Presynaptic Mechanisms of Lead Neurotoxicity

描述（由申请方提供）：众所周知，早期接触铅（Pb 2+）会对神经系统发育造成严重风险。现在越来越清楚的是，低浓度的Pb 2+会对儿童产生显著的有害影响，这就需要了解Pb 2+对大脑作用的性质和程度。我们的实验室最近发现，在体外暴露于非常低水平的Pb 2+产生长期损害突触前递质释放培养海马神经元，这些行动模仿动物缺乏营养因子脑源性神经营养因子（BDNF）中观察到的。我们建议在发育过程中暴露于低水平Pb 2+的大鼠海马切片中进行研究，以利用1）最先进的双光子成像方法来评估对完整突触中突触前Ca 2+内流和囊泡递质释放的长期影响，（2）用全细胞膜片钳技术记录低浓度Pb ~（2+）暴露对海马CA 1区锥体神经元突触后N-甲基-N-D-天冬氨酸受体（NMDAR）门控电流。我们的工作假设是，早期Pb 2+暴露会导致NMDAR功能受损，导致BDNF合成和释放减少，以及随后对正常认知功能至关重要的突触前传递受损。一个已知的增加BDNF水平和释放的操作是丰富的环境。为了验证我们的假设并确定保护大脑免受Pb 2+发育损伤的潜在方法，我们建议1）表征低[Pb 2 +]暴露对BDNF基因表达，启动子甲基化和TrkB受体活化的影响，以及2）测试富集环境，外源性脑室内BDNF输注或给予TrkB激动剂7的能力，8-二羟基黄酮预防Pb 2+诱导的NMDAR功能和递质释放的长期损伤。我们提出的研究计划解决了一个关键问题，即早期发育暴露于低水平的Pb 2+是否会对大脑功能产生长期的有害影响。这些研究将提供新的信息Pb 2+对突触前和突触后机制的认知和记忆存储的关键影响。此外，我们将研究新的治疗操作，提高BDNF的释放和TrKB受体的激活，以防止长期的有害影响Pb 2+暴露。 公共卫生相关性：众所周知，铅（Pb 2+）暴露会对儿童的长期认知功能产生严重影响。新的数据表明，早期暴露于低得多浓度的Pb 2+可能会产生长期甚至终身的重大有害影响。为了对这种对幼儿的环境风险设定合理的限制并制定治疗策略，我们需要更多地了解Pb 2+的这些低水平影响的范围。新的证据表明，极低水平的Pb 2+可导致突触前递质释放的长期损害，这些影响可能是由于N-甲基-d-天冬氨酸谷氨酸受体依赖的脑源性神经营养因子（BDNF）释放的损害。本申请中提出的研究将这些发现扩展到脑切片中的完整突触，直接测试关于Pb 2+对递质释放的作用机制的工作假设，并评估治疗操作的潜力，以帮助保护儿童免受此类影响。暴露于低水平的Pb 2+可以降低大脑中BDNF水平的新发现对整个生命周期的脑容量变化具有重要意义。很可能，大量儿童暴露于极低浓度的Pb 2+中，会产生微妙但终身的认知和结构性脑损伤，直到现在才开始被认识到。因此，需要了解这种危险的严重性和所涉及的关键机制是至关重要的，以处理相当大比例的公共卫生威胁。

DESCRIPTION (provided by applicant): The severe nervous system developmental risks of early life exposure to lead (Pb2+) are well known. It is now becoming increasingly clear that much lower concentrations of Pb2+ can produce significant detrimental effects in children, heightening the need to understand the properties and extent of Pb2+ actions on the brain. Our laboratory has recently discovered that in vitro exposure to very low levels of Pb2+ produces long-term impairments in presynaptic transmitter release in cultured hippocampal neurons, and that these actions mimic those observed in animals lacking the trophic factor brain-derived neurotrophic factor (BDNF). We propose studies in hippocampal slices from rats exposed to low levels of Pb2+ during development to utilize 1) state-of- the-art two-photon imaging methods to assess long-term effects on presynaptic Ca2+ influx and vesicular transmitter release in intact synapses, and 2) whole-cell patch-clamp recording from CA1 pyramidal neurons to characterize the long-term effects of low level Pb2+ exposure on postsynaptic N-methyl-D-aspartate receptor (NMDAR)-gated currents. Our working hypothesis is that early Pb2+ exposure produces impairments in NMDAR function that leads to reduced BDNF synthesis and release and subsequent impairments of presynaptic transmission that are critical to normal cognitive function. One manipulation known to increase BDNF levels and release is an enriched environment. To test our hypothesis and identify potential methods of protecting the brain from developmental damage from Pb2+, we propose to 1) characterize the effects of low [Pb2+] exposure on BDNF gene expression, promoter methylation and TrkB receptor activation, and 2) test the ability of an enriched environment, exogenous intraventricular BDNF infusion or administration of the TrkB agonist 7,8-dihydroxyflavone to prevent Pb2+-induced long-term damage to NMDAR function and transmitter release. The research program we propose addresses the critical question of whether early developmental exposure to low-levels of Pb2+ than previously thought have long-term detrimental effects on brain function. These studies will provide novel information about Pb2+ effects on both presynaptic and postsynaptic mechanisms critical to cognition and memory storage. Further, we will examine novel therapeutic manipulations that elevate BDNF release and TrKB receptor activation in order to protect against the long-term detrimental effects of Pb2+ exposure. PUBLIC HEALTH RELEVANCE: It is well recognized that exposure to lead (Pb2+) causes severe effects to children's long-term cognitive function. New data indicates the concerning possibility that early exposure to much lower concentrations of Pb2+ produces significant detrimental effects that can be long-term, perhaps even life-long. In order to set rational limits and develop therapeutic strategies to this environmental risk to young children, we need to know much more about the scope of these low-level effects of Pb2+. New evidence suggests that very low levels of Pb2+ can lead to long-term impairments in presynaptic transmitter release, and that these effects may be due to impairment of N-methyl-d-aspartate glutamate receptor-dependent release of brain-derived neurotrophic factor (BDNF). The studies proposed in this application will extend these findings to intact synapses in brain slices, directly test ths working hypothesis about the mechanism of action of Pb2+ on transmitter release, and evaluate the potential of therapeutic manipulations to help protect children from such effects. The novel finding that exposure to low levels of Pb2+ can reduced BDNF levels in the brain has important implications to brain volume changes throughout the lifespan. It is likely that large populations o children being exposed to very low concentrations of Pb2+ have subtle but life-long cognitive and structural brain damage that only now it is beginning to be recognized. Thus, the need to understand the severity of this danger and the key mechanisms involved is critical to dealing with a Public Health threat of significant proportion.