Proceedings of The Physiological Society
University of York (2002) J Physiol 539P, S008
Genes regulated 24 h after axotomy in identified 5-HT neurons in Hirudo medicinalis
W.-Z. Wang*, K. Christoffers*, R. Emes* and S.E. Blackshaw*
*Department of Human Anatomy & Genetics and MRC Functional Genetics Unit, University of Oxford, Oxford OX1 3QX, UK
The ability to make genetic libraries from small numbers of identified nerve cells in leech CNS, known to regenerate specific connections after damage, makes it feasible to study the molecular basis of successful repair in this system. In these experiments we have used probes constructed from regenerating ganglia at different stages after damage (Emes et al. 2002) to screen for genes that are up- as well as downregulated in a defined neuron type.
To determine genes regulated in a nerve cell during an early stage of regeneration we have examined genes that are present in identified serotonergic neurons of the leech, and which change their expression 24 h after axotomy. A serotonergic neuron (Retzius cell) library, constructed using polymerase chain reaction technology from 20-30 Retzius cells, was differentially screened using subtracted cDNA probes made from a regenerating ganglion 24 h after axotomy and a non-regenerating ganglion. We have screened 1.25 X 105 pfu from the Retzius cell cDNA library. Radioactive labelling was used to visualise the probe DNA that hybridised differentially to duplicate filter lifts of the Retzius cell library. The resulting autoradiographs were compared in order to identify those clones that were either up- or downregulated 24 h after injury, and were subsequently used to identify the relevant clone in the Retzius cell library. A Southern blot was performed to determine which of the regulated clones were ribosomal RNA (rRNA). Of 22 clones analysed, six were found to be 16s or 18s rRNA. The remaining 16 clones were sequenced and analysed using the Basic Local Alignment Search Tool (BLAST) at NCBI. Of these, four clones were upregulated and 12 were downregulated. All 12 downregulated clones were found to be partial sequences of a 28S ribosomal RNA. The four upregulated clones were homologous to known proteins thioredoxin, myohemerythrin, translocon-associated protein-δ (TRAP-δ) and a proton ATPase-associated protein (H+-ATPase). These proteins have various roles including redox regulation, oxygen transport, and energy conservation. Thioredoxin in particular has a variety of biological functions related to cell proliferation and apoptosis in mammalian CNS, including cellular defence against oxidative stress and facilitation of protein nucleic acid interacations.
In summary, our approach has enabled us to identify genes that are up- and downregulated at an early stage after injury in a defined cell type. These experiments also suggest that the genes whose expression changes 24 h after axotomy are distinct from those structural and neuron-specific genes upregulated at 7 days of regeneration (Korneev et al. 1998). Successful regeneration in the leech probably depends on a wide variety of factors that both enhance a cell's likelihood to survive after injury as well as genes that promote neurite extension and synapse formation. Simultaneous attention to each of these distinct steps may be essential for determining the underlying molecular mechanisms guiding effective repair.
This work was supported by the MRC and a HFSP grant to S.E.B.
Where applicable, experiments conform with Society ethical requirements