouse telencephalon aggregate cell culture system was developed to compare tissue from cannabinoid receptor 1 knockout mice with wildtype counterparts. Aggregate formation and neurofilament ⁄ myelin basic protein accumulation were dependent on the age of foetal dissection and species used. Following treatment with interferon-c, levels of myelin basic protein, neurofilament, neuronal dephosphorylation and caspase 3 activation were assessed in telencephalon tissue in vitro. Cytokine treatment resulted in significant loss of the neuronal marker neurofilament-H in cannabinoid receptor 1 knockout cultures but not in wildtypes, indicating that presence of the cannabinoid receptor 1 gene can be neuroprotective. Caspase 3 activation was higher in cultures from knockout animals, indicating an apoptotic mechanism of cell death. Dephosphorylated neurofilament levels were significantly elevated in knockout mice, lending support to the premise that neurofilament dephosphorylation is a marker for neuronal damage. Taken together, these results indicate that neuroprotection could be elicited through the cannabinoid receptor 1, and point towards a potential therapeutic role for cannabinoid compounds in demyelinating conditions such as multiple sclerosis. Introduction Degenerative diseases of the central nervous system (CNS) such as multiple sclerosis (MS) have been associated with loss of myelin and axonal damage triggered by inflammation. In MS, an inflam- matory insult, possibly coupled with other invasive and endogenous triggers, leads to autoimmune destruction of myelin (Keegan & Noseworthy, 2002). Loss of myelin may contribute to decreased signal conduction and symptoms; however, longer term disability in MS shows a strong correlation with axonal damage (Kieseier & Hartung, 2003). An in vitro model of demyelinating disease, in which aggregated foetal rat CNS tissue is demyelinated by cytokines or antibodies, has been previously described (Honegger & Richelson, 1976; Diemel et al., 2004). It comprises a reproducible and easily manipulated model of an acute demyelinative episode mediated by components of the autoimmune cascade. This provides an additional in vitro tool to other models such as experimental allergic encephalomyelitis (EAE) for studying aspects of the demyelinating process. Evidence from studies in EAE and MS patients have shown that cannabis or its active extracts can ameliorate signs or symptoms of disease (Baker et al., 2000; Wade et al., 2003; Zajicek et al., 2003), and suggest that cannabis could affect disease progression in EAE (Pryce et al., 2003). It has been found that cannabinoid compounds can exhibit neuroprotective effects by activation of the cannabinoid receptor 1 (CB 1 R) (Nagayama et al., 1999; Abood et al., 2001; Panikashvili et al., 2001; Marsicano et al., 2002; Pryce et al., 2003). CB 1 R gene knockout mice (ABH.Cnr1– ⁄ –) exhibit enhanced neurodegeneration following neurotoxic insults (Nagayama et al., 1999; Parmentier-Batteur et al., 2002; Pryce et al., 2003), and rapidly accumulate disability following the induction of EAE (Pryce et al., 2003). This is associated with significant axonal loss, as detected histologically and by quantative ELISA for neurofilament content. Abnormally phosphorylated neurofilaments have been identified as potential markers of neuronal damage in CNS disorders, using a range of monoclonal antibodies that selectively bind phosphorylated and non-phosphorylated epitopes on neurofilament heavy chain (NfH). Demyelination confers reactivity of one antibody, SMI 32, to axons (Sternberger & Sternberger, 1983). SMI 32 is a sensitive marker for early and chronic lesions in MS, conferring immuno- staining to demyelinating axons and, most strikingly, to axon- terminal spheroids, indicative of axonal transection in MS (Peterson et al., 2001). In this study, the effect of demyelination on the integrity of neurons and the myelin sheath was examined in aggregate cultures from ABH wildtype and ABH CB 1 R knockout mice. Correspondence: Dr S. J. Jackson, as above. E-mail: samuel.jackson@ucl.ac.uk Received 5 March 2004, revised 16 August 2004, accepted 31 August 2004 European Journal of Neuroscience, Vol. 20, pp. 2267–2275, 2004 ª Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2004.03711.x