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Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus [1]

['Hsiao-Tang Hu', 'Institute Of Molecular Biology', 'Academia Sinica', 'Taipei', 'Yung-Jui Lin', 'Ueh-Ting Tim Wang', 'Affiliated Senior High School Of National Taiwan Normal University', 'Research Center For Applied Sciences', 'Sue-Ping Lee', 'Yae-Huei Liou']

Date: 2023-09

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Funding: This work was supported by grants from Academia Sinica ( https://www.sinica.edu.tw , AS-IA-111-L01 and AS-TP-110-L10 to Y.-P.H.), and the National Science and Technology Council ( https://www.nstc.gov.tw/? , NSTC 108-2311-B-001-008-MY3 to Y.-P.H.). The funders had no role in study design, data collection and analysis, the decision to publish or the preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. The file of S1 Data contains the numerical value data of all figures. S2 Data contains all statistical results. S1 Raw Images contains uncropped blots.

Copyright: © 2023 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Nevertheless, how Klhl17 deficiency affects neuronal responses and functions remains elusive. We reported here that neuronal activation increases KLHL17 protein levels via glutamate receptor and protein synthesis. Using cultured mouse neurons, we demonstrate that KLHL17 is a critical factor involved in controlling activity-dependent dendritic spine enlargement. KLHL17 associates with synaptopodin (SYNPO), a marker of the spine apparatus (i.e., endoplasmic reticulum (ER) located at dendritic spines) [ 20 , 21 ]. SYNPO also associates with F-actin via its interaction with actinin [ 22 ]. We show that KLHL17 and SYNPO work together to control the synaptic clustering and distribution of the spine apparatus and calcium dynamics at dendritic spines. Ultimately, these functions influence neuronal activation, as reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression. Given that SYNPO is also linked to ASD [ 23 ], our study strengthens the relevance of ASD etiology to synaptic plasticity and the calcium dynamics controlled by the spine apparatus.

As a member of the Kelch-like protein family, KLHL17 contains a Bric-a-brac/Tramtrack/Broad complex (BTB) domain at its N-terminal region and 6 Kelch domains at the C-terminal half [ 11 , 15 – 17 ]. The BTB domain has been shown to mediate dimerization [ 18 ] and interaction with CUL3 ubiquitin E3 ligase [ 15 , 17 ]. The Kelch domains also act as a protein–protein interacting domain to recognize CUL3 substrates [ 15 , 16 , 19 ]. The interaction between KLHL17 and F-actin cytoskeletons is also mediated by the Kelch domains [ 11 , 13 ]. Knockdown or knockout of Klhl17 or disruption of the KLHL17 and F-actin interaction in mouse neurons impairs dendritic spine targeting of F-actin and disrupts dendritic spine enlargement during the developmental process [ 13 ]. Given that KLHL17 protein is specifically expressed in the brain [ 11 ], it has been suggested that KLHL17 exerts neuron-specific functions to control the morphology of excitatory synapses, i.e., neuron-specific and F-actin-enriched subcellular structures [ 13 ].

Among the various ASD-associated genes, Kelch-like protein 17 (KLHL17), also known as actinfilin [ 11 , 12 ], has been shown to contribute to dendritic spine enlargement during development [ 13 ]. Klhl17 +/– mice exhibit social deficits and hyperactive locomotion [ 13 ], echoing genetic evidence from patients that Klhl17 deficiency is associated with ASD [ 2 , 14 ]. Since KLHL17 controls dendritic spine enlargement, this protein may serve as a model to explore how morphological plasticity is relevant to ASD etiology.

Autism spectrum disorders (ASDs) are highly prevalent neuropsychiatric disorders characterized by impaired social and communication behaviors, abnormal sensations, and stereotyped activities [ 1 ]. Human genetic studies have identified hundreds of genes associated with ASD ( https://gene.sfari.org/database/human-gene/ ). Many of these disease-risk genes are directly or indirectly involved in synaptic formation, signaling, and plasticity [ 2 – 5 ]. Accordingly, it has been hypothesized that perturbation of those ASD-linked genes may increase or decrease synaptic number and/or strength, consequently promoting abnormal neuronal connectivity in the brain [ 5 – 10 ]. Thus, synaptopathy is highly relevant to ASD etiology.

Results

KLHL17 protein levels are regulated in development- and activity-dependent manners When we examined KLHL17 expression in mouse cortical and hippocampal mixed cultures, we observed that its protein levels gradually increased as the cultures matured (Fig 1A and 1B, upper). In mouse brains, KLHL17 protein levels also gradually increased from postnatal day 1 to 21 (Fig 1A and 1B, lower). However, in contrast to the increased protein levels, quantitative PCR revealed that levels of Klhl17 mRNAs were actually reduced in mouse brains and even more so in cultured neurons (Fig 1B), indicating that KLHL17 protein levels are posttranscriptionally up-regulated during development. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Increased neuronal activity up-regulates KLHL17 protein levels. (A, B) Protein levels, but not RNA levels, of KLHL17 are increased as neurons mature. Upper panel: Total cell lysate prepared from cultured neurons at different time points, i.e., 1, 7, 14, and 21 DIV. Lower panel: A total of 10 μg of tissue lysates isolated from whole brain of mice of different ages, i.e., postnatal days (P) 1, 7, 14, and 21. Quantifications of relative protein and RNA levels of the Klhl17 gene are shown in (B). (C, D) Protein levels of KLHL17 are increased by neuronal activation. Mature cultured neurons (18 DIV, upper) and immature cultured neurons (11 DIV, lower) were treated with TTX (1 μm), bicuculline (Bicu, 40 μm), and vehicle control (Ctrl) for 6 and 24 h, respectively. Neuro-2A (N2A) cell lysate transfected with KLHL17 acted a positive control. K17 and KLHL17 are interchangeable in the figure. Quantification is shown in (D). (E) Endogenous RNA levels of Klhl17 are reduced by bicuculline treatment. (F–I) Exogenous KLHL17 is sensitive to neuronal activity via NMDAR signaling and protein synthesis. Cultured neurons were transfected with Myc-KLHL17 and GFP at 12 DIV and then subjected to various treatments for 6 h at 18 DIV as indicated. NMDA, 10 μm; AP5, 100 μm; NBQX, 100 μm; CHX, 10 μm. (F, H) Representative images. GFP images shown in insets indicate transfected cells. (G, I) Quantification of exogenous Myc-KLHL17 signals. (J, K) The mTOR pathway and translation regulate KLHL17 protein levels. The effects of CHX (10 μm) or Rapamycin (10 nM) treatment were assessed. (J) Total cell lysates were analyzed by immunoblotting. (K) Quantification of relative protein levels of KLHL17. All immunoblots were performed using antibodies recognizing KLHL17 and internal control (HSP90 or actin, as indicated). The cultures were randomly assigned to treatments. For immunoblotting, each lane represents an independent sample. The sample sizes (N) of independent preparations or examined neurons are indicated in the panels. The data represent mean ± SEM. Individual data points are also shown. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant; one-way ANOVA. Scale bars: (F, H) 20 μm. The numerical value data and statistical results are available in S1 and S2 Data, respectively. DIV, day in vitro; KLHL17, Kelch-like protein 17; mTOR, mammalian target of rapamycin; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(f)quinoxaline; NMDAR, N-methyl-D-aspartate receptor. https://doi.org/10.1371/journal.pbio.3002274.g001 Given that increased neuronal activity is a key feature of maturing neurons, we investigated if the increased KLHL17 protein levels are relevant to enhanced neuronal activity. Under our culture conditions, cortical and hippocampal mixed cultures become fully mature at approximately 18 days in vitro (DIV) [24,25]. We treated the mature cultures with the sodium channel blocker tetrodotoxin (TTX) to inhibit neurotransmission and with the GABA A receptor antagonist bicuculline to enhance neuronal activity. Bicuculline-enhanced neuronal activity indeed increased KLHL17 protein levels, whereas limiting neuronal activity by means of TTX treatment reduced them (Fig 1C and 1D, upper). These alterations were not observed for immature cultures at 11 DIV (Fig 1C and 1D, lower), supporting that neuronal maturation is involved in controlling KLHL17 expression. In contrast to the increased protein levels upon neuronal activation, RNA levels of Klhl17 were reduced by bicuculline but remained unaltered upon TTX treatment (Fig 1E), revealing that neuronal activation exerts opposing control on the protein and RNA levels of Klhl17. Like endogenous KLHL17, expression levels of exogenous KLHL17 in mature neurons also responded in the same fashion to TTX and bicuculline treatments (Fig 1F and 1G), further supporting that KLHL17 proteins are controlled by neuronal activity. Taken together, these results show that neuronal activation up-regulates KLHL17 protein expression, even though RNA levels of Klhl17 are not increased or are even reduced. Thus, a complex regulatory mechanism is involved in controlling the RNA and protein levels of Klhl17 in opposing directions. Our findings also imply a critical role for synaptic stimulation in controlling KLHL17 expression.

NMDAR signaling and protein synthesis control KLHL17 proteins levels Next, we investigated if glutamate receptors, the major excitatory neurotransmitter receptors in mammalian brains, are involved in regulating KLHL17 protein levels. AP5, an N-methyl-D-aspartate receptor (NMDAR) blocker, but not NBQX, an antagonist of the AMPA receptor, completely prevented the increase in KLHL17 protein levels induced by bicuculline (Fig 1H and 1I). Consistently, NMDA treatment also increased KLHL17 protein levels (Fig 1H and 1I), supporting that the NMDAR pathway is critical for controlling protein levels of KLHL17. We then applied cycloheximide, an inhibitor of translational elongation, and rapamycin, a blocker of the mammalian target of rapamycin (mTOR) pathway for translation, to bicuculline-treated cultures. Inhibiting protein synthesis in this way blocked the ability of bicuculline to enhance both exogenous and endogenous KLHL17 protein levels (Fig 1H and 1K). Thus, neuronal activity via NMDAR signaling and protein synthesis tightly controls KLHL17 protein expression.

KLHL17 regulates calcium dynamics Activity-dependent spine enlargement is deemed relevant to spine apparatus-dependent calcium signaling [29–32]. We speculated that KLHL17 regulates spine apparatus-dependent calcium influx into the cytosol. To test that possibility, we first investigated if Klhl17 deficiency alters calcium dynamics in neurons. Cortical and hippocampal mixed cultures were transfected with GCaMP6s at 12 DIV and then we monitored relative changes in calcium concentrations in the cytoplasm of neurons based on GCaMP6s fluorescence signals at 18 DIV (Fig 4A). More specifically, we analyzed the frequency and amplitude of calcium events and the interval between events at dendritic spines. We found that the frequency of calcium events was reduced and the interval between calcium events was increased in Klhl17+/–neurons compared with wild-type neurons (Fig 4B–4D). However, the amplitude of calcium events was enhanced by Klhl17 deficiency (Fig 4E). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Klhl17 deficiency alters the frequency and amplitude of calcium events. Klhl17+/–neurons exhibit a reduced frequency and higher amplitude of spontaneous calcium events. Cultured neurons were transfected with GCaMP6s plasmids at 12 DIV and then calcium recording was performed by live-imaging at 18–19 DIV. (A) Representative images of recorded frames. Image series showing the change in calcium concentration based on GCaMP6s fluorescence signal intensity. The enlarged segment represents an example of an analyzed dendritic spine and the red circle indicates the ROI. The heat maps show the relative intensities of calcium signals. (B–E) Analysis of total calcium events at dendritic spines. (B) Representative patterns of calcium events. Scale units: amplitude (ΔF/F) and time (1 min). The calcium dynamics were analyzed based on the parameters of frequency (C), interval time (D), and calcium amplitude (E). Samples were randomly collected from multiple independent experiments. The sample size shown in (E) represents the number of examined neurons (N) and the number of examined dendritic spines (n) for the results shown in (C–E). (F–I) Paired analysis of calcium events at the spines and dendrites. (F) A representative dendritic segment with 2 ROIs; one in the spine (red) and the other in the dendrite (blue). Calcium events were categorized into 3 types: spine-only, dendrite-only, and both spine and dendrite with spikes at the same time (paired). Examples of these 3 types of events from wild-type neurons are shown. The relative amplitude (1, 1.5, 2, 2.5, 3) is also indicated at right. The percentage (G), frequency (H), and amplitude (I) of these 3 types of events are shown. The number (n) of examined paired ROIs was 50. In (I), the amplitude of all detected spine-only events was analyzed. The sample size is indicated. For paired events, the average amplitude of the paired ROIs at spines is shown. The data represent mean ± SEM. Individual data points are also shown. * P < 0.05, *** P < 0.001; unpaired two-tailed t test. Scale bars: (A) 5 μm, (F) 1 μm. The numerical value data and statistical results are available in S1 and S2 Data, respectively. DIV, day in vitro; KLHL17, Kelch-like protein 17; ROI, region of interest. https://doi.org/10.1371/journal.pbio.3002274.g004 We also performed a paired analysis of calcium events at the spines and dendrites (Fig 4F). The events were categorized into 3 groups, i.e., spine-only, dendrite-only, and paired (both spine and dendrite) responses (Fig 4F). The percentages of spine-only and dendrite-only events were very low, with a majority (>95%) of paired events in both Klhl17+/–and wild-type neurons under our experimental conditions (Fig 4G). In terms of calcium event frequency, only paired events were reduced in Klhl17+/–neurons (Fig 4H). For event amplitude, both spine-only and the spine element of paired events exhibited a bigger response in Klhl17+/–neurons relative to wild-type neurons (Fig 4I). We also noticed that the amplitudes of both spine- and dendrite-only events were obviously lower than those of paired events (Fig 4I). Thus, the differences in calcium events that we recorded are mainly attributable to the paired events, though spine-only events contributed somewhat to differences in amplitude. Together, these outcomes indicate that KLHL17 regulates both the frequency and amplitude of calcium dynamics in neurons.

SYNPO mediates the effect of KLHL17 on calcium dynamics, spine enlargement, and neuronal activation Based on our findings, we hypothesized that KLHL17 regulates ER distribution via SYNPO, thereby controlling calcium release from the ER to dendritic spines and consequently promoting dendritic spine enlargement and neuronal activation. To investigate that speculation, we first confirmed the involvement of the spine apparatus in the calcium events controlled by KLHL17. To do so, we treated neurons with ryanodine to inhibit calcium efflux from ER via the ryanodine receptor localized on ER. Ryanodine treatment of wild-type neurons reduced the frequency and increased the interval of calcium events at dendritic spines (Fig 7A–7C). The amplitude of calcium events was not obviously altered, perhaps because other types of calcium channels were involved (Fig 7D). Klhl17–/–neurons exhibited a reduced frequency but an increased amplitude of calcium events (Fig 7A–7D). Importantly, ryanodine treatment did not further alter the frequency or amplitude of the calcium events of Klhl17–/–neurons (Fig 7A–7D). Thus, this insensitivity of Klhl17–/–neurons to ryanodine treatment is consistent with our observation that Klhl17 deficiency impairs the synaptic distribution of ER. PPT PowerPoint slide

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TIFF original image Download: Fig 7. SYNPO overexpression rescues the deficits of Klhl17-deficient neurons. (A–D) Ryanodine treatment alters calcium dynamics in wild-type neurons but not Klhl17–/–neurons. (A) Representative patterns of calcium events. Scale units: amplitude (ΔF/F) and time (1 min). Quantification of the frequency (B), interval time (C), and amplitude (D) of calcium events. (E–H) SYNPO overexpression restores calcium dynamics in Klhl17-deficient neurons. Cultured neurons were co-transfected with GCaMP6s and SYNPO (or “S”) or vector control, as indicated, at 12 DIV, before performing calcium recording by means of live-imaging at 18–19 DIV. (E) Representative patterns of the calcium events. Scale units: amplitude (ΔF/F) and time (1 min). Quantification of frequency (F), interval time (G) and amplitude (H). (I–L) SYNPO overexpression also rescues the spine enlargement of Klhl17-deficient neurons upon bicuculline stimulation. Cultured neurons were transfected with GFP and SYNPO or vector control at 12 DIV, as indicated, and performed activity-induced spine enlargement at 18 DIV. (I) Representative images of dendritic segments. (J) Quantification of density, (K) width and (L) length of dendritic protrusions. In (J), the density of dendritic spines was analyzed on both a per-neuron and per-dendrite basis. (M, N) SYNPO overexpression increases ERK phosphorylation of Klhl17-deficient neurons upon bicuculline treatment. Cultured neurons were co-transfected with GFP and SYNPO or vector control at 12 DIV and treated with bicuculline at 18 DIV. (M) Representative images. GFP images are shown in insets, with transfected cells highlighted by yellow arrows. (N) Quantification of the relative intensity of phospho-ERK signals. All data were collected from at least 2 independent experiments. The sample sizes of examined neurons (N), dendritic segements (n), and dendritic spines (n) are indicated. For the same set of experiments, the sample size is labeled only in 1 panel. The data represent mean ± SEM and cumulative curves (K, L: right). Individual data points are also shown. ** P < 0.01; *** P < 0.001; ns, not significant. Two-way ANOVA (B–D, F–H, J–L: left, N); Kolmogorov–Smirnov test for cumulative probability (K, L: right). Scale bars: (I) 5 μm; (M) 20 μm. The numerical value data and statistical results are available in S1 and S2 Data, respectively. DIV, day in vitro; ERK, extracellular signal-regulated kinase; KLHL17, Kelch-like protein 17. https://doi.org/10.1371/journal.pbio.3002274.g007 We then examined if SYNPO overexpression ameliorates Klhl17 deficiency. In terms of calcium dynamics, we found that SYNPO overexpression improved all of the deficits of calcium dynamics prompted by Klhl17 deficiency, including the frequency, interval time, and amplitude of calcium events in cultured neurons (Fig 7E–7H). For the features of dendritic spines, we observed that SYNPO overexpression did not alter the density or length of dendritic spines, but it did specifically enhance the width of Klhl17+/–dendritic spines to values comparable to wild-type neurons (Fig 7I–7L). When cultured neurons were activated by means of bicuculline treatment, the spine width of SYNPO-overexpressing Klhl17+/–neurons was also fully rescued to the levels of wild-type neurons subjected to the same treatment (Fig 7I and 7K). These results reveal that SYNPO is involved in KLHL17-controlled dendritic spine enlargement. Moreover, the effect is specific because neither the density nor length of dendritic spines was affected upon SYNPO overexpression (Fig 7I–7L). Finally, SYNPO overexpression also improved neuronal activation of Klhl17+/–neurons because it enhanced ERK phosphorylation in Klhl17+/–neurons to levels comparable to wild-type neurons (Fig 7M and 7N). Thus, SYNPO overexpression ameliorates the perturbed calcium dynamics, dendritic spine enlargement, and neuronal activation of Klhl17-deficient neurons, supporting the involvement of SYNPO in KLHL17-dependent regulation.

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