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1.
Sci Rep ; 11(1): 19396, 2021 09 29.
Article in English | MEDLINE | ID: mdl-34588551

ABSTRACT

Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major hepatic fibrogenic cell population. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human livers. In conjunction with public single-cell data of fibrotic/cirrhotic human livers, these profiles enable the identification of potential intercellular signaling axes (e.g., ITGAV-LAMC1, TNFRSF11B-VWF and NOTCH2-DLL4) and master regulators (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH-related fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study highlights potential cell-cell interactions and master regulators that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis.


Subject(s)
Hepatic Stellate Cells , Non-alcoholic Fatty Liver Disease , Transcriptome , Animals , Healthy Volunteers , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Rats , Single-Cell Analysis
3.
Proc Natl Acad Sci U S A ; 115(2): E180-E189, 2018 01 09.
Article in English | MEDLINE | ID: mdl-29269392

ABSTRACT

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.


Subject(s)
CRISPR-Cas Systems , Gene Expression Regulation , Genome, Human/genetics , Mitophagy/genetics , Repressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Cell Line, Tumor , Cells, Cultured , HCT116 Cells , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Infant, Newborn , Neurons/metabolism , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Repressor Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism
4.
J Neurochem ; 119(2): 398-407, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21854390

ABSTRACT

Huntington's disease is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. This expansion produces a mutant form of the huntingtin protein, which contains an elongated polyglutamine stretch at its amino-terminus. Mutant huntingtin may adopt an aberrant, aggregation-prone conformation predicted to start the pathogenic process leading to neuronal dysfunction and cell death. Thus, strategies reducing mutant huntingtin may lead to disease-modifying therapies. We investigated the mechanisms and molecular targets regulating huntingtin degradation in a neuronal cell model. We first found that mutant and wild-type huntingtin displayed strikingly diverse turn-over kinetics and sensitivity to proteasome inhibition. Then, we show that autophagy induction led to accelerate degradation of mutant huntingtin aggregates. In our neuronal cell model, allosteric inhibition of mTORC1 by everolimus, a rapamycin analogue, did not induce autophagy or affect aggregate degradation. In contrast, this occurred in the presence of catalytic inhibitors of both mTOR complexes mTORC1 and mTORC2. Our data demonstrate the existence of an mTOR-dependent but everolimus-independent mechanism regulating autophagy and huntingtin-aggregate degradation in cells of neuronal origin.


Subject(s)
Autophagy/drug effects , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , Blotting, Western , Cells, Cultured , Everolimus , Exons/genetics , Humans , Huntingtin Protein , Immunoassay , Immunosuppressive Agents/pharmacology , Kinetics , Mechanistic Target of Rapamycin Complex 1 , Models, Neurological , Multiprotein Complexes , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Proteins/antagonists & inhibitors , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Sirolimus/analogs & derivatives , Sirolimus/pharmacology
5.
J Biol Chem ; 284(22): 15325-38, 2009 May 29.
Article in English | MEDLINE | ID: mdl-19346254

ABSTRACT

The tumor suppressor gene Lot1 is highly expressed during brain development. During cerebellar development, Lot1 is expressed by proliferating granule cells with a time course matching the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, a neuropeptide receptor that plays an important role in the regulation of granule cell proliferation/survival. Although it has become clear that Lot1 is a negative regulator of cell division in tumor cells, its role in neuronal proliferation is not understood. We previously demonstrated that in cerebellar granule cells Lot1 expression is regulated by the PACAP/cAMP system. The aim of this study was to investigate the role played by Lot1 in neuron proliferation/survival and to identify the molecular mechanisms underlying its actions. Using a Lot1-inducible expression system, we found that in PC12 cells Lot1 negatively regulates proliferation and favors differentiation by up-regulating the expression of the PACAP receptor. In cerebellar granule cells in culture, an increase in Lot1 expression was paralleled by inhibition of proliferation and up-regulation of the PACAP receptor, which in turn positively regulated Lot1 expression. Silencing of Lot1 leads to an increase in granule cell proliferation and a reduction in survival. Confirming the in vitro results, in vivo experiments showed that PACAP induced an increase in Lot1 expression that was paralleled by inhibition of cerebellar granule cell proliferation. These data show that Lot1 is a key element of the PACAP/cAMP pathway that negatively regulates neuronal precursor proliferation. The existence of a PACAP receptor/Lot1-positive feedback loop may powerfully regulate neural proliferation during critical phases of cerebellar development.


Subject(s)
Cell Cycle Proteins/metabolism , Cyclic AMP/metabolism , Neurons/cytology , Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Stem Cells/cytology , Transcription Factors/metabolism , Animals , Cell Cycle/drug effects , Cell Death/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cerebellum/cytology , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Genes, Tumor Suppressor , Mitogen-Activated Protein Kinase Kinases/metabolism , Models, Biological , Nerve Growth Factors/pharmacology , Neurons/drug effects , Neurons/enzymology , PC12 Cells , Rats , Rats, Wistar , Stem Cells/drug effects , Stem Cells/metabolism
6.
J Neurochem ; 103(2): 518-30, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17635667

ABSTRACT

The physiological role of alpha-synuclein, a protein found enriched in intraneuronal deposits characterizing Parkinson's disease, is debated. While its aggregation is usually considered linked to neuropathology, its normal function may be related to fundamental processes of synaptic transmission and plasticity. By using antisense oligonucleotide strategy, we report in this study that alpha-synuclein silencing in cultured cerebellar granule cells results in widespread death of these neurons, thus demonstrating an essential pro-survival role of the protein towards primary neurons. To study alpha-synuclein expression and processing in a Parkinson's disease model of neurotoxicity, we exposed differentiated cultures of cerebellar granule neurons to toxic concentrations of 6-hydroxydopamine (6-OHDA). This resulted in neuronal death accompanied by a decrease of the monomeric form of alpha-synuclein, which was due to both decreased synthesis of the protein and its increased mono-ubiquitination accompanied by nuclear translocation. The essential neuroprotective role of alpha-synuclein was confirmed by the fact that subchronic valproate treatment, which increases alpha-synuclein expression and prevents its nuclear translocation in cerebellar granule cells exposed to 6-OHDA, significantly protected these neurons from 6-OHDA insult. In agreement with the pro-survival role of alpha-synuclein in this model, subtoxic concentrations of alpha-synuclein antisense oligonucleotides, aggravated 6-OHDA toxicity towards granule neurons. Our results demonstrate that normal alpha-synuclein expression is essential for the viability of primary neurons and that its pro-survival role is abolished in 6-OHDA neurotoxic challenge. These results are relevant to more precisely define the role of alpha-synuclein in neuronal cells and to better understand its putative involvement in neurodegeneration.


Subject(s)
Cerebellum/cytology , Cerebellum/drug effects , Neurons/drug effects , Neuroprotective Agents , Oxidopamine/antagonists & inhibitors , Sympatholytics/antagonists & inhibitors , alpha-Synuclein/pharmacology , Animals , Apoptosis/drug effects , Blotting, Western , Cell Death/drug effects , Cell Death/genetics , Cell Nucleus/chemistry , Cell Survival/drug effects , Cytoplasmic Granules/drug effects , Cytosol/chemistry , Gene Silencing , Immunoprecipitation , Male , Oligonucleotides, Antisense/pharmacology , Oxidopamine/toxicity , Proteasome Endopeptidase Complex/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Sympatholytics/toxicity , Translocation, Genetic/drug effects , Valproic Acid/pharmacology , alpha-Synuclein/antagonists & inhibitors , alpha-Synuclein/genetics
7.
J Cell Sci ; 119(Pt 15): 3161-70, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16835271

ABSTRACT

The diffusible messenger, nitric oxide plays multiple roles in neuroprotection, neurodegeneration and brain plasticity. Its involvement in neurogenesis has been disputed, on the basis of results on models in vivo and in culture. We report here that pharmacological blockade of nitric oxide production in rat pups resulted, during a restricted time window of the first three postnatal days, in increased cerebellar proliferation rate, as assessed through tritiated thymidine or BrdU incorporation into DNA. This was accompanied by increased expression of Myc, a transcription factor essential for cerebellar development, and of the cell cycle regulating gene, cyclin D1. These effects were mediated downstream by the nitric oxide-dependent second messenger, cGMP. Schedules of pharmacological NO deprivation targeted to later developmental stages (from postnatal day 3 to 7), no longer increased proliferation, probably because of partial escape of the cGMP level from nitric oxide control. Though limited to a brief temporal window, the proliferative effect of neonatal nitric oxide deprivation could be traced into adulthood. Indeed, the number of BrdU-labeled surviving cells, most of which were of neuronal phenotype, was larger in the cerebellum of 60-day-old rats that had been subjected to NO deprivation during the first three postnatal days than in control rats. Experiments on cell cultures from neonatal cerebellum confirmed that nitric oxide deprivation stimulated proliferation of cerebellar precursor cells and that this effect was not additive with the proliferative action of sonic hedgehog peptide. The finding that nitric oxide deprivation during early cerebellar neurogenesis, stimulates a brief increase in cell proliferation may contribute to a better understanding of the controversial role of nitric oxide in brain development.


Subject(s)
Cell Proliferation , Cerebellum , Nitric Oxide/metabolism , Stem Cells/metabolism , Animals , Animals, Newborn , Cells, Cultured , Cerebellum/cytology , Cerebellum/growth & development , Cerebellum/metabolism , Cyclic GMP/metabolism , Enzyme Inhibitors/metabolism , Female , Guanylate Cyclase/metabolism , NG-Nitroarginine Methyl Ester/metabolism , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitric Oxide Synthase Type I/metabolism , Pregnancy , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Rats , Rats, Wistar , Signal Transduction/physiology , Stem Cells/cytology
8.
Neurochem Int ; 48(3): 201-7, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16290266

ABSTRACT

Natural polyamines (putrescine, spermidine and spermine) are ubiquitous molecules known to regulate a number of physiological processes and suspected to play a role also in various pathological conditions. Changes in polyamine levels and in their biosynthetic enzymes have been described for some neurodegenerative diseases but the available data are incomplete and somewhat contradictory. We report here alterations of the key enzyme of the polyamine pathway, ornithine decarboxylase (ODC) catalytic activity and polyamine levels in different CNS areas from SOD1 G39A transgenic mice, an animal model for amyotrophic lateral sclerosis (ALS). ODC catalytic activity, was found significantly increased both in the cervical and lumbar spinal cord and, to a lesser extent in the brain stem of transgenic mice at a symptomatic stage of the disease (125-day-old mice), while no differences were present at a pre-symptomatic stage (55-day-old mice). In parallel with the increase of ODC activity putrescine levels were several times increased in both cervical and lumbar spinal cord and in the brain stem of 125-day-old SOD1 G39A mice. Higher order polyamines were not increased except for a significant increase of spermidine in the cervical spinal cord. The present data demonstrate considerable alterations of the ODC/polyamine system in a reliable animal model of ASL, consistent with their role in neurodegeneration and in particular in motor neuron diseases.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Biogenic Polyamines/metabolism , Central Nervous System/metabolism , Ornithine Decarboxylase/metabolism , Amyotrophic Lateral Sclerosis/enzymology , Animals , Central Nervous System/enzymology , Mice , Mice, Transgenic
9.
Brain Res ; 1058(1-2): 183-8, 2005 Oct 05.
Article in English | MEDLINE | ID: mdl-16140286

ABSTRACT

Dietary supplementation with creatine has proven to be beneficial in models of acute and chronic neurodegeneration. We report here data on the neurochemical correlates of differential protection of long-term creatine supplementation in two models of excitotoxicity in rats, as well as in the mouse model for ALS (G93A mice). In rats, the fall in cholinergic and GABAergic markers due to the excitotoxic death of intrinsic neurons caused by intrastriatal infusion of the neurotoxin, ibotenic acid, was significantly prevented by long-term dietary supplementation with creatine. On the contrary, creatine was unable to recover a cholinergic marker in the cortex of rats subjected to the excitotoxic death of the cholinergic basal forebrain neurons. In G93A mice, long-term creatine supplementation marginally but significantly increased mean lifespan, as previously observed by others, and reverted the cholinergic deficit present in some forebrain areas at an intermediate stage of the disease. In both rats and mice, creatine supplementation increased the activity of the GABAergic enzyme, glutamate decarboxylase, in the striatum but not in other brain regions. The present data point at alterations of neurochemical parameters marking specific neuronal populations, as a useful way to evaluate neuroprotective effects of long-term creatine supplementation in animal models of neurodegeneration.


Subject(s)
Brain/drug effects , Creatine/pharmacology , Dietary Supplements , Neurodegenerative Diseases/drug therapy , Neuroprotective Agents/pharmacology , Neurotoxins/antagonists & inhibitors , Acetylcholine/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Basal Nucleus of Meynert/drug effects , Basal Nucleus of Meynert/metabolism , Basal Nucleus of Meynert/physiopathology , Biomarkers/analysis , Biomarkers/metabolism , Brain/metabolism , Brain/physiopathology , Cell Death/drug effects , Cell Death/physiology , Cholinergic Fibers/drug effects , Cholinergic Fibers/metabolism , Cholinergic Fibers/pathology , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Creatine/therapeutic use , Glutamate Decarboxylase/drug effects , Glutamate Decarboxylase/metabolism , Ibotenic Acid/antagonists & inhibitors , Male , Mice , Mice, Transgenic , Neurodegenerative Diseases/chemically induced , Neurodegenerative Diseases/metabolism , Neuroprotective Agents/therapeutic use , Neurotoxins/toxicity , Rats , Rats, Wistar , Survival Rate , Time , Treatment Outcome
10.
Neurochem Int ; 46(5): 357-68, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15737434

ABSTRACT

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.


Subject(s)
Acetylcholine/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Basal Nucleus of Meynert/metabolism , Cholinergic Fibers/metabolism , Down-Regulation/physiology , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Basal Nucleus of Meynert/pathology , Brain Stem/metabolism , Brain Stem/pathology , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Choline/metabolism , Choline/therapeutic use , Choline O-Acetyltransferase/metabolism , Cholinergic Fibers/pathology , Dietary Supplements , Disease Models, Animal , Female , Hippocampus/metabolism , Hippocampus/pathology , Male , Maternal-Fetal Exchange , Mice , Mice, Transgenic , Motor Neurons/metabolism , Motor Neurons/pathology , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Neural Pathways/metabolism , Neural Pathways/pathology , Pregnancy , Spinal Cord/metabolism , Spinal Cord/pathology , Superoxide Dismutase-1 , Treatment Outcome , gamma-Aminobutyric Acid/metabolism
11.
FASEB J ; 16(8): 761-70, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12039857

ABSTRACT

In analyzing the molecular mechanisms underlying glucocorticoid-induced apoptosis in neural cells, we observed that dexamethasone, by activating glucocorticoid receptors, causes arrest of HT-22 cells in the G1 phase of the cell cycle; upon withdrawal of the agonist, cells resume proliferation. Our investigations revealed that glucocorticoid treatment, although having no effects on endogenous p53 protein stability, induces rapid translocation of p53 to the nucleus and enhances its transcriptional activity. Consistently, transfection studies with p53-responsive promoters revealed a substantial stimulation of the trans-activation potential of exogenous p53 by dexamethasone. Cells arrested in G1 failed to show signs of apoptosis even after overexpression of p53. Although dexamethasone induced transcription of the proapoptotic gene bax, there was no increase of Bax protein levels. We conclude that glucocorticoid receptor-induced neural cell cycle arrest is associated with an increase in nuclear translocation and transcriptional activity of p53, and suggest that potentiation of p53 may serve as a brake on cell proliferation and may prime cells for differentiation or death induced by other signals.


Subject(s)
Neurons/metabolism , Receptors, Glucocorticoid/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Apoptosis/drug effects , Biological Transport/drug effects , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/drug effects , Cyclins/genetics , Cyclins/metabolism , Dexamethasone/pharmacology , Flow Cytometry , G1 Phase/drug effects , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Hormone Antagonists/pharmacology , Humans , In Situ Nick-End Labeling , Mifepristone/pharmacology , Neurons/cytology , Neurons/drug effects , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Glucocorticoid/drug effects , Tumor Cells, Cultured , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/metabolism
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