Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis

Holt, Leanne M.; Stoyanof, S. Tristan; Olsen, Michelle L.

doi:10.1002/cpns.71

Cited by 53 publications

(48 citation statements)

References 22 publications

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“…This result prompted us to evaluate total, full length, and truncated Ntrk2 mRNA expression in astrocytes relative to other CNS cell populations. Sequential isolation of oligodendrocytes, microglia, astrocytes, and neurons was performed as we have previously described (Holt and Olsen, 2016; Holt et al, 2019) again in PND 28 mice. Cellular purity was confirmed via qPCR by evaluating cell type specific gene expression (Figure 1—figure supplement 1).…”

Section: Resultsmentioning

confidence: 99%

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1

Holt

Hernandez

Pacheco

et al. 2019

eLife

125

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Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF’s receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

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Section: Resultsmentioning

confidence: 99%

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1

Holt

Hernandez

Pacheco

et al. 2019

eLife

125

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“…However, the question of how RTT astrocytes might exert apparent neurotoxic effects remains unanswered. Different methods of culturing astrocytes by use of magnetic cell sorting [53,73] or immunopanning [17] may be useful for these experiments.…”

Section: Astrocytes In Rett Syndromementioning

confidence: 99%

Glial Dysfunction in MeCP2 Deficiency Models: Implications for Rett Syndrome

Kahanovitch

Patterson

Hernandez

et al. 2019

IJMS

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Rett syndrome (RTT) is a rare, X-linked neurodevelopmental disorder typically affecting females, resulting in a range of symptoms including autistic features, intellectual impairment, motor deterioration, and autonomic abnormalities. RTT is primarily caused by the genetic mutation of the Mecp2 gene. Initially considered a neuronal disease, recent research shows that glial dysfunction contributes to the RTT disease phenotype. In the following manuscript, we review the evidence regarding glial dysfunction and its effects on disease etiology.

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“…Primary astrocyte cultures were obtained by magnetic sorting as previously described (Holt, Stoyanof, & Olsen, 2019), with some modifications. Briefly, 10–20 cortices of PN day 2–4 pups were dissociated using the MACS Neural Tissue Dissociation Kit‐T (Miltenyi Biotec, Cat# 130‐093‐231, Auburn, CA) at 37°C (5% CO 2 , 30 min).…”

Section: Methodsmentioning

confidence: 99%

Novel factor in olfactory ensheathing cell‐astrocyte crosstalk: Anti‐inflammatory protein α‐crystallin B

Saglam

Calof

Wray

2020

Glia

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Astrocytes are key players in CNS neuroinflammation and neuroregeneration that may help or hinder recovery, depending on the context of the injury. Although pro‐inflammatory factors that promote astrocyte‐mediated neurotoxicity have been shown to be secreted by reactive microglia, anti‐inflammatory factors that suppress astrocyte activation are not well‐characterized. Olfactory ensheathing cells (OECs), glial cells that wrap axons of olfactory sensory neurons, have been shown to moderate astrocyte reactivity, creating an environment conducive to regeneration. Similarly, astrocytes cultured in medium conditioned by cultured OECs (OEC‐CM) show reduced nuclear translocation of nuclear factor kappa‐B (NFκB), a pro‐inflammatory protein that induces neurotoxic reactivity in astrocytes. In this study, we screened primary and immortalized OEC lines to identify these factors and discovered that Alpha B‐crystallin (CryAB), an anti‐inflammatory protein, is secreted by OECs via exosomes, coordinating an intercellular immune response. Our results showed that: (a) OEC exosomes block nuclear NFκB translocation in astrocytes while exosomes from CryAB‐null OECs could not; (b) OEC exosomes could be taken up by astrocytes, and (c) CryAB treatment suppressed neurotoxicity‐associated astrocyte transcripts. Our results indicate CryAB, as well as other factors secreted by OECs, are potential agents that can ameliorate, or even reverse, the growth‐inhibitory environment created by neurotoxic reactive astrocytes following CNS injuries.

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Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis

Cited by 53 publications

References 22 publications

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1

Glial Dysfunction in MeCP2 Deficiency Models: Implications for Rett Syndrome

Novel factor in olfactory ensheathing cell‐astrocyte crosstalk: Anti‐inflammatory protein α‐crystallin B

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