Chang J W, Lee W Y, Milstien S, Kang U J
Department of Neurology and Neurobiology, Committee on Neurobiology, The University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.
J Neurochem. 2002 Oct;83(1):141-9. doi: 10.1046/j.1471-4159.2002.01120.x.
Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.
芳香族L-氨基酸脱羧酶（AADC）是L-多巴转化为多巴胺所必需的。因此，有人提出进行AADC基因治疗以增强递送L-多巴的药物治疗或基因治疗。然而，在帕金森病大鼠中，将AADC添加到表达酪氨酸羟化酶（TH）和GTP环水解酶1（GCH1）的转基因细胞移植物中（这些细胞可产生L-多巴），由于多巴胺对TH的反馈抑制，导致L-多巴和多巴胺的产量降低。已证明终产物反馈抑制是由TH的调节结构域介导的，丝氨酸40的位点特异性突变使TH对多巴胺抑制的敏感性降低。因此，我们在体外基因治疗模式下研究了使用丝氨酸40突变为亮氨酸的TH（mTH）的效果。用逆转录病毒转导Fischer 344大鼠的原代成纤维细胞（PF），以表达mTH或野生型大鼠TH cDNA（wtTH）。这两种细胞类型也都用GCH1进行转导，以提供必需的TH辅因子四氢生物蝶呤。转染了AADC的PF用作共培养和联合移植的伙伴。PFwtTHGC细胞和PFmTHGC细胞在培养中的TH活性和L-多巴产量相当。在与PFAADC细胞的共培养中，与PFwtTHGC细胞相比，PFmTHGC细胞显示出多巴胺抑制作用的显著降低。体内微透析测量表明，与PFAADC细胞和PFwtTHGC细胞的联合移植相比，PFAADC细胞与PFmTHGC细胞的联合移植导致L-多巴的降低幅度较小，且多巴胺水平没有降低，而PFAADC细胞与PFwtTHGC细胞的联合移植会使L-多巴和多巴胺水平均降低。用较低水平的L-多巴维持多巴胺水平将导致多巴胺更有针对性的局部递送，以及L-多巴扩散到其他结构中产生的潜在副作用更少。这些数据支持以下假设：丝氨酸40的突变在体内减弱了TH的终产物抑制作用，并说明了在基因治疗中仔细考虑生化途径和多个基因之间相互作用的重要性。
