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

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.

KLHL17 differentially controls the expression of AMPA- and KA-type glutamate receptors to regulate dendritic spine enlargement.

Kelch-like family member 17 (KLHL17), an actin-associated adaptor protein, is linked to neurological disorders, including infantile spasms and autism spectrum disorders. The key morphological feature of Klhl17-deficient neurons is impaired dendritic spine enlargement, resulting in the amplitude of calcium events being increased. Our previous studies have indicated an involvement of F-actin and the spine apparatus in KLHL17-mediated dendritic spine enlargement. Here, we show that KLHL17 further employs different mechanisms to control the expression of two types of glutamate receptors, that is, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KARs), to regulate dendritic spine enlargement and calcium influx. We deployed proteomics to reveal that KLHL17 interacts with N-ethylmaleimide-sensitive fusion protein (NSF) in neurons, with this interaction of KLHL17 and NSF enhancing NSF protein levels. Consistent with the function of NSF in regulating the surface expression of AMPAR, Klhl17 deficiency limits the surface expression of AMPAR, but not its total protein levels. The NSF pathway also contributes to synaptic F-actin distribution and the dendritic spine enlargement mediated by KLHL17. KLHL17 is known to act as an adaptor mediating degradation of the KAR subunit GluK2 by the CUL3 ubiquitin ligase complex, and Klhl17 deficiency impairs activity-dependent degradation of GluK2. Herein, we further demonstrate that GluK2 is critical to the increased amplitude of calcium influx in Klhl17-deficient neurons. Moreover, GluK2 is also involved in KLHL17-regulated dendritic spine enlargement. Thus, our study reveals that KLHL17 controls AMPAR and KAR expression via at least two mechanisms, consequently regulating dendritic spine enlargement. The regulatory effects of KLHL17 on these two glutamate receptors likely contribute to neuronal features in patients suffering from certain neurological disorders.

KLHL17/Actinfilin, a brain-specific gene associated with infantile spasms and autism, regulates dendritic spine enlargement.

BACKGROUND: Dendritic spines, the actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain. Many actin-regulating molecules modulate dendritic spine morphology. Since dendritic spines are neuron-specific structures, it is reasonable to speculate that neuron-specific or -predominant factors are involved in dendritic spine formation. KLHL17 (Kelch-like 17, also known as Actinfilin), an actin-binding protein, is predominantly expressed in brain. Human genetic study has indicated an association of KLHL17/Actinfilin with infantile spasms, a rare form of childhood epilepsy also resulting in autism and mental retardation, indicating that KLHL17/Actinfilin plays a role in neuronal function. However, it remains elusive if and how KLHL17/Actinfilin regulates neuronal development and brain function. METHODS: Fluorescent immunostaining and electrophysiological recording were performed to evaluate dendritic spine formation and activity in cultured hippocampal neurons. Knockdown and knockout of KLHL17/Actinfilin and expression of truncated fragments of KLHL17/Actinfilin were conducted to investigate the function of KLHL17/Actinfilin in neurons. Mouse behavioral assays were used to evaluate the role of KLHL17/Actinfilin in brain function. RESULTS: We found that KLHL17/Actinfilin tends to form circular puncta in dendritic spines and are surrounded by or adjacent to F-actin. Klhl17 deficiency impairs F-actin enrichment at dendritic spines. Knockdown and knockout of KLHL17/Actinfilin specifically impair dendritic spine enlargement, but not the density or length of dendritic spines. Both N-terminal Broad-Complex, Tramtrack and Bric-a-brac (BTB) domain and C-terminal Kelch domains of KLHL17/Actinfilin are required for F-actin remodeling and enrichment at dendritic spines, as well as dendritic spine enlargement. A reduction of postsynaptic and presynsptic markers at dendritic spines and altered mEPSC profiles due to Klhl17 deficiency evidence impaired synaptic activity in Klhl17-deficient neurons. Our behavioral assays further indicate that Klhl17 deficiency results in hyperactivity and reduced social interaction, strengthening evidence for the physiological role of KLHL17/Actinfilin. CONCLUSION: Our findings provide evidence that KLHL17/Actinfilin modulates F-actin remodeling and contributes to regulation of neuronal morphogenesis, maturation and activity, which is likely relevant to behavioral impairment in Klhl17-deficient mice. Trial registration Non-applicable.

The Involvement of Neuron-Specific Factors in Dendritic Spinogenesis: Molecular Regulation and Association with Neurological Disorders.

Dendritic spines are the location of excitatory synapses in the mammalian nervous system and are neuron-specific subcellular structures essential for neural circuitry and function. Dendritic spine morphology is determined by the F-actin cytoskeleton. F-actin remodeling must coordinate with different stages of dendritic spinogenesis, starting from dendritic filopodia formation to the filopodia-spines transition and dendritic spine maturation and maintenance. Hundreds of genes, including F-actin cytoskeleton regulators, membrane proteins, adaptor proteins, and signaling molecules, are known to be involved in regulating synapse formation. Many of these genes are not neuron-specific, but how they specifically control dendritic spine formation in neurons is an intriguing question. Here, we summarize how ubiquitously expressed genes, including syndecan-2, NF1 (encoding neurofibromin protein), VCP, and CASK, and the neuron-specific gene CTTNBP2 coordinate with neurotransmission, transsynaptic signaling, and cytoskeleton rearrangement to control dendritic filopodia formation, filopodia-spines transition, and dendritic spine maturation and maintenance. The aforementioned genes have been associated with neurological disorders, such as autism spectrum disorders (ASDs), mental retardation, learning difficulty, and frontotemporal dementia. We also summarize the corresponding disorders in this report.

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging.

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

Phase separation and zinc-induced transition modulate synaptic distribution and association of autism-linked CTTNBP2 and SHANK3.

Many synaptic proteins form biological condensates via liquid-liquid phase separation (LLPS). Synaptopathy, a key feature of autism spectrum disorders (ASD), is likely relevant to the impaired phase separation and/or transition of ASD-linked synaptic proteins. Here, we report that LLPS and zinc-induced liquid-to-gel phase transition regulate the synaptic distribution and protein-protein interaction of cortactin-binding protein 2 (CTTNBP2), an ASD-linked protein. CTTNBP2 forms self-assembled condensates through its C-terminal intrinsically disordered region and facilitates SHANK3 co-condensation at dendritic spines. Zinc binds the N-terminal coiled-coil region of CTTNBP2, promoting higher-order assemblies. Consequently, it leads to reduce CTTNBP2 mobility and enhance the stability and synaptic retention of CTTNBP2 condensates. Moreover, ASD-linked mutations alter condensate formation and synaptic retention of CTTNBP2 and impair mouse social behaviors, which are all ameliorated by zinc supplementation. Our study suggests the relevance of condensate formation and zinc-induced phase transition to the synaptic distribution and function of ASD-linked proteins.

Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

Interhemispheric Connectivity Potentiates the Basolateral Amygdalae and Regulates Social Interaction and Memory.

Impaired interhemispheric connectivity is commonly found in various psychiatric disorders, although how interhemispheric connectivity regulates brain function remains elusive. Here, we use the mouse amygdala, a brain region that is critical for social interaction and fear memory, as a model to demonstrate that contralateral connectivity intensifies the synaptic response of basolateral amygdalae (BLA) and regulates amygdala-dependent behaviors. Retrograde tracing and c-FOS expression indicate that contralateral afferents widely innervate BLA non-randomly and that some BLA neurons innervate both contralateral BLA and the ipsilateral central amygdala (CeA). Our optogenetic and electrophysiological studies further suggest that contralateral BLA input results in the synaptic facilitation of BLA neurons, thereby intensifying the responses to cortical and thalamic stimulations. Finally, pharmacological inhibition and chemogenetic disconnection demonstrate that BLA contralateral facilitation is required for social interaction and memory. Our study suggests that interhemispheric connectivity potentiates the synaptic dynamics of BLA neurons and is critical for the full activation and functionality of amygdalae.

Postsynaptic SDC2 induces transsynaptic signaling via FGF22 for bidirectional synaptic formation.

Functional synapse formation requires tight coordination between pre- and post-synaptic termini. Previous studies have shown that postsynaptic expression of heparan sulfate proteoglycan syndecan-2 (SDC2) induces dendritic spinogenesis. Those SDC2-induced dendritic spines are frequently associated with presynaptic termini. However, how postsynaptic SDC2 accelerates maturation of corresponding presynaptic termini is unknown. Because fibroblast growth factor 22 (FGF22), a heparan sulfate binding growth factor, has been shown to act as a presynaptic organizer released from the postsynaptic site, it seems possible that postsynaptic SDC2 presents FGF22 to the presynaptic FGF receptor to promote presynaptic differentiation. Here, we show that postsynaptic SDC2 uses its ectodomain to interact with and facilitate dendritic filopodial targeting of FGF22, triggering presynaptic maturation. Since SDC2 also enhances filopodial targeting of NMDAR via interaction with the CASK-mLIN7-MINT1 adaptor complex, presynaptic maturation promoted by FGF22 further feeds back to activate NMDAR at corresponding postsynaptic sites through increased neurotransmitter release and, consequently, promotes the dendritic filopodia-spines (F-S) transition. Meanwhile, via regulation of the KIF17 motor, CaMKII (activated by the NMDAR pathway) may further facilitate FGF22 targeting to dendritic filopodia that receive presynaptic stimulation. Our study suggests a positive feedback that promotes the coordination of postsynaptic and presynaptic differentiation.

Calcium influx and postsynaptic proteins coordinate the dendritic filopodium-spine transition.

Dendritic spines are the major locations of excitatory synapses in the mammalian central nervous system. The transformation from dendritic filopodia to dendritic spines has been recognized as one type of spinogenesis. For instance, syndecan-2 (SDC2), a synaptic heparan sulfate proteoglycan, is highly concentrated at dendritic spines and required for spinogenesis. It induces dendritic filopodia formation, followed by spine formation. However, the molecular regulation of the filopodium-spine transition induced by SDC2 is still unclear. In this report, we show that calcium is an important signal downstream of SDC2 in regulation of filopodium-spine transition but not filopodia formation. SDC2 interacted with the postsynaptic proteins calmodulin-dependent serine kinase (CASK) and LIN7 and further recruited NMDAR to the tips of filopodia induced by SDC2. Calcium influx via NMDAR promoted spine maturation because addition of EGTA or AP5 to the culture medium effectively prevented morphological change from dendritic filopodia to dendritic spines. Our data also indicated that F-actin rearrangement regulated by calcium influx is involved in the morphological change, because the knockdown of gelsolin, a calcium-activated F-actin severing molecule, impaired the filopodium-spine transition induced by SDC2. In conclusion, our study demonstrates that postsynaptic proteins coordinate to trigger calcium signalling and cytoskeleton rearrangement and consequently control filopodium-spine transition.

Sarm1 deficiency impairs synaptic function and leads to behavioral deficits, which can be ameliorated by an mGluR allosteric modulator.

Innate immune responses have been shown to influence brain development and function. Dysregulation of innate immunity is significantly associated with psychiatric disorders such as autism spectrum disorders and schizophrenia, which are well-known neurodevelopmental disorders. Recent studies have revealed that critical players of the innate immune response are expressed in neuronal tissues and regulate neuronal function and activity. For example, Sarm1, a negative regulator that acts downstream of Toll-like receptor (TLR) 3 and 4, is predominantly expressed in neurons. We have previously shown that Sarm1 regulates neuronal morphogenesis and the expression of inflammatory cytokines in the brain, which then affects learning ability, cognitive flexibility, and social interaction. Because impaired neuronal morphogenesis and dysregulation of cytokine expression may disrupt neuronal activity, we investigated whether Sarm1 knockdown affects the synaptic responses of neurons. We here show that reduced Sarm1 expression impairs metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) formation but enhances N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation production in hippocampal CA1 neurons. The expression levels of post-synaptic proteins, including NR2a, NR1, Shank1 and Shank3, are also altered in Sarm1 knockdown mice, suggesting a role for Sarm1 in the maintenance of synaptic homeostasis. The addition of a positive allosteric modulator of mGluR5, CDPPB, ameliorates the LTD defects in slice recording and the behavioral deficits in social interaction and associative memory. These results suggest an important role for mGluR5 signaling in the function of Sarm1. In conclusion, our study demonstrates a role for Sarm1 in the regulation of synaptic plasticity. Through these mechanisms, Sarm1 knockdown results in the impairment of associative memory and social interactions in mice.

CTTNBP2, but not CTTNBP2NL, regulates dendritic spinogenesis and synaptic distribution of the striatin-PP2A complex.

Cortactin-binding protein 2 (CTTNBP2) interacts with cortactin to regulate cortactin mobility and control dendritic spine formation. CTTNBP2 has also been associated with autistic spectrum disorder. The regulation of dendritic spinogenesis could explain the association of CTTNBP2 with autism. Sequence comparison has indicated that CTTNBP2 N-terminal-like protein (CTTNBP2NL) is a CTTNBP2 homologue. To confirm the specific effect of CTTNBP2 on dendritic spinogenesis, here we investigate whether CTTNBP2NL has a similar function to CTTNBP2. Although both CTTNBP2 and CTTNBP2NL interact with cortactin, CTTNBP2NL is associated with stress fibers, whereas CTTNBP2 is distributed to the cortex and intracellular puncta. We also provide evidence that CTTNBP2, but not CTTNBP2NL, is predominantly expressed in the brain. CTTNBP2NL does not show any activity in the regulation of dendritic spinogenesis. In addition to spine morphology, CTTNBP2 is also found to regulate the synaptic distribution of striatin and zinedin (the regulatory B subunits of protein phosphatase 2A [PP2A]), which interact with CTTNBP2NL in HEK293 cells. The association between CTTNBP2 and striatin/zinedin suggests that CTTNBP2 targets the PP2A complex to dendritic spines. Thus we propose that the interactions of CTTNBP2 and cortactin and the PP2A complex regulate spine morphogenesis and synaptic signaling.

Tie2-R849W mutant in venous malformations chronically activates a functional STAT1 to modulate gene expression.

Tie2 is an endothelial receptor tyrosine kinase. An amino-acid substitution of tryptophan for arginine at residue 849 (Tie2-R849W) leads to a ligand-independent activation of its kinase activity. This mutation has been associated with familial venous malformations (VMs), manifested by variable thickness or lack of smooth-muscle cells in the veins of patient lesions. The underlying mechanism for Tie2-R849W action in endothelial cells remains elusive. In this study, we used adenoviral infection to differentiate the effects of ectopic Tie2 (wild type, kinase-dead K855A, or constitutively active R849W) expression on endothelial cellular behaviors and Tie2-mediated downstream targets. Ectopic Tie2 reduced endothelial cell proliferation and serum withdrawal-induced apoptosis, while stimulating migration. When comparing R849W with K855A and its wild-type counterpart, a functional tyrosine kinase activity was required only for migration, and constitutively active Tie2-R849W conferred highest resistance to serum-induced apoptosis, but lowest ability to maintain tube-like structures formed on Matrigel. We further demonstrated that Tie2-R849W chronically induced STAT1 tyrosine phosphorylation and the promoter activity of STAT1-responsive IFN-regulatory factor 1 (IRF1). Although STAT1 phosphorylation required JNK and p38MAPK activation, only JNK activation was essential for IRF1 promoter activation by Tie2-R849W. Additional studies are needed to study the role of STAT1 activation in VMs.

Dynamic expression of Hsp27 in the presence of mutant ataxin-3.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. The molecular mechanisms underlying the selective neuronal death typical of MJD/SCA3 are unknown. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD with 78 CAG repeats were assayed for the dynamic expression of Hsp27, known as a suppressor of poly-Q mediated cell death, in the presence of mutant ataxin-3 in different passages of cultured cells. A dramatic decrease of Hsp27 expression was observed in the earlier passage of cultured SK-N-SH-MJD78 cells, however, the later passage of cells showed a significant increase of Hsp27 to almost the same level of the parental cells. Furthermore, immunohistochemical analysis of MJD transgenic mice and post-mortem human brain tissues showed increased expression of Hsp27 compared to normal control brain, suggesting an up-regulation of Hsp27 in the end stage of MJD. However, mutant cells of earlier passages were more susceptible to serum deprivation than mutant cells of later passages, indicating weak tolerance toward stress in cells with reduced Hsp27. While heat shock was used to assess the stress response, cells expressing mutant ataxin-3 displayed normal response upon heat shock stimuli when compared to the parental cells. Taken together, we proposed that during the early disease stage, the reduction of Hsp27 synthesis mitigated the ability of neuron cells to cope with cytotoxicity induced by mutant ataxin-3, triggering the cell death process during the disease progress. In the late stage of disease, after prolonged stressful conditions of polyglutamine cytotoxicity, the increased level of Hsp27 may reflect a dynamic process of the survived cells to unfold and remove mutant ataxin-3. However, this increased Hsp27 still cannot reverse the global dysfunction of cellular proteins due to accumulation of cytotoxic effects.