Partial images were systematically acquired for sections of each cell using the 20x objective until the entire cell was imaged. Neurite outgrowth is a fundamental event in the development and maintenance of synaptic connections in the nervous system. Through highly regulated mechanisms, young neurons undergo axonal/dendritic polarization, and subsequent outgrowth of these neurites is essential to the establishment of synaptic connections that lead to brain function (da Silva and Dotti, 2002). Cell adhesion molecules (CAMs) are a diverse class of proteins that function in neurite outgrowth, synaptic development PNPP and maintenance, and cell adhesion at synaptic and non-synaptic sites Mouse monoclonal antibody to PRMT1. This gene encodes a member of the protein arginine N-methyltransferase (PRMT) family. Posttranslationalmodification of target proteins by PRMTs plays an important regulatory role in manybiological processes, whereby PRMTs methylate arginine residues by transferring methyl groupsfrom S-adenosyl-L-methionine to terminal guanidino nitrogen atoms. The encoded protein is atype I PRMT and is responsible for the majority of cellular arginine methylation activity.Increased expression of this gene may play a role in many types of cancer. Alternatively splicedtranscript variants encoding multiple isoforms have been observed for this gene, and apseudogene of this gene is located on the long arm of chromosome 5 (Craig and Banker, 1994;Dalva et al., 2007). Several CAMs are enriched at growth cones and are required for normal neurite outgrowth. For example, neural cell adhesion molecule (NCAM), N-cadherin, and L1-CAMs have been shown to regulate neurite outgrowth through various mechanisms, including changes in intracellular calcium levels, associations with cytoskeletal proteins at growth cones, and the activation of FGFR and MAPK signaling cascades (Doherty et al., 2000;Francavilla et al., 2007;Meiri et al., 1998;Utton et al., 2001). In humans, mutations in L1-CAMs lead to various neurological disorders, including hydrocephalus and MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome, and expression of constructs encoding L1 with these known mutations leads to deficits in neurite outgrowth (Moulding et al., 2000). While a wealth of information implicates CAMs in neurite outgrowth, the mechanism is highly complex and not completely understood. Synaptic adhesion-like molecules (SALMs) are a family of CAMs that is largely restricted to the CNS and is involved in neurite outgrowth and synapse formation (Ko et al., 2006;Morimura et al., 2006;Wang et al., 2006). SALMs are also present in the adult where they may play a role in synaptic maintenance and other cellular interactions. Five family members have been identified: SALMs 15 (Ko et al., 2006;Morimura et al., 2006;Wang et al., 2006). The domain structure of SALMs includes extracellular leucine-rich repeats (LRR), an immunoglobulin C2-like domain (IgC2), a fibronectin type III (FN3) domain, PNPP a transmembrane (TM) region, and a PDZ-BD (PSD-95, Discs-large, ZO-1, binding domain; absent in SALMs 4 and 5). This domain structure is homologous with that of various related CAMs that function in outgrowth, including AMIGO, LINGO, NGL-1, and FLRT proteins (Chen et al., 2006). Over-expression of SALM1 in young (4 days in vitro, DIV4) primary hippocampal cultures promotes an increase in neurite outgrowth (Wang et al., 2006), while alterations in SALM2 expression affects synapse formation and may play a role in regulating the balance of excitatory and inhibitory synapses (Ko et al., 2006). PNPP Therefore, individual SALMs may have a range of different functions. Alternatively, all SALMs may have multiple roles and function in neurite outgrowth and synapse formation in developing animals, as well as maintenance of synapses in adults. To investigate these possibilities, we have studied the role of all SALMs in neurite outgrowth by using a combination of over-expression, RNAi-mediated knock-down of expression, and blocking of function with antibodies to extracellular domains. Our results show that all SALMs promote neurite outgrowth, but with various phenotypes. == Results == == Distribution of SALMs in neurons == SALM1 and SALM2 localize to both axons and dendrites (Ko et al., 2006;Wang et al., 2006). Additionally, SALM1 co-localizes with NMDA receptors (Wang et al., 2006), while SALM2 co-localizes with both pre- and post-synaptic proteins at excitatory synapses in mature neurons (Ko et al., 2006). To understand the roles of SALMs in neurite outgrowth, we began by characterizing the cellular localization and morphological effects of overexpressed SALMs early in neuronal development. Young primary hippocampal neurons (DIV4) were co-transfected with GFP and myc-SALM1, myc-SALM2, untagged SALM3, myc-SALM4, or HA-SALM5 cDNA constructs. Neurons transfected with GFP and pcDNA 3.1+empty vector were used as a control, and immunocytochemistry was performed 48 hours after transfection. Transfected SALM constructs over-expressed their respective proteins by about 300%, as compared to endogenous SALM levels (data not shown). Over-expressed SALMs are localized throughout the cell in the soma, axons, dendrites, and growth cones (Fig. 1) with a largely diffuse pattern. However, punctate staining is present and is particularly apparent when staining is restricted to SALMs present on the cell surface (supplemental Fig. 1). Therefore, SALMs are present in intracellular pools represented by the diffuse staining as well as on the surface where.
Categories