Nerve injury enhances rat neuronal glutamate transporter expression: identification by differential display PCR

Abstract

An increase in neuronal glutamate transporter expression after nerve injury was demonstrated by means of differential display PCR (DD-PCR) coupled with in situ hybridization. DD-PCR was carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the rat. The expression of several gene fragments were found to be increased following nerve injury; the full length cDNA corresponding to one fragment was cloned by subsequent rat cDNA library screening. The close homology of glutamate transporters with our rat cDNA led us to conclude that this clone corresponds to the rat neuronal glutamate transporter (rat EAAC1). We speculate that the upregulation of this glutamate uptake system may increase the resistance of these cells against neurotoxic glutamate accumulation during the process of nerve regeneration.

Damage-induced neuronal endopeptidase (DINE) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers

“…DD-PCR was carried out by using hypoglossal nuclei of the control and operated side obtained from 70 Wistar male rats as described (1). Subsequently, each pool of cDNA was amplified by PCR in the presence of [ 35 S] dATP (New England NuclearDuPont) by using a single primer, 5Ј-ACTGGCCGAGGG-3Ј, which was randomly selected from among 87 arbitrary primers.…”

“…Then, 20-m-thick sections were cut on a cryostat, thawmounted onto 3-aminopropyltriethoxysilane-coated slides, and stored at Ϫ80°C until use. In situ hybridization was performed as described (1).…”

“…For the last few years, we have attempted to identify molecules involved in this process by using a technique known as differential display PCR (DD-PCR) and random cloning with a specific cDNA library derived from nerve-injured hypoglossal nuclei (1,2).…”

A mRNA molecule encoding truncated excitatory amino acid carrier 1 (EAAC1) protein (EAAC2) is transcribed from an independent promoter but not an alternative splicing event

“…Excitatory amino acid carrier 1 (EAAC1) is a high-affinity Na + -dependent Lglutamate/D, L-aspartate cell-membrane transporter protein. EAAC1 cDNA was originally cloned from rabbit small jejunum [4], followed by finding its counterparts in rat [7][8][9], mouse [10] and human [11][12][13][14]. Human EAAC1 gene was mapped to 9p24, which was linked to dicarboxylic aminaciduria and neurodegenerative disorders [15].…”

Use of Two Reverse Transcriptases Eliminates False-Positive Results in Differential Display

“…It has been used to distinguish altered gene expression (i) between normal vs. cancer cells (10), normal vs. senescent cells (11) and normal vs. nerve-injured cells (9), (ii)in developing Xenopus embryos (1) and in developing neonatal rats (13) and (iii) following treatment with psychomotor stimulants (7), hormones (14) and dietary supplements (3,17). The method is straightforward and was initially designed to replace other more laborious methods of distinguishing differentially expressed mRNAs, namely subtractive hybridization and two-dimensional RNA fingerprinting.…”