Using primary hippocampal neurons, we demonstrate here that a 20-min stimulation with BDNF increases CaNAα and NFATc4 binding to AKAP6 (Fig. 1A, B), indicating neurotrophin-mediated anchoring of both proteins within the same signaling complex. Since NFAT activation requires CaN-mediated dephosphorylation, we anticipate that scaffolding of CaNAα and NFATc4 by AKAP6 can serve as a nodal point for BDNF-mediated regulation of NFAT transcriptional activity.

AKAP6 regulates NFAT activity for BDNF-mediated neuronal survival. A The AKAP6 antibody was used for immunoprecipitation of endogenous calcineurin (catalytic Aα subunit, CaN) or NFATc4 proteins from primary hippocampal neurons following incubation with BDNF. All presented blots are representative of experiments conducted at least three times. B Quantitative densitometric analysis of band intensity. The results are expressed in arbitrary units, defined as the optical density per milligram of protein relative to vehicle-treated cells, which were set as 100%, n = 3. C Primary hippocampal neurons were co-transduced with NFAT dual-reporter lentivirus and Lenti-AKAP6 shRNA (or an appropriate control), or alternatively, with D Lenti-NFATc4 shRNA (or an appropriate control). NFAT transcriptional activity was assessed in cell lysates following BDNF stimulation by quantifying luciferase activity (n = 5). E Hippocampal neurons were pretreated with 1 μM PLC inhibitor—U73122 or F 1 μM sarco(endo)plasmic Ca2+-ATPase inhibitor—thapsigargin or G 20 μM ryanodine receptor inhibitor—ryanodine (n = 5 for each) and luciferase activity was quantified 12 h following BDNF stimulation. H Colocalization of AKAP6 and ryanodine receptor 2 (RyR2) in primary hippocampal neurons. Fixed cells were immunostained with anti-AKAP6 primary and respective Alexa Fluor 594 conjugated secondary antibody (shown in red), as well as with anti-RyR2 primary antibody and respective Alexa Fluor 488 conjugated secondary antibody (shown in green). Colocalization percentage (fraction of AKAP6-positive pixels which are also positive for RyR2) was calculated using Leica LAS AF Lite software. Scale bars: 20 μM. I The design of CaN anchoring disruptor. The sequence corresponding to AKAP6 CaN binding domain (aa 1286–1345, CBD) was subcloned into rAAV-CAG-EGFPSV40 vector for AAV2 production. J Hippocampal neurons were co-transduced with NFAT dual-reporter lentivirus and AAV2-CBD-GFP (or AAV2-GFP control) for assessment of NFAT transcriptional activity (n = 5). K In vitro survival of RGC transduced with AAV2-CBD-GFP or AAV2-GFP alone, or L electroporated with NFATc4 or control siRNA and cultured in media with or without BDNF, normalized to appropriate control-treated RGCs cultured in minimal media (no forskolin, no BDNF). Representative data from repeated experiments shown (n = 3). M Experimental design of the optic nerve crush (ONC) in vivo model. BDNF was intravitreally injected immediately following ONC, and retinas were collected 7 days after. N Quantification of BDNF effect on RGC survival in vivo following ONC in wild-type (WT) or Nfatc4−/− mice. Retinal ganglion cells were stained with anti-RBPMS antibody and counted manually. Data are expressed as the ratio of RBPMS-positive cells in the BDNF-treated eye relative to control (PBS-injected) eye (n = 4 per group). The RGC densities (cells per mm2) in the Sham-operated groups are as follows: 3243 ± 89 for WT and 3159 ± 57 for Nfatc4−/− mice. O The schematic model of AKAP6-dependent regulation of NFAT transcriptional activity in response to BDNF stimulation. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate standard error of mean (± SEM)

To explore this possibility, we knockdown AKAP6 using lentiviral-mediated shRNA delivery (73 ± 4% knockdown efficiency as assessed by real time PCR, not showed) and observed a significant reduction in BDNF-mediated NFAT-dependent transcription (Fig. 1C). Next, we verified that the effect of BNDF on luciferase activity [6] was dependent on NFATc4. Knockdown of NFATc4, with 81 ± 6% efficiency (not showed) attenuated NFAT transcription by 60 ± 9%, highlighting NFATc4’s role in AKAP6-mediated BDNF action (Fig. 1D). Further, we examined signaling pathways upstream to AKAP6/NFATc4 with regard to their role in triggering NFAT-dependent transcription (Fig. 1E–G). Consistent with the canonical mode of BDNF action, pretreatment with phospholipase C (PLC) inhibitor U73122 decreased NFAT transcriptional activity. Similar effect was observed when intracellular Ca2+ stores were depleted with thapsigargin or ryanodine receptor (RyR)-mediated Ca2+ release was blocked by ryanodine. Furthermore, AKAP6 colocalized with RyR2 in hippocampal cultures (Fig. 1H) suggesting the importance of PLC-RyR signaling in the AKAP6-dependent orchestration of NFATc4 activation in response to BDNF.

To verify whether CaN anchoring to AKAP6 is necessary for BDNF-mediated NFATc4 activation we used a displacing peptide corresponding to CaN binding site in AKAP6 (aa 1286–1345, CBD, Fig. 1I). Transduction of hippocampal neurons with adeno-associated virus (AAV2)-GFP-CBD decreased both basal and BDNF-mediated NFAT activation (Fig. 1J). Because our results indicate that BDNF signals through AKAP6-CaN-NFATc4, we next assessed whether anchoring of CaN and NFATc4 to the scaffold is required for BDNF-mediated neuronal survival. For this purpose, we switched to RGCs, as their culturing and manipulation in vitro produces stress and subsequent ongoing cell death even in neurotrophic factor-rich growth media. No changes in RGC survival were seen in minimal media or in media with forskolin alone. However, the pro-survival effect of BDNF was significantly attenuated in neurons expressing AAV2-GFP-CBD when compared to GFP control (Fig. 1K). Similarly, siRNA-mediated NFATc4 knockdown resulted in significant decrease of BDNF effect (Fig. 1L).

Finally, to conclusively demonstrate that NFATc4 is required for BDNF-mediated neuroprotection in injured neurons, we performed ONC procedure in NFATc4−/− mice and collected retinas a week after BDNF injection (Fig. 1M) [7]. The ONC is an effective preclinical model for neuronal trauma and regeneration failure, as it similarly severs all of the RGC’s axons and induces time-dependent retrograde RGC death [8,9,10]. Staining of flat mount retinas with RBPMS demonstrated that survival of BDNF-treated RGCs, expressed as BDNF/control (PBS) ratio was increased 61 ± 9% in wild-type mice (Fig. 1N). However, no prosurvival effect of BDNF intravitreal injection was observed in NFATc4−/− mice after ONC.