Oleamide, a Sleep-Inducing Supplement, Upregulates Doublecortin in Hippocampal Progenitor Cells via PPARα

2021 ◽  
pp. 1-16
Author(s):  
Avik Roy ◽  
Madhuchhanda Kundu ◽  
Sudipta Chakrabarti ◽  
Dhruv R. Patel ◽  
Kalipada Pahan
Keyword(s):  
Progenitor Cells ◽  
Neuronal Differentiation ◽  
Chromatin Immunoprecipitation ◽  
Molecular Mechanisms ◽  
Hippocampal Neurogenesis ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Lipid Lowering ◽  
Dominant Negative Mutant ◽  
Wild Type

Background: Doublecortin (DCX), a microtubule associated protein, has emerged as a central biomarker of hippocampal neurogenesis. However, molecular mechanisms by which DCX is regulated are poorly understood. Objective: Since sleep is involved with the acquisition of memory and oleamide or 9-Octadecenamide (OCT) is a sleep-inducing supplement in human, we examined whether OCT could upregulate DCX in hippocampal progenitor cells (HPCs). Methods: We employed real-time PCR, western blot, immunostaining, chromatin immunoprecipitation, lentiviral transduction in HPCs, and the calcium influx assay. Results: OCT directly upregulated the transcription of Dcx in HPCs via activation of peroxisome proliferator-activated receptor α (PPARα), a lipid-lowering transcription factor. We observed that, HPCs of Ppara-null mice displayed significant impairment in DCX expression and neuronal differentiation as compared to that of wild-type mice. Interestingly, treatment with OCT stimulated the differentiation process of HPCs in wild-type, but not Ppara-null mice. Reconstruction of PPARα in mouse Ppara-null HPCs restored the expression of DCX, which was further stimulated with OCT treatment. In contrast, a dominant-negative mutant of PPARα significantly attenuated the stimulatory effect of OCT on DCX expression and suppressed neuronal differentiation of human neural progenitor cells. Furthermore, RNA microarray, STRING, chromatin immunoprecipitation, site-directed mutagenesis, and promoter reporter assay have identified DCX as a new target of PPARα. Conclusion: These results indicate that OCT, a sleep supplement, directly controls the expression of DCX and suggest that OCT may be repurposed for stimulating the hippocampal neurogenesis.

scholarly journals Cross-talk between RhoH and Rac1 in regulation of actin cytoskeleton and chemotaxis of hematopoietic progenitor cells

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2597-2605 ◽  
Author(s):  
Hee-Don Chae ◽  
Katherine E. Lee ◽  
David A. Williams ◽  
Yi Gu
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Green Fluorescence Protein ◽  
Dominant Negative ◽  
Hematopoietic Progenitor Cells ◽  
Dominant Negative Mutant ◽  
Active Member ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Hematopoietic Progenitor

RhoH, a hematopoietic-specific and constitutively active member of the Rho guanosine triphosphatase (GTPase) family, has been implicated in the negative regulation of Rac GTPase-mediated signaling in hematopoietic cells. However, the molecular mechanisms underlying the functional interaction between RhoH and Rac in primary cells are poorly understood. Here we show that deletion of Rhoh in hematopoietic progenitor cells (HPCs) leads to increased stromal-derived factor-1α (SDF-1α)–induced chemotaxis and chemokinesis (random migration). The abnormally enhanced migration of Rhoh−/− HPCs is associated with increased Rac1 activity and translocation of Rac1 protein to the cell membrane, where it colocalizes with cortical filamentous-actin (F-actin) and lipid rafts. Expression of the dominant-negative mutant Rac1N17 inhibits the cortical F-actin assembly and chemotaxis of wild-type and Rhoh−/− HPCs to the same extent. Conversely, overexpression of RhoH in HPCs blocks the membrane translocation of Rac1–enhanced green fluorescence protein (EGFP) and active Rac1V12–EGFP proteins and impairs cortical F-actin assembly and chemotaxis in response to SDF-1α stimulation. Furthermore, we demonstrate that the subcellular localization and inhibitory function of RhoH in HPCs are regulated by C-terminal motifs, including a CKIF prenylation site. Together, we have identified an antagonistic role of RhoH in regulation of cortical F-actin assembly and chemotaxis via suppressing Rac1 membrane targeting and activation in primary HPCs.

scholarly journals Wild-Type and Mutant FUS Expression Reduce Proliferation and Neuronal Differentiation Properties of Neural Stem Progenitor Cells

2021 ◽  
Vol 22 (14) ◽  
pp. 7566
Author(s):  
Eleonora Stronati ◽  
Stefano Biagioni ◽  
Mario Fiore ◽  
Mauro Giorgi ◽  
Giancarlo Poiana ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Neuronal Differentiation ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Nervous System Development ◽  
Mutant Form ◽  
Wild Type ◽  
Fused In Sarcoma ◽  
Glial Lineage

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.

scholarly journals Suppressor Mutations Bypass the Requirement of fluG for Asexual Sporulation and Sterigmatocystin Production in Aspergillus nidulans

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1083-1093
Author(s):  
Jeong-Ah Seo ◽  
Yajun Guan ◽  
Jae-Hyuk Yu
Keyword(s):  
Aspergillus Nidulans ◽  
Molecular Mechanisms ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Mutation ◽  
Site Mutation ◽  
Asexual Sporulation ◽  
Dominant Negative Mutation ◽  
Suppressor Mutations ◽  
Early Developmental

Abstract Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.

Mitochondrial dysfunction-induced KDM5A degradation impairs adult hippocampal neurogenesis in Alzheimer’s disease

2020 ◽  
Author(s):  
Dong Kyu Kim ◽  
Hyobin Jeong ◽  
Jingi Bae ◽  
Moon-Yong Cha ◽  
Moonkyung Kang ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Mitochondrial Dysfunction ◽  
Progenitor Cells ◽  
Neuronal Differentiation ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Hippocampal Neurogenesis ◽  
Adult Hippocampal Neurogenesis ◽  
Amyloid Pathology ◽  
Neural Stem Progenitor Cells

Abstract Background Adult hippocampal neurogenesis (AHN) is a process of continuously generating functional mature neurons from neural stem cells in the dentate gyrus. In Alzheimer’s disease (AD) brains, amyloid pathology has deleterious effects on AHN, but molecular mechanisms for dysregulated AHN are unclear. Mitochondria of neural stem/progenitor cells play crucial roles in determining cell fate. Since mitochondrial dysfunction by amyloid pathology is the typical symptom of AD pathogenesis, we aim to study whether mitochondrial dysfunction of neural stem/progenitor cells by amyloid pathology causes the impairment of AHN, and elucidate the molecular mechanism of the phenomenon. Methods To investigate the effect of mitochondrial dysfunction of neural stem/progenitor cells on neuronal differentiation, we expressed mitochondria-targeted amyloid beta (mitoAβ) in neural stem/progenitor cells in vitro and in vivo. Proteomic analysis of the hippocampal tissue implicated mitochondrial dysfunction by mitoAβ as a cause of AHN deficits. We identified epigenetic regulators of neural progenitor cells that are regulated by mitoAβ expression or drug-induced mitochondrial toxicity and proposed a link between mitochondria and AHN. Results Amyloid pathology characteristically inhibited the neuronal differentiation stage, not the proliferation of neural stem/progenitor cells during AHN in early AD model mice. Mitochondrial dysfunction in neural stem/progenitor cells by expressing mitoAβ inhibited the neuronal differentiation and AHN with cognitive impairment. Mechanistic studies revealed that lysine demethylase 5A (KDM5A) was involved in the neuronal differentiation and could be degraded by mitochondrial dysfunction in neural progenitor cells, thereby inhibiting the differentiation and cognitive functions. Conclusions These results reveal the new role of KDM5A as a mediator of retrograde signaling, reflecting mitochondrial status, and that the decrease of KDM5A in neural progenitor cells by mitochondrial dysfunction impairs the neuronal differentiation and AHN, finally leading to memory deficits. These findings and its relationship to mitochondrial dysfunction suggest that mitochondrial failure in neural progenitor cells by amyloid pathology closely associates with reduced AHN in AD.

scholarly journals Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

2007 ◽  
Vol 404 (2) ◽  
pp. 179-190 ◽  
Author(s):  
Mark Windheim ◽  
Christine Lang ◽  
Mark Peggie ◽  
Lorna A. Plater ◽  
Philip Cohen
Keyword(s):  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Muramyl Dipeptide ◽  
Dominant Negative ◽  
Signalling Pathways ◽  
Dominant Negative Mutant ◽  
Protein Kinase Activity ◽  
P38α Mapk

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2–RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1β (interleukin-1β) and TNFα (tumour necrosis factor-α). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-β-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38α MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38α MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1β and TNFα in these cells. Taken together, our results indicate that the MDP–NOD2/RIP2 and LPS (lipopolysaccharide)–TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

scholarly journals FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1581-1589 ◽  
Author(s):  
Marc Pellegrini ◽  
Sue Bath ◽  
Vanessa S. Marsden ◽  
David C. S. Huang ◽  
Donald Metcalf ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Caspase 8 ◽  
Liver Stem Cells ◽  
Hemopoietic Progenitor ◽  
Hemopoietic Progenitor Cells ◽  
Negative Mutant

Abstract The role of caspase-8 and its adaptor Fas-associated death domain (FADD) in lymphocyte apoptosis is well defined, but their functions in other hemopoietic lineages are not clear. We were unable to generate transgenic mice expressing dominant inhibitors of FADD or caspase-8 in hemopoietic cells, possibly because their expression may have precluded production of vital hemopoietic cells. When using a retroviral gene delivery system, fetal liver stem cells expressing a dominant-negative mutant of FADD (FADD-DN) were unable to generate myeloid or lymphoid cells upon transplantation into lethally irradiated mice. However, fetal liver stem cells expressing very low levels of the caspase-8 inhibitor cytokine response modifier A (CrmA) could reconstitute the hemopoietic system. This level of CrmA expression provided some protection against Fas ligand (FasL)–induced apoptosis and promoted accumulation of myeloid cells in the bone marrow, but it did not inhibit mitogen-induced proliferation of B or T lymphocytes. Using an in vitro colony formation assay, we found that fetal liver stem cells expressing FADD-DN, CrmA, or a dominant-negative mutant of caspase-8 could not proliferate in response to cytokine stimulation. These data demonstrate that the enzymatic activity of caspase-8 and its adaptor FADD are required for cytokine-induced proliferation of hemopoietic progenitor cells.

Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1808-1816 ◽  
Author(s):  
Erwan Le Scolan ◽  
Dimitri Pchejetski ◽  
Yoshiko Banno ◽  
Nicole Denis ◽  
Patrick Mayeux ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Recurrent Event ◽  
Dominant Negative Mutant ◽  
Apoptosis Resistance ◽  
Kinase Gene ◽  
Extracellular Signal

Abstract The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

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