WWC1/2 regulate spinogenesis and cognition in mice by stabilizing AMOT

Cao, Runyi; Zhu, Rui; Sha, Zhao; Qi, Sixian; Zhong, Zhenxing; Zheng, Fengyun; Lei, Yubin; Tan, Yanfeng; Zhu, Yuwen; Wang, Yu; Wang, Yi; Yu, Fa-Xing

doi:10.1038/s41419-023-06020-7

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Published: 01 August 2023

WWC1/2 regulate spinogenesis and cognition in mice by stabilizing AMOT

Cell Death & Disease volume 14, Article number: 491 (2023) Cite this article

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Abstract

WWC1 regulates episodic learning and memory, and genetic nucleotide polymorphism of WWC1 is associated with neurodegenerative diseases such as Alzheimer’s disease. However, the molecular mechanism through which WWC1 regulates neuronal function has not been fully elucidated. Here, we show that WWC1 and its paralogs (WWC2/3) bind directly to angiomotin (AMOT) family proteins (Motins), and recruit USP9X to deubiquitinate and stabilize Motins. Deletion of WWC genes in different cell types leads to reduced protein levels of Motins. In mice, neuron-specific deletion of Wwc1 and Wwc2 results in reduced expression of Motins and lower density of dendritic spines in the cortex and hippocampus, in association with impaired cognitive functions such as memory and learning. Interestingly, ectopic expression of AMOT partially rescues the neuronal phenotypes associated with Wwc1/2 deletion. Thus, WWC proteins modulate spinogenesis and cognition, at least in part, by regulating the protein stability of Motins.

Introduction

The WWC protein family, comprising WWC1 (WW and C2 domain-containing 1, also known as KIBRA for KIdney and BRAin), WWC2, and WWC3, have recently been identified as Hippo signaling pathway regulators [1,2,3]. Previous reports indicate that genetic perturbation of Wwc1 in rodent neuronal cells leads to reduced synaptic plasticity, episodic learning, and memory [4,5,6,7,8]. Moreover, WWC1 is associated with neurological disorders, including impaired memory, depression, post-traumatic stress disorder (PTSD), and Alzheimer’s disease [7, 9,10,11,12,13,14]. Despite these findings, the precise molecular mechanism underlying the role of WWC1 in brain function remains unclear.

WWC proteins contain multiple conserved domains that support protein-protein interactions and functions (Fig. 1A). For instance, the WW domains have been shown to interact with core components of Hippo pathway, such as LATS1/2 and PTPN14, to facilitate Hippo pathway activation [15,16,17,18,19]. In addition, as a key scaffolding protein enriched in postsynaptic sites, WWC1 can interact with several synaptic proteins including Dendrin and Synaptopodin [20,21,22]. The PDZ-binding motif and adjacent region of WWC proteins mediate interactions with protein interacting with PRKCA 1 (PICK1), atypical protein kinases (aPKC, PKMζ), and CAMD1, supporting its role in synaptic transmission and memory performance [5, 6, 23, 24]. The C2 domain of WWC1 binds to phosphatidylinositol-3-phosphate (PI3P) enriched on endosome membranes [25]. Moreover, WWC1 has been reported to inhibit the proteasomal degradation of Rab27a, hence playing a role in exosome trafficking and secretion [26]. Unlike WWC1, the roles of WWC2 and WWC3 in the nervous system have been largely unexplored.

**Fig. 1: WWC1/2/3 regulate protein stability of Motins.**

Motin family proteins, including AMOT, AMOTL1, and AMOTL2, are also Hippo signaling pathway components (Fig. 1A) [27,28,29,30,31]. AMOT gene produces two isoforms, AMOTp130 and AMOTp80, via alternative splicing (Fig. 1A). While AMOT is associated with autism spectrum disorder [32], its role in the nervous system remains poorly understood. In cultured hippocampal neurons, AMOT is enriched in dendritic spines, where it interacts with the actin cytoskeleton and postsynaptic scaffolds, such as multi-PDZ domain protein 1 (MUPP1) and postsynaptic density-95 (PSD-95), and these interactions are critical for actin turnover and stabilization of dendritic spines [33,34,35,36]. Moreover, AMOT is indispensable for dendrite growth and arborization in developing neurons, and conditional deletion of Amot in mouse neurons results in impaired dendritic network in Purkinje cells and impaired motor coordination [37]. These findings suggest that both WWC proteins and Motins are involved in the regulation of neuronal functions.

The interaction between WWC proteins and Motins has been previously reported [18, 38,39,40,41,42]. In mouse livers deficient in WWC proteins, AMOT protein expression is significantly reduced, although the underlying mechanism has not been studied [38]. Given their significant roles in brain function, it is reasonable to hypothesize that WWC proteins and Motins may coordinate with each other to regulate neuronal functions. Moreover, WWC1 and AMOT can activate Hippo signaling pathway by forming biomolecular condensates in various cellular contexts [43]. In this study, we demonstrate that the protein stability of Motins is regulated by deubiquitinase USP9X in a manner dependent on WWC proteins. Furthermore, we show that the defects in spinogenesis and cognition in mice with neuronal-specific deletion of Wwc1 and Wwc2 are partially rescued by ectopic expression of AMOT. Thus, the stabilization of Motins by WWC proteins may participate in the regulation of spinogenesis and cognitive behaviors.

Results

WWC1/2/3 regulate protein stability of Motins

To explore whether WWC proteins regulate Motins in different cell lines, we checked the expression of AMOT, AMOTL1, and AMOTL2 in WWC1/2/3 tKO HEK293A cells. Consistent with the previous report, AMOTp130, and AMOTL1 protein levels were significantly decreased in WWC1/2/3 tKO cells (Fig. 1B, C) [38]. AMOTp80 was also decreased in some experiments following Wwc1/2/3 deletion, but this was not consistently observed and might reflect an indirect and context-dependent regulation (Fig. 1B, C). Notably, the expression of AMOTL2 remained largely unchanged (Fig. 1B, C). Similar results were also observed in SH-SY5Y human neuroblastoma cells (Fig. S1A, B). Conversely, AMOTp130 protein level was induced by ectopic expression of WWC1, WWC2, or WWC3 in wild-type or WWC1/2/3 tKO HEK293A cells (Fig. 1D, S1C). These results indicate that the protein levels of Motins and WWC proteins are positively correlated.

To further investigate how Motins were regulated by WWCs, we examined if the transcription of Motins was perturbed in WWC1/2/3 tKO cells. As shown in Fig. 1E, the mRNA levels of AMOT and AMOTL1 were not changed upon WWC1/2/3 deletion, whereas the mRNA level of AMOTL2 was significantly increased. The increase in AMOTL2 mRNA level could compensate AMOTL2 protein levels in WWC1/2/3 tKO cells (Fig. 1B). Since AMOTL2 is a target gene of YAP/TAZ, this upregulation is likely due to the activation of YAP/TAZ in WWC1/2/3 tKO cells. Supporting this, we observed an increase in active YAP (aYAP, non-phosphorylated) and TAZ levels, as well as increased expression of canonical YAP/TAZ genes CTGF, CYR61, and ANKRD1 (Fig. 1B, E). These results suggest that the downregulation of AMOTp130 and AMOTL1 expression was regulated at a posttranscriptional level.

We then assessed the protein turnover of AMOT in wild-type and WWC1/2/3 tKO HEK293A cells treated with cycloheximide (CHX, a protein synthesis inhibitor). The half-life of AMOTp130 in wild-type cells was approximately 4 h, whereas it was markedly shortened to 2 h in WWC1/2/3 tKO cells (Fig. 1F, G). AMOTp80 was much more stable in both wild-type and WWC1/2/3-deficient cells (Fig. 1F). These results imply that AMOTp130 is destabilized in the absence of WWC proteins.

WWC proteins interact directly with and stabilize Motins

Direct interaction between AMOTp130 and WWC1 has been previously reported [18, 38,39,40,41,42]. Indeed, in reciprocal co-immunoprecipitation assays, AMOTp130 interacted with WWC1, WWC2, or WWC3 (Fig. 2A-C and S2A–C). This interaction was abolished when either WW domains in WWC1 were mutated or deleted, or when the third PPxY motif of AMOTp130 was mutated, indicating that the interaction between WWC proteins and Motins was mediated by WW domains in WWC proteins and PPxY motifs in Motins (Fig. 2D, E and S2D, F). On the other hand, the C2 domain and PDZ-binding domain (ADDV) of WWC1/2 were not required for binding with AMOTp130 (Fig. S2D, E). To test whether the interaction between WWC proteins and Motins regulates the protein stability of Motins, the half-life of AMOTp130 in control or WWC1-overexpressing cells was determined. The expression of wild-type WWC1, but not WW domain mutant WWC1, effectively stabilized AMOTp130 (Fig. 2F, G). The tryptophan residue in WWC1 at position 88 (W88) is critical for the hydrophobic interaction between WW domains and PPxY motifs. The W88C mutation in patients with Tourette syndrome has been reported to affect the formation of the WWC1-Dendrin complex [20]. We found that the interaction of WWC1 W88C mutant with AMOTp130 was reduced compared with wild-type WWC1 (Fig. S2F, G). Consistently, the WWC1 W88C mutant did not significantly boost the stability of AMOTp130 (Fig. 2H, I). Together, these results indicate that WWC proteins interact directly with Motins, and this interaction is crucial for regulating the protein stability of Motins.

WWC proteins recruit USP9X to deubiquitinate and stabilize Motins

The ubiquitin-proteasome system (UPS) is responsible for regulating cellular protein turnover and homeostasis [44, 45]. E3 ligases such as RNF146 and NEDD4 have been shown to promote ubiquitination and proteasomal degradation of AMOT [29, 46, 47]. Conversely, USP9X deubiquitinase stabilizes multiple Hippo pathway components, including LATS2, WWC1, and AMOTp130 [48,49,50,51]. Indeed, the protein levels of AMOTp130 and WWC proteins were decreased in USP9X knockdown HEK293 A cells (Fig. 3A). Since WWC proteins regulate the protein stability of Motins, and both are targeted by USP9X, we propose that USP9X may play a role in the stabilization of Motins by WWC proteins.

As indicated by co-immunoprecipitation assays, USP9X interacted with both AMOTp130 and WWC1 (Fig. 3B, S3A). Interestingly, in the presence of ectopic WWC1, the interaction between AMOTp130 and USP9X was significantly strengthened (Fig. 3B). Conversely, the interaction between AMOTp130 and USP9X was decreased in WWC1/2/3 tKO cells (Fig. 3C). Moreover, the ubiquitination of AMOTp130 was significantly induced in USP9X-knockdown cells, and ectopic expression of WWC1 was able to reduce the ubiquitination of AMOTp130 in wild-type but not USP9X-knockdown cells (Fig. 3D). Furthermore, we observed that the expression of wild-type WWC1, but not WW domain mutant WWC1, effectively reduced the ubiquitination of AMOTp130 (Fig. 3E). Consistently, the half-life of AMOTp130 was shortened in USP9X-deficient cells (Fig. S3B, C). These results indicate that WWC proteins, as a bridge between AMOTp130 and USP9X, reduce ubiquitination and then stabilize AMOTp130.

WWC proteins regulate stability of Motins in neurons and brain

Both WWC1 and AMOT are involved in regulating neuronal functions [4,5,6,7, 9, 11, 12, 20, 33, 37, 52]. We then asked whether the functions of WWC1 and AMOT in the nervous system were coupled. WWC1 is widely expressed in multiple brain regions, particularly in the hippocampus and cortex [53]. Nevertheless, it is unclear whether WWC2, USP9X, and Motins share similar expressions and functions in the nervous system, although abundant mRNA levels of these genes are detected in brain cells (Allen brain atlas) [37, 54, 55]. Western blot analysis revealed that WWC1/2 (no Wwc3 gene in mice), USP9X, and AMOT were ubiquitously expressed in the mouse brain, with relatively higher expression in cortex and hippocampus, regions highly related to memory function (Fig. S4A). These proteins were also highly expressed in primary cultured cortical neurons, and their expression gradually increased during neuronal maturation (Fig. S4B). Neuronal maturation is accompanied by spinogenesis, the process of dendritic spine development in neurons [56]. Supporting the role of WWC proteins and AMOT in spinogenesis, WWC proteins, and AMOT were mainly enriched in postsynaptic density (PSD) fractions following sucrose gradient fractionation of mouse cortex or hippocampus (Fig. S4C, D), which was consistent with previous reports [5, 33, 53, 57]. In mouse cortex extracts, WWC1 also interacted with AMOTp130 at the endogenous level, suggesting a conserved regulatory mechanism in the mouse brain (Fig. 4A).

**Fig. 4: WWC proteins regulate the stability of Motins in neurons and brain.**

To explore the role of WWC proteins in the stability of Motins in the mammalian brain, we first generated brain-specific Wwc1/2 knockout (KO) mice (Wwc1/2^Nes-cKO) by crossing Wwc1/2 flox mice with Nestin-Cre mice, the latter drove DNA recombination specifically in apical neural progenitor cells (NPCs) starting on E10.5 (Fig. 4B) [15, 58]. Wwc1/2^Nes-cKO mice were born normal but exhibited growth retardation, as indicated by smaller body size and brain size (Fig. 4C and S4E, F). We dissected the cortex and hippocampus of 8-week-old Wwc1/2^Nes-cKO and control mice, and observed efficient inactivation of WWC1/2 (a short WWC2 might be alternatively translated after DNA recombination, which lacks WW domains and should be inactive). Interestingly, in Wwc1/2^Nes-cKO mice, the protein level of AMOTp130, and to a less extent AMOTp80 and AMOTL1, was reduced, albeit no significant change at the mRNA level was observed (Fig. 4D–G and S4G, H). The expression of TAZ was also significantly induced in Wwc1/2^Nes-cKO mice, which may contribute to abnormal brain development and was not further investigated in this study (Fig. 4D, F). We isolated the primary neurons from Wwc1/2^Nes-cKO and control mice and observed a reduction in AMOT and AMOTL1 expression (Fig. 4H, I). Moreover, expression of wild-type WWC1, but not WW domain mutant and W88C WWC1, rescued AMOTp130 expression in Wwc1/2^Nes-cKO primary neurons (Fig. S4I, J). Collectively, these results indicate that the protein stability of Motins, especially AMOTp130, and AMOTL1, is regulated by WWC proteins in the brain and neurons.

Defective spinogenesis and cognition in WWC1/2-deficient mice

The growth defects of Wwc1/2^Nes-cKO mice prevented in-depth analysis of neuronal functions associated with Wwc1/2 deletion. To specifically delete Wwc1/2 in neurons, we crossed Wwc1/2 flox mice with transgenic mice expressing Cre recombinase under Synapsin 1 regulatory element (Syn-Cre mice) (Fig. 5A, B). Wwc1/2^Syn-Cre mice exhibited normal growth and brain size, and H&E staining on brain sections revealed no significant morphological differences including cortex thickness (Fig. S5A–D). Moreover, immunofluorescence staining of superficial and deep neocortical layers indicated that the thickness of the cortex and neuronal densities of Wwc1/2^Syn-cKO mice was comparable to that of control littermates (Fig. S5E), indicating that cortical development and neurogenesis were largely unaffected by Wwc1/2 deletion in neurons. We also analyzed the spine densities of neurons in the prefrontal cortex and hippocampal dentate gyrus in Wwc1/2^Syn-cKO mice using Golgi staining. Compared to the control mice, the dendritic spine density was reduced in Wwc1/2^Syn-cKO mice (Fig. 5C, D). These results suggest that mice with Wwc1/2 deletion in neurons have normal brain morphology, but the brain function is affected due to defective spinogenesis.

WWC1 (KIBRA) plays a role in synaptic plasticity, learning, and memory [4, 6, 20]. To determine the effect of Wwc1/2 deficiency on brain function, we performed multiple behavioral tests on Wwc1/2^Syn-cKO mice and control littermates (Fig. 5B). In the Y maze test, Wwc1/2^Syn-cKO mice exhibited reduced sequential alternations, indicating impaired associative working memory (Fig. 5E). Consistently, in the novel object recognition test, Wwc1/2^Syn-cKO mice showed no preference for the novel objects, indicating a defect in recognition memory (Fig. 5F). Previously, it has been shown that Wwc1 KO mice displayed extensive deficits in spatial learning and memory [6, 20]. We asked whether similar defects existed in Wwc1/2^Syn-cKO mice. In the Morris water maze test, Wwc1/2^Syn-cKO mice exhibited increased latency to reach a hidden platform, reduced time spent in the platform quadrant, and longer swim-path traces to reach the hidden platform on day 5 (Fig. 5G–I), suggesting impairment in spatial learning and memory. Together, these data support the role of WWC1/2 in regulating cognitive function in mice.

We also performed additional tests to monitor behaviors like depression and locomotor coordination, but no significant difference was observed between Wwc1/2^Syn-cKO mice and control mice. For instance, Wwc1/2^Syn-cKO and control mice showed comparable moving velocity and spent similar time at the center in the open field test, although Wwc1/2^Syn-cKO mice traveled a slightly longer distance (Fig. S5F-I). In the elevated plus maze test, Wwc1/2^Syn-cKO mice showed similar exploring time in the open arms, but relatively less time in the closed arms, compared with control mice (Fig. S5J, K). Moreover, Wwc1/2^Syn-cKO and control mice showed similar immobility time in the tail suspension test and latency to fall in the rotarod test (Fig. S5L, M). Hence, the poor performance in learning and memory-related assays observed in Wwc1/2^Syn-cKO mice was not due to a deficiency in motor ability. Taken together, these data suggest that Wwc1/2 deletion has no significant impact on neuropsychiatry-associated functions and motor coordination.

The phenotype of WWC1/2 loss is partially rescued by ectopic expression of AMOT

The reduced expression of AMOT in Wwc1/2 KO neurons may contribute to the phenotypes associated with Wwc1/2 deficiency (Fig. 4H). We tested whether ectopic expression of AMOT could rescue the impaired neuronal functions in Wwc1/2 KO mice. We injected AAV2/9-hSyn-ZsGreen viruses—which expressed ZsGreen under the control of a Synapsin promoter—into the lateral ventricles of neonatal (P0) pups. This led to a widespread and robust neuronal expression of ZsGreen in the cortex and hippocampus, and weak expression in cerebellar granule neurons (Fig. S6A). AAV2/9 viruses expressing either AMOTp130 (AAV2/9-AMOT) or control fluorescent protein ZsGreen (AAV2/9-Ctl) were then delivered to the lateral ventricles of Wwc1/2^Syn-cKO or control mice. Viral injection had no significant effect on the growth and brain development of mice (Fig. S6B, C). Immunoblotting analysis confirmed the expression of FLAG-tagged AMOTp130 in the cortex and hippocampus of most mice injected with AAV2/9-AMOT (Fig. S6D–G). Subsequently, we carried out a range of behavioral tests to determine the role of AMOTp130 in cognitive functions related to WWC1/2 (Fig. 6A, B). In the novel object recognition test, AAV2/9-AMOT injection could effectively normalize the impaired recognition memory in Wwc1/2^Syn-cKO mice (Fig. 6C). AAV2/9-AMOT injection in Wwc1/2^Syn-cKO mice also remarkably shortened the time spent in latency to find the hidden platform in the Morris water maze, suggesting that spatial learning and memory could be partially rescued by overexpression of AMOT (Fig. 6D). Moreover, AAV2/9-AMOT injection in Wwc1/2^Syn-cKO mice exhibited a tendency of improved performance under context condition, although the change was not statistically significant (Fig. 6E). On the other hand, AAV2/9-AMOT injection failed to restore working memory and associative memory, as indicated by the Y maze test (Fig. 6F). To assess the role of AMOT in WWC1/2-mediated spinogenesis, we also analyzed the spine density of neurons in the prefrontal cortex and hippocampal dentate gyrus from four groups of mice (Fig. 6G–J). In cortical neurons, AMOT overexpression significantly improved defects in spinogenesis (Fig. 6G, H). While the spine density of Wwc1/2^Syn-cKO mice was reduced in hippocampal neurons, there was also a tendency of recovery in mice injected with AAV2/9-AMOT (Fig. 6I, J). WWC1 is well known to regulate the activity and expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPA receptors), which are crucial for learning and memory [4, 5, 24, 59, 60]. Our results indicated that overexpression of AMOT in Wwc1/2 KO neurons induced GluA1 and GluA2 expression (Fig. S6H, I), indicating a potential role of AMOT in AMPAR regulation. Together, the expression of AMOTp130 could ameliorate impaired cognitive functions associated with WWC1/2 deficiency. However, likely due to the heterogeneous spatiotemporal expression of AMOTp130 following AAV2/9 injection, complete rescue of neuronal phenotypes caused by Wwc1/2 deletion was not achieved.

Discussion

In this study, we reveal a molecular mechanism underlying the stabilization of Motins by WWC proteins. WWC proteins positively regulate Motins, including AMOTp130 and AMOTL1, which is consistent with a previous study [38]. AMOTL2 might be regulated both post-translationally by WWC proteins and transcriptionally by YAP/TAZ. In addition, we have shown that WWC proteins bind directly to Motins through an interaction mediated by WW domains and PPxY motifs on WWC proteins and Motins, respectively. Moreover, WWC proteins are required for Motins to recruit protein deubiquitinase USP9X, remove polyubiquitination, and prevent protein degradation. USP9X also regulates the protein stability of WWC proteins [48] (Fig. 3). Thus, the cellular levels of WWC proteins and Motins are positively correlated, suggesting that these two proteins may jointly regulate common biological processes.

Dendritic spinogenesis and pruning are fundamental to synapse connections, neural circuit wiring, and subsequent higher brain function. In neurons, the protein stability of Motins is also tightly coupled with WWC proteins. Both Motins and WWC proteins are highly enriched in the dendritic spine, specifically in postsynaptic density (Fig. S4). Indeed, Wwc1/2 deletion in neurons leads to a sparse dendritic spine, which likely contributes to aberrant neurobehavioral features observed in Wwc1/2^Syn-cKO mice, in particular, impaired learning and memory. (Fig. 5). Notably, the defects caused by Wwc1/2 deficiency are partially rescued by ectopic expression of AMOTp130 (Fig. 6). Hence, Motins and WWC proteins coordinate the regulation of dendritic spine and cognitive functions.

The remodeling of actin-microtubule cytoskeleton is pivotal in regulating dendritic spine plasticity [61]. For instance, the nucleation, elongation, and branching of actin filaments are involved in the maturation and stabilization of the dendritic spine [62, 63]. Moreover, microtubules are present in dendritic spine and regulate their morphology and synaptic plasticity [64, 65]. Interestingly, Motins interact with actin filaments [27, 28, 66,67,68], and WWC1 has been shown to associate with the microtubule motor protein dynein light chain 1 [69], which is involved in endocytic recycling compartment transport by the SNX4-WWC1-dynein complex [70]. Hence, Motins and WWC proteins together may serve as an actin-microtubule adaptor to coordinate the dynamics of dendritic spine and synaptic plasticity [71]. In addition to Motins, WWC proteins also interact with additional components of the dendritic spine, such as dendrin, PICK1, synaptopodin, and aPKC isoforms [5, 6, 20, 22, 23, 72]. It is also possible that WWC proteins are involved in the transport of multiple regulators of the dendritic spine to defined subcellular locations. Moreover, a recent study has suggested that AMOT and WWC1 form condensates in response to distinct signals, it would be compelling to investigate whether such condensates are involved in spinogenesis [43].

Both WWC proteins and Motins are important Hippo pathway regulators, and their downregulation leads to activation of downstream effectors YAP/TAZ [1, 2, 15, 38, 48, 51, 73,74,75,76,77,78,79]. Our study found that YAP/TAZ target genes were significantly upregulated in Wwc1/2 KO neurons. However, Amot deletion in neurons did not significantly affect YAP target genes expression [37], suggesting a Hippo pathway-independent role of AMOT. Moreover, a WWC1 mutant defective in LATS binding was found to increase WWC1 abundance in AMPAR complexes, improving hippocampal-dependent learning and memory [59]. This suggests a potential link between the Hippo pathway, WWC1, and memory performance. It is currently unclear whether the coupling of the protein levels of WWC proteins and Motins regulates YAP/TAZ activity, and whether YAP/TAZ activation in Wwc1/2 KO mice accounts for the impaired dendritic spine and cognitive functions. A side-by-side comparison of YAP/TAZ loss-of-function and AMOT gain-of-function on the Wwc1/2 KO background should clarify these questions.

Materials and methods

DNA constructs, lentivirus production, and infection

The full-length open reading frame (ORF) and truncations with FLAG/HA/MYC tag were amplified by polymerase chain reaction (PCR) and cloned into pLVX vector (632164, Takara, Japan) using ClonExpress MultiS One Step Cloning Kit (C113–02, Vazyme, Nanjing, China). FLAG-tagged full-length Amot was constructed by cloning the corresponding mouse cDNA into pLVX vector. For CRISPR/Cas9 cloning, sgRNA oligos were cloned into the lentiCRISPR v2 vector provided by Dr. Feng Zhang [80], using T4 ligase (2011A, Takara, Shiga, Japan). PLVX or lentiCRISPR coupled with packaging vectors psPAX2 and pMD2.g were co-transfected into HEK293T cells to produce a high titer lentivirus. After transfection for 48 h, medium supernatant was harvested and filtered through a 0.45 μm filter (SLHP033RS, Millipore, MD, USA). To establish stable cell lines, cells were infected with the lentivirus concentrated by PEG8000 in the presence of 10 μg/ml polybrene (TR-1003-G, Sigma-Aldrich, MO, USA) and cells were selected with 2 μg/ml puromycin (ant-pr-1, InvivoGen, Toulouse, France) 48 h after infection.

Cell lines and DNA transfection

HEK293A cells were maintained in DMEM (GIBCO, Waltham, ME, USA) and SH-SY5Y cells in DMEM/F12 (GIBCO) media. Media were supplemented with 10% (vol/vol) fetal bovine serum (FBS, GIBCO) and 50 mg/ml P/S (penicillin/streptomycin, SV30010, Hyclone, Logan, UT, USA). All cell lines were authenticated according to the short tandem repeat (STR) profile. The sgRNA sequences targeting individual genes are listed in Supplementary Table 1. WWC1/2/3 tKO cells were described previously [15]. Cell lines were transfected with indicated plasmids using PolyJet Transfection Reagent (SL100688, SignaGen Laboratories, Rockville, MD, USA) according to the manufacturer’s instructions.

siRNA transfection

siRNA for USP9X and non-targeting control were synthesized by HANBIO (Shanghai, China) and prepared at a concentration of 20 μM in nuclease-free water. siRNA transfection was conducted using Lipofectamine RNAiMAX Transfection reagent (Cat. No. 13778075, Invitrogen, NY, Empire State, USA) according to the manufacturer’s instructions. The siRNA sequences are listed in Supplementary Table 1.

Primary cortical neuronal culture

Male and female C57BL/6 mice were used in this assay. Cortical tissues of E14.5-E15.5 Wwc1/2^fl/fl/Wwc1/2^Nes-cKO mice were dissected and then digested with papain (Cat. No. LS003126, Worthington Bio Corp, Lakewood, NJ, USA) for 30 min at 37 °C to generate dissociated neurons for cell culture. Cells were seeded at a density of 1 × 10⁵ per square centimeter onto 6-well plates pre-coated with poly-L-lysine containing Neurobasal medium (Cat. No. 21103049, Invitrogen, USA) supplemented with B-27 supplement (Cat. No. 17504044, Gibco, USA), 2 mM Glutamax (Cat. No. 35050061, Invitrogen, USA) and 1% penicillin/streptomycin (SV30010, Hyclone, USA). Primary neurons were then maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2), and half of the medium was refreshed every two days.

RNA extraction and real-time qPCR

Total RNA was isolated from cells or tissue samples using the MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan), and cDNA was synthesized using the First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Quantitative real-time PCR was conducted by using TB Green® Premix Ex Taq (TaKaRa, Japan) on a CFX96 Real-Time PCR system (Bio-Rad, USA). Primer sequences used in this study are listed in Supplementary Table 1. Relative mRNA levels were normalized to the housekeeping gene β-ACTIN. All reactions were performed in biological triplicates.

Immunoblotting

Cultured cells and brain tissues were lysed and homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM HEPES (pH7.5), 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 0.5% Sodium Deoxycholate, 1 mM PMSF, protease inhibitor cocktail (HY-K0010, MCE, NJ, USA) and phosphatase inhibitors (HY-K0021, MCE, USA)). Postsynaptic density fractions from adult brain tissues were isolated as previously described [4, 53]. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween, the membranes were incubated with indicated primary antibodies in 5% bovine serum albumin (BSA) overnight at 4 °C and then appropriate horseradish peroxidase-conjugated secondary antibodies in 5% non-fat milk for 1 h at room temperature. Bands were visualized using High-sig ECL Western Blotting Substrate (#180–501, Tanon, Shanghai, China) and Tanon 5200 S imaging system. The intensities of protein bands were quantified using ImageJ software (NIH).

Immunoprecipitation

Cultured cell lysates extracted from mild lysis buffer (50 mM HEPES at pH7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 50 mM NaF, 0.5% Sodium Deoxycholate, 1 mM PMSF, protease inhibitor cocktail (HY-K0010, MCE, USA) and phosphatase inhibitors (HY-K0021, MCE, USA)) were centrifuged at 12,000 rpm for 15 min at 4 °C, and then the supernatants were incubated with Anti-DYKDDDDK Affinity Beads (SA042001, Smart Lifesciences, Changzhou, China) with rotation overnight at 4 °C. After washing with ice-cold mild lysis buffer 4 times, the complexes were resuspended in SDS loading buffer (50 mM Tris-HCl at pH6.8, 2% SDS, 10% glycerol, 0.025% bromophenol blue and β-mercaptoethanol) at 95 °C for 5 min, followed by immunoblotting for analysis.

Mouse brain tissues were homogenized and lysed with RIPA buffer. The lysates were then rotated at 4 °C for 30 min, and centrifuged for 20 min at 12,000 rpm at 4 °C. The supernatants were incubated with the primary antibody for 1 h at 4 °C. Then protein A/G agarose beads (sc-2003, Santa Cruz, CA, USA) were added to the lysates and incubated with rotation at 4 °C for 2 h. After washing with ice-cold RIPA buffer, the complexes were resuspended in SDS loading buffer for 5 min at 95 °C, and the supernatant was subsequently subjected to SDS-PAGE for immunoblotting analysis.

Protein degradation and ubiquitination assay

To detect the protein degradation of AMOT, cells were treated with cycloheximide (CHX, 100 μg/ml, MCE, USA) at different time points before harvesting for immunoblotting analysis. To detect the ubiquitination level of AMOTp130, cells were transfected with pCDNA-HA-Ub, and harvested 48 h after transfection. Before harvest, cells were treated with MG132 (10 μM) for 4 h. Protein lysates were extracted by SDS lysis buffer (50 mM Tris-HCl at pH7.5; 1% SDS and 10 mM DTT) and boiled at 95 °C for 10 min. The lysates were diluted 10 times with mild lysis buffer and subjected to immunoprecipitation and immunoblotting.

Immunohistochemistry

For immunofluorescence staining, age-matched mice were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (wt/vol) in PBS. Brains were removed from the skull and kept in 4% paraformaldehyde (wt/vol) in PBS. After fixation, the brain was equilibrated in 30% sucrose at 4 °C, embedded in Tissue-Tek O.C.T. Compound (SAKURA, Japan), and coronally sectioned at 25 μm with a cryostat (Leica 1950 Ag Protect, Leica, Wetzlar, Germany). After incubation in blocking buffer (10% Goat serum and 0.3% TritonX-100 in PBS) for 1 h at room temperature, brain slices were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. After rinsing in PBS, sections were incubated with secondary antibodies in blocking buffer for 1–2 h at 37 °C, followed by DAPI staining for 15 min at room temperature. Primary antibodies and secondary antibodies are listed in Supplementary Table 1.

Animal work

All animal experiments were approved by the Animal Ethics Committee of Shanghai Medical College, Fudan University, and carried out in accordance with institutional guidelines. C57BL/6 background mice used in this study were kept under controlled temperature (21–23 °C), and on a standard 12 h light/dark cycle with ad libitum food and water. All of the mice were housed in a specific pathogen-free (SPF) animal facility at the Children’s Hospital of Fudan University. All age and sex-matched mice were randomly subjected to experimental analysis. For animal studies, grouping was performed based on animal genotype with no randomization or blinding used.

Wwc1/2 conditional knockout mice

The Wwc1/2^fl/fl conditional knockout mice were described previously [15]. Mice with conditional deletion of Wwc1/2 in neurons (Wwc1/2^Syn-cKO) were generated by first crossing Wwc1/2^fl/fl females with Syn-Cre transgenic mice (catalog no.110132, BIOCYTOGEN, Beijing, China). Then, Wwc1/2^Syn-het males were crossed with Wwc1/2^fl/fl to obtain homozygous cKO mice (Wwc1/2^Syn-cKO). Wwc1/2^fl/fl mice were used as controls. Wwc1/2^Nes-cKO were generated by crossing Wwc1/2^fl/fl females with Nestin-Cre mice (Shanghai Model Organisms Center Inc., Shanghai, China) as described above. The following primers were used for genotyping: Wwc1 (5’-TGAATATCTCCACTATTGCTCTCGC-3’ and 5’-CCATTCCCTTTCGTCTTCCTC-3’; band sizes for Wwc1^fl/+ mice are 218 base pairs (bp) (wild-type allele) and 331 bp (targeted allele with 3’ loxP)); Wwc2 (5’-CCTTCTGTGTGCTCAGTGGCT-3’ and 5’-AAGGTTCAGTGCTATTGGGAGC-3’; bands sizes for Wwc2^fl/+ mice are 307 bp (wild-type allele) and 420 bp (targeted allele with 3’ loxP)); Syn-Cre (5’-ATCGGGATCCACATTCGCCTCAGTCTCAGCTTC-3’ and 5’-ATCGCTCGAGAGAGCTCCAGGAGAGGATTCGAT-3’/5’-GCACACAGACAGGAGCATCTTC-3’; band sizes for Syn-Cre^Mut/+ mice are 726 bp (wild-type allele) and 582 bp (mutant-type allele)); Nestin-Cre (5’-TTGCTAAAGCGCTACATAGGA-3’ and 5’-GCCTTATTGTGGAAGGACTG-3’/ 5’-CCTTCCTGAAGCAGTAGAGCA-3’; band sizes for Nestin-Cre^Mut/+ mice are 246 bp (wild-type allele) and 150 bp (mutant-type allele)). Genotyping was carried out using standard PCR protocols. For timed pregnancies, the plug date was designated as E0.5, and the date of birth was defined as P0.

Behavioral tests

All mice used for behavioral tests were 8–10-week-old age-matched male littermates with comparable body weight. All behavioral tests were performed between 9:30 a.m. to 18:00 p.m. All mice (in their home cages) were habituated for 1–2 h in the testing room before any behavioral tests. After each test, the apparatus was cleaned with 75% ethanol. All behavioral assays were done blind to genotypes.

Open field

Open field test is used for evaluating spontaneous locomotion activity and the anxiety state of animals. The test mouse was gently placed in the corner of the open field apparatus (50 × 50 × 50 cm) in 30 lux and allowed to explore freely for 10 min. The mouse’s activity distance, velocity, and time spent in the central square region (20 × 20 cm) were recorded by overhead video camera and further analyzed with EthoVision XT 14.0 software (Noldus, Holland).

Elevated plus maze

The elevated plus maze apparatus is constructed of black Plexiglass and elevated about 40 cm above the ground, consisting of two open arms (30 × 6 cm) across from each other, perpendicular to two closed arms (30 × 6 × 16 cm), and a central platform (6 × 6 cm). To assess anxiety, the test mouse was initially positioned in the central platform facing an open arm, and the following 5 min of the video was collected. Total distance and the time spent in the open arms and closed arms were directly measured and analyzed with EthoVision XT 14.0 software (Noldus, Holland).

Tail suspension

The mouse was suspended by adhesive tape placed ~1 cm from the tip of the tail in the middle of a test cage. After 2 min of accommodation, the total time spent immobile, defined as the absence of struggling and just hanging passively without any movement of the whole body, was recorded during the last 4 min of the test.

Y maze

Spontaneous alternation of the Y maze is used to measure spatial working memory. The Y maze apparatus, made of black Plexiglass, consists of three opaque plastic arms (30 × 10 × 15 cm, at a 120° angle from each other), designated A, B, and C, and each arm ends with a wall. The mouse was initially placed in the distal end of arm A and allowed to freely explore the maze for 8 min. The movement of each mouse was recorded by the overhead video camera and further analyzed with EthoVision XT 14.0 software (Noldus, Holland). The arm entries were recorded and the percentage of spontaneous alternations (entry into an arm that differs from the previous two entries) was calculated with the following formula: (Alternations/Arm Entries-2) × 100.

Novel object recognition

The device used for the novel object recognition test was the same one used for the open field test. The test mice were handled before training and acclimated in the test box for 3 days, with 10 min habituation each day. On day 4, two identical objects were placed in the parallel corners of the arena 10 cm from the side walls. The test mouse was placed at the opposite side of the arena and allowed to freely explore the arena for 10 min. After 1 h, one object was randomly replaced with another novel object, which was of the same size but different in color and shape from the familiar one. Then, the same mouse was put back into the same box and allowed to freely explore the two different objects for another 10 min. The interaction time with the familiar and novel object was recorded by video camera above the test box and further analyzed with EthoVision XT 14.0 software (Noldus, Holland).

Rotarod

The test was carried out using an accelerating rotarod (Med Associates, Vermont, USA) to assess the motor coordination and balance ability of mice. The test consisted of four trials per day for four days, with a minimum of 20 min of recovery time between trials. The rotarod was started initially at 5 rpm and increased to 30 rpm within 5 min. The trial started once the test mouse was loaded into the partitioned compartment. The latency of each mouse to fall from the rotarod was recorded for further analysis.

Morris Water Maze

The test was performed in a circular tank (120 cm diameter) filled with opaque water (21–23 °C). A 10 cm-circular plexiglass platform submerged 1 cm below the surface of the water was placed at a fixed point at one quadrant. The device with matching software was purchased from Ji Liang Technology Co., Ltd (Shanghai, China). The test mouse was released from four possible starting locations and the order of starting locations was randomly determined. Each trial lasted 1 min and ended when the test mouse successfully climbed onto and remained on the platform for 10 s. The mouse that failed to reach the platform within the 60 s was guided to the platform and stayed for 20 s to remember location information. Four trials per day were conducted for 4 consecutive training days. The time spent by the mouse to reach the platform was recorded as its latency. Time for four trials was averaged and recorded as a result of each mouse. On day 5, the mouse was subjected to a single 60-s probe trial without a hidden platform to test memory retention. The test mouse was released from the distant point opposite the platform. The swimming path and time spent in the target quadrant were recorded automatically.

Fear conditioning

The test was performed in a fear conditioning apparatus (Med Associates, Vermont, USA; 25 × 30 × 25 cm). For the training phase, the test mouse was placed in the chamber for 6 min followed by 2 min of accumulation, recorded as a baseline, and five repeated pairings of conditional stimulus (auditory tone, 20 s, 90 dB) and unconditional stimulus (3-foot shocks of 0.75 mA during the last 2 s of tone) at a 60 s interval. Following 24 h of training, the test mouse was put back into the same chamber to evaluate the contextual fear memory, and 5 min of recordings were made (context freezing). After 2 h recovery, the test mouse was placed in a new and redecorated chamber (different walls and flooring) and given the conditioned cue (90 dB noise) for 5 min of recordings (cued freezing). Data were analyzed using Med Associates software (MED Associates, St. Albans, VT, USA).

Viral infection

Newborn pups (P0) were intracerebroventricularly injected with virally-encoded transgenes of AAV2/9-hSyn-AMOT (1.0 × 10⁸ particles/hemisphere) (HANBIO, Shanghai, China). Administer an average volume of 1 μl into each ventricle. Two weeks after viral infection, animals were anesthetized and intracardially perfused with 4% paraformaldehyde as described above, and brains were fixed and sectioned into 30 μm sagittal slices with a vibratome (Leica, VT1000S). Eight to ten weeks after viral infection, animals were used for behavioral tests.

Golgi staining

For studying the spine density and morphology of neurons, Golgi-Cox impregnation was performed using the FD Rapid GolgiStainTM Kit (PK401FD, NeuroTechnologies, Waltham, Maine, USA), according to the manufacturer’s instructions. In brief, age-matched mice were deeply anesthetized with 0.7% pentobarbital sodium, and brains were quickly removed and immersed into a mixture containing equal volumes of solution A and B at room temperature for 2 weeks. Then, the brains were transferred into solution C for at least 72 h. Serial coronal vibratome sections (130 μm) through the entire brain were mounted onto the gelatin-coated microscope slides (PO101FD, NeuroTechnologies) and stained using solutions D and E followed by image analysis.

Image acquisition and analysis

Confocal images were acquired using Zeiss LSM 880 with Airyscan with a ×10 objective at 1024 × 1024 pixel resolution. For dendritic spine density analysis, images were collected by Leica TCS SP8 with ×63/1.4 NA oil immersion objective at 3× optical zoom and 0.3 μm Z-interval. The spine density was analyzed with ImageJ (Fiji, Rawak Software Inc., Stuttgart, Germany) software blinded to the genotype.

Statistical analysis

All data quantifications are presented as the mean ± SD of at least three independent experiments. Results were analyzed using Prism 8.0 software (GraphPad, SanDiego, CA, USA). Statistical significance was determined using Student’s t test or one-way ANOVA between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates not significant.

Data availability

The data of this study are available from the corresponding author upon reasonable request.

References

Hoffken V, Hermann A, Pavenstadt H, Kremerskothen J. WWC proteins: important regulators of hippo signaling in cancer. Cancers (Basel). 2021;13:306.
Article PubMed Google Scholar
Baumgartner R, Poernbacher I, Buser N, Hafen E, Stocker H. The WW domain protein Kibra acts upstream of Hippo in drosophila. Dev Cell. 2010;18:309–16.
Article CAS PubMed Google Scholar
Genevet A, Wehr MC, Brain R, Thompson BJ, Tapon N. Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev Cell. 2010;18:300–8.
Article CAS PubMed PubMed Central Google Scholar
Heitz FD, Farinelli M, Mohanna S, Kahn M, Duning K, Frey MC, et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult brain. Neurobiol Learn Mem. 2016;135:100–14.
Article CAS PubMed Google Scholar
Makuch L, Volk L, Anggono V, Johnson RC, Yu Y, Duning K, et al. Regulation of AMPA receptor function by the human memory-associated gene KIBRA. Neuron 2011;71:1022–9.
Article CAS PubMed PubMed Central Google Scholar
Vogt-Eisele A, Kruger C, Duning K, Weber D, Spoelgen R, Pitzer C, et al. KIBRA (KIdney/BRAin protein) regulates learning and memory and stabilizes Protein kinase Mzeta. J Neurochem. 2014;128:686–700.
Article CAS PubMed Google Scholar
Papassotiropoulos A, Stephan DA, Huentelman MJ, Hoerndli FJ, Craig DW, Pearson JV, et al. Common Kibra alleles are associated with human memory performance. Science 2006;314:475–8.
Article CAS PubMed Google Scholar
Schwab LC, Luo V, Clarke CL, Nathan PJ. Effects of the KIBRA single nucleotide polymorphism on synaptic plasticity and memory: a review of the literature. Curr Neuropharmacol. 2014;12:281–8.
Article CAS PubMed PubMed Central Google Scholar
Galecki P, Szemraj J, Florkowski A, Talarowska M, Bienkiewicz M, Galecka E, et al. Single nucleotide polymorphism of the KIBRA gene in recurrent depressive disorders. Neuro Endocrinol Lett. 2010;31:97–102.
CAS PubMed Google Scholar
Rodriguez-Rodriguez E, Infante J, Llorca J, Mateo I, Sanchez-Quintana C, Garcia-Gorostiaga I, et al. Age-dependent association of KIBRA genetic variation and Alzheimer’s disease risk. Neurobiol Aging. 2009;30:322–4.
Article CAS PubMed Google Scholar
Talarowska M, Szemraj J, Kowalczyk M, Galecki P. Serum KIBRA mRNA and protein expression and cognitive functions in depression. Med Sci Monit. 2016;22:152–60.
Article CAS PubMed PubMed Central Google Scholar
Tracy TE, Sohn PD, Minami SS, Wang C, Min SW, Li Y, et al. Acetylated tau obstructs KIBRA-mediated signaling in synaptic plasticity and promotes tauopathy-related memory loss. Neuron 2016;90:245–60.
Article CAS PubMed PubMed Central Google Scholar
Han X, Wang C, Song L, Wang X, Tang S, Hou T, et al. KIBRA regulates amyloid beta metabolism by controlling extracellular vesicles secretion. EBioMedicine 2022;78:103980.
Article CAS PubMed PubMed Central Google Scholar
Vyas NS, Ahn K, Stahl DR, Caviston P, Simic M, Netherwood S, et al. Association of KIBRA rs17070145 polymorphism with episodic memory in the early stages of a human neurodevelopmental disorder. Psychiatry Res. 2014;220:37–43.
Article CAS PubMed Google Scholar
Qi S, Zhu Y, Liu X, Li P, Wang Y, Zeng Y, et al. WWC proteins mediate LATS1/2 activation by Hippo kinases and imply a tumor suppression strategy. Mol Cell. 2022;82:1850–64.e7.
Article CAS PubMed Google Scholar
Wilson KE, Yang N, Mussell AL, Zhang J. The regulatory role of KIBRA and PTPN14 in hippo signaling and beyond. Genes (Basel). 2016;7:23.
Article PubMed Google Scholar
Xiao L, Chen Y, Ji M, Dong J. KIBRA regulates Hippo signaling activity via interactions with large tumor suppressor kinases. J Biol Chem. 2011;286:7788–96.
Article CAS PubMed PubMed Central Google Scholar
Wang W, Li X, Huang J, Feng L, Dolinta KG, Chen J. Defining the protein-protein interaction network of the human hippo pathway. Mol Cell Proteomics. 2014;13:119–31.
Article CAS PubMed Google Scholar
Wilson KE, Li YW, Yang N, Shen H, Orillion AR, Zhang J. PTPN14 forms a complex with Kibra and LATS1 proteins and negatively regulates the YAP oncogenic function. J Biol Chem. 2014;289:23693–700.
Article CAS PubMed PubMed Central Google Scholar
Ji Z, Li H, Yang Z, Huang X, Ke X, Ma S, et al. Kibra modulates learning and memory via binding to dendrin. Cell Rep. 2019;26:2064–77.e7.
Article CAS PubMed Google Scholar
Duning K, Schurek EM, Schluter M, Bayer M, Reinhardt HC, Schwab A, et al. KIBRA modulates directional migration of podocytes. J Am Soc Nephrol. 2008;19:1891–903.
Article CAS PubMed PubMed Central Google Scholar
Kwok E, Rodriguez DJ, Kremerskothen J, Nyarko A. Intrinsic disorder and amino acid specificity modulate binding of the WW2 domain in kidney and brain protein (KIBRA) to synaptopodin. J Biol Chem. 2019;294:17383–94.
Article PubMed PubMed Central Google Scholar
Yoshihama Y, Sasaki K, Horikoshi Y, Suzuki A, Ohtsuka T, Hakuno F, et al. KIBRA suppresses apical exocytosis through inhibition of aPKC kinase activity in epithelial cells. Curr Biol. 2011;21:705–11.
Article CAS PubMed Google Scholar
Fukuda T, Nagashima S, Inatome R, Yanagi S. CAMDI interacts with the human memory-associated protein KIBRA and regulates AMPAR cell surface expression and cognition. PLoS One. 2019;14:e0224967.
Article CAS PubMed PubMed Central Google Scholar
Duning K, Wennmann DO, Bokemeyer A, Reissner C, Wersching H, Thomas C, et al. Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance. Transl Psychiatry. 2013;3:e272.
Article CAS PubMed PubMed Central Google Scholar
Song L, Tang S, Han X, Jiang Z, Dong L, Liu C, et al. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a. Nat Commun. 2019;10:1639.
Article PubMed PubMed Central Google Scholar
Chan SW, Lim CJ, Guo F, Tan I, Leung T, Hong W. Actin-binding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation. J Biol Chem. 2013;288:37296–307.
Article CAS PubMed PubMed Central Google Scholar
Ernkvist M, Aase K, Ukomadu C, Wohlschlegel J, Blackman R, Veitonmaki N, et al. p130-angiomotin associates to actin and controls endothelial cell shape. FEBS J. 2006;273:2000–11.
Article CAS PubMed Google Scholar
Wang Y, Zhu Y, Gu Y, Ma M, Wang Y, Qi S, et al. Stabilization of Motin family proteins in NF2-deficient cells prevents full activation of YAP/TAZ and rapid tumorigenesis. Cell Rep. 2021;36:109596.
Article CAS PubMed Google Scholar
Huang T, Zhou Y, Zhang J, Cheng ASL, Yu J, To KF. et al. The physiological role of Motin family and its dysregulation in tumorigenesis. J Transl Med. 2018;16:98.
Article CAS PubMed PubMed Central Google Scholar
Bratt A, Wilson WJ, Troyanovsky B, Aase K, Kessler R, Van Meir EG, et al. Angiomotin belongs to a novel protein family with conserved coiled-coil and PDZ binding domains. Gene 2002;298:69–77.
Article CAS PubMed Google Scholar
Schanzenbacher CT, Sambandan S, Langer JD, Schuman EM. Nascent proteome remodeling following homeostatic scaling at hippocampal synapses. Neuron 2016;92:358–71.
Article PubMed PubMed Central Google Scholar
Wigerius M, Quinn D, Diab A, Clattenburg L, Kolar A, Qi J, et al. The polarity protein Angiomotin p130 controls dendritic spine maturation. J Cell Biol. 2018;217:715–30.
Article CAS PubMed PubMed Central Google Scholar
Sugihara-Mizuno Y, Adachi M, Kobayashi Y, Hamazaki Y, Nishimura M, Imai T, et al. Molecular characterization of angiomotin/JEAP family proteins: interaction with MUPP1/Patj and their endogenous properties. Genes Cells. 2007;12:473–86.
Article CAS PubMed Google Scholar
Heller B, Adu-Gyamfi E, Smith-Kinnaman W, Babbey C, Vora M, Xue Y, et al. Amot recognizes a juxtanuclear endocytic recycling compartment via a novel lipid binding domain. J Biol Chem. 2010;285:12308–20.
Article CAS PubMed PubMed Central Google Scholar
Ernkvist M, Luna Persson N, Audebert S, Lecine P, Sinha I, Liu M, et al. The Amot/Patj/Syx signaling complex spatially controls RhoA GTPase activity in migrating endothelial cells. Blood 2009;113:244–53.
Article CAS PubMed PubMed Central Google Scholar
Rojek KO, Krzemien J, Dolezyczek H, Boguszewski PM, Kaczmarek L, Konopka W, et al. Amot and Yap1 regulate neuronal dendritic tree complexity and locomotor coordination in mice. PLoS Biol. 2019;17:e3000253.
Article CAS PubMed PubMed Central Google Scholar
Hermann A, Wennmann DO, Gromnitza S, Edeling M, Van Marck V, Sudol M, et al. WW and C2 domain-containing proteins regulate hepatic cell differentiation and tumorigenesis through the hippo signaling pathway. Hepatology 2018;67:1546–59.
Article CAS PubMed Google Scholar
Zhang L, Yang S, Wennmann DO, Chen Y, Kremerskothen J, Dong J. KIBRA: In the brain and beyond. Cell Signal. 2014;26:1392–9.
Article CAS PubMed PubMed Central Google Scholar
Couzens AL, Knight JD, Kean MJ, Teo G, Weiss A, Dunham WH, et al. Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions. Sci Signal. 2013;6:rs15.
Article PubMed Google Scholar
Hauri S, Wepf A, van Drogen A, Varjosalo M, Tapon N, Aebersold R, et al. Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1. Mol Syst Biol. 2013;9:713.
Article PubMed PubMed Central Google Scholar
Hirate Y, Hirahara S, Inoue K, Suzuki A, Alarcon VB, Akimoto K, et al. Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos. Curr Biol. 2013;23:1181–94.
Article CAS PubMed PubMed Central Google Scholar
Wang L, Choi K, Su T, Li B, Wu X, Zhang R, et al. Multiphase coalescence mediates Hippo pathway activation. Cell 2022;185:4376–93.e18.
Article CAS PubMed Google Scholar
Nandi D, Tahiliani P, Kumar A, Chandu D. The ubiquitin-proteasome system. J Biosci. 2006;31:137–55.
Article CAS PubMed Google Scholar
Pohl C, Dikic I. Cellular quality control by the ubiquitin-proteasome system and autophagy. Science 2019;366:818–22.
Article CAS PubMed Google Scholar
Campbell CI, Samavarchi-Tehrani P, Barrios-Rodiles M, Datti A, Gingras AC, Wrana JL. The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin. J Cell Sci. 2016;129:3396–411.
CAS PubMed Google Scholar
Wang C, An J, Zhang P, Xu C, Gao K, Wu D, et al. The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation. Biochem J. 2012;444:279–89.
Article CAS PubMed Google Scholar
Toloczko A, Guo F, Yuen HF, Wen Q, Wood SA, Ong YS, et al. Deubiquitinating enzyme USP9X suppresses tumor growth via LATS kinase and core components of the hippo pathway. Cancer Res. 2017;77:4921–33.
Article CAS PubMed PubMed Central Google Scholar
Zhu C, Ji X, Zhang H, Zhou Q, Cao X, Tang M, et al. Deubiquitylase USP9X suppresses tumorigenesis by stabilizing large tumor suppressor kinase 2 (LATS2) in the Hippo pathway. J Biol Chem. 2018;293:1178–91.
Article CAS PubMed Google Scholar
Thanh Nguyen H, Andrejeva D, Gupta R, Choudhary C, Hong X, Eichhorn PJ, et al. Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling angiomotin protein turnover. Cell Discov. 2016;2:16001.
Article CAS PubMed PubMed Central Google Scholar
Kim M, Kim M, Park SJ, Lee C, Lim DS. Role of angiomotin-like 2 mono-ubiquitination on YAP inhibition. EMBO Rep. 2016;17:64–78.
Article CAS PubMed Google Scholar
Song L, Tang S, Dong L, Han X, Cong L, Dong J, et al. The neuroprotection of KIBRA in promoting neuron survival and against amyloid beta-induced apoptosis. Front Cell Neurosci. 2019;13:137.
Article CAS PubMed PubMed Central Google Scholar
Johannsen S, Duning K, Pavenstadt H, Kremerskothen J, Boeckers TM. Temporal-spatial expression and novel biochemical properties of the memory-related protein KIBRA. Neuroscience 2008;155:1165–73.
Article CAS PubMed Google Scholar
Kasherman MA, Currey L, Kurniawan ND, Zalucki O, Vega MS, Jolly LA, et al. Abnormal behavior and cortical connectivity deficits in mice lacking Usp9x. Cereb Cortex. 2021;31:1763–75.
Article PubMed Google Scholar
Stegeman S, Jolly LA, Premarathne S, Gecz J, Richards LJ, Mackay-Sim A, et al. Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFbeta-mediated axonogenesis. PLoS One. 2013;8:e68287.
Article CAS PubMed PubMed Central Google Scholar
Bian WJ, Miao WY, He SJ, Qiu Z, Yu X. Coordinated spine pruning and maturation mediated by inter-spine competition for cadherin/catenin complexes. Cell 2015;162:808–22.
Article CAS PubMed Google Scholar
Yoon S, Parnell E, Penzes P. TGF-beta-induced phosphorylation of Usp9X stabilizes ankyrin-g and regulates dendritic spine development and maintenance. Cell Rep. 2020;31:107685.
Article CAS PubMed PubMed Central Google Scholar
Tronche F, Kellendonk C, Kretz O, Gass P, Anlag K, Orban PC, et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet. 1999;23:99–103.
Article CAS PubMed Google Scholar
Stepan J, Heinz DE, Dethloff F, Bajaj T, Zellner A, Hafner K, et al. Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning. Cell Rep. 2022;41:111766.
Article CAS PubMed Google Scholar
Mendoza ML, Quigley LD, Dunham T, Volk LJ. KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner. iScience. 2022;25:105623.
Article CAS PubMed PubMed Central Google Scholar
Coles CH, Bradke F. Coordinating neuronal actin-microtubule dynamics. Curr Biol. 2015;25:R677–91.
Article CAS PubMed Google Scholar
Chazeau A, Mehidi A, Nair D, Gautier JJ, Leduc C, Chamma I, et al. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion. EMBO J. 2014;33:2745–64.
Article CAS PubMed PubMed Central Google Scholar
Chazeau A, Garcia M, Czondor K, Perrais D, Tessier B, Giannone G, et al. Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines. Mol Biol Cell. 2015;26:859–73.
Article CAS PubMed PubMed Central Google Scholar
Hu X, Viesselmann C, Nam S, Merriam E, Dent EW. Activity-dependent dynamic microtubule invasion of dendritic spines. J Neurosci. 2008;28:13094–105.
Article CAS PubMed PubMed Central Google Scholar
Jaworski J, Kapitein LC, Gouveia SM, Dortland BR, Wulf PS, Grigoriev I, et al. Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity. Neuron 2009;61:85–100.
Article CAS PubMed Google Scholar
Gagne V, Moreau J, Plourde M, Lapointe M, Lord M, Gagnon E, et al. Human angiomotin-like 1 associates with an angiomotin protein complex through its coiled-coil domain and induces the remodeling of the actin cytoskeleton. Cell Motil Cytoskeleton. 2009;66:754–68.
Article CAS PubMed Google Scholar
Mana-Capelli S, Paramasivam M, Dutta S, McCollum D. Angiomotins link F-actin architecture to Hippo pathway signaling. Mol Biol Cell. 2014;25:1676–85.
Article PubMed PubMed Central Google Scholar
Hildebrand S, Hultin S, Subramani A, Petropoulos S, Zhang Y, Cao X, et al. The E-cadherin/AmotL2 complex organizes actin filaments required for epithelial hexagonal packing and blastocyst hatching. Sci Rep. 2017;7:9540.
Article PubMed PubMed Central Google Scholar
Rayala SK, den Hollander P, Manavathi B, Talukder AH, Song C, Peng S, et al. Essential role of KIBRA in co-activator function of dynein light chain 1 in mammalian cells. J Biol Chem. 2006;281:19092–9.
Article CAS PubMed Google Scholar
Traer CJ, Rutherford AC, Palmer KJ, Wassmer T, Oakley J, Attar N, et al. SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment. Nat Cell Biol. 2007;9:1370–80.
Article CAS PubMed Google Scholar
Zhang XL, Poschel B, Faul C, Upreti C, Stanton PK, Mundel P. Essential role for synaptopodin in dendritic spine plasticity of the developing hippocampus. J Neurosci. 2013;33:12510–8.
Article CAS PubMed PubMed Central Google Scholar
Falahati H, Wu Y, Feuerer V, Simon HG, De, Camilli P. Proximity proteomics of synaptopodin provides insight into the molecular composition of the spine apparatus of dendritic spines. Proc Natl Acad Sci USA. 2022;119:e2203750119.
Article CAS PubMed PubMed Central Google Scholar
Paramasivam M, Sarkeshik A, Yates JR 3rd, Fernandes MJ, McCollum D. Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor. Mol Biol Cell. 2011;22:3725–33.
Article CAS PubMed PubMed Central Google Scholar
Zhao B, Li L, Lu Q, Wang LH, Liu CY, Lei Q, et al. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev. 2011;25:51–63.
Article PubMed PubMed Central Google Scholar
Wang W, Huang J, Chen J. Angiomotin-like proteins associate with and negatively regulate YAP1. J Biol Chem. 2011;286:4364–70.
Article CAS PubMed Google Scholar
Chan SW, Lim CJ, Chong YF, Pobbati AV, Huang C, Hong W. Hippo pathway-independent restriction of TAZ and YAP by angiomotin. J Biol Chem. 2011;286:7018–26.
Article CAS PubMed PubMed Central Google Scholar
Chen B, Liu G. WWC3 inhibits intimal proliferation following vascular injury via the Hippo signaling pathway. Mol Med Rep. 2018;17:5175–83.
CAS PubMed PubMed Central Google Scholar
Hermann A, Wu G, Nedvetsky PI, Brucher VC, Egbring C, Bonse J, et al. The Hippo pathway component Wwc2 is a key regulator of embryonic development and angiogenesis in mice. Cell Death Dis. 2021;12:117.
Article CAS PubMed PubMed Central Google Scholar
Qi S, Zhong Z, Zhu Y, Wang Y, Ma M, Wang Y, et al. Two Hippo signaling modules orchestrate liver size and tumorigenesis. EMBO J. 2023;42:e112126.
Article CAS PubMed Google Scholar
Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783–4.
Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We would like to thank Drs. Yunli Xie and Rongfeng Hu for helpful discussion. This work was supported by grants from the Ministry of Science and Technology of China (National Key R&D program, 2018YFA0800304 and 2020YFA0803202), the Science and Technology Commission of Shanghai Municipality (21S11905000), and the Shanghai Municipal Health Commission (2022XD049) to F-XY, and Shanghai Municipal Science and Technology Major Project, International Human Phenome Program (Phase I) - Study of the Whole Phenome of Refractory Epilepsy (2017SHZDZX01) to Yi Wang. This work is also supported by the Medical Science Data Center in Shanghai Medical College of Fudan Univeristy.

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These authors contributed equally: Runyi Cao, Rui Zhu.

Authors and Affiliations

Institute of Pediatrics, Children’s Hospital of Fudan University, and the Shanghai Key Laboratory of Medical Epigenetics, The International Co-laboratory of Medical Epigenetics and Metabolism, the State Key Laboratory of Genetic Engineering, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China
Runyi Cao, Rui Zhu, Zhao Sha, Sixian Qi, Zhenxing Zhong, Fengyun Zheng, Yubin Lei, Yanfeng Tan, Yuwen Zhu, Yu Wang & Fa-Xing Yu
Department of Neurology, Children’s Hospital of Fudan University, National Children’s Medical Center, No. 399 Wanyuan Road, Shanghai, 201102, China
Yi Wang

Authors

Runyi Cao
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Rui Zhu
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Zhao Sha
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Sixian Qi
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Zhenxing Zhong
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Fengyun Zheng
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Yubin Lei
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Yanfeng Tan
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Yuwen Zhu
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Yu Wang
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Yi Wang
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Fa-Xing Yu
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Contributions

RC, RZ, ZS, SQ, ZZ, YL, YZ, and Yu Wang performed experiments. RC, Yu Wang, and F-XY analyzed the data. FZ and YT contributed to technical support. Yi Wang provided key resources for this work. RC and F-XY conceived and designed the study and wrote the manuscript. RC, RZ, Yu Wang, and F-XY revised the manuscript. F-XY coordinated and supervised the project. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yu Wang, Yi Wang or Fa-Xing Yu.

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The authors declare no competing interests.

Ethics

All mouse experiments were approved by the Animal Ethics Committee of Shanghai Medical College, Fudan University, and carried out in accordance with institutional guidelines.

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Cao, R., Zhu, R., Sha, Z. et al. WWC1/2 regulate spinogenesis and cognition in mice by stabilizing AMOT. Cell Death Dis 14, 491 (2023). https://doi.org/10.1038/s41419-023-06020-7

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Received: 17 December 2022
Revised: 16 July 2023
Accepted: 25 July 2023
Published: 01 August 2023
DOI: https://doi.org/10.1038/s41419-023-06020-7