Dystroglycan (Dag1) is a cell adhesion molecule that links the extracellular matrix to the actin cytoskeleton, and is critical for normal muscle and brain development. Mutations in Dag1 or the genes required for its functional glycosylation result in dystroglycanopathy, which is characterized by a wide range of phenotypes including muscle weakness, brain defects, and cognitive impairments. Whereas Dystroglycans role in muscle and early brain development are well defined, much less is known about its role at later stages of neural circuit development including synapse formation and refinement. Recent work has found that selective deletion of Dag1 from pyramidal neurons leads to a loss of presynaptic CCK+ inhibitory neurons (INs) early in development. In this study, we investigated how IN development is affected in multiple mouse models of dystroglycanopathy. Widespread forebrain deletion of Dag1 or Pomt2, which is required for Dystroglycan glycosylation, recapitulates brain phenotypes seen in severe forms of dystroglycanopathy. CCK+ INs were present in Dag1 and Pomt2 mutant mice, but their axons failed to properly target the somatodendritic compartment of pyramidal neurons in the hippocampus. In contrast, CCK+ IN axon targeting was largely normal in mouse models of mild dystroglycanopathy with partially reduced Dystroglycan glycosylation (B4Gat1,Fkrp). Furthermore, the intracellular domain of Dystroglycan appears to be dispensable for CCK+ IN axon targeting. Collectively, these data show that synaptic defects are a hallmark of severe dystroglycanopathy.

Oncogenic mutations in RAS genes, like KRAS G12D or NRAS G12D , trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals.In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem-and progenitor-cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRAS G12D or NRAS G12D , does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T cell leukemia.

Dystroglycan (Dag1) is a transmembrane glycoprotein that links the extracellular matrix to the actin cytoskeleton. Mutations in Dag1 or the genes required for its glycosylation result in dystroglycanopathy, a type of congenital muscular dystrophy characterized by a wide range of phenotypes including muscle weakness, brain defects, and cognitive impairment. We investigated interneuron (IN) development, synaptic function, and associated seizure susceptibility in multiple mouse models that reflect the wide phenotypic range of dystroglycanopathy neuropathology. Mice that model severe dystroglycanopathy due to forebrain deletion of Dag1 or POMT2, which is required for Dystroglycan glycosylation, show significant impairment of CCK+/CB1R+ IN development. CCK+/CB1R+IN axons failed to properly target the somatodendritic compartment of pyramidal neurons in the hippocampus, resulting in synaptic defects and increased seizure susceptibility. Mice lacking the intracellular domain of Dystroglycan have milder defects in CCK+/CB1R+ IN axon targeting, but exhibit dramatic changes in inhibitory synaptic function, indicating a critical postsynaptic role of this domain. In contrast, CCK+/CB1R+ IN synaptic function and seizure susceptibility was normal in mice that model mild dystroglycanopathy due to partially reduced Dystroglycan glycosylation. Collectively, these data show that inhibitory synaptic defects and elevated seizure susceptibility are hallmarks of severe dystroglycanopathy, and show that Dystroglycan plays an important role in organizing functional inhibitory synapse assembly.

Dystroglycan (Dag1) is a transmembrane glycoprotein that links the extracellular matrix to the actin cytoskeleton. Mutations in Dag1 or the genes required for its glycosylation result in dystroglycanopathy, a type of congenital muscular dystrophy characterized by a wide range of phenotypes including muscle weakness, brain defects, and cognitive impairment. We investigated interneuron (IN) development, synaptic function, and associated seizure susceptibility in multiple mouse models that reflect the wide phenotypic range of dystroglycanopathy neuropathology. Mice that model severe dystroglycanopathy due to forebrain deletion of Dag1 or POMT2, which is required for Dystroglycan glycosylation, show significant impairment of CCK+/CB1R+ IN development. CCK+/CB1R+IN axons failed to properly target the somatodendritic compartment of pyramidal neurons in the hippocampus, resulting in synaptic defects and increased seizure susceptibility. Mice lacking the intracellular domain of Dystroglycan have milder defects in CCK+/CB1R+ IN axon targeting, but exhibit dramatic changes in inhibitory synaptic function, indicating a critical postsynaptic role of this domain. In contrast, CCK+/CB1R+ IN synaptic function and seizure susceptibility was normal in mice that model mild dystroglycanopathy due to partially reduced Dystroglycan glycosylation. Collectively, these data show that inhibitory synaptic defects and elevated seizure susceptibility are hallmarks of severe dystroglycanopathy, and show that Dystroglycan plays an important role in organizing functional inhibitory synapse assembly.

24Oncogenic mutations in RAS genes, like KRAS G12D or NRAS G12D , trap Ras in the active 25 state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced 26 4 Keywords 43 Leukemia, bone marrow, T-ALL, RAS signaling, RASGRP1, cytokines, progenitor, stem 44 cells, hematopoiesis 45 46 47 5 48 49 Acute lymphoblastic leukemia (ALL) is an aggressive bone marrow (BM) malignancy. 50Approximately 15% of pediatric and 25% of adult cases of ALL are diagnosed as T-cell 51 ALL (T-ALL) cog. In the clinic, T-ALL patients receive combination chemotherapy, 52 sometimes combined with BM transplantation 2 . Understanding the molecularly altered 53 biochemical pathways implicated in T-ALL is critical to developing effective molecular 54 therapy (precision medicine). One commonly affected pathway is the RAS pathway with 55 roughly 50% of all patient T-ALL cases demonstrating hyperactive RAS signals in the 56 BM 3 . There are two major mechanisms of leukemogenic RAS signals in T-ALL patients; 57 the first consists of somatic activating mutations of small RAS GTPases themselves and 58 a second mechanism is through deregulated overexpression of the RAS activator 59 RASGRP1 (RAS guanine nucleotide releasing protein 1) 4,5 . 60 61Oncogenic mutations in RAS are among the most common somatic mutations in 62 cancer. Mutations in glycine at codon 12 in KRAS (KRAS G12 ) are prevalent in cancer 63 and result in severely impaired GTPase activity and elevated levels of constitutively 64 active, GTP-bound KRAS 6 . In hematopoietic malignancies, mutations in NRAS are 2 to 65 3 times more frequent than those in KRAS 7,8 , including pediatric T-ALL 9 . Generation of 66 Kras G12D mice aided the field of cancer research and oncogenic RAS signaling; these 67 mice express a mutation of glycine to aspartic acid at codon 12 from the endogenous 68Kras locus in a controlled and inducible manner via a LoxP-STOP-LoxP cassette 6 . In 69 BM cells, oncogenic KRAS G12D can be inducibly expressed using Mx1CRE transgenic 70 6 mice; in this model CRE is expressed from the IFN-α/β-inducible Mx1 promoter by 71 administration of polyinosinic-polycytidylic acid (pIpC) 10 . Such KRAS G12D mice develop 72 a lethal myeloproliferative disease (MPD) resulting in death around 35 days 11,12 . In the 73 background a T-ALL exists, which is suppressed by the MPD, but can be revealed via 74 transplantation of KRAS G12D hematopoietic stem cells into irradiated recipient mice [12][13][14] . 75Mx1CRE-driven NRAS G12D mutation does not lead to acute MPD 15 . Instead mice 76 develop a chronic myeloproliferative disorder and succumb to hematological disease by 77 15 months on a C57Bl/6 x 129/Sv.jae background but displaying a median survival of 78 588 days on a C57Bl/6 background 16 . 79 80We reported deregulated overexpression of the RAS guanine nucleotide exchange 81 factor RASGRP1 in T-ALL patients 4,5 . RASGRP1 has a growth promoting role in T-cell 82 leukemia 4 and skin cancer 17 . RASGRP1 overexpression through retroviral transduction 83 or via transgenic e...