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

Strategic Research Priority: Bioscience for Health
Abstract
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.