BDNF agonist treatment in experimental allergic encephalomyelitis

描述（由申请人提供）： 多发性硬化症（MS）的病理特征是炎症和神经变性。由于目前MS的治疗方法是免疫调节剂，不能预防神经元损失和残疾，因此一直在寻求神经保护性治疗。脑源性神经营养因子（BDNF）是一种具有神经保护特性的神经营养细胞因子，已被研究为潜在的治疗方法。在MS的动物模型中，实验性过敏性脑脊髓炎（EAE），BDNF由免疫细胞产生，并负责减少轴突损失。BDNF存在于MS病变中，并可能在病变发病机制中起保护作用。外源性给予BDNF的研究受到其血清半衰期短和血脑屏障（BBB）渗透性差的限制，因此我们进行了使用基因工程骨髓干细胞在EAE中递送BDNF的研究。BDNF减少炎症和神经变性，我们的观察结果已经被Linker等人（4）使用慢病毒递送系统证实。总之，这些研究表明，BDNF治疗减少了EAE的炎症和神经变性。 在2010年，Jang等人报道了7，8-二羟基黄酮（DHF）具有有效的BDNF激动剂活性，可以口服并容易穿过BBB。在培养的神经元中，它激活了BDNF、TrkB和下游信号的受体。它保护野生型，但不是TrkB缺陷的神经元凋亡。DHF激活小鼠脑中的TrkB，抑制红藻氨酸诱导的神经毒性，以TrkB依赖性方式减少实验性小鼠中风中的梗死体积，并且在帕金森病动物模型中具有神经保护作用。并非所有DHF效应都是TrkB依赖性的：DHF具有NF κ B依赖性的抗炎活性和可减少炎性损伤的抗氧化作用。我们在C57 Bl/6小鼠中测试了DHF在MOG诱导的EAE中的作用，并且显示即使当治疗延迟至症状发作时，治疗也导致临床和病理学严重程度降低。这些结果共同支持DHF作为MS的可能治疗的进一步研究。基于上述结果，DHF至少有四种可能的机制可以降低EAE的严重程度：它可以通过其抗氧化活性减少炎症损伤，可以通过非TrkB介导的途径减少炎症，它可以通过TrkB介导的对炎性细胞的作用减少炎症，或者它可以对神经元细胞具有直接的TrkB介导的保护作用。我们提出以下初步假设：DHF治疗C57 Bl/6小鼠MOG诱导的EAE可降低疾病的临床和病理严重程度。我们提出以下次要假设：DHF治疗C57 B1/6小鼠中MOG诱导的EAE通过TrkB依赖性机制降低疾病严重程度。我们将通过完成以下具体目标来测试这些假设：具体目标#1：优化患有MOG诱导的EAE的C57 B1/6小鼠的DHF给药。具体目标#2：确定在症状发作时开始DHF治疗对C57 B1/6小鼠中MOG诱导的EAE中CNS炎症、免疫、脱髓鞘、轴突损失和细胞凋亡的影响。 具体目标#3：确定在C57 B1/6小鼠中在MOG诱导的EAE中从第50天开始的DHF治疗对CNS炎症、免疫、脱髓鞘、轴突损失和细胞凋亡的影响。 具体目标#4：通过确定TrkBF 616 A敲入C57 B1/6小鼠对DHF在具有和不具有功能性TrkB的小鼠中的作用的敏感性，确定DHF在C57 B1/6小鼠中MOG诱导的EAE中的作用是否是TrkB依赖性的。 证实我们的假设将提供有关DHF治疗在MS模型中的作用的重要新信息，并可为DHF在MS患者中的研究提供理论基础。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is characterized pathologically by inflammation and neurodegeneration. Because current treatments for MS, which are immune modulators, do not prevent neuronal loss and disability, neuroprotective treatments have been sought. Brain derived neurotrophic factor (BDNF) is a phleotrophic cytokine with neuroprotective properties that has been studied as a potential treatment. In an animal model of MS, experimental allergic encephalomyelitis (EAE), BDNF is produced by immune cells and is responsible for reducing axonal loss. BDNF is present in MS lesions and may play a protective role in lesion pathogenesis. Studies of exogenously administered BDNF have been limited by its short serum half-life and poor blood brain barrier (BBB) penetration so we conducted studies using genetically engineered bone marrow stem cells to deliver BDNF in EAE. BDNF reduced inflammation and neurodegeneration and our observations have been confirmed by Linker et al (4) using a lentivirus delivery system. Together these studies suggest that BDNF treatment reduces inflammation and neurodegeneration in EAE. In 2010 Jang et al. reported that 7,8-dihydroxyflavone (DHF) has potent BDNF agonist activity, can be given orally and readily crosses the BBB. In cultured neurons it activated the receptor for BDNF, TrkB, and downstream signaling. It protected wild-type, but not TrkB-deficient neurons from apoptosis. DHF activated TrkB in the mouse brain, inhibited kainic acid-induced neurotoxicity, decreased infarct volumes in experimental murine stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson's disease. Not all DHF effects are TrkB dependent: DHF has anti-inflammatory activity that is NFKB dependent and antioxidant effects that could reduce inflammatory injury. We tested DHF in MOG-induced EAE in C57Bl/6 mice and showed that treatment resulted in reduced clinical and pathological severity even when treatment was delayed until the onset of symptoms. Together these results support further studies of DHF as a possible treatment for MS. Based on the results outlined above, there are at least four possible mechanisms through with DHF could reduce the severity of EAE: It could reduce inflammatory injury through its antioxidant activity, i could reduce inflammation through a non-TrkB mediated pathway, it could reduce inflammation through a TrkB mediated effect on inflammatory cells or it could have a direct TrkB mediated protective effect on neuronal cells. We propose the following Primary Hypothesis: DHF treatment of MOG-induced EAE in C57Bl/6 mice reduces the clinical and pathological severity of disease. We propose the following Secondary Hypothesis: DHF treatment of MOG-induced EAE in C57Bl/6 mice reduces disease severity by a TrkB dependent mechanism. We will test these hypotheses by completing the following specific aims: Specific Aim #1: Optimize DHF dosing of C57Bl/6 mice with MOG-induced EAE. Specific Aim #2: Determine the effect of DHF treatment started at the time of symptom onset on CNS inflammation, immunity, demyelination, axonal loss and apoptosis in the MOG-induced EAE in C57Bl/6 mice. Specific Aim #3: Determine the effect of DHF treatment started on day 50 on CNS inflammation, immunity, demyelination, axonal loss and apoptosis in MOG-induced EAE in C57Bl/6 mice. Specific Aim #4: Determine whether the effect of DHF in MOG-induced EAE in C57Bl/6 mice is TrkB dependent by determining the sensitivity of the TrkBF616A knock-in C57Bl/6 mouse to the effects of DHF in mice with and without functioning TrkB. Confirming our hypotheses will provide important new information about the effects of DHF treatment in a model of MS and could provide a rationale for studies of DHF in MS patients.