Dendritic Projections Research Articles

Structure and development of the subesophageal zone of the Drosophila brain. II. Sensory compartments.

The subesophageal zone (SEZ) of the Drosophila brain processes mechanosensory and gustatory sensory input from sensilla located on the head, mouth cavity and trunk. Motor output from the SEZ directly controls the movements involved in feeding behavior. In an accompanying paper (Hartenstein et al., ), we analyzed the systems of fiber tracts and secondary lineages to establish reliable criteria for defining boundaries between the four neuromeres of the SEZ, as well as discrete longitudinal neuropil domains within each SEZ neuromere. Here we use this anatomical framework to systematically map the sensory projections entering the SEZ throughout development. Our findings show continuity between larval and adult sensory neuropils. Gustatory axons from internal and external taste sensilla of the larva and adult form two closely related sensory projections, (a) the anterior central sensory center located deep in the ventromedial neuropil of the tritocerebrum and mandibular neuromere, and (b) the anterior ventral sensory center (AVSC), occupying a superficial layer within the ventromedial tritocerebrum. Additional, presumed mechanosensory terminal axons entering via the labial nerve define the ventromedial sensory center (VMSC) in the maxilla and labium. Mechanosensory afferents of the massive array of chordotonal organs (Johnston's organ) of the adult antenna project into the centrolateral neuropil column of the anterior SEZ, creating the antenno-mechanosensory and motor center (AMMC). Dendritic projections of dye back-filled motor neurons extend throughout a ventral layer of the SEZ, overlapping widely with the AVSC and VMSC. Our findings elucidate fundamental structural aspects of the developing sensory systems in Drosophila.

A crossroad of neuronal diversity to build circuitry.

Clustered protocadherin genes control convergence and divergence of neurons

Physical Exercise Restores the Generation of Newborn Neurons in an Animal Model of Chronic Epilepsy.

Neurogenesis impairment is associated with the chronic phase of the epilepsy in humans and also observed in animal models. Recent studies with animal models have shown that physical exercise is capable of improving neurogenesis in adult subjects, alleviating cognitive impairment and depression. Here, we show that there is a reduction in the generation of newborn granule cells in the dentate gyrus of adult rats subjected to a chronic model of epilepsy during the postnatal period of brain development. We also show that the physical exercise was capable to restore the number of newborn granule cells in this animals to the level observed in the control group. Notably, a larger number of newborn granule cells exhibiting morphological characteristics indicative of correct targeting into the hippocampal circuitry and the absence of basal dendrite projections was also observed in the epileptic animals subjected to physical exercise compared to the epileptic animals. The results described here could represent a positive interference of the physical exercise on the neurogenesis process in subjects with chronic epilepsy. The results may also help to reinterpret the benefits of the physical exercise in alleviating symptoms of depression and cognitive dysfunction.

Minocycline attenuates colistin-induced neurotoxicity via suppression of apoptosis, mitochondrial dysfunction and oxidative stress.

Neurotoxicity is an adverse effect patients experience during colistin therapy. The development of effective neuroprotective agents that can be co-administered during polymyxin therapy remains a priority area in antimicrobial chemotherapy. The present study investigates the neuroprotective effect of the synergistic tetracycline antibiotic minocycline against colistin-induced neurotoxicity. The impact of minocycline pretreatment on colistin-induced apoptosis, caspase activation, oxidative stress and mitochondrial dysfunction were investigated using cultured mouse neuroblastoma-2a (N2a) and primary cortical neuronal cells. Colistin-induced neurotoxicity in mouse N2a and primary cortical cells gives rise to the generation of reactive oxygen species (ROS) and subsequent cell death via apoptosis. Pretreatment of the neuronal cells with minocycline at 5, 10 and 20 μM for 2 h prior to colistin (200 μM) exposure (24 h), had an neuroprotective effect by significantly decreasing intracellular ROS production and by upregulating the activities of the anti-ROS enzymes superoxide dismutase and catalase. Minocycline pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation and subsequent apoptosis. Immunohistochemical imaging studies revealed colistin accumulates within the dendrite projections and cell body of primary cortical neuronal cells. To our knowledge, this is first study demonstrating the protective effect of minocycline on colistin-induced neurotoxicity by scavenging of ROS and suppression of apoptosis. Our study highlights that co-administration of minocycline kills two birds with one stone: in addition to its synergistic antimicrobial activity, minocycline could potentially ameliorate unwanted neurotoxicity in patients undergoing polymyxin therapy.

How Azobenzene Photoswitches Restore Visual Responses to the Blind Retina

Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates asvision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent from those with intact rods andcones. Here we show that P2X receptors mediate the entry of photoswitches into RGCs, where theyassociate with voltage-gated ion channels, enablinglight to control action-potential firing. All chargedphotoswitch compounds require permeation through P2X receptors, whose gene expression is upregulated in the blind retina. Photoswitches andmembrane-impermeant fluorescent dyes likewise penetrate through P2X receptors to label a subset of RGCs in the degenerated retina. Electrophysiological recordings and mapping of fluorescently labeledRGC dendritic projections together indicate that photosensitization is highly selective for OFF-RGCs. Hence, P2X receptors are a natural conduit allowing cell-type-selective and degeneration-specific delivery of photoswitches to restore visual function in blinding disease.

The X-Linked Autism Protein KIAA2022/KIDLIA Regulates Neurite Outgrowth via N-Cadherin and δ-Catenin Signaling.

Our previous work showed that loss of the KIAA2022 gene protein results in intellectual disability with language impairment and autistic behavior (KIDLIA, also referred to as XPN). However, the cellular and molecular alterations resulting from a loss of function of KIDLIA and its role in autism with severe intellectual disability remain unknown. Here, we show that KIDLIA plays a key role in neuron migration and morphogenesis. We found that KIDLIA is distributed exclusively in the nucleus. In the developing rat brain, it is expressed only in the cortical plate and subplate region but not in the intermediate or ventricular zone. Using in utero electroporation, we found that short hairpin RNA (shRNA)-mediated knockdown of KIDLIA leads to altered neuron migration and a reduction in dendritic growth and disorganized apical dendrite projections in layer II/III mouse cortical neurons. Consistent with this, in cultured rat neurons, a loss of KIDLIA expression also leads to suppression of dendritic growth and branching. At the molecular level, we found that KIDLIA suppression leads to an increase in cell-surface N-cadherin and an elevated association of N-cadherin with δ-catenin, resulting in depletion of free δ-catenin in the cytosolic compartment. The reduced availability of cytosolic δ-catenin leads to elevated RhoA activity and reduced actin dynamics at the dendritic growth cone. Furthermore, in neurons with KIDLIA knockdown, overexpression of δ-catenin or inhibition of RhoA rescues actin dynamics, dendritic growth, and branching. These findings provide the first evidence on the role of the novel protein KIDLIA in neurodevelopment and autism with severe intellectual disability.

NB‐3 signaling mediates the cross‐talk between post‐traumatic spinal axons and scar‐forming cells

Little is known about the molecules mediating the cross-talk between post-traumatic axons and scar-forming cells after spinal cord injury. We found that a sustained NB-3 induction was simultaneously present in the terminations of post-traumatic corticospinal axons and scar-forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced invivo with NB-3 deficiency or interruption of NB-3 trans-homophilic interactions. Biochemical, invitro and invivo evidence demonstrated that NB-3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar-forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB-3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB-3 trans-homophilic interactions mediate the cross-talk between post-traumatic axons and scar-forming cells and impair the intrinsic growth ability of injured axons.

PSD-95 regulates CRFR1 localization, trafficking and β-arrestin2 recruitment

Corticotropin-releasing factor (CRF) is a neuropeptide commonly associated with the hypothalamic–pituitary adrenal axis stress response. Upon release, CRF activates two G protein-coupled receptors (GPCRs): CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2). Although both receptors contribute to mood regulation, CRFR1 antagonists have demonstrated anxiolytic and antidepressant-like properties that may be exploited in the generation of new pharmacological interventions for mental illnesses. Previous studies have demonstrated CRFR1 capable of heterologously sensitizing serotonin 2A receptor (5-HT2AR) signaling: another GPCR implicated in psychiatric disease. Interestingly, this phenomenon was dependent on Postsynaptic density 95 (PSD-95)/Disc Large/Zona Occludens (PDZ) interactions on the distal carboxyl termini of both receptors. In the current study, we demonstrate that endogenous PSD-95 can be co-immunoprecipitated with CRFR1 from cortical brain homogenate, and this interaction appears to be primarily via the PDZ-binding motif. Additionally, PSD-95 colocalizes with CRFR1 within the dendritic projections of cultured mouse neurons in a PDZ-binding motif-dependent manner. In HEK 293 cells, PSD-95 overexpression inhibited CRFR1 endocytosis, whereas PSD-95 shRNA knockdown enhanced CRFR1 endocytosis. Although PSD-95 does not appear to play a significant role in CRF-mediated cAMP or ERK1/2 signaling, PSD-95 was demonstrated to suppress β-arrestin2 recruitment: providing a potential mechanism for PSD-95's inhibition of endocytosis. In revisiting previously documented heterologous sensitization, PSD-95 shRNA knockdown did not prevent CRFR1-mediated enhancement of 5-HT2AR signaling. In conclusion, we have identified and characterized a novel functional relationship between CRFR1 and PSD-95 that may have implications in the design of new treatment strategies for mental illness.

Intrinsic Biomarker for Oxidative Stress by FLIM

Reactive oxygen species (ROS), when present above normal physiological levels, causes oxidative stress (OS), leading to damage of proteins and DNA and lipid peroxidation. OS has been associated with myriad of diseases like inflammation, diabetes mellitus, cardiovascular diseases, and cancer. Hence an OS biomarker would have immense clinical importance. There is a dearth of nondestructive techniques to measure OS in vivo. This work shows non-invasive detection of OS by fluorescence lifetime imaging microscopy (FLIM) of an intrinsic probe. Recognition of this endogenous biomarker enables label-free OS imaging. We identified this as fluorescent product of lipid oxidation by ROS. Using FLIM-phasor analysis, we can identify the presence and spatial location these species with a characteristic long lifetime (LLS). We could correlate the LLS to lipid droplets in the oleic acid treated HeLa cells using third harmonic generation (THG) imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. For chemical analysis, we performed single point Raman spectroscopy which revealed typical lipid droplet Raman-bands. LLS FLIM signature was also observed in mouse perigonadal white adipose tissue (WAT) lipid droplets, which co-localized with THG signal. This further confirms the association of LLS to lipid droplets. We show application of LLS as OS biomarker in cardiology and cancer. An increase of LLS was observed in induced-pluripotent-stem-cell-derived cardiomyocytes under OS due to hypoxia. This correlates to the increase in ROS production due to hypoxia. LLS FLIM signature was also observed in melanoma, a ROS driven cancer. LLS were mostly located in the dendritic projection of the melanoma cells. Comparatively, only a small amount of LLS was displayed by melanocytes. This label-free, non-invasive FLIM-phasor method is a promising imaging technique for OS detection in biological systems, especially for in vivo measurements.Work supported in part by NIH grants P50GM076516,P41GM103540 and UH2TR000481-0.

Developmental changes in the morphology of mouse hypoglossal motor neurons.

Hypoglossal motor neurons (XII MNs) innervate tongue muscles important in breathing, suckling and vocalization. Morphological properties of 103 XII MNs were studied using Neurobiotin™ filling in transverse brainstem slices from C57/Bl6 mice (n = 34) from embryonic day (E) 17 to postnatal day (P) 28. XII MNs from areas thought to innervate different tongue muscles showed similar morphology in most, but not all, features. Morphological properties of XII MNs were established prior to birth, not differing between E17–18 and P0. MN somatic volume gradually increased for the first 2 weeks post-birth. The complexity of dendritic branching and dendrite length of XII MNs increased throughout development (E17–P28). MNs in the ventromedial XII motor nucleus, likely to innervate the genioglossus, frequently (42 %) had dendrites crossing to the contralateral side at all ages, but their number declined with postnatal development. Unexpectedly, putative dendritic spines were found in all XII MNs at all ages, and were primarily localized to XII MN somata and primary dendrites at E18–P4, increased in distal dendrites by P5–P8, and were later predominantly found in distal dendrites. Dye-coupling between XII MNs was common from E18 to P7, but declined strongly with maturation after P7. Axon collaterals were found in 20 % (6 of 28) of XII MNs with filled axons; collaterals terminated widely outside and, in one case, within the XII motor nucleus. These results reveal new morphological features of mouse XII MNs, and suggest that dendritic projection patterns, spine density and distribution, and dye-coupling patterns show specific developmental changes in mice.

Functional Integration of Adult-Born Hippocampal Neurons after Traumatic Brain Injury(1,2,3).

Traumatic brain injury (TBI) increases hippocampal neurogenesis, which may contribute to cognitive recovery after injury. However, it is unknown whether TBI-induced adult-born neurons mature normally and functionally integrate into the hippocampal network. We assessed the generation, morphology, and synaptic integration of new hippocampal neurons after a controlled cortical impact (CCI) injury model of TBI. To label TBI-induced newborn neurons, we used 2-month-old POMC-EGFP mice, which transiently and specifically express EGFP in immature hippocampal neurons, and doublecortin-CreERT2 transgenic mice crossed with Rosa26-CAG-tdTomato reporter mice, to permanently pulse-label a cohort of adult-born hippocampal neurons. TBI increased the generation, outward migration, and dendritic complexity of neurons born during post-traumatic neurogenesis. Cells born after TBI had profound alterations in their dendritic structure, with increased dendritic branching proximal to the soma and widely splayed dendritic branches. These changes were apparent during early dendritic outgrowth and persisted as these cells matured. Whole-cell recordings from neurons generated during post-traumatic neurogenesis demonstrate that they are excitable and functionally integrate into the hippocampal circuit. However, despite their dramatic morphologic abnormalities, we found no differences in the rate of their electrophysiological maturation, or their overall degree of synaptic integration when compared to age-matched adult-born cells from sham mice. Our results suggest that cells born after TBI participate in information processing, and receive an apparently normal balance of excitatory and inhibitory inputs. However, TBI-induced changes in their anatomic localization and dendritic projection patterns could result in maladaptive network properties.

Decrease in levels of the evolutionarily conserved microRNA miR-124 affects oligodendrocyte numbers in Zebrafish, Danio rerio.

Oligodendrocytes produce multi-lamellar myelin membranes that surround axons in the central nervous system (CNS). Preservation and generation of myelin are potential therapeutic targets for dysmyelinating and demyelinating diseases. MicroRNAs (miRNAs) play a vital role in oligodendrocyte differentiation and overall CNS development. miR-124 is a well-conserved neuronal miRNA with important roles in neuronal differentiation and function. miR-124 levels increase following loss of myelin in both human and rodent brains. While the role of neuronal miR-124 in neurogenesis has been established, its effects on axonal outgrowth and oligodendrocytes are not currently known. We therefore explored the possible effect of selective knockdown of miR-124 in Danio rerio using a morpholino-based knockdown approach. No morphological abnormalities or loss of motor neurons were detected despite loss of axonal outgrowth. Morpholino-based knockdown of miR-124 led to reciprocal increases in mRNA levels of target genes that inhibit axonal and dendritic projections. Importantly, loss of miR-124 led to decreased oligodendrocyte cell numbers and myelination of axonal projections in the ventral hindbrain. Taken together, our results add a new dimension to the existing complexity of neuron-glial relationships and highlight the utility of Danio rerio as a model system to investigate such interactions.

The Dielectric Breakdown Model applied to explain various morphologies of deposited metallic structures in thin gap metal electro-deposition

The phenomenon of metal electro-deposition in thin-gap geometry leads to very interesting and diverse two dimensional morphologies. This varies from dense ramified growth to thin dendritic projections. In this paper, we have proposed a stochastic model that incorporates such diversity. We carried out thin-gap electro-deposition of Copper and Zinc with varying electrolytic concentrations. A well known model, that until this work was used to explain dielectric breakdown patterns, was employed to explain the variation in deposition morphology with concentration. The sole parameter in the model was varied and the numerically obtained patterns was seen to correlate well with those obtained from electro-deposition. A linear relationship between the parameter and molar concentration was established. The established relationship was then analysed and interpreted.

Quantifying the osteocyte network in the human skeleton

Osteocytes form an extensive cellular network throughout the hard tissue matrix of the skeleton, which is known to regulate skeletal structure. However due to limitations in imaging techniques, the magnitude and complexity of this network remain undefined.We have used data from recent papers obtained by new imaging techniques, in order to estimate absolute and relative quantities of the human osteocyte network and form a more complete understanding of the extent and nature of this network.We estimate that the total number of osteocytes within the average adult human skeleton is ~42 billion and that the total number of osteocyte dendritic projections from these cells is ~3.7 trillion. Based on prior measurements of canalicular density and a mathematical model of osteocyte dendritic process branching, we calculate that these cells form a total of 23 trillion connections with each other and with bone surface cells. We estimate the total length of all osteocytic processes connected end-to-end to be 175,000km. Furthermore, we calculate that the total surface area of the lacuno-canalicular system is 215m2. However, the residing osteocytes leave only enough space for 24mL of extracellular fluid. Calculations based on measurements in lactation-induced murine osteocytic osteolysis indicate a potential total loss of ~16,000mm3 (16mL) of bone by this process in the human skeleton. Finally, based on the average speed of remodelling in the adult, we calculate that 9.1 million osteocytes are replenished throughout the skeleton on a daily basis, indicating the dynamic nature of the osteocyte network.We conclude that the osteocyte network is a highly complex communication network, and is much more vast than commonly appreciated. It is at the same order of magnitude as current estimates of the size of the neural network in the brain, even though the formation of the branched network differs between neurons and osteocytes. Furthermore, continual replenishment of large numbers of osteocytes in the process of remodelling allows therapeutic changes to the continually renewed osteoblast population to be rapidly incorporated into the skeleton.

Isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson's diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Parametric Anatomical Modeling: a method for modeling the anatomical layout of neurons and their projections.

Computational models of neural networks can be based on a variety of different parameters. These parameters include, for example, the 3d shape of neuron layers, the neurons' spatial projection patterns, spiking dynamics and neurotransmitter systems. While many well-developed approaches are available to model, for example, the spiking dynamics, there is a lack of approaches for modeling the anatomical layout of neurons and their projections. We present a new method, called Parametric Anatomical Modeling (PAM), to fill this gap. PAM can be used to derive network connectivities and conduction delays from anatomical data, such as the position and shape of the neuronal layers and the dendritic and axonal projection patterns. Within the PAM framework, several mapping techniques between layers can account for a large variety of connection properties between pre- and post-synaptic neuron layers. PAM is implemented as a Python tool and integrated in the 3d modeling software Blender. We demonstrate on a 3d model of the hippocampal formation how PAM can help reveal complex properties of the synaptic connectivity and conduction delays, properties that might be relevant to uncover the function of the hippocampus. Based on these analyses, two experimentally testable predictions arose: (i) the number of neurons and the spread of connections is heterogeneously distributed across the main anatomical axes, (ii) the distribution of connection lengths in CA3-CA1 differ qualitatively from those between DG-CA3 and CA3-CA3. Models created by PAM can also serve as an educational tool to visualize the 3d connectivity of brain regions. The low-dimensional, but yet biologically plausible, parameter space renders PAM suitable to analyse allometric and evolutionary factors in networks and to model the complexity of real networks with comparatively little effort.

Parametric anatomical modeling: a method for modeling the anatomical layout of neurons and their projections

Computational models of neural networks can be based on a variety of different parameters. These parameters include, for example, the 3d shape of neuron layers, the neurons' spatial projection patterns, spiking dynamics and neurotransmitter systems. While many well-developed approaches are available to model, for example, the spiking dynamics, there is a lack of approaches for modeling the anatomical layout of neurons and their projections. We present a new method, called Parametric Anatomical Modeling (PAM), to fill this gap. PAM can be used to derive network connectivities and conduction delays from anatomical data, such as the position and shape of the neuronal layers and the dendritic and axonal projection patterns. Within the PAM framework, several mapping techniques between layers can account for a large variety of connection properties between pre- and post-synaptic neuron layers. PAM is implemented as a Python tool and integrated in the 3d modeling software Blender. We demonstrate on a 3d model of the hippocampal formation how PAM can help reveal complex properties of the synaptic connectivity and conduction delays, properties that might be relevant to uncover the function of the hippocampus. Based on these analyses, two experimentally testable predictions arose: (i) the number of neurons and the spread of connections is heterogeneously distributed across the main anatomical axes, (ii) the distribution of connection lengths in CA3-CA1 differ qualitatively from those between DG-CA3 and CA3-CA3. Models created by PAM can also serve as an educational tool to visualize the 3d connectivity of brain regions. The low-dimensional, but yet biologically plausible, parameter space renders PAM suitable to analyse allometric and evolutionary factors in networks and to model the complexity of real networks with comparatively little effort.

ECM receptors in neuronal structure, synaptic plasticity, and behavior.

During central nervous system development, extracellular matrix (ECM) receptors and their ligands play key roles as guidance molecules, informing neurons where and when to send axonal and dendritic projections, establish connections, and form synapses between pre- and postsynaptic cells. Once stable synapses are formed, many ECM receptors transition in function to control the maintenance of stable connections between neurons and regulate synaptic plasticity. These receptors bind to and are activated by ECM ligands. In turn, ECM receptor activation modulates downstream signaling cascades that control cytoskeletal dynamics and synaptic activity to regulate neuronal structure and function and thereby impact animal behavior. The activities of cell adhesion receptors that mediate interactions between pre- and postsynaptic partners are also strongly influenced by ECM composition. This chapter highlights a number of ECM receptors, their roles in the control of synapse structure and function, and the impact of these receptors on synaptic plasticity and animal behavior.

The Beta-Subunit Of Heterotrimeric G Proteins Harbors Gain-Of-Function Mutations In Multiple Hematologic Malignancies

To identify new oncogene alleles directly from primary tumor specimens, we generate and screen cDNA libraries from patient samples for gain-of-function alterations that can substitute for cytokine signaling in cytokine-dependent cells. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia of plasmacytoid dendritic cells with a dismal prognosis. No driver oncogenes have been identified in cases of BPDCN. Screening of a cDNA library generated from a BPDCN resulted in multiple cytokine-independent clones that expressed a full-length transcript of GNB1 with a K89E mutation. GNB1 encodes a beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, including GNAQ/GNA11 in uveal melanoma and GNAS in pituitary tumors, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. ERK and AKT activation by GNB1 K89E were confirmed by western blotting. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex assay of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. To assay the transforming effects of GNB1 K89E in vivo, we transduced GNB1 (wild-type or K89E) into bone marrow from Cdkn2a-deficient donors after 5-FU treatment and transplanted into wild-type recipients. We opted to utilize Cdkn2a-deficient donors as the loss of CDKN2A is common in cases of BPDCN. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal malignancy characterized by pancytopenia and massive hepatosplenomegaly. Spleens were infiltrated by large, spindly cells with extensive dendritic projections, as well as extensive fibrosis that completely effaced the normal splenic architecture. The cells were negative for T-cell (CD2, CD3) and B-cell (CD19, B220) markers but positive for the dendritic cell/macrophage markers MAC-2 and MAC-3. Further characterization by flow cytometry demonstrated that the cells infiltrating the spleen were CD8, CD103, MHC class II, CD26, FLT3 and CD11c positive, consistent with neoplastic dendritic cells. Serial transplantation of splenic cells from five different GNB1 K89E-transplanted mice into secondary wild-type recipients resulted in 100% fatality within 50 days. We searched published datasets from exome, transcriptome and whole genome sequencing of hematologic malignancies for GNB1 mutations. We identified one case of K89E in B-cell acute lymphoblastic leukemia (ALL), four cases with I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, six cases with K57E/T in myeloid neoplasms, and D76G in T-cell ALL. Expression of any of these alleles but not wild-type GNB1 was sufficient to promote cytokine-independent growth of human TF1 cells. The published structure of GNB1 (Ford et al. Science 1998) reported a small number of residues, including K57, I80 and K89 that mediate interactions with both G-alpha subunits and effector proteins. In fact, affinity purification followed by MS using tagged GNB1 (wild-type, I80T and K89E) demonstrated that, unlike wild-type GNB1, the GNB1 mutants fail to bind distinct Gα subunits. The repertoire of protein interactors, which includes potential G protein effectors, also differed between different GNB1 alleles. Thus, gain-of-function mutations in GNB1 occur across a broad range of hematologic malignancies, modify essential interaction G-protein subunit interactions, can drive in vivo transformation, and activate targetable downstream kinases. Disclosures:Tyner:Incyte Corporation: Research Funding.