Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging

Reis, Ricardo Augusto de Melo; Schitine, Clarissa; Köfalvi, Attila; Grade, Sofia; Cortes, Luísa; Gardino, P.F.; Malva, João O.; Mello, Fernando G. de

doi:10.1007/s10571-011-9673-6

Cited by 21 publications

(17 citation statements)

References 49 publications

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“…Third, we used calcium responses to different cell-specific stimuli to characterize bipolar cells triggered by microPH medium. We used Fura-2 AM, a cell permeant calcium indicator: KCl, which triggers an intracellular Ca ϩ2 response in excitable cells (De Melo Reis et al, 2011); AMPA, which depolarizes neurons expressing the corresponding glutamate receptors (Bloodgood and Sabatini, 2008); ATP, which activates purinergic receptors in astrocytes and neurons (De Melo Reis et al, 2011); histamine, which triggers intracellular Ca ϩ2 response in immature cells through histamine receptor, highly expressed on immature/stem cells and embryonic stem cells (Eiriz et al, 2011); and NMDA ( Fig. 11A-D).…”

Section: The Secretome From Phagocytic Microglia Drives Neuroprogenitmentioning

confidence: 99%

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome

et al. 2020

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During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the longterm maintenance of adult hippocampal neurogenesis.

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Section: The Secretome From Phagocytic Microglia Drives Neuroprogenitmentioning

confidence: 99%

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome

et al. 2020

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“…Neurite growth was assessed via confocal microscopy after immunostaining for Tuj-1. Variations in the free intracellular calcium level ([Ca 2+ ]i) were evaluated in DRG neurons or Schwann cells obtained from P1 DRGs following an adaptation of the protocol reported by de Melo Reis et al, 2011 [18] . DRG neurons and Schwann cells were seeded on poly-L-lysine- and laminin-coated coverslips for 48 h. The cells were loaded for 40 min with 5 µM Fura-2/AM (Molecular Probes) in Krebs solution (132 mM NaCl, 4 mM KCl, 1.4 mM MgCl 2 , 2.5 mM CaCl 2 , 6 mM glucose, 10 mM HEPES, pH 7.4) containing 0.1% fatty acid-free bovine serum albumin (BSA) and 0.02% pluronic acid F-127 (Molecular Probes) in an incubator containing 5% CO 2 and 95% atmospheric air at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Mice Lacking GD3 Synthase Display Morphological Abnormalities in the Sciatic Nerve and Neuronal Disturbances during Peripheral Nerve Regeneration

et al. 2014

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The ganglioside 9-O-acetyl GD3 is overexpressed in peripheral nerves after lesioning, and its expression is correlated with axonal degeneration and regeneration in adult rodents. However, the biological roles of this ganglioside during the regenerative process are unclear. We used mice lacking GD3 synthase (Siat3a KO), an enzyme that converts GM3 to GD3, which can be further converted to 9-O-acetyl GD3. Morphological analyses of longitudinal and transverse sections of the sciatic nerve revealed significant differences in the transverse area and nerve thickness. The number of axons and the levels of myelin basic protein were significantly reduced in adult KO mice compared to wild-type (WT) mice. The G-ratio was increased in KO mice compared to WT mice based on quantification of thin transverse sections stained with toluidine blue. We found that neurite outgrowth was significantly reduced in the absence of GD3. However, addition of exogenous GD3 led to neurite growth after 3 days, similar to that in WT mice. To evaluate fiber regeneration after nerve lesioning, we compared the regenerated distance from the lesion site and found that this distance was one-fourth the length in KO mice compared to WT mice. KO mice in which GD3 was administered showed markedly improved regeneration compared to the control KO mice. In summary, we suggest that 9-O-acetyl GD3 plays biological roles in neuron-glia interactions, facilitating axonal growth and myelination induced by Schwann cells. Moreover, exogenous GD3 can be converted to 9-O-acetyl GD3 in mice lacking GD3 synthase, improving regeneration.

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“…Third, we examined the calcium response of bipolar, stellate, and freshly dissociated NPCs to different stimuli using Fura-2 AM, a cell permeant calcium indicator: KCl, which triggers an intracellular Ca +2 response in excitable cells (De Melo Reis et al, 2011); AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), which depolarizes neurons expressing the corresponding glutamate receptors (Bloodgood and Sabatini, 2008); ATP (adenosine triphosphate), which activates purinergic receptors in astrocytes and neurons (De Melo Reis et al, 2011); histamine, which triggers intracellular Ca +2 response in immature cells through histamine receptor, highly expressed on immature/stem cells and embryonic stem cells (Eiriz et al, 2011); and NMDA (N-methyl-D-aspartate) ( Figure 6A-D). We found that 69% of the freshly dissociated NPCs depolarized in response to ATP and histamine, and became hyperpolarized in response to KCl.…”

Section: The Secretome From Phagocytic Microglia Drives Neuroprogenitmentioning

confidence: 99%

“…In vivo no changes in the production of newborn astrocytes were observed in the neurogenic cascade, although on isolated NPCs the phagocytic microglial secretome gave rise to astrocytecommitted cells with a bipolar phenotype, reminiscent of radial glia (Encinas and Enikolopov, 2008). These cells presented several astrocytic features, including the expression of GFAP and S100β (Encinas and Enikolopov, 2008;Raponi et al, 2007); intracellular calcium response to ATP (De Melo Reis et al, 2011) and high phosphorylation of SMAD, which interacts with TGFβ to give rise to astrocytes/radial glia (Stipursky and Gomes, 2007). Nonetheless, our transcriptional assay did not show heterologous `gliogenic´ genes in phagocytic microglia, suggesting that rather than actively promoting astrogenesis, the acute phagocytosis secretome indirectly promote the default astrocytic lineage (Miller and Gauthier, 2007) by restricting the neuronal lineage.…”

Section: Microglia Regulates Neurogenesis Through the Phagocytosis Sementioning

confidence: 99%

Microglia actively remodels adult hippocampal neurogenesis through the phagocytosis secretome

Díaz-Aparicio

Paris

Sierra-Torre

et al. 2019

Preprint

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words)During adult hippocampal neurogenesis, the majority of newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of their intracellular contents. Here, we propose that phagocytosis is not merely a passive process of corpse removal but has an active role in maintaining adult hippocampal neurogenesis. First, we found that neurogenesis was disrupted in mice chronically deficient for two microglial phagocytosis pathways (P2Y12 and MerTK/Axl), but was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of microglial phagocytosis in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that reprogrammed phagocytic microglia act as a sensor of local cell death, modulating the balance between cell proliferation and cell survival in the neurogenic niche, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.

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Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging

Cited by 21 publications

References 49 publications

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome

Mice Lacking GD3 Synthase Display Morphological Abnormalities in the Sciatic Nerve and Neuronal Disturbances during Peripheral Nerve Regeneration

Microglia actively remodels adult hippocampal neurogenesis through the phagocytosis secretome

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