Localization and alternative splicing of agrin mRNA in adult rat brain: transcripts encoding isoforms that aggregate acetylcholine receptors are not restricted to cholinergic regions

Abstract

Agrin is a protein implicated in the formation and maintenance of the neuromuscular junction.In addition to motor neurons, agrin mRNA has been detected in the brains of embryonic rat and chick and adult marine ray, suggesting that this molecule may also be involved in the formation of synapses between neurons. As a step toward understanding agrin's role in the CNS, we utilized Northern blot and in situ hybridization techniques to analyze the regional distribution and cellular localization of agrin mRNA in the spinal cord and brain of adult rats. The results of these studies indicate that the agrin mRNA is expressed predominantly by neurons broadly distributed throughout the adult CNS. Moreover, expression of agrin mRNA is not restricted to cholinergic structures or regions of the brain receiving cholinergic input. Recently, RNA isolated from rat embryonic spinal cord was shown to contain four alternatively spliced agrin mRNAs, referred to as agrin,, agrin,, agrin,,, and agrin,,, each of which encodes agrin proteins that are active in acetylcholine receptor aggregating assays (Ferns et al., 1992). Using the polymerase chain reaction we demonstrate that all four of these agrin transcripts are expressed within the adult CNS. Agrin,, agrin,, and agrin,, were present in all regions analyzed. In contrast, agrin,, was detected only in forebrain. Results of these studies indicate that both the level of expression and pattern of alternative splicing of agrin mRNA are differentially regulated in the brain. The broad and predominantly neuronal distribution of agrin mRNA in the adult brain suggests that, in addition to its role at the neuromuscular junction, agrin may play a role in formation and maintenance of synapses between neurons in the CNS.

Selective Regulation of Agrin mRNA Induction and Alternative Splicing in PC12 Cells by Ras-dependent Actions of Nerve Growth Factor

“…2A), based on their electrophoretic mobility and confirmed by sequencing, PCR products representing two different agrin isoforms were amplified from treated and untreated cells. Consistent with previous analyses of agrin gene expression in vivo (6,15,16), transcripts lacking both exons at the Z site (agrin z0 ) were always the most abundant isoform detected. More striking, however, was the increase in the relative abundance of agrin z8 transcripts in response to NGF.…”

“…Consistent with the effect of NGF on neuronal differentiation, our studies show that NGF not only causes an increase in total agrin mRNA but also induces the expression of agrin mRNA splice variants with inserts at the Y and Z sites (agrin y4z8 , agrin y4z11 , and agrin y4z19 ), which are specifically expressed by neurons (7,8,15). The increases in agrin z8 mRNA were detected in several independent sublines of wild type PC12 cells, with the levels in NGF-treated cells similar to those found in adult rat olfactory bulb and cerebellum (6). Our results are in apparent contrast to a previous report that PC12 cells only express agrin splice variants lacking inserts at the Z site, even in the presence of NGF (15).…”

“…Reaction conditions were as described in our previous studies (6). PCR products were radiolabeled by inclusion of ϳ2 ϫ 10 5 cpm of 32 P-labeled forward primer in the reaction mixture, separated by electrophoresis on 8% polyacrylamide gels, and the relative abundance determined by analysis of the gel with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).…”

“…In developing and adult nervous tissue, agrin z0 mRNAs are the most abundant agrin transcripts, and generally account for more than 50% of the total agrin mRNA (4,6,15). However, the relative abundance of agrin Z region variants changes during development, with the levels of agrin z11 , agrin z19 , and agrin z8 mRNA peaking at early, intermediate, and late stages, respectively, in a pattern that appears to be reiterated throughout the nervous system (4,15,21).…”

Synaptic differentiation: the role of agrin in the formation and maintenance of the neuromuscular junction

“…Our current knowledge as summarized in this review, clearly shows that agrin plays a key role in the development of the NMJ. As agrin transcripts are expressed in the brain (e.g., OConnor et al 1994) and as agrin-like immunoreactivity is also found at synaptic sites in the chick retina (Mann and Kröger 1996), future studies should focus on whether agrin plays a role in the formation of interneuronal synapses.…”