Complementary mass spectrometry based detection of gangliosides and other lipids in the brain

理论基础：神经节苷脂是中枢神经系统(CNS)中富含的一类主要糖鞘糖脂，与大脑中的其他脂类一样，在一系列活动中发挥重要作用，包括膜功能、轴突稳定和再生、分化和神经退行性变。利用NSERC的资金，我们的实验室开发并使用了互补质谱学(MS)方法，包括成像质谱学(IMS)，以表明神经元对脂质微环境中的变化高度敏感。具体地说，我们已经发表了有害的神经节苷脂GM2和GM3在应激刺激小鼠和大鼠以及大脑中的神经元后瞬时增加，并且在体外和体内增强神经保护性神经节苷脂GM1保护神经元免受细胞死亡。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在神经元应激后一过性增加。我们进一步预测，阻止GM1分解为GM2和GM3可以防止神经退行性变。*目标：这项建议建立在我们目前NSERC资金的基础上，目标如下：*1)使用体外和体内的siRNA方法来靶向神经节苷脂降解途径中的酶，以瞬时增加神经元内的GM1。这将使我们能够检验这样的假设，即本质上和瞬时地增加GM1可以保护神经元免受细胞死亡的影响。*2)通过扩展成像质谱仪的功能来检测和成像其他类型的脂类，如甘油磷脂和神经鞘脂脂，并对其进行成像，从而建立和扩展我们目前的脂质计划。*方法：神经节苷脂，像其他脂类一样，包含在大脑的不同细胞区段中，能够改变其结构，以满足其外部和内部环境的需求。这种改变结构的能力使脂类既令人着迷，又具有技术挑战性，是识别和理解的目标。为了应对这一挑战，我们使用了互补质谱学(MS)技术，包括成像质谱学(IMS)来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介导的干预措施来防止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，我们将扩展我们的IMS能力以成像大脑中的额外脂质，以更好地了解脂质在调节神经退行性变易损性中的作用。*意义：这项建议将增加关于神经节苷脂和其他脂质在介导神经元脆弱性和神经退行性变中的作用的知识。这项提议还将推动基于IMS的完整组织样本中脂质检测的边界。*****

Rationale: Gangliosides are a major class of glycosphingolipids enriched within the central nervous system (CNS), and like other lipids in the brain play a major role in a wide range of activities including membrane function, axon stability and regeneration, differentiation and neurodegeneration. Using NSERC funding, our lab has developed and used complementary mass spectrometry (MS) approaches including imaging mass spectrometry (IMS) to show that neurons are highly sensitive to changes within the lipid microenvironment. Specifically, we've published that harmful gangliosides GM2 and GM3 are transiently increased following stressful stimuli to neurons in the mouse and rat and brain and that enhancing neuroprotective ganglioside GM1 protects neurons from cell death in vitro and in vivo. 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 siRNA approaches both in vitro and in vivo to target enzymes within the ganglioside degradation pathway to transiently increase GM1 within neurons. This will allow us to test the hypothesis that intrinsically and transiently increasing GM1 can protect neurons against cell death.******2) Build and expand our current lipidomic program by extending imaging mass spectrometry capabilities to detect and image other classes of lipids such as glycerophospholipids and sphingolipids in rodent models of aging and neurodegeneration******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 imaging mass spectrometry (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 will expand our IMS capabilities to image additional lipids within the brain to gain a better understanding on the role for lipids in mediating vulnerability to neurodegeneration.******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. *****