RESUMO
Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype five gene therapy under investigation for the treatment of hemophilia A. Herein, we assessed the potential for germline transmission of AAV5-hFVIII-SQ in mice. Male B6.129S6-Rag2tm1Fwa N12 mice received a single intravenous dose of vehicle or 6 × 1013 vg/kg AAV5-hFVIII-SQ. Vehicle and AAV5-hFVIII-SQ-treated mice were mated with naïve females 4 days after dosing, when the concentration of vector genomes was expected to be at its peak in semen, and 37 days after dosing, when a full spermatogenesis cycle was estimated to be complete. Quantitative PCR was used to evaluate the presence of transgene DNA in liver and testes from F0 males dosed with AAV5-hFVIII-SQ and liver tissue of F1 offspring. Transgene DNA was detected in liver and testes of all F0 males dosed with AAV5-hFVIII-SQ, confirming successful transduction. Importantly, no transgene DNA was detected in any tested F1 offspring derived from F0 males dosed with AAV5-hFVIII-SQ. Using a novel 2-stage statistical model that takes into account the number of males dosed with AAV5-hFVIII-SQ and the number of offspring sired by these males, we estimate that the risk of germline transmission is <5% with a 99.2% confidence level.
Assuntos
Fator VIII , Vetores Genéticos , Masculino , Animais , Camundongos , Fator VIII/genética , Vetores Genéticos/genética , Terapia Genética , Administração Intravenosa , Dependovirus/genéticaRESUMO
Replication-incompetent adeno-associated virus (AAV)-based vectors are nonpathogenic viral particles used to deliver therapeutic genes to treat multiple monogenic disorders. AAVs can elicit immune responses; thus, one challenge in AAV-based gene therapy is the presence of neutralizing antibodies against vector capsids that may prevent transduction of target cells or elicit adverse findings. We present safety findings from two 12-week studies in nonhuman primates (NHPs) with pre-existing or treatment-emergent antibodies. In the first study, NHPs with varying levels of naturally acquired anti-AAV5 antibodies were dosed with an AAV5-based vector encoding human factor VIII (hFVIII). In the second study, NHPs with no pre-existing anti-AAV antibodies were dosed with an AAV5-based vector carrying the beta subunit of choriogonadotropic hormone (bCG); this led to the induction of high-titer antibodies against the AAV5 capsid. Four weeks later, the same NHPs received an equivalent dose of an AAV5-based vector carrying human factor IX (hFIX). In both of these studies, the administration of vectors carrying hFVIII, bCG, and hFIX was well-tolerated in NHPs with no adverse clinical pathology or microscopic findings. These two studies demonstrate the safety of AAV-based vector administration in NHPs with either low-titer pre-existing anti-AAV5 antibodies or re-administration, even in the presence of high-titer antibodies.
Assuntos
Capsídeo , Dependovirus , Animais , Humanos , Dependovirus/genética , Anticorpos Neutralizantes/genética , Terapia GenéticaRESUMO
A therapeutic option for monogenic disorders is gene therapy with ex vivo-transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of ex vivo-modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34+ HSCs. Engrafted human CD34+ cells migrate to bone marrow and differentiate into various human hematopoietic lineages. A 12-week study was conducted in NSG™ mice to evaluate engraftment, differentiation, and safety of human CD34+ cells that were transduced (ex vivo) with a proprietary lentiviral vector encoding a human gene (BMRN-1) or a mock (green fluorescent protein) vector. Several mice intravenously injected with naive CD34+ cells or transduced CD34+ cells had variable lymphohistiocytic inflammatory cell infiltrates and microgranulomas in the liver and lungs consistent with graft-versus-host disease (GVHD). Spleen, bone marrow, stomach, reproductive tract, but not the skin had similar inflammatory changes. Ex vivo viral transduction of CD34+ cells did not impact engraftment or predispose to xenogeneic GVHD.
Assuntos
Antígenos CD34/genética , Terapia Genética/métodos , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Animais , Antígenos CD34/imunologia , Técnicas de Transferência de Genes , Doença Enxerto-Hospedeiro/imunologia , Proteínas de Fluorescência Verde/genética , Células-Tronco Hematopoéticas/imunologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Pesquisa Translacional BiomédicaRESUMO
This chapter describes various approaches for the preclinical assessment of drug-induced central nervous system (CNS) adverse effects. Traditionally, methods to evaluate CNS effects have consisted of observing and scoring behavioral responses of animals after drug is administered. Among several behavioral testing paradigms, the Irwin and the functional observational battery (FOB) are the most commonly used assays for the assessment of CNS effects. The Irwin and FOB are considered good first-tier assays to satisfy the ICH S7A guidance for the preclinical evaluation of new chemical entities (NCE) intended for humans. However, experts have expressed concern about the subjectivity and lack of quantitation that is derived from behavioral testing. More importantly, it is difficult to gain insight into potential mechanisms of toxicity by assessing behavioral outcomes. As a complement to behavioral testing, we propose using electrophysiology-based assays, both in vivo and in vitro, such as electroencephalograms and brain slice field-potential recordings. To better illustrate these approaches, we discuss the implementation of electrophysiology-based techniques in drug-induced assessment of seizure risk, sleep disruption, and cognitive impairment.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Animais , Comportamento Animal/efeitos dos fármacos , Sistema Nervoso Central/fisiologia , Transtornos Cognitivos/induzido quimicamente , Eletroencefalografia , Eletrofisiologia , Potenciais Evocados/efeitos dos fármacos , Humanos , Medição de Risco , Sono/efeitos dos fármacosRESUMO
Friedreich's ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of the cardiac phenotype of FRDA was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxnflox/null::MCK-Cre conditional knockout mouse (FXN-MCK) has an FXN gene ablation that prevents FXN expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss, and morbidity. FXN-MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN genes under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN-MCK control mice, AAV-treated FXN-MCK mice displayed increases in body weight, reversal of cardiac deficits, and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks postdose. FXN protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy.
RESUMO
Pompe disease is a severe disorder caused by loss of acid α-glucosidase (GAA), leading to glycogen accumulation in tissues and neuromuscular and cardiac dysfunction. Enzyme replacement therapy is the only available treatment. AT845 is an adeno-associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa-/- mice led to a dose-dependent increase in GAA activity, glycogen clearance in muscles and heart, and functional improvement. AT845 was tolerated in cynomolgus macaques at low doses, while high doses caused anti-GAA immune response, inflammation, and cardiac abnormalities resulting in unscheduled euthanasia of two animals. Conversely, a vector expressing the macaque GAA caused no detectable pathology, indicating that the toxicity observed with AT845 was an anti-GAA xenogeneic immune response. Western blot analysis showed abnormal processing of human GAA in cynomolgus muscle, adding to the species-specific effects of enzyme expression. Overall, these studies show that AAV-mediated GAA delivery to muscle is efficacious in Gaa-/- mice and highlight limitations in predicting the toxicity of AAV vectors encoding human proteins in non-human species.
Assuntos
Doença de Depósito de Glicogênio Tipo II , Animais , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by deficiency of the mitochondrial protein frataxin. Lack of frataxin causes neuronal loss in various areas of the CNS and PNS. In particular, cerebellar neuropathology in FRDA patients includes loss of large principal neurons and synaptic terminals in the dentate nucleus (DN), and previous studies have demonstrated early synaptic deficits in the Knockin-Knockout mouse model of FRDA. However, the exact correlation of frataxin deficiency with cerebellar neuropathology remains unclear. Here we report that doxycycline-induced frataxin knockdown in a mouse model of FRDA (FRDAkd) leads to synaptic cerebellar degeneration that can be partially reversed by AAV8-mediated frataxin restoration. Loss of cerebellar Purkinje neurons and large DN principal neurons are observed in the FRDAkd mouse cerebellum. Levels of the climbing fiber-specific glutamatergic synaptic marker VGLUT2 decline starting at 4 weeks after dox induction, whereas levels of the parallel fiber-specific synaptic marker VGLUT1 are reduced by 18-weeks. These findings suggest initial selective degeneration of climbing fiber synapses followed by loss of parallel fiber synapses. The GABAergic synaptic marker GAD65 progressively declined during dox induction in FRDAkd mice, while GAD67 levels remained unaltered, suggesting specific roles for frataxin in maintaining cerebellar synaptic integrity and function during adulthood. Expression of frataxin following AAV8-mediated gene transfer partially restored VGLUT1/2 levels. Taken together, our findings show that frataxin knockdown leads to cerebellar degeneration in the FRDAkd mouse model, suggesting that frataxin helps maintain cerebellar structure and function.
RESUMO
Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.
RESUMO
Acetylcholine and nicotine can modulate respiratory patterns by acting on nicotinic acetylcholine receptors (nAChRs) in the preBötzinger complex (preBötC). To further explore the molecular composition of these nAChRs, we studied a knock-in mouse strain with a leucine-to-alanine mutation in the M2 pore-lining region (L9'A) of the nAChR alpha4 subunit; this mutation renders alpha4-containing receptors hypersensitive to agonists. We recorded respiratory-related rhythmic motor activity from hypoglossal nerve (XIIn) and patch-clamped preBötC inspiratory neurons in an in vitro medullary slice preparation from neonatal mice. Nicotine affected respiratory rhythm at concentrations approximately 100-fold lower in the homozygous L9'A knock-in mice compared with wild-type mice. Bath application of 5 nm nicotine increased the excitability of preBötC inspiratory neurons, increased respiratory frequency, and induced tonic/seizure-like activities in XIIn in L9'A mice, effects similar to those induced by 1 microM nicotine in wild-type mice. In L9'A mice, microinjection of low nanomolar concentrations of nicotine into the preBötC increased respiratory frequency, whereas injection into the ipsilateral hypoglossal (XII) nucleus induced tonic/seizure-like activity. The alpha4*-selective nAChR antagonist dihydro-beta-erythroidine produced opposite effects and blocked the nicotinic responses. These data, showing that nAChRs in the preBötC and XII nucleus in L9'A mice are hypersensitive to nicotine and endogenous ACh, suggest that functional alpha4* nAChRs are present in the preBötC. They mediate cholinergic/nicotinic modulation of the excitability of preBötC inspiratory neurons and of respiratory rhythm. Furthermore, functional alpha4* nAChRs are present in XII nucleus and mediate cholinergic/nicotinic modulation of tonic activity in XIIn.
Assuntos
Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Respiração , Centro Respiratório/efeitos dos fármacos , Animais , Di-Hidro-beta-Eritroidina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/efeitos da radiação , Antagonistas Nicotínicos/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Periodicidade , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/metabolismo , Respiração/efeitos dos fármacos , Respiração/genética , Centro Respiratório/citologia , Centro Respiratório/fisiologia , Xenopus laevisRESUMO
The medial habenula (MHb) exhibits exceptionally high levels of nicotinic acetylcholine receptors (nAChRs), but it remains unclear whether all expressed nAChR subunit mRNAs are translated to form functional receptors. In particular alpha4 subunits have not been reported to have any functional role, despite strong alpha4 mRNA expression in the ventrolateral MHb. We studied a strain of knock-in mice expressing fluorescent alpha4* nAChRs (alpha4YFP), as well as a knock-in strain expressing hypersensitive alpha4* nAChRs (alpha4L9'A). In alpha4YFP mice, there was strong fluorescence in the ventrolateral MHb. In hypersensitive alpha4L9'A mice, injections of a low dose of nicotine (0.1 mg/kg) led to strong c-fos expression in only the ventrolateral region of the MHb, but not in the MHb of wild-type (WT) mice. In MHb slice recordings, ventrolateral neurons from alpha4L9'A mice, but not from WT mice, responded robustly to nicotine (1 microM). Neurons in the medial aspect of the MHb had >10-fold smaller responses. Thus alpha4* nAChRs contribute to the selective activation of a subset of MHb neurons. Subunit composition analysis based on gain-of-function knock-in mice provides a useful experimental paradigm.
Assuntos
Habenula/metabolismo , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Habenula/citologia , Habenula/efeitos dos fármacos , Técnicas In Vitro , Proteínas Luminescentes/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nicotina/farmacologia , Técnicas de Patch-Clamp , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismoRESUMO
Adeno-associated virus (AAV)-based vectors are widely used for gene therapy, but the effect of pre-existing antibodies resulting from exposure to wild-type AAV is unclear. In addition, other poorly defined plasma factors could inhibit AAV vector transduction where antibodies are not detected. To better define the relationship between various forms of pre-existing AAV immunity and gene transfer, we studied valoctocogene roxaparvovec (BMN 270) in cynomolgus monkeys with varying pre-dose levels of neutralizing anti-AAV antibodies and non-antibody transduction inhibitors. BMN 270 is an AAV5-based vector for treating hemophilia A that encodes human B domain-deleted factor VIII (FVIII-SQ). After infusion of BMN 270 (6.0 × 1013 vg/kg) into animals with pre-existing anti-AAV5 antibodies, there was a mean decrease in maximal FVIII-SQ plasma concentration (Cmax) and AUC of 74.8% and 66.9%, respectively, compared with non-immune control animals, and vector genomes in the liver were reduced. In contrast, animals with only non-antibody transduction inhibitors showed FVIII-SQ plasma concentrations and liver vector copies comparable with those of controls. These results demonstrate that animals without AAV5 antibodies are likely responders to AAV5 gene therapy, regardless of other inhibiting plasma factors. The biological threshold for tolerable AAV5 antibody levels varied between individual animals and should be evaluated further in clinical studies.
RESUMO
We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the alpha4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1-2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on alpha4S248Fbeta2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.
Assuntos
Nível de Alerta/genética , Modelos Animais de Doenças , Distúrbios Distônicos/genética , Epilepsia do Lobo Frontal/genética , Mutação , Nicotina/toxicidade , Animais , Nível de Alerta/efeitos dos fármacos , Distúrbios Distônicos/induzido quimicamente , Epilepsia do Lobo Frontal/induzido quimicamente , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Ratos , Especificidade da Espécie , XenopusRESUMO
A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.
Assuntos
Fenótipo , Subunidades Proteicas/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Convulsões/genética , Transtornos do Sono-Vigília/genética , Animais , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Transgênicos , Mutação , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/toxicidade , Subunidades Proteicas/biossíntese , Convulsões/induzido quimicamente , Convulsões/metabolismo , Transtornos do Sono-Vigília/metabolismo , XenopusRESUMO
We studied a strain of exon replacement mice ("L9'S knock-in") whose alpha4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9' position (Labarca et al., 2001); this mutation renders alpha4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9'S than in wild-type (WT) littermates. At these concentrations, L9'S but not WT showed increases in EEG amplitude and theta rhythm. L9'S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA(A) receptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9'S mice (0.1 mg/kg). The L9'S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9'S and WT mice in several brain regions. [(125)I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at approximately 25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. (86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9'S mice. These data support the possible involvement of gain-of-function alpha4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.
Assuntos
Hipocampo/fisiopatologia , Agonistas Nicotínicos/toxicidade , Receptores Nicotínicos/fisiologia , Convulsões/etiologia , Ritmo Teta , Animais , Comportamento Animal , Encéfalo/fisiopatologia , Eletroencefalografia , Feminino , Dosagem de Genes , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Nicotina , Fenótipo , Receptores Nicotínicos/análise , Receptores Nicotínicos/genética , Convulsões/induzido quimicamente , Convulsões/diagnóstico , Convulsões/patologia , CaudaRESUMO
This study analyzes the electrophysiological cause and behavioral consequence of dopaminergic cell loss in a knockin mouse strain bearing hypersensitive nicotinic alpha4-receptor subunits ("L9'S mice"). Adult brains of L9'S mice show moderate loss of substantia nigra dopaminergic neurons and of striatal dopaminergic innervation. Amphetamine-stimulated locomotion is impaired, reflecting a reduction of dopamine stored in presynaptic vesicles. Recordings from dopaminergic neurons in L9'S mice show that 10 microM nicotine depolarizes cells and increases spiking rates in L9'S cells but hyperpolarizes and decreases spiking rates in wild-type (WT) cells. Thus dopaminergic neurons of L9'S mice have an excitatory response to nicotine which is qualitatively different from that of WT neurons. The cause of dopaminergic cell death is therefore probably an increased sensitivity to acetylcholine or choline of alpha4-containing nicotinic receptors. Hypersensitive excitatory stimulation during activation of alpha4-containing receptors provides the first evidence for cholinergic excitotoxicity as a cause of dopaminergic neuron death. This novel concept may be relevant to the pathophysiology of Parkinson disease.
Assuntos
Morte Celular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptores Nicotínicos/metabolismo , Substância Negra/citologia , Substância Negra/fisiologia , Animais , Animais Recém-Nascidos , Agonistas de Dopamina/metabolismo , Feminino , Heterozigoto , Imuno-Histoquímica/métodos , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Mutação/fisiologia , Neurônios/química , Técnicas de Patch-Clamp/métodos , Receptores Dopaminérgicos/fisiologia , Receptores Nicotínicos/genética , Tirosina 3-Mono-Oxigenase/imunologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Two series of knockin mouse strains have been constructed with point mutations that result in hypersensitive neuronal nicotinic acetylcholine receptors containing alpha 4- or alpha 7-subunits. The full expression of the stronger alleles produces neonatal excitotoxic lethality; however, mice with attenuated expression or milder alleles are viable, and display a range of hypersensitive responses to nicotine. To date, measurements have been made on nicotine-induced seizures, Straub tail, hypothermia, antinociception, electroencephalograms and cellular electrophysiological responses. These strains are helping to define the occurrence of these important receptor subtypes, and their role in the acute and chronic actions of nicotine. The hypersensitive strains may be useful for the development of nicotinic drug therapy.
Assuntos
Camundongos Mutantes/genética , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Temperatura Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Camundongos , Modelos Animais , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Mutação Puntual , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiologia , Convulsões/induzido quimicamente , Convulsões/etiologia , Convulsões/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Trifosfato de Adenosina/biossíntese , Dopaminérgicos/metabolismo , Mitocôndrias/metabolismo , Compostos de Piridínio/metabolismo , Sinaptossomos/metabolismo , Animais , Antioxidantes/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Óxidos N-Cíclicos , Dopamina/metabolismo , Inibidores da Captação de Dopamina/metabolismo , Radicais Livres/metabolismo , Técnicas In Vitro , Masculino , Mazindol/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Óxidos de Nitrogênio/farmacologia , Compostos Organometálicos/farmacologia , Ratos , Ratos Sprague-Dawley , Rotenona/farmacologia , Salicilatos/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Desacopladores/farmacologiaRESUMO
Several mutations in α4 or ß2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the ß2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (ß2VL). Previous studies indicated that the ß2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of ß2VL mice. No differences in ß2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the ß2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.
Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Nicotina/farmacologia , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/genética , Temperatura Corporal/fisiologia , Sensibilização do Sistema Nervoso Central/genética , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Dopamina/metabolismo , Distonia/induzido quimicamente , Distonia/genética , Distonia/fisiopatologia , Técnicas de Introdução de Genes , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Atividade Motora/fisiologia , Mutação de Sentido Incorreto/genética , Nicotina/administração & dosagem , Terminações Pré-Sinápticas/efeitos dos fármacos , Ensaio Radioligante/métodos , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Radioisótopos de Rubídio , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Convulsões/fisiopatologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Cell transplantation is a promising treatment strategy for many neurological disorders, including stroke, which can target multiple therapeutic mechanisms in a sustained fashion. We investigated the ability of human mesenchymal stromal cells (MSCs) and MSC-derived SB623 cells to rescue neural cells via trophic support following an in vitro stroke model. Following oxygen glucose deprivation, cortical neurons or hippocampal slices were cocultured with either MSCs or SB623 cells separated by a semiporous membrane (prohibits cell-cell contact) or with MSC- or SB623 cell-conditioned medium. MSCs, SB623 cells, MSC-conditioned media, and SB623 cell-conditioned media all significantly reduced neural cell damage/death compared to untreated conditions, and the rescue effect of the conditioned media was dose dependent. We identified 11 neurotrophic factors secreted by MSCs and/or SB623 cells. This study emphasizes the importance of trophic support provided by marrow-derived cells, which likely contributes to the efficacy of cell therapy for brain injury.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/citologia , Adulto , Células da Medula Óssea/citologia , Isquemia Encefálica/terapia , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Hipocampo/citologia , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
INTRODUCTION: A novel automated blood sampling and telemetry (ABST) system was developed to integrate pharmacokinetic (PK), pharmacodynamic (PD) and toxicology studies. The goals of this investigation were to determine: 1) optimal feeding conditions and minimal acclimation times for recording PD parameters (blood pressure, heart rate, and temperature) after animals arrived on-site; 2) stress hormone levels in ABST-housed rats; 3) the feasibility of simultaneously recording cardiovascular parameters with electroencephalogram (EEG); 4) the equivalence of renal endpoints from ABST-housed rats with those in the metabolic cage, and 5) the cardiovascular responses to baclofen. METHODS: Body weight, blood pressure, temperature, stress biomarkers, urine chemistries, renal biomarkers and responses to vehicle or baclofen (10mg/kg) were compared in awake and freely moving rats housed in the ABST system, home cage (HC) or metabolic cage. RESULTS: Fasted rats lost 5+/-1% and 7+/-1% body weight when housed in ABST and metabolic cages, respectively. Weight loss was reversed by supplementing regular diet with hydration and nutritional supplements. Based on PD parameters, the minimum acclimation time required for both ABST and HC rats was 3days. The feasibility of simultaneously measuring multiple parameters, such as EEG with cardiovascular parameters in ABST was demonstrated. Renal function and biomarkers in rats continuously housed in the ABST and metabolic cages were equivalent (p>0.05) on days 1, 3, and 7. Baclofen-induced quantitatively and qualitatively similar (p>0.05) PK, mean arterial pressure, heart rate and temperature in ABST- and HC-housed rats. DISCUSSION: These studies demonstrate for the first time that drug-induced PD responses can be recorded simultaneously with time-matched pharmacokinetic, biochemical and metabolic parameters in the same animal. The ABST system has the added advantage of blood sampling without the need for satellite PK animals. ABST is a useful and novel tool for establishing efficacy and safety margins using an in vivo integrative pharmacology approach.