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Abstract(s)
O envelhecimento Ă© o principal fator de risco para a doença de Parkinson, a segunda doença neurodegenerativa mais comum, caracterizada principalmente pela perda progressiva e seletiva dos neurĂłnios dopaminĂ©rgicos da via nigroestriatal, resultando numa diminuição dos nĂveis de dopamina nesta regiĂŁo e consequentemente em sintomas motores. VĂĄrios sĂŁo os mecanismos que desempenham um papel importante na perda neuronal, estando entre eles o stress oxidativo, que leva Ă formação de radicais livres, e os processos neuroinflamatĂłrios. Um dos principais objetivos da investigação no Ăąmbito da DP Ă© o desenvolvimento de fĂĄrmacos que retardem ou parem o processo neurodegenerativo.
A Klotho Ă© uma proteĂna supressora do envelhecimento e a sua sobre-expressĂŁo em ratos estĂĄ associada a uma extensĂŁo do tempo de vida. Estudos anteriores mostraram que os nĂveis circulantes de Klotho diminuem com a idade e que existe uma forte correlação entre os nĂveis elevados da sua expressĂŁo e o estado saudĂĄvel dos animais. Apesar das mĂșltiplas funçÔes atribuĂdas a esta proteĂna, o seu mecanismo de ação no cĂ©rebro nĂŁo estĂĄ completamente compreendido. Contudo, tem sido relatado que esta proteĂna melhora a plasticidade sinĂĄptica, as funçÔes cognitivas e, para alĂ©m disso, pode influenciar uma variedade de estruturas e funçÔes do sistema nervoso central durante a maturação e o envelhecimento. Esta proteĂna Ă© altamente expressa no cĂ©rebro, principalmente no plexo corĂłide e nos neurĂłnios.
Estudos recentes tĂȘm atribuĂdo Ă Klotho um papel importante na neuroprotecção. Menores concentraçÔes de Klotho foram detetadas no lĂquido cefalorraquidiano de doentes de Alzheimer, quando comparado com indivĂduos saudĂĄveis da mesma idade. Esta proteĂna aumenta a resistĂȘncia ao stress oxidativo por ativação de algumas vias de sinalização e, quando adicionada exogenamente in vitro, protege os neurĂłnios do hipocampo da excitotoxicidade induzida pelo glutamato, atravĂ©s do sistema Trx/Prx, tendo sido proposta como um novo alvo terapĂȘutico para a doença de Alzheimer. No que diz respeito Ă doença de Parkinson, sabe-se apenas que em murganhos mutantes para o gene da Klotho existe uma diminuição no nĂșmero de neurĂłnios dopaminĂ©rgicos na substantia nigra e nos nĂveis de dopamina no estriado, e que animais transgĂ©nicos para a Klotho (que sobre-expressam esta proteĂna) sĂŁo menos vulnerĂĄveis ao stress oxidativo induzido pela exposição a MPTP.
Posto isto, o primeiro objetivo deste trabalho, foi averiguar uma possĂvel ação neuroprotetora da Klotho num modelo celular da doença de Parkinson. Para tal foram utilizadas culturas primĂĄrias enriquecidas em neurĂłnios e co-culturas de neurĂłnios-glia do mesencĂ©falo ventral, Ă s quais foi adicionada Klotho recombinante. A toxicidade dopaminĂ©rgica foi induzida por exposição ao 1-metil-4-fenilpiridina (MPP+) e a extensĂŁo da lesĂŁo foi analisada por imunocitoquĂmica. Os resultados obtidos mostraram que a Klotho, quando aplicada exogenamente e previamente Ă lesĂŁo, previne a perda dos neurĂłnios dopaminĂ©rgicos do mesencĂ©falo ventral induzida pela toxina dopaminĂ©rgica MPP+ e que a presença de astrĂłcitos Ă© importante para esta neuroprotecção. O mecanismo pelo qual exerce este efeito Ă© ainda desconhecido e merece uma investigação mais aprofundada. A ação neuroprotetora da Klotho Ă© abolida quando esta Ă© administrada apĂłs ou no momento da indução da lesĂŁo dopaminĂ©rgica.
O segundo objetivo deste trabalho, foi avaliar o papel da Klotho no controlo da inflamação mediada pela microglia. Utilizando culturas primĂĄrias enriquecidas em microglia do mesencĂ©falo ventral e o lipopolissacarĂdeo (LPS) como agente estimulante, verificĂĄmos que a Klotho consegue diminuir a libertação de NO pela microglia, induzida pelo LPS, sendo uma possĂvel via pela qual a Klotho modula a reatividade microglial e consequentemente poderĂĄ exercer um efeito neuroprotetor.
Conjuntamente, os resultados obtidos neste trabalho mostram pela primeira vez que a administração exĂłgena da Klotho Ă© neuroprotetora para os neurĂłnios dopaminĂ©rgicos do mesencĂ©falo ventral e exerce um efeito anti-inflamatĂłrio na microglia, podendo ser um ponto de partida na compreensĂŁo do papel da Klotho no contexto da doença de Parkinson. Os efeitos da administração exĂłgena desta proteĂna deverĂŁo ser investigados em modelos animais da doença de Parkinson a fim de avaliar o seu potencial terapĂȘutico nesta doença.
Aging is the main risk factor for Parkinson's disease, the second most common neurodegenerative disease, mainly characterized by progressive and selective loss of the dopaminergic neurons in the nigrostriatal pathway, resulting in a decrease of dopamine levels in this region and therefore in motor symptoms. There are several mechanisms that play an important role in neuronal loss, among them the oxidative stress, which leads to the formation of free radicals, and neuroinflammatory processes. One of the major research goals in the field of PD is the development of drugs that can retard or stop the neurodegenerative process. Klotho is an aging-suppressor protein and its overexpression in mice is associated with an extension of the lifetime. Previous studies have shown that circulating levels of Klotho decrease with age and there is a strong correlation between high levels of expression and animalâs health status. Besides the multiple functions attributed to this protein, the mechanism of action in the brain is not fully understood. However, it has been reported that this protein enhances synaptic plasticity, cognitive functions and can influence a variety of structures and functions in the central nervous system during maturation and aging. This protein is highly expressed in the brain, especially in the choroid plexus and neurons. Recent studies have assigned to Klotho an important role in neuroprotection. Low Klotho concentrations were detected in the cerebrospinal fluid of Alzheimer's disease patients compared with age-matched healthy individuals. This protein increases the resistance to oxidative stress by activation of some signaling pathways and, when added exogenously in vitro, protects hippocampal neurons from excitotoxicity induced by glutamate through the Trx / Prx system. Therefore it has been proposed as a new therapeutic target for Alzheimer's disease. Concerning Parkinson's disease, it is known that in mutant mice for the Klotho gene there is a decrease in the number of dopaminergic neurons in the substantia nigra and in dopamine levels in the striatum. Furthermore, Klotho transgenic mice, over-expressing this protein, are less vulnerable to the oxidative stress induced by MPTP exposure. Hence, the first objective of this work was to investigate a possible neuroprotective effect of Klotho in a cellular model of Parkinson's disease. To achieve this, neuron-enriched primary cultures as well as neuron-glia cocultures obtained from the ventral midbrain, were incubated with the recombinant Klotho protein. The dopaminergic toxicity was induced by exposure to 1-methyl-4-phenylpyridine (MPP+) and lesion extent of lesions was evaluated by immunocytochemistry. The results showed that Klotho, when applied exogenously and prior to the MPP+ incubation, prevented the loss of ventral midbrain dopaminergic neurons induced by dopaminergic toxin MPP+. Furthermore, our data suggested that the presence of astrocytes is required for Klothoâs neuroprotective effect. The mechanism by which Klotho exerts this effect is unknown and deserves further investigation. The neuroprotective effect of Klotho is abolished when this protein is administered simultaneously or after the induction of dopaminergic injury. The second goal of this study was to evaluate the role of Klotho in the control of microglia-mediated inflammation. Using ventral midbrain microglia-enriched cultures and the lipopolysaccharide (LPS) as a pro-inflammatory agent we observed that Klotho can prevent the LPS-induced release of NO by microglia. Therefore, Klotho is able to modulate microglial reactivity which may contribute for its neuroprotective effect. Taken together, the present results show for the first time that exogenous administration of Klotho is neuroprotective for dopaminergic neurons of the ventral mesencephalon and has an anti-inflammatory effect in microglia, contributing to a better undertanding of the role of Klotho in the context of Parkinson's disease. The effects of exogenously administered Klotho should be investigated in animal models of Parkinson's disease in order to assess their therapeutic potential use in this disease.
Aging is the main risk factor for Parkinson's disease, the second most common neurodegenerative disease, mainly characterized by progressive and selective loss of the dopaminergic neurons in the nigrostriatal pathway, resulting in a decrease of dopamine levels in this region and therefore in motor symptoms. There are several mechanisms that play an important role in neuronal loss, among them the oxidative stress, which leads to the formation of free radicals, and neuroinflammatory processes. One of the major research goals in the field of PD is the development of drugs that can retard or stop the neurodegenerative process. Klotho is an aging-suppressor protein and its overexpression in mice is associated with an extension of the lifetime. Previous studies have shown that circulating levels of Klotho decrease with age and there is a strong correlation between high levels of expression and animalâs health status. Besides the multiple functions attributed to this protein, the mechanism of action in the brain is not fully understood. However, it has been reported that this protein enhances synaptic plasticity, cognitive functions and can influence a variety of structures and functions in the central nervous system during maturation and aging. This protein is highly expressed in the brain, especially in the choroid plexus and neurons. Recent studies have assigned to Klotho an important role in neuroprotection. Low Klotho concentrations were detected in the cerebrospinal fluid of Alzheimer's disease patients compared with age-matched healthy individuals. This protein increases the resistance to oxidative stress by activation of some signaling pathways and, when added exogenously in vitro, protects hippocampal neurons from excitotoxicity induced by glutamate through the Trx / Prx system. Therefore it has been proposed as a new therapeutic target for Alzheimer's disease. Concerning Parkinson's disease, it is known that in mutant mice for the Klotho gene there is a decrease in the number of dopaminergic neurons in the substantia nigra and in dopamine levels in the striatum. Furthermore, Klotho transgenic mice, over-expressing this protein, are less vulnerable to the oxidative stress induced by MPTP exposure. Hence, the first objective of this work was to investigate a possible neuroprotective effect of Klotho in a cellular model of Parkinson's disease. To achieve this, neuron-enriched primary cultures as well as neuron-glia cocultures obtained from the ventral midbrain, were incubated with the recombinant Klotho protein. The dopaminergic toxicity was induced by exposure to 1-methyl-4-phenylpyridine (MPP+) and lesion extent of lesions was evaluated by immunocytochemistry. The results showed that Klotho, when applied exogenously and prior to the MPP+ incubation, prevented the loss of ventral midbrain dopaminergic neurons induced by dopaminergic toxin MPP+. Furthermore, our data suggested that the presence of astrocytes is required for Klothoâs neuroprotective effect. The mechanism by which Klotho exerts this effect is unknown and deserves further investigation. The neuroprotective effect of Klotho is abolished when this protein is administered simultaneously or after the induction of dopaminergic injury. The second goal of this study was to evaluate the role of Klotho in the control of microglia-mediated inflammation. Using ventral midbrain microglia-enriched cultures and the lipopolysaccharide (LPS) as a pro-inflammatory agent we observed that Klotho can prevent the LPS-induced release of NO by microglia. Therefore, Klotho is able to modulate microglial reactivity which may contribute for its neuroprotective effect. Taken together, the present results show for the first time that exogenous administration of Klotho is neuroprotective for dopaminergic neurons of the ventral mesencephalon and has an anti-inflammatory effect in microglia, contributing to a better undertanding of the role of Klotho in the context of Parkinson's disease. The effects of exogenously administered Klotho should be investigated in animal models of Parkinson's disease in order to assess their therapeutic potential use in this disease.
Description
Keywords
Doença de Parkinson Klotho Microglia Neuroinflamação Neurónios Dopaminérgicos Neuroproteção