The disruption of mitochondrial axonal transport is an early event in neuroinflammation

Background: in brain inflammatory diseases, axonal damage is one of the most critical steps in the cascade that leads to permanent disability. Thus, identifying the initial events triggered by inflammation or oxidative stress that provoke axonal damage is critical for the development of neuroprotective therapies. Energy depletion due to mitochondrial dysfunction has been postulated as an important step in the damage of axons. This prompted us to study the effects of acute inflammation and oxidative stress on the morphology, transport, and function of mitochondria in axons. Methods: mouse cerebellar slice cultures were challenged with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) ex vivo for 24 h. Axonal mitochondrial morphology was evaluated by transmission electron microscopy (TEM) and mitochondrial transportation by time-lapse imaging. In addition, mitochondrial function in the cerebellar slice cultures was analyzed through high-resolution respirometry assays and quantification of adenosine triphosphate (ATP) production. Results: both conditions promoted an increase in the size and complexity of axonal itochondria evident in electron microscopy images, suggesting a compensatory response. Such compensation was reflected at the tissue level as increased respiratory activity of complexes I and IV and as a transient increase in ATP production in response to acute inflammation. Notably, time-lapse microscopy indicated that mitochondrial transport (mean velocity) was severely impaired in axons, increasing the proportion of stationary mitochondria in axons after LPS challenge. Indeed, the two challenges used produced different effects: inflammation mostly reducing retrograde transport and oxidative stress slightly enhancing retrograde transportation. Conclusions: neuroinflammation acutely impairs axonal mitochondrial transportation, which would promote an inappropriate delivery of energy throughout axons and, by this way, contribute to axonal damage. Thus, preserving axonal mitochondrial transport might represent a promising avenue to exploit as a therapeutic target for neuroprotection in brain inflammatory diseases like multiple sclerosis

Mecanismos de degeneración axonal en neuroinflamación: papel de la disfunción mitocondrial

[spa] La Esclerosis Múltiple (EM) afecta preferentemente a la conectividad cerebral lesionando los axones de forma irreversible, siendo esta lesión la principal causa de la discapacidad permanente de los pacientes. El daño axonal se produce de forma aguda debido a la cascada inflamatoria y de forma crónica (degenerativa) debido a la falta de soporte trófico de la mielina, un microambiente alterado (gliótico) y persistencia de inflamación crónica (activación microglial). Los axones son dañados en la fase aguda por el estrés oxidativo y energético y mecanismos de citotoxicidad, produciéndose un fallo del transporte axonal y un fallo de la función mitocondrial. Todos estos procesos culminan en el déficit energético, que pone en marcha mecanismos activos de transección axonal que llevan a la lesión axonal aguda. Los mecanismos de daño axonal crónico serían en parte similares a los agudos (déficit energético) extendidos en el tiempo, junto con el fallo de soporte trófico de la mielina, la modificación de la función y distribución de los canales iónicos y la consecuente alteración del microambiente. Identificar los mecanismos y la dinámica del daño axonal agudo y crónico en la EM permitirá identificar dianas terapéuticas para las que se desarrollarían nuevas terapias neuroprotectoras que disminurían el daño axonal en la EM y por tanto las secuelas. El objetivo del proyecto es analizar distintos aspectos de la biología mitocondrial y de su función respiratoria y energética relacionados con la degeneración axonal en un entorno neuroinflamatorio agudo. Para conseguirlo se ha utilizado un modelo de neuroinflamación in vitro consistente en cultivos organotípicos de cerebelo de ratón estimulados con lipopolisacárido bacteriano (LPS) y los cambios se han evaluado mediante técnicas de microscopía avanzada y biología molecular. A lo largo del estudio de la biología mitocondrial también se ha utilizado un estímulo causante de estrés oxidativo, el peróxido de hidrógeno (H2O2), proceso que forma parte de la neuroinflamación y afecta directamente a la función de los complejos de la cadena respiratoria mitocondrial. Los resultados del estudio nos llevan a proponer el siguiente modelo de la respuesta mitocondrial en un entorno neuroinflamatorio agudo: mientras que a nivel tisular las mitocondrias responden aumentando su capacidad respiratoria y la producción de ATP, a nivel axonal las mitocondrias responden morfológicamente a la inflamación aumentando su tamaño y la complejidad de sus crestas mitocondriales, pero su transporte se ve completamente alterado. La paralización de las mitocondrias se produce en una etapa temprana y aguda de la inflamación, antes de que los daños axonales sean irreversibles. Por tanto, nuestro modelo indica que el mecanismo más crítico en la disfunción mitocondrial en la neuroinflamación aguda es la alteración del transporte axonal de mitocondrias (especialmente el retrógrado). Por tanto, nuestro estudio sugiere la preservación del transporte mitocondrial como una diana terapéutica de neuroprotección en lesiones agudas de EM.[eng] The main cause of permanent disability in Multiple Sclerosis (MS) patients is axonal degeneration. In acute phases of the disease axonal damage is a consequence of the inflammatory cascade and oxidative stress that cause direct damage to mitochondria, inhibiting OXPHOS complexes. In more chronic phases axonal degeneration is due to loss of myelin trophic support, gliosis, redistribution of axonal sodium channels and extended energetic deficiency. Mitochondrial dysfunction is a common player in both stages. Therefore, the elucidation of axonal degeneration mechanisms is important for the identification of new therapeutic targets and the development of neuroprotective therapies that decrease axonal damage in MS. The objective of the project is to analyze different aspects of mitochondrial biology and their respiratory and energetic functions related with axonal degeneration in an acute neuroinflammatory environment. Organotypic slice cultures of mouse cerebellum were stimulated with LPS to activate microglia and simulate neuroinflammation, and mitochondrial changes were evaluated with advanced techniques of molecular biology and microscopy. The results suggest that tissular mitochondria are responding to acute neuroinflammation increasing their respiratory capacity and their production of ATP, and axonal mitochondria react morphologically increasing their size and their cristae complexity. However, axonal mitochondrial transport is completely impaired, in an early stage of acute inflammation before axonal damage is irreversible. Therefore, we propose that the critical mechanism of mitochondrial dysfunction in acute neuroinflammation is the impairment of axonal mitochondrial transport. As a result, the preservation of axonal mitochondrial transport represents a therapeutic target for neuroprotection in acute MS lesions

The disruption of mitochondrial axonal transport is an early event in neuroinflammation

Background: in brain inflammatory diseases, axonal damage is one of the most critical steps in the cascade that leads to permanent disability. Thus, identifying the initial events triggered by inflammation or oxidative stress that provoke axonal damage is critical for the development of neuroprotective therapies. Energy depletion due to mitochondrial dysfunction has been postulated as an important step in the damage of axons. This prompted us to study the effects of acute inflammation and oxidative stress on the morphology, transport, and function of mitochondria in axons. Methods: mouse cerebellar slice cultures were challenged with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) ex vivo for 24 h. Axonal mitochondrial morphology was evaluated by transmission electron microscopy (TEM) and mitochondrial transportation by time-lapse imaging. In addition, mitochondrial function in the cerebellar slice cultures was analyzed through high-resolution respirometry assays and quantification of adenosine triphosphate (ATP) production. Results: both conditions promoted an increase in the size and complexity of axonal itochondria evident in electron microscopy images, suggesting a compensatory response. Such compensation was reflected at the tissue level as increased respiratory activity of complexes I and IV and as a transient increase in ATP production in response to acute inflammation. Notably, time-lapse microscopy indicated that mitochondrial transport (mean velocity) was severely impaired in axons, increasing the proportion of stationary mitochondria in axons after LPS challenge. Indeed, the two challenges used produced different effects: inflammation mostly reducing retrograde transport and oxidative stress slightly enhancing retrograde transportation. Conclusions: neuroinflammation acutely impairs axonal mitochondrial transportation, which would promote an inappropriate delivery of energy throughout axons and, by this way, contribute to axonal damage. Thus, preserving axonal mitochondrial transport might represent a promising avenue to exploit as a therapeutic target for neuroprotection in brain inflammatory diseases like multiple sclerosis