RESUMEN
The brain extracellular space (ECS) is a vast interstitial reticulum of extreme morphological complexity, composed of narrow gaps separated by local expansions, enabling interconnected highways between neural cells. Constituting on average 20% of brain volume, the ECS is key for intercellular communication, and understanding its diffusional properties is of paramount importance for understanding the brain. Within the ECS, neuroactive substances travel predominantly by diffusion, spreading through the interstitial fluid and the extracellular matrix scaffold after being focally released. The nanoscale dimensions of the ECS render it unresolvable by conventional live tissue compatible imaging methods, and historically diffusion of tracers has been used to indirectly infer its structure. Novel nanoscopic imaging techniques now show that the ECS is a highly dynamic compartment, and that diffusivity in the ECS is more heterogeneous than anticipated, with great variability across brain regions and physiological states. Diffusion is defined primarily by the local ECS geometry, and secondarily by the viscosity of the interstitial fluid, including the obstructive and binding properties of the extracellular matrix. ECS volume fraction and tortuosity both strongly determine diffusivity, and each can be independently regulated e.g. through alterations in glial morphology and the extracellular matrix composition. Here we aim to provide an overview of our current understanding of the ECS and its diffusional properties. We highlight emerging technological advances to respectively interrogate and model diffusion through the ECS, and point out how these may contribute in resolving the remaining enigmas of the ECS.
Asunto(s)
Encéfalo , Espacio Extracelular , Espacio Extracelular/metabolismo , Encéfalo/metabolismo , Matriz Extracelular/fisiología , Neuroglía/fisiología , Neuronas/fisiologíaRESUMEN
STED microscopy is one of several fluorescence microscopy techniques that permit imaging at higher spatial resolution than what the diffraction-limit of light dictates. STED imaging is unique among these super-resolution modalities in being a beam-scanning microscopy technique based on confocal or 2-photon imaging, which provides the advantage of superior optical sectioning in thick samples. Compared to the other super-resolution techniques that are based on widefield microscopy, this makes STED particularly suited for imaging inside live brain tissue, such as in slices or in vivo. Notably, the 50 nm resolution provided by STED microscopy enables analysis of neural morphologies that conventional confocal and 2-photon microscopy approaches cannot resolve, including all-important synaptic structures. Over the course of the last 20 years, STED microscopy has undergone extensive developments towards ever more versatile use, and has facilitated remarkable neurophysiological discoveries. The technique is still not widely adopted for live tissue imaging, even though one of its particular strengths is exactly in resolving the nanoscale dynamics of synaptic structures in brain tissue, as well as in addressing the complex morphologies of glial cells, and revealing the intricate structure of the brain extracellular space. Not least, live tissue STED microscopy has so far hardly been applied in settings of pathophysiology, though also here it shows great promise for providing new insights. This review outlines the technical advantages of STED microscopy for imaging in live brain tissue, and highlights key neurobiological findings brought about by the technique.
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Encéfalo/metabolismo , Espinas Dendríticas/metabolismo , Colorantes Fluorescentes/metabolismo , Microscopía Fluorescente/métodos , Sinapsis/metabolismo , Animales , Encéfalo/citología , Encéfalo/ultraestructura , Espinas Dendríticas/ultraestructura , Humanos , Microscopía Fluorescente/tendencias , Neuronas/metabolismo , Neuronas/ultraestructura , Sinapsis/ultraestructuraRESUMEN
The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.
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Encéfalo/diagnóstico por imagen , Espacio Extracelular/diagnóstico por imagen , Microscopía Intravital/métodos , Nanotubos de Carbono/química , Imagen Individual de Molécula/métodos , Animales , Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Ratones Endogámicos C57BL , Organoides/diagnóstico por imagen , Ratas , Ratas Sprague-Dawley , Reología , Espectroscopía Infrarroja Corta/métodosRESUMEN
Synaptic protein α-synuclein (α-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinson's disease. Here, we report that α-SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also show that premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking α-SYN resembles the effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic α-SYN. Interestingly, NSC loss in α-SYN-deficient mice can be prevented by viral delivery of human α-SYN into their sustantia nigra or by treatment with l-DOPA, suggesting that α-SYN regulates dopamine availability to NSCs. Our data indicate that α-SYN, present in dopaminergic nerve terminals supplying the subependymal zone, acts as a niche component to sustain the neurogenic potential of adult NSCs and identify α-SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain.SIGNIFICANCE STATEMENT We report an essential role for the protein α-synuclein present in dopaminergic nigral afferents in the regulation of adult neural stem cell maintenance, identifying the first synaptic regulator with an implication in stem cell niche biology. Although the exact role of α-synuclein in neural transmission is not completely clear, our results indicate that it is required for stemness and the preservation of neurogenic potential in concert with dopamine.
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Encéfalo/metabolismo , Neuronas Dopaminérgicas/metabolismo , Células-Madre Neurales/metabolismo , Nicho de Células Madre/fisiología , alfa-Sinucleína/metabolismo , Animales , Encéfalo/citología , Senescencia Celular/fisiología , Dopamina/metabolismo , Neuronas Dopaminérgicas/citología , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Células-Madre Neurales/citología , Neurogénesis/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismoRESUMEN
Mutations in the GBA1 gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) are important risk factors for Parkinson's disease (PD). In vitro, altered GBA1 activity promotes alpha-synuclein accumulation whereas elevated levels of alpha-synuclein compromise GBA1 function, thus supporting a pathogenic mechanism in PD. However, the mechanisms by which GBA1 deficiency is linked to increased risk of PD remain elusive, partially because of lack of aged models of GBA1 deficiency. As knocking-out GBA1 in the entire brain induces massive neurodegeneration and early death, we generated a mouse model of GBA1 deficiency amenable to investigate the long-term consequences of compromised GBA1 function in dopaminergic neurons. DAT-Cre and GBA1-floxed mice were bred to obtain selective homozygous disruption of GBA1 in midbrain dopamine neurons (DAT-GBA1-KO). Mice were followed for motor function, neuronal survival, alpha-synuclein phosphorylation and glial activation. Susceptibility to nigral viral vector-mediated overexpression of mutated (A53T) alpha-synuclein was assessed. Despite loss of GBA1 and substrate accumulation, DAT-GBA1-KO mice displayed normal motor performances and preserved dopaminergic neurons despite robust microglial activation in the substantia nigra, without accumulation of endogenous alpha-synuclein with respect to wild-type mice. Lysosomal function was only marginally affected. Screening of micro-RNAs linked to the regulation of GBA1, alpha-synuclein or neuroinflammation did not reveal significant alterations. Viral-mediated overexpression of A53T-alpha-synuclein yielded similar neurodegeneration in DAT-GBA1-KO mice and wild-type mice. These results indicate that loss of GBA1 function in mouse dopaminergic neurons is not critical for alpha-synuclein accumulation or neurodegeneration and suggest the involvement of GBA1 deficiency in other cell types as a potential mechanism.
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Neuronas Dopaminérgicas/metabolismo , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Animales , Encéfalo/metabolismo , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Vectores Genéticos , Mesencéfalo/metabolismo , Ratones , Ratones Noqueados , Microglía/metabolismo , Modelos Animales , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Most neurodegenerative disorders are characterized by deposits of misfolded proteins and neuronal degeneration in specific brain regions. Growing evidence indicates that lysosomal impairment plays a primary pathogenic role in these diseases, in particular, the occurrence of increased lysosomal pH. Thus, therapeutic development aiming at restoring lysosomal function represents a novel, precise, and promising strategy for the treatment of these pathologies. Herein we demonstrate that acidic oil-in-water nanoemulsions loaded with poly(dl-lactide- co-glycolide) (PLGA) are able to rescue impaired lysosomal pH in genetic cellular models of Parkinson's disease. For in vivo assays, nanoemulsions were labeled with an original synthetic hydrophobic far red-emitting dye to allow fluorescence monitoring. Following stereotaxic injection in the mouse brain, widespread diffusion of the nanocarrier was observed, up to 500 µm from the injection site, as well as internalization into the lysosomal compartment in brain cells. Finally, promising preliminary assays of systemic administration demonstrate that a fraction of the formulation crosses the blood brain barrier, penetrates the brain parenchyma, is internalized by cells, and colocalizes with lysosomal markers. Overall, these results suggest the feasibility and the therapeutic potential of this new nanoformulation as an effective drug delivery tool to the brain, with the potential to rescue pathological lysosomal deficits.
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Concentración de Iones de Hidrógeno , Lisosomas/metabolismo , Nanopartículas , Enfermedades Neurodegenerativas/tratamiento farmacológico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/uso terapéutico , Animales , Barrera Hematoencefálica , Línea Celular Tumoral , Portadores de Fármacos , Emulsiones , Endocitosis , Humanos , Ratones , Enfermedades Neurodegenerativas/metabolismo , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacocinéticaRESUMEN
The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP(+) astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g-ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381-1395.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Sistemas de Transporte de Aminoácidos Acídicos/antagonistas & inhibidores , Enfermedades Desmielinizantes/metabolismo , Vaina de Mielina/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Muerte Celular/fisiología , Células Cultivadas , Enfermedades Desmielinizantes/patología , Glutatión/deficiencia , Ratones , Microglía/metabolismo , Microglía/patología , Vaina de Mielina/patología , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/metabolismo , Nervio Ciático/patología , Médula Espinal/metabolismo , Médula Espinal/patología , Técnicas de Cultivo de TejidosRESUMEN
PURPOSE: Glutamate excitotoxicity contributes to oligodendroglial and axonal damage in multiple sclerosis pathology. Extracellular glutamate concentration in the brain is controlled by cystine/glutamate antiporter (system xc-), a membrane antiporter that imports cystine and releases glutamate. Despite this, the system xc(-) activity and its connection to the inflammatory reaction in multiple sclerosis (MS) is largely unknown. METHODS: Longitudinal in vivo magnetic resonance (MRI) and positron emission tomography (PET) imaging studies with 2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG), [(11)C]-(R)-(1-(2-chlorophenyl)-N-methyl-N-1(1-methylpropyl)-3-isoquinolinecarboxamide ([(11)C]PK11195) and (4S)-4-(3-(18)F-fluoropropyl)-L-glutamate ([(18)F]FSPG) were carried out during the course of experimental autoimmune encephalomyelitis (EAE) induction in rats. RESULTS: [(18)F]FSPG showed a significant increase of system xc(-) function in the lumbar section of the spinal cord at 14 days post immunization (dpi) that stands in agreement with the neurological symptoms and ventricle edema formation at this time point. Likewise, [(18)F]FDG did not show significant changes in glucose metabolism throughout central nervous system and [(11)C]PK11195 evidenced a significant increase of microglial/macrophage activation in spinal cord and cerebellum 2 weeks after EAE induction. Therefore, [(18)F]FSPG showed a major capacity to discriminate regions of the central nervous system affected by the MS in comparison to [(18)F]FDG and [(11)C]PK11195. Additionally, clodronate-treated rats showed a depletion in microglial population and [(18)F]FSPG PET signal in spinal cord confirming a link between neuroinflammatory reaction and cystine/glutamate antiporter activity in EAE rats. CONCLUSIONS: Altogether, these results suggest that in vivo PET imaging of system xc(-) could become a valuable tool for the diagnosis and treatment evaluation of MS.
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Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Imagen por Resonancia Magnética , Imagen Multimodal , Esclerosis Múltiple/diagnóstico por imagen , Esclerosis Múltiple/metabolismo , Tomografía de Emisión de Positrones , Animales , Proteínas Portadoras/metabolismo , Ventrículos Cerebrales/diagnóstico por imagen , Ventrículos Cerebrales/patología , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica , Glucosa/metabolismo , Masculino , Microglía/metabolismo , Microglía/patología , Tamaño de los Órganos , Ratas , Receptores de GABA-A/metabolismoRESUMEN
Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca(2+)](i), loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia-like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia-induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O(2) (reperfusion-like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high-affinity Zn(2+) chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high-affinity Zn(2+) probe, we showed that ischemia induced an intracellular Zn(2+) rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia-induced oligodendrocyte death. Moreover, UO126 blocked the ischemia-induced increase in poly-[ADP]-ribosylation of proteins, and the poly[ADP]-ribose polymerase 1 (PARP-1) inhibitor DPQ significantly inhibited ischemia-induced oligodendroglial cell death-demonstrating that PARP-1 was required downstream in the Zn(2+)-ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn(2+), blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia-reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucosa/metabolismo , Hipoxia/metabolismo , Oligodendroglía/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Daño por Reperfusión/metabolismo , Zinc/metabolismo , Animales , Calcio/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Oligodendroglía/patología , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patologíaRESUMEN
Cortical neuron-astrocyte communication in response to peripheral sensory stimulation occurs in a topographic-, frequency-, and intensity-dependent manner. However, the contribution of this functional interaction to the processing of sensory inputs and consequent behavior remains unclear. We investigate the role of astrocytes in sensory information processing at circuit and behavioral levels by monitoring and manipulating astrocytic activity in vivo. We show that astrocytes control the dynamic range of the cortical network activity, optimizing its responsiveness to incoming sensory inputs. The astrocytic modulation of sensory processing contributes to setting the detection threshold for tactile and thermal behavior responses. The mechanism of such astrocytic control is mediated through modulation of inhibitory transmission to adjust the gain and sensitivity of responding networks. These results uncover a role for astrocytes in maintaining the cortical network activity in an optimal range to control behavior associated with specific sensory modalities.
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Astrocitos , Corteza Somatosensorial , Astrocitos/fisiología , Vías Nerviosas , Calcio/metabolismo , Neuronas/fisiología , Electrofisiología , Animales , Ratones , Corteza Somatosensorial/citología , Corteza Somatosensorial/fisiología , Percepción Olfatoria , Percepción del TactoRESUMEN
Microglial phagocytosis of apoptotic debris prevents buildup damage of neighbor neurons and inflammatory responses. Whereas microglia are very competent phagocytes under physiological conditions, we report their dysfunction in mouse and preclinical monkey models of stroke (macaques and marmosets) by transient occlusion of the medial cerebral artery (tMCAo). By analyzing recently published bulk and single cell RNA sequencing databases, we show that the phagocytosis dysfunction was not explained by transcriptional changes. In contrast, we demonstrate that the impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrient deprivation (OND), which led to reduced process motility, lysosomal exhaustion, and the induction of a protective macroautophagy/autophagy response in microglia. Basal autophagy, in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using pharmacological and transgenic approaches. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro, where it even had a detrimental effect on microglia, suggesting that modulating microglial autophagy to optimal levels may be a hard to achieve goal. Nonetheless, our results show that pharmacological interventions, acting directly on microglia or indirectly on the brain environment, have the potential to recover phagocytosis efficiency in the diseased brain. We propose that phagocytosis is a therapeutic target yet to be explored in stroke and other brain disorders and provide evidence that it can be modulated in vivo using rapamycin.Abbreviations: AIF1/IBA1: allograft inflammatory factor 1; AMBRA1: autophagy/beclin 1 regulator 1; ATG4B: autophagy related 4B, cysteine peptidase; ATP: adenosine triphosphate; BECN1: beclin 1, autophagy related; CASP3: caspase 3; CBF: cerebral blood flow; CCA: common carotid artery; CCR2: chemokine (C-C motif) receptor 2; CIR: cranial irradiation; Csf1r/v-fms: colony stimulating factor 1 receptor; CX3CR1: chemokine (C-X3-C motif) receptor 1; DAPI: 4',6-diamidino-2-phenylindole; DG: dentate gyrus; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HI: hypoxia-ischemia; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCA: medial cerebral artery; MTOR: mechanistic target of rapamycin kinase; OND: oxygen and nutrient deprivation; Ph/A coupling: phagocytosis-apoptosis coupling; Ph capacity: phagocytic capacity; Ph index: phagocytic index; SQSTM1: sequestosome 1; RNA-Seq: RNA sequencing; TEM: transmission electron microscopy; tMCAo: transient medial cerebral artery occlusion; ULK1: unc-51 like kinase 1.
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Autofagia , Accidente Cerebrovascular , Animales , Ratones , Autofagia/fisiología , Microglía/metabolismo , Beclina-1/metabolismo , Fagocitosis/genética , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/metabolismo , Oxígeno/farmacología , Sirolimus/farmacologíaRESUMEN
Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, associated with the accumulation of misfolded α-synuclein and lysosomal impairment, two events deemed interconnected. Protein aggregation is linked to defects in degradation systems such as the autophagy-lysosomal pathway, while lysosomal dysfunction is partly related to compromised acidification. We have recently proven that acidic nanoparticles (aNPs) can re-acidify lysosomes and ameliorate neurotoxin-mediated dopaminergic neurodegeneration in mice. However, no lysosome-targeted approach has yet been tested in synucleinopathy models in vivo. Here, we show that aNPs increase α-synuclein degradation through enhancing lysosomal activity in vitro. We further demonstrate in vivo that aNPs protect nigral dopaminergic neurons from cell death, ameliorate α-synuclein pathology, and restore lysosomal function in mice injected with PD patient-derived Lewy body extracts carrying toxic α-synuclein aggregates. Our results support lysosomal re-acidification as a disease-modifying strategy for the treatment of PD and other age-related proteinopathies.
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Nanopartículas , Enfermedad de Parkinson , Animales , Humanos , Lisosomas/metabolismo , Ratones , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)â», an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)â» in glutamate homeostasis alterations in MS pathology. METHODS: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients. RESULTS AND DISCUSSION: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)â» and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes. CONCLUSIONS: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)â» in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Cistina/metabolismo , Ácido Glutámico/metabolismo , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Sistema de Transporte de Aminoácidos y+/genética , Sistemas de Transporte de Aminoácidos Acídicos , Animales , Línea Celular , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Glutatión/metabolismo , Homeostasis , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Nervio Óptico/patología , Ratas , Ratas Endogámicas Lew , Médula Espinal/patología , Adulto JovenRESUMEN
The brain extracellular space (ECS) is a continuous reticular compartment that lies between the cells of the brain. It is vast in extent relative to its resident cells, yet, at the same time the nano- to micrometer dimensions of its channels and reservoirs are commonly finer than the smallest cellular structures. Our conventional view of this compartment as largely static and of secondary importance for brain function is rapidly changing, and its active dynamic roles in signaling and metabolite clearance have come to the fore. It is further emerging that ECS microarchitecture is highly heterogeneous and dynamic and that ECS geometry and diffusional properties directly modulate local diffusional transport, down to the nanoscale around individual synapses. The ECS can therefore be considered an extremely complex and diverse compartment, where numerous physiological events are unfolding in parallel on spatial and temporal scales that span orders of magnitude, from milliseconds to hours, and from nanometers to centimeters. To further understand the physiological roles of the ECS and identify new ones, researchers can choose from a wide array of experimental techniques, which differ greatly in their applicability to a given sample and the type of data they produce. Here, we aim to provide a basic introduction to the available experimental techniques that have been applied to address the brain ECS, highlighting their main characteristics. We include current gold-standard techniques, as well as emerging cutting-edge modalities based on recent super-resolution microscopy. It is clear that each technique comes with unique strengths and limitations and that no single experimental method can unravel the unknown physiological roles of the brain ECS on its own.
RESUMEN
In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathology remains unknown. Here we study the nanoscale organization of pathological ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resolution imaging analysis, we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, experimental hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathology, unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.
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Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Ácido Hialurónico/metabolismo , Sinucleinopatías/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/ultraestructura , Microscopía Electrónica , Enfermedad de Parkinson/metabolismo , Espectroscopía Infrarroja CortaRESUMEN
Alpha-synuclein (α-syn) is involved in both familial and sporadic Parkinson's disease (PD). One of the proposed pathogenic mechanisms of α-syn mutations is mitochondrial dysfunction. However, it is not entirely clear the impact of impaired mitochondrial dynamics induced by α-syn on neurodegeneration and whether targeting this pathway has therapeutic potential. In this study we evaluated whether inhibition of mitochondrial fission is neuroprotective against α-syn overexpression in vivo. To accomplish this goal, we overexpressed human A53T-α- synuclein (hA53T-α-syn) in the rat nigrostriatal pathway, with or without treatment using the small molecule Mitochondrial Division Inhibitor-1 (mdivi-1), a putative inhibitor of the mitochondrial fission Dynamin-Related Protein-1 (Drp1). We show here that mdivi-1 reduced neurodegeneration, α-syn aggregates and normalized motor function. Mechanistically, mdivi-1 reduced mitochondrial fragmentation, mitochondrial dysfunction and oxidative stress. These in vivo results support the negative role of mutant α-syn in mitochondrial function and indicate that mdivi-1 has a high therapeutic potential for PD.
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Dinámicas Mitocondriales/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Quinazolinonas/farmacología , Degeneración Estriatonigral/tratamiento farmacológico , alfa-Sinucleína/genética , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Dinaminas/antagonistas & inhibidores , Dinaminas/genética , Dinaminas/metabolismo , Expresión Génica , Inyecciones Intraperitoneales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Actividad Motora/efectos de los fármacos , Mutación , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson Secundaria/genética , Enfermedad de Parkinson Secundaria/metabolismo , Enfermedad de Parkinson Secundaria/patología , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Porción Compacta de la Sustancia Negra/metabolismo , Porción Compacta de la Sustancia Negra/patología , Agregado de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Degeneración Estriatonigral/genética , Degeneración Estriatonigral/metabolismo , Degeneración Estriatonigral/patología , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Exosomes are extracellular nanovesicles (30-100 nm) generated from endosomal membranes and known to be released by all cell lineages of the Central Nervous System (CNS). They constitute important vesicles for the secretion and transport of multilevel information, including signaling, toxic, and regulatory molecules. Initially thought to have a function merely in waste disposal, the involvement of exosomes in neuronal development, maintenance, and regeneration through its paracrine and endocrine signaling functions has drawn particular attention in recent years. These vesicles, being involved in the clearance and cell-to-cell spreading of toxic molecules, have been naturally implicated in aging, and in several neurodegenerative diseases associated with pathological conversion of proteins, as well as in the transport of other disease-associated molecules, such as nucleic acids or pro-inflammatory cytokines. Our understanding of such unique form of communication may provide not only answers about (patho)physiological processes in the brain, but can also offer means to exploit these vesicles as vehicles for the delivery of biologically relevant molecules or as tools to monitor brain diseases in a non-invasive way. A promising field in expansion, the study of exosomes and related extracellular vesicles has just commenced to unveil their potential as therapeutic tools for brain disorders as well as biomarkers of disease state.
RESUMEN
Lysosomal impairment causes lysosomal storage disorders (LSD) and is involved in pathogenesis of neurodegenerative diseases, notably Parkinson disease (PD). Strategies enhancing or restoring lysosomal-mediated degradation thus appear as tantalizing disease-modifying therapeutics. Here we demonstrate that poly(DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restore impaired lysosomal function in a series of toxin and genetic cellular models of PD, i.e. ATP13A2-mutant or depleted cells or glucocerebrosidase (GBA)-mutant cells, as well as in a genetic model of lysosomal-related myopathy. We show that PLGA-aNP are transported to the lysosome within 24 h, lower lysosomal pH and rescue chloroquine (CQ)-induced toxicity. Re-acidification of defective lysosomes following PLGA-aNP treatment restores lysosomal function in different pathological contexts. Finally, our results show that PLGA-aNP may be detected after intracerebral injection in neurons and attenuate PD-related neurodegeneration in vivo by mechanisms involving a rescue of compromised lysosomes.
Asunto(s)
Ácidos/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/metabolismo , Nanopartículas/química , Enfermedad de Parkinson/patología , 1-Metil-4-fenilpiridinio , Álcalis/química , Animales , Muerte Celular , Neuronas Dopaminérgicas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inyecciones Intraventriculares , Ácido Láctico/química , Lisosomas/ultraestructura , Ratones , Modelos Biológicos , Modelos Genéticos , Nanopartículas/ultraestructura , Neostriado/patología , Degeneración Nerviosa/patología , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/fisiología , Isquemia Encefálica/etiología , Ácido Glutámico/metabolismo , Sistema de Transporte de Aminoácidos y+/deficiencia , Sistema de Transporte de Aminoácidos y+/genética , Animales , Benzoatos/farmacología , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Muerte Celular , Proteínas de Transporte de Glutamato en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Activación del Canal Iónico/fisiología , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Células Piramidales/efectos de los fármacos , Células Piramidales/patología , Células Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Transporte Vesicular de Glutamato/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Glutamato/fisiologíaRESUMEN
The aim of this research was to study how high water temperatures impair the reproductive activity of pejerrey Odontesthes bonariensis, an inland-water atherinopsid fish species from the pampasic region of Argentina. Adult pejerrey of both sexes were kept under a control-temperature regime (19 degrees C) and two experimental temperatures (23 degrees and 27 degrees C) for 8 d. The effect of elevated temperature on the pituitary-gonad axis was analyzed in terms of the expression of gonadotropin (GtH) subunits: follicle-stimulating hormone beta, luteinizing hormone beta (LH-beta), glycoprotein hormone alpha, and GtH receptors FSH-R and LH-R by semiquantitative reverse transcriptase polymerase chain reaction, plasma levels of sexual steroids by radioimmunoassay, and reproductive status by gonadal histology. The results of this work clearly indicate that short periods of exposure to high water temperatures disrupt pejerrey reproduction. This effect was observed in spawning activity, at the histological level, and in the reduction of plasma estradiol in females and testosterone in males. The mRNA levels of GtH subunits and GtH receptors generally decreased in proportion to the increase in temperature for both sexes. However, the differences between groups were statistically significant only for LH-beta and for FSH-R expression in pejerrey females. Thus, the gonads of pejerrey appear to be the primary target of high water temperature. Analysis of the air temperature in this region over the past 40 yr indicated an increase of 1.74 degrees C in mean annual temperature. This increase, coupled with the mechanism of high-temperature sensitivity shown in this study, may be one of the reasons for the decline in pejerrey populations observed in this region over recent decades.