ELSEVIER Molecular Brain Research 37 (1996) 49-62 MOLECULAR BRAIN RESEARCH Research report Differential subcellular localization of SNAP-25a and SNAP-25b RNA transcripts in spinal motoneurons and plasticity in expression after nerve injury Gunilla Jacobsson a, Fredrik Piehl a, I. Christina Bark b, XU Zhang c, BjSrn Meister a, * a The Berzelius Laboratory, Department ofNeuroscience, Karolinska Institute, I71 77 Stockholm, Sweden b Department of Developmental Neuroscience, Uppsala University, Uppsala, Sweden c Department of Anatomy, Fifth Military Medical College, Xian, China Accepted 3 October 1995 Abstract Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in the molecular regulation of neurotransmitter release. SNAP-25 exists in two isoforms, which arise from alternative splicing of exon 5. In situ hybridization was used to examine whether SNAP-25 isoform mRNA expression may be altered by experimental manipulations. The effect of unilateral nerve injury on SNAP-25 mRNA levels was studied in motoneurons of the rat lumbar spinal cord. In all animals, SNAP-25a RNA transcripts were demonstrated in the nucleus of motoneurons, whereas SNAP-25b mRNA was present mainly in the cytoplasm. Cloning of the rat Snap gene intron spacing the alternative exon 5a and 5b sequences and generation of an intron-specific oligonucleotide probe used for in situ hybridization did not point to the presence of unspliced variants of SNAP-25b mRNA. After unilateral sciatic nerve transection (axotomy), SNAP-25a and SNAP-25b expression decreased in axotomized motoneurons compared with corresponding motoneurons on the unlesioned side. A significant decrease was demonstrated 2 days after axotomy, which reached a maximum after 7 days (62% for SNAP-25a and 67% for SNAP-25b), while levels had slightly recovered by 14 and 28 days. Ventral root avulsion also induced a decrease in levels of SNAP-25 RNA transcripts, suggesting that the axonal injury in itself was responsible for the down-regulation of Snap gene expression. This study shows that, in spinal motoneurons, SNAP-25a and SNAP-25b RNA transcripts have different subcellular localization and that levels of SNAP-25 RNA transcripts are down-regulated after axonal injury. Keywords: Axotomy; Avulsion; Spinal cord; Ventral horn; GAP-43; Synaptic protein; Nucleus; Intron; In situ hybridization; mRNA; Gene; Rat; Chicken 1. Introduction SNAP-25 (s y naptosomal-associated protein of 25 kDa) [39] was originally identified as a neuron-specific protein with a wide but differential distribution in the nervous system that has an onset of expression that coincides with synaptogenesis [6,10,40]. The protein is primarily located at the presynaptic plasma membrane and is a substrate for palmitoylation, which implies that fatty acylation may be involved in membrane targeting of the protein [19]. SNAP- 25 has been identified as a component of the 20S protein complex that is involved in vesicular docking and fusion * Corresponding author. Fax: (46) (8) 33 16 92; E-mail: Bjorn.Meis- ter@neuro.ki.se. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0169-328X(95)00272-3 steps at the presynaptic plasma membrane [5,41,54,57]. Apart from being present in the nervous system, SNAP-25 has also been identified in non-neuronal cells [22,50,52], suggesting that the protein is of importance in molecular regulation of exocytosis not only in neurons. The Snap gene encodes two isoforms of SNAP-25: SNAP-25a and SNAP-25b, which differ by only 9 amino acids and are generated by obligate alternative splicing of the two simi- lar but distinct exons 5a and 5b [3,4]. The SNAP-25a isoform predominates during embryonic and early postna- tal development, whereas SNAP-25b becomes the predom- inant species after the first postnatal week and constitutes the majority of SNAP-25 mRNA in the adult brain [6]. Complementary DNA cloning has revealed that SNAP-25 amino acid sequences show a high degree of conservation between different vertebrate species [3,4,48,49]. A het-