Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
Sci Adv ; 6(2): eaaw8702, 2020 01.
Article in English | MEDLINE | ID: mdl-31934620

ABSTRACT

Impaired mitochondrial dynamics and function are hallmarks of many neurological and psychiatric disorders, but direct screens for mitotherapeutics using neurons have not been reported. We developed a multiplexed and high-content screening assay using primary neurons and identified 67 small-molecule modulators of neuronal mitostasis (MnMs). Most MnMs that increased mitochondrial content, length, and/or health also increased mitochondrial function without altering neurite outgrowth. A subset of MnMs protected mitochondria in primary neurons from Aß(1-42) toxicity, glutamate toxicity, and increased oxidative stress. Some MnMs were shown to directly target mitochondria. The top MnM also increased the synaptic activity of hippocampal neurons and proved to be potent in vivo, increasing the respiration rate of brain mitochondria after administering the compound to mice. Our results offer a platform that directly queries mitostasis processes in neurons, a collection of small-molecule modulators of mitochondrial dynamics and function, and candidate molecules for mitotherapeutics.


Subject(s)
Central Nervous System/cytology , High-Throughput Screening Assays , Mitochondria/metabolism , Neurons/cytology , Adenosine Triphosphate/biosynthesis , Animals , Cells, Cultured , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/pathology , Mitochondrial Dynamics/drug effects , Neurodegenerative Diseases/pathology , Neurons/drug effects , Neurons/metabolism , Phenotype , Propiophenones/pharmacology , Small Molecule Libraries/pharmacology , Synapses/drug effects , Synapses/metabolism
2.
PLoS One ; 13(12): e0209405, 2018.
Article in English | MEDLINE | ID: mdl-30576353

ABSTRACT

Cognitive decline is a common occurrence of the natural aging process in animals and studying age-related changes in gene expression in the brain might shed light on disrupted molecular pathways that play a role in this decline. The fruit fly is a useful neurobiological model for studying aging due to its short generational time and relatively small brain size. We investigated age-dependent changes in the Drosophila melanogaster whole-brain transcriptome by comparing 5-, 20-, 30- and 40-day-old flies of both sexes. We used RNA-Sequencing of dissected brain samples followed by differential expression, temporal clustering, co-expression network and gene ontology enrichment analyses. We found an overall decline in expression of genes from the mitochondrial oxidative phosphorylation pathway that occurred as part of aging. We also detected, in females, a pattern of continuously declining expression for many neuronal function genes, which was unexpectedly reversed later in life. This group of genes was highly enriched in memory-impairing genes previously identified through an RNAi screen. We also identified deficits in short-term olfactory memory performance in older flies of both sexes, some of which matched the timing of certain changes in the brain transcriptome. Our study provides the first transcriptome profile of aging brains from fruit flies of both sexes, and it will serve as an important resource for those who study aging and cognitive decline in this model.


Subject(s)
Aging/physiology , Brain/metabolism , Cognitive Dysfunction/physiopathology , Memory/physiology , Olfactory Perception/physiology , Age Factors , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster , Female , Gene Expression Profiling , Gene Regulatory Networks/physiology , Male , Mitochondria/metabolism , Models, Animal , Oxidative Phosphorylation , Sequence Analysis, RNA , Sex Factors , Transcriptome/physiology
4.
Cell Rep ; 22(1): 255-268, 2018 01 02.
Article in English | MEDLINE | ID: mdl-29298426

ABSTRACT

Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs) that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP), with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time.


Subject(s)
Cyclic AMP/metabolism , Molecular Imaging , Neurons/metabolism , Optogenetics , Receptors, G-Protein-Coupled/metabolism , Second Messenger Systems/physiology , Animals , Mice , Neurons/cytology
5.
Gene ; 606: 17-24, 2017 Mar 30.
Article in English | MEDLINE | ID: mdl-28042091

ABSTRACT

PDE10A is a cAMP/cGMP phosphodiesterase important in signal transduction within medium spiny neurons of the human striatum. This gene region has been associated with bipolar disorder via case-control and linkage studies. The three most studied human PDE10A isoforms differ in both their N-termini and trafficking within the cell with PDE10A2 found predominantly at the plasma membrane and PDE10A1 and PDE10A19 remaining primarily within the cytosol. RNA-sequencing and 5' RLM-RACE studies of the human putamen and caudate nucleus revealed 16 new exons and 12 novel transcripts of PDE10A, 3 of which are predicted to produce proteins with unique N-termini. The novel first exons of these transcripts are highly conserved in non-human primate species and are rarely found in other mammals. One hundred and eight previously classified intronic SNPs were found within the novel PDE10A exons of which 78% were classified as rare variants. Since most of the rare variants localize to 5' UTR regions, they may influence PDE10A transcription, translation, or mRNA stability. Dysregulation of cAMP signaling has been proposed as a cause of bipolar disorder and PDE10A inhibitors have been investigated as potential therapeutics for schizophrenia. Understanding the mechanisms contributing to PDE10A expression in the human striatum may provide evidence linking this gene to the phenotypes observed in neuropsychiatric disorders.


Subject(s)
Caudate Nucleus/metabolism , Phosphoric Diester Hydrolases/genetics , Putamen/metabolism , Aged , Animals , Bipolar Disorder/genetics , Female , Gene Expression Regulation , Humans , Male , Middle Aged , Neurons/metabolism , Nucleic Acid Amplification Techniques , Polymorphism, Single Nucleotide , Schizophrenia/genetics
6.
Diabetes ; 60(6): 1797-804, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21536946

ABSTRACT

OBJECTIVE: Congenital hyperinsulinemic hypoglycemia is a group of genetic disorders of insulin secretion most commonly associated with inactivating mutations of the ß-cell ATP-sensitive K(+) channel (K(ATP) channel) genes ABCC8 (SUR1) and KCNJ11 (Kir6.2). Recessive mutations of these genes cause hyperinsulinism that is unresponsive to treatment with diazoxide, a channel agonist. Dominant K(ATP) mutations have been associated with diazoxide-responsive disease. We hypothesized that some medically uncontrollable cases with only one K(ATP) mutation might have dominant, diazoxide-unresponsive disease. RESEARCH DESIGN AND METHODS: Mutations of the K(ATP) genes were identified by sequencing genomic DNA. Effects of mutations on K(ATP) channel function in vitro were studied by expression in COSm6 cells. RESULTS: In 15 families with diazoxide-unresponsive diffuse hyperinsulism, we found 17 patients with a monoallelic missense mutation of SUR1. Nine probands had de novo mutations, two had an affected sibling or parent, and four had an asymptomatic carrier parent. Of the 13 different mutations, 12 were novel. Expression of mutations revealed normal trafficking of channels but severely impaired responses to diazoxide or MgADP. Responses were significantly lower compared with nine SUR1 mutations associated with dominant, diazoxide-responsive hyperinsulinism. CONCLUSIONS: These results demonstrate that some dominant mutations of SUR1 can cause diazoxide-unresponsive hyperinsulinism. In vitro expression studies may be helpful in distinguishing such mutations from dominant mutations of SUR1 associated with diazoxide-responsive disease.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Congenital Hyperinsulinism/drug therapy , Congenital Hyperinsulinism/genetics , Diazoxide/therapeutic use , Potassium Channels, Inwardly Rectifying/genetics , Receptors, Drug/genetics , Antihypertensive Agents/therapeutic use , Humans , Models, Biological , Mutation , Mutation, Missense/genetics , Pedigree , Sulfonylurea Receptors
7.
Mod Pathol ; 19(1): 122-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16357843

ABSTRACT

Congenital hyperinsulinism is a rare pancreatic endocrine cell disorder that has been categorized histologically into diffuse and focal forms. In focal hyperinsulinism, the pancreas contains a focus of endocrine cell adenomatous hyperplasia, and the patients have been reported to possess paternally inherited mutations of the ABCC8 and KCNJ11 genes, which encode subunits of an ATP-sensitive potassium channel (K(ATP)). In addition, the hyperplastic endocrine cells show loss of maternal 11p15, where imprinted genes such as p57(kip2) reside. In order to evaluate whether all cases of focal hyperinsulinism are caused by this mechanism, 56 pancreatectomy specimens with focal hyperinsulinism were tested for the loss of maternal allele by two methods: immunohistochemistry for p57(kip2) (n=56) and microsatellite marker analysis (n=27). Additionally, 49 patients were analyzed for K(ATP) mutations. Out of 56 focal lesions, 48 demonstrated clear loss of p57(kip2) expression by immunohistochemistry. The other eight lesions similarly showed no nuclear labeling, but the available tissue was not ideal for definitive interpretation. Five of these eight patients had paternal K(ATP) mutations, of which four demonstrated loss of maternal 11p15 within the lesion by microsatellite marker analysis. All of the other three without a paternal K(ATP) mutation showed loss of maternal 11p15. K(ATP) mutation analysis identified 32/49 cases with paternal mutations. There were seven patients with nonmaternal mutations whose paternal DNA material was not available, and one patient with a mutation that was not present in either parent's DNA. These eight patients showed either loss of p57(kip2) expression or loss of maternal 11p15 region by microsatellite marker analysis, as did the remaining nine patients with no identifiable K(ATP) coding region mutations. The combined results from the immunohistochemical and molecular methods indicate that maternal 11p15 loss together with paternal K(ATP) mutation is the predominant causative mechanism of focal hyperinsulinism.


Subject(s)
Congenital Hyperinsulinism/surgery , Pancreas/pathology , Pancreatectomy , ATP-Binding Cassette Transporters/genetics , Adenosine Triphosphate/metabolism , Chromosomes, Human, Pair 11/genetics , Congenital Hyperinsulinism/genetics , Congenital Hyperinsulinism/metabolism , Cyclin-Dependent Kinase Inhibitor p57/biosynthesis , Haplotypes/genetics , Humans , Immunohistochemistry , Infant , Infant, Newborn , Microsatellite Repeats/genetics , Mutation , Pancreas/metabolism , Pancreas/surgery , Potassium Channels/genetics , Potassium Channels/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Receptors, Drug/genetics , Sulfonylurea Receptors
8.
Biochem J ; 363(Pt 1): 81-7, 2002 Apr 01.
Article in English | MEDLINE | ID: mdl-11903050

ABSTRACT

Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH.


Subject(s)
Glutamate Dehydrogenase/biosynthesis , Glutamate Dehydrogenase/isolation & purification , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Allosteric Site , Animals , Binding Sites , Cattle , Chaperonin 10/metabolism , Chaperonin 60/metabolism , Crystallography, X-Ray , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Glutamate Dehydrogenase/chemistry , Glutamic Acid/metabolism , Guanosine Triphosphate/metabolism , Humans , Ketoglutaric Acids/metabolism , Mitochondria/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutation
SELECTION OF CITATIONS
SEARCH DETAIL
...