Immunological and pharmacological characterisation of imidazoline receptors and neuroprotective effects of imidazoline drugs in the kainic acid-model of excitotoxicity in mice

Abstract

The present Ph.D. dissertation in Neuropharmacology comprises three studies that aimed 1) to detect, characterise and compare immunoreactive imidazoline receptor (IR) proteins in different preparations, 2) to assess the regulation of apoptotic and neurotoxic signalling in the mouse brain after treatment with the glutamate analogue kainic acid (KA), and 3) to investigate the potential of repeated pretreatment with I1-, I2- and mixed I1-/I2-IR agonists to inhibt KA-induced excitotoxicity in mice. The existence of three IR subtypes (I1-3-IR) is generally accepted, but the molecular nature of such receptors remains to be established to date. In this Ph.D. work, three different IR antibodies were used in various immunological and pharmacological approaches to characterise two subtypes of imidazoline receptors (I1- and I2-IRs) in the mammalian brain and in cells. The reported findings indicate that brain-type I1-IRs constitute a heterogeneous group of proteins, which involve nischarin-related peptides of 167 kDa, 105/115 kDa and 85 kDa, whereas immunoreactive I2-IRs are related to 66 kDa, 45 kDa and 30 kDa proteins. Notably, full-length nischarin or IRAS-M (167 kDa), but apparently no other immunoreactive IR peptide, forms part of higher order complexes and is sensitive to strong reducing conditions. The excitotoxin KA has been shown to induce neuronal death mainly through activation of the intrinsic/mitochondrial pathway, and less is known about the contribution of extrinsic/death receptor-mediated apoptosis to KA-induced cell death. To explore this issue, the effect of a single high dose KA on the main components of the extrinsic apoptotic pathway (Fas ligand, Fas receptor, FADD, caspase-8, FLIP) and associated regulatory proteins was studied in the mouse brain. The results of this study show that KA (45 mg/kg) leads to delayed downregulation (at 72 hours) of the extrinsic apoptotic machinery (Fas ligand and receptor aggregates, FADD, caspase-8) with concomitant increases of anti-apoptotic mediators (p-FADD, FLIPS, p-Akt) in mice, indicating the induction of contraregulatory processes to counteract KA neurotoxicity. Based on previous studies that revealed IR ligand-mediated inhibition of both Ca2+ influx and apoptotic signalling, it was hypothesised that I1-/I2-selective compounds might also be protective in the KA model of excitotoxicity. Thus, using the aforementioned experimental design (monophasic KA-induced insult with neurochemical analyses at 72 hours after administration), the potential of repeated pretreatment with I1- (moxonidine), I2- (BU224) and mixed I1-/I2-IR (LSL61122) agonists to inhibt KA-induced excitotoxic signalling was assessed in the third part of this Ph.D. dissertation. These experiments were complemented by acute treatments and by drug combination studies with putative antagonists for the I1- and I2-IR (efaroxan and idazoxan, respectively), to study the acute effects of these compounds and to prove if the effects are mediated by IRs. The results presented in this Ph.D. work show that I1- and I2-IR-selective agonists (moxonidine, BU224 and LSL61122) inhibit central mediators of excitotoxic signalling (such as JNK, calpain and p25/Cdk5) at basal level and after KA in the mouse brain, while only LSL61122 attenuated the Kainduced behavioural syndrome. Further, I1- and I2-IR agonists regulate the activity and localisation of cdk5 through inhibition of p35 cleavage into neurotoxic p25, and alter PSD-95 expression, which, according to the regulatory role of this important scaffold protein at the postsynaptic density, is likely to affect the strength of glutamatergic synapses. Additionally, the drug combination experiments challenged the suitability of the classical IR antagonists efaroxan (I1) and idazoxan (I2) to antagonise the effects of moxonidine and LSL61122 in the present experimental paradigm (due to agonistic properties of the antagonists). Together, these findings indicate that the observed neuroprotective effects of imidazoline compounds (agonists) involve both IR activation and allosteric regulation of non-IR entities.