Activation of nicotinic receptors protects brainstem motoneurons from exitotoxic death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscular paralysis reflecting patchy motoneuron degeneration. The exact causes are still unknown, but it is thought to be a complex interplay among multiple pathological mechanisms, such as excitotoxicity, mitochondrial dysfunction, increased reactive oxygen species, and endoplasmic reticulum stress. High levels of glutamate have been detected in the cerebrospinal fluid of ALS patients due to decreased function of the excitatory aminoacid transporter (EAAT) proteins, responsible for glutamate re-uptake. We, therefore, hypothesize that glutamate-induced excitotoxicity is one major provoking mechanism of ALS. During disease progression, the hypoglossal nucleus is one of the most affected motor nuclei due to some peculiarities of its motoneurons, like Ca2+ permeable AMPA receptors, low Ca2+ buffer capacity and poor EAAT expression. Thus, these characteristics make hypoglossal motoneurons (HMs) readily vulnerable to glutamate excitotoxicity. To investigate the early pathological mechanisms activated by excitotoxic stress, we used an established in vitro model of the neonatal rat brainstem slice containing the nucleus hypoglossus in which excitotoxicity is induced by the glutamate uptake blocker DL-threo-β-benzyloxyaspartate, TBOA. Because the activation of nicotinic acetylcholine receptors (nAChRs) by nicotine has manifested neuroprotective function in certain brain neurons, we investigated if nicotine could arrest the progression of the excitotoxic damage to highly vulnerable HMs. On about 50% of HMs, TBOA evoked intense network bursting activity and large Ca2+ transients that were inhibited by 1-10 μM nicotine, whereas nAChR antagonists facilitated burst emergence in non-bursting cells. Moreover, nicotine inhibited glutamatergic transmission and enhanced GABA and glycine release. The strong neuroprotection given by nicotine prevented cell loss after 4h of continuous TBOA exposure. This neuroprotective action was due to block of downstream processes of neurotoxixcity such as impaired energy metabolism, increased intracellular reactive oxygen species, upregulated genes involved in the endoplasmic reticulum stress, and increased level of the apoptotic inducing factor (AIF). We hypothesised that the neuroprotective role of nicotine was mediated by its effect on gap junctions and, in particular, on connexin 36, which supports excitotoxicity spread. Moreover, nicotine raised the expression levels of the heat shock protein 70 (Hsp70), a protective molecule that binds AIF preventing its nuclear translocation associated with cell death. Our results suggest that activation of nAChRs should be a potential target for inhibiting excitotoxic damage of motoneurons at an early stage of the neurodegenerative process.