RESUMO
Liposomal formulations are hypothesized to alleviate anthracycline cardiotoxicity, although this has only been documented clinically for doxorubicin. We developed an in vitro multiparametric model using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to assess the relative toxicity of anthracyclines across formulations. Proof of concept was established by treating hiPSC-CM with equivalent concentrations of free and liposomal doxorubicin. The study was then repeated with free daunorubicin plus cytarabine and CPX-351, a dual-drug liposomal encapsulation of daunorubicin/cytarabine. hiPSC-CM were treated with free-drug or liposomal formulations for 24 h on Days 1, 3, and 5 at equivalent concentrations ranging from 0 to 1000 ng/mL and assessed on subsequent days. Free-drug treatment resulted in concentration-dependent cumulative cytotoxicity (microscopy), more profound decrease in ATP levels, and significant time- and concentration-dependent decreases in oxygen consumption versus liposomal formulations (p < 0.01). Repeated free-drug exposure also resulted in greater release of biomarkers (cardiac troponin I, FABP3) and lactate dehydrogenase, as well as in a biphasic rhythmicity response (initial increase followed by slowing/quiescence of beating) indicating significant injury, which was not observed after repeated exposure to liposomal formulations. Overall, liposomal formulations were considerably less toxic to hiPSC-CM than their free-drug counterparts. Clinical data will be needed to confirm findings for CPX-351.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Cardiotoxicidade , Miócitos Cardíacos , Daunorrubicina/toxicidade , Citarabina/toxicidade , Antraciclinas , Antibióticos Antineoplásicos/toxicidade , Inibidores da Topoisomerase II , Combinação de Medicamentos , LipossomosRESUMO
The development of pharmacological therapies for mitochondrial diseases is hampered by the lack of tissue-level and circulating biomarkers reflecting effects of compounds on endothelial and mitochondrial function. This phase 0 study aimed to identify biomarkers differentiating between patients with mitochondrial disease and healthy volunteers (HVs). In this cross-sectional case-control study, eight participants with mitochondrial disease and eight HVs matched on age, sex, and body mass index underwent study assessments consisting of blood collection for evaluation of plasma and serum biomarkers, mitochondrial function in peripheral blood mononuclear cells (PBMCs), and an array of imaging methods for assessment of (micro)circulation. Plasma biomarkers GDF-15, IL-6, NT-proBNP, and cTNI were significantly elevated in patients compared to HVs, as were several clinical chemistry and hematology markers. No differences between groups were found for mitochondrial membrane potential, mitochondrial reactive oxygen production, oxygen consumption rate, or extracellular acidification rate in PBMCs. Imaging revealed significantly higher nicotinamide-adenine-dinucleotide-hydrogen (NADH) content in skin as well as reduced passive leg movement-induced hyperemia in patients. This study confirmed results of earlier studies regarding plasma biomarkers in mitochondrial disease and identified several imaging techniques that could detect functional differences at the tissue level between participants with mitochondrial disease and HVs. However, assays of mitochondrial function in PBMCs did not show differences between participants with mitochondrial disease and HVs, possibly reflecting compensatory mechanisms and heterogeneity in mutational load. In future clinical trials, using a mix of imaging and blood-based biomarkers may be advisable, as well as combining these with an in vivo challenge to disturb homeostasis.
Assuntos
Leucócitos Mononucleares , Doenças Mitocondriais , Humanos , Leucócitos Mononucleares/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Mitocôndrias , Biomarcadores , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismoRESUMO
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is linked to Parkinson's disease (PD) and memory and is selectively expressed in neurons at high levels. Its expression pattern suggests a function distinct from that of its widely expressed homolog UCH-L3. We report here that, in contrast to UCH-L3, UCH-L1 exists in a membrane-associated form (UCH-L1(M)) in addition to the commonly studied soluble form. C-terminal farnesylation promotes the association of UCH-L1 with cellular membranes, including the endoplasmic reticulum. The amount of UCH-L1(M) in transfected cells is shown to correlate with the intracellular level of alpha-synuclein, a protein whose accumulation is associated with neurotoxicity and the development of PD. Reduction of UCH-L1(M) in cell culture models of alpha-synuclein toxicity by treatment with a farnesyltransferase inhibitor (FTI-277) reduces alpha-synuclein levels and increases cell viability. Proteasome function is not affected by UCH-L1(M), suggesting that it may negatively regulate the lysosomal degradation of alpha-synuclein. Therefore, inhibition of UCH-L1 farnesylation may be a therapeutic strategy for slowing the progression of PD and related synucleinopathies.
Assuntos
Membrana Celular/enzimologia , Neurônios/efeitos dos fármacos , Doença de Parkinson/enzimologia , Doença de Parkinson/terapia , Prenilação/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , alfa-Sinucleína/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Células COS , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Chlorocebus aethiops , Cisteína/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
BACKGROUND: Amyloid plaque deposition and axonal degeneration are early events in AD pathogenesis. Aß disrupts microtubules in presynaptic dystrophic neurites, resulting in the accumulation of impaired endolysosomal and autophagic organelles transporting ß-site amyloid precursor protein cleaving enzyme (BACE1). Consequently, dystrophic neurites generate Aß42 and significantly contribute to plaque deposition. Farnesyltransferase inhibitors (FTIs) have recently been investigated for repositioning toward the treatment of neurodegenerative disorders and block the action of farnesyltransferase (FTase) to catalyze farnesylation, a post-translational modification that regulates proteins involved in lysosome function and microtubule stability. In postmortem AD brains, FTase and its downstream signaling are upregulated. However, the impact of FTIs on amyloid pathology and dystrophic neurites is unknown. METHODS: We tested the effects of the FTIs LNK-754 and lonafarnib in the 5XFAD mouse model of amyloid pathology. RESULTS: In 2-month-old 5XFAD mice treated chronically for 3 months, LNK-754 reduced amyloid plaque burden, tau hyperphosphorylation, and attenuated the accumulation of BACE1 and LAMP1 in dystrophic neurites. In 5-month-old 5XFAD mice treated acutely for 3 weeks, LNK-754 reduced dystrophic neurite size and LysoTracker-Green accumulation in the absence of effects on Aß deposits. Acute treatment with LNK-754 improved memory and learning deficits in hAPP/PS1 amyloid mice. In contrast to LNK-754, lonafarnib treatment was less effective at reducing plaques, tau hyperphosphorylation and dystrophic neurites, which could have resulted from reduced potency against FTase compared to LNK-754. We investigated the effects of FTIs on axonal trafficking of endolysosomal organelles and found that lonafarnib and LNK-754 enhanced retrograde axonal transport in primary neurons, indicating FTIs could support the maturation of axonal late endosomes into lysosomes. Furthermore, FTI treatment increased levels of LAMP1 in mouse primary neurons and in the brains of 5XFAD mice, demonstrating that FTIs stimulated the biogenesis of endolysosomal organelles. CONCLUSIONS: We show new data to suggest that LNK-754 promoted the axonal trafficking and function of endolysosomal compartments, which we hypothesize decreased axonal dystrophy, reduced BACE1 accumulation and inhibited amyloid deposition in 5XFAD mice. Our results agree with previous work identifying FTase as a therapeutic target for treating proteinopathies and could have important therapeutic implications in treating AD.
Assuntos
Amiloide , Amiloidose , Farnesiltranstransferase , Doença de Alzheimer/metabolismo , Amiloide/efeitos dos fármacos , Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Ácido Aspártico Endopeptidases/metabolismo , Axônios/efeitos dos fármacos , Axônios/patologia , Modelos Animais de Doenças , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/metabolismo , Camundongos , Camundongos Transgênicos , Placa Amiloide/patologiaRESUMO
Age-related neurodegenerative disorders are characterized by a slow, persistent accumulation of aggregated proteins. Although cells can elicit physiological responses to enhance cellular clearance and counteract accumulation, it is unclear how pathogenic proteins evade this process in disease. We find that Parkinson's disease α-synuclein perturbs the physiological response to lysosomal stress by impeding the SNARE protein ykt6. Cytosolic ykt6 is normally autoinhibited by a unique farnesyl-mediated regulatory mechanism; however, during lysosomal stress, it activates and redistributes into membranes to preferentially promote hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, thereby disabling the lysosomal stress response and facilitating protein accumulation. Activating ykt6 by small-molecule farnesyltransferase inhibitors restores lysosomal activity and reduces α-synuclein in patient-derived neurons and mice. Our findings indicate that α-synuclein creates a permissive environment for aggregate persistence by inhibiting regulated cellular clearance and provide a therapeutic strategy to restore protein homeostasis by harnessing SNARE activity.
Assuntos
Lisossomos/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Proteínas R-SNARE/metabolismo , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transporte Proteico/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.
Assuntos
Metabolismo Energético , Imunidade , Mitocôndrias/metabolismo , Algoritmos , Biomarcadores , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Espaço Extracelular/metabolismo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Metaboloma , Metabolômica/métodos , Mitocôndrias/imunologia , Consumo de OxigênioRESUMO
Tolvaptan is a selective vasopressin V2 receptor antagonist, approved in several countries for the treatment of hyponatremia and autosomal dominant polycystic kidney disease (ADPKD). No liver injury has been observed with tolvaptan treatment in healthy subjects and in non-ADPKD indications, but ADPKD clinical trials showed evidence of drug-induced liver injury (DILI). Although all DILI events resolved, additional monitoring in tolvaptan-treated ADPKD patients is required. In vitro assays identified alterations in bile acid disposition and inhibition of mitochondrial respiration as potential mechanisms underlying tolvaptan hepatotoxicity. This report details the application of DILIsym software to determine whether these mechanisms could account for the liver safety profile of tolvaptan observed in ADPKD clinical trials. DILIsym simulations included physiologically based pharmacokinetic estimates of hepatic exposure for tolvaptan and2 metabolites, and their effects on hepatocyte bile acid transporters and mitochondrial respiration. The frequency of predicted alanine aminotransferase (ALT) elevations, following simulated 90/30 mg split daily dosing, was 7.9% compared with clinical observations of 4.4% in ADPKD trials. Toxicity was multifactorial as inhibition of bile acid transporters and mitochondrial respiration contributed to the simulated DILI. Furthermore, simulation analysis identified both pre-treatment risk factors and on-treatment biomarkers predictive of simulated DILI. The simulations demonstrated that in vivo hepatic exposure to tolvaptan and the DM-4103 metabolite, combined with these 2 mechanisms of toxicity, were sufficient to account for the initiation of tolvaptan-mediated DILI. Identification of putative risk-factors and potential novel biomarkers provided insight for the development of mechanism-based tolvaptan risk-mitigation strategies.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/efeitos adversos , Benzazepinas/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Biológicos , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacocinética , Benzazepinas/farmacocinética , Suscetibilidade a Doenças , Humanos , TolvaptanRESUMO
In this study, we demonstrate that angiotensin II (Ang II) protects dopamine (DA) neurons from rotenone toxicity in vitro. Primary ventral mesencephalic (VM) cultures from E15 rats were grown for 5 days and then cultured in the presence of the mitochondrial complex I inhibitor, rotenone. Acute exposure (20 h) to 20 nM rotenone reduced the number of tyrosine hydroxylase-positive (TH+) neurons by 50 +/- 6% when compared to untreated cultures. Pre-treatment of VM cultures with 100 nM Ang II decreased TH+ neuronal loss to 25 +/- 10% at the 20-nM rotenone concentration. Ang II in the presence of the angiotensin type 1 receptor (AT1R) antagonist, losartan, was even more effective in protecting DA neurons showing a loss of only 13 +/- 4% at 20 nM rotenone. Conversely, the AT2R antagonist, PD123319, abolished the protective effects of Ang II. Furthermore, both the NMDA receptor antagonist, MK801, and the antioxidant, alpha-tocopheryl succinate (vitamin E analogue), prevented rotenone-induced toxicity. Here, we show that acute exposure of VM cultures to the pesticide rotenone leads to dopaminergic neuronal cell death and that angiotensin acting through the AT2 receptor protects dopamine neurons from rotenone toxicity.
Assuntos
Angiotensina II/farmacologia , Dopamina/metabolismo , Neurônios/efeitos dos fármacos , Rotenona/antagonistas & inibidores , Substância Negra/efeitos dos fármacos , Angiotensina II/uso terapêutico , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Antioxidantes/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Inseticidas/antagonistas & inibidores , Losartan/farmacologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/tratamento farmacológico , Degeneração Neural/prevenção & controle , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurotoxinas/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/prevenção & controle , Ratos , Ratos Sprague-Dawley , Substância Negra/patologia , Substância Negra/fisiopatologia , Tirosina 3-Mono-Oxigenase/metabolismoAssuntos
Processamento Alternativo , Hipertensão Pulmonar Primária Familiar/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Glicólise , Humanos , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Inquéritos e QuestionáriosRESUMO
While it has been shown that the angiotensin type-2 (AT(2)) receptor plays an important role in the development and differentiation of many tissues, the second messengers involved in its signaling pathways are just beginning to be understood. To further determine the signaling pathways for the AT(2) receptor, we have investigated whether human angiotensin type-2 receptor transfected into Chinese hamster ovary (CHO) cells can modulate insulin-induced extracellular signal-related protein kinase (ERK-2) phosphorylation via a G-protein coupled mechanism. Our results indicate that the human AT(2) receptor decreases insulin-induced ERK-2 phosphorylation through a G-protein mediated pathway since inhibition was attenuated by pertussis toxin (a G(i)/G(0) inhibitor). Our findings further indicate that the inhibitory response was insensitive to sodium orthovanadate (a PTPase inhibitor), but sensitive (attenuated) to okadaic acid, suggesting an important role for protein phosphatase 2A (PP2A). We have also shown that alanine substitution of the putative G-protein coupling DRY(141-143) motif of the second intracellular loop significantly decreases the human AT(2) receptor's ability to inhibit insulin-induced ERK-2 phosphorylation. Our results support the hypothesis that the AT(2) receptor inhibits insulin-induced ERK-2 activity via a G-protein coupled pathway involving the up-regulation of PP2A.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Insulina/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Receptor Tipo 2 de Angiotensina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting/métodos , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Modelos Biológicos , Mutagênese/fisiologia , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo , TransfecçãoRESUMO
The signaling pathways for the seven transmembrane G-protein coupled angiotensin II receptors (AT(1) and AT(2)) are just beginning to be understood. While these receptors play an important role in the development and differentiation of many tissues, including the cardiovascular and central nervous systems, information about amino acid motifs involved in angiotensin II-mediated signaling is only available for the AT(1) receptor subtype. In the present study, we mutated the conserved DRY(141-143) motif in the AT(2) receptor, which is thought to be involved in G-protein recruitment. Expression of wild type and mutant receptors in CHO-K1 cell plasma membranes was confirmed using radioligand binding analyses. Our findings indicate a significant change in the binding affinities (kD) and capacities (B(max)) of the mutant receptors relative to wild type. Alanine substitutions of D(141) and DRY(141-143) resulted in a significant decrease of binding affinity for both Sar(1)Ile(8)-angiotensin II (SarIle-Ang II) (mixed agonist/antagonist) and angiotensin II (agonist). The binding affinities following alanine substitutions of R(142) and Y(143) were not significantly different from wild type receptor. Interestingly, the R(142)-A and Y(143)-A mutants revealed a significant decrease in binding levels from wild type with SarIle-Ang II, but not angiotensin II. The effect of GTPgammaS on angiotensin II binding affinity between wild type and mutant receptors was similarly significant. The D(141)-A, Y(143)-A, and DRY(141-143)-AAA mutant receptors showed a marked decrease in GTPgammaS-induced angiotensin II affinity shift. The R(142)-A GTPgammaS binding affinity shift was not different from the wild type receptor. Our results support the hypothesis that the DRY motif plays a significant role in the binding affinity, structural stability and G-protein recruiting of the AT(2) receptor.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Motivos de Aminoácidos , Angiotensina II/metabolismo , Animais , Células CHO , Cricetinae , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Ensaio Radioligante , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/químicaRESUMO
We have previously reported that angiotensin II (Ang II) protects cortical neurons from chemical-induced hypoxia through activation of the angiotensin type 2 (AT(2)) receptor. Here, we show in mouse primary neuronal cultures that the AT(2) receptor neuroprotection results from the activation of the delayed rectifier K(+) channel as well as the involvement of the Na(+)/Ca(2+) exchanger (NCX) and Na(+)/K(+) ATPase (ATPase). Roles of the K(+) channel, NCX and ATPase were determined using the specific blockers alpha-dendrotoxin, KB-R7943 and ouabain, respectively. Sodium azide (10mM) induced apoptosis in 40% of neurons. Inhibition of the AT(1) receptor with losartan (1 microM) facilitated angiotensin II mediated neuroprotection by reducing sodium azide-induced apoptosis 61.8 +/- 5.6%, while inhibition of the AT(2) receptor with PD123319 (1 microM) showed no neuroprotection. These results suggest that angiotensin II neuroprotection is mediated through the AT(2) receptor and requires inhibition of the AT(1) receptor in order to facilitate its effect. To determine the roles of delayed rectifier K(+) channel, NCX and ATPase cultures were pretreated with alpha-dendrotoxin (10nM), KB-R7943 (100 nM) and ouabain (100 nM), which significantly attenuated AT(2) receptor mediated neuroprotection. These findings further suggest that the mechanism of AT(2) receptor mediated neuroprotection is coupled to activation of the delayed rectifier K(+) channel, NCX and ATPase.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Células Cultivadas , Córtex Cerebral/metabolismo , Canais de Potássio de Retificação Tardia , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos ICR , Fármacos Neuroprotetores/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidoresRESUMO
In this study we determined whether caspase-3 is required in mouse cortical neurons for sodium azide-mediated apoptosis. Primary cortical neuronal cultures were treated with a cell permeable caspase-3 inhibitor, DEVD (1 nM-100 fM), prior to sodium azide-induced hypoxia. Treatment with the caspase-3 inhibitor resulted in a dose-dependent decrease in apoptosis, suggesting that sodium azide-induced apoptosis is mediated through a caspase-3 dependent pathway. Levels of cytochrome-c release and caspase-3 cleavage were assayed by Western analysis. Cytochrome-c release and caspase-3 cleavage were observed at 5 h (85.3+/-5.8%) and 8 h (53.4+/-14.9%), respectively. We have previously reported that angiotensin II, acting through the AT(2) receptor subtype, protects cultured mouse cortical neurons from sodium azide-induced apoptosis. We also examined whether the protective effect of angiotensin II is mediated through modulation of caspase-3. Pre-treatment of cells with angiotensin II and the AT(1) receptor antagonist, losartan, reduced levels of sodium azide-induced caspase-3 cleavage by 95.0+/-4.0%. Cells pre-treated with the AT(2) receptor antagonist, PD123319 showed a smaller reduction of caspase-3 cleavage (53.8+/-3.4%). Our findings indicate that sodium azide-induced apoptosis is caspase-3 dependent and that angiotensin II protects cortical neurons from chemical-induced apoptosis by reducing caspase-3 cleavage.
Assuntos
Angiotensina II/farmacologia , Caspases/metabolismo , Córtex Cerebral/enzimologia , Neurônios/enzimologia , Animais , Caspase 3 , Inibidores de Caspase , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacosRESUMO
INTRODUCTION: Regulated neurotransmitter actions in the mammalian central nervous system determine brain function and control peripheral organs and behavior. Although drug-seeking behaviors, including alcohol consumption, depend on central neurotransmission, modification of neurotransmitter actions in specific brain nuclei remains challenging. Herein, we report a novel approach for neurotransmission modification in vivo by transplantation of stem cells engineered to take up the neurotransmitter dopamine (DA) efficiently through the action of the human dopamine transporter (hDAT). As a functional test in mice, we used voluntary alcohol consumption, which is known to release DA in nucleus accumbens (NAC), an event hypothesized to help maintain drug-seeking behavior. We reasoned that reducing extracellular DA levels, by engrafting into NAC DA-sequestering stem cells expressing hDAT, would alter alcohol intake. METHODS: We have generated a neural stem cell line stably expressing the hDAT. Uptake kinetics of DA were determined to select a clone for transplantation. These genetically modified stem cells (or cells transfected with a construct lacking the hDAT sequence) were transplanted bilaterally into the NAC of wild-type mice trained to consume 10% alcohol in a two-bottle free-choice test for alcohol consumption. Alcohol intake was then ascertained for 1 week after transplantation, and brain sections through the NAC were examined for surviving grafted cells. RESULTS: Modified stem cells expressed hDAT and uptaken DA selectively via hDAT. Mice accustomed to drinking 10% ethanol by free choice reduced their alcohol consumption after being transplanted with hDAT-expressing stem cells. By contrast, control stem cells lacked that effect. Histologic examination revealed surviving stem cells in the NAC of all engrafted brains. CONCLUSIONS: Our findings represent proof of principle suggesting that genetically engineered stem cells can be useful for exploring the role of neurotransmitters (or other signaling molecules) in alcohol consumption and potentially in other aspects of brain function.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Comportamento de Procura de Droga/fisiologia , Células-Tronco Neurais/metabolismo , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Células Cultivadas , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/transplante , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção , beta-Galactosidase/metabolismoRESUMO
Among various types of neurons affected in Parkinson's disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2(+) neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH(+)) neurons and MAP2(+) neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.
Assuntos
Apoptose/efeitos dos fármacos , Dopamina/farmacologia , Neurônios/efeitos dos fármacos , Rotenona/toxicidade , Animais , Dopamina/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Neurônios/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-DawleyRESUMO
In this study, we examined the effects of angiotensin II (AngII) in a genetic in vitro PD model produced by alpha-synuclein (alpha-syn) overexpression in the human neuroglioma H4 cell line. We observed a maximal decrease in alpha-syn-induced toxicity of 85% and reduction in inclusion formation by 19% when cultures were treated with AngII in the presence of the angiotensin type 1 (AT1) receptor antagonist losartan and AT2 receptor antagonist PD123319. When compared to AngII, the AT4 receptor agonist AngIV was moderately effective in protecting H4 cells against alpha-syn toxicity and did not significantly reduce inclusion formation. Here we show that AngII is protective against genetic, as well as neurotoxic models of PD. These data support the view that agents acting on the renin-angiotensin-system (RAS) may be useful in the prevention and/or treatment of Parkinson's disease.
Assuntos
Angiotensina II/farmacologia , Receptores de Angiotensina/metabolismo , alfa-Sinucleína/fisiologia , Angiotensina II/análogos & derivados , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Humanos , Imidazóis/farmacologia , Immunoblotting , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Losartan/farmacologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/prevenção & controle , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Vasoconstritores/farmacologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Treatments based on pharmacological inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) have been suggested for a broad variety of human disorders, including Parkinson's disease (PD). The neuroprotective effects underlying the efficacy of PARP-1 inhibitors in PD models suggest a role for PARP-1 in neurodegeneration. In this study, we assessed the efficacy of PARP-1 inhibition in two distinct PD models. First, we tested a panel of small molecule PARP-1 inhibitors in alpha-synuclein (aSyn) cytotoxicity assay, where we observed compound-dependent ameliorating effects. Next, we tested the same panel in primary ventral mesencephalic neuronal cultures, treated with MPP(+). Dopaminergic neurons, the primary cells affected in PD, were selected and subjected to analysis. A significant ameliorating effect was achieved only with a highly potent PARP-1 inhibitor. Our data implicates aberrant PARP-1 function in different pathways of neurodegeneration. Further, our results suggest a rationale for the development of highly potent, bio-available, brain-penetrable PARP-1 inhibitors to provide therapeutic benefits for Parkinson's patients.
Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , alfa-Sinucleína/biossíntese , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fenantrenos/química , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1RESUMO
BACKGROUND: Recent attention has focused on understanding the role of the brain-renin-angiotensin-system (RAS) in stroke and neurodegenerative diseases. Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT1) receptor, losartan, protects dopaminergic (DA) neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary ventral mesencephalic (VM) cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice (Fig. 1). RESULTS: In the presence of exogenous Ang II, losartan reduced MPP+ (5 muM) induced DA neuronal loss by 72% in vitro. Mice challenged with MPTP showed a 62% reduction in the number of DA neurons in the SNpc and a 71% decrease in tyrosine hydroxylase (TH) immunostaining of the striatum, whereas daily treatment with losartan lessened MPTP-induced loss of DA neurons to 25% and reduced the decrease in striatal TH+ immunostaining to 34% of control. CONCLUSION: Our study demonstrates that the brain-RAS plays an important neuroprotective role in the MPTP model of PD and points to AT1 receptor as a potential novel target for neuroprotection.
RESUMO
Several transgenic mouse lines with altered alpha-synuclein expression have been developed that show a variety of Parkinson's disease-like symptoms without specific loss of dopaminergic neurons. Targeted over-expression of human alpha-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates leads to dopaminergic cell loss and the formation of alpha-synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno-associated virus (AAV) to over-express wild type human alpha-synuclein in the substantia nigra of mice. We hypothesized that this over-expression would recapitulate pathological hallmarks of Parkinson's disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing alpha-synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress-related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over-expression of alpha-synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson's disease pathogenesis can be further elucidated.
Assuntos
Dopamina/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Substância Negra/citologia , Substância Negra/metabolismo , Regulação para Cima/fisiologia , alfa-Sinucleína/metabolismo , Animais , Morte Celular/fisiologia , Dependovirus/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/genéticaRESUMO
The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.