Tetrahydrobiopterin and pain

描述(由申请人提供)：周围神经损伤后，两种四氢生物蝶呤(BH4)合成酶，GTP环水解酶1(GCH1)和sepiapterin还原酶，以及BH4循环酶奎宁二氢蝶啶还原酶的转录本在背根(DRG)神经元中上调。这种诱导与DRG中BH4的增加有关。BH4是产生5-羟色胺、去甲肾上腺素和多巴胺的芳香胺羟基酶以及所有一氧化氮合酶的重要辅因子。全身应用GCH1抑制剂二氨基羟基嘧啶(DAHP)可防止神经损伤引起的BH4增加。DAHP对Naove动物没有止痛作用，但对有周围神经损伤和炎症的啮齿动物的疼痛相关行为有显着减少。鞘内注射BH4本身会产生急性疼痛过敏。利用单核苷酸多态分析，我们还在人类GCH1中确定了一种单倍型，该单倍型在慢性背痛手术后的患者中具有疼痛保护作用，并与健康受试者的疼痛敏感性降低有关。 基于这些数据，我们假设BH4有助于神经病理性和炎症性疼痛的启动和维持。本研究的目的是：1.研究BH4在痛相关啮齿动物模型的DRG中何时何地发生诱导，以及何种刺激负责；2.表征GCH1选择性缺失、抑制或过度表达在成年初级感觉神经元中的行为后果；3.确定BH4在感觉神经元中产生疼痛的机制。我们将研究组织炎症和部分周围神经损伤时，DRG中BH4合成酶和循环酶的表达和活性变化的时间进程和细胞定位(目标1)。为了阐明BH4在感觉神经元中的特定作用，我们将使用三苯氧胺诱导的DRG神经元特异性Cre重组酶技术(Aim 2)，使用在成人初级传入细胞中选择性过度表达或缺失GCH1的小鼠，以及过度表达GCH1的内源性抑制物GFRP的小鼠(目标2)。NNOS随着BH4的增加而上调，我们将测试BH4是否通过产生NO和钙内流的增加而产生疼痛。我们将使用原代培养的DRG神经元来探索BH4对这些神经元和负责的下游效应器的直接作用。该提案旨在探索导致疼痛的分子机制，并为开发新的止痛药确定新的靶点。相关性：持续性疼痛是一个巨大的问题，这既是由于患者所经历的痛苦，也是由于社会的高经济成本。由于目前的治疗方法往往无效或与副作用相关，因此需要更有效的止痛药。这项拨款的目的既是为了了解疼痛的机制，也是为了验证一种特定的酶，GCH1，作为开发新型止痛药的目标。

DESCRIPTION (provided by applicant): Transcripts for two tetrahydrobiopterin (BH4) synthetic enzymes, GTP cyclohydrolase 1 (GCH1) and sepiapterin reductase, and a BH4 recycling enzyme quinoid dihydroypteridine reductase, are upregulated in dorsal root (DRG) neurons after peripheral nerve injury. This induction is associated with an increase in BH4 in the DRG. BH4 is an essential cofactor for the aromatic amine hydroxylases that produce 5- hyrdoxytryptamine, norepinephrine and dopamine, and for all nitric oxide synthases. The nerve injury-induced increase in BH4 is prevented by systemic administration of a GCH1 inhibitor, diamino hydroxypyrimidine (DAHP). DAHP has no analgesic effects in naove animals but produces a marked reduction in pain-related behavior in rodents with peripheral nerve lesions and inflammation. Intrathecal BH4 itself produces acute pain hypersensitivity. Using an analysis of single nucleotide polymorphisms we have also identified a haplotype in human GCH1 that is pain protective in patients after surgery for chronic back pain and associated with reduced pain sensitivity in healthy subjects. Based on these data we hypothesize that BH4 contributes to the initiation and maintenance of neuropathic and inflammatory pain. The aim of this proposal is to: 1. Study where and when BH4 induction occurs in the DRG in pain-related rodent models and what kinds of stimuli are responsible, 2. Characterize the behavioral consequences of deletion, inhibition or overexpression of GCH1 selectively in adult primary sensory neurons, and 3. Identify the mechanisms in sensory neurons by which BH4 produces pain. We will study the time course and cellular localization of changes in the expression and activity of BH4 synthetic and recycling enzymes in the DRG in response to tissue inflammation and partial peripheral nerve injury (Aim 1). To elucidate the specific role of BH4 in sensory neurons we will employ mice that selectively overexpress, or have a deletion of GCH1 in adult primary afferents, as well as mice that overexpress the endogenous inhibitor of GCH1, GFRP, using tamoxifen-inducible DRG neuron-specific Cre-recombinase technology (Aim 2). nNOS is upregulated in association with the increase in BH4 and we will test if BH4 produces pain by producing an increase in NO and calcium influx. We will use primary cultures of DRG neurons to explore the direct action of BH4 on these neurons and the downstream effectors responsible. The proposal is designed to explore the molecular mechanisms responsible for pain and identify novel targets for the development of new analgesics. Relevance: Persistent pain is an enormous problem due both to the suffering experienced by patients and the high economic cost to society. Because current therapy is often ineffective or associated with adverse side effects, more efficacious analgesics are required. This grant aims both to understand the mechanisms responsible for pain, and validate a particular enzyme, GCH1, as a target for developing novel analgesics.