Hippocampal CA1-specific Naa10 cKO (Naa10 cKO) induces anxiety and reduces neurite complexity in vivo.

(A) Naa10 cKO induces anxious behavior. Control (Ctrl) and Naa10-cKO (cKO) mice were subjected to the elevated plus maze (EPM) test. Left: individual data points and mean ± SD for each group (Ctrl: 6 mice, cKO: 12 mice). P-values analyzed by unpaired two-tailed t test are indicated. Right: representative track sheets.

(B) Naa10 cKO suppresses dendrite formation in the CA1. Left: The hippocampal CA1 regions of 12-week-old control (Ctrl) and Naa10-cKO mice were subjected to immunostaining using the indicated Abs. Map2, microtubule-associated protein 2. Gfap, glial fibrillary acid protein. Right: quantification of Map2 and Gfap fluorescent intensity in the hippocampus. DAPI stains for the nucleus. Shown are individual data points and mean ± SD for each group (Ctrl: 5 mice, cKO: 5 mice). P-values analyzed by unpaired two-tailed t test are indicated.

(C) Naa10 cKO reduces branching of dendrites in the CA1 pyramidal neurons. The hippocampi of a 12-week-old control and a Naa10-cKO mouse were subjected to silver staining. Arrow indicates the soma of the single neuron in the image; white broken line represents the dendritic branching area.

(D) Naa10 cKO reduces dendritic complexity in the CA1. Map2 immunostaining of whole brains of a 12-week-old control (Ctrl) and a Naa10-cKO mouse was imaged using light-sheet fluorescent microscopy.

Naa10 promotes neurite outgrowth by N-acetylating Btbd3.

(A) WT but not acetyltransferase-dead Naa10 enhances neurite outgrowth. HT-22 cells, either wild-type (WT) or Naa10 knockout (Naa10-KO), were subjected to lentiviral infection with constructs expressing WT or mutant Naa10. Following differentiation into neurons, western blotting (upper left) confirmed protein expression. Representative images of Map2 staining (right) and neurite length quantification (lower left) were performed. Neurite length was measured using Olympus cellSens software from nine randomly selected fields across three independent experiments, totaling a cell count of ≥ 400. Data points represent individual measurements, and mean ± SD values are displayed. Statistical significance was determined by two-way ANOVA with Sidak’s post-hoc test (P-values indicated).

(B) Clinically relevant NAA10 mutants fail to rescue neurite outgrowth. Similarly, HT-22 cells were transduced with lentiviruses expressing either WT or the specified mutant NAA10 constructs, followed by neuronal differentiation. Western blotting (upper left) confirmed protein expression, while representative images of F-actin staining (right) were obtained using phalloidin-conjugated Alexa Fluor 488. Neurite length was quantified (lower left) using the same method described in (A). Data presentation and statistical analysis were performed as described for (A).

(C) Naa10 acetylates Btbd3 N-terminus in vitro. Btbd3 N-terminal peptides were incubated with acetyl-CoA with or without recombinant Naa10. Acetylated Btbd3 was quantified by mass spectrometry after the acetyltransferase assay. Data points represent individual measurements, and mean ± SD values are shown. Statistical significance was determined by unpaired two-tailed t test (p-values indicated in the figure).

(D) Btbd3 N-acetylation promotes neurite outgrowth. Btbd3-KO HT-22 cells were infected with lentiviruses expressing GFP, WT, or V1P mutant Btbd3 and differentiated into neurons. Map2 immunostaining (top), western blot analysis (lower left), and neurite length quantification based on Map2 staining (lower right) were performed. Data points represent individual measurements, and mean ± SD values are shown (N=3). Neurite length was determined using Olympus cellSens software from three randomly selected fields across three independent experiments with a total cell count of ≥ 150. P-values determined by two-way ANOVA with Sidak’s post-hoc test are indicated.

(A-B, D) DAPI was used to stain DNA. Scale bar is indicated.

Naa10-mediated Btbd3 N-α-acetylation promotes CapZb binding to F-actin.

(A) Btbd3 binding to CapZb and β-actin depends on Naa10 and Btbd3 N-α-acetylation. Btbd3-Flag immunoprecipitation (IP) was performed in WT or Naa10-KO HT-22 neurons expressing the indicated proteins, followed by western blotting using antibodies against Flag, CapZb, β-actin, and Naa10. Input represents 10% of total cell lysates. The experiment was replicated twice, with the additional replicate shown in Figure S4C.

(B) Naa10 KO reduces the association of CapZb and F-actin.

(C) Acetyltransferase activity of Naa10 promotes CapZb association with F-actin.

(D) Btbd3 N-α-acetylation enhances CapZb association with F-actin. Western blotting was performed using Abs against CapZb, β-actin, and Btbd3.

(B-D) Total cell lysates of WT, Naa10-KO or Btbd3-KO HT-22 neurons expressing the indicated proteins were separated into globular actin (G-actin) and F-actin fractions, followed by western blotting. The bands of CapZb and β-actin in F-actin fraction were quantified using ImageJ. The relative ratios of CapZb to β-actin of each lane compared to lanes 3 (B), 4 (C) and 5 (D) are shown. The bar charts display the relative ratios of CapZb to β-actin in the F-actin fraction with individual data points and mean ± SD for each group from three independent experiments (see Figure S4D-F for replicates). (B) analyzed by unpaired two-tailed t test. (C-D) analyzed by two-way ANOVA plus Sidak’s post-hoc.

Naa10-Btbd3-CapZb-F-actin axis regulates neurite outgrowth in primary hippocampal neurons.

(A) Naa10 KO reduces neurite complexity. Left: Representative images of Map2 immunostaining of hippocampal neurons from control (Ctrl) and Naa10-cKO P0 mice. DAPI stains for the nucleus. Middle and right: Sholl analysis.

(B) Btbd3 N-α-acetylation promotes neurite complexity. Upper left: Experimental scheme. Lower left: Representative images of hippocampal neurons from WT mice cultured in vitro and infected at DIV 3 with lentivirus containing indicated genes. Right: Sholl analysis. Four groups show significant differences (e.g., at radii 20, 40, and 60 μm): scramble vs shBtbd3 (p-values: <0.0001, <0.0001, and 0.0006, respectively); shBtbd3 vs shBtbd3 + Btbd3WT (p-values: <0.0001, <0.0001, and 0.0015, respectively); shBtbd3 + Btbd3WT vs shBtbd3 + Btbd3V1P (p-values: <0.0001, <0.0001, and 0.0008, respectively); scramble vs shBtbd3 + Btbd3V1P (p-values: <0.0001, <0.0001, and 0.0003, respectively).

(C) Cytochalasin D (CytoD) rescues Naa10 KO-induced neurite reduction. Left: Representative images of hippocampal neurons from WT or whole-body Naa10-KO P0 mice treated with 20 pM CytoD or DMSO at DIV3. Middle and right: Sholl analysis. Three groups show significant differences (e.g., at radii 20, 40, 60 and 80 μm): WT + DMSO vs Naa10 KO + DMSO (p-values: <0.0001, <0.0001, <0.0001, and 0.0045, respectively); Naa10 KO + DMSO vs Naa10 KO + CytoD (p-values: <0.0001, <0.0001, <0.0001, and 0.0044, respectively); WT + CytoD vs Naa10 KO + DMSO (p-values: <0.0001, <0.0001, <0.0001, and 0.0027, respectively).

(A-C) Total neurite intersections and individual intersections at specified radii were assessed using Sholl analysis, with analyses conducted on 30 primary neurons from three independent hippocampal cultures for each group at Day 12 in vitro (DIV 12). Total neurite intersections were analyzed by two-way ANOVA along with Sidak’s post-hoc test, and individual intersections at specified radii were analyzed by two-way ANOVA repeat measures along with Sidak’s post-hoc test.

(related to Figure 2C). Potential substrates of Naa10 screened by subtiligase assay in HT-22 cells

(related to Figure 3). Btbd3-interacting proteins in HT-22 cells

(related to Figure 3). Proteins associated with Naa10 N-acetylated Btbd3 in HT-22 cells

(related to Figure 1). Depletion of Naa10 from CA1 does not affect global brain morphology, spatial memory, exploration or induce depression.

(A) The mRNAs of the NatA complex are enriched in the mouse hippocampus. Shown are RNA in situ hybridization images of Naa10, Naa15 and Hypk genes in the brain (sagittal section) of an 8-week-old male B6 mouse from the Allen Brain Atlas (https://mouse.brain-map.org). Colors ranging from blue to red indicate the relative expression levels from low to high, respectively. OLF, olfactory bulb; CTX, cerebral cortex; HIP, hippocampus; CB, cerebellum; BS, brain stem. The numbers of experiments conducted by the Allen Brain Atlas database are 1 for Naa10, 1 for Naa15 and 2 for HYPK (using two different antisense RNAs), respectively.

(B) The Naa10 Ab used in the study fails to detect Naa10 in the hippocampus of an 8-week-old Naa10-KO mouse. The scale bar is indicated.

(C) Naa10 protein is enriched in the hippocampal CA1 and DG of a 16-week-old mouse. The scale bar is indicated. The images shown are representative of three independent experiments.

(D) Naa10 is enriched in the mature neurons of an 8-week-old mouse. Immunohistochemistry fluorescent staining of NeuN and Nestin serves as a mature neuronal marker and a marker for neural stem cells, respectively. The scale bar is indicated.

(E) Schematic illustration of the experimental strategy for hippocampal CA1-specific Naa10 depletion. Naa10-cKO mice were generated by crossing male mice containing Camk2a-promoter-driven Cre and female mice carrying loxP-flanked Naa10 allele.

(F) Naa10 is completely depleted in CA1 in a 12-week-old Naa10-cKO mouse. The hippocampi of a control (Ctrl) and a Naa10-cKO mouse were subjected to immunohistochemistry fluorescent staining using Naa10 Ab. DAPI was used to stain DNA. The scale bar is indicated.

(G) Sagittal views of brain morphology of 2 control (Ctrl) and 3 Naa10-cKO mice by computed tomography. The scale bar is indicated.

(H) Coronal views of brain morphology in control (Ctrl) and Naa10-cKO mice by computed tomography. Representative images from 2 control and 3 Naa10-cKO mice are shown.

(I) Depletion of Naa10 from CA1 does not affect spatial memory in the Y maze test.

(J) Depletion of Naa10 from CA1 does not induce depressive-like behaviors in the tail suspension test.

(K) Depletion of Naa10 from CA1 does not result in difference in open field test.

(I-K) Shown are individual data points and mean ± SD for each group (Ctrl: 6 mice, cKO: 12 mice). ns, not significant by unpaired two-tailed t test.

(related to Figure 2) WT but not acetyltransferase-dead Naa10 promotes neurite outgrowth.

(A) Schematic illustration of HT-22 neuronal differentiation, which was used in independent experiments shown in figures 2A, 2B, 2D, and S2B. HT-22 cells before and after differentiation were examined using phase-contrast microscopy (bottom). Scale bar is indicated.

(B) HT-22 cells can be differentiated into Map2- and NeuN-positive neurons. HT-22 neurons after differentiation were subjected to immunostaining with Abs against the indicated proteins. DAPI was used to stain DNA. Scale bar is indicated.

(C) Acetyltransferase activity of Naa10 promotes neurite outgrowth. Naa10-KO HT-22 cells were infected with lentiviruses expressing WT or R82A mutant Naa10 and differentiated into neurons in neurobasal medium supplemented with N2 for 48 hr, followed by phase-contrast microscopy (left) and western blot analysis (lower right). Upper right: quantification of neurite length. Shown are mean ± SD for each group (N = 150 neurons from 3 independent experiments). P-values were analyzed by two-way ANOVA plus Sidak’s post-hoc. Scale bar is indicated.

(related to Figures 2C and 2D) Naa10 acetylates the N-terminus of Btbd3 in vitro.

(A) Btbd3 is highly expressed in the mouse hippocampus. Shown is RNA in situ hybridization of the Btbd3 gene in the brain (sagittal section) of an 8-week-old mouse analyzed from the Allen Brain Atlas (https://mouse.brain-map.org). Colors ranging from blue to red indicate the relative expression level from low to high, respectively.

(B-C) Recombinant Naa10 protein directly acetylates the N-terminus of Btbd3 in vitro. (B) MS spectrum shows a distinct peak at 2670.34 m/z with Naa10 (1.5 μg) compared to control (2628.33 m/z), indicating acetylation of Btbd3 by Naa10. (C) Representative MS/MS spectra confirm Naa10-mediated acetylation at the N-terminus of Btbd3 peptide.

(D) Schematic illustration of HT-22 neuronal differentiation used in figure 2D. Bottom: Btbd3 is completely depleted in Btbd3-KO HT-22 neurons. WT and Btbd3-KO HT-22 neurons were subjected to western blotting using the indicated Abs.

(related to Figure 3) Naa10-mediated Btbd3 N-α-acetylation increases interactions of CapZb and F-actin.

(A) Btbd3-interacting proteins are enriched in cytoskeleton proteins. Left: A silver staining gel of total lysates of HT-22 cells with or without expressing Btbd3-Flag subjected to immunoprecipitation (IP) with Flag beads, followed by SDS-PAGE. In-gel digestion was then performed, followed by mass spectrometry analysis. Right: Molecular function analysis of Btbd3-interacting proteins analyzed using the online tool THE GENE ONTOLOGY RESOURCE.

(B) N-acetylated Btbd3-interacting proteins. Pie charts show proteins associated with Btbd3 depending on Naa10 and Btbd3 N-acetylation. Flag-bead pull-down experiments in WT HT-22 cells expressing Btbd3WT-Flag or Btbd3V1P-Flag or in Naa10-KO HT-22 cells expressing Btbd3WT-Flag were performed. The list of proteins in each group is shown in Table S3. Bottom: Molecular function analysis of 13 N-acetylated Btbd3-interacting proteins analyzed using the online tool THE GENE ONTOLOGY RESOURCE.

(C) Btbd3 binding to CapZb and β-actin depends on Naa10 and Btbd3 N-α-acetylation. Btbd3-Flag IP was performed in WT or Naa10-KO HT-22 cells expressing the indicated proteins, followed by western blotting. Input comprises 10% of the total cell lysates.

(D) Naa10 KO reduces the association of CapZb and F-actin.

(E) Acetyltransferase activity of Naa10 promotes CapZb association with F-actin.

(F) Btbd3 N-α-acetylation enhances CapZb association with F-actin.

(D-F) Total cell lysates of WT, Naa10-KO or Btbd3-KO HT-22 cells expressing the indicated proteins were separated into globular actin (G-actin) and F-actin fractions, followed by western blotting. The bands of CapZb and β-actin in F-actin fraction were quantified using ImageJ. The relative ratios of CapZb to β-actin of each lane compared to lanes 3 (D), 4 (E) and 5 (F) are shown.

(C-F) Repeats of western blotting experiments from figure 3.