A Structural Network Analysis of Neuronal ArhGAP21/23 Interactors by Computational Modeling.

RhoGTPase-activating proteins (RhoGAPs) play multiple roles in neuronal development; however, details of their substrate recognition system remain elusive. ArhGAP21 and ArhGAP23 are RhoGAPs that contain N-terminal PDZ and pleckstrin homology domains. In the present study, the RhoGAP domain of these ArhGAPs was computationally modeled by template-based methods and the AlphaFold2 software program, and their intrinsic RhoGTPase recognition mechanism was analyzed from the domain structures using the protein docking programs HADDOCK and HDOCK. ArhGAP21 was predicted to preferentially catalyze Cdc42, RhoA, RhoB, RhoC, and RhoG and to downregulate RhoD and Tc10 activities. Regarding ArhGAP23, RhoA and Cdc42 were deduced to be its substrates, whereas RhoD downregulation was predicted to be less efficient. The PDZ domains of ArhGAP21/23 possess the FTLRXXXVY sequence, and similar globular folding consists of antiparalleled ß-sheets and two α-helices that are conserved with PDZ domains of MAST-family proteins. A peptide docking analysis revealed the specific interaction of the ArhGAP23 PDZ domain with the PTEN C-terminus. The pleckstrin homology domain structure of ArhGAP23 was also predicted, and the functional selectivity for the interactors regulated by the folding and disordered domains in ArhGAP21 and ArhGAP23 was examined by an in silico analysis. An interaction analysis of these RhoGAPs revealed the existence of mammalian ArhGAP21/23-specific type I and type III Arf- and RhoGTPase-regulated signaling. Multiple recognition systems of RhoGTPase substrates and selective Arf-dependent localization of ArhGAP21/23 may form the basis of the functional core signaling necessary for synaptic homeostasis and axon/dendritic transport regulated by RhoGAP localization and activities.

Function of SYDE C2-RhoGAP family as signaling hubs for neuronal development deduced by computational analysis.

Recent investigations of neurological developmental disorders have revealed the Rho-family modulators such as Syde and its interactors as the candidate genes. Although the mammalian Syde proteins are reported to possess GTPase-accelerating activity for RhoA-family proteins, diverse species-specific substrate selectivities and binding partners have been described, presumably based on their evolutionary variance in the molecular organization. A comprehensive in silico analysis of Syde family proteins was performed to elucidate their molecular functions and neurodevelopmental networks. Predicted structural modeling of the RhoGAP domain may account for the molecular constraints to substrate specificity among Rho-family proteins. Deducing conserved binding motifs can extend the Syde interaction network and highlight diverse but Syde isoform-specific signaling pathways in neuronal homeostasis, differentiation, and synaptic plasticity from novel aspects of post-translational modification and proteolysis.

Monoacylglycerol lipase promotes Fcγ receptor-mediated phagocytosis in microglia but does not regulate LPS-induced upregulation of inflammatory cytokines.

Monoacylglycerol lipase (MAGL) is important for neuroinflammation. However, the regulatory mechanisms underlying its expression and function remain unknown. Lipopolysaccharide (LPS) treatment post-translationally upregulated MAGL expression, whereas it downregulated MAGL transcription through a Stat6-mediated mechanism in microglia. Neither MAGL knockdown nor JZL-184, a selective MAGL inhibitor, suppressed LPS-induced upregulation of inflammatory cytokines in microglia. Moreover, exogenous expression of MAGL in BV-2 microglial cell line, which lacks endogenous MAGL, did not promote the induction of inflammatory cytokines by LPS treatment. Interestingly, MAGL knockdown reduced Fcγ receptor-mediated phagocytosis in primary microglia, and introduction of MAGL into the BV-2 cells increased Fcγ receptor-mediated phagocytosis. Collectively, these results suggest that MAGL regulates phagocytosis, but not LPS-mediated cytokine induction in microglia.

Niemann-Pick disease type C1 predominantly involving the frontotemporal region, with cortical and brainstem Lewy bodies: an autopsy case.

Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage disorder. Two disease-causing genes (NPC1 and NPC2) have been identified. NPC is characterized by neuronal and glial lipid storage and NFTs. Here, we report a man with juvenile-onset progressive neurological deficits, including pyramidal signs, ataxia, bulbar palsy, vertical supranuclear ophthalmoplegia, and psychiatric symptoms; death occurred at age 37 before definitive clinical diagnosis. Post mortem gross examination revealed a unique distribution of brain atrophy, predominantly in the frontal and temporal lobes. Microscopically, lipid storage in neurons and widely distributed NFTs were observed. Lipid storage cells appeared in systemic organs and filipin staining indicated intracellular cholesterol accumulation in hepatic macrophages. Electron microscopy revealed accumulation of lipids and characteristic oligolamellar inclusions. These findings suggested an NPC diagnosis. Neuronal loss and gliosis were frequently accompanied by NFTs and occurred in the frontal and temporal cortices, hippocampus, amygdala, basal forebrain, basal ganglia, thalamus, substantia nigra and brain stem nuclei. Lewy bodies (LBs) were observed in most, but not all, regions where NFTs were evident. In contrast, neuronal lipid storage occurred in more widespread areas, including the parietal and occipital cortices where neurodegeneration with either NFTs or LBs was minimal. Molecular genetic analysis demonstrated that the patient had compound heterozygous mutations in the cysteine-rich loop (A1017T and Y1088C) of the NPC1 gene. To our knowledge there has been no previous report of the A1017T mutation. The pathological features of this patient support the notion that NPC has an aspect of α-synucleinopathy, and long-term survivors of NPC may develop a frontotemporal-predominant distribution of brain atrophy.

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation.

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells.

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) has anti-cancer activity in several colon cancers. 1α,25(OH)(2)D(3) induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIß) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)(2)D(3)-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P(2) production mediates E-cadherin expression through PIPKIIß in a VDR-dependent manner. PIPKIIß is also involved in the suppression of the cell motility induced by 1α,25(OH)(2)D(3). These results indicate that PIPKIIß-mediated PI(4,5)P(2) signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth.

Phospholipase Cδ3 (PLCδ3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLCδ3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLCδ3 in neuronal migration and outgrowth. PLCδ3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLCδ3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLCδ3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLCδ3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLCδ3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLCδ3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLCδ3KD cells. We also found that exogenous expression of PLCδ3 down-regulated RhoA protein, and constitutively active PLCδ3 promotes the RhoA down-regulation more significantly than PLCδ3 upon differentiation. These results indicate that PLCδ3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.

Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Ialpha are required for invadopodia formation in human breast cancer cells.

Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and PI(4,5)P(2)-producing enzymes in invadopodia formation in MDA-MB-231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P(2) accumulates at invadopodia on the ventral cell surface. Injection of an anti-PI(4,5)P(2) antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P(2) by overexpression of the phospholipase C (PLC) delta1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P(2), also blocked invadopodia formation, while a mutated PLCdelta1-PH domain that lacks PI(4,5)P(2)-binding activity had no effect. Cellular PI(4,5)P(2) production is mainly mediated by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) family proteins, which include PIP5KIalpha, Ibeta, and Igamma. Real-time quantitative PCR analysis showed that PIP5KIalpha is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KIalpha by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIalpha protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KIalpha protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P(2) by PIP5KIalpha is required for invadopodia formation and ECM degradation by human breast cancer cells.

Phospholipase C-eta2 is highly expressed in the habenula and retina.

Phospholipase C (PLC), a key enzyme involved in phosphoinositide turnover, hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. PLCeta2 (PLCeta2), a neuron-specific isozyme of PLC, is abundantly expressed in the postnatal brain, suggesting the importance of PLCeta2 in the formation and maintenance of the neuronal network in the postnatal brain. However, the detailed expression patterns of PLCeta2 in the brain and other neuronal tissues remain to be clarified. Here, we generated PLCeta2 knockout/LacZ knockin (plch2(lacZ)(/)(lacZ)) mice-the first mice to lack full-length PLCeta2. Although the plch2(lacZ)(/)(lacZ) mice exhibited no obvious abnormalities, the LacZ reporter revealed unexpected and abundant expressions of PLCeta2 in the habenula and retina. We confirmed these PLCeta2 expression patterns by in situ hybridization and immunohistochemical analyses. In the retina, strong PLCeta2 expression was detected in the photoreceptor (rod/cone), outer nuclear layer, outer plexiform layer, and inner nuclear layer, suggesting that PLCeta2 is expressed in rods and cones, and also in horizontal, bipolar, and amacrine cells, but not in ganglion cells. Interestingly PLCeta2 exhibited a dynamic expression pattern during postnatal retinal development, strongly suggesting that this isozyme might be involved in the development and maturation of the retina. Since both the habenula and retina are thought to play important roles in the regulation of circadian rhythms, our results suggest that PLCeta2 may be involved in the function of habenula and retina.

Lipid rafts and caveolin-1 are required for invadopodia formation and extracellular matrix degradation by human breast cancer cells.

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA-mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP-dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion.

p120 catenin recruits cadherins to gamma-secretase and inhibits production of Abeta peptide.

The gamma-secretase complex cleaves many transmembrane proteins, including amyloid precursor protein, EphB and ErbB tyrosine kinase receptors, Notch1 receptors, and adhesion factors. Presenilin 1, the catalytic subunit of gamma-secretase, associates with the cadherin/catenin cell-cell adhesion/communication system and promotes cadherin processing (Georgakopoulos, A., et al. (1999) Mol. Cell 4, 893-902; Marambaud, P., et al. (2002) EMBO J. 21, 1948-1956), but the mechanism by which gamma-secretase and cadherins associate is unclear. Here we report that p120 catenin (p120ctn), a component of the cadherin-catenin complex, recruits gamma-secretase to cadherins, thus stimulating their processing while inhibiting production of Abeta peptide and the amyloid precursor protein intracellular domain. This function of p120ctn depends on both p120ctn-cadherin and p120ctn-presenilin 1 binding, indicating that p120ctn is the central factor that bridges gamma-secretase and cadherin-catenin complexes. Our data show that p120ctn is a unique positive regulator of the gamma-secretase processing of cadherins and a negative regulator of the amyloid precursor protein processing. Furthermore, our data suggest that specific members of the gamma-secretase complex may be used to recruit different substrates and that distinct PS1 sequences are required for processing of APP and cadherins.

FAD mutants unable to increase neurotoxic Abeta 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta.

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.

Cadherins mediate both the association between PS1 and beta-catenin and the effects of PS1 on beta-catenin stability.

Presenilin1 (PS1), a protein involved in cellular development, forms functional complexes with beta-catenin, a regulator of Wnt signaling and cell-cell adhesion. In addition, both proteins have been shown to play important roles in disease including cancer and Alzheimer disease. Although PS1 and beta-catenin are found in the same complexes, it is not clear whether they bind directly to each other or a third complex component, like cadherin, may mediate their interactions. Here we show that PS1 and beta-catenin form no detectable complexes in cells that express no cadherin. In contrast, these complexes are readily found in E-cadherin containing cells. Furthermore, binding of both PS1 and beta-catenin to E-cadherin is necessary for the formation of PS1/beta-catenin complexes. Importantly, our data show that binding of PS1 to cadherin mediates the effects of PS1 on the phosphorylation, ubiquitination, and destabilization of beta-catenin. Thus, cadherins mediate both the association of PS1 and beta-catenin and the effects of PS1 on the cellular levels of beta-catenin.

The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Czeta.

Sperm-specific phospholipase C-zeta (PLCzeta) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCzeta has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at approximately 100 nM Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408-10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCzeta activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCzeta activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCzeta activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCzeta.

Ca2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation.

Sperm-specific phospholipase C zeta (PLC zeta) is known to induce intracellular Ca(2+) oscillations and egg activation when expressed in mouse eggs by injection of RNA encoding PLC zeta. We investigated the expression level and spatial distribution of PLC zeta in the egg in real time and in relation to the initiation and termination of Ca(2+) oscillations by monitoring fluorescence of a yellow fluorescent protein 'Venus' fused with PLC zeta. Ca(2+) oscillations similar to those at fertilization were induced at 40-50 min after RNA injection, when expressed PLC zeta reached 10-40 x 10(-15) g in the egg. PLC zeta-Venus increased up to 3 h and attained a steady level at 4-5 h. Interestingly, PLC zeta-Venus is accumulated to the pronucleus (PN) formed at 5-6 h and continuously increased there. Ca(2+) oscillations stopped in most eggs before initiation of the accumulation. A variant of PLC zeta that lacks three EF hand domains was much less effective in induction of Ca(2+) oscillations and little accumulated in the pronucleus, indicating a critical role of those domains. The ability of the accumulation to the pronucleus qualifies PLC zeta for a strong candidate of the Ca(2+) oscillation-inducing sperm factor, which is introduced into the ooplasm upon sperm-egg fusion and concentrated to the pronucleus after inducing egg activation.

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs.

Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.