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1.
Hum Mol Genet ; 22(10): 2067-82, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23393156

RESUMO

Mutations in ATP13A2 (PARK9), encoding a lysosomal P-type ATPase, are associated with both Kufor-Rakeb syndrome (KRS) and neuronal ceroid lipofuscinosis (NCL). KRS has recently been classified as a rare genetic form of Parkinson's disease (PD), whereas NCL is a lysosomal storage disorder. Although the transport activity of ATP13A2 has not been defined, in vitro studies show that its loss compromises lysosomal function, which in turn is thought to cause neuronal degeneration. To understand the role of ATP13A2 dysfunction in disease, we disrupted its gene in mice. Atp13a2(-/-) and Atp13a2(+/+) mice were tested behaviorally to assess sensorimotor and cognitive function at multiple ages. In the brain, lipofuscin accumulation, α-synuclein aggregation and dopaminergic pathology were measured. Behaviorally, Atp13a2(-/-) mice displayed late-onset sensorimotor deficits. Accelerated deposition of autofluorescent storage material (lipofuscin) was observed in the cerebellum and in neurons of the hippocampus and the cortex of Atp13a2(-/-) mice. Immunoblot analysis showed increased insoluble α-synuclein in the hippocampus, but not in the cortex or cerebellum. There was no change in the number of dopaminergic neurons in the substantia nigra or in striatal dopamine levels in aged Atp13a2(-/-) mice. These results show that the loss of Atp13a2 causes sensorimotor impairments, α-synuclein accumulation as occurs in PD and related synucleinopathies, and accumulation of lipofuscin deposits characteristic of NCL, thus providing the first direct demonstration that null mutations in Atp13a2 can cause pathological features of both diseases in the same organism.


Assuntos
Adenosina Trifosfatases , Envelhecimento/metabolismo , Encéfalo/metabolismo , Retroalimentação Sensorial , Proteínas de Membrana , Lipofuscinoses Ceroides Neuronais/enzimologia , Transtornos Parkinsonianos/enzimologia , alfa-Sinucleína/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Animais , Comportamento Animal , Encéfalo/patologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Camundongos , Camundongos Mutantes , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , ATPases Translocadoras de Prótons , alfa-Sinucleína/genética
2.
Molecules ; 18(9): 11537-52, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24048286

RESUMO

Dendrimers are highly customizable nanopolymers with qualities that make them ideal for drug delivery. The high binding affinity of biotin/avidin provides a useful approach to fluorescently label synthesized dendrimer-conjugates in cells and tissues. In addition, biotin may facilitate delivery of dendrimers through the blood-brain barrier (BBB) via carrier-mediated endocytosis. The purpose of this research was to: (1) measure toxicity using lactate dehydrogenase (LDH) assays of generation (G)4 biotinylated and non-biotinylated poly(amidoamine) (PAMAM) dendrimers in a co-culture model of the BBB, (2) determine distribution of dendrimers in the rat brain, kidney, and liver following systemic administration of dendrimers, and (3) conduct atomic force microscopy (AFM) on rat brain sections following systemic administration of dendrimers. LDH measurements showed that biotinylated dendrimers were toxic to cell co-culture after 48 h of treatment. Distribution studies showed evidence of biotinylated and non-biotinylated PAMAM dendrimers in brain. AFM studies showed evidence of dendrimers only in brain tissue of treated rats. These results indicate that biotinylation does not decrease toxicity associated with PAMAM dendrimers and that biotinylated PAMAM dendrimers distribute in the brain. Furthermore, this article provides evidence of nanoparticles in brain tissue following systemic administration of nanoparticles supported by both fluorescence microscopy and AFM.


Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Dendrímeros/farmacocinética , Portadores de Fármacos/farmacocinética , Animais , Biotinilação , Células Cultivadas , Técnicas de Cocultura , Dendrímeros/toxicidade , Portadores de Fármacos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Modelos Biológicos , Ratos , Distribuição Tecidual
3.
Exp Clin Psychopharmacol ; 16(2): 178-89, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18489022

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder in which loss of dopaminergic (DA) neurons (>50%) in the substantia nigra (SN) precedes most of the overt motor symptoms, making early diagnosis and treatment interventions difficult. Because PD has been associated with free radicals generated by nitric oxide, this study tested whether treatments of 7-nitroindazole (7NI), a nitric-oxide-synthase inhibitor, could reduce cognitive deficits that often emerge before overt motor symptoms in a presymptomatic rat model of PD. Rats were given intraperitoneal injections of 50 mg/kg 7NI (or vehicle) just before receiving bilateral, intrastriatal injections of the DA-toxin, 6-hydroxydopamine (6-OHDA). The rats were then given a battery of motor tasks, and their learning ability was assessed using a spatial reversal task in a water-T maze. Results indicate that 7NI treatments attenuate 6-OHDA-induced spatial learning deficits and protect against DA cell loss in the SN, suggesting that 7NI may have potential as an early, presymptomatic pharmacotherapy for PD.


Assuntos
Dopamina/metabolismo , Indazóis/uso terapêutico , Deficiências da Aprendizagem/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Oxidopamina , Comportamento Espacial/efeitos dos fármacos , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Deficiências da Aprendizagem/induzido quimicamente , Deficiências da Aprendizagem/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , NADP/metabolismo , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Comportamento Espacial/fisiologia , Substância Negra/patologia
4.
J Drug Target ; 24(7): 635-44, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26707984

RESUMO

Background Poly(butylcyanoacrylate) (PBCA) nanoparticles (NPs) loaded with doxorubicin (DOX) and coated with polysorbate 80 (PS80) have shown efficacy in the treatment of rat glioblastoma. However, cytotoxicity of this treatment remains unclear. Purpose The purpose of this study was to investigate cytotoxicity and apoptotic gene expression using a proven in vitro co-culture model of the blood-brain barrier. Methods The co-cultures were exposed to uncoated PBCA NPs, PBCA-PS80 NPs or PBCA-PS80-DOX NPs at varying concentrations and evaluated using a resazurin-based cytotoxicity assay and an 84-gene apoptosis RT-PCR array. Results The cytotoxicity assays showed PBCA-PS80-DOX NPs exhibited a decrease in metabolic function at lower concentrations than uncoated PBCA NPs and PBCA-PS80 NPs. The apoptosis arrays showed differential expression of 18 genes in PBCA-PS80-DOX treated cells compared to the untreated control. Discussion As expected, the cytotoxicity assays demonstrated enhanced dose-dependent toxicity in the DOX loaded NPs. The differentially expressed apoptotic genes participate in both the tumor necrosis factor receptor-1 and mitochondria-associated apoptotic pathways implicated in current DOX chemotherapeutic toxicity. Conclusion The following data suggest that the cytotoxic effect may be attributed to DOX and not the NPs themselves, further supporting the use of PBCA-PS80 NPs as an effective drug delivery vehicle for treating central nervous system conditions.


Assuntos
Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Portadores de Fármacos/toxicidade , Embucrilato/toxicidade , Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Nanopartículas/toxicidade , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/toxicidade , Apoptose/genética , Astrócitos/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Portadores de Fármacos/química , Embucrilato/química , Células Endoteliais/efeitos dos fármacos , Nanopartículas/química , Ratos Sprague-Dawley
5.
Int J Anal Chem ; 2012: 341260, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22505915

RESUMO

Immunoimaging scanning probe microscopy was utilized for the low-level detection and quantification of biotinylated G4 poly(amidoamine) PAMAM dendrimers. Results were compared to those of high-performance liquid chromatography (HPLC) and found to provide a vastly improved analytical method for the low-level detection of dendrimers, improving the limit of detection by a factor of 1000 (LOD = 2.5 × 10(-13) moles). The biorecognition method is reproducible and shows high specificity and good accuracy. In addition, the capture assay platform shows a promising approach to patterning dendrimers for nanotechnology applications.

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