Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum
Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum
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DOI：
10.1111/j.1471-4159.2007.04529.x
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发表时间：
2007-07-01
影响因子：
4.7
通讯作者：
Civelli, O.
中科院分区：
文献类型：
--
作者：
Clark, S. D.;Alderson, H. L.;Civelli, O.
尿紧张素II是一种神经肽，最初从鱼类中分离出来，后来在哺乳动物中发现：它对心血管、内分泌和行为有强大的影响。在大鼠脑中，尿紧张素II受体（UII-R）主要表达于桥足部（PPTg）和外侧背侧被盖核的胆碱能神经元。通常，PPTg的功能是用兴奋毒素来检测的，它破坏了胆碱能和非胆碱能神经元，这使解释混乱。我们利用ui - r独特的表达谱，通过将ui与白喉毒素结合，设计了一种针对PPTg胆碱能神经元的毒素。在体外，两种不同的毒素结构被证明可以选择性地激活UII-R（平均EC50约为30 nmol/L；钙迁移率测定），并且对表达UII-R的CHO细胞的毒性比野生型细胞（平均LD50约为2 nmol/L；细胞活力）高10,000倍。在体内，将压力注射到大鼠的PPTg中，导致已知表达uir的胆碱转运体和NADPH diaphorase阳性神经元的特异性损失。随着时间的推移，病变逐渐发展，导致超过80%的胆碱能神经元在21天的损失，周围的神经元几乎没有损伤。这是第一个高选择性的分子工具，用于消耗中桥碱胆碱能神经元。毒素将有助于功能性解剖桥脚核和背外侧被盖核，并促进对这些结构功能的理解。
Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximate to 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximate to 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.