RESUMO
Alzheimer's disease (AD) is characterized by the accumulation of aggregated amyloid peptides in the brain parenchyma and within the walls of cerebral vessels. The hippocampus-a complex brain structure with a pivotal role in learning and memory-is implicated in this disease. However, there is limited data on vascular changes during AD pathological degeneration in this susceptible structure, which has distinctive vascular traits. Our aim was to evaluate vascular alterations in the hippocampus of AD patients and PDAPP-J20 mice-a model of AD-and to determine the impact of Aß40 and Aß42 on endothelial cell activation. We found a loss of physical astrocyte-endothelium interaction in the hippocampus of individuals with AD as compared to non-AD donors, along with reduced vascular density. Astrocyte-endothelial interactions and levels of the tight junction protein occludin were altered early in PDAPP-J20 mice, preceding any signs of morphological changes or disruption of the blood-brain barrier in these mice. At later stages, PDAPP-J20 mice exhibited decreased vascular density in the hippocampus and leakage of fluorescent tracers, indicating dysfunction of the vasculature and the BBB. In vitro studies showed that soluble Aß40 exposure in human brain microvascular endothelial cells (HBMEC) was sufficient to induce NFκB translocation to the nucleus, which may be linked with an observed reduction in occludin levels. The inhibition of the membrane receptor for advanced glycation end products (RAGE) prevented these changes in HBMEC. Additional results suggest that Aß42 indirectly affects the endothelium by inducing astrocytic factors. Furthermore, our results from human and mouse brain samples provide evidence for the crucial involvement of the hippocampal vasculature in Alzheimer's disease.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Astrócitos , Hipocampo , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Humanos , Hipocampo/patologia , Hipocampo/metabolismo , Peptídeos beta-Amiloides/metabolismo , Masculino , Idoso , Camundongos Transgênicos , Feminino , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Idoso de 80 Anos ou mais , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/metabolismo , Camundongos , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
Intracellular transport of large cargoes, such as organelles, vesicles, or large proteins, is a complex dynamical process that involves the interplay of adenosine triphosphate-consuming molecular motors, cytoskeleton filaments, and the viscoelastic cytoplasm. In this work we investigate the motion of pigment organelles (melanosomes) driven by myosin-V motors in Xenopus laevis melanocytes using a high-spatio-temporal resolution tracking technique. By analyzing the obtained trajectories, we show that the melanosomes mean-square displacement undergoes a transition from a subdiffusive to a superdiffusive behavior. A stochastic theoretical model, which explicitly considers the collective action of the molecular motors, is introduced to generalize the interpretation of our data. Starting from a generalized Langevin equation, we derive an analytical expression for the mean square displacement, which also takes into account the experimental noise. By fitting theoretical expressions to experimental data we were able to discriminate the exponents that characterize the passive and active contributions to the dynamics and to estimate the "global" motor forces correctly. Then, our model gives a quantitative description of active transport in living cells with a reduced number of parameters.
Assuntos
Actinas/metabolismo , Difusão , Espaço Intracelular/metabolismo , Modelos Biológicos , Miosina Tipo V/metabolismo , Animais , Transporte Biológico , Elasticidade , Melanossomas/metabolismo , Reologia , Processos Estocásticos , Viscosidade , Xenopus laevis/metabolismoRESUMO
P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.
Assuntos
Cálcio/metabolismo , Membrana Celular/química , Técnicas de Química Analítica/métodos , Trifosfato de Adenosina/metabolismo , Autorradiografia , ATPases Transportadoras de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Cinética , Fosforilação , Fatores de TempoRESUMO
Ca(2+) pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions.
Assuntos
ATPases Transportadoras de Cálcio/química , Sítios de Ligação , ATPases Transportadoras de Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Dimerização , Membrana Eritrocítica/enzimologia , Fluorescência , Humanos , Cinética , Ligação Proteica , Dobramento de Proteína , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Thermal stability of plasma membrane Ca(2+) pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca(2+) pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K(LD)) showing that those phospholipids with higher affinity provide greater stability to the Ca(2+) pump. We developed a method for directly measure K(LD) by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K(LD) values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.