Análise óptica da junção neuromuscular de diafragma em modelo de camundongo transgênico para doença de Huntington

Abstract

The Huntingtons disease (HD) is a neurodegenerative disorder characterized by a progressive decline of motor and cognitive functions including involuntary movements of the limbs and face, personality changes and dementia. It is caused by a CAG trinucleotide repetition that leads to a polyglutamine expansion in the huntingtin protein. It is known that huntingtin is highly expressed in the central and peripheral nervous systems. Within neurons, this protein binds to synaptic vesicles and interacts with several proteins associated with vesicular transport. Thus, it is suggested that the mutant huntingtin may interfere with the release of neurotransmitters causing synaptic dysfunction, which may lead to motor impairment characteristic of the disease. Previous data indicate a reduction in cholinergic neurotransmission, as well as motor impairment in animal models of HD. Based on this evidence, this study aims to assess the possible changes in the neuromuscular junction of diaphragm muscle of mice BACHDQ97, a transgenic model for HD, which disease progression is slower when compared to other previously established strains. This model represents a new and robust in vivo paradigm for studying the pathogenesis of HD. Briefly, for optical analysis, neuromuscular preparations were isolated and stained with the dye FM1-43fx (4 M) and -bungarotoxin-Alexa 594 (12 M) to visualize presynaptic terminals and acetylcholine receptors clusters, respectively. The styryl dye FM1-43 is a powerful tool to track exocytosis, endocytosis and recycling of synaptic vesicles. Through confocal and fluorescence microscopy, morphological parameters were analyzed in BACHDQ97 and WT animals. We observed that pre and post-synaptic elements presented less fluorescent in BACHDQ97 animals when compared to WT. We also notice that in the nerve terminals previously labeled with FM1-43 the destaining/excocytosis rates were lower in the transgenic animals. Moreover, ultrastructural studies were performed using electron microscopy transmission (TEM) technique to characterize the morphological aspect of neuromuscular synapses in these animals. Concerning the TEM, we saw that the synpatic elements of the neuromuscular junctions in BACHDQ97 mice show ultrastructural morphology resembling the WT animals. Thereby, our evidences suggest a quantitative and functional impairment of BACHDQ97 mice presynaptic elements what might affect motor function.