A new in vitro mouse oligodendrocyte precursor cell migration assay reveals a role for integrin-linked kinase in cell motility

O’Meara, Ryan W.; Cummings, Sarah E.; Michalski, John-Paul; Kothary, Rashmi

doi:10.1186/s12868-016-0242-2

Cited by 11 publications

(12 citation statements)

References 80 publications

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“…Oligodendrocyte progenitor cells in the adult brain and spinal cord are not readily accessible and functional analysis or live imaging are very challenging tasks in vivo . Therefore, we decided to establish a novel in vitro platform that would allow us to study adult zebrafish OPC proliferation, migration, differentiation and myelination, combined with the possibility to perform live imaging, e.g., in the context of OPC migration studies ( O’Meara et al, 2016 ). Furthermore, this method can be used to generate cultures for electrophysiological measurements.…”

Section: Discussionmentioning

confidence: 99%

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

Kroehne

Tsata

Marrone

et al. 2017

Front. Cell. Neurosci.

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Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC)-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable analysis up to 10 days in vitro. Finally, we demonstrate that zebrafish OPCs differentiate into Myelin Basic Protein (MBP)-expressing OLs when co-cultured with human motor neurons differentiated from induced pluripotent stem cells (iPSCs). This shows that the basic mechanisms of oligodendrocyte differentiation are conserved across species and that understanding the regulation of zebrafish OPCs can contribute to the development of new treatments to human diseases.

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Section: Discussionmentioning

confidence: 99%

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

Kroehne

Tsata

Marrone

et al. 2017

Front. Cell. Neurosci.

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“…The cerebral cortex was dissociated from 2-day-old post-natal C57BL/6 mice. The isolated cells were cultured and purified as previously described [ 50 ]. Briefly, cells were replanted in poly-D lysine coated 75 cm 2 flasks, and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) medium containing 20% horse serum and 1% penicillin/streptomycin for 10 days.…”

Section: Methodsmentioning

confidence: 99%

Administration of Downstream ApoE Attenuates the Adverse Effect of Brain ABCA1 Deficiency on Stroke

Wang

Zacharek

et al. 2018

IJMS

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The ATP-binding cassette transporter member A1 (ABCA1) and apolipoprotein E (ApoE) are major cholesterol transporters that play important roles in cholesterol homeostasis in the brain. Previous research demonstrated that specific deletion of brain-ABCA1 (ABCA1−B/−B) reduced brain grey matter (GM) and white matter (WM) density in the ischemic brain and decreased functional outcomes after stroke. However, the downstream molecular mechanism underlying brain ABCA1-deficiency-induced deficits after stroke is not fully understood. Adult male ABCA1−B/−B and ABCA1-floxed control mice were subjected to distal middle-cerebral artery occlusion and were intraventricularly infused with artificial mouse cerebrospinal fluid as vehicle control or recombinant human ApoE2 into the ischemic brain starting 24 h after stroke for 14 days. The ApoE/apolipoprotein E receptor 2 (ApoER2)/high-density lipoprotein (HDL) levels and GM/WM remodeling and functional outcome were measured. Although ApoE2 increased brain ApoE/HDL levels and GM/WM density, negligible functional improvement was observed in ABCA1-floxed-stroke mice. ApoE2-administered ABCA1−B/−B stroke mice exhibited elevated levels of brain ApoE/ApoER2/HDL, increased GM/WM density, and neurogenesis in both the ischemic ipsilateral and contralateral brain, as well as improved neurological function compared with the vehicle-control ABCA1−B/−B stroke mice 14 days after stroke. Ischemic lesion volume was not significantly different between the two groups. In vitro supplementation of ApoE2 into primary cortical neurons and primary oligodendrocyte-progenitor cells (OPCs) significantly increased ApoER2 expression and enhanced cholesterol uptake. ApoE2 promoted neurite outgrowth after oxygen-glucose deprivation and axonal outgrowth of neurons, and increased proliferation/survival of OPCs derived from ABCA1−B/−B mice. Our data indicate that administration of ApoE2 minimizes the adverse effects of ABCA1 deficiency after stroke, at least partially by promoting cholesterol traffic/redistribution and GM/WM remodeling via increasing the ApoE/HDL/ApoER2 signaling pathway.

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“…OPC aggregates were collected and plated as described previously [ 26 ]. In brief, following shaking of the mixed glial cultures, medium containing suspended OLs was filtered through 0.45 μm mesh.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we take advantage of a recently developed method for primary culture of mouse OLs [ 25 ], as well as an assay to measure OL migratory capacity [ 26 ] to characterize the morphological and molecular differentiation of OLs from the more severe Dst dt-27J model of Dst dt , which lacks all neuronal DST isoforms. While we show that both OPCs and OLs do express all three neuronal isoforms of Dst endogenously, OLs isolated from Dst dt-27J animals do not show a deficiency in morphological or molecular maturity in vitro .…”

Section: Introductionmentioning

confidence: 99%

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

et al. 2016

Self Cite

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Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.

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A new in vitro mouse oligodendrocyte precursor cell migration assay reveals a role for integrin-linked kinase in cell motility

Cited by 11 publications

References 80 publications

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

Administration of Downstream ApoE Attenuates the Adverse Effect of Brain ABCA1 Deficiency on Stroke

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

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