Protein variants as blood based biomarkers for diagnosing and staging AD

摘要 美国有500多万阿尔茨海默病(AD)病例，预计病例数量将 在接下来的15年里几乎翻一番，每年的总经济成本超过225亿美元。尽管如此 费用巨大，目前仍没有有效的治疗AD的方法。淀粉样蛋白-β(A？)与 AD已经有很多年了，但尽管有数以千计的出版物关于A？和AD之间的联系，仍然有 人们对阿伯在阿尔茨海默病中的作用以及针对阿伯的治疗方法存在很多困惑，但取得的成功非常有限。 Tau蛋白也与阿尔茨海默病有关。与Aü类似，tau也以各种不同的形式存在于 因此，tau在AD的发生和发展中的作用也不清楚。另外两个神经元 α-突触核蛋白(a-syn)和Tar-DNA结合蛋白(TDP-43)等蛋白质也与此有关 神经退行性疾病在帕金森病和肌萎缩侧索硬化症中起主要作用 (ALS)。所有这四种关键的神经元蛋白，A？、tau、a-syn和TDP-43都容易发生错误折叠 而这些蛋白质的聚集和小的有毒聚集被认为在发病中起到了作用， 一些不同的神经退行性疾病的进展和扩散。有毒物质引起的细胞应激 一种蛋白质的聚集体可以诱导其他蛋白质的有毒聚集体的形成，最终导致 神经退行性疾病的谱系。明显地产生和积累了有毒的tau蛋白变体， A？、TDP-43和a-syn AD在神经退行性疾病的发生发展中起重要作用 包括公元。因为这些蛋白变体在不同的神经退行性变中起着重要的作用 疾病，我们假设有不同的毒性蛋白变异谱来区分AD和 在AD的进展过程中，这些图谱中的关键蛋白质变体的水平会发生变化，也会从 病人对病人。我们假设，通过确定每个个体的有毒蛋白质变体 使用血清或血浆样本中存在的生物标记物的指纹，我们可以促进症状前期 AD的诊断和分期，为每个患者确定适当的治疗策略，以及 还可以实时监测不同疗法的效果。这项提案的目标是确定 血液生物标记物指纹用于区分不同阶段的AD，包括症状前AD， 并为每个患者确定最合适的治疗策略。我们开发了 使我们能够产生选择性结合疾病的抗体片段(纳米体)的新技术 相关的蛋白质变体。我们有一个由15个纳米体组成的小组，分别针对不同的有毒变种A？、tau、a-syn和 TDP-43，并表明我们可以在AD、PD患者的血液样本中检测到这些变体 和其他神经退行性疾病，但不是在年龄匹配的认知正常病例中。 这项提议的目的是利用我们的纳米机构小组选择性地识别不同的 疾病相关形式A？、tau、a-syn和TDP-43用于表征一组纵向血浆样本 来自AD患者和年龄匹配的对照组患者。这些研究将定义蛋白质变异指纹，不仅 区分AD和认知正常对照，但会为AD的不同阶段定义不同的指纹。 然后，我们将从临床人群中鉴定一组更大的样本，以确定蛋白质变异 指纹与不同的临床症状相关，如帕金森症、失语症和共济失调。定义 阿尔茨海默病不同分期和临床表现的不同蛋白质变异指纹图谱 提供一个非常强大的工具来促进AD的症状前和个性化诊断，提示大多数 为每个患者提供适当的治疗策略，并提供一种监测疗效的手段 所选治疗方案的实时记录。

ABSTRACT There are over 5 million cases of Alzheimer's Disease (AD) in the US, the number of cases is expected to nearly double over the next 15 years, and the total annual economic costs are over $225 billion. Despite these tremendous costs, there is still no effective treatment for AD. The protein amyloid-beta (Aß) has been linked to AD for many years, but despite thousands of publications on the connection between Aß and AD, there is still much confusion over the role of Aß in AD, and therapeutics targeting Aß have met with very limited success. The protein tau has also been linked to AD. Similar to Aß, tau also exists in a wide variety of different forms in the human brain, so the role of tau in the onset and progression of AD is also not clear. Two other neuronal proteins, alpha-synuclein (a-syn) and Tar-DNA binding protein (TDP-43), have also been implicated in neurodegenerative diseases and play major roles in Parkinson's disease and Amyotrophic lateral sclerosis (ALS), respectively. All four of these key neuronal proteins, Aß, tau, a-syn and TDP-43, are prone to misfolding and aggregation and small toxic aggregates of each of these proteins are thought to play a role in the onset, progression and spread of a number of different neurodegenerative diseases. Cellular stress caused by toxic aggregates of one protein can induce formation of toxic aggregates of other proteins ultimately resulting in a spectrum of neurodegenerative diseases. Clearly generation and accumulation of toxic protein variants of tau, Aß, TDP-43 and a-syn AD play an important role in the onset and progression of neurodegenerative diseases including AD. Because of the important role of these protein variants in different neurodegenerative disease, we hypothesize that there are distinct toxic protein variant profiles that distinguish AD, and that levels of key protein variants in these profiles will change during progression of AD and also from patient to patient. We hypothesize that by determining each individual's toxic protein variant fingerprint using biomarkers present in sera or plasma samples, we can facilitate pre-symptomatic diagnosis and staging of AD, identify appropriate therapeutic strategies for each individual patient, and also monitor effectiveness of different therapeutics in real time. The goal of this proposal is to identify blood based biomarker fingerprints that distinguish different stages AD, including presymptomatic AD, and that identify the most appropriate therapeutic strategy for each individual patient. We developed novel technology that enables us to generate antibody fragments (nanobodies) that selectively bind disease related protein variants. We have a panel of 15 nanobodies to different toxic variants of Aß, tau, a-syn and TDP-43 and have shown that we can detect these variants in blood samples of patients suffering from AD, PD and other neurodegenerative diseases, but not in age-matched cognitively normal cases. The aims of this proposal are to utilize our panel of nanobodies that selectively recognize different disease related forms of Aß, tau, a-syn and TDP-43 to characterize a set of longitudinal plasma samples taken from AD and age-matched control patients. These studies will define protein variant fingerprints that not only distinguish AD from cognitively normal controls, but will define different fingerprints for different stages of AD. We will then characterize a larger set of samples from a clinical population to determine protein variant fingerprints that correlate with different clinical symptoms, such as parkinsonism, aphasia and ataxia. Defining the various protein variant fingerprints associated with different stages and clinical manifestations of AD will provide a very powerful tool to facilitate presymptomatic and personalized diagnoses of AD, indicate the most appropriate therapeutic strategy for each individual patient, and provide a means to monitor the effectiveness of the selected therapeutic in real time.