RESUMO
OBJECTIVE: Germline loss-of-function mutations in DEPDC5, and in its binding partners (NPRL2/3) of the mammalian target of rapamycin (mTOR) repressor GATOR1 complex, cause focal epilepsies and increase the risk of sudden unexpected death in epilepsy (SUDEP). Here, we asked whether DEPDC5 haploinsufficiency predisposes to primary cardiac defects that could contribute to SUDEP and therefore impact the clinical management of patients at high risk of SUDEP. METHODS: Clinical cardiac investigations were performed in 16 patients with pathogenic variants in DEPDC5, NPRL2, or NPRL3. Two novel Depdc5 mouse strains, a human HA-tagged Depdc5 strain and a Depdc5 heterozygous knockout with a neuron-specific deletion of the second allele (Depdc5c/- ), were generated to investigate the role of Depdc5 in SUDEP and cardiac activity during seizures. RESULTS: Holter, echocardiographic, and electrocardiographic (ECG) examinations provided no evidence for altered clinical cardiac function in the patient cohort, of whom 3 DEPDC5 patients succumbed to SUDEP and 6 had a family history of SUDEP. There was no cardiac injury at autopsy in a postmortem DEPDC5 SUDEP case. The HA-tagged Depdc5 mouse revealed expression of Depdc5 in the brain, heart, and lungs. Simultaneous electroencephalographic-ECG records on Depdc5c/- mice showed that spontaneous epileptic seizures resulting in a SUDEP-like event are not preceded by cardiac arrhythmia. INTERPRETATION: Mouse and human data show neither structural nor functional cardiac damage that might underlie a primary contribution to SUDEP in the spectrum of DEPDC5-related epilepsies. ANN NEUROL 2022;91:101-116.
Assuntos
Epilepsias Parciais/complicações , Proteínas Ativadoras de GTPase/genética , Coração , Morte Súbita Inesperada na Epilepsia/etiologia , Adolescente , Adulto , Animais , Eletrocardiografia , Eletroencefalografia , Epilepsias Parciais/genética , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
The increase of cholesterol synthesis after a status epilepticus may lead to excitotoxic processes, neuronal loss and favor the appearance of spontaneous epileptic seizures. Lowering cholesterol content could be a neuroprotective strategy. Here, we evaluated the protective effect of simvastatin administrated daily for 14 days, after the induction of a status epilepticus by intrahippocampal injection of kainic acid in mice. The results were compared to those obtained from mice showing a kainic acid-induced status epilepticus, treated daily with a saline solution, and from mice injected with a control phosphate-buffered solution without any status epilepticus. We first assessed the antiseizure effects of simvastatin by performing video-electroencephalographic recordings during the first three hours after kainic acid injection and continuously between the fifteenth and the thirty-first days. Mice treated with simvastatin had significantly fewer generalized seizures during the first three hours without a significant effect on generalized seizures after two weeks. There was a trend for fewer hippocampal electrographic seizures after two weeks. Secondly, we evaluated the neuroprotective and anti-inflammatory effects of simvastatin by measuring the fluorescence of neuronal and astrocyte markers on the thirtieth day after status onset. We found that simvastatin reduced CA1 reactive astrocytosis, demonstrated by a significant 37% decrease in GFAP-positive cells, and that simvastatin prevented the neuronal loss in CA1, demonstrated by a significant 42% increase in the NeuN-positive cells, as compared to the findings in mice with kainic acid-induced status epilepticus treated by a saline solution. Our study confirms the interest of cholesterol-lowering agents, and in particular simvastatin, in status epilepticus and paves the way for a clinical pilot study to prevent neurological sequelae after status epilepticus. This paper was presented at the 8th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures held in September 2022.
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Fármacos Neuroprotetores , Estado Epiléptico , Camundongos , Animais , Ácido Caínico/farmacologia , Fármacos Neuroprotetores/farmacologia , Sinvastatina/uso terapêutico , Sinvastatina/farmacologia , Projetos Piloto , Solução Salina/efeitos adversos , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/complicações , Estado Epiléptico/tratamento farmacológico , Convulsões/induzido quimicamente , HipocampoRESUMO
BACKGROUND: Botulinum toxin A (BTX-A) has a variety of uses in medicine. Some evidence suggests that intracavernosal (ic) BTX-A injection administered in addition to phosphodiesterase type 5 inhibitors (PDE5-Is) could effectively treat erectile dysfunction (ED) in insufficient responders to PDE5-Is. AIM: To provide experimental pharmacological evidence for the use of onabotulinumtoxinA ic alone or in combination with PDE5-Is for difficult-to-treat ED. We thus compared the effects of BTX-A ic alone and BTX-A ic combined with PDE5-I iv, and a placebo treatment ic or iv. METHODS: Erectile function was evaluated following cavernous nerve electrical stimulation (6 V, 1-millisecond pulse, 45-second duration) at different frequencies (0, 2, 3, 4, 5, 7.5, and 10 Hz) in 4 groups (n = 8 / group) of anesthetized, spontaneously hypertensive rats, a robust animal model of ED of vascular origin. Rats were treated by onabotulinumtoxinA 10U or saline ic 1 week prior to erectile function testing and sildenafil (0.3 mg/kg) or saline iv 4 minutes prior to testing. Frequency-response curves were compared with a 2 way ANOVA. OUTCOMES: Both onabotulinumtoxinA ic, and sildenafil iv significantly improved erectile responses in spontaneously hypertensive rats, however the effect was greatly amplified when the treatments were combined. RESULTS: Intracavernosal pressure and/or mean arterial pressure ratios were significantly increased by sildenafil and onabotulinumtoxinA ic versus the control condition. OnabotulinumtoxinA 10U ic combined with sildenafil iv significantly potentiated erectile responses. Area under the curve and/or mean arterial pressure ratio increased by 19% with sildenafil iv, by 15% with onabotulinumtoxinA ic and by 58% with the combined treatment following cavernous nerve electrical stimulation at 6V, 1 ms, 10 Hz: these stimulation parameters elicited the maximal erectile response. CLINICAL TRANSLATION: These data provide a pharmacological rationale for the combined administration of onabotulinumtoxinA ic and sildenafil iv since the effects of both treatments were potentiated when their administration was combined. STRENGTHS & LIMITATIONS: First evidence of a synergistic pro-erectile effect of BTX-A combined with PDE5-I, however the mechanism behind the pro-erectile effect of BTX-A ic remains hypothetical. CONCLUSIONS: These results support further studies into the mechanisms behind the pro-erectile effect of BTX-A ic, as well as multicenter randomized control trials to evaluate the safety and efficacy of BTX-A ic combined with sildenafil for difficult-to-treat ED. Giuliano F., Joussain C., Denys P., et al. Intracavernosal OnabotulinumtoxinA Exerts a Synergistic Pro-Erectile Effect When Combined With Sildenafil in Spontaneously Hypertensive Rats. J Sex Med 2022;19:899-906.
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Toxinas Botulínicas Tipo A , Disfunção Erétil , Animais , Toxinas Botulínicas Tipo A/farmacologia , Disfunção Erétil/tratamento farmacológico , Masculino , Ereção Peniana , Inibidores da Fosfodiesterase 5/farmacologia , Inibidores da Fosfodiesterase 5/uso terapêutico , Ratos , Ratos Endogâmicos SHR , Citrato de Sildenafila/farmacologia , Citrato de Sildenafila/uso terapêuticoRESUMO
Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.
Assuntos
Esclerose Lateral Amiotrófica , Profilinas , Proteínas rab de Ligação ao GTP , Esclerose Lateral Amiotrófica/metabolismo , Animais , Autofagia/genética , Homeostase , Humanos , Camundongos , Mitocôndrias/metabolismo , Mutação , Profilinas/genética , Profilinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The understanding of the excitotoxic processes associated with a severe status epilepticus (SE) is of major importance. Changes of brain cholesterol homeostasis is an emerging candidate for excitotoxicity. We conducted an overall analysis of the cholesterol homeostasis both (i) in fluids and tissues from patients with SE: blood (n = 63, n = 87 controls), CSF (n = 32, n = 60 controls), and post-mortem brain tissues (n = 8, n = 8 controls) and (ii) in a mouse model of SE induced by an intrahippocampal injection of kainic acid. 24-hydroxycholesterol levels were decreased in kainic acid mouse hippocampus and in human plasma and post-mortem brain tissues of patients with SE when compared with controls. The decrease of 24-hydroxycholesterol levels was followed by increased cholesterol levels and by an increase of the cholesterol synthesis. Desmosterol levels were higher in human CSF and in mice and human hippocampus after SE. Lanosterol and dihydrolanosterol levels were higher in plasma from SE patients. Our results suggest that a CYP46A1 inhibition could occur after SE and is followed by a brain cholesterol accumulation. The excess of cholesterol is known to be excitotoxic for neuronal cells and may participate to neurological sequelae observed after SE. This study highlights a new pathophysiological pathway involved in SE excitotoxicity.
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Encéfalo/metabolismo , Colesterol/metabolismo , Hidroxicolesteróis/metabolismo , Estado Epiléptico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estudos Prospectivos , Estado Epiléptico/patologiaRESUMO
BACKGROUND: The main purpose of this study is to investigate the effect of silodosin on the urodynamic consequences in a previously established model of lower urinary tract symptoms associated with benign prostate hyperplasia, the spontaneously hypertensive rats (SHR) supplemented with testosterone. METHODS: Three groups of animals (8-week-old; n = 10/group) were considered: Wistar Kyoto (control) rats (WKY), SHR supplemented with testosterone at 3 mg/kg/day and treated with either vehicle (SHR-T, n = 10) or silodosin at 0.1 mg/kg/day (SHR-T + silodosin, n = 10) by oral gavage for 6 weeks. Cystometry experiments were performed. The bladder was harvested, weighed and paraffin-embedded for morphometric analysis. The prostate was also harvested and weighed. RESULTS: The number of animals included in the analysis were n = 10/10 for WKY and n = 7-8/10 for each SHR rats supplemented with testosterone group. SHR-T displayed a significant decrease in the intercontraction interval, infused volume and mean flow rate whereas the frequency of non-voiding contractions was increased. Silodosin improved the voiding behavior of SHR-T by significantly increasing the intercontraction interval, the infused volume and the mean flow rate and decreasing the number of non-voiding contractions. SHR-T displayed a significant increase in prostate and bladder weights and a 15% increase in the detrusor wall area compared to WKY. CONCLUSIONS: Chronic silodosin treatment relieved storage symptoms in SHR supplemented with testosterone and decreased the frequency of non-voiding detrusor contractions during the filling phase.
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Antagonistas de Receptores Adrenérgicos alfa 1/uso terapêutico , Indóis/uso terapêutico , Hiperplasia Prostática/tratamento farmacológico , Testosterona/uso terapêutico , Obstrução do Colo da Bexiga Urinária/tratamento farmacológico , Animais , Masculino , Estudo de Prova de Conceito , Hiperplasia Prostática/complicações , Distribuição Aleatória , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Resultado do Tratamento , Obstrução do Colo da Bexiga Urinária/etiologiaRESUMO
INTRODUCTION: Following the results of the EMPA-REG Outcome trial, we hypothesized that empagliflozin, a highly potent and specific sodium/glucose cotransporteur 2 inhibitor, could improve type 2 diabetes mellitus (T2DM)-associated erectile dysfunction (ED), a highly prevalent complication of T2DM, very often coexisting with cardiovascular complications and considered as a prognostic factor of cardiovascular disease in men with diabetes. AIM: To investigate the effects of chronic treatment with empagliflozin on ED in a T2DM rat model in the presence or absence of sildenafil. METHODS: Male Goto-Kakizaki (GK), a model of T2DM, and age-matched Wistar rats received placebo or empagliflozin treatment at 25.3 ± 0.9 mg/kg/d for 4 weeks. Then, the in vivo effect of empagliflozin on erectile function was assessed by electrical stimulation of the cavernous nerve at different frequencies under anesthesia in the presence or absence of acute intravenous injection of sildenafil. Endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from the rats were studied. MAIN OUTCOME MEASURES: Body weight, food consumption, metabolic parameters, plasma inflammation biomarkers, and in vivo erectile responses elicited by electrical stimulation of the cavernous nerve in empagliflozin-treated and untreated GK rats and control Wistar rats were assessed and followed by concentration or frequency response curves to endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from these rats. RESULTS: Chronic empagliflozin followed by acute sildenafil significantly improved erectile responses in adult GK rats (n = 12-15/group). Ratios of intracavernous pressure and area under the curve/mean arterial pressure during the electrical stimulation were significantly increased in empagliflozin-treated vs untreated GK rats. Nitrergic relaxations of cavernosal strips from GK rats were significantly increased with empagliflozin compared with placebo. Moreover, the effect of sildenafil on erectile function was not altered by empagliflozin treatment. CLINICAL IMPLICATIONS: Empagliflozin may benefit T2DM patient with ED. STRENGTHS & LIMITATIONS: The mechanism(s) by which empagliflozin shows favorable effect on erectile function in GK rats needs to be further elucidated. CONCLUSION: Empagliflozin shows favorable effect on erectile function in diabetic GK rats mediated by an improvement of nitrergic relaxation of erectile tissue. Whether this favorable effect on ED in the experimental context of T2DM is due to better glycemic control or to another effect of empagliflozin deserves further investigation. Assaly R, Gorny D, Compagnie S, et al. The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes. J Sex Med 2018;15:1224-1234.
Assuntos
Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Disfunção Erétil/tratamento farmacológico , Glucosídeos/uso terapêutico , Ereção Peniana/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Animais , Compostos Benzidrílicos/administração & dosagem , Compostos Benzidrílicos/farmacologia , Modelos Animais de Doenças , Disfunção Erétil/complicações , Glucosídeos/administração & dosagem , Glucosídeos/farmacologia , Masculino , Ratos , Ratos Endogâmicos , Ratos Wistar , Inibidores do Transportador 2 de Sódio-Glicose/administração & dosagem , Inibidores do Transportador 2 de Sódio-Glicose/farmacologiaRESUMO
DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms.
Assuntos
Córtex Cerebral/anormalidades , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Complexos Multiproteicos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Animais Geneticamente Modificados , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Genótipo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Neurônios/patologia , Neurônios/fisiologia , Fosforilação , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
PURPOSE: Erectile dysfunction is highly prevalent in type II diabetes mellitus. Low intensity extracorporeal shock wave therapy improves erectile function in patients with erectile dysfunction of vasculogenic origin, including diabetes. However, its mode of action remains unknown. We investigated the effects of low intensity extracorporeal shock wave therapy compared to or combined with sildenafil on erectile dysfunction in a type II diabetes mellitus model. Our purpose was to test our hypothesis of a mode of action targeting the cavernous nitric oxide/cyclic guanosine monophosphate pathway. MATERIALS AND METHODS: GK rats, a validated model of type II diabetes mellitus, and age matched Wistar rats were treated with low intensity extracorporeal shock wave therapy twice weekly for 3 weeks. Treatment was repeated after a 3-week no-treatment interval. The penis was stretched and dipped in a specifically designed water-filled cage. Shock waves were delivered by a calibrated probe yielding a controlled energy flux density (0.09 mJ/mm(2)). The probe was attached to an electrohydraulic unit with a focused shock wave source, allowing for accurate extrapolation to humans. Following a 4-week washout period erectile function was assessed as well as endothelium dependent and independent, and nitrergic relaxations of the corpus cavernosum of GK rats. RESULTS: Low intensity extracorporeal shock wave therapy significantly improved erectile function in GK rats to the same extent as sildenafil. Treatment effects were potentiated when combined with sildenafil. Shock wave effects were not associated with improved cavernous endothelium dependent or independent, or nitrergic reactivity. CONCLUSIONS: Low intensity extracorporeal shock wave therapy improved erectile function in GK rats. Unexpectedly, this was not mediated by a nitric oxide/cyclic guanosine monophosphate dependent mechanism. Sildenafil increased shock wave efficacy. This preclinical paradigm to deliver low intensity extracorporeal shock wave therapy to the rat penis should help further exploration of the mode of action of this therapy on erectile tissue.
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GMP Cíclico/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicações , Disfunção Erétil/terapia , Tratamento por Ondas de Choque Extracorpóreas/métodos , Óxido Nítrico/metabolismo , Ereção Peniana/fisiologia , Animais , Modelos Animais de Doenças , Disfunção Erétil/complicações , Disfunção Erétil/metabolismo , Masculino , Ratos Wistar , Transdução de SinaisRESUMO
Neuroglobin (NGB) is considered as an endogenous neuroprotective molecule against stroke, since the protein alleviates the adverse effects of hypoxic and ischemic insults. We previously demonstrated the functional link between NGB and mitochondria since it is required for respiratory chain function. Thus, here, we evaluated the relevance of this effect in the Harlequin (Hq) mouse strain, which exhibits retinal ganglion cell (RGC) loss and optic atrophy due to a respiratory chain complex I (CI) defect. A twofold decrease of NGB amounts was observed in Hq retinas. We constructed a recombinant adeno-associated virus which combines to the mouse NGB open reading frame, its 5' and 3'UTR, for guarantying mRNA stability and translation capacity. The vector was administrated intravitreally to Hq mice and NGB expression was stable for up to 7 months without negative effect on retinal architecture or function. On the contrary, RGCs and their axons were substantially preserved from degeneration; consequently, CI activity in optic nerves was protected conferring improvements in vision. Hence, we established that NGB prevents respiratory chain impairment, therefore, protecting visual function otherwise compromised by mitochondrial energetic failure.
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Complexo I de Transporte de Elétrons/deficiência , Globinas/genética , Proteínas do Tecido Nervoso/genética , Atrofia Óptica/prevenção & controle , Atrofia Óptica/terapia , Células Ganglionares da Retina/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/administração & dosagem , Gliose/patologia , Gliose/prevenção & controle , Globinas/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neuroglobina , Atrofia Óptica/genética , Atrofia Óptica/patologia , Células Ganglionares da Retina/patologiaRESUMO
Neuroglobin is a member of the globin superfamily proposed to be only expressed in neurons and involved in neuronal protection from hypoxia or oxidative stress. A significant fraction of the protein localizes within the mitochondria and is directly associated with mitochondrial metabolism and integrity. The retina is the site of the highest concentration for neuroglobin and has been reported to be up to 100-fold higher than in the brain. Since neuroglobin was especially abundant in retinal ganglion cell layer, we investigated its abundance in optic nerves. Remarkably in optic nerves, neuroglobin is observed, as expected, in retinal ganglion cell axon profiles but also astrocyte processes, in physiological conditions, possess high levels of the protein. Neuroglobin mRNA and protein levels are ~10-fold higher in optic nerves than in retinas, indicating an important accumulation of neuroglobin in these support cells. Additionally, neuroglobin levels increase in Müller cells during reactive gliosis in response to eye injury. This suggests the pivotal role of neuroglobin in retinal glia involved in neuronal support and/or healing. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
Assuntos
Gliose/metabolismo , Globinas/metabolismo , Cristalino/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Vias Visuais/metabolismo , Animais , Western Blotting , Gliose/patologia , Globinas/genética , Cristalino/lesões , Cristalino/patologia , Masculino , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neuroglobina , Nervo Óptico/citologia , RNA Mensageiro/genética , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vias Visuais/patologiaRESUMO
Despite the fact that urogenito-sexual complications significantly impact the quality of life of diabetic patients, a robust in vivo experimental model is lacking. Bladder and erectile function in the Type 2 diabetic Goto-Kakizaki (GK) rat and responses to standard-of-care treatments for each disorder have been assessed. GK rats (n = 25, 18-wk-old, GK/Par colony) and age-matched Wistar rats (n = 23), characterized for their metabolic parameters, were used. Bladder function was assessed by cystometry in conscious rats treated by intravenous solifenacin (1 mg/kg). Subsequently, erectile function was assessed under anesthesia following electrical stimulation of the cavernous nerve in presence of intravenous sildenafil (0.3 mg/kg). GK rats displayed detrusor overactivity with a significant increase in frequency/amplitude of nonvoiding contractions during the filling phase, together with an increase in bladder capacity, intercontraction interval, voided volume, and maximal pressure of voiding contraction. Solifenacin significantly decreased parameters characterizing voiding contractions without modifying voiding efficiency. Erectile function in GK rats was markedly impaired and remained so after sildenafil treatment despite a significant improvement. GK rats display both bladder and erectile dysfunctions and respond at least partially to standard-of-care treatments for each disorder, thus representing a suitable model to investigate the pathophysiology and assess the efficacy of new therapeutic agents for Type 2 diabetes-associated bladder and erectile complications.
Assuntos
Complicações do Diabetes/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Disfunção Erétil/etiologia , Doenças da Bexiga Urinária/etiologia , Animais , Diabetes Mellitus Tipo 2/fisiopatologia , Masculino , Ereção Peniana/efeitos dos fármacos , Piperazinas/farmacologia , Purinas/farmacologia , Quinuclidinas/farmacologia , Ratos , Ratos Wistar , Citrato de Sildenafila , Succinato de Solifenacina , Sulfonas/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Bexiga Urinária/fisiologia , Doenças da Bexiga Urinária/tratamento farmacológico , Doenças da Bexiga Urinária/fisiopatologia , Micção/efeitos dos fármacos , Micção/fisiologia , Agentes Urológicos/farmacologia , Vasodilatadores/farmacologiaRESUMO
RATIONALE: Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel regulating fluid homeostasis at epithelial surfaces. Its loss of function induces hypohydration, mucus accumulation, and bacterial infections in CF and potentially other lung chronic diseases. OBJECTIVES: To test whether neutrophil elastase (NE) and neutrophil-mediated inflammation negatively impact CFTR structure and function, in vitro and in vivo. METHODS: Using an adenovirus-CFTR overexpression approach, we showed that NE degrades wild-type (WT)- and ΔF508-CFTR in vitro and WT-CFTR in mice through a new pathway involving the activation of intracellular calpains. MEASUREMENTS AND MAIN RESULTS: CFTR degradation triggered a loss of function, as measured in vitro by channel patch-clamp and in vivo by nasal potential recording in mice. Importantly, this mechanism was also shown to be operative in a Pseudomonas aeruginosa lung infection murine model, and was NE-dependent, because CFTR integrity was significantly protected in NE(-/-) mice compared with WT mice. CONCLUSIONS: These data provide a new mechanism and show for the first time a link between NE-calpains activation and CFTR loss of function in bacterial lung infections relevant to CF and to other chronic inflammatory lung conditions.
Assuntos
Calpaína/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Elastase de Leucócito/fisiologia , Animais , Calpaína/metabolismo , Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epitélio/fisiologia , Humanos , Elastase de Leucócito/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Pneumonia Bacteriana/etiologia , Pneumonia Bacteriana/fisiopatologia , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/fisiopatologiaRESUMO
Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated ß-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.
Assuntos
Convulsões , Serina-Treonina Quinases TOR , Animais , Serina-Treonina Quinases TOR/metabolismo , Camundongos , Humanos , Convulsões/tratamento farmacológico , Senoterapia/farmacologia , Senescência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Epilepsia/tratamento farmacológico , Masculino , Malformações do Desenvolvimento Cortical/tratamento farmacológico , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , FemininoRESUMO
Diffuse bronchiectasis is a common problem in respiratory clinics. We hypothesized that mutations in the solute carrier 26A9 (SLC26A9) gene, encoding for a chloride (Cl(-)) transporter mainly expressed in lungs, may lead to defects in mucociliary clearance. We describe two missense variants in the SLC26A9 gene in heterozygote patients presenting with diffuse idiopathic bronchiectasis : p.Arg575Trp, identified in a patient also heterozygote for p.Phe508del in the CFTR gene; and p.Val486Ile. Expression of both mutants in Xenopus laevis oocytes abolished SLC26A9-mediated Cl(-) conductance without decreasing protein membrane expression. Coexpression of CFTR with SLC26A9-p.Val486Ile resulted in a significant increase in the Cl(-) current induced by PKA stimulation, similar to that obtained in oocytes expressing CFTR and SLC26A9-WT. In contrast, coexpression of CFTR with SLC26A9-p.Arg575Trp inhibited SLC26A9-enhanced CFTR activation upon PKA. Further structure-function analyses led us to propose a site encompassing Arg575 in the SLC26A9-STAS domain for CFTR-SLC26A9 interaction. We hypothesize that SLC26A9-p.Arg575Trp prevented SLC26A9-mediated functional activation of CFTR by altering SLC26A9-CFTR interaction. Although we cannot confirm that these mutations by themselves are deleterious, we propose that they trigger the pathogenic role of a single CFTR mutation and provide insight into a novel mechanism of Cl(-) transport alteration across the respiratory mucosa, based on functional inhibition of CFTR.
Assuntos
Antiporters/genética , Pneumopatias/diagnóstico , Pneumopatias/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antiporters/química , Antiporters/metabolismo , Estudos de Casos e Controles , Criança , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Éxons , Feminino , Expressão Gênica , Humanos , Pneumopatias/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Oócitos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transportadores de Sulfato , Tomografia Computadorizada por Raios X , Xenopus laevis , Adulto JovemRESUMO
Autoimmune encephalitis (AIE) associated with antibodies directed against the leucine-rich glioma inactivated 1 (LGI1) protein is the second most common AIE and is responsible for deleterious neocortical and limbic epileptic seizures. Previous studies demonstrated a pathogenic role of anti-LGI1 antibodies via alterations in the expression and function of Kv1 channels and AMPA receptors. However, the causal link between antibodies and epileptic seizures has never been demonstrated. Here, we attempted to determine the role of human anti-LGI1 autoantibodies in the genesis of seizures by analyzing the impact of their intracerebral injection in rodents. Acute and chronic injections were performed in rats and mice in the hippocampus and primary motor cortex, the two main brain regions affected by the disease. Acute infusion of CSF or serum IgG of anti-LGI1 AIE patients did not lead to the emergence of epileptic activities, as assessed by multisite electrophysiological recordings over a 10 h period after injection. A chronic 14 d injection, coupled with continuous video-EEG monitoring, was not more effective. Overall, these results demonstrate that acute and chronic injections of CSF or purified IgG from LGI1 patients are not able to generate epileptic activity by themselves in the different animal models tested.
Assuntos
Epilepsia , Peptídeos e Proteínas de Sinalização Intracelular , Humanos , Ratos , Camundongos , Animais , Leucina , Roedores , Convulsões/induzido quimicamente , Epilepsia/induzido quimicamente , Hipocampo , Imunoglobulina GRESUMO
OBJECTIVE: It is unknown whether ultrasound-induced blood-brain barrier (BBB) disruption can promote epileptogenesis and how BBB integrity changes over time after sonication. METHODS: To gain more insight into the safety profile of ultrasound (US)-induced BBB opening, we determined BBB permeability as well as histological modifications in C57BL/6 adult control mice and in the kainate (KA) model for mesial temporal lobe epilepsy in mice after sonication with low-intensity pulsed ultrasound (LIPU). Microglial and astroglial changes in ipsilateral hippocampus were examined at different time points following BBB disruption by respectively analyzing Iba1 and glial fibrillary acidic protein immunoreactivity. Using intracerebral EEG recordings, we further studied the possible electrophysiological repercussions of a repeated disrupted BBB for seizure generation in nine non-epileptic mice. RESULTS: LIPU-induced BBB opening led to transient albumin extravasation and reversible mild astrogliosis, but not to microglial activation in the hippocampus of non-epileptic mice. In KA mice, the transient albumin extravasation into the hippocampus mediated by LIPU-induced BBB opening did not aggravate inflammatory processes and histologic changes that characterize the hippocampal sclerosis. Three LIPU-induced BBB opening did not induce epileptogenicity in non-epileptic mice implanted with depth EEG electrodes. CONCLUSION: Our experiments in mice provide persuasive evidence of the safety of LIPU-induced BBB opening as a therapeutic modality for neurological diseases.
Assuntos
Barreira Hematoencefálica , Epilepsia do Lobo Temporal , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Epilepsia do Lobo Temporal/terapia , Epilepsia do Lobo Temporal/induzido quimicamente , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Albuminas , HipocampoRESUMO
The GGGGCC intronic repeat expansion within C9ORF72 is the most common genetic cause of ALS and FTD. This mutation results in toxic gain of function through accumulation of expanded RNA foci and aggregation of abnormally translated dipeptide repeat proteins, as well as loss of function due to impaired transcription of C9ORF72. A number of in vivo and in vitro models of gain and loss of function effects have suggested that both mechanisms synergize to cause the disease. However, the contribution of the loss of function mechanism remains poorly understood. We have generated C9ORF72 knockdown mice to mimic C9-FTD/ALS patients haploinsufficiency and investigate the role of this loss of function in the pathogenesis. We found that decreasing C9ORF72 leads to anomalies of the autophagy/lysosomal pathway, cytoplasmic accumulation of TDP-43 and decreased synaptic density in the cortex. Knockdown mice also developed FTD-like behavioral deficits and mild motor phenotypes at a later stage. These findings show that C9ORF72 partial loss of function contributes to the damaging events leading to C9-FTD/ALS.
RESUMO
UNLABELLED: What's known on the subject? and What does the study add? Lower urinary tract symptoms (LUTS) resulting from benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) are common problems in the aging male population. Moreover, several recent studies have shown that ED is closely associated with the presence and severity of LUTS independently of co-morbidities. However, the pathophysiological mechanisms linking LUTS/BPH and ED remain largely unexplored. The major difficulty in studying such relationships between ED and LUTS/BPH, and of exploring the impact of new therapeutic approaches for both LUTS/BPH and ED, is the lack of experimental model combining ED, prostate enlargement and bladder dysfunction all at once. The present study describes a new model of BPH, the SHR supplemented with testosterone which is the first animal model which displays all at once the key features of BPH: prostate enlargement and an increased sympathetic tone of bladder outlet mimicking the static and the dynamic components of voiding symptoms of BPH, a significant impairment of bladder function which reflects the storage symptoms of BPH and finally, ED. This model could be very relevant to better characterize the close relationship that exists between BPH/LUTS and ED, and to evaluate new therapeutic strategies for BPH together with their side effect profile on sexual function on the same animal, thus allowing a reduction of the number of animals to be used in such studies. Study Type - Aetiology (case control) Level of Evidence 3b. OBJECTIVE: ⢠To design a new experimental model combining erectile dysfunction, prostate enlargement and urodynamic impairment characteristic of lower urinary tract symptoms (LUTS) associated with benign prostate hyperplasia (BPH). MATERIALS AND METHODS: ⢠Three groups of animals (12-week-old; n= 7/group) were considered: Wistar Kyoto (control) rats (WKY), untreated spontaneously hypertensive rats (SHR) and SHR treated with testosterone (SHR-T, 3 mg/kg/day) for 3 weeks. ⢠Cystometry experiments and evaluation of erectile function were performed. Prostate enlargement was evaluated. RESULTS: ⢠SHR displayed a significant decrease in the intercontraction interval (ICI) and in the voided volume (VV) whereas non-voiding contractions (NVC) were increased. SHR-T exhibited a further decreased ICI and VV and an increased frequency of NVC. ⢠Erectile responses to electrical stimulation of the cavernous nerve were significantly impaired in both SHR (-66%) and SHR-T (-58%). ⢠The prostate weight was similar in WKY and SHR, but significantly increased in SHR-T. CONCLUSIONS: ⢠The testosterone-supplemented SHR represents an experimental model for urodynamic impairment combining both static and dynamic components of voiding symptoms with erectile dysfunction and prostate enlargement. ⢠This model is suitable for the assessment of sexual side effects of LUTS/BPH treatments and efficacy of new therapeutic agents in LUTS/BPH and associated erectile dysfunction.
Assuntos
Androgênios/farmacologia , Modelos Animais de Doenças , Disfunção Erétil/etiologia , Sintomas do Trato Urinário Inferior/etiologia , Hiperplasia Prostática/complicações , Testosterona/farmacologia , Análise de Variância , Androgênios/administração & dosagem , Animais , Pressão Arterial/fisiologia , Estimulação Elétrica , Disfunção Erétil/fisiopatologia , Sintomas do Trato Urinário Inferior/fisiopatologia , Masculino , Pressão , Hiperplasia Prostática/fisiopatologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Testosterona/administração & dosagemRESUMO
Autoimmune encephalitis associated with antibodies directed against the leucine-rich glioma inactivated 1 (LGI1) protein is responsible for specific tonic-dystonic motor seizures. Although dysfunctions in neuronal excitability have been associated with anti-LGI1 autoantibodies, their relation to seizures remain inconclusive. We developed a new in vivo experimental rat model to determine whether inhibition of Kv1.1 channels by dentrotoxin-K (DTX) in the primary motor cortex (M1) could recapitulate the human seizures and to elucidate their subtending cortical mechanisms. Comparing electro-clinical features of DTX-induced seizures in rats with those recorded from a cohort of anti-LGI1 encephalitis patients revealed striking similarities in their electroencephalographic (EEG) signature, frequency of recurrence and semiology. By combining multi-site extracellular and intracellular recordings of M1 pyramidal neurons in DTX rats, we demonstrated that the blockade of Kv1.1 channels induced a sequence of changes in neuronal excitability and synaptic activity, leading to massive suprathreshold membrane depolarizations underlying the paroxysmal EEG activity. Our results suggest the central role of Kv1.1 channels disruption in the emergence of anti-LGI1-associated seizures and suggest that this new rodent model could serve future investigations on ictogenesis in autoimmune encephalitis.