Dulbecco’s Modified Eagle Medium (DMEM, Gibco™, UK), Horse Serum (HS, Biowest, France), Neurobasal (Gibco™, UK), B-27 supplement (Gibco™, UK), Insulin (Sigma-Aldrich, USA), Sodium pyruvate (Gibco™, UK), Glutamine (Biowest, France), Hanks’ Balanced Salt Solution (1X) (HBSS) without calcium and magnesium (GibcoTM, UK), Hanks’ Balanced Salt Solution (1X) (HBSS) with calcium and magnesium (GibcoTM, UK), Poly-L-lysine hydrobromide (Sigma-Aldrich, USA), Arabinosyl cytosine (Sigma-Aldrich, USA), Lipofectamine LTX Reagent (ThermoFisher, USA), Pierce™ RIPA buffer (ThermoFisher, USA), Bovine Serum Albumin (BSA, Millipore, USA), BCIP/NBT and ECL Western Blot System (Bio-Rad Laboratories, USA), Protein A/G PLUS-Agarose (Millipore, USA), EverBrite™ Hardset Mounting Medium (VWR, USA), FITC Annexin V Apoptosis Detection Kit (Sigma-Aldrich, USA), Trizol (Santa Cruz Biotechnology, USA), HOT FIREPol® EvaGreen® qPCR Mix Plus (Cytogen, Poland), Dual-Glo Luciferase Assay System (Promega, USA). NFAT luciferase reporter lentivirus and firefly luciferase lentivirus were sourced from BPS Bioscience (USA). Primers used in the Real Time PCR Reaction were provided by the BioCat GmbH (Germany).

Plasmids and lentiviral vectors were constructed by Genewiz (Germany) using the company’s proprietary techniques. The pmCherry-C1-CBD expression vector contains cDNA encoding a peptide corresponding to mAKAPα amino acids 1,286–1,345 (Calcineurin Binding Domain – “CBD”) fused to the C-terminus of mCherry. The pEGFP plasmid includes a fragment of the N-terminal mAKAPα domain that is not expressed in mAKAPβ (aa3-196) – GFP-(aa3-196), or a fragment of mAKAPα encoding the sequence for MEF2 transcription factor binding (aa301-500) – GFP-(aa301-500) inserted downstream of GFP. The mCherry plasmid (#176016, Addgene) and EGFP plasmid (#165830, Addgene) were used as backbones. NFATc4 was overexpressed using a ready-to-use pEGFP-C1 NFATc4 vector (#10961, Addgene). VIVIT peptide was expressed using GFP-VIVIT plasmid (#11106, Addgene). Endogenous MEF2A expression was silenced using MEF2A-shRNA plasmid (#32899, Addgene). Before transfection into primary neurons, plasmids were sequenced and expressed in HeLa or HEK293 cells.

A lentivirus expression system was used to deliver mAKAPα shRNA to the neuronal cultures. The sequences were as follows: scrambled shRNA: 5′-GACGAACCCCTGTTCCGAATT CAAGAGATTCGGAACAGGGGTTCGTCTTTTT-3′ and mAKAPα shRNA – 5′-GACGAACCTT CCTTCCGAATTCAAGAGATTCGGAAGGAAGGTTC GTCTTTTT-3′, as published in Pare et al. (2005b). Both vectors contain the U6 promoter and an mCherry coding sequence. The titer of both lentiviruses was ≥10×10⁸ TU/mL. Hippocampal neurons were infected with MOI 5 and cultured for 48 h in growth medium before further analysis. The efficiency of lentiviral-mediated silencing was tested in PC12 cells overexpressing full-length rat mAKAPα protein. PC12 cells were maintained and transfected as described previously (Boczek et al., 2017).

Adeno-associated virus serotype 2 (AAV2) used to express the GFP-tagged CBD domain of mAKAPα (rAAV-CAG-1286-1345 CBD-EGFPSV40 polyA) was constructed based on the mCherry-CBD plasmid and produced by BioHippo Inc. (USA). rAAV-CAG-EGFP was used as the control. The titer of both AAVs was ≥2.00×10¹² vg/mL. Hippocampal neurons were transduced with MOI 1000 and cultured for 48 h in growth medium before experiments. The antibodies used in this study are listed in Table 1.

All procedures for animal handling were approved by the Institutional Animal Care and Use Committee at the Medical University of Lodz. Hippocampal neurons were isolated from embryonic day 17 (E17) Sprague–Dawley rat embryos of either sex. In brief, hemispheres were dissected in PBS medium on ice and digested with 0.05% trypsin–EDTA in HBSS without calcium and magnesium for 30 min at 37°C. The separated tissues were centrifuged at 250 × g for 2 min and then triturated with a fire-polished glass pipette in HBSS with calcium and magnesium. The dissociated neurons were seeded on nitric acid-treated 25 mm cover glasses coated with poly-L-lysine in plating medium (10% v/v horse serum in DMEM). Four hours after plating, the medium was replaced with maintenance Neurobasal defined medium supplemented with 2% B27, 1 mM glutamine, 1 mM sodium pyruvate, and 5 μg/mL insulin. On DIV3, 4 μM arabinosyl cytosine was added to prevent glial proliferation. To extend cultures beyond 4 days, half of the medium was exchanged with fresh medium on either DIV3 or DIV4.

For the neurite extension assay, on DIV3-4, hippocampal neurons were transfected or co-transfected with appropriate plasmids using Lipofectamine LTX with Plus Reagent, or transduced with lentiviruses. To promote neurite elongation, 30 mM KCl was added directly to the growth medium. Cell imaging was performed 24–48 h after transfection/transduction. Images were acquired on a Leica DMi8 inverted microscope, and the longest neurite per cell (~20 cells on average in each experiment) was measured using ImageJ with the Simple Neurite Tracer plugin.

Cells were lysed using RIPA buffer supplemented with a protease inhibitor cocktail. Following centrifugation, total protein lysate was quantified colorimetrically with the Bio-Rad Protein Assay Kit. Next, 10–50 μg of the protein samples were fractionated in 8% or 10% polyacrylamide gels and transferred to a nitrocellulose membrane using a semi-dry method. The membranes were immersed in blocking buffer (5% BSA) in TBS-T (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween-20) for 1 h at room temperature and incubated with primary antibodies for 24 h at 4°C. The primary antibodies used are as follows: Tpa/Plat (1:1000), Mif (1:1000), iNOS (1:1000), Prkar1a (1:1000), and GAPDH (1:1000). Following three washes in TBS-T buffer, the membrane was probed with secondary antibodies conjugated with alkaline phosphatase (AP) (1:5000) for 2 h or conjugated with horseradish peroxidase (HRP) (1:20,000) for 1 h at room temperature. BCIP/NBT or ECL Western Blot System was used to detect immunoreactive bands. The membranes were scanned densitometrically, and the optical density of the bands was quantified using ImageJ. The results are expressed as arbitrary units after normalization to the endogenous GAPDH level, providing a quantitative assessment of protein expression levels.

For the co-immunoprecipitation assay, cells were treated with 30 mM KCl for 20 min. Additionally, to inhibit calcineurin activity, neurons were treated with 5 μM cyclosporine (CsA), and 1 mM ethylenediaminetetraacetic acid (EDTA) was used to chelate Ca²⁺. Next, 200 μg of proteins was incubated with anti-mAKAPα antibodies (~2 μg of antibodies/200 μg of lysate proteins) overnight at 4°C, followed by incubation with 25 μL of Protein A/G PLUS-Agarose beads at 4°C for 2 h. The immunocomplexes were recovered by centrifugation at 8130 x g for 5 min, washed three times with PBS, eluted with 60 μL of SDS-PAGE sample buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.001% bromophenol blue) containing 5% β-mercaptoethanol, and subjected to immunoblotting. The membranes with immunoprecipitated mAKAPα protein were probed with anti-NFATc4 (1:500), anti-phospho-NFATc4 (Ser289) (1:1000), anti-CaNAα (1:500), and MEF2A (1:1000) antibodies. For interaction in heterologous cells, aa3-196 peptide was immunoprecipitated with anti-GFP antibody and immunoblotted with anti-Flag antibody (1:1000). Next, immunoblots were incubated with secondary antibodies (1:5000 or 1:20,000) conjugated to AP or HRP. Bands were visualized using the AP system or ECL substrates (Bio-Rad, USA) following the manufacturer’s protocol. The results were normalized to OD/mg protein and are presented as the fold change in relation to control cells.

Isolated primary neurons were transfected with mCherry or mCherry-CBD and treated with 30 mM KCl for 24 h. For immunostaining, a day after transfection, cells were washed three times with cold PBS and fixed in 4% PFA for 10 min at room temperature. After several washes with PBS, neurons were permeabilized with Triton X-100 and immersed in blocking buffer (1% BSA in PBS-T) for 1 h at room temperature. Subsequently, cells were incubated overnight at 4°C with a primary anti-Bcl2 antibody (1:200) in 1% BSA in PBS-T. Following washing with PBS, neuronal cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:1000) for 2 h at room temperature. Next, coverslips with hippocampal neurons were mounted in mounting medium on glass slides. Images of transfected cells and immunolabeled Bcl-2 were acquired on a Leica DMi8 inverted microscope. Fluorescence intensity was measured using ImageJ.

For Annexin V staining, 24 h after transfection, neurons were washed two times with PBS and 1X Binding Buffer (FITC Annexin V Apoptosis Detection Kit I). Next, cells were incubated with Annexin V diluted 1:100 in 1X Binding Buffer for 15 min at room temperature in the dark. After incubation, images of positive transfected neurons were acquired on a Leica DMi8 inverted microscope. Fluorescence intensity was measured using ImageJ.

Total RNA was extracted from the isolated hippocampal neurons using Trizol reagent according to the manual provided by the manufacturer. Single-stranded cDNA was synthesized from 1 μg of isolated RNA and oligo (dT) primers with M-MLV reverse transcriptase. The Real-time PCR conditions included an initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s using the Abi Prism 7,000 sequence detection system using Eva Green Master Mix. Primers used in the reactions: Akap6 set#1 (NM_022618), Akap6 set#2 (NM_022618), Akap6 set#3 (NM_022618). The specificity of the product was assessed by agarose gel electrophoresis.

For microarray screening, total RNA was extracted from the primary neurons 48 h after transduction with AAV2-GFP or AAV2-GFP-CBD, using Trizol Reagent according to the manual provided by the manufacturer. cDNA amplified from 2 μg of neuronal RNA was hybridized with RT² Profiler™ PCR Array Rat calcium/cAMP signaling, and the reaction was performed using HOT FIREPol® EvaGreen® qPCR Mix Plus. Real-time PCR reactions were carried out under the following conditions: an initial cycle at 95°C for 10 min, followed by cycles at 95°C for 15 s, 60°C for 1 min, and a dissociation curve at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s. The fold change was calculated by a method of Livak and Schmittgen (2001). Data were analyzed using Qiagen PCR Array Data Analysis Web Portal. The microarray analysis was run in triplicate, and the RT² software averaged the triplicate normalized expression for each gene (ΔCt) before calculating ΔΔCt between the control (AAV2-GFP) and experimental group (AAV2-GFP-CBD). Housekeeping genes used for normalization were selected based on the recommendations of Vandesompele et al. (2002). Two Microarray Quality Control studies demonstrated that a p-value calculation based on fold change could be considered sufficient for obtaining reproducible results across microarray analyses, including RT² Profiler™ PCR Arrays (Shi et al., 2006; Shi et al., 2010).

NFAT transcriptional activity was done essentially as described previously (Na et al., 2010; Mackiewicz et al., 2024) with some modifications. Briefly, lentiviral particles were engineered to carry a firefly luciferase gene under the control of the NFAT response element positioned upstream of the minimal TATA promoter. Primary hippocampal neurons were transduced with Lenti-NFAT luciferase reporter and Lenti-luciferase at DIV0, and the neurons were cultured for 3 days. NFAT transcriptional activity in control GFP-expressing cells or GFP-(aa3-196)-expressing cells was measured in cell lysates using the Dual-Glo Luciferase Assay System (Promega, USA) according to the manufacturer’s instructions. The expression of the NFAT luciferase reporter was normalized to the expression of firefly luciferase. The fold increase of normalized NFAT luciferase reporter expression was then calculated relative to the baseline values. Ca²⁺-dependent increase in NFAT transcriptional activity was determined following a 20-min stimulation with 30 mM KCl. CsA (10 μM) was added 1 h prior the experiment.

Statistical analysis was conducted using GraphPad Prism version 8.0.1. Single comparisons were done using a two-tailed Student’s t-test. All data involving multiple comparisons were analyzed using one- or two-way ANOVA with post-hoc correction. All datasets are expressed as means of at least three independent experiments with standard error of the mean (SEM). Significance is represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Previous studies have indicated that mAKAPα is expressed in the brain, including the retina (Wang et al., 2015; Michel et al., 2005). Consistent with our earlier report (Boczek et al., 2019), we confirmed mAKAPα expression in primary rat hippocampal neurons isolated from E18 embryos. The presence of mAKAPα mRNA was detected with different sets of primers, producing PCR products between 111 bp and 163 bp (Figure 1A). Additionally, mAKAPα was immunoblotted in extracts prepared from isolated primary neurons (Figure 1B). The antibody raised against the LTMSVTLSPLRSQ sequence, corresponding to the N-terminus of mAKAPα (NP_004265.3) which is absent in mAKAPβ, detected a band around 250 kDa, similar to the previously reported mAKAPα mobility in the brain (Wang et al., 2015). A band of ~190 kDa was also detected in the neuronal extract, consistent with the size declared by the manufacturer. However, since the ~190 kDa band is smaller than the molecular weight of mAKAPα, this protein is likely a degradation product of the 250 kDa mAKAPα.

Given that mAKAPα is indispensable for RGC survival and outgrowth in vitro (Wang et al., 2015), we evaluated whether it is also required for KCl-mediated elongation of hippocampal neurons. To address this, hippocampal neurons were transduced with lentivirus carrying shRNA against mAKAPα in the presence or absence of KCl (30 mM), which has been shown to induce neuronal outgrowth in a cAMP- and PKA-dependent manner (Goldberg et al., 2002). The specificity and efficiency of shRNA were evaluated in our previous paper, demonstrating compromised cAMP synthesis in the perinuclear space in the absence of the scaffold (Boczek et al., 2019). Two days after transduction, the mAKAPα protein level decreased by ~90% relative to the scrambled Lenti-shRNA control group (Figures 1C,D). Following mAKAPα depletion, the mean length of the longest neurite per cell was markedly less compared to the control (Figures 1E,F) in depolarizing conditions. No effect of mAKAPα downregulation on axonal length was observed in resting conditions (5 mM KCl) (Figures 1E,F). Together, these data indicate that mAKAPα is required for depolarization-mediated axonal elongation but is dispensible for basal neuronal outgrowth.

Having shown that mAKAPα function is relevant to activity-dependent neuronal elongation, we investigated the mAKAPα-regulated signaling that underlies the attenuated KCl effect. The Ca²⁺-activated protein phosphatase calcineurin (CaN) has previously been demonstrated to interact with mAKAP in cardiac myocytes (Pare et al., 2005b; Li et al., 2010). To determine whether this interaction occurs in primary hippocampal neurons, we probed mAKAPα immunoprecipitates for CaN by using an antibody that predominantly recognizes the catalytic subunit of calcineurin Aα (CaNAα). A significant amount of CaNAα co-immunoprecipitated with mAKAPα in a steady-state conditions (Figure 2A). Further experiments were performed to evaluate whether neuronal depolarization can affect CaNAα/mAKAPα association. Co-immunoprecipitation of both proteins following a 20-min depolarization with 30 mM KCl increased CaNAα immunoreactivity in mAKAPα immunoprecipitates, suggesting that neuronal activity enhances CaN tethering to the scaffold.

Based on this observation, we aimed to test for potential CaN substrates that might also dock on the mAKAPα scaffold. Our initial approach involved NFATc4 protein, a well-known downstream target of CaN involve in neurotrophin-mediated survival of hippocampal neurons (Groth and Mermelstein, 2003). To determine whether mAKAPα and NFATc4 interact in hippocampal neurons, mAKAPα immunocomplexes were probed for co-immunoprecipitation of NFATc4. Western blot analysis demonstrated that NFATc4 binding to the mAKAPα signalosome was enhanced following KCl depolarization (Figure 2B) and blocked in the presence of extracellular Ca²⁺ chelators, i.e., EDTA (Figure 2C).

Given that both CaNAα and NFATc4 bound more strongly to the scaffold in the presence of Ca²⁺, we suspected that CaNAα may retain its catalytic activity within the complex and regulate NFATc4 binding. mAKAPα/NFATc4 co-immunoprecipitations were repeated in the presence of cyclosporin A – a calcineurin inhibitor, demonstrating a significant decrease in the protein–protein interaction with the inhibitor (Figure 2D). We anticipated that increased neuronal activity recruits active CaNAα to the scaffold, which dephosphorylates bound NFATc4. Following KCl treatment, phosphorylated NFATc4 (S289) co-immunoprecipitated less with mAKAPα (Figure 2E), supporting a decreased phosphorylation state of NFATc4 with the scaffold.

Previous studies have demonstrated that CaNAβ binds directly to a unique site within mAKAP (residues 1,286–1,345), which is both necessary and sufficient for phosphatase binding (Dodge-Kafka et al., 2005; Pare et al., 2005b). To address the significance of CaN scaffolding by mAKAPα for axonal outgrowth, we generated a genetically encoded anchoring disruptor by fusing residues 1,286–1,345 of mAKAPα (referred to here as the Calcineurin Binding Domain – “CBD”) to the C-terminus of mCherry. Hippocampal neurons were transfected with expression plasmids for either mCherry or mCherry-CBD, cultured for 24 h in the presence or absence of KCl, and assayed for neurite extension. Consistent with the slower axon growth of mAKAPα-depleted neurons (see Figure 1F), mCherry-CBD expression abolished KCl-dependent neuronal extension compared to mCherry-expressing neurons (Figures 3A,B). These findings indicate that depolarization-induced CaN recruitment to the mAKAPα scaffold is required for axonal outgrowth during chronic KCl exposure.

To understand the phenomenon of inhibited axon growth in the presence of mCherry-CBD under stimulated conditions, we tested whether CaN displacement may result in neuronal apoptosis. Staining with annexin V, which detects the apoptosis-related shift of phosphatidylserine to the outer leaflet of the plasma membrane, revealed no significant differences between mCherry- and mCherry-CBD-expressing neurons (Supplementary Figure S1). Interestingly, immunocytochemical staining of Bcl-2, a member of the anti-apoptotic protein family, showed increased expression of this protein in neurons expressing mCherry-CBD compared to the control (Figures 4C,D), suggesting a plausible adaptive response to potentially counterbalance apoptotic signaling.

Based our previous work demonstrating that mAKAPα selectively coordinates Ca²⁺-dependent cAMP signaling for axonal outgrowth in vitro and neuroprotection in vivo (Boczek and Kapiloff, 2020; Mackiewicz and Boczek, 2025; Tomczak et al., 2024; Boczek et al., 2019), we hypothesized that the loss of KCl effect on axon elongation in the presence of the mCherry-CBD peptide could be due to peptide-related deficits in signal transduction and gene expression. To explore this possibility, hippocampal neurons were transduced with AAV2 for GFP-CBD or GFP control. A day after transfection, 30 mM KCl was added, and neurons were chronically depolarized for another 24 h. Microarray screening of genes involved in cAMP/Ca²⁺ signaling (the full list of genes can be found in Supplementary Table S1) showed no significant changes in the average Ct values between AAV2-GFP-CBD and AAV2-GFP control (Figures 4A,B), suggesting that displacing CaN from the mAKAPα signalosome does not affect global gene expression. Considering a 1.5-fold change as a minimum and p < 0.05, we identified five genes with significantly upregulated expression: Nos2, Mif, Plat, Tnf, Cyr61 and one with markedly downregulated expression: Prkar1a (Figure 4C). We further confirmed that AAV2-GFP-CBD treatment affected the levels of four of the corresponding proteins (Figures 4D–H).

Given our new findings that neuronal activity promotes both CaN and NFATc4 tethering to mAKAPα, we asked whether inhibition of NFAT binding would also affect neurite outgrowth. Considering the difference in the N-terminal domain between mAKAPα and β isoforms, we constructed a GFP-tagged peptide corresponding to amino acids 3–196 of mAKAPα. Expression of this peptide is expected to displace NFAT from the N-terminus of mAKAPα. The GFP-(aa3-196) and Flag-NFATc4 were co-expressed in COS-7 cells and analyzed by co-immunoprecipitation. Immunoprecipitation using anti-GFP antibodies resulted in co-purification of NFATc4 (Figure 5A).

We next tested the ability of the GFP-(aa3-196) peptide to regulate NFAT transcriptional activity in primary hippocampal neurons using a transient luciferase reporter assay (Figure 5B). To ensure effective expression, we constructed a Lenti-GFP-(aa3-196) vector and used lentiviral particles to deliver it along with a Lenti-NFAT luciferase reporter. As expected, stimulation with 30 mM KCl resulted in a 3.1-fold increase in NFAT transcriptional activity, which was completely inhibited by cyclosporine A. Remarkably, expression of GFP-(aa3-196) decreased NFAT activity by ~40% compared to neurons expressing GFP only. These results indicate that N-terminus of mAKAPα binds NFAT to regulate its transcriptional activity in response to neuronal depolarization. This raises the possibility that NFATc4 may be downstream of CaN/mAKAPα signaling in activity-dependent neuronal outgrowth.

To explore this hypothesis, we first overexpressed NFATc4-GFP and cultured hippocampal neurons in the absence or presence of 30 mM KCl for 24 h. We did not observe any effects on neuronal outgrowth under either basal or stimulated conditions (Figures 6A,B). Next, we assessed the relevance of the NFATc4/mAKAPα interaction to the inhibitory effect of the CaN-delocalizing peptide on neuronal outgrowth (Figures 6C,D). However, in contrast to the mCherry-CBD peptide (see Figures 3A,B), the expression of the GFP-(aa3-196) peptide did not affect axonal length, indicating a mechanism independent of NFAT binding to the N-terminal mAKAPα domain.

Because the GFP-(aa3-196) peptide only partially inhibited NFAT transcriptional activity, suggesting the possibility of multiple NFAT binding sites on mAKAPα, we transfected neurons with GFP-VIVIT (Figures 6E,F). VIVIT is a selective inhibitor of CaN-mediated dephosphorylation of NFAT that does not disrupt other CaN-dependent pathways. The efficacy of VIVIT in blocking NFAT activation is comparable to that of CsA, as we previously demonstrated in hippocampal neurons (Mackiewicz et al., 2024). The axon length of neurons expressing GFP-VIVIT was similar to that of neurons expressing GFP alone, indicating that NFAT activity is dispensable for hippocampal neuron growth in vitro.

Another transcription factor which activation is controlled by CaN and it is tethered to mAKAPα signalosome is MEF2. Moreover, a growing body of evidence clearly indicates that MEF2 plays a critical role in neuronal development (Lisek et al., 2023). To determine whether mAKAPα and MEF2 interact in hippocampal neurons, mAKAPα immunocomplexes were probed for co-immunoprecipitation of MEF2A. Similar to CaN and NFATc4, Western blot analysis showed that MEF2A binds to the mAKAPα signalosome in a Ca²⁺-dependent manner (Figure 7A).

To determine whether mAKAPα is the scaffold required for the recruitment of MEF2A to the perinuclear compartment, MEF2A was immunoblotted in mAKAPα immunocomplexes from hippocampal neurons transduced with either control or mAKAPα shRNA (Figure 7B). Endogenous mAKAPα strongly co-immunoprecipitated with the MEF2A antibody only in cells expressing the scaffold. Furthermore, the KCl-enhanced MEF2A/mAKAPα association was significantly reduced in neurons expressing mAKAPα shRNA.

We next used the AAV2-GFP-CBD virus, which disrupts the full-length mAKAPα/CaN interaction, to assess whether CaN binding to the scaffold is a prerequisite for MEF2A association. The CBD peptide does not compete with MEF2 binding, as MEF2 and CaN bind to different mAKAPα fragments. As shown in Figure 7C, the binding of MEF2A to mAKAPα, both in the presence and absence of KCl, was reduced when AAV2-GFP-CBD was expressed. These results demonstrate that mAKAPα functions to anchor both CaN and MEF2A in the perinuclear space, and that CaN tethering is a necessary step for MEF2 binding during neuronal activity.

Based on these findings, we sought to investigate whether MEF2 scaffolding by mAKAPα is critical for neuronal outgrowth in hippocampal neurons. We constructed a GFP-tagged peptide that specifically displaces MEF2 from the mAKAPα complex (amino acids 301–500), as mapped elsewhere (Vargas et al., 2012). Neurons were transfected with expression plasmids for either control GFP or GFP-(aa301-500), cultured for 24 h in the presence or absence of KCl, and then assayed for neurite extension (Figures 8A,B). Cells expressing the GFP-(aa301-500) peptide exhibited significantly shorter axons under both basal and stimulated conditions. Similar phenotypic changes were observed in neurons transfected with MEF2A shRNA (Figures 8C,D). These data, along with the results from mAKAPα depletion and CaN/mAKAPα disruption studies, conclusively demonstrate that the CaN/MEF2 interaction at mAKAPα is required for activity-induced signaling with MEF2A playing a crucial role in the axonal elongation of primary hippocampal neurons.