RESUMO
The amyloid ß-peptide (Aß) is transported across the blood-brain barrier (BBB) by binding with the receptor for advanced glycation end products (RAGE). Previously, we demonstrated that the Aß fraction 25-35 (Aß25-35) increases RAGE expression in the rat hippocampus, likely contributing to its neurotoxic effects. However, it is still debated if the interaction of Aß with RAGE compromises the BBB function in Alzheimer' disease (AD). Here, we evaluated the effects of Aß25-35 in an established in vitro model of the BBB. Rat brain microvascular endothelial cells (rBMVECs) were treated with 20 µM active Aß25-35 or the inactive Aß35-25 (control), for 24 h. Exposure to Aß25-35 significantly decreased cell viability, increased cellular necrosis, and increased the production of reactive oxygen species (ROS), which triggered a decrease in the enzyme glutathione peroxidase when compared to the control condition. Aß25-35 also increased BBB permeability by altering the expression of tight junction proteins (decreasing zonula occludens-1 and increasing occludin). Aß25-35 induced monolayer disruption and cellular disarrangement of the BBB, with RAGE being highly expressed in the zones of disarrangement. Together, these data suggest that Aß25-35-induces toxicity by compromising the functionality and integrity of the BBB in vitro. Graphical abstract Aß25-35 induces BBB dysfunction in vitro, wich is likely mediated by OS and ultimately leads to disruption of BBB integrity and cell death.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Glutationa Peroxidase/metabolismo , Masculino , Camundongos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas de Junções Íntimas/metabolismoRESUMO
Graphene-based nanomaterials hold the potential to be used in a wide variety of applications, including biomedical devices. Pristine graphene (PG) is an un-functionalized, defect-free type of graphene that could be used as a material for neural interfacing. However, the neurotoxic effects of PG, particularly to the blood-brain barrier (BBB), have not been fully studied. The BBB separates the brain tissue from the circulating substances in the blood and is essential to maintain the brain homeostasis. The principal components of the BBB are brain microvascular endothelial cells (BMVECs), which maintain a protectively low permeability due to the expression of tight junction proteins. Here we analyzed the effects of PG on BMVECs in an in vitro model of the BBB. BMVECs were treated with PG at 0, 10, 50 and 100 µg/mL for 24 hours and viability and functional analyses of BBB integrity were performed. PG increased lactate dehydrogenase release at 50 and 100 µg/mL, suggesting the induction of necrosis. Surprisingly, 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) conversion was increased at 10 and 50 µg/mL. In contrast, XTT conversion was decreased at 100 µg/mL, suggesting the induction of cell death. In addition, 100 µg/mL PG increased DNA fragmentation, suggesting induction of apoptosis. At the same time, 50 and 100 µg/mL of PG increased the endothelial permeability, which corresponded with a decrease in the expression of the tight junction protein occludin at 100 µg/mL. In conclusion, these results suggest that PG negatively affects the viability and function of the BBB endothelial cells in vitro.
Assuntos
Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Grafite/toxicidade , Microvasos/efeitos dos fármacos , Animais , Apoptose/genética , Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/patologia , Encéfalo/irrigação sanguínea , Permeabilidade Capilar/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Grafite/farmacocinética , L-Lactato Desidrogenase/metabolismo , Microvasos/enzimologia , Microvasos/patologia , RatosRESUMO
Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson's disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson's disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson's disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules.
RESUMO
Mutations in parkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Intoxicação por MPTP/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilcisteína/farmacologia , Animais , Benzamidas , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Linhagem Celular , Modelos Animais de Doenças , Dopamina/farmacologia , Esquema de Medicação , Sequestradores de Radicais Livres/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Mesilato de Imatinib , Imunoprecipitação/métodos , Intoxicação por MPTP/induzido quimicamente , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/patologia , Masculino , Metaloporfirinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Piperazinas/toxicidade , Polietilenoglicóis/farmacologia , Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/toxicidade , RNA Interferente Pequeno/farmacologia , Estatísticas não Paramétricas , Transfecção/métodos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacosRESUMO
The pathophysiology underlying the loss of dopaminergic neurons in Parkinson's disease (PD) is unclear. A gap of knowledge in the molecular and cellular events leading to degeneration of the nigrostriatal DA system is a major barrier to the development of effective therapies for PD. 1-methyl-4-phenylpyridinium (MPP+) is used as a reliable in vitro model of PD in dopaminergic neurons; however, the molecular mechanisms that lead to cell death with this model are not fully understood. Additionally, there is a lack of translational in vitro models to fully understand progressive dopaminergic neurotoxicity. Here, we propose cultures of primary human dopaminergic neuronal precursor cells (HDNPCs) as a model to study progressive dopaminergic toxicity and neuronal damage in PD. We evaluated the concentration-response of MPP+ (0-10 mM) at 24 h, using cell viability and mitochondrial activity assays (LDH, XTT, Live/Dead staining, and MitoTracker). Based on concentration-response data, we chose two concentrations (1.0 and 2.5 mM) of MPP+ to evaluate markers of autophagy and dopaminergic status [tyrosine hydroxylase (TH)] after a 24-h exposure. Exposure to MPP+ induced cytotoxicity, reduced cell viability, and decreased mitochondrial activity. MPP+ at 1.0 and 2.5 mM also induced expression of lysosome-associated membrane protein 1 (LAMP-1) and increased the ratio of light chain 3 (LC3), LC3BII/LC3BI. The expression of TH also decreased. Furthermore, α-synuclein (α-SYN) and parkin were evaluated by immunofluorescence (IF) at 1.0 and 2.5 mM MPP+ after 24 h. A qualitative analysis revealed decreased parkin expression while α-SYN aggregation was observed in the cytoplasm and the nucleus. These data suggest that in HDNPCs MPP+ can cause cytotoxicity and neuronal damage. This damage may be mediated by autophagy, dopamine synthesis, and protein aggregation. The combination of HDNPCs and MPP+ may serve as valuable in vitro model of progressive dopaminergic neurotoxicity for research into potential treatments for PD.
RESUMO
Glial cell line-derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinson's disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood-brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell-derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH(+) terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.
Assuntos
Dopamina/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Macrófagos/metabolismo , Degeneração Neural/terapia , Doença de Parkinson/terapia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ingestão de Alimentos/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Neurotoxinas/farmacologia , Doença de Parkinson/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
The Cockayne Syndrome group B (CSB) protein plays important roles in transcription, transcription-coupled nucleotide excision repair and base excision DNA repair. c-Abl kinase also plays a role in DNA repair as a regulator/coordinator of the DNA damage response. This study presents evidence that the N-terminal region of CSB interacts with the SH3 domain of c-Abl in vitro and in vivo. In addition, c-Abl kinase phosphorylates CSB at Tyr932. The subcellular localization of CSB to the nucleus and nucleolus is altered after phosphorylation by c-Abl. c-Abl-dependent phosphorylation of CSB increased in cells treated with hydrogen peroxide and decreased in cells pre-treated with STI-571, a c-Abl-specific protein kinase inhibitor. Activation of the c-Abl kinase in response to oxidative damage is not observed in CSB null cells. These results suggest that c-Abl and CSB may regulate each other in a reciprocal manner in response to oxidative stress.
Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Linhagem Celular , Células Cultivadas , DNA Helicases/análise , DNA Helicases/química , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/química , Humanos , Camundongos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Proto-Oncogênicas c-abl/análise , Tirosina/metabolismoRESUMO
The Cockayne syndrome B (CSB) protein--defective in a majority of patients suffering from the rare autosomal disorder CS--is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP-DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.
Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Linhagem Celular Transformada , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Genoma Humano , Humanos , Metanossulfonato de Metila/toxicidade , Proteínas de Ligação a Poli-ADP-Ribose , Timidina/análogos & derivados , Timidina/toxicidadeRESUMO
The ends of linear chromosomes are capped and protected by protein-DNA complexes termed telomeres. Consequences of telomere dysfunction include genomic instability that can contribute to neoplastic transformation and progression. Telomere binding proteins interact with numerous proteins involved in DNA repair, underscoring the importance of regulating DNA repair pathways at telomeres. Telomeric DNA is particularly susceptible to oxidative damage, and such damage is repaired primarily via the base excision repair (BER) pathway. Using a screen for potential interactions between telomere repeat binding factor 2 (TRF2) and proteins involved in BER of oxidized bases in vitro, we found that TRF2 physically bound DNA polymerase beta (Pol beta) and flap endonuclease 1 (FEN-1). The interactions with endogenous proteins in human cell extracts were confirmed by coimmunoprecipitation experiments. The primary binding sites for both Pol beta and FEN-1 mapped to the TRF2 NH2-terminal and COOH-terminal domains. We further tested the ability of TRF2 to modulate BER protein partners individually on a variety of substrates in vitro. TRF2 stimulated Pol beta primer extension DNA synthesis on telomeric and nontelomeric primer/template substrates, resulting in up to a 75% increase in the proportion of longer products. TRF2 also stimulated Pol beta strand displacement DNA synthesis in reconstituted BER reactions and increased the percent of long-patch BER intermediates on both telomeric and nontelomeric substrates. Potential roles of TRF2 in cooperation with BER proteins for DNA repair pathways at telomeres, as well as other genomic regions, are discussed.
Assuntos
DNA Polimerase beta/metabolismo , Reparo do DNA/fisiologia , DNA de Neoplasias/biossíntese , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Dano ao DNA , Endonucleases Flap/metabolismo , Células HeLa , Humanos , ImunoprecipitaçãoRESUMO
IMPACT STATEMENT: Attrition in drug discovery and development remains a major challenge. Safety/toxicity is the most prevalent reason for failure with cardiovascular and CNS toxicities predominating. Non-invasive biomarkers of neurotoxicity would provide significant advantage by allowing earlier prediction of likely neurotoxicity in preclinical studies as well as facilitating clinical trials of new therapies for neurodegenerative conditions such as Parkinson's disease (PD) and multiple sclerosis (MS).
Assuntos
Biomarcadores/análise , Lesões Encefálicas/tratamento farmacológico , Descoberta de Drogas , Doenças Neurodegenerativas/tratamento farmacológico , Síndromes Neurotóxicas/tratamento farmacológico , Animais , Modelos Animais de Doenças , HumanosRESUMO
Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24â¯h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro.
Assuntos
Barreira Hematoencefálica/metabolismo , Concussão Encefálica/metabolismo , Células Endoteliais/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Claudina-5/metabolismo , Ocludina/metabolismo , Permeabilidade , Ratos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismoRESUMO
Traumatic brain injury (TBI) is one of the major causes of disability in the USA. It occurs when external mechanical forces induce brain damage that causes deformation of brain tissue. TBI is also associated with alterations of the blood-brain barrier (BBB). Using primary rat brain microvascular endothelial cells as an in vitro BBB model, the effects of biaxial stretch were characterized at 5, 10, 15, 25, and 50% deformation using a commercially available system. The results were compared to the effects of mild and moderate TBI in vivo, induced by the weight-drop method in mice. In vitro, live/dead cells, lactate dehydrogenase (LDH) release, caspase 3/7 staining, and tight junction (TJ) protein expression were evaluated 24 h after a single stretch episode. In vivo, Evans blue extravasation, serum levels of S100ß, and TJ protein expression were evaluated. Stretch induced a deformation-dependent increase in LDH release, cell death, and activation of caspase 3/7, suggesting the induction of apoptosis. Interestingly, low magnitudes of deformation increased the expression of TJ proteins, likely in an attempt to compensate for stretch damage. High magnitudes of deformation decreased the expression of TJ proteins, suggesting that the damage was too severe to counteract. In vivo, mild TBI did not affect BBB permeability or the expression of TJ proteins. However, moderate TBI significantly increased BBB permeability and decreased the expression of these proteins, similar to the results obtained with a high magnitude deformation. These data support the use biaxial stretch as valuable tool in the study of TBI in vitro.
Assuntos
Barreira Hematoencefálica/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Permeabilidade Capilar/fisiologia , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Junções Íntimas/metabolismo , Animais , Barreira Hematoencefálica/patologia , Lesões Encefálicas Traumáticas/patologia , Endotélio Vascular/patologia , Ratos , Ratos Sprague-Dawley , Junções Íntimas/patologiaRESUMO
Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.
Assuntos
Biomarcadores , Líquidos Corporais/química , Sistema Nervoso Central/patologia , MicroRNAs/análise , Neurônios/patologia , Síndromes Neurotóxicas/diagnóstico , Compostos de Trimetilestanho/toxicidade , Aminoácidos/análise , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/urina , Humanos , Imageamento por Ressonância Magnética , Masculino , Metaboloma/fisiologia , MicroRNAs/genética , Projetos Piloto , Ratos , Ratos Sprague-DawleyRESUMO
Oxoguanine DNA glycosylase (OGG1) initiates the repair of 8-oxoguanine (8-oxoG), a major oxidative DNA base modification that has been directly implicated in cancer and aging. OGG1 functions in the base excision repair pathway, for which a molecular hand-off mechanism has been proposed. To date, only one functional and a few physical protein interactions have been reported for OGG1. Using the yeast two-hybrid system and a protein array membrane, we identified two novel protein interactions of OGG1, with two different protein kinases: Cdk4, a serine-threonine kinase, and c-Abl, a tyrosine kinase. We confirmed these interactions in vitro using recombinant proteins and in vivo by co-immunoprecipitation from whole cell extracts. OGG1 is phosphorylated in vitro by Cdk4, resulting in a 2.5-fold increase in the 8-oxoG/C incision activity of OGG1. C-Abl tyrosine phosphorylates OGG1 in vitro; however, this phosphorylation event does not affect OGG1 8-oxoG/C incision activity. These results provide the first evidence that a post-translational modification of OGG1 can affect its catalytic activity. The distinct functional outcomes from serine/threonine or tyrosine phosphorylation may indicate that activation of different signal transduction pathways modulate OGG1 activity in different ways.
Assuntos
DNA Glicosilases/metabolismo , Linhagem Celular , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Humanos , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismoRESUMO
Bath salts, or synthetic cathinones, have cocaine-like or amphetamine-like properties and induce psychoactive effects via their capacity to modulate serotonin (5-HT) and dopamine (DA). Structurally distinct synthetic cathinones are continuously being generated to skirt existing drug laws. One example of these modified compounds is cathinone phthalimide (CP), which has already appeared on the global market. The lack of toxicological studies on the effects of CP on monoaminergic systems led to the development of the present study in order to generate an acute toxicity profile for CP, and to clarify whether it primarily affects both dopamine and serotonin, like the synthetic cathinones mephedrone and methylone, or primarily affects dopamine, like 3, 4-methylenedioxypyrovalerone (MDPV). For the first time, the toxicity profile of CP (10µM-1000µM) is reported. In pheochromocytoma cells, exposure to CP induced cell death, and altered mitochondrial function, as well as intracellular DA and 5-HT levels; at the same time, reduced glutathione (GSH) levels remained unaffected. This seems to indicate that CP functions like mephedrone or methylone. The role of CP metabolites, the effect of CP induced hyperthermia on neurotoxicity, and its ability to traverse the blood-brain barrier warrant further consideration.
Assuntos
Estimulantes do Sistema Nervoso Central/toxicidade , Dopamina/metabolismo , Ftalimidas/toxicidade , Propiofenonas/toxicidade , Serotonina/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Glutationa/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Células PC12 , RatosRESUMO
Knocking out of Nurr1 gene, a member of nuclear receptor superfamily, causes selective agenesis of dopaminergic neurons in midbrain. Reduced expression of Nurr1 increases the vulnerability of mesencephalic dopamine neurons to dopaminergic toxins. We evaluated the role of nitric oxide as a possible mechanism for this increased susceptibility. Increased expression of neuronal nitric oxide synthase and increased 3-nitrotyrosine were observed in striatum of Nurr1 heterozygous (Nurr1 +/-) mice as compared with wild-type. Increased cytochrome C activation and consecutive release of Smac/DIABLO were also observed in Nurr1 +/- mice. An induction of active Caspase-3 and p53, cleavage of poly-ADP (RNase) polymerase and reduced expression of bcl-2 were observed in Nurr1 +/- mice. Methamphetamine significantly increased these markers in Nurr1 +/- mice as compared with wild-type. The present data therefore suggest that nitric oxide plays a role as a modulating factor for the increased susceptibility, but not the potentiation, of the dopaminergic terminals in Nurr1 +/- mice. We also report that this increased neuronal nitric oxide synthase expression and increased nitration in Nurr1 +/- mice led to the activation of apoptotic cascade via the differential alterations in the DNA binding activity of transcription factors responsible for the propagation of growth arrest as well as apoptosis.
Assuntos
Corpo Estriado/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Óxido Nítrico/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Corpo Estriado/metabolismo , Citocromos c/análise , Dano ao DNA , Proteína 1 de Resposta de Crescimento Precoce/genética , Masculino , Metanfetamina/toxicidade , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Óxido Nítrico Sintase Tipo I/biossíntese , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteína Supressora de Tumor p53/análise , alfa-Sinucleína/metabolismo , Proteína bcl-X/análiseRESUMO
Cocaine is a widely used drug of abuse and psychostimulant that acts on the central nervous system by blocking the dopamine reuptake sites. PC12 cells, a rat pheochromocytoma clonal line, in the presence of nerve growth factor (NGF), multiply and differentiate into competent neurons that can synthesize, store, and secrete the neurotransmitter dopamine (DA). In the present study, we evaluated the effect of increasing doses of cocaine on the expression of immediate early genes (IEGs), c-fos and c-jun, and closely related transcription factors, SP-1 and NF-kbeta, at 24 h after the exposure to cocaine (50, 100, 200, 500, 1000, 2500 microM) in NGF-differentiated PC12 cells. Cocaine (50-500 microM) resulted in significant induction of the expression of c-fos, c-jun, SP-1, and NF-kbeta. However, higher concentrations of cocaine (1000 and 2500 microM) resulted in the downregulation of these expressions after 24 h. To further understand the role of dose-dependent changes in the mechanisms of cell death, we evaluated the protein expression of apoptotic markers. A concentration-dependent increase in the expression of caspase-9 and -3 was observed up to 500 microM cocaine. However, the higher dose did not show any expression. We also evaluated the effect of increasing doses of cocaine on DA concentration and the expression of dopamine transporter (DAT). A significant dose-dependent decrease in the concentration of DA as well as the expression of DAT was observed 24 h after the exposure of PC12 cells to cocaine. Therefore, in the present study, we reported that cocaine has both upstream and downstream regulatory actions on some IEGs and transcription factors that can regulate the mechanism of cell death, and these effects on gene expression are independent of its action on the dopaminergic system.
Assuntos
Caspases/biossíntese , Transtornos Relacionados ao Uso de Cocaína/patologia , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Genes Precoces/genética , Fatores de Transcrição/biossíntese , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Cromatografia Líquida de Alta Pressão , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/biossíntese , NF-kappa B/genética , Células PC12 , Ratos , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Various iron-oxide nanoparticles have been in use for a long time as therapeutic and imaging agents and for supplemental delivery in cases of iron-deficiency. While all of these products have a specified size range of â¼ 40 nm and above, efforts are underway to produce smaller particles, down to â¼ 1 nm. Here, we show that after a 24-h exposure of SHSY-5Y human neuroblastoma cells to 10 µg/ml of 10 and 30 nm ferric oxide nanoparticles (Fe-NPs), cellular dopamine content was depleted by 68 and 52 %, respectively. Increases in activated tyrosine kinase c-Abl, a molecular switch induced by oxidative stress, and neuronal α-synuclein expression, a protein marker associated with neuronal injury, were also observed (55 and 38 % percent increases, respectively). Inhibition of cell-proliferation, significant reductions in the number of active mitochondria, and a dose-dependent increase in reactive oxygen species (ROS) were observed in neuronal cells. Additionally, using a rat in vitro blood-brain barrier (BBB) model, a dose-dependent increase in ROS accompanied by increased fluorescein efflux demonstrated compromised BBB integrity. To assess translational implications, in vivo Fe-NP-induced neurotoxicity was determined using in vivo MRI and post-mortem neurochemical and neuropathological correlates in adult male rats after exposure to 50 mg/kg of 10 nm Fe-NPs. Significant decrease in T 2 values was observed. Dynamic observations suggested transfer and retention of Fe-NPs from brain vasculature into brain ventricles. A significant decrease in striatal dopamine and its metabolites was also observed, and neuropathological correlates provided additional evidence of significant nerve cell body and dopaminergic terminal damage as well as damage to neuronal vasculature after exposure to 10 nm Fe-NPs. These data demonstrate a neurotoxic potential of very small size iron nanoparticles and suggest that use of these ferric oxide nanoparticles may result in neurotoxicity, thereby limiting their clinical application.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Nanopartículas de Magnetita/toxicidade , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Caspases/metabolismo , Catecolaminas/análise , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Corpo Estriado/química , Corpo Estriado/efeitos dos fármacos , Neurônios Dopaminérgicos/química , Neurônios Dopaminérgicos/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanosferas , Neuroblastoma/patologia , Estresse Oxidativo , Tamanho da Partícula , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/análise , Espectrometria por Raios XRESUMO
Methamphetamine (METH) is a widely abused psychomotor stimulant known to cause dopaminergic neurotoxicity in rodents, nonhuman primates, and humans. METH administration selectively damages the dopaminergic nerve terminals, which is hypothesized to be due to release of dopamine from synaptic vesicles within the terminals. This process is believed to be mediated by the production of free radicals. The current study evaluates METH-induced dopaminergic toxicity in pheochromocytoma 12 (PC12) cells cultured in the presence or absence of nerve growth factor (NGF). Dopaminergic changes and the formation of 3-nitrotyrosine (3-NT), a marker for peroxynitrite production, were studied in PC12 cell cultures grown in the presence or absence of NGF after different doses of METH (100-1,000 microM). METH exposure did not cause significant alterations in cell viability and did not produce significant dopaminergic changes or 3-NT production in PC12 cells grown in NGF-negative media after 24 hours. However, cell viability of PC12 cells grown in NGF-positive media was decreased by 45%, and significant dose-dependent dopaminergic alteration and 3-NT production were observed 24 hours after exposure to METH. The current study supports the hypothesis that METH acts at the dopaminergic nerve terminals and produces dopaminergic damage by the production of free radical peroxynitrite.
Assuntos
Dopamina/metabolismo , Metanfetamina/toxicidade , Fator de Crescimento Neural/farmacologia , Neurotoxinas , Ácido Peroxinitroso/metabolismo , Tirosina/análogos & derivados , Animais , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Cinética , Células PC12 , Feocromocitoma , Ratos , Fatores de Tempo , Tirosina/metabolismoRESUMO
Paramethoxyamphetamine (PMA) is a methoxylated phenethylamine derivative that has been used illicitly in Australia since 1994. PMA is also becoming popular at rave parties in the United States. PMA raised concern when a series of fatalities resulted after its use in South Australia, where it was marketed as "ecstasy," which is the colloquial name for MDMA. In the present study, we evaluated the comparative neurotoxicity of substituted amphetamines in rats. Extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were assayed in the caudate of freely moving rats using microdialysis and HPLC-EC. Dialysates were assayed every 20 minutes for 4 hours after an intraperitoneal (i.p.) injection of PMA (2.5, 5, 10, 20 mg/kg), MDMA (10 and 20 mg/kg), or METH (2.5 mg/kg). METH produced a significant increase in extracellular DA (700%), and significant decreases in extracellular DOPAC and HVA (30% and 50%), with no detectable changes in either 5-HT or 5-HIAA. MDMA produced significant increases in DA (700% at 10 mg/kg and 950% at 20 mg/kg) and decreases in DOPAC (15% for both 10 and 20 mg/kg), and HVA (50% at 10 mg/kg and 35% at 20 mg/kg). MDMA also increased 5-HT (350% at 10, and 575% at 20 mg/kg), and decreased 5-HIAA to 60% for both dose levels. PMA produced no detectable increases in DA at dose levels of 2.5, 5, or 10 mg/kg, but significantly increased DA (975%) at a dose of 20 mg/kg. However, PMA significantly decreased DOPAC at all dose levels (75% at 2.5; 40% at 5; 30% at 10; 10% at 20 mg/kg), with comparable decreases in HVA at all dose levels. PMA also produced significant increases in 5-HT at 10 and 20 mg/kg (350% for both dose levels), with no detectable changes in 5-HT at 2.5 or 5 mg/kg. All dose levels of PMA significantly decreased 5-HIAA (50 to 70%). These data suggest that PMA, like MDMA and METH, is capable of producing dopaminergic and serotonergic neurotoxicity.