Establishing the earthworm as a model to study natural processes of neuronal regeneration

: new insights from histological, transcriptomic and biophysical studies

Abstract

The hallmark of neurodegenerative disease in humans, including Alzheimer’s, Amyotrophic Lateral Sclerosis and Parkinson’s, lies in our inability to efficiently restore damaged neurons. Contrary to widespread belief, humans can regenerate neurons within the Peripheral Nervous System (PNS) and some areas of the Central Nervous System (CNS), however the underlying mechanisms which drive or repress complete functional and structural neuronal regeneration remain elusive. To develop our understanding of this natural phenomenon one should focus on species which are capable of efficient neuronal regeneration following neurotrauma. The earthworm species Eisenia fetida can regenerate their Cerebral Ganglion (CG) - loosely defined as the brain, within a few weeks following surgical removal. The characterization of fundamental aspects of neuronal regeneration in the earthworm promises to provide an insight as to why humans have largely lost that capability. Here we present a detailed micro-dissection protocol that has been developed to excise the CG and study the progression of its regeneration. The Ventral Nerve Cord (VNC) of the worm is a tissue which connects the CG to the rest of the nervous system via the Circumpharyngeal Connectives (CC). Exploration of molecular dynamics through changes in the transcriptome were determined in the VNC and CC at 1 week and 5-week post-decerebration using an RNAseq approach (90 million reads/condition, 100bp Paired End). RNAseq established that specific groups of genes are up- or downregulated in the regenerating VNC. More than 500 significantly enriched biological processes were identified throughout the regeneration process, including vascular development, neurogenesis and extracellular matrix organization, as well as more than 100 significantly enriched molecular functions, including calcium ion binding, metal ion binding and metalloendopeptidase activity. Differential expression of transcripts was confirmed via qPCR. Examples of transcripts which have been validated at 1w post-decerebration, include catalase (~34-fold), superoxide dismutase 1 (sod-1) (~12-fold) and transcription factor jun-B (junb) (~3-fold). On the other hand, examples of transcripts which have been validated at 5w post-decerebration, include Glial Fibrillary Acidic Protein (GFAP) (~101-fold), adam19 (~17-fold) and Bone Morphogenetic Protein 1 (BMP1) (~3-fold). Moreover, the large number of differentially expressed metalloproteins (ADAMs, BMPs, MMPs, metallothionein) identified in the transcriptome, led to the hypothesis that metal trafficking could play a role in the course of regeneration. This was confirmed using a synchrotron-based approach, namely X-Ray Fluorescence (XRF) spectroscopy, where Zinc (Zn) and Iron (Fe) show a differential distribution pattern across different stages (at 1 week to 10-weeks post-decerebration) of regeneration. Lastly, various histological techniques were implemented to characterize/describe neuronal structures during the regenerating process, including immunohistochemistry and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL). Furthermore, numerous novel markers and biological processes have been identified which, to date, have not been linked to neuronal regeneration. In summary, the results suggest that the increase of axon growth promoting factors as well the decrease of growth inhibitory factors act in conjunction to ensure efficient neuronal regeneration in the earthworm.

Date of Award	1 Mar 2020
Original language	English
Awarding Institution	King's College London
Supervisor	Stephen Sturzenbaum (Supervisor)