scholarly journals Single Cell ADNP Predictive of Human Muscle Disorders: Mouse Knockdown Results in Muscle Wasting

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2320
Author(s):  
Oxana Kapitansky ◽  
Gidon Karmon ◽  
Shlomo Sragovich ◽  
Adva Hadar ◽  
Meishar Shahoha ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Muscle ◽  
Motor Dysfunction ◽  
Major Constituent ◽  
Interacting Protein ◽  
Muscle Disorders ◽  
Gene Transcripts ◽  
Personalized Diagnosis ◽  
Mouse Transcriptome ◽  
Activity Dependent

Activity-dependent neuroprotective protein (ADNP) mutations are linked with cognitive dysfunctions characterizing the autistic-like ADNP syndrome patients, who also suffer from delayed motor maturation. We thus hypothesized that ADNP is deregulated in versatile myopathies and that local ADNP muscle deficiency results in myopathy, treatable by the ADNP fragment NAP. Here, single-cell transcriptomics identified ADNP as a major constituent of the developing human muscle. ADNP transcript concentrations further predicted multiple human muscle diseases, with concentrations negatively correlated with the ADNP target interacting protein, microtubule end protein 1 (EB1). Reverting back to modeling at the single-cell level of the male mouse transcriptome, Adnp mRNA concentrations age-dependently correlated with motor disease as well as with sexual maturation gene transcripts, while Adnp expressing limb muscle cells significantly decreased with aging. Mouse Adnp heterozygous deficiency exhibited muscle microtubule reduction and myosin light chain (Myl2) deregulation coupled with motor dysfunction. CRISPR knockdown of adult gastrocnemius muscle Adnp in a Cas9 mouse resulted in treadmill (male) and gait (female) dysfunctions that were specifically ameliorated by treatment with the ADNP snippet, microtubule interacting, Myl2—regulating, NAP (CP201). Taken together, our studies provide new hope for personalized diagnosis/therapeutics in versatile myopathies.

Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count

Blood ◽  
2021 ◽  
Author(s):  
Melissa M Lee-Sundlov ◽  
Robert Thomas Burns ◽  
Taylor Olmsted Kim ◽  
Renata Grozovsky ◽  
Silvia Giannini ◽  
...  
Keyword(s):  
Platelet Count ◽  
Sialic Acid ◽  
Single Cell ◽  
Immune Cells ◽  
Plasmacytoid Dendritic Cell ◽  
Type I ◽  
Targeted Deletion ◽  
Platelet Disorder ◽  
Gene Transcripts ◽  
Antigen Antibody

Immune thrombocytopenia (ITP) is a common platelet disorder in pediatric patients. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF-antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF-antigen in these patients. The O-glycan sialyltransferase St3gal1 add sialic acid specifically on the TF-antigen. To understand if TF-antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF-antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon (IFN-I) secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following and inhibition of interferon and Siglec H receptors. Single cell RNAseq determined that TF-antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release IFN-I. Single cell RNAseq further revealed a new population of immune cells with a plasmacytoid dendritic cell (pDC)-like signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF-antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.-

scholarly journals HIV-1 provirus transcription and translation in macrophages differs from pre-integrated cDNA complexes and requires E2F transcriptional programs

2021 ◽  
Author(s):  
Albebson L. Lim ◽  
Philip Moos ◽  
Christopher D. Pond ◽  
Erica C. Larson ◽  
Laura J. Martins ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Immunological Response ◽  
Virus Production ◽  
Cell Analysis ◽  
Gene Transcripts ◽  
Nuanced Understanding ◽  
New Perspective ◽  
Hiv 1

AbstractHIV-1 cDNA pre-integration complexes have been shown to persist for weeks in macrophages and to be transcriptionally active. Early and late gene transcripts are produced, along with some viral proteins, yet whole virus is not. While previous work has focused on the transcription and translation of HIV-1 genes; our understanding of cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single cell RNA sequencing (scRNA-seq) to compare the transcriptomes of uninfected cells, cells harboring pre-integration HIV-1 complexes (PIC) and those containing integrated provirus and actively making late HIV proteins. These are also compared to control cells, not exposed to virus.Several observations provide new perspective on the effects of HIV-1 transcription from pre-integrated cDNA versus from integrated provirus. First, HIV-1 transcript levels do not necessarily correlate with virus production, cells harboring PIC cDNA have transcript loads comparable to cells transcribing from provirus and making p24, mCherry, and vpu proteins. Second, all HIV-1 transcripts are easily detectable in abundance from PIC cDNA transcription, as is the case with cells transcribing from provirus, although the frequency of PIC cells with detectable gag-pol, tat, env, and nef transcripts is higher than the corresponding frequencies observed for “Provirus cells”. Third, the background transcriptomes of cells harboring pre- integrated HIV-1 cDNA are not otherwise detectably altered from cells not containing any HIV- 1 transcript. Fourth, integration and production of p24, mCherry, and Vpu proteins is accompanied by a switch from transcriptomes characterized by NFkB and AP-1 promoted transcription to a transcriptome characterized by E2F family transcription products. While some of these observations may seem heretical, single cell analysis provides a more nuanced understanding of PIC cDNA transcription and the transcriptomic changes that support HIV-1 protein production from integrated provirus.Author SummarySingle cell analysis is able to distinguish between HIV-1 infected macrophage cells that are transcribing pre-integrated HIV-1 cDNA and those transcribing HIV-1 provirus. Only cells transcribing HIV-1 provirus are making p24, marker mCherry and Vpu proteins, which corresponds with a change in the host cell’s background transcriptome from one expressing viral restriction and immunological response genes to one that is expressing genes associated with cell replication and oxidative phosphorylation.

scholarly journals NudCL2 regulates cell migration by stabilizing both myosin-9 and LIS1 with Hsp90

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Wenwen Chen ◽  
Wei Wang ◽  
Xiaoxia Sun ◽  
Shanshan Xie ◽  
Xiaoyang Xu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Single Cell ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Heat Shock Protein 90 ◽  
Underlying Mechanism ◽  
Collective Cell Migration ◽  
Interacting Protein ◽  
Protein Levels ◽  
Protein Instability

Abstract Cell migration plays pivotal roles in many biological processes; however, its underlying mechanism remains unclear. Here, we find that NudC-like protein 2 (NudCL2), a cochaperone of heat shock protein 90 (Hsp90), modulates cell migration by stabilizing both myosin-9 and lissencephaly protein 1 (LIS1). Either knockdown or knockout of NudCL2 significantly increases single-cell migration, but has no significant effect on collective cell migration. Immunoprecipitation–mass spectrometry and western blotting analyses reveal that NudCL2 binds to myosin-9 in mammalian cells. Depletion of NudCL2 not only decreases myosin-9 protein levels, but also results in actin disorganization. Ectopic expression of myosin-9 efficiently reverses defects in actin disorganization and single-cell migration in cells depleted of NudCL2. Interestingly, knockdown of myosin-9 increases both single and collective cell migration. Depletion of LIS1, a NudCL2 client protein, suppresses both single and collective cell migration, which exhibits the opposite effect compared with myosin-9 depletion. Co-depletion of myosin-9 and LIS1 promotes single-cell migration, resembling the phenotype caused by NudCL2 depletion. Furthermore, inhibition of Hsp90 ATPase activity also reduces the Hsp90-interacting protein myosin-9 stability and increases single-cell migration. Forced expression of Hsp90 efficiently reverses myosin-9 protein instability and the defects induced by NudCL2 depletion, but not vice versa. Taken together, these data suggest that NudCL2 plays an important role in the precise regulation of cell migration by stabilizing both myosin-9 and LIS1 via Hsp90 pathway.

scholarly journals Precise Regulation of the Basal PKCγ Activity by DGKγ Is Crucial for Motor Coordination

2020 ◽  
Vol 21 (21) ◽  
pp. 7866
Author(s):  
Ryosuke Tsumagari ◽  
Kenta Maruo ◽  
Sho Kakizawa ◽  
Shuji Ueda ◽  
Minoru Yamanoue ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Motor Coordination ◽  
Receptor Potential ◽  
Motor Dysfunction ◽  
Interacting Protein ◽  
Lipid Kinase ◽  
Transient Receptor ◽  
Type 3 ◽  
Channel Type

Diacylglycerol kinase γ (DGKγ) is a lipid kinase to convert diacylglycerol (DG) to phosphatidic acid (PA) and indirectly regulates protein kinase C γ (PKCγ) activity. We previously reported that the basal PKCγ upregulation impairs cerebellar long-term depression (LTD) in the conventional DGKγ knockout (KO) mice. However, the precise mechanism in impaired cerebellar LTD by upregulated PKCγ has not been clearly understood. Therefore, we first produced Purkinje cell-specific DGKγ KO (tm1d) mice to investigate the specific function of DGKγ in Purkinje cells and confirmed that tm1d mice showed cerebellar motor dysfunction in the rotarod and beam tests, and the basal PKCγ upregulation but not PKCα in the cerebellum of tm1d mice. Then, the LTD-induced chemical stimulation, K-glu (50 mM KCl + 100 µM, did not induce phosphorylation of PKCα and dissociation of GluR2 and glutamate receptor interacting protein (GRIP) in the acute cerebellar slices of tm1d mice. Furthermore, treatment with the PKCγ inhibitor, scutellarin, rescued cerebellar LTD, with the phosphorylation of PKCα and the dissociation of GluR2 and GRIP. In addition, nonselective transient receptor potential cation channel type 3 (TRPC3) was negatively regulated by upregulated PKCγ. These results demonstrated that DGKγ contributes to cerebellar LTD by regulation of the basal PKCγ activity.

scholarly journals Multimodal Single-Cell Characterization of the Human Granulocyte Lineage

2021 ◽  
Author(s):  
Jingjing Qi ◽  
Darwin D'Souza ◽  
Travis Dawson ◽  
Daniel Geanon ◽  
Hiyab Stefanos ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cell ◽  
Gene Transcript ◽  
Diverse Range ◽  
Human Whole Blood ◽  
Significant Challenge ◽  
Human Granulocytes ◽  
Gene Transcripts ◽  
Transcript Detection

High throughput single cell transcriptomics (scRNA-seq) has been successfully applied to characterize immune cell heterogeneity across a diverse range of settings; however, analysis of human granulocytes remains a significant challenge due to their low gene expression transcript detection. Consequently, granulocytes are typically either absent or highly under-represented and inaccurately enumerated in most human scRNA-seq datasets. Here, we apply multi-modal CITE-seq profiling to characterize granulocytes in human whole blood and bone marrow, and we show that these populations can be accurately detected and analyzed using the antibody-based modality, and that their frequencies and phenotype align well with antibody-based characterization of the same samples using CyTOF. These analyses also clearly highlight extremely low gene transcript detection across the entire granulocyte lineage including the earliest neutrophil progenitor populations when using the 10X Genomics platform. By contrast, when performing parallel analyses of the same samples using the BD Rhapsody platform, we recovered a much higher proportion of granulocyte gene transcripts, enabling true multi-modal characterization of human granulocyte heterogeneity.

scholarly journals Activity-dependent synaptic GRIP1 accumulation drives synaptic scaling up in response to action potential blockade

2015 ◽  
Vol 112 (27) ◽  
pp. E3590-E3599 ◽  
Author(s):  
Melanie A. Gainey ◽  
Vedakumar Tatavarty ◽  
Marc Nahmani ◽  
Heather Lin ◽  
Gina G. Turrigiano
Keyword(s):  
Action Potential ◽  
Ampa Receptors ◽  
Scaling Up ◽  
Receptor Trafficking ◽  
Neuronal Firing ◽  
Homeostatic Plasticity ◽  
Synaptic Scaling ◽  
Interacting Protein ◽  
Excitatory Postsynaptic Currents ◽  
Activity Dependent

Synaptic scaling is a form of homeostatic plasticity that stabilizes neuronal firing in response to changes in synapse number and strength. Scaling up in response to action-potential blockade is accomplished through increased synaptic accumulation of GluA2-containing AMPA receptors (AMPAR), but the receptor trafficking steps that drive this process remain largely obscure. Here, we show that the AMPAR-binding protein glutamate receptor-interacting protein-1 (GRIP1) is essential for regulated synaptic AMPAR accumulation during scaling up. Synaptic abundance of GRIP1 was enhanced by activity deprivation, directly increasing synaptic GRIP1 abundance through overexpression increased the amplitude of AMPA miniature excitatory postsynaptic currents (mEPSCs), and shRNA-mediated GRIP1 knockdown prevented scaling up of AMPA mEPSCs. Furthermore, knockdown and replace experiments targeting either GRIP1 or GluA2 revealed that scaling up requires the interaction between GRIP1 and GluA2. Finally, GRIP1 synaptic accumulation during scaling up did not require GluA2 binding. Taken together, our data support a model in which activity-dependent trafficking of GRIP1 to synaptic sites drives the forward trafficking and enhanced synaptic accumulation of GluA2-containing AMPAR during synaptic scaling up.

scholarly journals MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIPL

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Osamu Nakabayashi ◽  
Hirotaka Takahashi ◽  
Kenta Moriwaki ◽  
Sachiko Komazawa-Sakon ◽  
Fumiaki Ohtake ◽  
...  
Keyword(s):  
Complex I ◽  
Kinase Activity ◽  
Notch Signalling ◽  
Caspase 8 ◽  
Inhibitory Protein ◽  
Interacting Protein ◽  
Polyubiquitin Chains ◽  
Long Form ◽  
Induced Apoptosis ◽  
Activity Dependent

AbstractMind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48– and K63–linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.

scholarly journals A Novel Functional In Vitro Model that Recapitulates Human Muscle Disorders

2018 ◽  
Author(s):  
Iván Toral-Ojeda ◽  
Garazi Aldanondo ◽  
Jaione Lasa-Elgarresta ◽  
Haizpea Lasa-Fernandez ◽  
Camila Vesga-Castro ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Human Muscle ◽  
Muscle Disorders ◽  
Vitro Model

scholarly journals PDZ protein mediated activity-dependent LTP/LTD developmental switch at rat retinocollicular synapses

2010 ◽  
Vol 298 (6) ◽  
pp. C1572-C1582 ◽  
Author(s):  
Lei Xue ◽  
Fan Zhang ◽  
Xianhua Chen ◽  
Junji Lin ◽  
Jian Shi
Keyword(s):  
Ampa Receptors ◽  
Long Term Potentiation ◽  
Basic Mechanism ◽  
Long Term Effects ◽  
Interacting Protein ◽  
Term Potentiation ◽  
Retinal Input ◽  
Pdz Protein ◽  
Activity Dependent

The insertion of amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors into the plasma membrane and removal via internalization are essential for regulating synaptic strength, which underlies the basic mechanism of learning and memory. The retinocollicular pathway undergoes synaptic refinement during development and shows a wide variety of long-term synaptic changes; however, still little is known about its underlying molecular regulation. Here we report a rapid developmental long-term potentiation (LTP)/long-term depression (LTD) switch and its intracellular mechanism at the rat retinocollicular pathway from postnatal day 5 (P5) to P14. Before P9, neurons always exhibited LTP, whereas LTD was observed only after P10. Blockade of GluR2/3-glutamate receptor-interacting protein (GRIP)/AMPA-receptor-binding protein (ABP)/protein interacting with C kinase 1 (PICK1) interactions with pep2-SVKI could sustain the LTP after P10. This suggests that the LTP/LTD switch relied on PDZ protein activities. Selective interruption of GluR2/3-PICK1 binding by pep2-EVKI blocked the long-lasting effects of both LTP and LTD, suggesting a role for PICK1 in the maintenance of long-term synaptic plasticity. Interestingly, synaptic expression of GRIP increased more than twofold from P7 to P11, whereas ABP and PICK1 expression levels remained stable. Blockade of spontaneous retinal input suppressed this increase and abolished the LTP/LTD switch. These results suggest that the increased GRIP synaptic expression may be a key regulatory factor in mediating the activity-dependent developmental LTP/LTD switch, whereas PICK1 may be required for both LTP and LTD to maintain their long-term effects.

scholarly journals Epimedium koreanumExtract and Its Constituent Icariin Improve Motor Dysfunction in Spinal Cord Injury

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Chihiro Tohda ◽  
Aiko Nagata
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Chronic Treatment ◽  
Methanol Extract ◽  
Motor Dysfunction ◽  
Major Constituent ◽  
Active Constituent ◽  
Short Period ◽  
Cord Injury ◽  
Recovery Of Motor Function

Although cell transplantation strategies for spinal cord injury (SCI) using sources such as iPS cells and neural stem cells are focused as expectative therapies for SCI, the possibility of medication as more accessible and practical way should not be given up. We, therefore, aimed to develop medical sources for SCI. In this paper, we evaluated effects of a famous tonic herb,Epimedium koreanum, on motor dysfunction in spinal cord injury (SCI). The spinal cord was injured by contusion after laminectomy at T10 level. Oral administration of the methanol extract ofE. koreanumsignificantly enhanced hindlimb function in SCI mice by short period treatment (for initial 3 days) and chronic treatment (21 days), although chronic treatment recovered the function more potently. Since it is well known that icariin is the major constituent inE. koreanum, icariin was administered orally to SCI mice for initial 3 days. Motor dysfunction was ameliorated by icariin treatment similarly to the methanol extract ofE. koreanum. This paper is the first report to indicateE. koreanumis effective for recovery of motor function in SCI, and at least icariin is an active constituent.

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