RESUMO
Dysregulation of protein expression, function and/or aggregation is a hallmark of a number of neuropathological conditions. Among them, upregulation and/or de novo expression of the neuronal isoform of nitric oxide (NO) synthase (nNOS) commonly occurs in diverse neurodegenerative diseases and in axotomized motoneurons. We used adenoviral (AVV) and lentiviral (LVV) vectors to study the effects of de novo nNOS expression on the functional properties and synaptic array of motoneurons. AVV-nNOS injection into the genioglossus muscle retrogradely transduced neonatal hypoglossal motoneurons (HMNs). Ratiometric real-time NO imaging confirmed that transduced HMNs generated NO gradients in brain parenchyma (space constant: 12.3 µm) in response to a glutamatergic stimulus. Unilateral AVV-nNOS microinjection in the hypoglossal nucleus of adult rats induced axotomy-like changes in HMNs. Specifically, we found alterations in axonal conduction properties and the recruitment order of motor units and reductions in responsiveness to synaptic drive and in the linear density of synaptophysin-positive puncta opposed to HMN somata. Functional alterations were fully prevented by chronic treatment with nNOS or soluble guanylyl cyclase inhibitors. Synaptic and functional changes were also completely avoided by prior intranuclear injection of a neuron-specific LVV system for miRNA-mediated nNOS knock-down (LVV-miR-shRNA/nNOS). Furthermore, synaptic and several functional changes evoked by XIIth nerve injury were to a large extent prevented by intranuclear administration of LVV-miR-shRNA/nNOS. We suggest that nNOS up-regulation creates a repulsive NO gradient for synaptic boutons underlying most of the functional impairment undergone by injured motoneurons. This further strengthens the case for nNOS targeting as a plausible strategy for treatment of peripheral neuropathies and neurodegenerative disorders.
