Novel therapy for monoamine neurotransmitter deficiency in PKU

描述（由申请人提供）： 我们项目的目标是调查苯丙酮尿症（PKU）中慢性高苯丙氨酸血症相关神经行为症状的原因并评估潜在的新疗法，PKU是通过新生儿筛查发现的最常见的先天性代谢缺陷之一。许多患有PKU的青少年和成年人难以坚持推荐的饮食疗法。慢性高苯丙氨酸血症通常与焦虑、抑郁和执行功能受损（如注意力集中和短期记忆困难）有关。脑内单胺类神经递质多巴胺和5-羟色胺的缺乏可能是苯丙酮尿症神经行为障碍的近端原因。提出的多巴胺和5-羟色胺缺乏的原因包括L-酪氨酸和L-色氨酸（分别为多巴胺和5-羟色胺合成的底物）的脑含量降低，以及苯丙氨酸介导的酪氨酸羟化酶（TH）和色氨酸羟化酶（TPH）（多巴胺和5-羟色胺合成途径中的限速酶）的竞争性抑制。我们的首要假设是，恢复脑多巴胺和5-羟色胺含量将成功地改善与慢性高苯丙氨酸血症相关的神经行为症状。我们的初步数据记录了严重的单胺神经递质缺乏症Pahenu 2小鼠，一个模型的人PKU，与短期记忆的缺陷，如测量的Morris水迷宫测试。在我们的第一个具体目标中，我们将进一步确定Pahenu 2小鼠的行为表型，并使用分子和生物化学方法全面评估脑中氨基酸和单胺神经递质代谢。我们项目的第二个目标将集中在评估几种新疗法在克服脑单胺神经递质缺乏症和改善小鼠行为异常方面的疗效。单独和联合评价的治疗将是大量中性氨基酸（包括L-酪氨酸和L-色氨酸）补充剂，以治疗脑氨基酸缺乏症，酪氨酸激动剂的药理学抑制，以提高脑酪氨酸含量，或给予5-羟基色氨酸和L-DOPA，以绕过TH和TPH，恢复脑单胺神经递质合成。将结果与通过饮食苯丙氨酸限制或重组腺相关病毒（rAAV）介导的肝脏定向基因治疗降低血液和脑苯丙氨酸的效果进行比较。在该项目的最终目的，我们将调查，使用微透析方法，是否急性管理（6 R）-5，6，7，8-四氢生物蝶呤（BH 4），所需的辅助因子TH和TPH的活动，可以克服苯丙氨酸介导的竞争性抑制这些酶和恢复多巴胺和5-羟色胺的合成。我们的目标是在Pahenu 2小鼠中彻底评估这些新的治疗方法，并为未来的PKU临床试验选择特定的干预措施。

DESCRIPTION (provided by applicant): The goal of our project is to investigate the causes of and evaluate potential novel therapies for the neurobehavioral symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU), one of the most common inborn errors of metabolism detected through newborn screening. Many adolescents and adults with PKU struggle with adherence to recommended dietary therapy. Chronic hyperphenylalaninemia is frequently associated with anxiety, depression, and impaired executive function such as difficulties with concentration and short-term memory. Deficiency of the monoamine neurotransmitters, dopamine and serotonin, in brain has been implicated as a probable proximal cause of neurobehavioral difficulties in PKU. The proposed causes of dopamine and serotonin deficiencies include decreased brain content of L-tyrosine and L-tryptophan, the substrates for dopamine and serotonin synthesis respectively, and phenylalanine- mediated competitive inhibition of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate- limiting enzymes in the dopamine and serotonin synthetic pathways. Our overarching hypothesis is that restoration of brain dopamine and serotonin content will successfully ameliorate the neurobehavioral symptoms associated with chronic hyperphenylalaninemia. Our preliminary data document severe monoamine neurotransmitter deficiency in Pahenu2 mice, a model of human PKU, in association with a deficit in short-term memory as measured by a Morris Water Maze test. In our first specific aim, we will further define the behavioral phenotype of Pahenu2 mice and use molecular and biochemical methods to comprehensively evaluate amino acid and monoamine neurotransmitter metabolism in brain. The second aim of our project will focus upon evaluating the efficacy of several novel therapies in overcoming brain monoamine neurotransmitter deficiency and ameliorating behavioral abnormalities in the mice. The therapies to be evaluated individually and in combination will be large neutral amino acid (including L-tyrosine and L-tryptophan) supplementation to treat brain amino acid deficiencies, pharmacologic inhibition of tyrosine catabolism to raise brain tyrosine content, or administration of 5-hydroxytryptophan and L-DOPA to bypass TH and TPH and restore brain monoamine neurotransmitter synthesis. The outcomes will be compared to the effects of lowering blood and brain phenylalanine through dietary phenylalanine restriction or recombinant adeno-associated virus (rAAV)- mediated liver-directed gene therapy. In the final aim of the project, we will investigate, using microdialysis methods, whether acute administration of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), the required cofactor for TH and TPH activity, can overcome phenylalanine-mediated competitive inhibition of these enzymes and restore dopamine and serotonin synthesis. Our goal is to thoroughly evaluate these novel therapeutic approaches in Pahenu2 mice and to choose specific interventions for a future clinical trial in humans with PKU.