Live-cell metabolic analysis of oligodendroglia isolated from postnatal mouse brain and spinal cord

Khandker, Luipa; Wood, Teresa L.

doi:10.1016/j.xpro.2022.101655

Cited by 2 publications

(2 citation statements)

References 10 publications

(17 reference statements)

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“…Details can be referred to other seahorse protocols. 4 , 5 Note: General manufacturer’s instructions for performing a mitochondrial stress test can be found at https://www.agilent.com/cs/library/usermanuals/public/XF_Cell_Mito_Stress_Test_Kit_User_Guide.pdf . Note: There are two sets of plates included in the seahorse assay.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…As a result, the final unit of OCR for each sample is normalized into pmol/min/μg. 5 In case that cell number and protein content can hardly be measured due to cell culture limitation or cell 3D structure (i.e., organoids), genomic DNA can be isolated from samples and gDNA content per well can thus be utilized for OCR normalization. 11 …”

Section: Quantification and Statistical Analysismentioning

confidence: 99%

See 1 more Smart Citation

Analyzing mitochondrial respiration of human induced pluripotent stem cell-derived myeloid progenitors using Seahorse technology

Fan¹,

Mizoguchi²,

Tatematsu³

et al. 2023

STAR Protocols

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Quantification and Statistical Analysismentioning

confidence: 99%

Analyzing mitochondrial respiration of human induced pluripotent stem cell-derived myeloid progenitors using Seahorse technology

Fan¹,

Mizoguchi²,

Tatematsu³

et al. 2023

STAR Protocols

View full text Add to dashboard Cite

mTORC2 Loss in Oligodendrocyte Progenitor Cells Results in Regional Hypomyelination in the Central Nervous System

et al. 2022

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In the CNS, oligodendrocyte progenitor cells (OPCs) differentiate into mature oligodendrocytes to generate myelin, an essential component for normal nervous system function. OPC differentiation is driven by signaling pathways, such as mTOR, which functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), containing Raptor or Rictor, respectively. In the current studies, mTORC2 signaling was selectively deleted from OPCs in PDGFRα-Cre X Rictorfl/flmice. This study examined developmental myelination in male and female mice, comparing the impact of mTORC2 deletion in the corpus callosum and spinal cord. In both regions, Rictor loss in OPCs resulted in early reduction in myelin RNAs and proteins. However, these deficits rapidly recovered in spinal cord, where normal myelin was noted at P21 and P45. By contrast, the losses in corpus callosum resulted in severe hypomyelination and increased unmyelinated axons. The hypomyelination may result from decreased oligodendrocytes in the corpus callosum, which persisted in animals as old as postnatal day 350. The current studies focus on uniquely altered signaling pathways following mTORC2 loss in developing oligodendrocytes. A major mTORC2 substrate is phospho-Akt-S473, which was significantly reduced throughout development in both corpus callosum and spinal cord at all ages measured, yet this had little impact in spinal cord. Loss of mTORC2 signaling resulted in decreased expression of actin regulators, such as gelsolin in corpus callosum, but only minimal loss in spinal cord. The current study establishes a regionally specific role for mTORC2 signaling in OPCs, particularly in the corpus callosum.SIGNIFICANCE STATEMENTmTORC1 and mTORC2 signaling has differential impact on myelination in the CNS. Numerous studies identify a role for mTORC1, but deletion of Rictor (mTORC2 signaling) in late-stage oligodendrocytes had little impact on myelination in the CNS. However, the current studies establish that deletion of mTORC2 signaling from oligodendrocyte progenitor cells results in reduced myelination of brain axons. These studies also establish a regional impact of mTORC2, with little change in spinal cord in these conditional Rictor deletion mice. Importantly, in both brain and spinal cord, mTORC2 downstream signaling targets were impacted by Rictor deletion. Yet, these signaling changes had little impact on myelination in spinal cord, while they resulted in long-term alterations in myelination in brain.

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Live-cell metabolic analysis of oligodendroglia isolated from postnatal mouse brain and spinal cord

Cited by 2 publications

References 10 publications

Analyzing mitochondrial respiration of human induced pluripotent stem cell-derived myeloid progenitors using Seahorse technology

Analyzing mitochondrial respiration of human induced pluripotent stem cell-derived myeloid progenitors using Seahorse technology

mTORC2 Loss in Oligodendrocyte Progenitor Cells Results in Regional Hypomyelination in the Central Nervous System

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