Distinct role of 5′UTR sequences in dendritic trafficking of BDNF mRNA: additional mechanisms for the BDNF splice variants spatial code

Figures

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  Fig. 1. Trafficking of GFP, exon 1-GFP, and exon 4-GFP transcripts in hippocampal neurons. a Representative image of an in situ hybridization against the GFP coding sequence on one hippocampal pyramidal neuron transfected with pEGFP-N1 plasmid before (upper panel) and after straightening (central panel). Bottom panels represent two control in situ hybridization on pEGFP-N1 transfected neurons using GFP coding sequence sense probe (left) and on untransfected neurons using antisense probe. Scale bar: 10 μm. b–d Densitometric analysis on in situ hybridization of apical dendrites of neurons transfected with GFP (b), exon 1-GFP (c), and exon 4-GFP (d) untreated (gray boxes) or treated with 10 mM KCl for 3 h (white boxes). Below each graph, representative straightened neurons labeled by in situ hybridization are reported. Lines in graphs C and D represent the plot of the median gray value of GFP densitometric analysis untreated (solid line) and after KCl stimulation (dashed line). Scale bar: 10 μm

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  Fig. 2. Trafficking of exon 2a-GFP, exon 2b-GFP, exon 2c-GFP, and exon 6-GFP transcripts in hippocampal pyramidal neurons. a–d Densitometric analysis on in situ hybridization of apical dendrites of pyramidal neurons transfected with exon 2a-GFP (a), exon 2b-GFP (b), exon 2c-GFP (c) and exon 6-GFP (d) untreated (gray boxes) or treated with 10 mM KCl for 3 h (white boxes). Below each graph, representative straightened neurons labeled by in situ hybridization are reported. Lines in graphs represent the plot of the median gray value of GFP densitometric analysis untreated (solid line) and after KCl stimulation (dashed line). Scale bars: 10 μm. *p ≤ 0.05 exon-GFP ctrl respect to GFP control and exon-GFP KCl vs GFP KCl

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  Fig. 3. Trafficking of exon 3-GFP, exon 5-GFP, exon 7-GFP, and exon 8-GFP transcripts in hippocampal neurons. a–d Densitometric analysis of in situ hybridization on apical dendrites of neurons transfected with exon 3-GFP (a), exon 5-GFP (b), exon 7-GFP (c) and exon 8-GFP (d) untreated (gray boxes) or treated with 10 mM KCl for 3 h (white boxes). Below each graph, representative straightened neurons labeled by in situ hybridization are reported. Lines in graphs represent the plot of the median gray value of GFP densitometric analysis untreated (solid line) and after KCl stimulation (dashed line). Scale bars: 10 μm. *p ≤ 0.05 exon-GFP ctrl respect to GFP control and exon-GFP KCl vs GFP KCl. #p ≤ 0.05 exon-GFP ctrl vs exon-GFP KCl

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  Fig. 4. Predicted folding of exon 2 mRNA variants. a–c Predicted secondary structures using the RNAfold platform from the University of Vienna (https://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) of BDNF mRNA 5′UTR splicing variants exon 2a (a), 2b (b) and 2c (c). The overall secondary structures of each exon are reported on the right. The insets on the left highlight a common region displaying a putative binding site for two clusters of RNA binding proteins: CPEB2/CPEB4, HNRNPC/CL1, HuR, TIA1 cluster (green curved line) and HNRNPA2/B1 cluster (orange straight line)