Insights into Repeated Renal Injury Using RNA-Seq with Two New RPTEC Cell Lines.

Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25-2.5 µM cisplatin (CisPt) or 12.5-100 µM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 µM CisPt from 3 to 96 h expressing 2000-5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.

Endogenous and Therapeutic 25-Hydroxycholesterols May Worsen Early SARS-CoV-2 Pathogenesis in Mice.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-Hydroxycholesterol (25HC), a product of the activity of cholesterol-25-hydroxylase (CH25H) on cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, 25HC can also amplify inflammation and be converted by CYP7B1 (cytochrome P450 family 7 subfamily B member 1) to 7α,25-dihydroxycholesterol, a lipid with chemoattractant activity, via the G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2)/GPR183 (G protein-coupled receptor 183). Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that although 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 (angiotensin-converting enzyme 2) mouse model in vivo. Treatment with 25HC also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma proinflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points but no change in weight loss. Consistent with these findings, although Ch25h and 25HC were upregulated in the lungs of SARS-CoV-2-infected wild-type mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the ß variant were similar to those in control animals. Taken together, endogenous 25HCs do not significantly regulate early SARS-CoV-2 replication or pathogenesis, and supplemental 25HC may have proinjury rather than therapeutic effects in SARS-CoV-2 pneumonia.

TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication.

Coronaviruses are positive-strand RNA viruses with 3' polyadenylated genomes and subgenomic transcripts. The lengths of the viral poly(A) tails change during infection by mechanisms that remain poorly understood. Here, we use a splint-ligation method to measure the poly(A) tail length and poly(A) terminal uridylation and guanylation of the mouse hepatitis virus (MHV) RNAs. Upon infection of 17-CL1 cells with MHV, a member of the Betacoronavirus genus, we observe two populations of terminally uridylated viral transcripts, one with poly(A) tails ~44 nucleotides long and the other with poly(A) tails shorter than ~22 nucleotides. The mammalian terminal uridylyl-transferase 4 (TUT4) and terminal uridylyl-transferase 7 (TUT7), referred to as TUT4/7, add non-templated uracils to the 3'-end of endogenous transcripts with poly(A) tails shorter than ~30 nucleotides to trigger transcript decay. Here we find that depletion of the host TUT4/7 results in an increased replication capacity of the MHV virus. At late stages of infection, the population of uridylated subgenomic RNAs with tails shorter than ~22 nucleotides is reduced in the absence of TUT4/7 while the viral RNA load increases. Our findings indicate that TUT4/7 uridylation marks the MHV subgenomic RNAs for decay and delays viral replication.

DUX4 double whammy: The transcription factor that causes a rare muscular dystrophy also kills the precursors of the human nose.

SMCHD1 mutations cause congenital arhinia (absent nose) and a muscular dystrophy called FSHD2. In FSHD2, loss of SMCHD1 repressive activity causes expression of double homeobox 4 (DUX4) in muscle tissue, where it is toxic. Studies of arhinia patients suggest a primary defect in nasal placode cells (human nose progenitors). Here, we show that upon SMCHD1 ablation, DUX4 becomes derepressed in H9 human embryonic stem cells (hESCs) as they differentiate toward a placode cell fate, triggering cell death. Arhinia and FSHD2 patient-derived induced pluripotent stem cells (iPSCs) express DUX4 when converted to placode cells and demonstrate variable degrees of cell death, suggesting an environmental disease modifier. HSV-1 may be one such modifier as herpesvirus infection amplifies DUX4 expression in SMCHD1 KO hESC and patient iPSC. These studies suggest that arhinia, like FSHD2, is due to compromised SMCHD1 repressive activity in a cell-specific context and provide evidence for an environmental modifier.

Imaging Inflammasome Activation in Microglia.

Inflammasomes are multiprotein complexes that play key roles in the host's innate immune response to insult. The assembly of an inflammatory complex is initiated with the oligomerization of the upstream inflammasome-forming sensor and then follows a well-orchestrated multi-step process leading to downstream effector functions that are critical in the innate immune response. The final assembly of these steps provides a detectable readout of inflammasome complex activation in the form of an apoptosis-associated speck-like protein containing a CARD (ASC) speck. Inflammasome activation-and the release of IL-1ß and ASC specks from the microglia, the brain resident immune cell-have been implicated in various neurological and neurodegenerative disorders. Protocols exist for the generation of fluorescent inflammasome indicator peripheral macrophages. Building upon these protocols, we describe here a protocol that details the generation of fluorescent inflammasome indicator microglia cells using recombinant retroviruses to transduce murine BV-2 cells. In this protocol, the cells are established in a manner to allow for experimental control of the initial priming step of the inflammasome activation process. We then provide a series of steps for using these reporter cells within an inflammasome activation assay and use real-time imaging of ASC-speck formation as an indicator of inflammasome activation. In addition, we describe strategies for using these cells for examining the effects of a test substance on inflammasome activation. This protocol offers an effective approach conducive to screening for and examining modifications of microglia inflammasome activation due to exposure to chemicals or pharmacological agents. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Production of retroviruses to express inflammasome indicator Basic Protocol 2: Generation of inflammasome indicator BV-2 cells Basic Protocol 3: Priming and activation of BV-2-ASC-Cerulean cells for inflammasome activation assay Basic Protocol 4: Examining modifications to inflammasome activation by test substances Basic Protocol 5: Imaging and analysis of ASC speck formation.

Evaluation of endogenous and therapeutic 25-hydroxycholesterols in murine models of pulmonary SARS-CoV-2 infection.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7α,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo. 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor-binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.Abbreviations: ACE2 - angiotensin converting enzyme 2BSA - buried surface areaCDR - complementary determining regionRBD - receptor binding domainRBM - receptor-binding motifSARS-CoV-2 - severe acute respiratory syndrome coronavirus 2.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses, including the currently dominant Delta variant. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme.

There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12-15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1-5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

A Simple, Two-Step, Small-Scale Purification of Recombinant Adeno-Associated Viruses.

Recombinant adeno-associated viruses (rAAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in rAAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. rAAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for the production and purification of rAAVs. However, most platforms rely heavily on large amounts of starting material and multiple purification steps to produce highly purified viral particles. Often, researchers require several small-scale purified rAAVs. Here, we describe a simple and efficient technique for purification of recombinant rAAVs from small amounts of starting material in a two-step purification method. In this method, rAAVs are released into the packaging cell medium using high salt concentration, pelleted by ultracentrifugation to remove soluble impurities. Then, the resuspended pellet is purified using a protein spin-concentrator. In this protocol, we modify the conventional rAAV purification methods to eliminate the need for fraction collection and the labor-intensive steps for evaluating the titer and purity of individual fractions. The resulting rAAV preparations are comparable in titer and purity to commercially available samples. This simplified process can be used to generate highly purified rAAV particles on a small scale, thereby saving resources, generating less waste, and reducing a laboratory's environmental footprint.

ESR1 Mutations Associated With Estrogen Insensitivity Syndrome Change Conformation of Ligand-Receptor Complex and Altered Transcriptome Profile.

Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor-α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to its rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and deoxyribonucleic acid methylome from stable cell lines of both Q375H and R394H clinical mutants shows a differential profile compared with wild-type ERα, resulting in loss of estrogen responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand-receptor complexes. Furthermore, we generated a mouse model Esr1-Q harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the patient with Q375H. Finally, we explore the effects of a synthetic progestogen and a gonadotropin-releasing hormone inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.

AAV diffuses across zona pellucida for effortless gene delivery to fertilized eggs.

Gene delivery to fertilized eggs is often the first step in creation of transgenic animals, CRISPR knock-out, or early developmental studies. The zona pellucida, a hardened glycoprotein matrix surrounding the mammalian fertilized eggs, often complicates gene delivery by forming a barrier against transfection reagents and viruses. High efficiency techniques to perforate or penetrate the zona allow for access and gene delivery to fertilized eggs. However, these techniques often rely on highly skilled technologists, are costly, and require specialized equipment for micromanipulation, laser perforation, or electroporation. Here, we report that adenoassociated viruses (AAVs) with serotypes 1 or DJ can efficiently diffuse across the zona to deliver genes without any manipulations to fertilized eggs. We observe lowered rates of embryo development after treatment of embryos with all AAV serotypes. However, we were able to reduce adverse effects on embryo development by exposing embryos to AAVs at later stages of in vitro development. AAVs have low immune response and do not incorporate into their host chromosomes to cause insertional mutations. Hence, AAVs can serve as a highly effective tool for transient delivery of genes to fertilized mammalian eggs.

Recombinant Viral Vectors as Neuroscience Tools.

Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Viral vectors expand the neurobiology toolbox to include direct and rapid anterograde, retrograde, and trans-synaptic delivery of tracers, sensors, and actuators to the mammalian brain. Each viral type offers unique advantages and limitations. To establish strategies for selecting a suitable viral type, this article aims to provide readers with an overview of viral recombinant technology, viral structure, tropism, and differences between serotypes and pseudotypes for three of the most commonly used vectors in neurobiology research: adeno-associated viruses, retro/lentiviruses, and glycoprotein-deleted rabies viruses. © 2019 by John Wiley & Sons, Inc.

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics.

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida.

Lentiviruses are highly efficient vehicles for delivering genes into cells. They readily transduce primary and immortalized cells in vivo and in vitro. Genes delivered by lentiviruses are incorporated and replicated as part of their host genome and therefore offer a powerful tool for creation of stable cell lines and transgenic animals. However, the zona pellucida surrounding the fertilized eggs acts as a barrier and hinders lentiviral transduction of embryos. Here, we utilize a laser, typically used to perforate the zona pellucida for in vitro fertilization, to permeabilize the zona for lentiviral gene delivery. A single hole in the zona is sufficient for the lentivirus to gain access to fertilized eggs without the need for microinjection for en masse gene delivery. Embryos generated by this method elicit no damage and can develop to term for creation of transgenic animals.

A rapid cytoplasmic mechanism for PI3 kinase regulation by the nuclear thyroid hormone receptor, TRß, and genetic evidence for its role in the maturation of mouse hippocampal synapses in vivo.

Several rapid physiological effects of thyroid hormone on mammalian cells in vitro have been shown to be mediated by the phosphatidylinositol 3-kinase (PI3K), but the molecular mechanism of PI3K regulation by nuclear zinc finger receptor proteins for thyroid hormone and its relevance to brain development in vivo have not been elucidated. Here we show that, in the absence of hormone, the thyroid hormone receptor TRß forms a cytoplasmic complex with the p85 subunit of PI3K and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TRß that are not conserved in TRα. When hormone is added, TRß dissociates and moves to the nucleus, and phosphatidylinositol (3, 4, 5)-trisphosphate production goes up rapidly. Mutating either tyrosine to a phenylalanine prevents rapid signaling through PI3K but does not prevent the hormone-dependent transcription of genes with a thyroid hormone response element. When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of Thrb, circulating hormone levels, TRß expression, and direct gene regulation by TRß in the pituitary and liver were all unaffected. However, the mutation significantly impaired maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TRß with PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases.

Intersectin (ITSN) family of scaffolds function as molecular hubs in protein interaction networks.

Members of the intersectin (ITSN) family of scaffold proteins consist of multiple modular domains, each with distinct ligand preferences. Although ITSNs were initially implicated in the regulation of endocytosis, subsequent studies have revealed a more complex role for these scaffold proteins in regulation of additional biochemical pathways. In this study, we performed a high throughput yeast two-hybrid screen to identify additional pathways regulated by these scaffolds. Although several known ITSN binding partners were identified, we isolated more than 100 new targets for the two mammalian ITSN proteins, ITSN1 and ITSN2. We present the characterization of several of these new targets which implicate ITSNs in the regulation of the Rab and Arf GTPase pathways as well as regulation of the disrupted in schizophrenia 1 (DISC1) interactome. In addition, we demonstrate that ITSN proteins form homomeric and heteromeric complexes with each other revealing an added level of complexity in the function of these evolutionarily conserved scaffolds.

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway.

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.