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1.
Nat Neurosci ; 24(3): 391-400, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33589832

RESUMO

Experimental research controls for past experience, yet prior experience influences how we learn. Here, we tested whether we could recruit a neural population that usually encodes rewards to encode aversive events. Specifically, we found that GABAergic neurons in the lateral hypothalamus (LH) were not involved in learning about fear in naïve rats. However, if these rats had prior experience with rewards, LH GABAergic neurons became important for learning about fear. Interestingly, inhibition of these neurons paradoxically enhanced learning about neutral sensory information, regardless of prior experience, suggesting that LH GABAergic neurons normally oppose learning about irrelevant information. These experiments suggest that prior experience shapes the neural circuits recruited for future learning in a highly specific manner, reopening the neural boundaries we have drawn for learning of particular types of information from work in naïve subjects.


Assuntos
Condicionamento Clássico/fisiologia , Medo/fisiologia , Neurônios GABAérgicos/fisiologia , Região Hipotalâmica Lateral/fisiologia , Aprendizagem/fisiologia , Animais , Sinais (Psicologia) , Feminino , Masculino , Vias Neurais/fisiologia , Ratos , Ratos Long-Evans , Ratos Transgênicos , Recompensa
2.
Am J Physiol Renal Physiol ; 320(3): F351-F358, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33459166

RESUMO

A heterozygous mutation (c.643C.A; p.Q215X) in the creatine transporter SLC16A12 has been proposed to cause a syndrome with juvenile cataracts, microcornea, and glucosuria in humans. To further explore the role of SLC16A12 in renal physiology and decipher the mechanism underlying the phenotype of humans with the SLC16A12 mutation, we studied Slc16a12 knockout (KO) rats. Slc16a12 KO rats had lower plasma levels and increased absolute and fractional urinary excretion of creatine and its precursor guanidinoacetate (GAA). Slc16a12 KO rats displayed lower plasma and urinary creatinine levels, but the glomerular filtration rate was normal. The phenotype of heterozygous rats was indistinguishable from wild-type (WT) rats. Renal artery to vein (RAV) concentration differences in WT rats were negative for GAA and positive for creatinine. However, RAV differences for GAA were similar in Slc16a12 KO rats, indicating incomplete compensation of urinary GAA losses by renal GAA synthesis. Together, our results reveal that Slc16a12 in the basolateral membrane of the proximal tubule is critical for the reabsorption of creatine and GAA. Our data suggest a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation. Furthermore, in the absence of Slc16a12, urinary losses of GAA are not adequately compensated by increased tubular synthesis, likely caused by feedback inhibition of the rate-limiting enzyme l-arginine:glycine amidinotransferase by creatine in proximal tubular cells.NEW & NOTEWORTHY SLC16A12 is a recently identified creatine transporter of unknown physiological function. A heterozygous mutation in the human SLC16A12 gene causes juvenile cataracts and reduced plasma guanidinoacetate (GAA) levels with an increased fractional urinary excretion of GAA. Our study with transgenic SLC16A12-deficient rats reveals that SLC16A12 is critical for tubular reabsorption of creatine and GAA in the kidney. Our data furthermore indicate a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation.


Assuntos
Creatinina/urina , Glicina/análogos & derivados , Túbulos Renais Proximais/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Reabsorção Renal , Animais , Creatinina/sangue , Técnicas de Inativação de Genes , Genótipo , Glicina/sangue , Glicina/urina , Fígado/metabolismo , Transportadores de Ácidos Monocarboxílicos/deficiência , Transportadores de Ácidos Monocarboxílicos/genética , Fenótipo , Ratos Endogâmicos F344 , Ratos Transgênicos
3.
Am J Physiol Renal Physiol ; 320(1): F97-F113, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33308016

RESUMO

We recently reported that the enhanced susceptibility to chronic kidney disease (CKD) in the fawn-hooded hypertensive (FHH) rat is caused, at least in part, by a mutation in γ-adducin (ADD3) that attenuates renal vascular function. The present study explored whether Add3 contributes to the modulation of podocyte structure and function using FHH and FHH.Add3 transgenic rats. The expression of ADD3 on the membrane of primary podocytes isolated from FHH was reduced compared with FHH.Add3 transgenic rats. We found that F-actin nets, which are typically localized in the lamellipodia, replaced unbranched stress fibers in conditionally immortalized mouse podocytes transfected with Add3 Dicer-substrate short interfering RNA (DsiRNA) and primary podocytes isolated from FHH rats. There were increased F/G-actin ratios and expression of the Arp2/3 complexes throughout FHH podocytes in association with reduced synaptopodin and RhoA but enhanced Rac1 and CDC42 expression in the renal cortex, glomeruli, and podocytes of FHH rats. The expression of nephrin at the slit diaphragm and the levels of focal adhesion proteins integrin-α3 and integrin-ß1 were decreased in the glomeruli of FHH rats. Cell migration was enhanced and adhesion was reduced in podocytes of FHH rats as well as in immortalized mouse podocytes transfected with Add3 DsiRNA. Mean arterial pressures were similar in FHH and FHH.Add3 transgenic rats at 16 wk of age; however, FHH rats exhibited enhanced proteinuria associated with podocyte foot process effacement. These results demonstrate that reduced ADD3 function in FHH rats alters baseline podocyte pathophysiology by rearrangement of the actin cytoskeleton at the onset of proteinuria in young animals.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Hipertensão/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Insuficiência Renal Crônica/metabolismo , Citoesqueleto de Actina/patologia , Animais , Pressão Arterial , Proteínas de Ligação a Calmodulina/genética , Adesão Celular , Linhagem Celular , Movimento Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Adesões Focais/metabolismo , Adesões Focais/patologia , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Integrinas/metabolismo , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Podócitos/patologia , Proteinúria/genética , Proteinúria/patologia , Proteinúria/fisiopatologia , Ratos Endogâmicos , Ratos Transgênicos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Transdução de Sinais
4.
Anesthesiology ; 134(2): 219-233, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33332534

RESUMO

BACKGROUND: The general anesthetic propofol induces frontal alpha rhythm in the cerebral cortex at a dose sufficient to induce loss of consciousness. The authors hypothesized that propofol-induced facilitation of unitary inhibitory postsynaptic currents would result in firing synchrony among postsynaptic pyramidal neurons that receive inhibition from the same presynaptic inhibitory fast-spiking neurons. METHODS: Multiple whole cell patch clamp recordings were performed from one fast-spiking neuron and two or three pyramidal neurons with at least two inhibitory connections in rat insular cortical slices. The authors examined how inhibitory inputs from a presynaptic fast-spiking neuron modulate the timing of spontaneous repetitive spike firing among pyramidal neurons before and during 10 µM propofol application. RESULTS: Responding to activation of a fast-spiking neuron with 150-ms intervals, pyramidal cell pairs that received common inhibitory inputs from the presynaptic fast-spiking neuron showed propofol-dependent decreases in average distance from the line of identity, which evaluates the coefficient of variation in spike timing among pyramidal neurons: average distance from the line of identity just after the first activation of fast-spiking neuron was 29.2 ± 24.1 (mean ± SD, absolute value) in control and 19.7 ± 19.2 during propofol application (P < 0.001). Propofol did not change average distance from the line of identity without activating fast-spiking neurons and in pyramidal neuron pairs without common inhibitory inputs from presynaptic fast-spiking neurons. The synchronization index, which reflects the degree of spike synchronization among pyramidal neurons, was increased by propofol from 1.4 ± 0.5 to 2.3 ± 1.5 (absolute value, P = 0.004) and from 1.5 ± 0.5 to 2.2 ± 1.0 (P = 0.030) when a presynaptic fast-spiking neuron was activated at 6.7 and 10 Hz, respectively, but not at 1, 4, and 13.3 Hz. CONCLUSIONS: These results suggest that propofol facilitates pyramidal neuron firing synchrony by enhancing inhibitory inputs from fast-spiking neurons. This synchrony of pyramidal neurons may contribute to the alpha rhythm associated with propofol-induced loss of consciousness.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Interneurônios/efeitos dos fármacos , Propofol/farmacologia , Células Piramidais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Masculino , Modelos Animais , Ratos , Ratos Transgênicos , Sinapses/efeitos dos fármacos
5.
PLoS One ; 15(9): e0239125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32991590

RESUMO

A mesoscale network model is proposed for the development of spike and wave discharges (SWDs) in the cortico-thalamo-cortical (C-T-C) circuit. It is based on experimental findings in two genetic models of childhood absence epilepsy-rats of WAG/Rij and GAERS strains. The model is organized hierarchically into two levels (brain structures and individual neurons) and composed of compartments for representation of somatosensory cortex, reticular and ventroposteriomedial thalamic nuclei. The cortex and the two thalamic compartments contain excitatory and inhibitory connections between four populations of neurons. Two connected subnetworks both including relevant parts of a C-T-C network responsible for SWD generation are modelled: a smaller subnetwork for the focal area in which the SWD generation can take place, and a larger subnetwork for surrounding areas which can be only passively involved into SWDs, but which is mostly responsible for normal brain activity. This assumption allows modeling of both normal and SWD activity as a dynamical system (no noise is necessary), providing reproducibility of results and allowing future analysis by means of theory of dynamical system theories. The model is able to reproduce most time-frequency changes in EEG activity accompanying the transition from normal to epileptiform activity and back. Three different mechanisms of SWD initiation reported previously in experimental studies were successfully reproduced in the model. The model incorporates also a separate mechanism for the maintenance of SWDs based on coupling analysis from experimental data. Finally, the model reproduces the possibility to stop ongoing SWDs with high frequency electrical stimulation, as described in the literature.


Assuntos
Epilepsia Tipo Ausência/fisiopatologia , Modelos Neurológicos , Neurônios/fisiologia , Córtex Somatossensorial/fisiopatologia , Núcleos Talâmicos/fisiopatologia , Animais , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia Tipo Ausência/genética , Epilepsia Tipo Ausência/terapia , Masculino , Vias Neurais/fisiopatologia , Ratos , Ratos Transgênicos , Córtex Somatossensorial/citologia , Núcleos Talâmicos/citologia , Estimulação Transcraniana por Corrente Contínua/métodos
6.
Am J Physiol Renal Physiol ; 319(4): F624-F635, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32830539

RESUMO

Recently, we reported a mutation in γ-adducin (ADD3) was associated with an impaired myogenic response of the afferent arteriole and hypertension-induced chronic kidney disease (CKD) in fawn hooded hypertensive (FHH) rats. However, the mechanisms by which altered renal blood flow (RBF) autoregulation promotes hypertension-induced renal injury remain to be determined. The present study compared the time course of changes in renal hemodynamics and the progression of CKD during the development of DOCA-salt hypertension in FHH 1BN congenic rats [wild-type (WT)] with an intact myogenic response versus FHH 1BN Add3KO (Add3KO) rats, which have impaired myogenic response. RBF was well autoregulated in WT rats but not in Add3KO rats. Glomerular capillary pressure rose by 6 versus 14 mmHg in WT versus Add3KO rats when blood pressure increased from 100 to 150 mmHg. After 1 wk of hypertension, glomerular filtration rate increased by 38% and glomerular nephrin expression decreased by 20% in Add3KO rats. Neither were altered in WT rats. Proteinuria doubled in WT rats versus a sixfold increase in Add3KO rats. The degree of renal injury was greater in Add3KO than WT rats after 3 wk of hypertension. RBF, glomerular filtration rate, and glomerular capillary pressure were lower by 20%, 28%, and 19% in Add3KO rats than in WT rats, which was associated with glomerular matrix expansion and loss of capillary filtration area. The results indicated that impaired RBF autoregulation and eutrophic remodeling of preglomerular arterioles increase the transmission of pressure to glomeruli, which induces podocyte loss and accelerates the progression of CKD in hypertensive Add3KO rats.


Assuntos
Pressão Sanguínea , Taxa de Filtração Glomerular , Hipertensão/complicações , Glomérulos Renais/irrigação sanguínea , Proteinúria/etiologia , Circulação Renal , Insuficiência Renal Crônica/etiologia , Animais , Arteríolas/metabolismo , Arteríolas/fisiopatologia , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Acetato de Desoxicorticosterona , Modelos Animais de Doenças , Progressão da Doença , Homeostase , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Desenvolvimento Muscular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Ratos Transgênicos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Cloreto de Sódio na Dieta , Remodelação Vascular
7.
Proc Natl Acad Sci U S A ; 117(31): 18661-18669, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675242

RESUMO

Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.


Assuntos
Proteína Huntingtina , Doença de Huntington , Ubiquitinação/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lisina/química , Lisina/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma , Agregação Patológica de Proteínas/metabolismo , Ratos , Ratos Transgênicos
8.
Am J Physiol Heart Circ Physiol ; 319(2): H349-H358, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32589443

RESUMO

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models.NEW & NOTEWORTHY The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.


Assuntos
Antígenos CD/genética , Caderinas/genética , Células Endoteliais/metabolismo , Genes Reporter , Integrases/genética , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Fatores Etários , Animais , Feminino , Regulação da Expressão Gênica , Integrases/metabolismo , Proteínas Luminescentes/biossíntese , Masculino , Microcirculação , Ratos Sprague-Dawley , Ratos Transgênicos , Distribuição Tecidual , Transposases/genética , Transposases/metabolismo
10.
Psychopharmacology (Berl) ; 237(8): 2555-2568, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32533210

RESUMO

RATIONALE: Selective serotonin reuptake inhibitor (SSRI) antidepressants are increasingly prescribed during pregnancy. Changes in serotonergic signaling during human fetal development have been associated with changes in brain development and with changes in affective behavior in adulthood. The suprachiasmatic nucleus (SCN) is known to be modulated by serotonin and it is therefore assumed that SSRIs may affect circadian rhythms. However, effects of perinatal SSRI treatment on circadian system functioning in the offspring are largely unknown. OBJECTIVE: Our aim was to investigate the effects of perinatal exposure to the SSRI fluoxetine (FLX) on circadian behavior, affective behavior, and 5-HT1A receptor sensitivity in female rats. In addition, we studied the expression of clock genes and the 5-HT1A receptor in the SCN, as they are potentially involved in underlying mechanisms contributing to changes in circadian rhythms. RESULTS: Perinatal FLX exposure shortened the free-running tau in response to the 5-HT1A/7 agonist 8-OH-DPAT. However, FLX exposure did not alter anxiety, stress coping, and 5-HT1A receptor sensitivity. No differences were found in 5-HT1A receptor and clock genes Per1, Per2, Cry1, and Cry2 SCN gene expression. CONCLUSIONS: Perinatal FLX exposure altered the response to a phase-shifting challenge in female rats, whether this may pose health risks remains to be investigated.


Assuntos
Adaptação Psicológica/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Fluoxetina/farmacologia , Inibidores de Captação de Serotonina/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Adaptação Psicológica/fisiologia , Animais , Antidepressivos/farmacologia , Ritmo Circadiano/fisiologia , Feminino , Ratos , Ratos Transgênicos , Ratos Wistar , Serotonina/metabolismo , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologia
11.
J Vis Exp ; (159)2020 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-32478714

RESUMO

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.


Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Animais , Camundongos , Ratos , Ratos Transgênicos
12.
Stroke ; 51(6): 1835-1843, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32397936

RESUMO

Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1-deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T2 weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T2 weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading (P<0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1-deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.


Assuntos
Barreira Hematoencefálica , Isquemia Encefálica , Deleção de Genes , Hipertensão , Receptores Depuradores Classe E/deficiência , Acidente Vascular Cerebral , Animais , Barreira Hematoencefálica/lesões , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Isquemia Encefálica/sangue , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , MicroRNA Circulante , Hipertensão/sangue , Hipertensão/genética , Hipertensão/patologia , MicroRNAs/sangue , MicroRNAs/genética , Ratos , Ratos Endogâmicos SHR , Ratos Transgênicos , Receptores Depuradores Classe E/metabolismo , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia
13.
Epilepsia ; 61(6): 1291-1300, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32415786

RESUMO

OBJECTIVE: Sodium valproate (VPA), the most effective antiepileptic drug for patients with genetic generalized epilepsy (GGE), is a potent human teratogen that increases the risk of a range of congenital malformations, including spina bifida. The mechanisms underlying this teratogenicity are not known, but may involve genetic risk factors. This study aimed to develop an animal model of VPA-induced birth defects. METHODS: We used three different rat strains: inbred Genetic Absence Epilepsy Rats From Strasbourg (GAERS), a model of GGE with absence seizures; inbred Non-Epileptic Controls (NEC); and outbred nonepileptic Wistars. Female rats were fed standard chow or VPA (20 g/kg food) mixed in standard chow for 2 weeks prior to conception, and then mated with same-strain males. Treatment continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 and examined for birth defects, including external assessment and spinal measurements. RESULTS: VPA-exposed pups showed significant reductions in weight, length, and whole-body development compared with controls of all three strains (P < .0001). Gestational VPA treatment altered intravertebral distances, and resulted in underdeveloped vertebral arches between thoracic region T11 and caudal region C2 in most pups (GAERS, 100%; NEC, 95%; Wistar, 80%), more frequently than in controls (9%, 13%, 19%). SIGNIFICANCE: Gestational VPA treatment results in similar developmental and morphological abnormalities in three rat strains, including one with GGE, indicating that the genetic underpinnings of epilepsy do not contribute markedly to VPA-induced birth defects. This model may be used in future studies to investigate mechanisms involved in the pathogenesis of antiepileptic drug-induced birth defects.


Assuntos
Anticonvulsivantes/toxicidade , Modelos Animais de Doenças , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Teratogênios/toxicidade , Ácido Valproico/toxicidade , Anormalidades Induzidas por Medicamentos/patologia , Administração Oral , Animais , Feminino , Masculino , Gravidez , Ratos , Ratos Transgênicos , Ratos Wistar
14.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L1097-L1108, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233792

RESUMO

We previously demonstrated that the combined exposure of human pulmonary microvascular endothelial cells (HPMECs) to morphine and viral protein(s) results in the oxidative stress-mediated induction of autophagy, leading to shift in the cells from early apoptotic to apoptosis-resistant proliferative status associated with the angioproliferative remodeling observed in pulmonary arterial hypertension (PAH). In this study, we tried to delineate the major source of HIV-1 protein Tat and morphine induced oxidative burst in HPMECs and its consequences on vascular remodeling and PAH in an in vivo model. We observed switch from the initial increased expression of NADPH oxidase (NOX) 2 in response to acute treatment of morphine and HIV-Tat to later increased expression of NOX4 on chronic treatment in the endoplasmic reticulum of HPMECs without any alterations in the mitochondria. Furthermore, NOX-dependent induction of autophagy was observed to play a pivotal role in regulating the endothelial cell survival. Our in vivo findings showed significant increase in pulmonary vascular remodeling, right ventricular systolic pressure, and Fulton index in HIV-transgenic rats on chronic administration of morphine. This was associated with increased oxidative stress in lung tissues and rat pulmonary microvascular endothelial cells. Additionally, endothelial cells from morphine-treated HIV-transgenic rats demonstrated increased expression of NOX2 and NOX4 proteins, inhibition of which ameliorated their increased survival upon serum starvation. In conclusion, this study describes NADPH oxidases as one of the main players in the oxidative stress-mediated endothelial dysfunction on the dual hit of HIV-viral protein(s) and opioids.


Assuntos
Analgésicos Opioides/farmacologia , Células Endoteliais/efeitos dos fármacos , Morfina/farmacologia , NADPH Oxidase 2/genética , NADPH Oxidase 4/genética , Hipertensão Arterial Pulmonar/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , HIV-1/genética , HIV-1/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Estresse Oxidativo , Hipertensão Arterial Pulmonar/induzido quimicamente , Hipertensão Arterial Pulmonar/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
15.
Proc Natl Acad Sci U S A ; 117(11): 6075-6085, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123074

RESUMO

MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden following N-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P = 0.038) and miR-31 gene knockout abrogated development of ESCC (P = 1 × 10-6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB-controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31-associated EGLN3/NF-κB-controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31-/- rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development.


Assuntos
Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , MicroRNAs/metabolismo , Neoplasias Experimentais/genética , Animais , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Suplementos Nutricionais , Neoplasias Esofágicas/induzido quimicamente , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/prevenção & controle , Carcinoma de Células Escamosas do Esôfago/induzido quimicamente , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/prevenção & controle , Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , NF-kappa B/metabolismo , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Nitrosaminas/toxicidade , Ratos , Ratos Transgênicos , Transdução de Sinais/genética , Zinco/administração & dosagem , Zinco/deficiência
16.
PLoS One ; 15(3): e0230083, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160266

RESUMO

Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease caused by mutations in the DMD gene encoding dystrophin, expressed mainly in muscles but also in other tissues like retina and brain. Non-progressing cognitive dysfunction occurs in 20 to 50% of DMD patients. Furthermore, loss of expression of the Dp427 dystrophin isoform in the brain of mdx mice, the most used animal model of DMD, leads to behavioral deficits thought to be linked to insufficiencies in synaptogenesis and channel clustering at synapses. Mdx mice where the locomotor phenotype is mild also display a high and maladaptive response to stress. Recently, we generated Dmdmdx rats carrying an out-of frame mutation in exon 23 of the DMD gene and exhibiting a skeletal and cardiac muscle phenotype similar to DMD patients. In order to evaluate the impact of dystrophin loss on behavior, we explored locomotion parameters as well as anhedonia, anxiety and response to stress, in Dmdmdx rats aged from 1.5 to 7 months, in comparison to wild-type (WT) littermates. Pattern of dystrophin expression in the brain of WT and Dmdmdx rats was characterized by western-blot analyses and immunohistochemistry. We showed that dystrophin-deficient Dmdmdx rats displayed motor deficits in the beam test, without association with depressive or anxiety-like phenotype. However, Dmdmdx rats exhibited a strong response to restraint-induced stress, with a large increase in freezings frequency and duration, suggesting an alteration in a functional circuit including the amygdala. In brain, large dystrophin isoform Dp427 was not expressed in mutant animals. Dmdmdx rat is therefore a good animal model for preclinical evaluations of new treatments for DMD but care must be taken with their responses to mild stress.


Assuntos
Encéfalo/metabolismo , Distrofina/genética , Distrofia Muscular Animal/patologia , Animais , Ansiedade/patologia , Sistema Nervoso Central/metabolismo , Distrofina/deficiência , Distrofina/metabolismo , Locomoção , Aprendizagem em Labirinto , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Transgênicos , Estresse Psicológico
17.
Proc Natl Acad Sci U S A ; 117(12): 6844-6854, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32144141

RESUMO

Chronic inflammation during Alzheimer's disease (AD) is most often attributed to sustained microglial activation in response to amyloid-ß (Aß) plaque deposits and cell death. However, cytokine release and microgliosis are consistently observed in AD transgenic animal models devoid of such pathologies, bringing into question the underlying processes that may be at play during the earliest AD-related immune response. We propose that this plaque-independent inflammatory reaction originates from neurons burdened with increasing levels of soluble and oligomeric Aß, which are known to be the most toxic amyloid species within the brain. Laser microdissected neurons extracted from preplaque amyloid precursor protein (APP) transgenic rats were found to produce a variety of potent immune factors, both at the transcript and protein levels. Neuron-derived cytokines correlated with the extent of microglial activation and mobilization, even in the absence of extracellular plaques and cell death. Importantly, we identified an inflammatory profile unique to Aß-burdened neurons, since neighboring glial cells did not express similar molecules. Moreover, we demonstrate within disease-vulnerable regions of the human brain that a neuron-specific inflammatory response may precede insoluble Aß plaque and tau tangle formation. Thus, we reveal the Aß-burdened neuron as a primary proinflammatory agent, implicating the intraneuronal accumulation of Aß as a significant immunological component in the AD pathogenesis.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patologia , Inflamação/patologia , Neurônios/imunologia , Placa Amiloide/patologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Amiloidose , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Neurônios/metabolismo , Neurônios/patologia , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Ratos , Ratos Transgênicos
18.
PLoS Biol ; 18(3): e3000618, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32182233

RESUMO

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacologia , Membrana Celular/metabolismo , Animais , Sítios de Ligação , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/genética , Células Cultivadas , Cristalografia por Raios X , Feminino , Gangliosídeos/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paralisia/induzido quimicamente , Engenharia de Proteínas , Ratos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Sinaptotagminas/metabolismo , Triptofano/química , Triptofano/metabolismo
19.
Endocrinology ; 161(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32141511

RESUMO

Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of estrogen receptor ß (ESR2) signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wild-type and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2 signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1, indicating that the regulation of primordial follicle activation is ESR2 specific. Follicle activation was also increased in Esr2 mutants lacking the DNA binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2, suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK, and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles.


Assuntos
Hormônio Antimülleriano/sangue , Estradiol/sangue , Receptor beta de Estrogênio/genética , Folículo Ovariano/metabolismo , Reserva Ovariana/fisiologia , Animais , Moduladores de Receptor Estrogênico/farmacologia , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina , Nitrilos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Reserva Ovariana/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais/efeitos dos fármacos
20.
Oxid Med Cell Longev ; 2020: 6834236, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190176

RESUMO

Objective: To investigate the protective effects and mechanisms of human tissue kallikrein 1 (hKLK1) on type 1 diabetes mellitus- (DM-) induced erectile dysfunction in rats. Materials and Methods. The homozygous transgenic rats (TGR) harboring the hKLK1 gene and age-matched wild-type Sprague Dawley rats (WTR) were involved, and intraperitoneal injection of streptozotocin was utilized to induce diabetes in rats. Forty-eight-week-old male rats were randomly divided into a WTR group, TGR group, diabetic WTR group (WTDM), diabetic TGR group (TGDM), and TGDM with HOE140 group (TGDMH), with eight rats in each group. Twelve weeks later, the erectile response of all rats was detected by cavernous nerve electric stimulation, and corpus cavernosums were harvested to evaluate the levels of cavernous oxidative stress (OS), apoptosis, fibrosis, and involved pathways. Moreover, cavernous smooth muscle cells (CSMC) and endothelial cells (EC) were primarily isolated to build a coculture system for a series of in vitro verification. Results: The hKLK1 gene and age-matched wild-type Sprague Dawley rats (WTR) were involved, and intraperitoneal injection of streptozotocin was utilized to induce diabetes in rats. Forty-eight-week-old male rats were randomly divided into a WTR group, TGR group, diabetic WTR group (WTDM), diabetic TGR group (TGDM), and TGDM with HOE140 group (TGDMH), with eight rats in each group. Twelve weeks later, the erectile response of all rats was detected by cavernous nerve electric stimulation, and corpus cavernosums were harvested to evaluate the levels of cavernous oxidative stress (OS), apoptosis, fibrosis, and involved pathways. Moreover, cavernous smooth muscle cells (CSMC) and endothelial cells (EC) were primarily isolated to build a coculture system for a series of. Conclusions: hKLK1 preserves erectile function of DM rats through its antitissue excessive OS, apoptosis, and fibrosis effects, as well as activation of the PI3K/AKT/eNOS/cGMP pathway in the penis. Moreover, hKLK1 promotes relaxation and prevents high glucose-induced injuries of CSMC mediated by EC-CSMC crosstalk.


Assuntos
Diabetes Mellitus Experimental/complicações , Disfunção Erétil/tratamento farmacológico , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Calicreínas Teciduais/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Peso Corporal , Cálcio/metabolismo , GMP Cíclico/metabolismo , Diabetes Mellitus Experimental/patologia , Estimulação Elétrica , Disfunção Erétil/complicações , Disfunção Erétil/patologia , Jejum/sangue , Fibrose , Glucose/toxicidade , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pênis/efeitos dos fármacos , Pênis/patologia , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais/efeitos dos fármacos , Estreptozocina , Calicreínas Teciduais/farmacologia
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