Characterization of the neuroprotective activity of rasagiline in cerebellar granule cells

Abstract

Rasagiline (N-propargyl-1-R-aminoindan) is a new selective inhibitor of MAO-B which is in development for the treatment of Parkinson's disease. The aim of the present study was to evaluate the neuroprotective properties of rasagiline and characterize the mechanism by which it exerts its neuroprotective effect in cerebellar granule cells. Cerebellar granule cells were prepared from 7 to 8 days postnatal Sprague-Dawley rats and maintained in high K⁺ (25 mM) medium. Rasagiline increased the survival of cerebellar granule cells treated with cytosine β-d-arabinofuranoside (Ara-C), l-buthionine-(S,R)-sulfoximine (BSO) or glutamate (100 μM) but did not reduce cell death induced by transferring the cells to physiological K⁺ concentration (5 mM) or by serum deprivation. Examination of different derivatives of rasagiline showed that the propargyl moiety is essential to the neuroprotective effect of these molecules, as the compound 1-R-aminoindan (a major metabolite of rasagiline) was devoid of neuroprotective effect in this model system and a rasagiline derivative with a double bond in place of the acetylenic propargyl triple bond was much less effective. The S(−)-enantiomer of rasagiline was also significantly less active than R(+)-rasagiline, as was 6-fluoro rasagiline. Addition of rasagiline (0.1–10 μM) to cerebellar granule cells grown in medium containing a physiological concentration of K⁺ did not have an effect on neurite outgrowth as measured by synapsin expression level but increased the density of glial cell processes. The neuroprotective effects of rasagiline may include a direct action on the neurons through inhibition of neuronal death as well as an indirect effect mediated by the astrocytes.

Introduction

Rasagiline (N-propargyl-1-R-aminoindan) is a new selective inhibitor of MAO-B which has been developed for the treatment of Parkinson's disease. Like selegiline (deprenyl), the only selective MAO-B inhibitor currently in clinical use for the treatment of Parkinson's disease, rasagiline exerts symptomatic anti-Parkinsonian effect by blocking the oxidative metabolism of dopamine (DA), thus prolonging its physiological action in control of movement (Rabey et al., 2000, Kieburtz and the Parkinson Study Group, 2001). In addition to inhibition of DA catabolism, however, both compounds have been shown to possess neuroprotective properties in a variety of in vivo and in vitro animal models, an effect which is not dependent on MAO inhibition (Boulton et al., 1997, Finberg et al., 1998, Finberg et al., 1999, Youdim et al., 2001a, Tatton et al., 2003). In vivo systems in which propargylamines including rasagiline have been found effective as neuroprotectants include dopaminergic neurotoxicity caused by MPTP in mice and non-human primates (Heikkila et al., 1985, Kupsch et al., 2001), closed head injury in mice (Huang et al., 1999), protection from cerebral focal ischemia and post-natal anoxia in rats (Speiser et al., 1998, Speiser et al., 1999), and protection of facial nerves from apoptosis following axotomy in immature, post-natal rats (Salo and Tatton, 1992). In isolated cell systems, both rasagiline and selegiline protect fetal rat mesencephalic neurons, human neuroblastoma SH-SY5Y, and NGF-differentiated rat pheochromocytoma PC-12 cells from a variety of challenges (Roy and Bedard, 1993, Tatton et al., 1994, Finberg et al., 1998, Maruyama et al., 2000a, Maruyama et al., 2000b, Maruyama et al., 2001, Youdim et al., 2001a, Abu-Raya et al., 2002). A major potential advantage of rasagiline as a neuroprotective anti-Parkinsonian drug is that it is not metabolized to amphetamines, as is selegiline, since amphetamines are potentially neurotoxic and antagonize the neuroprotective effect of selegiline (Tatton and Chalmers-Redman, 1996, Abu-Raya et al., 2002, Bar-Am et al., 2004).

Apart from the case of the MPTP model, the neuroprotective activity of rasagiline does not result from MAO-B inhibition, since its S-enantiomer, TV-1022, which has over 1000 fold weaker MAO-B inhibitory activity, exhibits similar neuroprotective properties as shown by improving the sequelae of brain trauma injury in mice (Huang et al., 1999) and ischemic brain injury in rats (Speiser et al., 1999). In addition, in vitro neuroprotective activity of rasagiline is seen at concentrations below those effective for inhibition of MAO in the cells under test (Maruyama et al., 2002).

A number of different mechanisms have been proposed to explain the neuroprotective action of rasagiline at the cellular level. These include a direct effect to prevent opening of the mitochondrial permeability transition pore (Akao et al., 2002a), and alterations in the expression of various proteins involved in apoptosis or antiapoptosis such as Bcl-2, cJUN, Bax, SOD and GAPDH (Tatton et al., 2002, Akao et al., 2002b). These actions of rasagiline lead to maintenance of the mitochondrial membrane potential following various challenges, decreased activation of caspase 3 and decreased translocation of GAPDH from the cytoplasm to the nucleus (Carlile et al., 2000, Youdim et al., 2001a). Binding of neuroprotective propargylamines to GAPDH blocks its dimerisation and nuclear translocation at the onset of apoptotic cell death (Carlile et al., 2000).

To further characterize and understand the mechanisms underlying the neuroprotective activity of rasagiline, we elected to use the model of isolated rat cerebellar granule cells treated with different neurotoxic insults. Selegiline has been shown to protect these cells from apoptotic death caused by cytosine β-d-arabinofuranoside (Ara-C; Paterson et al., 1998) indicating their suitability for studying the action of propargylamine neuroprotective drugs. Primary neuronal cultures are a superior model for intact CNS neurons than actively replicating cells such as neuroblastoma-derived SH-SY5Y or rat pheochromocytoma-derived PC-12, which have been studied more extensively. Cerebellar cells also have the advantage of not producing catecholamines, as do SH-SY5Y and PC-12 cell lines, and dopaminergic mesencephalic cells. Although rasagiline is a selective inhibitor of MAO-B, yet higher concentrations of rasagiline (above 0.4 μM, the IC-50 value for rat brain MAO-A; Youdim et al., 2001b) will substantially inhibit MAO-A, and blockade of this form of the enzyme will increase endogenous catecholamine levels, which themselves have neuroprotective properties (Amano et al., 1994, Noh et al., 1999, Troadec et al., 2001). Establishing a neuroprotective action of rasagiline in a non-catecholaminergic primary neuronal culture is important in understanding the potential application of this compound as a neuroprotectant in various types of neurotrauma or neurodegeneration.