Antifungal activity of amyloid beta as a driver of dementia and AD pathogenesis.

项目摘要 血脑屏障(BBB)是一种保护中枢神经系统的高度限制性结构。 (CNS)。很少有真菌病原体，如新生隐球菌(CN)，能破坏血脑屏障，入侵 中枢神经系统。真菌孢子在我们的环境中很普遍，我们在吸入 雾化孢子。一旦吸入，CN的孢子就会在肺组织中增殖并传播到中枢神经系统，这是由于 它们的嗜神经性。CN是成人真菌性脑膜脑炎的主要病因，通常 由于类似的神经症状被误诊为阿尔茨海默病(AD)。的确，认知的 61%的受试者在被诊断为CN感染后至少一年内出现了损害。在公元后， 炎症和感染过程促进血脑屏障功能障碍，强调了 淀粉样前体蛋白(APP)在血脑屏障内皮炎症及其后续功能障碍中的作用。 我们解决了感染人脑微血管内皮细胞的转录特征 与CN.用CN攻击的BMEC显示出几个基因的显著失调，这些基因对于正常的 脑内皮细胞(即血脑屏障)的功能，包括血脑屏障的关键介质EphA2受体酪氨酸激酶 CN挑战期间的功能障碍(基于我们父母资助的研究)。我们还鉴定了APBB3(淀粉样蛋白 β前体结合蛋白3)作为血脑屏障功能障碍的潜在介体。APBB3是一种未刻画的 在APP加工/调节中的作用从未被研究过的蛋白质。我们的结果与 在AD发病机制中，血脑屏障的异常也有类似的变化。血脑屏障完整性降低与 神经炎症、神经元损伤、氧化应激和淀粉样β蛋白(A)清除错误。基于我们的 我们的数据表明，真菌脑感染导致血脑屏障功能障碍的部分原因是通过失调APP和减少 的许可，这将进一步损害英国广播公司。我们的数据与最近的报告一致，这些报告发现 AD患者尸检中感染不同种类真菌的不同脑区。真菌DNA， 在AD患者的冰冻脑组织中发现了蛋白质和结构，而在对照组中没有 组织。此外，在AD患者的神经元细胞内和细胞外检测到真菌物质。甲壳素 真菌细胞壁的关键成分--多糖也被鉴定出来，并与人类AD脑脊椎 液体样本进一步证实了真菌的存在，包括隐球菌和念珠菌。事实上，以下是 这些最新的发现，以及提出的的抗微生物特性是一个新的AD假说 作为“抗菌保护假说”应运而生。这一假说表明斑块的沉积 在大脑中可以启动早期的先天免疫反应，其中A捕获并中和入侵的真菌 病原体在正常情况下。拟议的补充研究项目将解决两个具体目标 这是父项目的延伸，与阿尔茨海默病的发病机制基本相关。这个 以下特定目标将检验脑真菌感染通过APBB3和APBB3调节APP失调的假设 淀粉样β蛋白(A)的抗真菌免疫活性是由EphA2信号介导的。特定目标 1将在真菌脑感染的小鼠模型中研究APP在血脑屏障功能障碍中的作用。APP和APBB3 将在感染CN的野生型和ephA2-/-小鼠中检测活性及其对血脑屏障完整性的影响。 多肽与真菌细胞的物理相互作用以及A的直接作用将在体内进行研究 关于血脑屏障完整性的多肽将在血脑屏障球体中进行评估，这是一个与血脑屏障高度相关的体外模型。 特定目标2将确定是否具有抗真菌活性或诱导抗真菌免疫活性 通过EphA2介导的信号转导。我们已经进行了几项动物研究，以解决真菌的机制 通过检测EphA2活性在血脑屏障功能障碍中的脑发病机制，但我们还没有检查 隐球菌脑感染背景下AD的神经学标记物。鉴于我们在动物模型方面的专业知识 关于真菌感染和使用体外血脑屏障模型的经验，下面建议的研究是 探索AD发病机制和潜在突破性领域的自然片段 治疗方面的发展。

PROJECT ABSTRACT The blood-brain barrier (BBB) is a highly-restrictive structure that protects the central nervous system (CNS). Few fungal pathogens, such as Cryptococcus neoformans (Cn), can breach the BBB and invade the CNS. Fungal spores are prevalent in our environment and we become infected following inhalation of aerosolized spores. Once inhaled, spores of Cn proliferate in lung tissue and disseminate to the CNS due to their neurotropic nature. Cn is the leading cause of fungal meningoencephalitis in adults and it is often misdiagnosed as Alzheimer’s Disease (AD) due to similar neurological symptoms. Indeed, cognitive impairment was reported in 61% of subjects at least one year following Cn infection diagnosis. In AD, inflammatory and infectious processes promote BBB dysfunction underscoring the potentially critical role of the amyloid precursor protein (APP) in endothelial inflammation of the BBB and its subsequent dysfunction. We resolved the transcriptional signature of human brain microvascular endothelial cells (BMECs) infected with Cn. BMECs challenged with Cn showed significant dysregulation of several genes essential to the proper function of the brain endothelium (i.e. BBB), including EphA2-receptor tyrosine kinase, a key mediator of BBB dysfunction during Cn challenge (based on studies from our parent grant). We also identified APBB3 (amyloid beta precursor binding protein 3) as a potential mediator of BBB dysfunction. APBB3 is an uncharacterized protein whose role in APP processing/regulation has never been examined. Our results are consistent with similar changes reported in the abnormal BBB in AD pathogenesis. Reduced BBB integrity is associated with neuroinflammation, neuronal injury, oxidative stress and faulty clearance of amyloid beta (A). Based on our data we propose that fungal brain infection causes BBB dysfunction in part by dysregulating APP and reducing A clearance which would further damage the BBB. Our data are consistent with recent reports that found various brain regions from AD patients at autopsy infected with different species of fungi. Fungal DNA, proteins and structures were identified in frozen brain tissue from AD patients, but not from control patient tissue. Moreover fungal material was detected intra- and extracellularly in neurons from AD patients. Chitin polysaccharides, a key component of fungal cell walls, were also identified and human AD cerebral spinal fluid samples further confirmed the presence of fungi, including Cryptococcus and Candida. Indeed, following these recent discoveries, and the proposed antimicrobial properties of A a new AD hypothesis referred to as “antimicrobial protection hypothesis” has emerged. This hypothesis suggests that deposition of A plaque in brain can initiate early innate immune responses, where A entraps and neutralizes the invading fungal pathogen under normal conditions. The proposed supplement research project will address 2 specific aims that are an extension of the parent project and are fundamentally relevant to the pathogenesis of AD. The following specific aims will test the hypothesis that brain fungal infection dysregulates APP via APBB3 and the resulting antifungal immunity activity of amyloid beta (A) is mediated by EphA2- signaling. Specific aim 1 will examine the role of APP in BBB dysfunction in a mouse model of fungal brain infection. APP and APBB3 activity and their effects on BBB integrity will be examined in wild type and ephA2-/- mice infected with Cn. The physical interaction of A peptides with fungal cells will be examined in vivo and direct effects of A peptides on BBB integrity will be assessed in BBB spheroids, a highly-relevant in vitro model of the BBB. Specific aim 2 will determine whether A exhibits antifungal activity or induces antifungal immunity activity via EphA2-mediated signaling. We have performed several animal studies to resolve mechanisms of fungal brain pathogenesis by examining EphA2 activity in BBB dysfunction, however we have not examined neurological markers of AD in the context of cryptococcal brain infection. Given our expertise in animal models of fungal infection and experience working with in vitro models of the BBB, the studies proposed below are a natural segue to exploring an exciting and potentially ground breaking area of AD pathogenesis and therapeutic development.