Val66Met polymorphism of BDNF alters prodomain structure to induce neuronal growth cone retraction

Abstract

A common single-nucleotide polymorphism (SNP) in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This SNP is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism, we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75(NTR) and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand, which modulates neuronal morphology.

Figure 1

The BDNF prodomain is detected at high levels in vivo. (a) Detection of BDNF prodomain (15.5 kDa.) from mice hippocampus after glutaraldehyde fixation of the transfer membrane following SDS-PAGE. (b) The prodomain levels were reduced by half in Bdnf^+/− mice hippocampal lysates, compared to wild type animals (WT). (c) Treatment of hippocampal lysates with N-glycanase (+Gly) reduced the molecular weight of the prodomain to 12.3 kDa. (d) BDNF prodomain levels at embryonic day 18 (E18), postnatal days 0 and 5 (P0 and P5), and 1, 2, 3 or 9 months (M) in hippocampal lystes of C57BL/6 mice. (e) Quantification of (d). Bars represent mean ± s.e.m. of prodomain densitometry data normalized to β tubulin. n=3 per group. (f) Comparison of BDNF prodomain levels from hippocampi of Bdnf^Val/Val and Bdnf^Met/Met mice at P0, P5, 1 and 2 month of age. (g) Quantification of (f). Bars represent mean ± s.e.m. of prodomain densitometry data normalized to β tubulin. n=4 per group. Statistical comparisons were made by one way analysis of variance test. * p < 0.05.

Figure 2

The BDNF prodomain is secreted from cultured neurons. (a) Prodomain was detected in the media of day 6 in vitro rat hippocampal cultures. Cultures were treated with or without α-2 anti-plasmin and/or MMP inhibitor II (MMP1, 3, 7 and 9 inhibitor) in order to prevent extracellular cleavage of secreted proBDNF. Activity-dependent secretion was achieved by depolarization with KCl. (b) Quantification of (a). Bars represent mean ± s.e.m. of prodomain densitometry data. n=3 per group. (c) Treatment with N-glycanase reduced the molecular mass of the secreted prodomain. (d) Prodomain was detected in the media of day 6 in vitro hippocampal-cortical neurons from E18 pups obtained from Bdnf^Met/+ × Bdnf^Met/+ mice litters. Level of secreted prodomain differ between Bdnf^Val/Val and Bdnf^Met/Met cultures. (e) Quantification of (d). Bars represent mean ± s.e.m. of prodomain densitometry data. n=4 per group. Statistical comparisons were made by one way analysis of variance test. * p < 0.05.

Figure 3

Impact of the Val66Met substitution on the structure of the BDNF prodomain. (a) Overlay of the heteronuclear single-quantum coherence (HSQC) spectrum on the Val66 (blue) and Met66 (red) prodomains. Each cross-peak (chemical shift) corresponds to one residue within the sequence of the prodomain (one chemical shift for each covalently bonded pair of ¹H-¹⁵N atoms assigned to specific amides within the prodomain sequence). Full HSQC spectra of Val66 and Met66 prodomains are available in Supplementary Fig. S5. The backbone chemical shifts for the prodomains are deposited in the Biological Magnetic Resonance Bank: Val66 ID number: 19358. Met66 ID number: 19357. (b) Chemical shift deviation (Δδ) between BDNF Val66 and Met66 prodomains showed that changes induced by the substitution are localized to seven residues (E64, H65, I67, E68, E69, L70, L71) neighboring the Val66Met substitution site. The variation in Δδ for residues 23-55, and 77-113 outside the display window was between 0.007 and 0.0037 ppm.

Figure 4

Val66 and Met66 prodomains differ in transient secondary structure.(a) Secondary structure propensity (SSP) score obtained using the backbone ¹H, ¹⁵N, ¹³C chemical shifts from the BDNF prodomain Val66 (blue bars) and Met66 (red bars). The SSP score identified regions of transient structure formation (positive values = α-helix, negative values = β-sheet). (b)(c) Graphs illustrating secondary structure prediction by TALOS+ analysis using the heteronuclear backbone chemical shifts of Val66 (b) and Met66 (c) prodomains. α-helix in blue spheres, β-strand in red squares, and disorder in green triangles. TALOS+ analysis showed decreased β-sheet propensity in the Met66 prodomain compared to the Val66 prodomain, result that is consistent with the SSP score. (d) Ultraviolet circular dichroism (CD) spectra of 30 μM of the Val66 (blue) and Met66 (red) prodomains collected in10 mM NaH₂PO₄ and 50 mM NaCl pH 7.0 at 23°C. The negative peak around 200 nm revealed the natively unfolded conformation of both prodomains. However, the lower absorption at 222 nm for Met66 prodomain is consistent with increased tendency to helical conformation compared to the Val66 prodomain. Each spectrum is representative of 4 averaged scans and is normalized to the spectrum of buffer alone.

Figure 5

The Met66 prodomain induces growth cone retraction in cultured hippocampal neurons. (a) Neurons were treated with Val66 or Met66 prodomains (10 ng/ml) for 20 min, fixed, and stained for actin and p75^NTR. Arrows indicate retracted growth cones; asterisks indicate intact growth cones. Scale bar: 20 μm. (b) Quantification of growth cone retraction in p75^NTR positive cells shown in (a), compared to p75^NTR negative cells. Prodomain abbreviated as prod. (b) n= 4 independent experiments. Bars represent mean ± s.e.m. Statistical comparisons were made by one way analysis of variance test. * p <0.05.

Figure 6

The Val66 and Met66 prodomains binds differently to SorCS2 but do not bind to p75^NTR. (a, b) HEK293T cells were transfected with the indicated constructs, the lysates were immunoprecipitated with anti-HA antibody, followed by detection with indicated antibodies. V=Val66 prodomain and M=Met66 prodomain constructs. (a) We were unable to detect interaction of either prodomains with p75^NTR by co-immunoprecipitation. Representative blot of 4 independent experiments. (b) Both Val66 and Met66 prodomains co-immunoprecipitated with SorCS2, however Met66 prodomain interacted ~23% more than the Val66 as quantified in (c). (c) n=9 independent experiments. Bars represent mean ± s.e.m. Statistical comparisons were made by one way analysis of variance test. * p <0.05. Interaction of Val66 and Met66 prodomains with SorCS2 (d) or p75^NTR (e) was also assessed by NMR spectroscopy. Δδ = chemical shift deviation between Val66 and Met66 prodomains together with the receptors. Interaction of the prodomain with SorCS2 (d) was assessed at 4.5μM for both proteins and data were collected at 600 MHz. The Met66 prodomain displayed greater chemical shift changes upon interaction to SorCS2 between residues 65 and 71 (shown under #) compared to the Val66 prodomain. Interaction of the prodomain with p75^NTR (e) was assessed at 6.6μM for both proteins and data were collected at 800 MHz. Interaction of the prodomain with p75^NTR was undetectable by NMR. Standard deviation (s.d.) was calculated for all the analysis and a threshold line equivalent to 2 s.d. was drawn to show the limit of what is considered interaction.

Figure 7

Met66-SorCS2 interaction is required to induce growth cone retraction. (a) SorCS2 antibodies were able to block the Met66 prodomain-induced growth cone retraction. Neurons were pre-incubated with anti-SorCS2 (or control IgGs), treated with Val66 or Met66 prodomains (10 ng/ml) for 20 min, and followed by growth cone retraction analysis. Quantification assessed in p75^NTR positive cells from 3 independent experiments. (b) Representative blot showing SorCS2 expression in cultured hippocampal neurons after knock-down using SorCS2 shRNA (sh-SorCS2) lentivirus infection, as compared to uninfected controls or scramble shRNA (sh-Scr) infected cells. (c) SorCS2 partial down regulation achieved with SorCS2 shRNA was able to partially prevent the Met66 prodomain-induced growth cone retraction. Quantification of 4 independent experiments. (d) Only Met66 prodomain administration, but not the Val66, induced a decrease in Rac activity in cultured hippocampal neurons. Hippocampal neurons were incubated with Val66 or Met66 prodomain for 20 min, and cell lysates were incubated with GST-PAK-CRIB beads to isolate activated Rac (Rac-GTP). (e) Quantification of (d). Activated Rac was measured by densitometry and normalized to total Rac in the input. Quantification assessed in 4 independent experiments. Bars represent mean ± s.e.m. Statistical comparisons were made by one way analysis of variance test. * p <0.05.