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

项目摘要 血脑屏障（blood-brain barrier，BBB）是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，如新型隐球菌（Cn），可以突破血脑屏障并侵入血脑屏障。 CNS。真菌孢子在我们的环境中很普遍，我们在吸入 气溶胶化的孢子一旦吸入，Cn孢子在肺组织中增殖，并由于呼吸道感染而扩散到CNS。 它们的亲神经性CN是成人真菌性脑膜脑炎的主要原因， 由于类似的神经系统症状，被误诊为阿尔茨海默病（AD）。事实上，认知 在Cn感染诊断后至少一年，61%的受试者报告了损伤。在AD中， 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。 我们解析了感染病毒的人脑微血管内皮细胞（BMEC）的转录特征， 关于Cn用Cn攻击的BMEC显示了几个对适当的细胞增殖至关重要的基因的显著失调。 脑内皮（即BBB）的功能，包括EphA 2受体酪氨酸激酶，BBB的关键介质 Cn挑战期间的功能障碍（基于我们母基金的研究）。我们还鉴定了APBB 3（淀粉样蛋白 β前体结合蛋白3）作为BBB功能障碍的潜在介质。APBB 3是一种未表征的 在APP加工/调节中的作用从未被研究过的蛋白质。我们的研究结果是一致 在AD发病机制中，在异常BBB中报道了类似的变化。血脑屏障完整性降低与 神经炎症、神经元损伤、氧化应激和淀粉样蛋白β（A β）的错误清除。基于我们 我们提出，真菌性脑感染部分通过APP失调和降低血脑屏障功能， 会进一步损害BBB的间隙。我们的数据与最近的报告一致， AD患者尸检时不同脑区感染不同种类的真菌。真菌DNA， 在AD患者的冷冻脑组织中鉴定了蛋白质和结构，但在对照患者中没有 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 多糖，真菌细胞壁的关键成分，也被确定和人类AD脑脊髓 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，继 这些最近的发现，以及提出的抗微生物特性的A β，一个新的AD假说提到， 因为“抗菌保护假说”已经出现。这一假说表明，A β斑块的沉积 可以启动早期的先天免疫反应，其中A β捕获并中和入侵的真菌 正常情况下的病原体。拟议的补充研究项目将解决2个具体目标 是母项目的延伸，与AD的发病机制基本相关。的 以下具体目标将检验脑真菌感染通过APBB 3失调APP的假设， 淀粉样蛋白β（A β）的抗真菌免疫活性由EphA 2信号传导介导。具体目标 1将研究APP在真菌脑感染小鼠模型中BBB功能障碍中的作用。APP和APBB 3 活性及其对BBB完整性的影响将在感染Cn的野生型和ephA 2-/-小鼠中进行检测。 将在体内检查A β肽与真菌细胞的物理相互作用，并将A β肽与真菌细胞的直接作用进行比较。 将在BBB球状体（一种高度相关的BBB体外模型）中评估肽对BBB完整性的影响。 具体目标2将决定A β是否具有抗真菌活性或诱导抗真菌免疫活性 通过EphA 2介导的信号传导。我们已经进行了几项动物研究，以解决真菌感染的机制， 通过检查BBB功能障碍中EphA 2活性的脑发病机制，然而，我们还没有检查 隐球菌脑感染背景下AD的神经学标志物。鉴于我们在动物模型方面的专业知识 真菌感染和血脑屏障体外模型的工作经验，下面提出的研究是一个 探索AD发病机制的一个令人兴奋和潜在的突破性领域的自然发展， 治疗发展

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.