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Rho GTPase westerns, RhoA inhibitor synaptic transmission data, and model schematic summary a, Western blots of hippocampal lysates from Kctd13 mutants reveal no change in RhoB, RhoC, or Rac1. b, Combined data from Fig. 2c, d. c, Combined data from Fig. 2e, f. d, Slice incubation (3.5 h) with rhosin had no effect on mEPSC amplitude; P = 0.396 (WT vehicle n = 12/4 cells/mice; KO vehicle n = 17/4; WT rhosin n = 17/4; KO rhosin n = 18/4). Inset: representative mEPSC averaged traces. Scale bar, 2 pA, 5 ms. e, Under normal neuronal conditions, KCTD13 (probably acting with the ubiquitin E3 ligase CUL3, not shown) inhibits RhoA levels and allows for normal synaptic function (left). Heterozygous or homozygous deletion of Kctd13 leads to increased RhoA levels, causing decreased synaptic transmission (right). RhoA inhibition (centre) normalizes RhoA activity to restore synaptic function to normal. Mean ± s.e.m. in a–c. Box (interquartile range), whiskers (5th–95th percentile confidence intervals), line (median) in box and whisker plot in d. Source data
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Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders and are among the most prevalent in autism spectrum disorders. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the...
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Citations
... Many susceptibility genes for autism are involved in regulating synapse formation, maintenance, or function (Kwon et al., 2006;Tabuchi et al., 2007;Blundell et al., 2010;Kouser et al., 2013;De Rubeis et al., 2014;Iossifov et al., 2014;Geschwind and State, 2015;Sanders et al., 2015;Araujo et al., 2017;Escamilla et al., 2017). Although little is known about the role of Cul3 in mammalian brain, deletion of the gene Kctd13, a gene in the 16p11.2 ...
... We have used developing neuronal cultures in vitro from an fCul3 model paired with AAV-Cre transduction due to the embryonic lethal nature of constitutive Cul3 homozygous deletion (Singer et al., 1999). For genes such as Cul3 for which neuronal function remains underexplored, homozygous deletion is among the best tools to elucidate function, particularly for autism risk genes Etherton et al., 2009;Zhou et al., 2009;Blundell et al., 2010;Espinosa et al., 2015;Escamilla et al., 2017;Jaramillo et al., 2017). Thus, our approach adds to the existing literature demonstrating effects of homozygous Cul3 deletion on neuronal function (Rapanelli et al., 2019;Dong et al., 2020;Fischer et al., 2020;Amar et al., 2021;Morandell et al., 2021). ...
Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/ PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.
... However, the role of KCTD13 in regulating NPC proliferation and dif ferentiation remains inconsistent. Dis crepancies in previous reports examining KCTD13 function might reflect knockdown and/or off-target effects related to the method of KCTD13 inactivation (24,56). Our functional screening suggests that KCTD13 overexpression phenocopies Kctd10 deficiency, sup porting the model of KCTD10 regulating cortical development by controlling KCTD13 protein levels. ...
KCTD10 belongs to the KCTD (potassiumchannel tetramerization domain) family, many members of which are associated with neuropsychiatric disorders. However, the biological function underlying the association with brain disorders remains to be explored. Here, we reveal that Kctd10 is highly expressed in neuronal progenitors and layer V neurons throughout brain development. Kctd10 deficiency triggers abnormal proliferation and differentiation of neuronal progenitors, reduced deep-layer (especially layer V) neurons, increased upper-layer neurons, and lowered brain size. Mechanistically, we screened and identified a unique KCTD10-interacting protein, KCTD13, associated with neurodevelopmental disorders. KCTD10 mediated the ubiquitination-dependent degradation of KCTD13 and KCTD10 ablation resulted in a considerable increase of KCTD13 expression in the developing cortex. KCTD13 overexpression in neuronal progenitors led to reduced proliferation and abnormal cell distribution, mirroring KCTD10 deficiency. Notably, mice with brain-specific Kctd10 knockout exhibited obvious motor deficits. This study uncovers the physiological function of KCTD10 and provides unique insights into the pathogenesis of neurodevelopmental disorders.
... locus, systematic morpholino-mediated knockdown methods in the zebrafish model identified KCTD13 (potassium channel tetramerization domain containing 13) as a main driver of brain size [20]. However, studies in the mouse model did not confirm this association [21,22]. Intriguingly, MVP, which encodes the 100-kDa major vault protein in the interval, was found to modify NAPs via cis genetic epistasis with KCTD13 [20,21] and had synergistic effects in combination with Kctd13 and Mapk3 for craniofacial measurements in mice [23]. ...
... Four genes (Coro1a, Ino80e, Mapk3, and Kctd13) were categorized as non-NAP, both in males and females. Considering the discrepancies in the literature [20][21][22], heterozygous Kctd13 +/− mice were assessed twice using two independent allelic constructions, which gave identical results with no NAPs (Additional file 9). We also studied the homozygous (hom) Kctd13 −/− mice and found a reduction in the size of the hippocampus by 10% (p = 0.015) for males (Additional file 1: Fig. S2 and Additional file 1: Fig. S4C-D) and 6% (p = 0.014) for females (Additional file 1: Fig. S3). ...
... We also studied the homozygous (hom) Kctd13 −/− mice and found a reduction in the size of the hippocampus by 10% (p = 0.015) for males (Additional file 1: Fig. S2 and Additional file 1: Fig. S4C-D) and 6% (p = 0.014) for females (Additional file 1: Fig. S3). This reinforces the existing link between Kctd13 and hippocampal biology [21,22]. ...
Background
Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus.
Results
We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp+/−;Mapk3+/− mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms.
Conclusions
Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice.
... The intensity of the Caspase-3-positive signals was signi cantly decreased in the optic tecta and the telencephalons of paroxetine-treated embryos, indicating the suppression of apoptosis in the paroxetine-treated brains ( Fig. 1j-l). We then examined the expression of Ras homolog gene family member A (RhoA) because the GTP-binding protein suppresses apoptosis in zebra sh embryos and also exhibits increased expression in the brain of ASD model, Kctd13-de cient mice [39][40][41] . RhoA proteins were signi cantly increased in the optic tecta of paroxetinetreated embryos compared to the controls (Fig. 1m, n). ...
... The increased expression of RhoA proteins in the paroxetine-treated optic tectum is supported by the observation that RhoA prevents apoptosis during embryonic development in zebra sh 39 . These ndings raise the possibility that anti-apoptotic signaling through RhoA is a common pathway shared among ASD models, including Kctd13-de cient mice, in which increased RhoA reduces synaptic transmission 40,41 . ...
Autism spectrum disorder (ASD) is a neurodevelopmental condition caused by various genetic and environmental factors. This disorder has the cardinal symptoms including impaired social behavior involving the amygdala. Antidepressants such as paroxetine in early pregnancy increase the risk of ASD in offspring. However, a comprehensive picture of the underlying pathogenic mechanisms remains elusive. Here, we demonstrate that early exposure of zebrafish embryos to paroxetine suppresses neurogenesis in the optic tectum and the dorsal telencephalon which corresponds to the human amygdala. Paroxetine-treated embryos exhibit impaired growth, with small heads and short body lengths resulting from transient apoptosis. This is reminiscent of the early-onset fetal growth restriction (FGR) associated with ASD. Interestingly, the suppressed neurogenesis in the small heads was found to be restored after the cessation of paroxetine. This was accompanied by extended retinotectal projections, suggesting brain-preferential remodeling. Finally, the paroxetine-treated fish exhibited impaired social behavior, further supporting the correspondence with ASD. Our findings offer new insights into the early neurodevelopmental etiology of ASD.
... The CULΔ474-477 patient had recurrent pulmonary infections that could have been the result of diminished functional KCTD9 and deficient immunological response to pulmonary pathogens neonatally. A kctd13 deletion in mice resulted in reduced synaptic transmission due to an increase in RhoA, a CUL3-KCTD13 substrate [87]. It is possible that aberrant CUL3 and KCTD13 binding sequesters KCTD13, leading to excess RhoA and neurological development issues seen in several CUL3 patients. ...
Familial hyperkalemic hypertension (FHHt), also known as Pseudohypoaldosteronism type II (PHAII) or Gordon syndrome is a rare Mendelian disease classically characterized by hyperkalemia, hyperchloremic metabolic acidosis, and high systolic blood pressure. The most severe form of the disease is caused by autosomal dominant variants in CUL3 (Cullin 3), a critical subunit of the multimeric CUL3-RING ubiquitin ligase complex. The recent identification of a novel FHHt disease variant of CUL3 revealed intricacies within the underlying disease mechanism. When combined with studies on canonical CUL3 variant-induced FHHt, these findings further support CUL3’s role in regulating renal electrolyte transport and maintaining systemic vascular tone. However, the pathophysiological effects of CUL3 variants are often accompanied by diverse systemic disturbances in addition to classical FHHt symptoms. Recent global proteomic analyses provide a rationale for these systemic disturbances, paving the way for future mechanistic studies to reveal how CUL3 variants dysregulate processes outside of the renovascular axis.
... In our set of genes, we found that these chameleon transitions could be induced just by the presence or absence of a distant spliced region with the rest of the structural and sequence neighborhood context of the protein being nearly identical. A notable example of an alphabeta switch was found in the gene Kctd13 (Fig. 3F), encoding a component of a complex required for synaptic transmission (Escamilla et al., 2017) and implicated in neurodevelopmental disorders such as macrocephaly (Golzio et al., 2012) (although a subsequent study could not confirm the association with this latter phenotype (Escamilla et al., 2017)). This example highlights how identical sequences can adopt different structural conformations as a result of an AS event occurring 10 amino acids away. ...
... In our set of genes, we found that these chameleon transitions could be induced just by the presence or absence of a distant spliced region with the rest of the structural and sequence neighborhood context of the protein being nearly identical. A notable example of an alphabeta switch was found in the gene Kctd13 (Fig. 3F), encoding a component of a complex required for synaptic transmission (Escamilla et al., 2017) and implicated in neurodevelopmental disorders such as macrocephaly (Golzio et al., 2012) (although a subsequent study could not confirm the association with this latter phenotype (Escamilla et al., 2017)). This example highlights how identical sequences can adopt different structural conformations as a result of an AS event occurring 10 amino acids away. ...
Regulation of gene expression is critical for fate commitment of stem and progenitor cells during tissue formation. In the context of mammalian brain development, a plethora of studies have described how changes in the expression of individual genes characterize cell types across ontogeny and phylogeny. However, little attention was paid to the fact that different transcripts can arise from any given gene through alternative splicing (AS). Considered a key mechanism expanding transcriptome diversity during evolution, assessing the full potential of AS on isoform diversity and protein function has been notoriously difficult. Here we capitalize on the use of a validated reporter mouse line to isolate neural stem cells, neurogenic progenitors and neurons during corticogenesis and combine the use of short- and long-read sequencing to reconstruct the full transcriptome diversity characterizing neurogenic commitment. Extending available transcriptional profiles of the mammalian brain by nearly 50,000 new isoforms, we found that neurogenic commitment is characterized by a progressive increase in exon inclusion resulting in the profound remodeling of the transcriptional profile of specific cortical cell types. Most importantly, we computationally infer the biological significance of AS on protein structure by using AlphaFold2 and revealing how radical protein conformational changes can arise from subtle changes in isoforms sequence. Together, our study reveals that AS has a greater potential to impact protein diversity and function than previously thought independently from changes in gene expression.
... This protein family also includes two members, KCTD10 and KCTD13. Studies have shown that KCTD10 is involved in early cardiac morphogenesis and vascular development [3][4][5]; KCTD13 is involved in the regulation of early embryonic neurodevelopment [6][7][8] and is considered to be an important neurodevelopmental regulator. TNFAIP1 interacts with Rnd2 and Rnd3 to regulate the lateral migration of neuronal cells and influence the dynamics of neuronal branching and dendritic spines, suggesting Figure 5. Yeast two-hybrid point-to-point validation of Tnfaip1-interacting proteins. ...
... This protein family also includes two members, KCTD10 and KCTD13. Studies have shown that KCTD10 is involved in early cardiac morphogenesis and vascular development [3][4][5]; KCTD13 is involved in the regulation of early embryonic neurodevelopment [6][7][8] and is considered to be an important neurodevelopmental regulator. TNFAIP1 interacts with Rnd2 and Rnd3 to regulate the lateral migration of neuronal cells and influence the dynamics of neuronal branching and dendritic spines, suggesting the role of TNFAIP1 in early embryonic neural development [9,10]. ...
TNFAIP1 regulates cellular biological functions, including DNA replication, DNA repair, and cell cycle, by binding to target proteins. Identification of Tnfaip1-interacting proteins contributes to the understanding of the molecular regulatory mechanisms of their biological functions. In this study, 48 hpf, 72 hpf, and 96 hpf wild-type zebrafish embryo mRNAs were used to construct yeast cDNA library. The library titer was 1.12 × 107 CFU/mL, the recombination rate was 100%, and the average length of the inserted fragments was greater than 1000 bp. A total of 43 potential interacting proteins of Tnfaip1 were identified using zebrafish Tnfaip1 as a bait protein. Utilizing GO functional annotation and KEGG signaling pathway analysis, we found that these interacting proteins are mainly involved in translation, protein catabolic process, ribosome assembly, cytoskeleton formation, amino acid metabolism, and PPAR signaling pathway. Further yeast spotting analyses identified four interacting proteins of Tnfaip1, namely, Ubxn7, Tubb4b, Rpl10, and Ybx1. The Tnfaip1-interacting proteins, screened from zebrafish embryo cDNA in this study, increased our understanding of the network of Tnfaip1-interacting proteins during the earliest embryo development and provided a molecular foundation for the future exploration of tnfaip1’s biological functions.
... DA abnormalities associated with SZ and ASD suggest the importance of CUL3, an overlapped susceptible gene in both disorders, in DA systems. It has been shown that CUL3-deficient mice displayed anxiety-like behavior, impaired social ability, and dysregulated sensory gating Rapanelli et al., 2021Rapanelli et al., , 2023, and CUL3 deficiencies impaired neuronal excitability and synaptic transmission (Escamilla et al., 2017;Kikuma et al., 2019;Dong et al., 2020;Rapanelli et al., 2021Rapanelli et al., , 2023. CUL3 may regulate DA neuronal activity, but its specific function in DA system remains unknown because of the dominant effects of CUL3 deficiency in cell types studied in these mouse models. ...
The dopaminergic neuromodulator system is fundamental to brain functions. Abnormal dopamine (DA) pathway is implicated in psychiatric disorders including schizophrenia (SZ) and autism spectrum disorder (ASD). Mutations in Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex, have been associated with SZ and ASD. However, little is known about the function and mechanism of CUL3 in the DA system. Here, we show that CUL3 is critical for the function of DA neurons and DA-relevant behaviors in male mice. CUL3-deficient mice exhibited hyperactive locomotion, deficits in working memory and sensorimotor gating, and increased sensitivity to psychostimulants. In addition, enhanced DA signaling and elevated excitability of the ventral tegmental area (VTA) DA neurons were observed in CUL3-deficient animals. Behavioral impairments were attenuated by dopamine D2 receptor antagonist haloperidol and chemogenetic inhibition of DA neurons. Furthermore, we identified HCN2, a hyperpolarization-activated and cyclic nucleotide-gated channel, as a potential target of CUL3 in DA neurons. Our study indicates that CUL3 controls DA neuronal activity by maintaining ion channel homeostasis and provides insight into the role of CUL3 in the pathogenesis of psychiatric disorders.
Significance Statement
This study provides evidence that Cullin 3, a core component of the Cullin-RING ubiquitin E3 ligase complex that has been associated with ASD and SZ, controls the excitability of DA neurons in mice. Its DA-specific heterozygous deficiency increased spontaneous locomotion, impaired working memory and sensorimotor gating, and elevated response to psychostimulants. We showed that CUL3 deficiency increased the excitability of VTA DA neurons, and inhibiting D2 receptor or DA neuronal activity attenuated behavioral deficits of CUL3-deficient mice. We found HCN2, a hyperpolarization-activated channel, as a target of CUL3 in DA neurons. Our findings reveal CUL3’s role in DA neurons and offer insights into the pathogenic mechanisms of ASD and SZ.
... For instance, more than 40 unique mutations in KCTD7 have been identified in more than 50 patients with progressive myoclonic epilepsy (PME) (17). Copy-number variations in KCTD13 are associated with neurodevelopmental disorders such as autism and schizophrenia (19)(20)(21). The BTB domains in most, but not all, members of the KCTD family bind Cullin3 (Cul3) as 5:5 heterodecamers (22)(23)(24) and assemble into Cul3-RING E3 ligase (CRL3) with the RING domain protein Rbx1, while CTDs of KCTDs serve as substrate receptors. ...
... In addition to Gβγ, RhoA that can be regulated by some GPCRs through the Gα 12/13 pathway is targeted by KCTD13/BACURD for degradation (51). The KCTD13-Cul3-RhoA pathway has been shown to be associated with neurodevelopment and psychiatric diseases (19)(20)(21). However, it remains to be elucidated whether other KCTD members can regulate GPCR signaling since the substrates of most other KCTD members have not been characterized. ...
G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gβγ signaling is negatively regulated by a Cullin3 (Cul3)-dependent E3 ligase, KCTD5, which triggers ubiquitination and degradation of free Gβγ. Here, we report the cryo-electron microscopy structures of the KCTD5-Gβγ fusion complex and the KCTD7-Cul3 complex. KCTD5 in pentameric form engages symmetrically with five copies of Gβγ through its C-terminal domain. The unique pentameric assembly of the KCTD5/Cul3 E3 ligase places the ubiquitin-conjugating enzyme (E2) and the modification sites of Gβγ in close proximity and allows simultaneous transfer of ubiquitin from E2 to five Gβγ subunits. Moreover, we show that ubiquitination of Gβγ by KCTD5 is important for fine-tuning cyclic adenosine 3´,5´-monophosphate signaling of GPCRs. Our studies provide unprecedented insights into mechanisms of substrate recognition by unusual pentameric E3 ligases and highlight the KCTD family as emerging regulators of GPCR signaling.
... Previously, we found that the loss of Kctd13, a gene in the 16p11.2 recurrent Copy Number Variant (CNV) region [6][7][8][9][10][11][12][13][14][15] encoding a binding partner of CUL3, reduces hippocampal synaptic transmission via the RhoA pathway [16], and decreases synapse numbers in the hippocampus, among other brain regions [16]. Interestingly, KCTD13 is an adaptor protein for CUL3, a ubiquitin ligase that has been characterized as a high-risk autism gene through genome-wide association studies [17]. ...
... Previously, we found that the loss of Kctd13, a gene in the 16p11.2 recurrent Copy Number Variant (CNV) region [6][7][8][9][10][11][12][13][14][15] encoding a binding partner of CUL3, reduces hippocampal synaptic transmission via the RhoA pathway [16], and decreases synapse numbers in the hippocampus, among other brain regions [16]. Interestingly, KCTD13 is an adaptor protein for CUL3, a ubiquitin ligase that has been characterized as a high-risk autism gene through genome-wide association studies [17]. ...
... RhoA is a regulator of the actin cytoskeleton and has recently been implicated in autism [18]. RhoA is also a target of KCTD13/CUL3 ubiquitination [19], however, we found that Kctd13 deletion does not generate elevated RhoA until after P7 [16]. Because KCTD13 performs its function in part by binding to CUL3, we were interested in how Cul3 deletion affects hippocampal synaptic function, behavior, and protein expression. ...
Autism Spectrum Disorder (ASD) is a developmental disorder in which children display repetitive behavior, restricted range of interests, and atypical social interaction and communication. CUL3, coding for a Cullin family scaffold protein mediating assembly of ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors, has been identified as a high-risk gene for autism. Although complete knockout of Cul3 results in embryonic lethal-ity, Cul3 heterozygous mice have reduced CUL3 protein, demonstrate comparable body weight, and display minimal behavioral differences including decreased spatial object recognition memory. In measures of reciprocal social interaction, Cul3 heterozygous mice behaved similarly to their wild-type littermates. In area CA1 of hippocampus, reduction of Cul3 significantly increased mEPSC frequency but not amplitude nor baseline evoked syn-aptic transmission or paired-pulse ratio. Sholl and spine analysis data suggest there is a small yet significant difference in CA1 pyramidal neuron dendritic branching and stubby spine density. Unbiased proteomic analysis of Cul3 heterozygous brain tissue revealed dys-regulation of various cytoskeletal organization proteins, among others. Overall, our results suggest that Cul3 heterozygous deletion impairs spatial object recognition memory, alters cytoskeletal organization proteins, but does not cause major hippocampal neuronal morphology , functional, or behavioral abnormalities in adult global Cul3 heterozygous mice.
