. 2017 Jan 18;93(2):379-393.
Epub 2017 Jan 5.
TAOK2 Kinase Mediates PSD95 Stability and Dendritic Spine Maturation Through Septin7 Phosphorylation
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TAOK2 Kinase Mediates PSD95 Stability and Dendritic Spine Maturation Through Septin7 Phosphorylation
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Abnormalities in dendritic spines are manifestations of several neurodevelopmental and psychiatric diseases. TAOK2 is one of the genes in the 16p11.2 locus, copy number variations of which are associated with autism and schizophrenia. Here, we show that the kinase activity of the serine/threonine kinase encoded by TAOK2 is required for spine maturation. TAOK2 depletion results in unstable dendritic protrusions, mislocalized shaft-synapses, and loss of compartmentalization of NMDA receptor-mediated calcium influx. Using chemical-genetics and mass spectrometry, we identified several TAOK2 phosphorylation targets. We show that TAOK2 directly phosphorylates the cytoskeletal GTPase Septin7, at an evolutionary conserved residue. This phosphorylation induces translocation of Septin7 to the spine, where it associates with and stabilizes the scaffolding protein PSD95, promoting dendritic spine maturation. This study provides a mechanistic basis for postsynaptic stability and compartmentalization via TAOK2-Sept7 signaling, with implications toward understanding the potential role of TAOK2 in neurological deficits associated with the 16p11.2 region.
Septin7; TAOK2; chemical genetics; dendritic spine; kinase signaling; neurodevelopment; postsynaptic stability.
Copyright © 2017 Elsevier Inc. All rights reserved.
TAOK2 Localizes to Dendritic Spines and Its Kinase Activity Is Required for Spine Maturation (A) Representative immunofluorescence image of a DIV18 hippocampal neuron stained using an antibody against TAOK2 and Phalloidin to visualize F-actin in dendritic shaft and spines. Magnification of the boxed area (white) is shown below. The scale bar represents 3 µm. (B) Hippocampal neurons transfected with either human TAOK2-WT (left) or TAOK2-K57A (right) along with EGFP to visualize morphology of dendritic spines. The lower images are higher magnification images. The scale bar represents 5 µm. (C) Bar graph depicts percent of mature mushroom spine-shaped dendritic protrusions in TAOK2-WT and TAOK2-K57A transfected neurons, respectively (n =15 per condition, p < 0.0001, and t test). (D) Montage of time-lapse images depicting spine dynamics was created by confocal imaging of dendritic spines of neurons transfected with either control shRNA or shRNA against TAOK2. The green arrow marks a morphing event, the blue arrow marks retraction, and the red arrow marks an extension event. (E) Number of morphing (green), extension (red), and retraction (blue) events per spine in neurons transfected with either control or TAOK2 shRNA. The five movies from independent experiments were analyzed with at least ten spines per experiment in each condition. (F) Spine morphology of control and TAOK2 shRNA treated neurons quantified as a percent of total protrusions binned into spine, filopodia, stubby, or atypical protrusions (n = 15 per condition, p < 0.0001, and multiple t tests). (G) Spine density of control and TAOK2 shRNA treated neurons defined as number of protrusions per unit dendritic length (n = 15 per condition, p < 0.0001, and t test). (H) Quantification of the length of protrusion from the base of the dendritic shaft (n = 15 per condition, p < 0.0001, and t test with Welch correction). All of the error bars represent SEM.
Synapses Are Formed Directly on the Dendritic Shaft in Absence of TAOK2 (A) Hippocampal neurons transfected with either control or TAOK2 shRNA along with RFP-tagged PSD95 to visualize postsynaptic localization of PSD95. The scale bar represents 10 µm. (B) Percent dendritic protrusions positive for PSD95 (n =15 per condition, p < 0.0001, and t test). (C) DIV18 neurons transfected with either control or shRNA against TAOK2 were fixed and immunostained for presynaptic Bassoon and postsynaptic Homer1a. The co-localization of Bassoon/Homer1 a in confocal images was considered as a synapse. The scale bar represents 1 µm. (D) Number of synapses made on the dendritic protrusions as a fraction of total protrusions (n = 10 neurons per condition, p < 0.0001, and t test). (E) Number of synapses made directly on the dendritic shaft per unit dendritic length (n = 10 neurons per condition, p < 0.0001, and t test). The error bars represent SEM. (F) Representative confocal images of hippocampal neurons transfected with either control or TAOK2 shRNA (expresses tdTomato under separate promoter) along with pH-sensitive SEP-GluR1. The scale bar represents 5 µm. (G) Percent of GluR1 positive protrusions (n = 15 per condition, p < 0.0001, and t test). All of the error bars represent SEM.
Loss of Dendritic Spines Leads to Defect in Calcium Compartmentalization (A) Representative traces of miniature excitatory postsynaptic currents measured from DIV16–18 neurons transfected with either control or TAOK2 shRNA. The inset depicts a typical event in higher magnification. (B) Amplitude of mEPSCs in control and TAOK2 shRNA treated neurons. (C) Frequency of excitatory events in control and TAOK2 shRNA treated neurons (n = 11–15 neurons from three independent experiments per condition, p > 0.05, and t test). The error bars represent SD. (D) Montages depict sequential frames acquired every 300 ms in time-lapse imaging of DIV16–18 neurons transfected with GCaMP6f along with either control or TAOK2 shRNA. The calcium transients are denoted by higher fluorescence intensity values in the pseudo-color scaled images. A maximum projection image (MaxProj) of all the time frame reveals all the calcium spike events in green. The tdTomato channel is used as a reference for dendritic spine morphology. The scale bar represents 2 µm. (E) The percent of total calcium transients observed in spine or the dendritic shaft. Three movies per condition were acquired as independent experiments and each spike during the movie was counted (****p < 0.0001 and one-way ANOVA with multiple comparisons). (F) Mean of the distance traversed by the calcium spike during distinct events (n = 20–25 per condition, p < 0.0001, and t test with Welch correction). The error bars represent SEM.
Mass Spectrometry Based Identification of TAOK2 Substrates in the Brain (A) Kinase activity of the engineered TAOK2 mutants was measured to screen for the analog sensitive mutation using the autophosphorylation of TAOK2 as a readout. HA-tagged TAOK2 kinase domain (1–320 amino acids) harboring the depicted mutations was incubated with either ATPγS or the analogs N6-Benzyl-ATPγS (BN) and N6-Furfuryl-ATPγS(FF) in an in vitro kinase reaction followed by alkylation using p-nitrobenzyl mesylate (PNBM)to produce thiophosphate-ester. Western blot of the phosphorylated TAOK2 mutants was probed with HA antibody and thiophosphate-ester specific antibody to visualize the total amount of TAOK2 mutant and the kinase activity of the mutant, respectively. The kinase dead (K57A), gatekeeper mutation (M105G), and the analog-sensitive mutation (M105G+G168A+R97Y) are highlighted. (B) Brain lysate from P13 mouse was incubated with either the kinase-dead (KD) or the analog-sensitive (AS) TAOK2 and probed for total amount of phosphorylated proteins using the thiophosphate-ester specific antibody. (C) Schematic depicts the workflow for TAOK2 substrate labeling and subsequent covalent capture prior to identification through mass spectrometry. (D) Representative spectrum obtained from the mass spectrometry analysis of phosphorylated peptides enriched in the analog sensitive condition, but not in the kinase dead reaction. This spectrum was obtained by HCD fragmentation from a precursor ion with m/z value 813.3971
+3, corresponding to a phosphorylated tryptic peptide spanning residues I417 to K429 of mouse septin7, phosphorylated on T426 (phosphorylated threonine is shown in the sequence as T). The observed b and y product ion peaks are labeled accordingly with the subscripts denoting their position in the identified peptide. (E) Gene Ontology analysis of proteins identified as direct substrates of TAOK2 reveals enrichment in processes such as dendritic spine formation/synaptogenesis, axon growth, cytoskeletal remodeling, and ciliogenesis. P
Phosphorylation of Sept7 by TAOK2 Is Required for Dendritic Spine Formation (A) Identified candidate substrates of TAOK2 from the mouse brain, with the threonine phosphorylation sites (bold) identified by mass spectrometry and the consensus sequence for phosphorylation by TAOK2. (B) Representative images of dendritic spines of DIV18 hippocampal neurons transfected with GFP alone and GFP along with the Septin7 phosphomutant (T426A). The scale bar represents 5 mm. (C) Percent of mature mushroom shaped spine protrusions in control neurons and in neurons transfected with the identified phosphomutants (n = 10–12 from three independent experiments, ****p = 0.0001, n.s. = p > 0.05, and one-way ANOVA). (D) Classification of the dendritic protrusions in control and Sept7
expressing neurons on the basis of mature mushroom spines or long filopodia as a percent of total dendritic protrusions. Three experiments with ten neurons per group were analyzed using multiple t tests to compare the two groups in each category. (E) Average length of individual dendritic protrusions in control and Sept7 phosphomutant expressing neurons where each dot represents individual data points. The lengths from three independent experiments were pooled and analyzed (n = 50 protrusions per condition, ****p < 0.0001, and unpaired t test with Welch correction). All of the error bars represent SEM. (F) Multiple sequence alignment of the C-terminal tail of Sept7 shows highly conserved sequence and conserved site of phosphorylation from fish to human. The site of phosphorylation is shown in red. (G) Purified TAOK2 wild-type (WT) or kinase dead (K57A) was incubated either alone, with GST-tagged wild-type, or phosphomutant Sept7 C-terminal tails (Sept7C T426A or Sept7C WT ). Phosphorylation of Sept7 C-terminal tail was detected in a western blot by probing with a pan phosphorserine/threonine (pS/T) antibody. The phosphorylated TAOK2 representing the active kinase is detected by antibody against pTAOK2 and total kinase by an antibody against the His-tag. T426A
Sept7 Is Directly Phosphorylated by TAOK2 at Its C-terminal Tail and Is Required for Synapse Location and Calcium Compartmentalization (A) Hippocampal neurons transfected with control or shRNA against TAOK2 were immunostained using an antibody against phosphorylated Sept7 (pT426) to detect the endogenous levels of phosphorylated Sept7. The transfected cells are detected by tdTomato expression. (B) Mean pSept7 fluorescence intensity in TAOK2 knockdown neuronal soma normalized to the mean of the fluorescence in the control treated soma (n=10 neurons per condition, p < 0.0001, and t test). (C) Hippocampal neurons at DIV15 were transfected with TAOK2 shRNA (tdTomato) along with co-expression of GFP-tagged Sept7-WT or Sept7-T426D. The scale bar represents 3 µm. (D) Percent of dendritic protrusions that were mushroom spines upon co-expression of GFP-tagged Sept7-WT, Sept7-T426A, or Sept7-T426D with either control or TAOK2 shRNA (n = 15 per experimental condition, **** = p < 0.0001, ** = 0.01 > p > 0.001, and * = 0.05 > p > 0.01 oneway ANOVA with Holm-Sidak’s multiple comparison test). The significance values are shown in respect to the first column. (E) Synaptic localization in neurons expressing GFP-tagged Sept7 T426A or Sept7 T426D along with RFP-PSD95 was determined by immunofluorescence assay with antibodies against GFP and presynaptic protein Bassoon. (F) The synaptic localization was quantified as a percent of total synaptic punctae that were present along the dendritic shaft. The error bars represent SEM. (G) DIV14 hippocampal neurons were transfected with GFP-tagged Sept7 T426A or Sept7 T426D along with the genetically encoded red calcium indicator RGeco1.2 and imaged at DIV16 in Mg+2 free media to visualize calcium transients. (H) The calcium transients were quantified as the percent of total calcium transients observed in the shaft or protrusions in neurons expressing Sept7T426A or Sept7T426D. The error bars represent SEM.
Sept7 Localization and Mobility Is Regulated by TAOK2 Mediated Phosphorylation (A) Representative images of hippocampal neurons transfected with TdTomato along with GFP-tagged Sept7
or Sept7 T426A show an increase in Sept7 localization in spines on expression of the phosphomimetic T426D mutant. The scale bar represents 3 µm. (B) Montage of images from hippocampal neurons expressing GFP-tagged Sept7 T426D or Sept7 T426A depict prebleach image, photobleached image, and fluorescence recovery at 20 min after bleaching of the Sept7 fluorescence in the region of interest (ROI) marked by the circle. (C) Graph depicts the mean normalized fluorescence intensity in ROI of neurons expressing either GFP-tagged Sept7 T426D (blue) or Sept7 T426A (green) over 20 min where fluorescence intensity was measured every min. The error bars are SEM and n = 6 per condition. The scale bar represents 3 µm. (D) Western blot of neuronal lysates obtained from cultured hippocampal neurons transduced with virus expressing control shRNA or shRNA against MST3 and TAOK2 depicts the changes in levels of phosphorylated Sept7 (T426) and phosphorylated TAOK2 (T441) on knock down of either TAOK2 or MST3. (E) Hippocampal neurons were transfected at DIV9 with control or MST3 shRNA and the levels of phosphorylated Sept7 were visualized at DIV12 by immunofluorescence assay using the pSept7 T426 antibody. (F) Phosphorylation level of Sept7 was quantified as a percent of the total normalized fluorescence intensity. The error bars are SEM and n > 7 per condition. (G) Higher magnification images comparing localization of GFP-tagged Sept7 T426D and Sept7 WT along with RFP-tagged PSD95 (red). The merged image highlights co-localization between Sept7 T426D and PSD95. The scale bar represents 2 µm. (H) Percent dendriticspines positive for Sept7 in neurons transfected with either Sept7 T426D or Sept7 WT mutant (n = 15–20 neurons per condition, p < 0.0001, and t test). The error bars are SEM. T426D
Phosphorylated Sept7 Interacts with PSD95 and Restricts Its Mobility (A) Purified TAOK2
or TAOK2 WT was incubated with either purified GST-Sept7 K57A or GST-Sept7 WT in a kinase reaction, followed by incubation with mouse brain lysate. Glutathione beads were used to pull down the GST-tagged proteins and the co-immunoprecipitates were probed in a western blot for presence of PSD95 and phosphorylated Sept7. Total GST protein was detected by GST antibody. (B) Montage depicts time-lapse images of neurons expressing GFP-tagged PSD95 along with control and TAOK2 shRNA taken before (pre), immediately after (0’), and then after every 5 min of photobleaching of the region of interest marked by the circle. The scale bar represents 3 µm. (C) Montage depicts time-lapse images of neurons expressing GFP-tagged PSD95 along with Sept7 T426A (blue) or Sept7 T426A (green) taken before (pre), immediately after (0’), and then after every 5 min of photobleaching of the region of interest marked by the circle. The scale bar represents 3 µm. (D) Graph plots the normalized PSD95-GFP fluorescence intensity of the region of interest as a percent of the prebleach intensity over 20 min in control (gray), TAOK2 knockdown (orange), or expression of phosphomimetic (green) and phosphomutant Sept7 (blue). (E) Schematic summarizes how Sept7 phosphorylation by TAOK2 mediates dendritic spine maturation. TAOK2 phosphorylated by MST3 kinase then phosphorylates Sept7, which translocates to the dendritic spine and, through its association with the synapse scaffolding protein PSD95, restricts its mobility. Stabilization of PSD95 at the spine leads to spine synapse maturation likely through accumulation of glutamate receptors, cell adhesion molecules, and other synaptic regulators. In absence of TAOK2 or MST3, Sept7 exists at the base of the protrusions in the non-phosphorylated form unable to interact with PSD95. In the absence of any stabilizing force, PSD95 along with associated receptors accumulates in the dendritic shaft leading to mislocalization of synapses to the dendritic shaft. T426D
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Dendritic Spines / metabolism*
Disks Large Homolog 4 Protein
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Protein-Serine-Threonine Kinases / genetics
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