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Human BDNF Isoforms are Differentially Expressed in Cocaine Addicts and are Sorted to the Regulated Secretory Pathway Independent of the Met66 Substitution

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Abstract

Differential BDNF gene (BDNF) promoter use leads to protein isoforms differing by 8 or 15 N-terminal residues (BDNF1 and BDNF2) whose regulation and function are not completely understood versus the well-known 247-aa BDNF “short” form. To describe how BDNF isoform levels were regulated by chronic drug use, we measured BDNF isoform-specific mRNA levels in different human brain regions from cocaine addicts relative to age, race, and gender-matched controls. The cocaine group had threefold higher levels of exon 4-specific (BDNF Short) mRNAs in cerebellum versus controls (P < 0.01). In cortex, exon 4 and exon 1-specific BDNF mRNA levels (BDNF1) were significantly reduced in the cocaine group relative to controls (40%, P < 0.01). We also tested the hypothesis that the signal peptides of isoforms BDNF1 and BDNF2 confer different functional properties and determined if the functional Val66Met polymorphism influenced these functions. In contrast to transfected AtT-20 cells producing BDNF Short, regulated secretion of BDNF1 or BDNF2 was not affected by the Met66 substitution. Hippocampal neurons producing BDNF1 or BDNF2 on either the Val66 or Met66 background were similarly distributed in dendrites and had similar colocalization patterns with the secretory granule marker Sec II. This pattern differed from neurons producing BDNF Short Met66, which had impaired trafficking. Together, these findings support a mechanism by which variant BDNF proteins can overcome the functional defect of the Met66 substitution and suggest how functional differences in BDNF may impact brain responses in disease.

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Acknowledgments

This study was supported by NIH intramural grant NIAAA Z01-AA00325 (RHL) and extramural grant F-192EG-C1 from the Defense Brain and Spinal Cord Injury Program (AMM).

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Authors and Affiliations

  1. Section on Molecular Genetics, Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, Room 3S32, MSC 9412, Bethesda, MD, 20892-9412, USA

    Xueying Jiang, Jian Zhou & Robert H. Lipsky

  2. Department of Neurology, University of Miami Medical School, Miami, FL, USA

    Deborah C. Mash

  3. Department of Neurology and Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, MD, USA

    Ann M. Marini

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  1. Xueying Jiang
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Correspondence to Robert H. Lipsky.

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Fig. S1

BDNF–GFP fusion protein predicts a furin proprotein convertase cleavage site. The pre-BDNF–GFP cDNA initially used in functional studies contained a furin site which destabilized the fusion protein. Mutations in of the original plasmid construct were created, replacing two arginine residues with two glycine residues (TIFF 139 kb)

Fig. S2

Enhanced stability of BDNF–GFP fusion proteins following site-directed mutagenesis. Left panel AtT-20 cells were transfected with each BDNF-GFP isoform (S = BDNF Short, 1 = BDNF1, 2 = BDNF2). Cell lysates were used for protein blots. BDNF Short-GFP and BDNF2-GFP were unstable prior mutagenesis. To visualize free GFP, cells were also transfected with pGFP-N1 (Clontech/Takara Bio, Mountain View, CA). Position of BDNF–GFP fusion proteins and free GFP detected with anti-GFP antibody are indicated by the arrows. Right panel: Stripped blots were reincubated with anti-β-actin antibody to show equal loading (TIFF 206 kb)

Fig. S3

Detecting pre-proBDNF isoform-specific cDNAs in postmortem human cerebellum. RT-PCR using specific primer sets for each BDNF isoform cDNA. RT-PCR with a primer pair 7A resulted in a band (molecular size 264 bp) for BDNF2 in addition to a smaller band (210 bp) for BDNF Short Form, due to alternative splicing of exon 7. RT-PCR with primer pair exon 7B/terminal coding exon (exon 9, Pruunsild et al. 2007) produced only the 264 bp band for BDNF 2. Each BDNF cDNA was verified by sequence analysis (TIFF 154 kb)

Fig. S4

Expression of BDNF by transfected AtT-20 cells. AtT-20 cells were transfected with BDNF-GFP Short Val, BDNF 1 Val, BDNF2 Val, BDNF Short Met, BDNF1 Met, or BDNF2 Met constructs. Protein blots were developed with an anti-GFP antibody. Upper panel Cell lysates were prepared 48 h posttransfection and BDNF–GFP fusion proteins visualized. Lower panel Blot from upper panel was stripped of protein and re-incubated with anti-β-actin to show equal loading. Both pro and mature forms of BDNF1 and BDNF2 were increased compared with AtT-20 cells expressing the BDNF Short (S). Presence of the Met66 substitution in the prodomain did not affect expression of any of the BDNF isoforms (TIFF 111 kb)

Fig. S5

Representative image of a BDNF–GFP expressing neuron. Arrows show distribution of the fusion protein (TIFF 479 kb)

Fig. S6

Signal P prediction of signal peptides, nonsignal sequences, and cleavage sites in BDNF1. Probabilities for the N-region, H-region, and C-region are shown as peaks above the primary amino acid sequence for the BDNF1 signal peptide. The predicted signal peptidase cleavage site is shown by a vertical red line with a probability of cleavage of 0.681 between residues 26 and 27 (TIFF 189 kb)

Fig. S7

Signal P prediction of signal peptides, nonsignal sequences, and cleavage sites in BDNF2. Probabilities for the N-region, H-region, and C-region are shown as peaks above the primary amino acid sequence for the BDNF2 signal peptide. The predicted cleavage site by signal peptidase, shown by the vertical red line, has a site selection probability of 0.043 for cleavage between residues 33 and 34 (TIFF 181 kb)

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Jiang, X., Zhou, J., Mash, D.C. et al. Human BDNF Isoforms are Differentially Expressed in Cocaine Addicts and are Sorted to the Regulated Secretory Pathway Independent of the Met66 Substitution. Neuromol Med 11, 1–12 (2009). https://doi.org/10.1007/s12017-008-8051-0

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