Brain regeneration, an earthworm phenomenon: from molecular characterization to bioengineering a functional "brain in a dish"

战略研究重点：生物科学促进健康摘要大型无脊椎动物蚯蚓能够在手术切除后2-3周内完全再生其大脑。这种生物现象的分子遗传学基础还没有得到详细的研究，机制驱动因素在很大程度上是难以捉摸的。拟议的项目将通过识别和表征参与大脑再生的关键分子转录本和蛋白质来弥补这一不足。这些细胞和分子线索将成为基于生物电喷雾的方法的后续开发的基础，以在体外重建具有3D功能的蚯蚓大脑，即在碟子中。项目拟议的研究汇集了世界领先的蚯蚓分子遗传学(KCL主管斯特森鲍姆教授)、再生生物学/医学(UCL主管Jayasinghe博士)和蚯蚓免疫学(来自匈牙利佩克斯大学的项目合作伙伴Laszlo Molnar教授)的实验室。同时，将研究两个要点：1)分子遗传学将被应用于确定在蚯蚓大脑的发育和再生过程中涉及哪些细胞和分子。特定的分子和细胞靶点被假设参与脑再生，即GABA受体、CAPA多肽、FVRIamide、Neuromacins和体腔细胞(免疫细胞)。此外，手术切除和体内再生方案已经建立，并将与莫尔纳尔教授联系提供。然而，更多的驱动因素有待发现和定性。Sturzenbaum教授将监督靶标识别，这将通过消减杂交(例如使用PCR-Select cDNA消减试剂盒Clontech)和RNA-SEQ，即下一代全转录组鸟枪测序(WTSS)方法来实现。确定的再生生物标志物将通过定量聚合酶链式反应(QPCR)在分离的不同程度再生的大脑中得到验证。2)上述知识库随后将转移到UCL团队。单个细胞和分子将以排列/组合的方式混合，包括用于生物电喷的各种细胞外基质(ECM)材料。这将有助于随后形成包含发育中大脑的所有成分的隔间珠子。伦敦大学学院创造的这些珠子内细胞和分子的3D接近程度将被用于详细研究KCL的细胞和分子线索，最终目标是在体外开发再生大脑的功能组织。

Strategic Research Priority: Bioscience for HealthAbstract The macro invertebrate earthworm is capable of fully regenerating its brain within 2-3 weeks of surgically removal. The molecular genetic basis of this biological phenomenon has not been studied in fine detail and the mechanistic drivers are largely elusive. The proposed project will redress this short-fall by identifying and characterizing key molecular transcripts and proteins involved in brain regeneration. These cellular and molecular cues will form the basis of the subsequent development of a bio-electrospray based approach to recreate a 3D functional earthworm brain ex vivo, i.e. in a dish.Project The proposed study brings together world-leading laboratories in earthworm molecular genetics (Prof Sturzenbaum, the supervisor at KCL), regenerative biology/medicine (Dr Jayasinghe, the supervisor at UCL) and earthworm immunology (Prof Laszlo Molnar, a project partner from the University of Pecs, Hungary). In concert, the two overarching points will be studied: 1) Molecular genetics will be applied to define what cells and molecules are involved during the development and regeneration of the earthworm brain. Specific molecular and cellular targets have been hypothesized to be involved in brain regeneration, namely the GABA-receptors, CAPA peptides, FVRIamides, Neuromacins, and coelomocytes (the immune cells), respectively. Furthermore the surgical removal and in vivo regeneration protocols are established and will be provided in liaison with Prof Molnár. However, many more drivers await discovery and characterization. Prof Sturzenbaum will supervise the target identification, which will be achieved by Subtractive Hybridization (e.g. using the PCR-Select cDNA Subtraction Kit, Clontech) and RNA-seq, the next-generation Whole Transcriptome Shotgun Sequencing (WTSS) approach. Identified biomarkers of regeneration will be validated by quantitative PCR (qPCR) in brains isolated at varying degrees of regeneration. 2) This above knowledge-base will be subsequently transferred to the UCL team. Individual cells and molecules will be mixed in a permutation / combination approach including a wide range of extracellular matrix (ECM) materials for bio-electrospraying. This will facilitate the subsequent formation of a compartmentalized bead containing all the constituents of the developing brain. The 3D proximity of the cells and molecules within these beads created at UCL will be used to study in detail the cellular and molecular cues at KCL, with the ultimate goal of developing, ex vivo, functional tissue of a regenerating brain.