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1.
Nat Commun ; 11(1): 4949, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009388

RESUMO

Electron microscopy (EM) is widely used for studying cellular structure and network connectivity in the brain. We have built a parallel imaging pipeline using transmission electron microscopes that scales this technology, implements 24/7 continuous autonomous imaging, and enables the acquisition of petascale datasets. The suitability of this architecture for large-scale imaging was demonstrated by acquiring a volume of more than 1 mm3 of mouse neocortex, spanning four different visual areas at synaptic resolution, in less than 6 months. Over 26,500 ultrathin tissue sections from the same block were imaged, yielding a dataset of more than 2 petabytes. The combined burst acquisition rate of the pipeline is 3 Gpixel per sec and the net rate is 600 Mpixel per sec with six microscopes running in parallel. This work demonstrates the feasibility of acquiring EM datasets at the scale of cortical microcircuits in multiple brain regions and species.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Transmissão , Rede Nervosa/ultraestrutura , Neurônios/ultraestrutura , Animais , Automação , Camundongos , Neocórtex/diagnóstico por imagem , Software
2.
Pain ; 161(11): 2434-2436, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33065697
3.
Cancer Epidemiol ; 68: 101778, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32674053

RESUMO

BACKGROUND: Mitochondrial DNA copy number (mtDNAcn) is considered a biomarker for mitochondrial function and oxidative stress. Although previous studies have suggested a potential relationship between mtDNAcn at the time of colorectal cancer (CRC) diagnosis and CRC prognosis, findings have been inconsistent, and no study has specifically investigated the association of pre-diagnostic mtDNAcn with CRC survival. METHODS: We examined the association of pre-diagnostic leukocyte mtDNAcn (measured by qPCR) with overall and CRC-specific survival among 587 patients in Nurses' Health Study and Health Professionals Follow-Up Study. Cox models were constructed to estimate hazard ratios (HRs) and 95 % confidence intervals (95 % CIs). RESULTS: During a mean follow-up of 10.5 years, 395 deaths were identified; 180 were due to CRC. Overall, we did not observe significant associations between mtDNAcn and either overall or CRC-specific survival among all cases or by cancer location, grade, or stage. In an exploratory stratified analysis, a suggestive inverse association of mtDNAcn and overall death risk appeared among current smokers [HR (95 % CI) for 1 SD decrease in mtDNAcn = 1.50 (0.98, 2.32), P-trend = 0.06]. Reduced mtDNAcn and lower CRC-specific death risk was observed among patients aged ≤ 70.5 at diagnosis [HR (95 % CI) for 1 SD decrease of mtDNAcn = 0.71 (0.52, 0.97), P-trend = 0.03], ≤ 5 years from blood collection to diagnosis [HR (95 % CI) for 1 SD decrease in mtDNAcn = 0.65 (0.44, 0.96), P-trend = 0.03] and those consuming a low-inflammatory diet [HR (95 % CI) for 1 SD decrease in mtDNAcn = 0.61 (0.42, 0.88), P-trend = 0.009]. CONCLUSION: no significant associations between pre-diagnostic leukocyte mtDNAcn and either overall or CRC-specific survival appeared but exploratory analysis identified potential sub-group associations.

4.
J Nutr ; 150(8): 2156-2163, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32492151

RESUMO

BACKGROUND: Poor lifestyles have been linked to insulin insensitivity/hyperinsulinemia, which may contribute to downstream changes such as inflammation and oxidative damage and the development of chronic diseases. As a biomarker of intracellular oxidative stress, leukocyte mitochondrial DNA copy number (mtDNA-CN) has been related to lifestyle factors including diet and weight. No epidemiologic study has examined the relation between combined insulinemic potential of lifestyle and mtDNA-CN. OBJECTIVES: Our aim was to examine the association between Empirical Lifestyle Index for Hyperinsulinemia (ELIH) and leukocyte mtDNA-CN in US men and women. METHODS: This cross-sectional analysis included 2835 white adults without cancers, diabetes, or cardiovascular disease at blood collection, including 2160 women from the Nurses' Health Study and 675 men from the Health Professionals Follow-Up Study. ELIH is an index based on plasma C-peptide that characterizes the insulinemic potential of lifestyle (diet, body weight, and physical activity). Relative mtDNA-CN in peripheral blood leukocytes was measured by qPCR-based assay. RESULTS: We found a significant inverse association between ELIH and mtDNA-CN. In multivariable-adjusted linear models, absolute least squares means ± SDs of mtDNA-CN z score across ELIH quintiles in women were as follows: Q1: 0.14 ± 0.05; Q2: 0.04 ± 0.06; Q3: 0.008 ± 0.05; Q4: 0.01 ± 0.05; and Q5: -0.06 ± 0.05 (P-trend = 0.006). Means ± SDs in men were as follows: Q1: 0.25 ± 0.09; Q2: 0.23 ± 0.09; Q3: 0.07 ± 0.09; Q4: 0.02 ± 0.09; and Q5: -0.04 ± 0.09 (P-trend = 0.007). Means ± SDs in all participants were as follows: Q1: 0.16 ± 0.05; Q2: 0.07 ± 0.05; Q3: 0.01 ± 0.05; Q4: 0.01 ± 0.05; and Q5: -0.05 ± 0.05 (P-trend = 0.0004). CONCLUSIONS: Hyperinsulinemic lifestyles (i.e., higher ELIH) were associated with lower leukocyte mtDNA-CN among subjects without major diseases, suggesting that the difference in lifestyle insulinemic potential may be related to excessive oxidative stress damage.


Assuntos
Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Grupo com Ancestrais do Continente Europeu/genética , Hiperinsulinismo , Leucócitos , Estilo de Vida , Adulto , Estudos Transversais , Feminino , Predisposição Genética para Doença , Humanos , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Estados Unidos
5.
Transl Psychiatry ; 10(1): 176, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32488052

RESUMO

Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi-platform targeted mass spectrometry-based metabolomics. Ndufs4GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4GT/GT from WT mice. Specifically, we observed a reversed TCA cycle flux and rewiring of amino acid metabolism in the prefrontal cortex. Next, exposing mice to chronic variable stress (a model for depression-like behavior), we found that Ndufs4GT/GT mice showed altered stress response and coping strategies with a robust stress-associated reprogramming of amino acid metabolism. Our data suggest that impaired mitochondrial CI function is a candidate driver for altered stress reactivity and stress-induced brain metabolic reprogramming. These changes result in unique phenomic and metabolomic signatures distinguishing groups based on their mitochondrial genotype.

6.
Artigo em Inglês | MEDLINE | ID: mdl-32426895

RESUMO

We report on a 26-year-old male with extreme short stature, microcephaly, macroglossia, other dysmorphic features, severe intellectual disability, and a bone dysplasia. The patient had an extensive genetic and biochemical evaluation that was all normal or noninformative. Recently, the proband died following a period of not eating. He likely had a previously undescribed syndrome of unknown etiology.

7.
Brain Res ; 1734: 146540, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704081

RESUMO

This article reviews the contributions of the English neurophysiologist, Charles Scott Sherrington [1857-1952], and his Australian PhD trainee and collaborator, John Carew Eccles [1903-1997], to the concept of central inhibition in the spinal cord and brain. Both were awarded Nobel Prizes; Sherrington in 1932 for "discoveries regarding the function of neurons," and Eccles in 1963 for "discoveries concerning the ionic mechanisms involved in excitation and inhibition in central portions of the nerve cell membrane." Both spoke about central inhibition at their Nobel Prize Award Ceremonies. The subsequent publications of their talks were entitled "Inhibition as a coordinative factor" and "The ionic mechanism of postsynaptic inhibition", respectively. Sherrington's work on central inhibition spanned 41 years (1893-1934), and for Eccles 49 years (1928-1977). Sherrington first studied central inhibition by observing hind limb muscle responses to electrical (peripheral nerve) and mechanical (muscle) stimulation. He used muscle length and force measurements until the early 1900s and electromyography in the late 1920s. Eccles used these techniques while working with Sherrington, but later employed extracellular microelectrode recording in the spinal cord followed in 1951 by intracellular recording from spinal motoneurons. This considerably advanced our understanding of central inhibition. Sherrington's health was poor during his retirement years but he nonetheless made a small number of largely humanities contributions up to 1951, one year before his death at the age of 94. In contrast, Eccles retained his health and vigor until 3 years before his death and published prolifically on many subjects during his 22 years of official retirement. His last neuroscience article appeared in 1994 when he was 91. Despite poor health he continued thinking about his life-long interest, the mind-brain problem, and was attempting to complete his autobiography in the last years of his life.

8.
Elife ; 82019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31713514

RESUMO

Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.


Assuntos
Calbindina 2/genética , Interneurônios/metabolismo , Neuralgia/fisiopatologia , Neurônios/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Animais , Calbindina 2/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Halorrodopsinas/genética , Halorrodopsinas/metabolismo , Interneurônios/efeitos dos fármacos , Interneurônios/patologia , Camundongos , Camundongos Transgênicos , Morfina/farmacologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Neuralgia/tratamento farmacológico , Neuralgia/genética , Neuralgia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Optogenética/métodos , Limiar da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp , Estimulação Luminosa , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Corno Dorsal da Medula Espinal/patologia , Técnicas de Cultura de Tecidos , Transgenes
9.
Am J Med Genet A ; 179(12): 2357-2364, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31512387

RESUMO

Coffin-Lowry syndrome (CLS) is a rare X-linked disorder characterized by moderate to severe intellectual disability, hypotonia, craniofacial features, tapering digits, short stature, and skeletal deformities. Using whole exome sequencing and high-resolution targeted comparative genomic hybridization array analysis, we identified a novel microduplication encompassing exons five through nine of RPS6KA3 in three full brothers. Each brother presented with intellectual disability and clinical and radiographic features consistent with CLS. qRT-PCR analyses performed on mRNA from the peripheral blood of the three siblings revealed a marked reduction of RPS6KA3 levels suggesting a loss-of-function mechanism. PCR analysis of the patients' cDNA detected a band greater than expected for an exon 4-10 amplicon, suggesting this was likely a direct duplication that lies between exons 4 through 10, which was later confirmed by Sanger sequencing. This microduplication is only the third intragenic duplication of RPS6KA3, and the second and smallest reported to date thought to cause CLS. Our study further supports the clinical utility of methods such as next-generation sequencing and high-resolution genomic arrays to detect small intragenic duplications. These methods, coupled with expression studies and cDNA structural analysis have the capacity to confirm the diagnosis of CLS in these rare cases.

10.
Artigo em Inglês | MEDLINE | ID: mdl-31518899

RESUMO

Plasma elevations of the amino acids alloisoleucine and argininosuccinic acid (ASA) are pathognomonic for maple syrup urine disease and argininosuccinate lyase deficiency, respectively. Reliable detection of these biomarkers is typically achieved using methods with tedious sample preparations or long chromatographic separations, and many published amino acid assays report poor specificity and/or sensitivity for one or both of these compounds. This report describes a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method that provides rapid quantification of alloisoleucine and ASA in human plasma. The basis of this method is a mixed-mode solid phase separation that achieves baseline resolution of alloisoleucine from isobaric interferents without the use of derivatization or ion pairing agents. The inject-to-inject time is 6 min including elution, column washing and re-equilibration. Validation studies demonstrate excellent limits of quantification (1 µmol/L), linearity (r = 0.999 from 1 to 250 µmol/L), accuracy (bias = -3.8% and -10.1%), and inter-assay imprecision (CV < 8.06%) for plasma analyses. Data from long-term clinical application confirms chromatographic consistency equivalent to more traditional reversed-phase or HILIC based columns. Additional matrix studies indicate low suppression (<10%) for a wide range of amino acids and compatibility with other matrixes such as blood spot analyses. Finally, analysis of our first 257 clinical specimens demonstrates high analytic specificity and sensitivity, allowing the detection of subtle but clinically relevant elevations of alloisoleucine and ASA that may be missed by other less sensitive methods. In conclusion, the novel LC-MS/MS method reported here overcomes a number of the challenges associated with alloisoleucine and ASA quantification. Combining this approach with published incomplete amino acid quantification methods allows, for the first time, a rapid and comprehensive LC-MS/MS analysis of underivatized amino acids without the use of ion pairing agents.


Assuntos
Ácido Argininossuccínico/sangue , Cromatografia Líquida/métodos , Isoleucina/sangue , Espectrometria de Massas em Tandem/métodos , Ácido Argininossuccínico/química , Humanos , Isoleucina/química , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
Am J Hum Genet ; 105(3): 493-508, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31447100

RESUMO

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.


Assuntos
Senescência Celular/fisiologia , Histonas/fisiologia , Aneuploidia , Nucléolo Celular/metabolismo , Criança , Cromatina/metabolismo , Metilação de DNA , Feminino , Histonas/química , Humanos , Lactente , Masculino , Pessoa de Meia-Idade
12.
Am J Med Genet A ; 179(10): 2138-2143, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31290619

RESUMO

Autosomal recessive COX4I1 deficiency has been previously reported in a single individual with a homozygous pathogenic variant in COX4I1, who presented with short stature, poor weight gain, dysmorphic features, and features of Fanconi anemia. COX4I1 encodes subunit 4, isoform 1 of cytochrome c oxidase. Cytochrome c oxidase is a respiratory chain enzyme that plays an important role in mitochondrial electron transport and reduces molecular oxygen to water leading to the formation of ATP. Defective production of cytochrome c oxidase leads to a variable phenotypic spectrum ranging from isolated myopathy to Leigh syndrome. Here, we describe two siblings, born to consanguineous parents, who presented with encephalopathy, developmental regression, hypotonia, pathognomonic brain imaging findings resembling Leigh-syndrome, and a novel homozygous variant on COX4I1, expanding the known clinical phenotype associated with pathogenic variants in COX4I1.

13.
Cell Rep ; 28(2): 526-540.e6, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31291586

RESUMO

Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I-IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.

14.
Genome Med ; 11(1): 12, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819258

RESUMO

BACKGROUND: Neurodevelopmental disorders are genetically and phenotypically heterogeneous encompassing developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), structural brain abnormalities, and neurological manifestations with variants in a large number of genes (hundreds) associated. To date, a few de novo mutations potentially disrupting TCF20 function in patients with ID, ASD, and hypotonia have been reported. TCF20 encodes a transcriptional co-regulator structurally related to RAI1, the dosage-sensitive gene responsible for Smith-Magenis syndrome (deletion/haploinsufficiency) and Potocki-Lupski syndrome (duplication/triplosensitivity). METHODS: Genome-wide analyses by exome sequencing (ES) and chromosomal microarray analysis (CMA) identified individuals with heterozygous, likely damaging, loss-of-function alleles in TCF20. We implemented further molecular and clinical analyses to determine the inheritance of the pathogenic variant alleles and studied the spectrum of phenotypes. RESULTS: We report 25 unique inactivating single nucleotide variants/indels (1 missense, 1 canonical splice-site variant, 18 frameshift, and 5 nonsense) and 4 deletions of TCF20. The pathogenic variants were detected in 32 patients and 4 affected parents from 31 unrelated families. Among cases with available parental samples, the variants were de novo in 20 instances and inherited from 4 symptomatic parents in 5, including in one set of monozygotic twins. Two pathogenic loss-of-function variants were recurrent in unrelated families. Patients presented with a phenotype characterized by developmental delay, intellectual disability, hypotonia, variable dysmorphic features, movement disorders, and sleep disturbances. CONCLUSIONS: TCF20 pathogenic variants are associated with a novel syndrome manifesting clinical characteristics similar to those observed in Smith-Magenis syndrome. Together with previously described cases, the clinical entity of TCF20-associated neurodevelopmental disorders (TAND) emerges from a genotype-driven perspective.


Assuntos
Anormalidades Craniofaciais/genética , Deficiências do Desenvolvimento/genética , Mutação INDEL , Deficiência Intelectual/genética , Hipotonia Muscular/genética , Síndrome de Smith-Magenis/genética , Fatores de Transcrição/genética , Adolescente , Criança , Pré-Escolar , Anormalidades Craniofaciais/patologia , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Hipotonia Muscular/patologia , Síndrome de Smith-Magenis/patologia , Fatores de Transcrição/metabolismo , Adulto Jovem
16.
Dev Cell ; 48(5): 685-696.e5, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30713071

RESUMO

Lysosomes and mitochondria are both crucial cellular organelles for metabolic homeostasis and organism health. However, mechanisms linking their metabolic activities to promote organism longevity remain poorly understood. We discovered that the induction of specific lysosomal signaling mediated by a LIPL-4 lysosomal acid lipase and its lipid chaperone LBP-8 increases mitochondrial ß-oxidation to reduce lipid storage and promote longevity in Caenorhabditis elegans. We further discovered that increased mitochondrial ß-oxidation reduces mitochondrial electron transport chain complex II activity, contributing to the induction of reactive oxygen species in mitochondria (mtROS) and the longevity effect conferred by LIPL-4-LBP-8 signaling. Moreover, by activating the JUN-1 transcription factor downstream of mtROS, the LIPL-4-LBP-8 signaling pathway induces antioxidant targets and oxidative stress tolerance. Together, these results reveal regulatory mechanisms by which lysosomal signaling triggers adjustments in mitochondrial activity and suggest the significance of these metabolic adjustments for improving metabolic fitness, redox homeostasis, and longevity.


Assuntos
Homeostase/fisiologia , Longevidade/fisiologia , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição
17.
Genet Med ; 21(9): 1977-1986, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30670878

RESUMO

PURPOSE: Untargeted metabolomic analysis is increasingly being used in the screening and management of individuals with inborn errors of metabolism (IEM). We aimed to test whether untargeted metabolomic analysis in plasma might be useful for monitoring the disease course and management of urea cycle disorders (UCDs). METHODS: Untargeted mass spectrometry-based metabolomic analysis was used to generate z-scores for more than 900 metabolites in plasma from 48 individuals with various UCDs. Pathway analysis was used to identify common pathways that were perturbed in each UCD. RESULTS: Our metabolomic analysis in plasma identified multiple potentially neurotoxic metabolites of arginine in arginase deficiency and, thus, may have utility in monitoring the efficacy of treatment in arginase deficiency. In addition, we were also able to detect multiple biochemical perturbations in all UCDs that likely reflect clinical management, including metabolite alterations secondary to dietary and medication management. CONCLUSION: In addition to utility in screening for IEM, our results suggest that untargeted metabolomic analysis in plasma may be beneficial for monitoring efficacy of clinical management and off-target effects of medications in UCDs and potentially other IEM.


Assuntos
Biomarcadores/sangue , Erros Inatos do Metabolismo/sangue , Metabolômica , Distúrbios Congênitos do Ciclo da Ureia/sangue , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Ureia/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/genética , Distúrbios Congênitos do Ciclo da Ureia/patologia , Adulto Jovem
18.
Neuropharmacology ; 154: 22-33, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30253175

RESUMO

The perifornical/lateral hypothalamic area (LHA) orexin (hypocretin) system is involved in drug-seeking behavior elicited by drug-associated stimuli. Cocaine exposure is associated with presynaptic plasticity at LHA orexin cells such that excitatory input to orexin cells is enhanced acutely and into withdrawal. These changes may augment orexin cell reactivity to drug-related cues during abstinence and contribute to relapse-like behavior. Studies in hypothalamic slices from drug-naïve animals indicate that agonism of group III metabotropic glutamate receptors (mGluRs) reduces presynaptic glutamate release onto orexin cells. Therefore, we examined the group III mGluR system as a potential target to reduce orexin cell excitability in-vivo, including in animals with cocaine experience. First, we verified that group III mGluRs regulate orexin cell activity in behaving animals by showing that intra-LHA infusions of the selective agonist L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) reduces c-fos expression in orexin cells following 24 h food deprivation. Next, we extended these findings to show that intra-LHA L-AP4 infusions reduced discriminative stimulus-driven cocaine-seeking following withdrawal. Importantly, L-AP4 had no effect on lever pressing for sucrose pellets or general motoric behavior. Finally, using whole-cell patch-clamp recordings from identified orexin cells in orexin-GFP transgenic mice, we show enhanced presynaptic drive to orexin cells following 14d withdrawal and that this plasticity can be normalized by L-AP4. Together, these data indicate that activation of group III mGluRs in LHA reduces orexin cell activity in vivo and may be an effective strategy to suppress cocaine-seeking behavior following withdrawal. These effects are likely mediated, at least in part, by normalization of presynaptic plasticity at orexin cells that occurs as a result of cocaine exposure. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.


Assuntos
Comportamento Aditivo/metabolismo , Cocaína/administração & dosagem , Região Hipotalâmica Lateral/metabolismo , Orexinas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Comportamento Aditivo/prevenção & controle , Região Hipotalâmica Lateral/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microinjeções , Propionatos/administração & dosagem , Ratos , Receptores de Glutamato Metabotrópico/agonistas , Autoadministração
19.
Mitochondrion ; 44: 58-64, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29307858

RESUMO

Elevations of specific acylcarnitines in blood reflect carboxylase deficiencies, and have utility in newborn screening for life-threatening organic acidemias and other inherited metabolic diseases. In this report, we describe a newly-identified association of biochemical features of multiple carboxylase deficiency in individuals harboring mitochondrial DNA (mtDNA) mutations in MT-ATP6 and in whom organic acidemias and multiple carboxylase deficiencies were excluded. Using retrospective chart review, we identified eleven individuals with abnormally elevated propionylcarnitine (C3) or hydroxyisovalerylcarnitine (C5OH) with mutations in MT-ATP6, most commonly m.8993T>G in high heteroplasmy or homoplasmy. Most patients were ascertained on newborn screening; most had normal enzymatic or molecular genetic testing to exclude biotinidase and holocarboxylase synthetase deficiencies. MT-ATP6 is associated with some cases of Leigh disease; clinical outcomes in our cohort ranged from death from neurodegenerative disease in early childhood to clinically and developmentally normal after several years of follow-up. These cases expand the biochemical phenotype associated with MT-ATP6 mutations, especially m.8993T>G, to include acylcarnitine abnormalities mimicking carboxylase deficiency states. Clinicians should be aware of this association and its implications for newborn screening, and consider mtDNA sequencing in patients exhibiting similar acylcarnitine abnormalities that are biotin-unresponsive and in whom other enzymatic deficiencies have been excluded.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/genética , Deficiência Múltipla de Carboxilase/genética , Deficiência Múltipla de Carboxilase/patologia , Mutação , Adolescente , Carnitina/análogos & derivados , Carnitina/sangue , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Adulto Jovem
20.
Am J Hum Genet ; 103(6): 1030-1037, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30503518

RESUMO

FUK encodes fucokinase, the only enzyme capable of converting L-fucose to fucose-1-phosphate, which will ultimately be used for synthesizing GDP-fucose, the donor substrate for all fucosyltransferases. Although it is essential for fucose salvage, this pathway is thought to make only a minor contribution to the total amount of GDP-fucose. A second pathway, the major de novo pathway, involves conversion of GDP-mannose to GDP-fucose. Here we describe two unrelated individuals who have pathogenic variants in FUK and who presented with severe developmental delays, encephalopathy, intractable seizures, and hypotonia. The first individual was compound heterozygous for c.667T>C (p.Ser223Pro) and c.2047C>T (p.Arg683Cys), and the second individual was homozygous for c.2980A>C (p.Lys994Gln). Skin fibroblasts from the first individual confirmed the variants as loss of function and showed significant decreases in total GDP-[3H] fucose and [3H] fucose-1-phosphate. There was also a decrease in the incorporation of [5,6-3H]-fucose into fucosylated glycoproteins. Lys994 has previously been shown to be an important site for ubiquitin conjugation. Here, we show that loss-of-function variants in FUK cause a congenital glycosylation disorder characterized by a defective fucose-salvage pathway.


Assuntos
Anormalidades Congênitas/genética , Variação Genética/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Encefalopatias/genética , Criança , Deficiências do Desenvolvimento/genética , Feminino , Fibroblastos/patologia , Fucosiltransferases/genética , Glicosilação , Guanosina Difosfato Fucose/genética , Guanosina Difosfato Manose/genética , Humanos , Masculino , Hipotonia Muscular/genética , Convulsões/genética , Alinhamento de Sequência , Pele/patologia , Ubiquitina/genética
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