Experimental and Regenerative Neuroscience, School of Animal Biology, The University of Western Australia, Perth, Australia.
PLoS One. 2012;7(2):e31061. doi: 10.1371/journal.pone.0031061. Epub 2012 Feb 8.
Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.
重组腺相关病毒（rAAV）载体可用于将神经营养基因导入受损的中枢神经系统神经元，促进其存活和轴突再生。基因治疗为神经创伤和神经退行性疾病的治疗带来了很大的希望；然而，已知神经营养因子会改变树突结构，因此我们着手确定这些转基因是否也会改变转导神经元的形态。我们比较了长期转导 rAAV2 后再生成年大鼠视网膜神经节细胞（RGC）的树突形态变化，rAAV2 编码：（i）绿色荧光蛋白（GFP），或（ii）双顺反子载体编码 GFP 和睫状神经营养因子（CNTF）、脑源性神经营养因子（BDNF）或生长相关蛋白-43（GAP43）。为了增强再生，大鼠接受自体周围神经移植到每个 rAAV2 注射眼的切断视神经上。5-8 个月后，用荧光金（FG）逆行标记有再生轴突的 RGC。制备活视网膜全层，并用电离子导入法将 2%荧光素黄注入 GFP 阳性（转导）或 GFP 阴性（非转导）的 RGC。使用 Neurolucida 软件分析树突形态。在转导和非转导群体中均发现树突结构发生了显著变化。多变量分析显示转基因 BDNF 增加了树突野面积，而 GAP43 增加了树突复杂性。CNTF 降低了复杂性，但仅在一部分 RGC 中。Sholl 分析显示 rAAV2-BDNF-GFP 和 rAAV2-CNTF-GFP 组的树突分支发生变化，与对照组相比，这些组中 FG 阳性 RGC 的异常形态比例增加了两倍。所有转基因组的 RGC 均显示出异常分层。因此，除了促进细胞存活和轴突再生外，载体介导的神经营养因子表达对受伤成年神经元的形态具有可测量的、基因特异性的影响。这些变化可能会改变神经元的功能特性，在设计基于载体的神经创伤和神经退行性疾病治疗方案时需要考虑这些变化。
