Microglial phagocytosis: unraveling the role of GPR34-LYSOPS signaling and phagocytic microglia on metabolism and neurogenesis

200 p.Las células de microglía constituyen los fagocitos residentes del cerebro. Una de las principales funciones de las células de microglía consiste en la fagocitosis de células apoptóticas. La fagocitosis depende de diversos receptores anclados a la membrana de la microglía y que permiten el encapsulamiento y posterior degradación de los cuerpos apoptóticos en su interior. En esta tesis pretendemos profundizar en el estudio de uno de estos receptores, llamado GPR34, que presenta un único ligando conocido, lysoPS. Los resultados muestran que GPR34 regula la fagocitosis microglial in vivo e in vitro, mientras que lysoPS parece ejercer un papel relevante solo en condiciones in vitro. Por otra parte, también se ha estudiado el posible impacto de la fagocitosis microglial tanto en el metabolismo microglial así como su posible influencia en la neurogénesis en el hipocampo. Los resultados mostraron que la microglía fagocítica sufría adaptaciones metabólicas en las vías mitocondriales así como en la morfología de las mitocondrias. Sin embargo, no se observaron efectos de la inhibición de la fagocitosis microglial sobre la neurogénesis hipocampal. En conjunto, nuestros datos reflejan la importancia de la fagocitosis microglial en diversos procesos celulares y el papel relevante de GPR34

From the Cajal alumni Achucarro and Rio-Hortega to the rediscovery of never-resting microglia

Under the guidance of Ramon y Cajal, a plethora of students flourished and began to apply his silver impregnation methods to study brain cells other than neurons: the neuroglia. In the first decades of the twentieth century, Nicolas Achucarro was one of the first researchers to visualize the brain cells with phagocytic capacity that we know today as microglia. Later, his pupil Pio del Rio-Hortega developed modifications of Achucarro's methods and was able to specifically observe the fine morphological intricacies of microglia. These findings contradicted Cajal's own views on cells that he thought belonged to the same class as oligodendroglia (the so called "third element" of the nervous system), leading to a long-standing discussion. It was only in 1924 that Rio-Hortega's observations prevailed worldwide, thus recognizing microglia as a unique cell type. This late landing in the Neuroscience arena still has repercussions in the twenty first century, as microglia remain one of the least understood cell populations of the healthy brain. For decades, microglia in normal, physiological conditions in the adult brain were considered to be merely "resting," and their contribution as "activated" cells to the neuroinflammatory response in pathological conditions mostly detrimental. It was not until microglia were imaged in real time in the intact brain using two-photon in vivo imaging that the extreme motility of their fine processes was revealed. These findings led to a conceptual revolution in the field: "resting" microglia are constantly surveying the brain parenchyma in normal physiological conditions. Today, following Cajal's school of thought, structural and functional investigations of microglial morphology, dynamics, and relationships with neurons and other glial cells are experiencing a renaissance and we stand at the brink of discovering new roles for these unique immune cells in the healthy brain, an essential step to understand their causal relationship to diseases.The authors are grateful to Alain Bessis (Institut de Biologic, Bcole Normale Superieure, Institut National de la Sante et de la Recherche Medicale, Paris, France) and Luis Miguel Garcia-Segura (Cajal Institute, CSIC, Madrid, Spain) for their critical reading of the manuscript. They also thank Maria Angeles Langa for her help obtaining Rio-Hortega's original reprints. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness with FEDER funds to AS (BFU2012-32089) and from the National Sciences and Engineering Research Council of Canada (NSERC) to MET and LS was awarded of a summer scholarship from the Faculte de medecine of Universite Laval, and VS is recipient of a predoctoral fellowship from the Spanish Ministry of Economy and Competitivity

Marco teórico en el diseño industrial

En el campo del diseño, y por la propia naturaleza del trabajo disciplinar, los conceptos que se construyen y que son propios del discurso proyectual, son los sistemas que generan formas estéticas, formas perceptuales que satisfacen necesidades, que solucionan problemas antropológicos, que conforman la cultura material y que por supuesto abonan al conocimiento del diseño. Por eso al nombrar distintos objetos como: silla, mesa, sofá, estufa, refrigerador, plancha, ordenador, escritorio, radio, grabadora, entre muchos otros que rodean la vida cotidiana de nosotros los humanos, no sólo nos referimos a la cosa tangible, material y presente, también nos referimos a la idea, al concepto, al sistema, a la imagen que formamos mentalmente y que tiene para nosotros una estructura y una apariencia exterior, una figura que nos posibilita si ya existe, o posibilitará en el porvenir la estética y con ello la asignación de significados según la circunstancia en que se encuentre

Neuronal hyperactivity disturbs ATP microgradients, impairs microglial motility, and reduces phagocytic receptor expression triggering apoptosis/microglial phagocytosis uncoupling

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders

Microglial phagocytic response during in vivo acute and chronic inflammatory challenge.

<p>(<b>A</b>) Experimental design and apoptosis in the DG of c57BL/6 fms-EGFP 1-mo mice injected systemically with LPS (1mg/kg; <i>n</i> = 5) or vehicle (saline; <i>n</i> = 4) 8 h prior to sacrifice. Apoptotic cells were identified by pyknosis/karryorhexis. <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g002" target="_blank">Fig 2A</a></b> was generated from data that was originally published as part of [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. (<b>B</b>) Weighted Ph capacity of microglia (in parts per unit, ppu) in control and LPS mice. (<b>C</b>) Number of microglial cells in control and LPS mice. (<b>D</b>) Ph/A coupling in the 1-mo mouse hippocampus (in fold change) during acute inflammatory challenge. (<b>E</b>) Experimental design and representative confocal z-stacks of the DG of PND21 Swiss mice fed during gestation and lactation with a diet balanced (Ω3 bal; <i>n</i> = 7) or deficient (Ω3 def; <i>n</i> = 7) in the omega 3 polyunsaturated fatty acid, a diet that induces chronic inflammation in the hippocampus. Microglia were labeled with Iba1 (cyan) and apoptotic nuclei were detected by pyknosis/karyorrhexis (white, DAPI). Arrows point to apoptotic cells engulfed by microglia (M). Scale bars = 50 μm; z = 22.5μm. (<b>F</b>) Number of apoptotic (pyknotic/karyorrhectic) cells in mice fed with Ω3 balanced and deficient diets. (<b>G</b>) Ph index in the PND21 hippocampus (in % of apoptotic cells) in mice fed with Ω3 balanced and deficient diets. (<b>H</b>) Weighted Ph capacity of microglia (in ppu) in PND21 mice. (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in PND21 mice. (<b>J</b>) Total number of microglial cells (Iba1⁺) in PND21 mice. (<b>K</b>) Ph/A coupling in PND21 mice. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.01 by one-tail Student´s <i>t</i> test. Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p

Microglial phagocytic impairment leads to delayed clearance of apoptotic cells at 1 dpi.

<p>(<b>A</b>) Experimental design used to analyze the survival of 3 do cells after the injection of saline (<i>n</i> = 7) or KA (<i>n</i> = 8) in mice. (<b>B</b>) Representative confocal z-stacks of the DG of control and KA-injected mice (1 dpi). The damage induced by KA was evidenced by the presence of cells with abnormal nuclear morphology (DAPI, white), and the altered morphology of microglia (fms-EGFP⁺, cyan). (<b>C</b>) Representative confocal images of 3 do apoptotic (pyknotic, DAPI, white) cells labeled with BrdU (red; arrows) in the SGZ of the hippocampus of saline and KA-injected mice at 1 dpi. In the saline mouse, the BrdU⁺ apoptotic cell, next to a cluster of BrdU⁺ cells, was phagocytosed by a terminal branch of a nearby microglia (fms-EGFP, cyan), whose nucleus was also positive for BrdU. In the KA mouse, the apoptotic BrdU⁺ cell was not phagocytosed by microglia. A nearby apoptotic cell (BrdU^-; arrowhead) was partially engulfed by microglia. (<b>D</b>) Total number of live 3 do BrdU⁺ cells (nonapoptotic) in the septal hippocampus after treatment with KA. The total number of 3 do and 8 do BrdU⁺ cells by a single BrdU injection in saline and KA-injected mice is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s026" target="_blank">S13A and S13B Fig</a></b>. (<b>E</b>) Total number of apoptotic 3 do BrdU⁺ cells in the septal hippocampus after treatment with KA. (<b>F</b>) Percentage of 3 do BrdU⁺ cells that re-enter cell cycle, assessed by their colabeling with the proliferation marker Ki67 after treatment with KA. Representative confocal z-stacks of BrdU/Ki67 cells are found in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s026" target="_blank">S13C Fig</a></b>. (<b>G</b>) Percentage of apoptotic BrdU⁺ cells over total apoptotic cells in the septal hippocampus. (<b>H</b>) Estimated clearance of apoptotic cells in the septal hippocampus. The total number of apoptotic BrdU⁺ (from E) present in the tissue was added to the number of estimated apoptotic BrdU⁺ cells that had been cleared. In saline mice, this number was calculated using the clearance time formula shown in Methods with a clearance time of 1.5 h [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. As the total number of cells should be identical in saline and KA mice, the number of cleared apoptotic cells in KA mice was calculated as the difference between the total (in saline) and the number of present apoptotic cells (in KA). From here, we calculated a new clearance time using the same formula as in saline mice, of 6.3 h. (<b>I</b>) Linear regression analysis of the relationship between apoptosis and phagocytosis (Ph index) in saline and KA-injected mice (6 hpi and 1 dpi). (<b>J</b>) Experimental design used to compare SGZ apoptosis induced by KA at 1 dpi in young (2 mo) and mature (6 mo) mice. (<b>K</b>) Representative epifluorescent tiling image of the hippocampus and surrounding cortex of 2 and 6 mo mice injected with KA at 1 dpi stained with the neuronal activation marker c-fos. The same pattern of expression was found in young and mature mice throughout the DG, CA2, CA1 and the above cortex. (<b>L</b>) Representative confocal z-stacks of the apoptotic (pyknotic, white; act-casp3⁺, red) cells in the SGZ of the hippocampus of 2 mo and 6 mo mice injected with KA (1 dpi). The microglial phagocytosis impairment was similar in the two age groups (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s026" target="_blank">S13D Fig</a></b>). (<b>M</b>) Total number of apoptotic cells in the SGZ of 2 and 6 mo mice treated with saline or KA (1 dpi; <i>n</i> = 4–5 per group). Bars show mean ± SEM. * indicates <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001 by Student´s <i>t</i> test (E, G) or by Holm-Sidak posthoc test after one-way ANOVA (M) was significant at <i>p</i> < 0.05. Scale bars = 50 μm (B), 20 μm (C), 500 μm (K), 25 μm (L). z = 14 μm (B), 12.6 μm (C, sal), 15.4 μm (C, KA), 25 μm (L). Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p