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1.
Cell Rep Med ; 5(7): 101623, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-38936368

ABSTRACT

In rodents with unilateral ablation of neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA induces a progressive increase of behavioral responses, a process known as behavioral sensitization. This sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of these mice, we find that the restoration of arrestin-3 fully rescues behavioral sensitization, whereas its mutant defective in c-Jun N-terminal kinase (JNK) activation does not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in direct pathway striatal neurons, also fully rescues sensitization, whereas an inactive homologous arrestin-2-derived peptide does not. Behavioral rescue is accompanied by the restoration of JNK3 activity, as reflected by JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-assisted JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization upon dopamine depletion and chronic L-DOPA treatment.


Subject(s)
Arrestins , Behavior, Animal , Dopamine , Mice, Knockout , Mitogen-Activated Protein Kinase 10 , Animals , Mitogen-Activated Protein Kinase 10/metabolism , Mitogen-Activated Protein Kinase 10/genetics , Mice , Dopamine/metabolism , Behavior, Animal/drug effects , Arrestins/metabolism , Arrestins/genetics , Corpus Striatum/metabolism , Corpus Striatum/drug effects , Levodopa/pharmacology , Phosphorylation/drug effects , Enzyme Activation/drug effects , Mice, Inbred C57BL , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/drug effects , Humans
2.
bioRxiv ; 2023 Oct 30.
Article in English | MEDLINE | ID: mdl-37961199

ABSTRACT

In rodents with unilateral ablation of the substantia nigra neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA or dopamine agonists induces a progressive increase of behavioral responses, a process known as behavioral sensitization. The sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of arrestin-3 knockout mice, we found that the restoration of arrestin-3 fully rescued behavioral sensitization, whereas its mutant defective in JNK activation did not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in the direct pathway striatal neurons, fully rescued sensitization, whereas an inactive homologous arrestin-2-derived peptide did not. Behavioral rescue was accompanied by the restoration of JNK3 activity and of JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-dependent JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization.

3.
Clin Lab ; 67(7)2021 Jul 01.
Article in English | MEDLINE | ID: mdl-34258959

ABSTRACT

BACKGROUND: MicroRNA-21 (miR-21) is a small non-coding RNA which influences tumorigenesis by inhibiting the expression of target genes. Ki-67 is a nucleolar antigen highly correlated with the rate of proliferating cells. In this study, we aimed to evaluate the prognostic impact of miR-21 and Ki-67 in DLBCL disease in a cohort of Egyptian patients. METHODS: We prospectively enrolled 53 newly diagnosed DLBCL patients. RT-PCR was used to evaluate the plasma expression levels of miR-21. Tissue Ki-67 was assessed using immunohistochemistry (IHC) of lymph node biopsy sections. Overall survival (OS) and progression free survival (PFS) were the primary outcomes. RESULTS: miR-21 expression was significantly higher in patients with DLBCL in comparison to controls (p < 0.001). The median Ki-67 expression was 70% and positivity ranged from 25% to 100%. Response to treatment was achieved in 23 patients (43.4%). Higher miR-21 was associated with poor response to treatment (p = 0.03). Although patients' age was a significant predictor of OS in univariate analysis, none of the studied factors could predict OS in multivariate analysis. However, we found that Ki-67 expression was a significant predictor of PFS in both univariate and multivariate analyses. CONCLUSIONS: The study suggested that plasma miR-21 might be a valuable non-invasive prognostic marker of response to treatment in DLBCL patients. Moreover, Ki-67 is a potential significant predictor of both OS and PFS in those patients.


Subject(s)
Lymphoma, Large B-Cell, Diffuse , MicroRNAs , Antineoplastic Combined Chemotherapy Protocols , Disease-Free Survival , Egypt , Humans , Ki-67 Antigen/genetics , Lymphoma, Large B-Cell, Diffuse/diagnosis , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/genetics , MicroRNAs/genetics , Prognosis
4.
Sci Rep ; 10(1): 5028, 2020 03 19.
Article in English | MEDLINE | ID: mdl-32193420

ABSTRACT

Loss-of-function mutations in the E3 ubiquitin ligase parkin have been implicated in the death of dopaminergic neurons in the substantia nigra, which is the root cause of dopamine deficit in the striatum in Parkinson's disease. Parkin ubiquitinates proteins on mitochondria that lost membrane potential, promoting the elimination of damaged mitochondria. Neuroprotective activity of parkin has been linked to its critical role in the mitochondria maintenance. Here we report a novel regulatory mechanism: another E3 ubiquitin ligase Mdm2 directly binds parkin and enhances its enzymatic activity in vitro and in intact cells. Mdm2 translocates to damaged mitochondria independently of parkin, enhances parkin-dependent ubiquitination of the outer mitochondria membrane protein mitofusin1. Mdm2 facilitates and its knockdown reduces parkin-dependent mitophagy. Thus, ubiquitously expressed Mdm2 might enhance cytoprotective parkin activity. The data suggest that parkin activation by Mdm2 could be targeted to increase its neuroprotective functions, which has implications for anti-parkinsonian therapy.


Subject(s)
Mitophagy/genetics , Mitophagy/physiology , Neuroprotective Agents , Parkinson Disease/genetics , Proto-Oncogene Proteins c-mdm2/physiology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Dopaminergic Neurons/pathology , GTP Phosphohydrolases/metabolism , HEK293 Cells , Humans , Loss of Function Mutation , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Targeted Therapy , Parkinson Disease/etiology , Parkinson Disease/pathology , Parkinson Disease/therapy , Protein Binding , Proto-Oncogene Proteins c-mdm2/metabolism , Ubiquitin-Protein Ligases/physiology , Ubiquitination
5.
Sci Rep ; 5: 10920, 2015 Jun 04.
Article in English | MEDLINE | ID: mdl-26043205

ABSTRACT

Degeneration of dopaminergic neurons causes Parkinson's disease. Dopamine replacement therapy with L-DOPA is the best available treatment. However, patients develop L-DOPA-induced dyskinesia (LID). In the hemiparkinsonian rat, chronic L-DOPA increases rotations and abnormal involuntary movements modeling LID, via supersensitive dopamine receptors. Dopamine receptors are controlled by G protein-coupled receptor kinases (GRKs). Here we demonstrate that LID is attenuated by overexpression of GRK3 in the striatum, whereas knockdown of GRK3 by microRNA exacerbated it. Kinase-dead GRK3 and its separated RGS homology domain (RH) suppressed sensitization to L-DOPA, whereas GRK3 with disabled RH did not. RH alleviated LID without compromising anti-akinetic effect of L-DOPA. RH binds striatal Gq. GRK3, kinase-dead GRK3, and RH inhibited accumulation of ∆FosB, a marker of LID. RH-dead mutant was ineffective, whereas GRK3 knockdown exacerbated ∆FosB accumulation. Our findings reveal a novel mechanism of GRK3 control of the dopamine receptor signaling and the role of Gq in LID.


Subject(s)
Dyskinesias/metabolism , G-Protein-Coupled Receptor Kinase 3/metabolism , Levodopa/metabolism , Parkinson Disease/metabolism , Protein Interaction Domains and Motifs , RGS Proteins/metabolism , Animals , Behavior, Animal , Corpus Striatum/metabolism , Disease Models, Animal , Dyskinesias/etiology , G-Protein-Coupled Receptor Kinase 3/chemistry , G-Protein-Coupled Receptor Kinase 3/genetics , Gene Expression , Gene Knockdown Techniques , Parkinson Disease/complications , Parkinson Disease/genetics , RNA Interference , RNA, Small Interfering/genetics , Rats , Signal Transduction
6.
PLoS One ; 7(11): e48912, 2012.
Article in English | MEDLINE | ID: mdl-23139825

ABSTRACT

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.


Subject(s)
Arrestin/metabolism , G-Protein-Coupled Receptor Kinases/metabolism , Neostriatum/cytology , Neostriatum/enzymology , Animals , Cell Nucleus/enzymology , Cholinergic Neurons/cytology , Cholinergic Neurons/enzymology , Female , Humans , Interneurons/cytology , Interneurons/enzymology , Isoenzymes/metabolism , Male , Middle Aged , Phosphorylation , Protein Transport , Rats , Rats, Sprague-Dawley , Subcellular Fractions/enzymology
7.
Sci Transl Med ; 2(28): 28ra28, 2010 Apr 21.
Article in English | MEDLINE | ID: mdl-20410529

ABSTRACT

Parkinson's disease is caused primarily by degeneration of brain dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine in the striatum. Dopamine replacement therapy with the dopamine precursor l-dopa is the mainstay of current treatment. After several years, however, the patients develop l-dopa-induced dyskinesia, or abnormal involuntary movements, thought to be due to excessive signaling via dopamine receptors. G protein-coupled receptor kinases (GRKs) control desensitization of dopamine receptors. We found that dyskinesia is attenuated by lentivirus-mediated overexpression of GRK6 in the striatum in rodent and primate models of Parkinson's disease. Conversely, reduction of GRK6 concentration by microRNA delivered with lentiviral vector exacerbated dyskinesia in parkinsonian rats. GRK6 suppressed dyskinesia in monkeys without compromising the antiparkinsonian effects of l-dopa and even prolonged the antiparkinsonian effect of a lower dose of l-dopa. Our finding that increased availability of GRK6 ameliorates dyskinesia and increases duration of the antiparkinsonian action of l-dopa suggests a promising approach for controlling both dyskinesia and motor fluctuations in Parkinson's disease.


Subject(s)
Dyskinesias/complications , Dyskinesias/prevention & control , G-Protein-Coupled Receptor Kinases/therapeutic use , Genetic Therapy , Lentivirus/genetics , Parkinsonian Disorders/complications , Parkinsonian Disorders/therapy , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Animals , Antiparkinson Agents/pharmacology , Antiparkinson Agents/therapeutic use , Behavior, Animal/drug effects , Dose-Response Relationship, Drug , Endocytosis/drug effects , G-Protein-Coupled Receptor Kinases/genetics , Gene Knockdown Techniques , Humans , Levodopa , Macaca , Oxidopamine/pharmacology , Parkinsonian Disorders/drug therapy , Parkinsonian Disorders/genetics , Rats , Rats, Sprague-Dawley , Receptors, Dopamine D1/agonists , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/agonists , Receptors, Dopamine D2/metabolism , Rotation , Signal Transduction/drug effects
8.
Gene Expr ; 13(4-5): 255-69, 2007.
Article in English | MEDLINE | ID: mdl-17605299

ABSTRACT

Disruption of the genes encoding for the transcription coactivators, peroxisome proliferator-activated receptor (PPAR)-interacting protein (PRIP/ASC-2/RAP250/TRBP/NRC) and PPAR-binding protein (PBP/TRAP220/DRIP205/MED1), results in embryonic lethality by affecting placental and multiorgan development. Targeted deletion of coactivator PBP gene in liver parenchymal cells (PBP(LIV-/-)) results in the near abrogation of the induction of PPARalpha and CAR (constitutive androstane receptor)-regulated genes in liver. Here, we show that targeted deletion of coactivator PRIP gene in liver (PRIP(LIV-/-)) does not affect the induction of PPARalpha-regulated pleiotropic responses, including hepatomegaly, hepatic peroxisome proliferation, and induction of mRNAs of genes involved in fatty acid oxidation system, indicating that PRIP is not essential for PPARalpha-mediated transcriptional activity. We also provide additional data to show that liver-specific deletion of PRIP gene does not interfere with the induction of genes regulated by nuclear receptor CAR. Furthermore, disruption of PRIP gene in liver did not alter zoxazolamine-induced paralysis, and acetaminophen-induced hepatotoxicity. Studies with adenovirally driven EGFP-CAR expression in liver demonstrated that, unlike PBP, the absence of PRIP does not prevent phenobarbital-mediated nuclear translocation/retention of the receptor CAR in liver in vivo and cultured hepatocytes in vitro. These results show that PRIP deficiency in liver does not interfere with the function of nuclear receptors PPARalpha and CAR. The dependence of PPARalpha- and CAR-regulated gene transcription on coactivator PBP but not on PRIP attests to the existence of coactivator selectivity in nuclear receptor function.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , PPAR alpha/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Acetaminophen/toxicity , Active Transport, Cell Nucleus/drug effects , Analgesics, Non-Narcotic/toxicity , Animals , Anticonvulsants/pharmacology , Cells, Cultured , Constitutive Androstane Receptor , Gene Expression Regulation , Gene Targeting , Hepatocytes/cytology , Hepatocytes/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Liver/cytology , Male , Mice , Mice, Transgenic , Muscle Relaxants, Central/metabolism , Nuclear Receptor Coactivators , Organ Size , PPAR alpha/genetics , Phenobarbital/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Zoxazolamine/metabolism
9.
J Biol Chem ; 282(23): 17053-60, 2007 Jun 08.
Article in English | MEDLINE | ID: mdl-17438330

ABSTRACT

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (Pbp(DeltaLiv)) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G(2)/M phase in Pbp(DeltaLiv) hepatocytes after partial hepatectomy. Pbp(DeltaLiv) hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl(4)-induced hepatocellular proliferation and hepatotoxicity occurred in Pbp(DeltaLiv) mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl(4) activation. Pbp(DeltaLiv) mice, chronically exposed to Wy-14,643, a PPARalpha ligand, revealed a striking proliferative response and clonal expansion of a few Pbp(fl/fl) hepatocytes that escaped Cre-mediated gene deletion in Pbp(DeltaLiv) livers, but no proliferative expansion of PBP null hepatocytes was observed. In these Pbp(DeltaLiv) mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBP(DeltaLiv) hepatocytes; all liver tumors developing in Pbp(DeltaLiv) mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.


Subject(s)
Liver Neoplasms, Experimental/physiopathology , Liver Regeneration/physiology , PPAR alpha/metabolism , Transcription Factors/physiology , Animals , Base Sequence , DNA Primers , Hepatectomy , Ligands , Liver Neoplasms, Experimental/pathology , Mediator Complex Subunit 1 , Mice
10.
Proc Natl Acad Sci U S A ; 104(9): 3125-8, 2007 Feb 27.
Article in English | MEDLINE | ID: mdl-17360618

ABSTRACT

Arrestins (Arrs) are ubiquitous regulators of the most numerous family of signaling proteins, G protein-coupled receptors. Two models of the Arr-receptor interaction have been proposed: the binding of one Arr to an individual receptor or to two receptors in a dimer. To determine the binding stoichiometry in vivo, we used rod photoreceptors where rhodopsin (Rh) and Arr are expressed at comparably high levels and where Arr localization in the light is determined by its binding to activated Rh. Genetic manipulation of the expression of both proteins shows that the maximum amount of Arr that moves to the Rh-containing compartment exceeds 80%, but not 100%, of the molar amount of Rh present. In vitro experiments with purified proteins confirm that Arr "saturates" Rh at a 1:1 ratio. Thus, a single Rh molecule is necessary and sufficient to bind Arr. Remarkable structural conservation among receptors and Arrs strongly suggests that all Arr subtypes bind individual molecules of their cognate receptors.


Subject(s)
Arrestin/metabolism , Models, Molecular , Protein Binding , Retinal Rod Photoreceptor Cells/metabolism , Rhodopsin/metabolism , Animals , Arrestin/genetics , Dimerization , Immunohistochemistry , Mice , Mice, Knockout , Protein Transport , Rhodopsin/genetics
11.
Biochem Biophys Res Commun ; 347(2): 485-95, 2006 Aug 25.
Article in English | MEDLINE | ID: mdl-16828057

ABSTRACT

The constitutive androstane receptor (CAR) regulates transcription of phenobarbital-inducible genes that encode xenobiotic-metabolizing enzymes in liver. CAR is localized to the hepatocyte cytoplasm but to be functional, it translocates into the nucleus in the presence of phenobarbital-like CAR ligands. We now demonstrate that adenovirally driven EGFP-CAR, as expected, translocates into the nucleus of normal wild-type hepatocytes following phenobarbital treatment under both in vivo and in vitro conditions. Using this approach we investigated the role of transcription coactivators PBP and PRIP in the translocation of EGFP-CAR into the nucleus of PBP and PRIP liver conditional null mouse hepatocytes. We show that coactivator PBP is essential for nuclear translocation of CAR but not PRIP. Adenoviral expression of both PBP and EGFP-CAR restored phenobarbital-mediated nuclear translocation of exogenously expressed CAR in PBP null livers in vivo and in PBP null primary hepatocytes in vitro. CAR translocation into the nucleus of PRIP null livers resulted in the induction of CAR target genes such as CYP2B10, necessary for the conversion of acetaminophen to its hepatotoxic intermediate metabolite, N-acetyl-p-benzoquinone imine. As a consequence, PRIP-deficiency in liver did not protect from acetaminophen-induced hepatic necrosis, unlike that exerted by PBP deficiency. These results establish that transcription coactivator PBP plays a pivotal role in nuclear localization of CAR, that it is likely that PBP either enhances nuclear import or nuclear retention of CAR in hepatocytes, and that PRIP is redundant for CAR function.


Subject(s)
Cell Nucleus/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Acetaminophen/toxicity , Active Transport, Cell Nucleus/drug effects , Adenoviridae/genetics , Animals , Blotting, Northern , Cells, Cultured , Constitutive Androstane Receptor , Cytoplasm/metabolism , Gene Expression/drug effects , Genetic Vectors/genetics , Genotype , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Immunoblotting , Intracellular Signaling Peptides and Proteins/genetics , Liver/drug effects , Liver/pathology , Mediator Complex Subunit 1 , Mice , Mice, Inbred C57BL , Mice, Knockout , Necrosis/chemically induced , Necrosis/genetics , Necrosis/metabolism , Nuclear Receptor Coactivators , Phenobarbital/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics
12.
J Laryngol Otol ; 119(1): 2-7, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15807953

ABSTRACT

Intratympanic steroids offer direct access to the inner ear with high concentration and without systemic effects. In this study, the efficacy of intratympanic methylprednisolone acetate (IT-MPA) was evaluated in a guinea-pig model of drill-induced inner ear trauma. Twenty-five guinea pigs were divided into a control group to document the baseline distortion product otoacoustic emissions (DPOAEs) and the normal scanning electron microscopic (SEM) morphology of the inner ear. The animals in the study group were subdivided into a steroid-only group (S), a trauma-only group (T), a trauma-plus-time group (TT), and a trauma-plus-steroid (TS) group. IT-MPA was found to have no damaging effect on the inner ear. Twelve days after trauma, there was spontaneous although incomplete recovery of the DPOAEs amplitudes and SEM morphology with scar tissue replacing lost outer hair cells. Statistically higher DPOAEs amplitudes (p < 0.05) were recorded in the TS group that had nearly normal SEM morphology compared to the TT group. The authors conclude that IT-MPA significantly improves drill-induced sensorineural hearing loss and inner ear morphological changes in guinea pigs.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Cochlea/pathology , Hearing Loss, Sensorineural/drug therapy , Methylprednisolone/analogs & derivatives , Methylprednisolone/administration & dosage , Animals , Drug Administration Routes , Drug Evaluation, Preclinical/methods , Female , Guinea Pigs , Hair Cells, Auditory, Inner/pathology , Hair Cells, Auditory, Outer/pathology , Hearing Loss, Sensorineural/pathology , Male , Methylprednisolone Acetate , Microscopy, Electron, Scanning/methods , Models, Animal , Noise , Organ of Corti/pathology , Otoacoustic Emissions, Spontaneous , Tympanic Membrane
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