Complementary mass spectrometry based detection of gangliosides within the brain

原理：神经节苷脂是鞘糖脂的主要种类，在中枢神经系统（CNS）中富集。神经节苷类具有广泛的中枢神经系统功能，包括膜功能、轴突稳定性和再生、分化和神经变性。在NSERC的资助下，我们的实验室已经证明神经元对脂质微环境的变化高度敏感。具体来说，我们已经发表了有害的神经节苷类GM2和GM3，它们都是GM1的前体和分解产物，在小鼠和大鼠和大脑的神经元受到应激刺激后会短暂增加。此外，我们最近发表了外源性递送的GM1可以保护初级神经元免受兴奋性毒性的影响。基于这些已发表的和初步的研究，我们假设神经节苷脂GM2和GM3在神经元应激后短暂增加。我们进一步预测，防止GM1分解为GM2和GM3可以预防神经退行性变。******目标：该提案建立在我们目前的NSERC资助的基础上，目标如下：******1)在体外和体内使用药理学和siRNA方法来靶向神经节苷脂合成和降解途径中的酶，以增加神经元内的GM1。这将使我们能够验证一个假设，即内在地增加GM1可以保护神经元免受细胞死亡。******2)继续发展方法，以增强基于成像质谱（IMS）检测神经节苷脂和其他脂质的能力，特别是在固定的啮齿动物和人类大脑样本中。迄今为止，在甲醛和/或福尔马林固定组织内进行IMS的研究取得了有限的成功。将IMS图像与组织病理学结果叠加的能力将对IMS领域做出重大贡献。******途径：神经节苷脂，像其他脂类一样，包含在大脑的不同细胞区室中，并具有改变其结构以满足其外部和内部环境需求的能力。这种改变结构的能力使得脂质既迷人又具有技术挑战性，难以识别和理解。为了应对这一挑战，我们采用互补质谱（MS）技术，包括IMS直接在脑组织中检测和成像神经节苷脂。我们将使用药理学和siRNA介导的干预来阻止GM1分解为GM2和GM3，以进一步验证GM2和GM3是神经退行性变的基本介质。最后，我们通过开发在福尔马林固定组织样本中成像神经节苷脂的方法来推动IMS技术的界限。******意义：这一提议将增加对神经节苷脂和其他脂质在介导神经元易感性和神经变性中的作用的认识。该建议还将推动基于IMS的完整组织样本中脂质检测的边界。**

Rationale: Gangliosides are a major class of glycosphingolipids and are enriched within the central nervous system (CNS). Gangliosides have a wide range of CNS functions including membrane function, axon stability and regeneration, differentiation and neurodegeneration. Using NSERC funding, our lab has shown that neurons are highly sensitive to changes within the lipid micro-environment. Specifically, we've published that harmful gangliosides GM2 and GM3, which are both precursors and breakdown products of GM1, are transiently increased following stressful stimuli to neurons in the mouse and rat and brain. Furthermore, we have recently published that exogenously delivered GM1 can protect primary neurons from excitotoxicity. Based on these published and preliminary studies we hypothesize that gangliosides GM2 and GM3 are transiently increased following neuronal stress. We further predict that preventing the breakdown of GM1 into GM2 and GM3 can prevent neurodegeneration.******Objectives: This proposal builds upon our current NSERC funding with the following objectives:******1) Use pharmacological and siRNA approaches both in vitro and in vivo to target enzymes within the ganglioside synthetic and degradation pathways to increase GM1 within neurons. This will allow us to test the hypothesis that intrinsically increasing GM1 can protect neurons against cell death.******2) Continue to develop methodology to enhance the capabilities of imaging mass spectrometry (IMS) based detection of gangliosides and other lipids, specifically in fixed rodent and human brain samples. To date, IMS within formaldehyde and/or formalin fixed tissues has been met with limited success. The ability to overlay IMS images with histopathological findings would be a significant contribution to the field of IMS. ******Approach: Gangliosides, like other lipids, are contained within different cellular compartments of the brain and have the ability to change their structure to meet their external and internal environmental demands. This ability to change structure makes lipids both fascinating and technologically challenging targets to identify and understand. To meet this challenge, we employ complementary mass spectrometry (MS) techniques, including IMS to detect and image gangliosides directly within brain tissue. We will use pharmacological and siRNA mediated intervention to prevent the breakdown of GM1 into GM2 and GM3 to further validate that GM2 and GM3 are fundamental mediators of neurodegeneration. Finally, we push the boundaries of IMS technology by developing methodology to image gangliosides within formalin-fixed tissue samples. ******Significance: This proposal will add knowledge on the role of gangliosides and other lipids in mediating neuronal vulnerability and neurodegeneration. This proposal will also push the boundaries of IMS based detection of lipids within intact tissue samples. **