Bio

Kimberley Tolias, PhD, is a Professor in the Department of Anesthesiology, Perioperative and Pain Medicine at Stanford University School of Medicine. The Tolias laboratory studies the molecular and cellular mechanisms that govern neural circuit formation, plasticity, and repair in the mammalian central nervous system (CNS). In particular, the lab investigates how neurons sense extracellular cues and convert them into intracellular signaling pathways that control synapse development and remodeling, dendritic and axonal growth, and cell migration. The laboratory also examines how disruption of these pathways contributes to neurological and neuropsychiatric disorders including intellectual disability, autism spectrum disorder, Alzheimer’s disease, depression, and chronic pain, and whether targeting specific signaling mechanisms can promote recovery following CNS injury or disease. To address these questions, the laboratory combines mouse genetics with state-of-the-art molecular, cellular, biochemical, electrophysiological, imaging, genomic, and behavioral approaches.

Research from the Tolias laboratory has provided fundamental insights into Rho GTPase signaling pathways that regulate synapse development and plasticity through control of cytoskeletal dynamics, as well as novel mechanisms that provide precise spatiotemporal regulation of Rho GTPase signaling in neurons and glia during CNS development. Her group has also identified important roles for the Adhesion-GPCR BAI1 in synapse and dendritic arbor development, defined functions for RhoA signaling in locomotor circuit assembly and cerebellar morphogenesis, and developed new approaches for preventing pathological synaptic remodeling caused by traumatic brain injury and radiation therapy. More recently, the laboratory has expanded into developing tools to identify synapses undergoing remodeling during learning or following injury, investigating mechanisms underlying chronic pain, opioid tolerance, and chronic pain-associated mood disorders, and exploring how the gut microbiome influences brain development, synaptic function, and behavior.

Dr. Tolias received her BS in Biochemistry from the University of Minnesota, graduating summa cum laude. She earned her PhD in Cell and Developmental Biology at Harvard Medical School, where she trained in the laboratory of Dr. Lewis Cantley studying signaling pathways that regulate the actin cytoskeleton and cell morphogenesis. She subsequently completed postdoctoral training in neurobiology in the laboratory of Dr. Michael Greenberg at Harvard Medical School, where she investigated molecular mechanisms linking neural activity to synaptic development and plasticity. Prior to joining Stanford, Dr. Tolias served for nearly 20 years on the faculty at Baylor College of Medicine, where she was a tenured Professor in the Departments of Neuroscience and Biochemistry and Molecular Pharmacology. Her research has been continuously supported by the National Institutes of Health and other major foundations and has been recognized by awards including the McKnight Memory and Cognitive Disorders Award and the NIH EUREKA Award.

Academic Appointments

  • Professor (Research), Anesthesiology, Perioperative and Pain Medicine
  • Member, Bio-X

Honors & Awards

  • McKnight Memory and Cognitive Disorders Award, McKnight Foundation (2016 - 2019)
  • NIH EUREKA Award, NIH (2013 - 2017)
  • Damon Runyon Cancer Research Fellowship, Damon Runyon (1999 - 2002)

Professional Education

  • Postdoctoral Fellowship, Harvard Medical School, Neurobiology, Lab of Michael E. Greenberg (2006)
  • PhD, Harvard Medical School, Cell and Developmental Biology, Lab of Lewis Cantley (1998)
  • BS, University of Minnesota, Biochemistry (1992)

Contact

  • Academic
    ktolias@stanford.edu
    University - Faculty Department: Anesthesia - Department Research Position: Professor-Research
    • 3176 Porter Dr # 220F
    • Stanford School Of Medicine
    • Palo Alto,  California  94304-1212 

Additional Info

Links

Current Research and Scholarly Interests

The human brain is composed of billions of neurons connected through trillions of synapses that form the neural circuits underlying thought, emotion, learning, memory, and behavior. These synaptic connections are not static - they are continuously shaped and remodeled throughout life in response to experience, a processes critical for learning and memory. When neural circuits fail to develop or function properly, the consequences can be devastating, contributing to conditions ranging from autism spectrum disorder and intellectual disability to chronic pain, depression, schizophrenia, Alzheimer's disease, and cognitive decline.

Research in the Tolias laboratory seeks to understand the fundamental molecular and cellular mechanisms that govern how neural circuits form, adapt, and repair themselves in the mammalian central nervous system (CNS). Our work focuses on how neurons sense signals from their environment and convert them into intracellular signaling pathways that drive synapse formation and remodeling, dendritic and axonal growth, and cell migration. We are also interested in how disruption of these pathways contributes to neurological and neuropsychiatric disease, and whether targeting specific signaling mechanisms can promote recovery following CNS injury or disease.

The laboratory combines mouse genetics with molecular, cellular, biochemical, electrophysiological, genomic, and behavioral approaches to bridge fundamental neuroscience with translational discovery.

Current Research Areas:

- Molecular mechanisms regulating synapse development, neural circuit formation, and synaptic plasticity during learning and memory

- Roles of Rho GTPase signaling pathways in brain development, circuit remodeling, and neurological disease

- Functions of Adhesion-GPCRs in synapse development, dendritic and axonal growth, and neural circuit function

- Mechanisms underlying chronic pain, opioid tolerance, and chronic pain-associated mood and cognitive disorders

- Synaptic and circuit dysfunction following traumatic brain injury, radiotherapy, and other CNS injuries

- Development of new tools to visualize and map synaptic remodeling during learning and disease

- Interactions between the gut microbiome, brain development, neural circuit function, and behavior

Graduate and Fellowship Programs

All Publications

  • Infant gut microbiomes contribute to metabolic states that impact brain function. bioRxiv : the preprint server for biology Midani, F. S., Lee, D. H., Moon, Y., Seale, M., Horvath, T. D., Ardis, A. K., Cantú, J., Coles, E., Pizzini, J. D., Zhu, D., Dooling, S. W., Ahern, G. J., Ardis, C. K., Beckford, A., Ruggiero, N. M., Shin, J., Joos, R., Stanton, C., Ross, R. P., Dai, D. L., Mandhane, P. J., Petersen, C., Turvey, S. E., Kiely, M. E., Murray, D. M., Costa-Mattioli, M., Tolias, K. F., Britton, R. A., Danhof, H. A. 2026

    Abstract

    Alterations in the gut microbiome are associated with neurodevelopmental disorders, but causal mechanisms and therapeutic strategies remain undefined. Here, we demonstrate that human infant microbiomes isolated during the first six months of life drive behavioral impairments in mice and that microbiota-based interventions restore mice to normal behavior. Early-life microbiomes from twelve infants who later exhibited cognitive deficits at 2 years old (low-scoring) transferred adverse metabolic, brain, and behavioral phenotypes to mice, in contrast to microbiomes from twenty-three cognitively typical or high-scoring infants. Deficits in mice were rescued by fecal microbiota transplant from high-scoring infants or a rationally designed consortium that promoted amino acid levels. We confirmed lower fecal amino acid concentrations in low-scoring infants and replicated the association between early-life microbiome composition and cognitive outcomes in a second geographically independent infant cohort. Altogether, we discovered an early-life microbiome-mediated metabolic state causally linked to cognitive deficits and amenable to microbial intervention.

    View details for DOI 10.64898/2026.03.09.710596

    View details for PubMedID 41959053

    View details for PubMedCentralID PMC13060970

  • Adhesion G protein-coupled receptors. Pharmacological reviews Langenhan, T., Anderson, G. R., Araç, D., Aust, G., Avila-Zozaya, M., Bagger, S. M., Barth, P., Berndt, S., Blacklow, S. C., Blanco-Redondo, B., Boucard, A. A., Bridges, J. P., Brodmerkel, L. S., Caron, K. M., Chung, Y. K., Dates, A. N., de Araujo Farias, V., Del Toro, D., Duman, J. G., Engel, F. B., Favara, D. M., Formstone, C. J., Fu, C., Garcia De Las Bayonas, A., Georgiadi, A., Gloriam, D. E., Hall, R. A., Hamann, J., Hildebrand, P. W., Hsiao, C. C., Huang, B. X., Javitch, J. A., Kim, H. Y., Kittel, R. J., Kleinau, G., Leduc, R., Liebscher, I., Lin, H. H., Linnert, J., Ludwig, M. G., Martinelli, D. C., Mathiasen, S., Matúš, D., Melkumyan, M., Moreno-Salinas, A. L., Mulder, J., Nash, M. A., Pal, K., Pederick, D. T., Perry-Hauser, N. A., Piao, X., Ping, Y. Q., Placantonakis, D. G., Pohl, F., Prömel, S., Rosenkilde, M. M., Sabbagh, L., Sando, R. C., Scheerer, P., Schöneberg, T., Seiradake, E., Selcho, M., Seufert, F., Singh, A. K., Skiniotis, G., Spiess, K., Sträter, N., Strutt, D., Südhof, T. C., Sun, J., Tall, G. G., Thor, D., Tilley, D. G., Tolias, K. F., Vallon, M., Van Meir, E. G., Vanhollebeke, B., Wiggin, G. R., Wolfrum, U., Yan, J., Zaidman, N. A., Zou, Y., Scholz, N. 2026; 78 (3): 100116

    Abstract

    Adhesion G protein-coupled receptors (aGPCRs) constitute a structurally and functionally distinct group within the superfamily of GPCRs. In 2015, the International Union of Pharmacology invited the Adhesion GPCR Consortium to publish a comprehensive review about aGPCRs and establish a unified nomenclature. Since then, substantial progress has been made in delineating the biological roles, molecular architecture, biochemical properties, expression profiles, ligand repertoire, and activation and signaling strategies of aGPCRs. Commensurate with these advances, their relevance to human pathophysiology has become increasingly apparent. In a coordinated effort, the Adhesion GPCR Consortium has reviewed recent progress in this field and provides a comprehensive assessment of the current understanding of aGPCR biology, including a focus on human and mammalian aGPCRs, their evolutionary origins, methodological approaches, and model systems for their investigation, as well as emerging approaches for their therapeutic targeting. SIGNIFICANCE STATEMENT: Adhesion G protein-coupled receptors are versatile cell-surface proteins that integrate structural, biochemical, and physiological functions, with major roles in health and disease. This review summarizes current knowledge of their molecular features, functions in diverse model systems, and emerging opportunities for therapeutic targeting, providing a comprehensive resource that connects basic biology with translational applications across multiple scientific disciplines.

    View details for DOI 10.1016/j.pharmr.2026.100116

    View details for PubMedID 41895071

  • Primary cortical neurons precipitate and extrude large mitochondria-associated calcium-phosphate sheets with a bone-precursor-like ultrastructure. Molecular brain Anderson, E. D., Cronkite, C. A., Baldwin, P. R., Abella, C. P., Duman, J. G., Simmonds, A. N., Waxham, M. N., Tolias, K. F., Ludtke, S. J. 2026; 19 (1): 11

    Abstract

    Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimer's disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 days in vitro. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntington's disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimer's disease.

    View details for DOI 10.1186/s13041-025-01272-0

    View details for PubMedID 41508094

    View details for PubMedCentralID PMC12882532

  • Restoration of sFRP3 Preserves the Neural Stem Cell Pool and Spatial Discrimination Ability in a Mouse Model of Alzheimer's Disease. The Journal of neuroscience : the official journal of the Society for Neuroscience Fu, C. H., Park, J., Tosi, U., Blanco, F. A., Silva-Pérez, M., Muralidharan, K., You, J. C., Lee, M., Stephens, G. S., Zhang, X., Zheng, Y., Scharfman, H., Tolias, K. F., Chin, J. 2025; 45 (49)

    Abstract

    Individuals with Alzheimer's disease (AD) have an increased incidence of seizures, which worsen cognitive decline. Using a transgenic mouse model of AD neuropathology that exhibits spontaneous seizures, we previously found that seizure activity stimulates and accelerates depletion of the hippocampal neural stem cell (NSC) pool, which was associated with deficits in neurogenesis-dependent spatial discrimination. However, the precise molecular mechanisms that drive seizure-induced activation and depletion of NSCs are unclear. Here, using mice of both sexes, we performed RNA-sequencing on the hippocampal dentate gyrus and identified differentially expressed regulators of neurogenesis in the Wnt signaling pathway that regulates many aspects of cell proliferation. We found that the expression of sFRP3, a Wnt signaling inhibitor, is altered in a seizure-dependent manner and might be regulated by ΔFosB, a seizure-induced transcription factor. Increasing sFRP3 expression prevented NSC depletion and improved spatial discrimination, suggesting that the loss of sFRP3 might mediate seizure-driven impairment in cognition in AD model mice and perhaps also in AD.

    View details for DOI 10.1523/JNEUROSCI.0049-25.2025

    View details for PubMedID 41136336

    View details for PubMedCentralID PMC12677132

  • Primary cortical neurons precipitate and extrude large mitochondria-associated calcium-phosphate sheets with a bone-precursor-like ultrastructure. bioRxiv : the preprint server for biology Anderson, E. D., Cronkite, C. A., Baldwin, P. R., Abella, C. P., Duman, J. G., Simmonds, A. N., Waxham, M. N., Tolias, K. F., Ludtke, S. J. 2025

    Abstract

    Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimer's disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 DIV. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntington's disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimer's disease.

    View details for DOI 10.1101/2025.08.04.668590

    View details for PubMedID 40799591

    View details for PubMedCentralID PMC12340797

  • Bright and photostable yellow fluorescent proteins for extended imaging. Nature communications Lee, J., Lai, S., Yang, S., Zhao, S., Blanco, F. A., Lyons, A. C., Merino-Urteaga, R., Ahrens, J. F., Nguyen, N. A., Liu, H., Liu, Z., Lambert, G. G., Shaner, N. C., Chen, L., Tolias, K. F., Zhang, J., Ha, T., St-Pierre, F. 2025; 16 (1): 3241

    Abstract

    Fluorescent proteins are indispensable molecular tools for visualizing biological structures and processes, but their limited photostability restricts the duration of dynamic imaging experiments. Yellow fluorescent proteins (YFPs), in particular, photobleach rapidly. Here, we introduce mGold2s and mGold2t, YFPs with up to 25-fold greater photostability than mVenus and mCitrine, two commonly used YFPs, while maintaining comparable brightness. These variants were identified using a high-throughput pooled single-cell platform, simultaneously screening for high brightness and photostability. Compared with our previous benchmark, mGold, the mGold2 variants display a ~4-fold increase in photostability without sacrificing brightness. mGold2s and mGold2t extend imaging durations across diverse modalities, including widefield, total internal reflection fluorescence (TIRF), super-resolution, single-molecule, and laser-scanning confocal microscopy. When incorporated into fluorescence resonance energy transfer (FRET)-based biosensors, the proposed YFPs enable more reliable, prolonged imaging of dynamic cellular processes. Overall, the enhanced photostability of mGold2s and mGold2t enables high-sensitivity imaging of subcellular structures and cellular activity over extended periods, broadening the scope and precision of biological imaging.

    View details for DOI 10.1038/s41467-025-58223-5

    View details for PubMedID 40185748

    View details for PubMedCentralID PMC11971446

  • The contribution of de novo coding mutations to meningomyelocele NATURE Ha, Y., Nisal, A., Tang, I., Lee, C., Jhamb, I., Wallace, C., Howarth, R., Schroeder, S., Vong, K., Meave, N., Jiwani, F., Barrows, C., Lee, S., Jiang, N., Patel, A., Bagga, K., Banka, N., Friedman, L., Blanco, F. A., Yu, S., Rhee, S., Jeong, H., Plutzer, I., Major, M. B., Benoit, B., Pous, C., Heffner, C., Kibar, Z., Bot, G., Northrup, H., Au, K., Strain, M., Ashley-Koch, A. E., Finnell, R. H., Le, J. T., Meltzer, H. S., Araujo, C., Machado, H. R., Stevenson, R. E., Yurrita, A., Mumtaz, S., Ahmed, A., Khara, M., Mutchinick, O. M., Medina-Bereciartu, J., Hildebrandt, F., Melikishvili, G., Marwan, A. I., Capra, V., Noureldeen, M. M., Salem, A. M. S., Issa, M. Y., Zaki, M. S., Xu, L., Lee, J., Shin, D., Alkelai, A., Shuldiner, A. R., Kingsmore, S. F., Murray, S. A., Gee, H., Miller, W., Tolias, K. F., Wallingford, J. B., Kim, S., Gleeson, J. G., Spina Bifida Sequencing Consortium, J. T., Koch, A., Lupo, P. J., Magana, T., Kolvenbach, C. M., Shril, S., Takahashi, Y., Salimi-Dafsari, H., Phillips, H., Hanak, B., Kara, B., Gunes, A., Gonda, D. D., Kirmani, S., Tkemaladze, T. 2025

    Abstract

    Meningomyelocele (also known as spina bifida) is considered to be a genetically complex disease resulting from a failure of the neural tube to close. Individuals with meningomyelocele display neuromotor disability and frequent hydrocephalus, requiring ventricular shunting. A few genes have been proposed to contribute to disease susceptibility, but beyond that it remains unexplained1. We postulated that de novo mutations under purifying selection contribute to the risk of developing meningomyelocele2. Here we recruited a cohort of 851 meningomyelocele trios who required shunting at birth and 732 control trios, and found that de novo likely gene disruption or damaging missense mutations occurred in approximately 22.3% of subjects, with 28% of such variants estimated to contribute to disease risk. The 187 genes with damaging de novo mutations collectively define networks including actin cytoskeleton and microtubule-based processes, Netrin-1 signalling and chromatin-modifying enzymes. Gene validation demonstrated partial or complete loss of function, impaired signalling and defective closure of the neural tube in Xenopus embryos. Our results indicate that de novo mutations make key contributions to meningomyelocele risk, and highlight critical pathways required for neural tube closure in human embryogenesis.

    View details for DOI 10.1038/s41586-025-08676-x

    View details for Web of Science ID 001453394800001

    View details for PubMedID 40140573

    View details for PubMedCentralID 7321880

  • Targeting Tiam1 Enhances Hippocampal-Dependent Learning and Memory in the Adult Brain and Promotes NMDA Receptor-Mediated Synaptic Plasticity and Function. The Journal of neuroscience : the official journal of the Society for Neuroscience Blanco, F. A., Saifullah, M. A., Cheng, J. X., Abella, C., Scala, F., Firozi, K., Niu, S., Park, J., Chin, J., Tolias, K. F. 2025; 45 (6)

    Abstract

    Excitatory synapses and the actin-rich dendritic spines on which they reside are indispensable for information processing and storage in the brain. In the adult hippocampus, excitatory synapses must balance plasticity and stability to support learning and memory. However, the mechanisms governing this balance remain poorly understood. Tiam1 is an actin cytoskeleton regulator prominently expressed in the dentate gyrus (DG) throughout life. Previously, we showed that Tiam1 promotes dentate granule cell synapse and spine stabilization during development, but its role in the adult hippocampus remains unclear. Here, we deleted Tiam1 from adult forebrain excitatory neurons (Tiam1fKO ) and assessed the effects on hippocampal-dependent behaviors. Adult male and female Tiam1fKO mice displayed enhanced contextual fear memory, fear extinction, and spatial discrimination. Investigation into underlying mechanisms revealed that dentate granule cells from Tiam1fKO brain slices exhibited augmented synaptic plasticity and N-methyl-D-aspartate-type glutamate receptor (NMDAR) function. Additionally, Tiam1 loss in primary hippocampal neurons blocked agonist-induced NMDAR internalization, reduced filamentous actin levels, and promoted activity-dependent spine remodeling. Notably, strong NMDAR activation in wild-type hippocampal neurons triggered Tiam1 loss from spines. Our results suggest that Tiam1 normally constrains hippocampal-dependent learning and memory in the adult brain by restricting NMDAR-mediated synaptic plasticity in the DG. We propose that Tiam1 achieves this by limiting NMDAR availability at synaptic membranes and stabilizing spine actin cytoskeleton and that these constraints can be alleviated by activity-dependent degradation of Tiam1. These findings reveal a previously unknown mechanism restricting hippocampal synaptic plasticity and highlight Tiam1 as a therapeutic target for enhancing cognitive function.

    View details for DOI 10.1523/JNEUROSCI.0298-24.2024

    View details for PubMedID 39725519

    View details for PubMedCentralID PMC11800756

  • Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia. Brain : a journal of neurology Yao, C., Fang, X., Ru, Q., Li, W., Li, J., Mehsein, Z., Tolias, K. F., Li, L. 2024; 147 (7): 2507-2521

    Abstract

    Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network. Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signalling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signalling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management. These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

    View details for DOI 10.1093/brain/awae106

    View details for PubMedID 38577773

    View details for PubMedCentralID PMC11224607

  • Correlative Cryo-FIB and Cryo-ET of Dendritic Spines and Synaptic Connections. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Anderson, E. D., Cronkite, C. A., Tolias, K. F., Ludtke, S. J. 2023; 29 (29 Suppl 1): 1086

    View details for DOI 10.1093/micmic/ozad067.559

    View details for PubMedID 39479569

    View details for PubMedCentralID PMC11094623

  • Tiam1 coordinates synaptic structural and functional plasticity underpinning the pathophysiology of neuropathic pain. Neuron Li, L., Ru, Q., Lu, Y., Fang, X., Chen, G., Saifullah, A. B., Yao, C., Tolias, K. F. 2023; 111 (13): 2038-2050.e6

    Abstract

    Neuropathic pain is a common, debilitating chronic pain condition caused by damage or a disease affecting the somatosensory nervous system. Understanding the pathophysiological mechanisms underlying neuropathic pain is critical for developing new therapeutic strategies to treat chronic pain effectively. Tiam1 is a Rac1 guanine nucleotide exchange factor (GEF) that promotes dendritic and synaptic growth during hippocampal development by inducing actin cytoskeletal remodeling. Here, using multiple neuropathic pain animal models, we show that Tiam1 coordinates synaptic structural and functional plasticity in the spinal dorsal horn via actin cytoskeleton reorganization and synaptic NMDAR stabilization and that these actions are essential for the initiation, transition, and maintenance of neuropathic pain. Furthermore, an antisense oligonucleotides (ASO) targeting spinal Tiam1 persistently alleviate neuropathic pain sensitivity. Our findings suggest that Tiam1-coordinated synaptic functional and structural plasticity underlies the pathophysiology of neuropathic pain and that intervention of Tiam1-mediated maladaptive synaptic plasticity has long-lasting consequences in neuropathic pain management.

    View details for DOI 10.1016/j.neuron.2023.04.010

    View details for PubMedID 37146610

    View details for PubMedCentralID PMC10330505

  • Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination. Cell discovery Sharma, J., Mulherkar, S., Chen, U. I., Xiong, Y., Bajaj, L., Cho, B. K., Goo, Y. A., Leung, H. E., Tolias, K. F., Sardiello, M. 2023; 9 (1): 32

    Abstract

    Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3-KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease.

    View details for DOI 10.1038/s41421-023-00533-3

    View details for PubMedID 36964131

    View details for PubMedCentralID PMC10038992

  • TIAM1-mediated synaptic plasticity underlies comorbid depression-like and ketamine antidepressant-like actions in chronic pain. The Journal of clinical investigation Ru, Q., Lu, Y., Saifullah, A. B., Blanco, F. A., Yao, C., Cata, J. P., Li, D. P., Tolias, K. F., Li, L. 2022; 132 (24)

    Abstract

    Chronic pain often leads to depression, increasing patient suffering and worsening prognosis. While hyperactivity of the anterior cingulate cortex (ACC) appears to be critically involved, the molecular mechanisms underlying comorbid depressive symptoms in chronic pain remain elusive. T cell lymphoma invasion and metastasis 1 (Tiam1) is a Rac1 guanine nucleotide exchange factor (GEF) that promotes dendrite, spine, and synapse development during brain development. Here, we show that Tiam1 orchestrates synaptic structural and functional plasticity in ACC neurons via actin cytoskeleton reorganization and synaptic N-methyl-d-aspartate receptor (NMDAR) stabilization. This Tiam1-coordinated synaptic plasticity underpins ACC hyperactivity and drives chronic pain-induced depressive-like behaviors. Notably, administration of low-dose ketamine, an NMDAR antagonist emerging as a promising treatment for chronic pain and depression, induces sustained antidepressant-like effects in mouse models of chronic pain by blocking Tiam1-mediated maladaptive synaptic plasticity in ACC neurons. Our results reveal Tiam1 as a critical factor in the pathophysiology of chronic pain-induced depressive-like behaviors and the sustained antidepressant-like effects of ketamine.

    View details for DOI 10.1172/JCI158545

    View details for PubMedID 36519542

    View details for PubMedCentralID PMC9753999

  • Rac-maninoff and Rho-vel: The symphony of Rho-GTPase signaling at excitatory synapses. Small GTPases Duman, J. G., Blanco, F. A., Cronkite, C. A., Ru, Q., Erikson, K. C., Mulherkar, S., Saifullah, A. B., Firozi, K., Tolias, K. F. 2022; 13 (1): 14-47

    Abstract

    Synaptic connections between neurons are essential for every facet of human cognition and are thus regulated with extreme precision. Rho-family GTPases, molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state, comprise a critical feature of synaptic regulation. Rho-GTPases are exquisitely controlled by an extensive suite of activators (GEFs) and inhibitors (GAPs and GDIs) and interact with many different signalling pathways to fulfill their roles in orchestrating the development, maintenance, and plasticity of excitatory synapses of the central nervous system. Among the mechanisms that control Rho-GTPase activity and signalling are cell surface receptors, GEF/GAP complexes that tightly regulate single Rho-GTPase dynamics, GEF/GAP and GEF/GEF functional complexes that coordinate multiple Rho-family GTPase activities, effector positive feedback loops, and mutual antagonism of opposing Rho-GTPase pathways. These complex regulatory mechanisms are employed by the cells of the nervous system in almost every step of development, and prominently figure into the processes of synaptic plasticity that underlie learning and memory. Finally, misregulation of Rho-GTPases plays critical roles in responses to neuronal injury, such as traumatic brain injury and neuropathic pain, and in neurodevelopmental and neurodegenerative disorders, including intellectual disability, autism spectrum disorder, schizophrenia, and Alzheimer's Disease. Thus, decoding the mechanisms of Rho-GTPase regulation and function at excitatory synapses has great potential for combatting many of the biggest current challenges in mental health.

    View details for DOI 10.1080/21541248.2021.1885264

    View details for PubMedID 33955328

    View details for PubMedCentralID PMC9707551

  • Ketamine: Neuroprotective or Neurotoxic? Frontiers in neuroscience Choudhury, D., Autry, A. E., Tolias, K. F., Krishnan, V. 2021; 15: 672526

    Abstract

    Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, has been employed clinically as an intravenous anesthetic since the 1970s. More recently, ketamine has received attention for its rapid antidepressant effects and is actively being explored as a treatment for a wide range of neuropsychiatric syndromes. In model systems, ketamine appears to display a combination of neurotoxic and neuroprotective properties that are context dependent. At anesthetic doses applied during neurodevelopmental windows, ketamine contributes to inflammation, autophagy, apoptosis, and enhances levels of reactive oxygen species. At the same time, subanesthetic dose ketamine is a powerful activator of multiple parallel neurotrophic signaling cascades with neuroprotective actions that are not always NMDAR-dependent. Here, we summarize results from an array of preclinical studies that highlight a complex landscape of intracellular signaling pathways modulated by ketamine and juxtapose the somewhat contrasting neuroprotective and neurotoxic features of this drug.

    View details for DOI 10.3389/fnins.2021.672526

    View details for PubMedID 34566558

    View details for PubMedCentralID PMC8461018

  • The Rac-GEF Tiam1 Promotes Dendrite and Synapse Stabilization of Dentate Granule Cells and Restricts Hippocampal-Dependent Memory Functions. The Journal of neuroscience : the official journal of the Society for Neuroscience Cheng, J., Scala, F., Blanco, F. A., Niu, S., Firozi, K., Keehan, L., Mulherkar, S., Froudarakis, E., Li, L., Duman, J. G., Jiang, X., Tolias, K. F. 2021; 41 (6): 1191-1206

    Abstract

    The dentate gyrus (DG) controls information flow into the hippocampus and is critical for learning, memory, pattern separation, and spatial coding, while DG dysfunction is associated with neuropsychiatric disorders. Despite its importance, the molecular mechanisms regulating DG neural circuit assembly and function remain unclear. Here, we identify the Rac-GEF Tiam1 as an important regulator of DG development and associated memory processes. In the hippocampus, Tiam1 is predominantly expressed in the DG throughout life. Global deletion of Tiam1 in male mice results in DG granule cells with simplified dendritic arbors, reduced dendritic spine density, and diminished excitatory synaptic transmission. Notably, DG granule cell dendrites and synapses develop normally in Tiam1 KO mice, resembling WT mice at postnatal day 21 (P21), but fail to stabilize, leading to dendrite and synapse loss by P42. These results indicate that Tiam1 promotes DG granule cell dendrite and synapse stabilization late in development. Tiam1 loss also increases the survival, but not the production, of adult-born DG granule cells, possibly because of greater circuit integration as a result of decreased competition with mature granule cells for synaptic inputs. Strikingly, both male and female mice lacking Tiam1 exhibit enhanced contextual fear memory and context discrimination. Together, these results suggest that Tiam1 is a key regulator of DG granule cell stabilization and function within hippocampal circuits. Moreover, based on the enhanced memory phenotype of Tiam1 KO mice, Tiam1 may be a potential target for the treatment of disorders involving memory impairments.SIGNIFICANCE STATEMENT The dentate gyrus (DG) is important for learning, memory, pattern separation, and spatial navigation, and its dysfunction is associated with neuropsychiatric disorders. However, the molecular mechanisms controlling DG formation and function remain elusive. By characterizing mice lacking the Rac-GEF Tiam1, we demonstrate that Tiam1 promotes the stabilization of DG granule cell dendritic arbors, spines, and synapses, whereas it restricts the survival of adult-born DG granule cells, which compete with mature granule cells for synaptic integration. Notably, mice lacking Tiam1 also exhibit enhanced contextual fear memory and context discrimination. These findings establish Tiam1 as an essential regulator of DG granule cell development, and identify it as a possible therapeutic target for memory enhancement.

    View details for DOI 10.1523/JNEUROSCI.3271-17.2020

    View details for PubMedID 33328293

    View details for PubMedCentralID PMC7888217

  • Cell type composition and circuit organization of clonally related excitatory neurons in the juvenile mouse neocortex. eLife Cadwell, C. R., Scala, F., Fahey, P. G., Kobak, D., Mulherkar, S., Sinz, F. H., Papadopoulos, S., Tan, Z. H., Johnsson, P., Hartmanis, L., Li, S., Cotton, R. J., Tolias, K. F., Sandberg, R., Berens, P., Jiang, X., Tolias, A. S. 2020; 9

    Abstract

    Clones of excitatory neurons derived from a common progenitor have been proposed to serve as elementary information processing modules in the neocortex. To characterize the cell types and circuit diagram of clonally related excitatory neurons, we performed multi-cell patch clamp recordings and Patch-seq on neurons derived from Nestin-positive progenitors labeled by tamoxifen induction at embryonic day 10.5. The resulting clones are derived from two radial glia on average, span cortical layers 2-6, and are composed of a random sampling of transcriptomic cell types. We find an interaction between shared lineage and connection type: related neurons are more likely to be connected vertically across cortical layers, but not laterally within the same layer. These findings challenge the view that related neurons show uniformly increased connectivity and suggest that integration of vertical intra-clonal input with lateral inter-clonal input may represent a developmentally programmed connectivity motif supporting the emergence of functional circuits.

    View details for DOI 10.7554/eLife.52951

    View details for PubMedID 32134385

    View details for PubMedCentralID PMC7162653

  • RhoA-ROCK Signaling as a Therapeutic Target in Traumatic Brain Injury. Cells Mulherkar, S., Tolias, K. F. 2020; 9 (1)

    Abstract

    Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBIs, which range in severity from mild to severe, occur when a traumatic event, such as a fall, a traffic accident, or a blow, causes the brain to move rapidly within the skull, resulting in damage. Long-term consequences of TBI can include motor and cognitive deficits and emotional disturbances that result in a reduced quality of life and work productivity. Recovery from TBI can be challenging due to a lack of effective treatment options for repairing TBI-induced neural damage and alleviating functional impairments. Central nervous system (CNS) injury and disease are known to induce the activation of the small GTPase RhoA and its downstream effector Rho kinase (ROCK). Activation of this signaling pathway promotes cell death and the retraction and loss of neural processes and synapses, which mediate information flow and storage in the brain. Thus, inhibiting RhoA-ROCK signaling has emerged as a promising approach for treating CNS disorders. In this review, we discuss targeting the RhoA-ROCK pathway as a therapeutic strategy for treating TBI and summarize the recent advances in the development of RhoA-ROCK inhibitors.

    View details for DOI 10.3390/cells9010245

    View details for PubMedID 31963704

    View details for PubMedCentralID PMC7016605

  • The adhesion-GPCR BAI1 shapes dendritic arbors via Bcr-mediated RhoA activation causing late growth arrest ELIFE Duman, J. G., Mulherkar, S., Tu, Y., Erikson, K. C., Tzeng, C. P., Mavratsas, V. C., Ho, T., Tolias, K. F. 2019; 8

    Abstract

    Dendritic arbor architecture profoundly impacts neuronal connectivity and function, and aberrant dendritic morphology characterizes neuropsychiatric disorders. Here, we identify the adhesion-GPCR BAI1 as an important regulator of dendritic arborization. BAI1 loss from mouse or rat hippocampal neurons causes dendritic hypertrophy, whereas BAI1 overexpression precipitates dendrite retraction. These defects specifically manifest as dendrites transition from growth to stability. BAI1-mediated growth arrest is independent of its Rac1-dependent synaptogenic function. Instead, BAI1 couples to the small GTPase RhoA, driving late RhoA activation in dendrites coincident with growth arrest. BAI1 loss lowers RhoA activation and uncouples it from dendrite dynamics, causing overgrowth. None of BAI1's known downstream effectors mediates BAI1-dependent growth arrest. Rather, BAI1 associates with the Rho-GTPase regulatory protein Bcr late in development and stimulates its cryptic RhoA-GEF activity, which functions together with its Rac1-GAP activity to terminate arborization. Our results reveal a late-acting signaling pathway mediating a key transition in dendrite development.

    View details for DOI 10.7554/eLife.47566

    View details for Web of Science ID 000483057500001

    View details for PubMedID 31461398

    View details for PubMedCentralID PMC6713510

  • An autism-linked missense mutation in SHANK3 reveals the modularity of Shank3 function. Molecular psychiatry Wang, L., Pang, K., Han, K., Adamski, C. J., Wang, W., He, L., Lai, J. K., Bondar, V. V., Duman, J. G., Richman, R., Tolias, K. F., Barth, P., Palzkill, T., Liu, Z., Holder, J. L., Zoghbi, H. Y. 2019

    Abstract

    Genome sequencing has revealed an increasing number of genetic variations that are associated with neuropsychiatric disorders. Frequently, studies limit their focus to likely gene-disrupting mutations because they are relatively easy to interpret. Missense variants, instead, have often been undervalued. However, some missense variants can be informative for developing a more profound understanding of disease pathogenesis and ultimately targeted therapies. Here we present an example of this by studying a missense variant in a well-known autism spectrum disorder (ASD) causing gene SHANK3. We analyzed Shank3's in vivo phosphorylation profile and identified S685 as one phosphorylation site where one ASD-linked variant has been reported. Detailed analysis of this variant revealed a novel function of Shank3 in recruiting Abelson interactor 1 (ABI1) and the WAVE complex to the post-synaptic density (PSD), which is critical for synapse and dendritic spine development. This function was found to be independent of Shank3's other functions such as binding to GKAP and Homer. Introduction of this human ASD mutation into mice resulted in a small subset of phenotypes seen previously in constitutive Shank3 knockout mice, including increased allogrooming, increased social dominance, and reduced pup USV. Together, these findings demonstrate the modularity of Shank3 function in vivo. This modularity further indicates that there is more than one independent pathogenic pathway downstream of Shank3 and correcting a single downstream pathway is unlikely to be sufficient for clear clinical improvement. In addition, this study illustrates the value of deep biological analysis of select missense mutations in elucidating the pathogenesis of neuropsychiatric phenotypes.

    View details for DOI 10.1038/s41380-018-0324-x

    View details for PubMedID 30610205

  • The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors. Annals of the New York Academy of Sciences Morgan, R. K., Anderson, G. R., Araç, D. n., Aust, G. n., Balenga, N. n., Boucard, A. n., Bridges, J. P., Engel, F. B., Formstone, C. J., Glitsch, M. D., Gray, R. S., Hall, R. A., Hsiao, C. C., Kim, H. Y., Knierim, A. B., Kusuluri, D. K., Leon, K. n., Liebscher, I. n., Piao, X. n., Prömel, S. n., Scholz, N. n., Srivastava, S. n., Thor, D. n., Tolias, K. F., Ushkaryov, Y. A., Vallon, M. n., Van Meir, E. G., Vanhollebeke, B. n., Wolfrum, U. n., Wright, K. M., Monk, K. R., Mogha, A. n. 2019

    Abstract

    The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

    View details for DOI 10.1111/nyas.14094

    View details for PubMedID 31168816

  • The Adhesion-GPCR BAI1 Promotes Excitatory Synaptogenesis by Coordinating Bidirectional Trans-synaptic Signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience Tu, Y. K., Duman, J. G., Tolias, K. F. 2018; 38 (39): 8388-8406

    Abstract

    Excitatory synapses are specialized cell-cell contacts located on actin-rich dendritic spines that mediate information flow and storage in the brain. The postsynaptic adhesion-G protein-coupled receptor (A-GPCR) BAI1 is a critical regulator of excitatory synaptogenesis, which functions in part by recruiting the Par3-Tiam1 polarity complex to spines, inducing local Rac1 GTPase activation and actin cytoskeletal remodeling. However, a detailed mechanistic understanding of how BAI1 controls synapse and spine development remains elusive. Here, we confirm that BAI1 is required in vivo for hippocampal spine development, and we identify three distinct signaling mechanisms mediating BAI1's prosynaptogenic functions. Using in utero electroporation to sparsely knock down BAI1 expression in hippocampal pyramidal neurons, we show that BAI1 cell-autonomously promotes spinogenesis in the developing mouse brain. BAI1 appears to function as a receptor at synapses, as its extracellular N-terminal segment is required for both its prospinogenic and prosynaptogenic functions. Moreover, BAI1 activation with a Stachel-derived peptide, which mimics a tethered agonist motif found in A-GPCRs, drives synaptic Rac1 activation and subsequent spine and synapse development. We also reveal, for the first time, a trans-synaptic function for BAI1, demonstrating in a mixed-culture assay that BAI1 induces the clustering of presynaptic vesicular glutamate transporter 1 (vGluT1) in contacting axons, indicative of presynaptic differentiation. Finally, we show that BAI1 forms a receptor complex with the synaptogenic cell-adhesion molecule Neuroligin-1 (NRLN1) and mediates NRLN1-dependent spine growth and synapse development. Together, these findings establish BAI1 as an essential postsynaptic A-GPCR that regulates excitatory synaptogenesis by coordinating bidirectional trans-synaptic signaling in cooperation with NRLN1.SIGNIFICANCE STATEMENT Adhesion-G protein-coupled receptors are cell-adhesion receptors with important roles in nervous system development, function, and neuropsychiatric disorders. The postsynaptic adhesion-G protein-coupled receptor BAI1 is a critical regulator of dendritic spine and excitatory synapse development. However, the mechanism by which BAI1 controls these functions remains unclear. Our study identifies three distinct signaling paradigms for BAI1, demonstrating that it mediates forward, reverse, and lateral signaling in spines. Activation of BAI1 by a Stachel-dependent mechanism induces local Rac1 activation and subsequent spinogenesis/synaptogenesis. BAI1 also signals trans-synaptically to promote presynaptic differentiation. Furthermore, BAI1 interacts with the postsynaptic cell-adhesion molecule Neuroligin-1 (NRLN1) and facilitates NRLN1-dependent spine growth and excitatory synaptogenesis. Thus, our findings establish BAI1 as a functional synaptogenic receptor that promotes presynaptic and postsynaptic development in cooperation with synaptic organizer NRLN1.

    View details for DOI 10.1523/JNEUROSCI.3461-17.2018

    View details for PubMedID 30120207

    View details for PubMedCentralID PMC6158688

  • Memantine prevents acute radiation-induced toxicities at hippocampal excitatory synapses. Neuro-oncology Duman, J. G., Dinh, J., Zhou, W., Cham, H., Mavratsas, V. C., Paveškovic, M., Mulherkar, S., McGovern, S. L., Tolias, K. F., Grosshans, D. R. 2018; 20 (5): 655-665

    Abstract

    Memantine has shown clinical utility in preventing radiation-induced cognitive impairment, but the mechanisms underlying its protective effects remain unknown. We hypothesized that abnormal glutamate signaling causes radiation-induced abnormalities in neuronal structure and that memantine prevents synaptic toxicity.Hippocampal cultures expressing enhanced green fluorescent protein were irradiated or sham-treated and their dendritic spine morphology assessed at acute (minutes) and later (days) times using high-resolution confocal microscopy. Excitatory synapses, defined by co-localization of the pre- and postsynaptic markers vesicular glutamate transporter 1 and postsynaptic density protein 95, were also analyzed. Neurons were pretreated with vehicle, the N-methyl-d-aspartate-type glutamate receptor antagonist memantine, or the glutamate scavenger glutamate pyruvate transaminase to assess glutamate signaling. For animal studies, Thy-1-YFP mice were treated with whole-brain radiotherapy or sham with or without memantine.Unlike previously reported long-term losses of dendritic spines, we found that the acute response to radiation is an initial increase in spines and excitatory synapses followed by a decrease in spine/synapse density with altered spine dynamics. Memantine pre-administration prevented this radiation-induced synaptic remodeling.These results demonstrate that radiation causes rapid, dynamic changes in synaptic structural plasticity, implicate abnormal glutamate signaling in cognitive dysfunction following brain irradiation, and describe a protective mechanism of memantine.

    View details for DOI 10.1093/neuonc/nox203

    View details for PubMedID 29112734

    View details for PubMedCentralID PMC5892158

  • RhoA-ROCK Inhibition Reverses Synaptic Remodeling and Motor and Cognitive Deficits Caused by Traumatic Brain Injury. Scientific reports Mulherkar, S., Firozi, K., Huang, W., Uddin, M. D., Grill, R. J., Costa-Mattioli, M., Robertson, C., Tolias, K. F. 2017; 7 (1): 10689

    Abstract

    Traumatic brain injury (TBI) causes extensive neural damage, often resulting in long-term cognitive impairments. Unfortunately, effective treatments for TBI remain elusive. The RhoA-ROCK signaling pathway is a potential therapeutic target since it is activated by TBI and can promote the retraction of dendritic spines/synapses, which are critical for information processing and memory storage. To test this hypothesis, RhoA-ROCK signaling was blocked by RhoA deletion from postnatal neurons or treatment with the ROCK inhibitor fasudil. We found that TBI impairs both motor and cognitive performance and inhibiting RhoA-ROCK signaling alleviates these deficits. Moreover, RhoA-ROCK inhibition prevents TBI-induced spine remodeling and mature spine loss. These data argue that TBI elicits pathological spine remodeling that contributes to behavioral deficits by altering synaptic connections, and RhoA-ROCK inhibition enhances functional recovery by blocking this detrimental effect. As fasudil has been safely used in humans, our results suggest that it could be repurposed to treat TBI.

    View details for DOI 10.1038/s41598-017-11113-3

    View details for PubMedID 28878396

    View details for PubMedCentralID PMC5587534

  • RhoGTPases Spread the Word for Synaptic Crosstalk. Developmental cell Duman, J. G., Tolias, K. F. 2016; 39 (2): 136-138

    Abstract

    Excitatory synaptic strengthening and the corresponding enlargement of dendritic spines are thought to be essential for learning and memory. In two recent Nature papers, Harward et al. (2016) and Hedrick et al. (2016) provide insight into the mechanisms that regulate these processes and illuminate the molecular basis of crosstalk between synapses.

    View details for DOI 10.1016/j.devcel.2016.10.007

    View details for PubMedID 27780038

  • Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nature biotechnology Cadwell, C. R., Palasantza, A., Jiang, X., Berens, P., Deng, Q., Yilmaz, M., Reimer, J., Shen, S., Bethge, M., Tolias, K. F., Sandberg, R., Tolias, A. S. 2016; 34 (2): 199-203

    Abstract

    Despite the importance of the mammalian neocortex for complex cognitive processes, we still lack a comprehensive description of its cellular components. To improve the classification of neuronal cell types and the functional characterization of single neurons, we present Patch-seq, a method that combines whole-cell electrophysiological patch-clamp recordings, single-cell RNA-sequencing and morphological characterization. Following electrophysiological characterization, cell contents are aspirated through the patch-clamp pipette and prepared for RNA-sequencing. Using this approach, we generate electrophysiological and molecular profiles of 58 neocortical cells and show that gene expression patterns can be used to infer the morphological and physiological properties such as axonal arborization and action potential amplitude of individual neurons. Our results shed light on the molecular underpinnings of neuronal diversity and suggest that Patch-seq can facilitate the classification of cell types in the nervous system.

    View details for DOI 10.1038/nbt.3445

    View details for PubMedID 26689543

    View details for PubMedCentralID PMC4840019

  • Emerging Roles of BAI Adhesion-GPCRs in Synapse Development and Plasticity. Neural plasticity Duman, J. G., Tu, Y. K., Tolias, K. F. 2016; 2016: 8301737

    Abstract

    Synapses mediate communication between neurons and enable the brain to change in response to experience, which is essential for learning and memory. The sites of most excitatory synapses in the brain, dendritic spines, undergo rapid remodeling that is important for neural circuit formation and synaptic plasticity. Abnormalities in synapse and spine formation and plasticity are associated with a broad range of brain disorders, including intellectual disabilities, autism spectrum disorders (ASD), and schizophrenia. Thus, elucidating the mechanisms that regulate these neuronal processes is critical for understanding brain function and disease. The brain-specific angiogenesis inhibitor (BAI) subfamily of adhesion G-protein-coupled receptors (adhesion-GPCRs) has recently emerged as central regulators of synapse development and plasticity. In this review, we will summarize the current knowledge regarding the roles of BAIs at synapses, highlighting their regulation, downstream signaling, and physiological functions, while noting the roles of other adhesion-GPCRs at synapses. We will also discuss the relevance of BAIs in various neurological and psychiatric disorders and consider their potential importance as pharmacological targets in the treatment of these diseases.

    View details for DOI 10.1155/2016/8301737

    View details for PubMedID 26881134

    View details for PubMedCentralID PMC4736325

  • Molecular Mechanisms of Dendritic Spine Development and Plasticity NEURAL PLASTICITY Lai, K., Jordan, B. A., Ma, X., Srivastava, D. P., Tolias, K. F. 2016; 2016: 2078121

    View details for DOI 10.1155/2016/2078121

    View details for Web of Science ID 000374422500001

    View details for PubMedID 27127656

    View details for PubMedCentralID PMC4834162

  • Mechanisms for spatiotemporal regulation of Rho-GTPase signaling at synapses. Neuroscience letters Duman, J. G., Mulherkar, S., Tu, Y. K., X Cheng, J., Tolias, K. F. 2015; 601: 4-10

    Abstract

    Synapses mediate information flow between neurons and undergo plastic changes in response to experience, which is critical for learning and memory. Conversely, synaptic defects impair information processing and underlie many brain pathologies. Rho-family GTPases control synaptogenesis by transducing signals from extracellular stimuli to the cytoskeleton and nucleus. The Rho-GTPases Rac1 and Cdc42 promote synapse development and the growth of axons and dendrites, while RhoA antagonizes these processes. Despite its importance, many aspects of Rho-GTPase signaling remain relatively unknown. Rho-GTPases are activated by guanine nucleotide exchange factors (GEFs) and inhibited by GTPase-activating proteins (GAPs). Though the number of both GEFs and GAPs greatly exceeds that of Rho-GTPases, loss of even a single GEF or GAP often has profound effects on cognition and behavior. Here, we explore how the actions of specific GEFs and GAPs give rise to the precise spatiotemporal activation patterns of Rho-GTPases in neurons. We consider the effects of coupling GEFs and GAPs targeting the same Rho-GTPase and the modular pathways that connect specific cellular stimuli with a given Rho-GTPase via different GEFs. We discuss how the creation of sharp borders between Rho-GTPase activation zones is achieved by pairing a GEF for one Rho-GTPase with a GAP for another and the extensive crosstalk between different Rho-GTPases. Given the importance of synapses for cognition and the fundamental roles that Rho-GTPases play in regulating them, a detailed understanding of Rho-GTPase signaling is essential to the progress of neuroscience.

    View details for DOI 10.1016/j.neulet.2015.05.034

    View details for PubMedID 26003445

    View details for PubMedCentralID PMC4513188

  • The small GTPases RhoA and Rac1 regulate cerebellar development by controlling cell morphogenesis, migration and foliation. Developmental biology Mulherkar, S., Uddin, M. D., Couvillon, A. D., Sillitoe, R. V., Tolias, K. F. 2014; 394 (1): 39-53

    Abstract

    The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology.

    View details for DOI 10.1016/j.ydbio.2014.08.004

    View details for PubMedID 25128586

    View details for PubMedCentralID PMC4163514

  • Dynamic Control of Excitatory Synapse Development by a Rac1 GEF/GAP Regulatory Complex DEVELOPMENTAL CELL Um, K., Niu, S., Duman, J. G., Cheng, J. X., Tu, Y., Schwechter, B., Liu, F., Hiles, L., Narayanan, A. S., Ash, R. T., Mulherkar, S., Alpadi, K., Smirnakis, S. M., Tolias, K. F. 2014; 29 (6): 701–15

    Abstract

    The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here, we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.

    View details for DOI 10.1016/j.devcel.2014.05.011

    View details for Web of Science ID 000338174600008

    View details for PubMedID 24960694

    View details for PubMedCentralID PMC4111230

  • The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity. Molecular biology of the cell Narayanan, A. S., Reyes, S. B., Um, K., McCarty, J. H., Tolias, K. F. 2013; 24 (24): 3857-68

    Abstract

    Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCζ) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear. We identify the Rac-specific GTPase-activating protein (GAP) breakpoint cluster region protein (Bcr) as a novel regulator of the Par-Tiam1 complex. We show that Bcr interacts with members of the Par complex and inhibits both Rac1 and PKCζ signaling. Loss of Bcr results in faster, more random migration and striking polarity defects in astrocytes. These polarity defects are rescued by reducing PKCζ activity or by expressing full-length Bcr, but not an N-terminal deletion mutant or the homologous Rac-GAP, Abr, both of which fail to associate with the Par complex. These results demonstrate that Bcr is an integral member of the Par-Tiam1 complex that controls polarized cell migration by locally restricting both Rac1 and PKCζ function.

    View details for DOI 10.1091/mbc.E13-06-0333

    View details for PubMedID 24152735

    View details for PubMedCentralID PMC3861082

  • Cytoskeletal mechanisms for synaptic potentiation. Communicative & integrative biology Schwechter, B., Tolias, K. F. 2013; 6 (6): e27343

    Abstract

    Excitatory synaptic transmission takes place at actin-rich protrusions called dendritic spines. Strong synaptic input activates NMDA-type glutamate receptors and induces calcium flux into these structures, initiating a program of cytoskeletal rearrangement that results in larger spines with stronger synapses. These changes in synaptic strength are thought to be the primary cellular mechanism underlying learning and memory. We recently reported that the dual Ras/Rac1 guanine nucleotide exchange factor (GEF) RasGRF2 links calcium flux to both spine enlargement and synaptic strengthening through its Rac-GEF activity. Additionally, we demonstrated that acute Rac1 activation is sufficient to enhance synaptic transmission. Since Rac1 is a major regulator of the actin cytoskeleton, these results suggest that the cytoskeleton itself regulates synaptic strengthening. Here we discuss models for how cytoskeletal modifications may enhance synaptic AMPA-type glutamate receptor abundance during long-term potentiation.

    View details for DOI 10.4161/cib.27343

    View details for PubMedID 24505509

    View details for PubMedCentralID PMC3914911

  • RasGRF2 Rac-GEF activity couples NMDA receptor calcium flux to enhanced synaptic transmission. Proceedings of the National Academy of Sciences of the United States of America Schwechter, B., Rosenmund, C., Tolias, K. F. 2013; 110 (35): 14462-7

    Abstract

    Dendritic spines are the primary sites of excitatory synaptic transmission in the vertebrate brain, and the morphology of these actin-rich structures correlates with synaptic function. Here we demonstrate a unique method for inducing spine enlargement and synaptic potentiation in dispersed hippocampal neurons, and use this technique to identify a coordinator of these processes; Ras-specific guanine nucleotide releasing factor 2 (RasGRF2). RasGRF2 is a dual Ras/Rac guanine nucleotide exchange factor (GEF) that is known to be necessary for long-term potentiation in situ. Contrary to the prevailing assumption, we find RasGRF2's Rac-GEF activity to be essential for synaptic potentiation by using a molecular replacement strategy designed to dissociate Rac- from Ras-GEF activities. Furthermore, we demonstrate that Rac1 activity itself is sufficient to rapidly modulate postsynaptic strength by using a photoactivatable derivative of this small GTPase. Because Rac1 is a major actin regulator, our results support a model where the initial phase of long-term potentiation is driven by the cytoskeleton.

    View details for DOI 10.1073/pnas.1304340110

    View details for PubMedID 23940355

    View details for PubMedCentralID PMC3761609

  • The small GTPase RhoA is required for proper locomotor circuit assembly. PloS one Mulherkar, S., Liu, F., Chen, Q., Narayanan, A., Couvillon, A. D., Shine, H. D., Tolias, K. F. 2013; 8 (6): e67015

    Abstract

    The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior.

    View details for DOI 10.1371/journal.pone.0067015

    View details for PubMedID 23825607

    View details for PubMedCentralID PMC3692541

  • The adhesion-GPCR BAI1 regulates synaptogenesis by controlling the recruitment of the Par3/Tiam1 polarity complex to synaptic sites. The Journal of neuroscience : the official journal of the Society for Neuroscience Duman, J. G., Tzeng, C. P., Tu, Y. K., Munjal, T., Schwechter, B., Ho, T. S., Tolias, K. F. 2013; 33 (16): 6964-78

    Abstract

    Excitatory synapses are polarized structures that primarily reside on dendritic spines in the brain. The small GTPase Rac1 regulates the development and plasticity of synapses and spines by modulating actin dynamics. By restricting the Rac1-guanine nucleotide exchange factor Tiam1 to spines, the polarity protein Par3 promotes synapse development by spatially controlling Rac1 activation. However, the mechanism for recruiting Par3 to spines is unknown. Here, we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a synaptic adhesion GPCR that is required for spinogenesis and synaptogenesis in mice and rats. We show that BAI1 interacts with Par3/Tiam1 and recruits these proteins to synaptic sites. BAI1 knockdown results in Par3/Tiam1 mislocalization and loss of activated Rac1 and filamentous actin from spines. Interestingly, BAI1 also mediates Rac-dependent engulfment in professional phagocytes through its interaction with a different Rac1-guanine nucleotide exchange factor module, ELMO/DOCK180. However, this interaction is dispensable for BAI1's role in synapse development because a BAI1 mutant that cannot interact with ELMO/DOCK180 rescues spine defects in BAI1-knockdown neurons, whereas a mutant that cannot interact with Par3/Tiam1 rescues neither spine defects nor Par3 localization. Further, overexpression of Tiam1 rescues BAI1 knockdown spine phenotypes. These results indicate that BAI1 plays an important role in synaptogenesis that is mechanistically distinct from its role in phagocytosis. Furthermore, our results provide the first example of a cell surface receptor that targets members of the PAR polarity complex to synapses.

    View details for DOI 10.1523/JNEUROSCI.3978-12.2013

    View details for PubMedID 23595754

    View details for PubMedCentralID PMC3670686

  • αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion MOLECULAR BIOLOGY OF THE CELL Reyes, S. B., Narayanan, A. S., Lee, H., Tchaicha, J. H., Aldape, K. D., Lang, F. F., Tolias, K. F., McCarty, J. H. 2013; 24 (4): 474-482

    Abstract

    The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin coimmunoprecipitates with Rho-GDP dissociation inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, regulates activation of Rho proteins to promote GBM cell invasiveness. Hence targeting the αvβ8 integrin-RhoGDI1 signaling axis might be an effective strategy for blocking GBM cell invasion.

    View details for DOI 10.1091/mbc.E12-07-0521

    View details for Web of Science ID 000321117800005

    View details for PubMedID 23283986

    View details for PubMedCentralID PMC3571870

  • Control of synapse development and plasticity by Rho GTPase regulatory proteins. Progress in neurobiology Tolias, K. F., Duman, J. G., Um, K. 2011; 94 (2): 133-48

    Abstract

    Synapses are specialized cell-cell contacts that mediate communication between neurons. Most excitatory synapses in the brain are housed on dendritic spines, small actin-rich protrusions extending from dendrites. During development and in response to environmental stimuli, spines undergo marked changes in shape and number thought to underlie processes like learning and memory. Improper spine development, in contrast, likely impedes information processing in the brain, since spine abnormalities are associated with numerous brain disorders. Elucidating the mechanisms that regulate the formation and plasticity of spines and their resident synapses is therefore crucial to our understanding of cognition and disease. Rho-family GTPases, key regulators of the actin cytoskeleton, play essential roles in orchestrating the development and remodeling of spines and synapses. Precise spatio-temporal regulation of Rho GTPase activity is critical for their function, since aberrant Rho GTPase signaling can cause spine and synapse defects as well as cognitive impairments. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and inhibited by GTPase-activating proteins (GAPs). We propose that Rho-family GEFs and GAPs provide the spatiotemporal regulation and signaling specificity necessary for proper Rho GTPase function based on the following features they possess: (i) existence of multiple GEFs and GAPs per Rho GTPase, (ii) developmentally regulated expression, (iii) discrete localization, (iv) ability to bind to and organize specific signaling networks, and (v) tightly regulated activity, perhaps involving GEF/GAP interactions. Recent studies describe several Rho-family GEFs and GAPs that uniquely contribute to spinogenesis and synaptogenesis. Here, we highlight several of these proteins and discuss how they occupy distinct biochemical niches critical for synaptic development.

    View details for DOI 10.1016/j.pneurobio.2011.04.011

    View details for PubMedID 21530608

    View details for PubMedCentralID PMC3129138

  • Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors. Neuron Zhou, P., Porcionatto, M., Pilapil, M., Chen, Y., Choi, Y., Tolias, K. F., Bikoff, J. B., Hong, E. J., Greenberg, M. E., Segal, R. A. 2007; 55 (1): 53-68

    Abstract

    During development, neural precursors migrate in response to positional cues such as growth factor gradients. However, the mechanisms that enable precursors to sense and respond to such gradients are poorly understood. Here we show that cerebellar granule cell precursors (GCPs) migrate along a gradient of brain-derived neurotrophic factor (BDNF), and we demonstrate that vesicle trafficking is critical for this chemotactic process. Activation of TrkB, the BDNF receptor, stimulates GCPs to secrete BDNF, thereby amplifying the ambient gradient. The BDNF gradient stimulates endocytosis of TrkB and associated signaling molecules, causing asymmetric accumulation of signaling endosomes at the subcellular location where BDNF concentration is maximal. Thus, regulated BDNF exocytosis and TrkB endocytosis enable precursors to polarize and migrate in a directed fashion along a shallow BDNF gradient.

    View details for DOI 10.1016/j.neuron.2007.05.030

    View details for PubMedID 17610817

    View details for PubMedCentralID PMC2661852

  • The Rac1 guanine nucleotide exchange factor Tiam1 mediates EphB receptor-dependent dendritic spine development. Proceedings of the National Academy of Sciences of the United States of America Tolias, K. F., Bikoff, J. B., Kane, C. G., Tolias, C. S., Hu, L., Greenberg, M. E. 2007; 104 (17): 7265-70

    Abstract

    Dendritic spines are small, actin-rich protrusions on the surface of dendrites that receive the majority of excitatory synaptic inputs in the brain. The formation and remodeling of spines, processes that underlie synaptic development and plasticity, are regulated in part by Eph receptor tyrosine kinases. However, the mechanism by which Ephs regulate actin cytoskeletal remodeling necessary for spine development is not fully understood. Here, we report that the Rac1 guanine nucleotide exchange factor Tiam1 interacts with the EphB2 receptor in a kinase-dependent manner. Activation of EphBs by their ephrinB ligands induces the tyrosine phosphorylation and recruitment of Tiam1 to EphB complexes containing NMDA-type glutamate receptors. Either knockdown of Tiam1 protein by RNAi or inhibition of Tiam1 function with a dominant-negative Tiam1 mutant blocks dendritic spine formation induced by ephrinB1 stimulation. Taken together, these findings suggest that EphBs regulate spine development in part by recruiting, phosphorylating, and activating Tiam1. Tiam1 can then promote Rac1-dependent actin cytoskeletal remodeling required for dendritic spine morphogenesis.

    View details for DOI 10.1073/pnas.0702044104

    View details for PubMedID 17440041

    View details for PubMedCentralID PMC1855368

  • The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines. Neuron Tolias, K. F., Bikoff, J. B., Burette, A., Paradis, S., Harrar, D., Tavazoie, S., Weinberg, R. J., Greenberg, M. E. 2005; 45 (4): 525-38

    Abstract

    NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. We report that the Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with RNAi and dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines.

    View details for DOI 10.1016/j.neuron.2005.01.024

    View details for PubMedID 15721239

  • BTK regulates PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity. Immunity Saito, K., Tolias, K. F., Saci, A., Koon, H. B., Humphries, L. A., Scharenberg, A., Rawlings, D. J., Kinet, J. P., Carpenter, C. L. 2003; 19 (5): 669-78

    Abstract

    Intracellular signaling by most cell surface receptors requires the generation of two major second messengers, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) and inositol-1,4,5-trisphosphate (IP3). The enzymes that produce these second messengers, phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC), utilize a common substrate, phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2). Until now, it has not been clear whether de novo PtdIns-4,5-P2 synthesis is necessary for PtdIns-3,4,5-P3 and IP3 production. Here we show that BTK, a member of the Tec family of cytoplasmic protein tyrosine kinases, associates with phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), the enzymes that synthesize PtdIns-4,5-P2. Upon B cell receptor activation, BTK brings PIP5K to the plasma membrane as a means of generating local PtdIns-4,5-P2 synthesis. This enzyme-enzyme interaction provides a shuttling mechanism that allows BTK to stimulate the production of the substrate required by both its upstream activator, PI3K, and its downstream target, PLC-gamma2.

    View details for DOI 10.1016/s1074-7613(03)00297-8

    View details for PubMedID 14614854

  • BTK regulates Ptdlns-4,5-P<sub>2</sub> synthesis:: Importance for calcium signaling and PI3K activity IMMUNITY Saito, K., Tolias, K. F., Saci, A., Koon, H. B., Humphries, L. A., Scharenberg, A., Rawlings, D. J., Kinet, J. P., Carpenter, C. L. 2003; 19 (5): 669-678

    Abstract

    Intracellular signaling by most cell surface receptors requires the generation of two major second messengers, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) and inositol-1,4,5-trisphosphate (IP3). The enzymes that produce these second messengers, phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC), utilize a common substrate, phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2). Until now, it has not been clear whether de novo PtdIns-4,5-P2 synthesis is necessary for PtdIns-3,4,5-P3 and IP3 production. Here we show that BTK, a member of the Tec family of cytoplasmic protein tyrosine kinases, associates with phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), the enzymes that synthesize PtdIns-4,5-P2. Upon B cell receptor activation, BTK brings PIP5K to the plasma membrane as a means of generating local PtdIns-4,5-P2 synthesis. This enzyme-enzyme interaction provides a shuttling mechanism that allows BTK to stimulate the production of the substrate required by both its upstream activator, PI3K, and its downstream target, PLC-gamma2.

    View details for DOI 10.1016/S1074-7613(03)00297-8

    View details for Web of Science ID 000186672200007

    View details for PubMedID 14614854

  • Type Iα phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly CURRENT BIOLOGY Tolias, K. F., Hartwig, J. H., Ishihara, H., Shibasaki, Y., Cantley, L. C., Carpenter, C. L. 2000; 10 (3): 153-156

    Abstract

    Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.

    View details for DOI 10.1016/S0960-9822(00)00315-8

    View details for Web of Science ID 000085354100019

    View details for PubMedID 10679324

  • <i>In vitro</i> Interaction of phosphoinositide-4-phosphate 5-kinases with Rac REGULATORS AND EFFECTORS OF SMALL GTPASES, PT D Tolias, K., Carpenter, C. L. edited by Balch, W. E., Der, C. J., Hall, A. 2000; 325: 190-200

    View details for Web of Science ID 000165500200018

    View details for PubMedID 11036604

  • Rac homologues and compartmentalized phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate polar pollen tube growth JOURNAL OF CELL BIOLOGY Kost, B., Lemichez, E., Spielhofer, P., Hong, Y., Tolias, K., Carpenter, C., Chua, N. H. 1999; 145 (2): 317-330

    Abstract

    Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

    View details for DOI 10.1083/jcb.145.2.317

    View details for Web of Science ID 000079858700010

    View details for PubMedID 10209027

    View details for PubMedCentralID PMC2133117

  • Pathways for phosphoinositide synthesis Tolias, K. F., Cantley, L. C. ELSEVIER IRELAND LTD. 1999: 69-77

    Abstract

    In eukaryotic cells, phosphatidylinositol can be phosphorylated on the inositol ring by a series of kinases to produce at least seven distinct phosphoinositides. These lipids have been implicated in a variety of cellular processes, including calcium regulation, actin rearrangement, vesicle trafficking, cell survival and mitogenesis. The phosphorylated lipids can act as precursors of second messengers or act directly to recruit specific signaling proteins to the membrane. A number of the kinases responsible for producing these lipids have been purified and their cDNA clones have been isolated. The most well characterized of these enzymes are the phosphoinositide 3-kinases. However, progress has also been made in the characterization of phosphatidylinositol 4-kinases and phosphatidylinositol-4-phosphate 5-kinases. In addition, new pathways involving phosphatidylinositol-5-phosphate 4-kinases, phosphatidylinositol-3-phosphate 5-kinases and phosphatidylinositol-3-phosphate 4-kinases have recently been described. The various enzymes and pathways involved in the synthesis of cellular phosphoinositides will be discussed.

    View details for DOI 10.1016/S0009-3084(99)00019-5

    View details for Web of Science ID 000081515100008

    View details for PubMedID 10358929

  • Lipid kinases are novel effectors of the GTPase Rac1 Carpenter, C. L., Tolias, K. F., Van Vugt, A., Hartwig, J. edited by Weber, G. PERGAMON PRESS LTD. 1999: 299-312

    Abstract

    We have found that a complex consisting of a type I PIPK and a DGK associates with the GTPase Rac1. Binding of the lipid kinase complex is through the C-terminus of Rac. Complex formation is augmented in the presence of specific phospholipids. The complex also associates with Rho GDI, through Rac. Based on the role of PtdIns-4,5-P2 in regulating proteins that influence actin structures we propose that the Rac-associated lipid kinase complex functions to generate locally high concentrations of PtdIns-4,5-P2 in a Rac-dependent manner. There are many possible roles PtdIns-4,5-P2 might play. A likely role is binding to barbed-end actin capping proteins. This would release the capping protein, providing free barbed ends for actin polymerization. Uncapping would occur at the membrane so that additional actin polymerization would result in membrane protrusions and lamellapodia, in a Brownian ratchet model. It is also possible that PtdIns-4,5-P2 has other roles, such as promoting the release of G actin from profilin or promoting the cross-linking of actin or its anchorage to the plasma membrane. Studies are currently underway to determine the role of this lipid kinase complex in Rac signaling and actin regulation in vivo.

    View details for DOI 10.1016/S0065-2571(98)00009-0

    View details for Web of Science ID 000082191200018

    View details for PubMedID 10470380

  • Type I phosphatidylinositol-4-phosphate 5-kinases synthesize the novel lipids phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 5-phosphate JOURNAL OF BIOLOGICAL CHEMISTRY Tolias, K. F., Rameh, L. E., Ishihara, H., Shibasaki, Y., Chen, J., Prestwich, G. D., Cantley, L. C., Carpenter, C. L. 1998; 273 (29): 18040-18046

    Abstract

    Inositol phospholipids regulate a variety of cellular processes including proliferation, survival, vesicular trafficking, and cytoskeletal organization. Recently, two novel phosphoinositides, phosphatidylinositol-3,5-bisphosphate (PtdIns-3,5-P2) and phosphatidylinositol- 5-phosphate (PtdIns-5-P), have been shown to exist in cells. PtdIns-3,5-P2, which is regulated by osmotic stress, appears to be synthesized by phosphorylation of PtdIns-3-P at the D-5 position. No evidence yet exists for how PtdIns-5-P is produced in cells. Understanding the regulation of synthesis of these molecules will be important for identifying their function in cellular signaling. To determine the pathway by which PtdIns-3,5-P2 and Ptd-Ins-5-P might be synthesized, we tested the ability of the recently cloned type I PtdIns-4-P 5-kinases (PIP5Ks) alpha and beta to phosphorylate PtdIns-3-P and PtdIns at the D-5 position of the inositol ring. We found that the type I PIP5Ks phosphorylate PtdIns-3-P to form PtdIns-3,5-P2. The identity of the PtdIns-3,5-P2 product was determined by anion exchange high performance liquid chromatography analysis and periodate treatment. PtdIns-3,4-P2 and PtdIns-3,4,5-P3 were also produced from PtdIns-3-P phosphorylation by both isoforms. When expressed in mammalian cells, PIP5K Ialpha and PIP5K Ibeta differed in their ability to synthesize PtdIns-3,5-P2 relative to PtdIns-3,4-P2. We also found that the type I PIP5Ks phosphorylate PtdIns to produce PtdIns-5-P and phosphorylate PtdIns-3,4-P2 to produce PtdIns-3,4,5-P3. Our findings suggest that type I PIP5Ks synthesize the novel phospholipids PtdIns-3,5-P2 and PtdIns-5-P. The ability of PIP5Ks to produce multiple signaling molecules indicates that they may participate in a variety of cellular processes.

    View details for DOI 10.1074/jbc.273.29.18040

    View details for Web of Science ID 000074828500014

    View details for PubMedID 9660759

  • Characterization of a Rac1- and RhoGDI-associated lipid kinase signaling complex MOLECULAR AND CELLULAR BIOLOGY Tolias, K. F., Couvillon, A. D., Cantley, L. C., Carpenter, C. L. 1998; 18 (2): 762-770

    Abstract

    Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.

    View details for DOI 10.1128/MCB.18.2.762

    View details for Web of Science ID 000071716000011

    View details for PubMedID 9447972

    View details for PubMedCentralID PMC108787

  • A new pathway for synthesis of phosphatidylinositol-4,5-bisphosphate NATURE Rameh, L. E., Tolias, K. F., Duckworth, B. C., Cantley, L. C. 1997; 390 (6656): 192-196

    Abstract

    Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2), a key molecule in the phosphoinositide signalling pathway, was thought to be synthesized exclusively by phosphorylation of PtdIns-4-P at the D-5 position of the inositol ring. The enzymes that produce PtdIns-4,5-P2 in vitro fall into two related subfamilies (type I and type II PtdInsP-5-OH kinases, or PIP(5)Ks) based on their enzymatic properties and sequence similarities'. Here we have reinvestigated the substrate specificities of these enzymes. As expected, the type I enzyme phosphorylates PtdIns-4-P at the D-5 position of the inositol ring. Surprisingly, the type II enzyme, which is abundant in some tissues, phosphorylates PtdIns-5-P at the D-4 position, and thus should be considered as a 4-OH kinase, or PIP(4)K. The earlier error in characterizing the activity of the type II enzyme is due to the presence of contaminating PtdIns-5-P in commercial preparations of PtdIns-4-P. Although PtdIns-5-P was previously thought not to exist in vivo, we find evidence for the presence of this lipid in mammalian fibroblasts, establishing a new pathway for PtdIns-4,5-P2 synthesis.

    View details for DOI 10.1038/36621

    View details for Web of Science ID A1997YF49400059

    View details for PubMedID 9367159

  • A novel link between integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase JOURNAL OF BIOLOGICAL CHEMISTRY Berditchevski, F., Tolias, K. F., Wong, K., Carpenter, C. L., Hemler, M. E. 1997; 272 (5): 2595-2598

    Abstract

    Enzymatic and immunochemical assays show a phosphatidylinositol 4-kinase in novel and specific complexes with proteins (CD63 and CD81) of the transmembrane 4 superfamily (TM4SF) and an integrin (alpha3beta1). The size (55 kDa) and other properties of the phosphatidylinositol 4-kinase (PI 4-K) (stimulated by nonionic detergent, inhibited by adenosine, inhibited by monoclonal antibody 4CG5) are consistent with PI 4-K type II. Not only was PI 4-K associated with alpha3beta1-CD63 complexes in alpha3-transfected K562 cells, but also it could be co-purified from CD63 in untransfected K562 cells lacking alpha3beta1. Thus, TM4SF proteins may link PI 4-K activity to the alpha3beta1 integrin. The alpha5beta1 integrin, which does not associate with TM4SF proteins, was not associated with PI 4-K. Notably, alpha3beta1-CD63-CD81-PI 4-K complexes are located in focal complexes at the cell periphery rather than in focal adhesions. The novel linkage between integrins, transmembrane 4 proteins, and phosphoinositide signaling at the cell periphery may play a key role in cell motility and provides a signaling pathway distinct from conventional integrin signaling through focal adhesion kinase.

    View details for DOI 10.1074/jbc.272.5.2595

    View details for Web of Science ID A1997WE66700004

    View details for PubMedID 9006891

  • Signal transduction pathways involving the small G proteins rac and CDC42 and phosphoinositide kinases ADVANCES IN ENZYME REGULATION, VOL 37 Carpenter, C. L., Tolias, K. F., Couvillon, A. C., Hartwig, J. H. edited by Weber, G. 1997; 37: 377-390

    Abstract

    We found that rac specifically binds to a type I PtdIns-4-P 5-kinase and that both rac and Cdc42 in the activated forms associate with PI 3-kinase. The association of PI 3-kinase with rac was stimulated by PDGF in vivo. Rac is constitutively associated with a PtdIns-4-P 5-kinase and stimulates PtdIns-4,5-P2 production in permeabilized platelets. These data suggest a model in which the initial step in the activation of rac is release from rho GDI (Fig. 7). Rac in the GDP bound form can associate with the PtdIns-4-P 5-kinase and also interact with an exchange factor. GTP bound rac may then localize to sites of actin reorganization, bringing the PtdIns-4-P 5-kinase with it. Locally synthesized PtdIns-4,5-P2 binds to actin capping proteins, leading to their release and the production of actin free ends. Actin polymerization can then occur from the free ends. Many other factors must be involved to regulate the type and extent of actin polymerization that is necessary in such complex processes as cell movement and membrane ruffling. The rac-associated PtdIns-4-P 5-kinase and its product PtdIns-4,5-P2 may act at a crucial regulatory point that permits polymerization to begin.

    View details for DOI 10.1016/S0065-2571(96)00005-2

    View details for Web of Science ID A1997BJ54D00019

    View details for PubMedID 9381982

  • Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kolluri, R., Tolias, K. F., Carpenter, C. L., Rosen, F. S., Kirchhausen, T. 1996; 93 (11): 5615-5618

    Abstract

    Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets. The disease arises from mutations in the gene encoding the WAS protein. T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization. We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42. This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins. A weaker interaction was also detected with Rac1 using WAS protein from cell lysates. It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal. Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.

    View details for DOI 10.1073/pnas.93.11.5615

    View details for Web of Science ID A1996UN25300080

    View details for PubMedID 8643625

    View details for PubMedCentralID PMC39296

  • RHO-FAMILY GTPASES BIND TO PHOSPHOINOSITIDE KINASES JOURNAL OF BIOLOGICAL CHEMISTRY TOLIAS, K. F., CANTLEY, L. C., CARPENTER, C. L. 1995; 270 (30): 17656-17659

    Abstract

    Rho family GTPases appear to play an important role in the regulation of the actin cytoskeleton, but the mechanism of regulation is unknown. Since phosphoinositide 3-kinase and phosphatidylinositol 4,5-bisphosphate have also been implicated in actin reorganization, we investigated the possibility that Rho family members interact with phosphoinositide kinases. We found that both GTP- and GDP-bound Rac1 associate with phosphatidylinositol-4-phosphate 5-kinase in vitro and in vivo. Phosphoinositide 3-kinase also bound to Rac1 and Cdc42Hs, and these interactions were GTP-dependent. Stimulation of Swiss 3T3 cells with platelet-derived growth factor induced the association of PI 3-kinase with Rac in immunoprecipitates. PI 3-kinase activity was also detected in Cdc42 immunoprecipitates from COS7 cells. These results suggest that phosphoinositide kinases are involved in Rho family signal transduction pathways and raise the possibility that the effects of Rho family members on the actin cytoskeleton are mediated in part by phosphoinositide kinases.

    View details for DOI 10.1074/jbc.270.30.17656

    View details for Web of Science ID A1995RM26600005

    View details for PubMedID 7629060

https://orcid.org/0000-0002-2092-920X