Publications by authors named "Lino Tessarollo"

193 Publications

Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress.

EMBO Rep 2021 May 5:e52122. Epub 2021 May 5.

Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.
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http://dx.doi.org/10.15252/embr.202052122DOI Listing
May 2021

Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants.

bioRxiv 2021 Mar 4. Epub 2021 Mar 4.

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.
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http://dx.doi.org/10.1101/2021.03.04.433768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941635PMC
March 2021

SSSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis.

J Biol Chem 2021 Mar 1:100491. Epub 2021 Mar 1.

Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702. Electronic address:

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the LinSca1c-Kit (LSK) stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function and since Sptlc1 and ssSPTa mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.
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http://dx.doi.org/10.1016/j.jbc.2021.100491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047174PMC
March 2021

Delayed onset of inherited ALS by deletion of the BDNF receptor TrkB.T1 is non-cell autonomous.

Exp Neurol 2021 Mar 24;337:113576. Epub 2020 Dec 24.

Neural Development Section, Mouse Cancer Genetics Program, CCR, NCI, NIH, USA. Electronic address:

The pathophysiology of Amyotrophic Lateral Sclerosis (ALS), a disease caused by the gradual degeneration of motoneurons, is still largely unknown. Insufficient neurotrophic support has been cited as one of the causes of motoneuron cell death. Neurotrophic factors such as BDNF have been evaluated in ALS human clinical trials, but yielded disappointing results attributed to the poor pharmacokinetics and pharmacodynamics of BDNF. In the inherited ALS G93A SOD1 animal model, deletion of the BDNF receptor TrkB.T1 delays spinal cord motoneuron cell death and muscle weakness through an unknown cellular mechanism. Here we show that TrkB.T1 is expressed ubiquitously in the spinal cord and its deletion does not change the SOD1 mutant spinal cord inflammatory state suggesting that TrkB.T1 does not influence microglia or astrocyte activation. Although TrkB.T1 knockout in astrocytes preserves muscle strength and co-ordination at early stages of disease, its specific conditional deletion in motoneurons or astrocytes does not delay motoneuron cell death during the early stage of the disease. These data suggest that TrkB.T1 may limit the neuroprotective BDNF signaling to motoneurons via a non-cell autonomous mechanism providing new understanding into the reasons for past clinical failures and insights into the design of future clinical trials employing TrkB agonists in ALS.
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http://dx.doi.org/10.1016/j.expneurol.2020.113576DOI Listing
March 2021

is a critical regulator of adult long-term hematopoietic stem cell quiescence.

Proc Natl Acad Sci U S A 2020 12 2;117(50):31945-31953. Epub 2020 Dec 2.

Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of in adult HSCs remain unclear. To investigate the role of in adult HSCs, we generated a novel conditional knockout mouse model and deleted in adult mouse hematopoietic system using the IFN-inducible Our results show that deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without deletion showed that loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which and were identified as direct targets of Our results suggest that preserves the function of adult LT-HSCs by promoting their quiescence.
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http://dx.doi.org/10.1073/pnas.2017626117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749346PMC
December 2020

Phox2a Defines a Developmental Origin of the Anterolateral System in Mice and Humans.

Cell Rep 2020 11;33(8):108425

Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC H2W 1R7, Canada; Integrated Program in Neuroscience, McGill University, Montréal, QC H3A 2B4, Canada; Department of Anatomy and Cell Biology, McGill University, Montréal, QC H3A 0C7, Canada; Division of Experimental Medicine, McGill University, Montréal, QC H3A 2B2, Canada. Electronic address:

Anterolateral system neurons relay pain, itch, and temperature information from the spinal cord to pain-related brain regions, but the differentiation of these neurons and their specific contribution to pain perception remain poorly defined. Here, we show that most mouse spinal neurons that embryonically express the autonomic-system-associated Paired-like homeobox 2A (Phox2a) transcription factor innervate nociceptive brain targets, including the parabrachial nucleus and the thalamus. We define the Phox2a anterolateral system neuron birth order, migration, and differentiation and uncover an essential role for Phox2a in the development of relay of nociceptive signals from the spinal cord to the brain. Finally, we also demonstrate that the molecular identity of Phox2a neurons is conserved in the human fetal spinal cord, arguing that the developmental expression of Phox2a is a prominent feature of anterolateral system neurons.
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http://dx.doi.org/10.1016/j.celrep.2020.108425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713706PMC
November 2020

Generation of Functional Mouse Hippocampal Neurons.

Bio Protoc 2020 Aug;10(15)

Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States.

Primary culture of mouse hippocampal neurons is a very useful model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos' isolation, culturing conditions and assessment of culture's purity and differentiation. Evaluation of neuronal activity was performed by analysis of calcium imaging dynamics at six days in culture.
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http://dx.doi.org/10.21769/bioprotoc.3702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518151PMC
August 2020

Novel metabolic role for BDNF in pancreatic β-cell insulin secretion.

Nat Commun 2020 04 23;11(1):1950. Epub 2020 Apr 23.

Mouse Cancer Genetics Program, CCR, NCI, NIH, Frederick, USA.

BDNF signaling in hypothalamic circuitries regulates mammalian food intake. However, whether BDNF exerts metabolic effects on peripheral organs is currently unknown. Here, we show that the BDNF receptor TrkB.T1 is expressed by pancreatic β-cells where it regulates insulin release. Mice lacking TrkB.T1 show impaired glucose tolerance and insulin secretion. β-cell BDNF-TrkB.T1 signaling triggers calcium release from intracellular stores, increasing glucose-induced insulin secretion. Additionally, BDNF is secreted by skeletal muscle and muscle-specific BDNF knockout phenocopies the β-cell TrkB.T1 deletion metabolic impairments. The finding that BDNF is also secreted by differentiated human muscle cells and induces insulin secretion in human islets via TrkB.T1 identifies a new regulatory function of BDNF on metabolism that is independent of CNS activity. Our data suggest that muscle-derived BDNF may be a key factor mediating increased glucose metabolism in response to exercise, with implications for the treatment of diabetes and related metabolic diseases.
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http://dx.doi.org/10.1038/s41467-020-15833-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181656PMC
April 2020

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Immunity 2020 01 24;52(1):83-95.e4. Epub 2019 Dec 24.

Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.
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http://dx.doi.org/10.1016/j.immuni.2019.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962539PMC
January 2020

Assaying Homodimers of NF-κB in Live Single Cells.

Front Immunol 2019 7;10:2609. Epub 2019 Nov 7.

Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States.

NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as "NF-κB" in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired.
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http://dx.doi.org/10.3389/fimmu.2019.02609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853996PMC
November 2020

Sptlc1 is essential for myeloid differentiation and hematopoietic homeostasis.

Blood Adv 2019 11;3(22):3635-3649

Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD.

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity.
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http://dx.doi.org/10.1182/bloodadvances.2019000729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880889PMC
November 2019

Knockout of the non-essential gene SUGCT creates diet-linked, age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype.

Cell Mol Life Sci 2020 Sep 13;77(17):3423-3439. Epub 2019 Nov 13.

Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, Proteos #3-09, Singapore, 138673, Republic of Singapore.

SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as "crown-like" structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.
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http://dx.doi.org/10.1007/s00018-019-03359-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426296PMC
September 2020

Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels.

Elife 2019 08 20;8. Epub 2019 Aug 20.

Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States.

Brain-derived neurotrophic factor (BDNF) is a potent modulator of brain synaptic plasticity. Signaling defects caused by dysregulation of its Ntrk2 (TrkB) kinase (TrkB.FL) and truncated receptors (TrkB.T1) have been linked to the pathophysiology of several neurological and neurodegenerative disorders. We found that upregulation of Rbfox1, an RNA binding protein associated with intellectual disability, epilepsy and autism, increases selectively hippocampal TrkB.T1 isoform expression. Physiologically, increased Rbfox1 impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels. RNA-seq analysis of hippocampi with upregulation of in conjunction with the specific increase of TrkB.T1 isoform expression also shows that the genes affected by Rbfox1 gain of function are surprisingly different from those influenced by deletion. These findings not only identify TrkB as a major target of Rbfox1 pathophysiology but also suggest that gain or loss of function of Rbfox1 regulate different genetic landscapes.
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http://dx.doi.org/10.7554/eLife.49673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715404PMC
August 2019

Parkin interacting substrate zinc finger protein 746 is a pathological mediator in Parkinson's disease.

Brain 2019 08;142(8):2380-2401

Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson's disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson's disease, there is evidence that parkin is inactivated in sporadic Parkinson's disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson's disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson's disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson's disease and related α-synucleinopathies.
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http://dx.doi.org/10.1093/brain/awz172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658849PMC
August 2019

Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis.

Elife 2019 02 12;8. Epub 2019 Feb 12.

Ludwig Institute for Cancer Research, University of California, San Diego, San Diego, United States.

Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.
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http://dx.doi.org/10.7554/eLife.40811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389288PMC
February 2019

The Emergence and Functional Fitness of Memory CD4 T Cells Require the Transcription Factor Thpok.

Immunity 2019 01 9;50(1):91-105.e4. Epub 2019 Jan 9.

Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Electronic address:

Memory CD4 T cells mediate long-term immunity, and their generation is a key objective of vaccination strategies. However, the transcriptional circuitry controlling the emergence of memory cells from early CD4 antigen-responders remains poorly understood. Here, using single-cell RNA-seq to study the transcriptome of virus-specific CD4 T cells, we identified a gene signature that distinguishes potential memory precursors from effector cells. We found that both that signature and the emergence of memory CD4 T cells required the transcription factor Thpok. We further demonstrated that Thpok cell-intrinsically protected memory cells from a dysfunctional, effector-like transcriptional program, similar to but distinct from the exhaustion pattern of cells responding to chronic infection. Mechanistically, Thpok- bound genes encoding the transcription factors Blimp1 and Runx3 and acted by antagonizing their expression. Thus, a Thpok-dependent circuitry promotes both memory CD4 T cells' differentiation and functional fitness, two previously unconnected critical attributes of adaptive immunity.
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http://dx.doi.org/10.1016/j.immuni.2018.12.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503975PMC
January 2019

Regulation of Kit Expression in Early Mouse Embryos and ES Cells.

Stem Cells 2019 03 28;37(3):332-344. Epub 2019 Jan 28.

Dipartimento di Biomedicina e Prevenzione, Università degli Studi di Roma Tor Vergata, Rome, Italy.

Kit is a growth factor receptor that regulates proliferation and/or survival of many embryonic and postnatal stem cell types. When mutated, it can induce malignant transformation of the host cells. To dissect the Kit role in the control of ESC pluripotency, we studied its expression during early mouse embryogenesis and during the process of ESC derivation from inner cell mass (ICM) cells. We followed the in vitro development of early mouse embryos obtained from transgenic mice carrying Kit promoter regions fused to EGFP (Kit-EGFP) and found that they initiate EGFP expression at morula stage. EGFP expression is then maintained in the blastocyst, within the ICM, and its levels increase when cultured in the presence of MAPK and GSK3β inhibitors (2i) plus LIF compared with the LIF-only condition. Kit-EGFP ESCs showed nonhomogeneous EGFP expression pattern when cultured in LIF condition, but they upregulated EGFP expression, as well as that of Sox2, Nanog, Prdm14, when shifted to 2i-LIF culture. Similarly, primordial germ cells (PGCs) in the process of embryonic germ cell (EGC) conversion showed enhanced EGFP expression in 2i-LIF. Kit expression was affected by manipulating Sox2 levels in ESCs. Chromatin immunoprecipitation experiments confirmed that Sox2 binds Kit regulatory regions containing Sox2 consensus sequences. Finally, Kit constitutive activation induced by the D814Y mutation increased ESC proliferation and cloning efficiency in vitro and in teratoma assays in vivo. Our results identify Kit as a pluripotency-responsive gene and suggest a role for Kit in the regulation of ESC proliferation. Stem Cells 2019;37:332-344.
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http://dx.doi.org/10.1002/stem.2960DOI Listing
March 2019

ALS/FTD-Linked Mutation in FUS Suppresses Intra-axonal Protein Synthesis and Drives Disease Without Nuclear Loss-of-Function of FUS.

Neuron 2018 11 18;100(4):816-830.e7. Epub 2018 Oct 18.

Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

Through the generation of humanized FUS mice expressing full-length human FUS, we identify that when expressed at near endogenous murine FUS levels, both wild-type and ALS-causing and frontotemporal dementia (FTD)-causing mutations complement the essential function(s) of murine FUS. Replacement of murine FUS with mutant, but not wild-type, human FUS causes stress-mediated induction of chaperones, decreased expression of ion channels and transporters essential for synaptic function, and reduced synaptic activity without loss of nuclear FUS or its cytoplasmic aggregation. Most strikingly, accumulation of mutant human FUS is shown to activate an integrated stress response and to inhibit local, intra-axonal protein synthesis in hippocampal neurons and sciatic nerves. Collectively, our evidence demonstrates that human ALS/FTD-linked mutations in FUS induce a gain of toxicity that includes stress-mediated suppression in intra-axonal translation, synaptic dysfunction, and progressive age-dependent motor and cognitive disease without cytoplasmic aggregation, altered nuclear localization, or aberrant splicing of FUS-bound pre-mRNAs. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2018.09.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277851PMC
November 2018

Id1 Ablation Protects Hematopoietic Stem Cells from Stress-Induced Exhaustion and Aging.

Cell Stem Cell 2018 Aug 28;23(2):252-265.e8. Epub 2018 Jun 28.

Mouse Cancer Genetics Program, Center for Cancer Research, NCI, Frederick, MD 21702, USA; Basic Science Program and Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address:

Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1 HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1 HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging.
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http://dx.doi.org/10.1016/j.stem.2018.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149219PMC
August 2018

Deficiency in Fpr2 results in reduced numbers of LincKitSca1 myeloid progenitor cells.

J Biol Chem 2018 08 17;293(35):13452-13463. Epub 2018 Jul 17.

From the Cancer and Inflammation Program and

The Linc-Kit Sca-1 cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31Ly6C (granulocytes and monocytes), CD31/Ly6C (granuloid cells), and CD31/Ly6C (predominantly monocytes) cells. In particular, the number of Linc-KitSca-1 (LKS) cells was reduced in Fpr2 mouse BM. This was supported by observations of the reduced incorporation of intraperitoneally injected bromodeoxyuridine by cells in the c-Kit population from Fpr2 mouse BM. Purified c-Kit cells from Fpr2 mice showed reduced expansion when cultured with stem cell factor (SCF). SCF/c-Kit-mediated phosphorylation of P38, STAT1, Akt (Thr-308), and Akt (Ser-473) was also significantly reduced in c-Kit cells from Fpr2 mice. Furthermore, Fpr2 agonists enhanced SCF-induced proliferation of c-Kit cells. Colony-forming unit assays revealed that CFU-granulocyte-macrophage formation of BM cells from Fpr2 mice was significantly reduced. After heat-inactivated bacterial stimulation in the airway, the expansion of c-kit Sca-1 cells in BM and recruitment of Ly6G cells to the lungs and CD11bLy6CTNFα cells to the spleen of Fpr2 mice was significantly reduced. These results demonstrate an important role for Fpr2 in the development of myeloid lineage precursors in mouse BM.
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http://dx.doi.org/10.1074/jbc.RA118.002683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120191PMC
August 2018

POGZ Is Required for Silencing Mouse Embryonic β-like Hemoglobin and Human Fetal Hemoglobin Expression.

Cell Rep 2018 06;23(11):3236-3248

Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Bldg. 560/12-70, 1050 Boyles Street, Frederick, MD 21702, USA; Basic Research Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Bldg. 560/32-31D, 1050 Boyles Street, Frederick, MD 21702, USA. Electronic address:

Fetal globin genes are transcriptionally silenced during embryogenesis through hemoglobin switching. Strategies to derepress fetal globin expression in the adult could alleviate symptoms in sickle cell disease and β-thalassemia. We identified a zinc-finger protein, pogo transposable element with zinc-finger domain (POGZ), expressed in hematopoietic progenitor cells. Targeted deletion of Pogz in adult hematopoietic cells in vivo results in persistence of embryonic β-like globin expression without affecting erythroid development. POGZ binds to the Bcl11a promoter and erythroid-specific intragenic regulatory regions. Pogz mice show elevated embryonic β-like globin expression, suggesting that partial reduction of Pogz expression results in persistence of embryonic β-like globin expression. Knockdown of POGZ in primary human CD34 progenitor cell-derived erythroblasts reduces BCL11A expression, a known repressor of embryonic β-like globin expression, and increases fetal hemoglobin expression. These findings are significant, since new therapeutic targets and strategies are needed to treat β-globin disorders.
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http://dx.doi.org/10.1016/j.celrep.2018.05.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301966PMC
June 2018

Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels.

J Neurosci 2018 06 16;38(23):5415-5428. Epub 2018 May 16.

Department of Cell Biology and Pathology, Instituto de Neurociencias de Castilla y León, University of Salamanca, Salamanca 37007, Spain,

BDNF is a growth factor with important roles in the nervous system in both physiological and pathological conditions, but the mechanisms controlling its secretion are not completely understood. Here, we show that ARMS/Kidins220 negatively regulates BDNF secretion in neurons from the CNS and PNS. Downregulation of the ARMS/Kidins220 protein in the adult mouse brain increases regulated BDNF secretion, leading to its accumulation in the striatum. Interestingly, two mouse models of Huntington's disease (HD) showed increased levels of ARMS/Kidins220 in the hippocampus and regulated BDNF secretion deficits. Importantly, reduction of ARMS/Kidins220 in hippocampal slices from HD mice reversed the impaired regulated BDNF release. Moreover, there are increased levels of ARMS/Kidins220 in the hippocampus and PFC of patients with HD. ARMS/Kidins220 regulates Synaptotagmin-IV levels, which has been previously observed to modulate BDNF secretion. These data indicate that ARMS/Kidins220 controls the regulated secretion of BDNF and might play a crucial role in the pathogenesis of HD. BDNF is an important growth factor that plays a fundamental role in the correct functioning of the CNS. The secretion of BDNF must be properly controlled to exert its functions, but the proteins regulating its release are not completely known. Using neuronal cultures and a new conditional mouse to modulate ARMS/Kidins220 protein, we report that ARMS/Kidins220 negatively regulates BDNF secretion. Moreover, ARMS/Kidins220 is overexpressed in two mouse models of Huntington's disease (HD), causing an impaired regulation of BDNF secretion. Furthermore, ARMS/Kidins220 levels are increased in brain samples from HD patients. Future studies should address whether ARMS/Kidins220 has any function on the pathophysiology of HD.
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http://dx.doi.org/10.1523/JNEUROSCI.1653-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990986PMC
June 2018

Deletion of the endogenous TrkB.T1 receptor isoform restores the number of hippocampal CA1 parvalbumin-positive neurons and rescues long-term potentiation in pre-symptomatic mSOD1(G93A) ALS mice.

Mol Cell Neurosci 2018 06 24;89:33-41. Epub 2018 Mar 24.

Physiological Science Section, Department of Experimental and Clinical Medicine, University of Florence, Italy. Electronic address:

Amyotrophic lateral sclerosis (ALS) causes rapidly progressive paralysis and death within 5 years from diagnosis due to degeneration of the motor circuits. However, a significant population of ALS patients also shows cognitive impairments and progressive hippocampal pathology. Likewise, the mutant SOD1(G93A) mouse model of ALS (mSOD1), in addition to loss of spinal motor neurons, displays altered spatial behavior and hippocampal abnormalities including loss of parvalbumin-positive interneurons (PVi) and enhanced long-term potentiation (LTP). However, the cellular and molecular mechanisms underlying these morpho-functional features are not well understood. Since removal of TrkB.T1, a receptor isoform of the brain-derived neurotrophic factor, can partially rescue the phenotype of the mSOD1 mice, here we tested whether removal of TrkB.T1 can normalize the number of PVi and the LTP in this model. Stereological analysis of hippocampal PVi in control, TrkB.T1, mSOD1, and mSOD1 mice deficient for TrkB.T1 (mSOD1/T1) showed that deletion of TrkB.T1 restored the number of PVi to physiological level in the mSOD1 hippocampus. The rescue of PVi neuron number is paralleled by a normalization of high-frequency stimulation-induced LTP in the pre-symptomatic mSOD1/T1 mice. Our experiments identified TrkB.T1 as a cellular player involved in the homeostasis of parvalbumin expressing interneurons and, in the context of murine ALS, show that TrkB.T1 is involved in the mechanism underlying structural and functional hippocampal degeneration. These findings have potential implications for hippocampal degeneration and cognitive impairments reported in ALS patients at early stages of the disease.
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http://dx.doi.org/10.1016/j.mcn.2018.03.010DOI Listing
June 2018

The Antimicrobial Peptide CRAMP Is Essential for Colon Homeostasis by Maintaining Microbiota Balance.

J Immunol 2018 03 12;200(6):2174-2185. Epub 2018 Feb 12.

Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702;

Commensal bacteria are critical for physiological functions in the gut, and dysbiosis in the gut may cause diseases. In this article, we report that mice deficient in cathelin-related antimicrobial peptide (CRAMP) were defective in the development of colon mucosa and highly sensitive to dextran sulfate sodium (DSS)-elicited colitis, as well as azoxymethane-mediated carcinogenesis. Pretreatment of CRAMP mice with antibiotics markedly reduced the severity of DSS-induced colitis, suggesting CRAMP as a limiting factor on dysbiosis in the colon. This was supported by observations that wild-type (WT) mice cohoused with CRAMP mice became highly sensitive to DSS-induced colitis, and the composition of fecal microbiota was skewed by CRAMP deficiency. In particular, several bacterial species that are typically found in oral microbiota, such as , , and , were increased in feces of CRAMP mice and were transferred to WT mice during cohousing. When littermates of CRAMP parents were examined, the composition of the fecal microbiota of WT pups and heterozygous parents was similar. In contrast, although the difference in fecal microbiota between CRAMP and WT pups was small early on after weaning and single mouse housing, there was an increasing divergence with prolonged single housing. These results indicate that CRAMP is critical in maintaining colon microbiota balance and supports mucosal homeostasis, anti-inflammatory responses, and protection from carcinogenesis.
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http://dx.doi.org/10.4049/jimmunol.1602073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931736PMC
March 2018

Robust kinase- and age-dependent dopaminergic and norepinephrine neurodegeneration in LRRK2 G2019S transgenic mice.

Proc Natl Acad Sci U S A 2018 02 31;115(7):1635-1640. Epub 2018 Jan 31.

Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205;

Mutations in LRRK2 are known to be the most common genetic cause of sporadic and familial Parkinson's disease (PD). Multiple lines of LRRK2 transgenic or knockin mice have been developed, yet none exhibit substantial dopamine (DA)-neuron degeneration. Here we develop human tyrosine hydroxylase (TH) promoter-controlled tetracycline-sensitive LRRK2 G2019S (GS) and LRRK2 G2019S kinase-dead (GS/DA) transgenic mice and show that LRRK2 GS expression leads to an age- and kinase-dependent cell-autonomous neurodegeneration of DA and norepinephrine (NE) neurons. Accompanying the loss of DA neurons are DA-dependent behavioral deficits and α-synuclein pathology that are also LRRK2 GS kinase-dependent. Transmission EM reveals that that there is an LRRK2 GS kinase-dependent significant reduction in synaptic vesicle number and a greater abundance of clathrin-coated vesicles in DA neurons. These transgenic mice indicate that LRRK2-induced DA and NE neurodegeneration is kinase-dependent and can occur in a cell-autonomous manner. Moreover, these mice provide a substantial advance in animal model development for LRRK2-associated PD and an important platform to investigate molecular mechanisms for how DA neurons degenerate as a result of expression of mutant LRRK2.
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http://dx.doi.org/10.1073/pnas.1712648115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816154PMC
February 2018

regulates cell cycle progression, the mode of cell division and the DNA-damage response in Purkinje neuron progenitors.

Development 2017 10 11;144(20):3686-3697. Epub 2017 Sep 11.

Division of Neuroscience, San Raffaele Scientific Institute, Milan 20132, Italy

The gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null mutants develop cerebellar malformations, the underlying mechanism remains unknown. mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.
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http://dx.doi.org/10.1242/dev.155077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675449PMC
October 2017

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

Am J Hum Genet 2017 Sep;101(3):391-403

Orthopedic Hospital of Speising, Speisinger Strasse 109, 1130 Vienna, Austria.

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.
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http://dx.doi.org/10.1016/j.ajhg.2017.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591019PMC
September 2017

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

Mol Cell 2017 Aug 10;67(4):566-578.e10. Epub 2017 Aug 10.

Cell Biology of Genomes, NCI, NIH, Bethesda, MD 20892, USA.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
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http://dx.doi.org/10.1016/j.molcel.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854204PMC
August 2017