Adenoviral GDNF gene therapy against motoneuron death

胶质细胞源性神经营养因子(GDNF)对颅脑和脊髓运动神经元具有保护作用，在脊髓损伤和运动神经元病的治疗中具有潜在的应用前景。我们检测了腺病毒载体(AxCAhGDNF)编码的人GDNF对受损成年大鼠面神经和脊髓运动神经元死亡的神经保护作用。取成年Fisher 344大鼠面神经或颈7段(C7)前、后根和背根神经节，分别从茎乳突或椎孔取出，分别接种AxCAhGDNF、AxCALacZ(编码β-半乳糖苷酶基因的腺病毒)或PBS。撕脱和AxCALacZ治疗一周后，动物的面部和脊髓运动神经元中显示出β-半乳糖苷酶活性的表达。撕脱和AxCAhGDNF治疗的动物在受损的面和脊髓运动神经元中显示出强烈的GDNF免疫标记，并在受损的脑干和脊髓组织中表达病毒诱导的人GDNF mRNA转录本。尼氏染色细胞计数显示，与AxCALacZ或PBS治疗相比，AxCAhGDNF治疗显著防止了撕脱后2-8周受损运动神经元的丢失。此外，AxCAhGDNF治疗可改善撕脱后损伤运动神经元胆碱乙酰转移酶的免疫反应性，抑制一氧化氮合酶的诱导。这些结果表明，腺病毒介导的GDNF基因转移可以预防运动神经元损伤和运动神经元病的成人运动神经元变性。

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, that suggests potential uses of GDNF in the treatment of spinal cord injury and motor neuron diseases. We examined neuroprotective effect of human GDNF encoded by an adenovirus vector (AxCAhGDNF) on the death of lesioned adult rat facial and spinal motoneurons. The facial nerve or the seventh cervical segment (C7) ventral and dorsal roots and dorsal root ganglia of adult Fisher 344 rats were avulsed and removed from the stylomastoid or vertebral foramen, respectively, and AxCAhGDNF, AxCALacZ (adenovirus encoding b-galactosidase gene) or PBS was inoculated in the lesioned foramen. One week after the avulsion and treatment with AxCALacZ, the animals showed expression of beta-galactosidase activity in lesioned facial and spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned facial and spinal motoneurons and expression of virus-induced human GDNF mRNA transcripts in the lesioned brain stem and spinal cord tissues. Nissl-stained cell counts revealed that the treatment with AxCAhGDNF significantly prevented the loss of lesioned motoneurons 2 to 8 weeks after avulsion, as compared to AxCALacZ or PBS treatment. Furthermore, the AxCAhGDNF treatment ameliorated choline acetyltransferase immunoreactivity and suppressed the induction of nitric oxide synthase activity in the lesioned motoneurons after avulsion. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.