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In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.

Morsch M, Radford R, Lee A, Don EK, Badrock AP, Hall TE, Cole NJ, Chung R - Front Cell Neurosci (2015)

Bottom Line: In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord.This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation.This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

View Article: PubMed Central - PubMed

Affiliation: Motor Neuron Disease Research Group, Faculty of Medicine and Health Sciences, Macquarie University Sydney, NSW, Australia.

ABSTRACT
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

No MeSH data available.


Related in: MedlinePlus

UV ablation activates annexinV-mediated neuronal apoptosis. UV ablation of a single neuron in a transgenic fish expressing the neuronal marker mnx1:mKO2 as well as the apoptotic marker AnnexinV (ubiq:secAnnexinV-mVenus). Shortly, after UV ablation of the soma of the neuron (A; circle), phosphatidylserine (PS) gets switched to the outer leaflet of the plasma membrane and trapped by the AnnexinV marker indicated by the yellow fluorescence (B,C, arrowheads). Throughout the time-course of neuronal degeneration, AnnexinV puncta (yellow fluorescence) were present within the degenerating cell body (C,D) as well as along the axon, progressing anterogradely from the site of ablation (D–F). Scale bars = 20 μm. Supplementary Video 2 shows the time-lapse video of this process.
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Figure 4: UV ablation activates annexinV-mediated neuronal apoptosis. UV ablation of a single neuron in a transgenic fish expressing the neuronal marker mnx1:mKO2 as well as the apoptotic marker AnnexinV (ubiq:secAnnexinV-mVenus). Shortly, after UV ablation of the soma of the neuron (A; circle), phosphatidylserine (PS) gets switched to the outer leaflet of the plasma membrane and trapped by the AnnexinV marker indicated by the yellow fluorescence (B,C, arrowheads). Throughout the time-course of neuronal degeneration, AnnexinV puncta (yellow fluorescence) were present within the degenerating cell body (C,D) as well as along the axon, progressing anterogradely from the site of ablation (D–F). Scale bars = 20 μm. Supplementary Video 2 shows the time-lapse video of this process.

Mentions: When cells undergo stress or death, they release “eat-me” signals that mediate the rapid recognition and engulfment of these dying neurons and neuronal debris to avoid a spread of inflammation. Eat-me signals, such as the phospholipid phosphatidylserine (PS) act as crucial detection signals for the recognition and ultimately the efficient digestion of these cells (Davalos et al., 2005; Takahashi et al., 2005; Ravichandran, 2011; Brown and Neher, 2014). We utilized AnnexinV, a protein that binds to PS lipids exposed on apoptotic cells (Vermes et al., 1995), to visualize this apoptotic/phagocytic signaling process in vivo. We generated a stable transgenic line (ubiq:secAnnexinV-mVenus; see Materials and Methods) and crossed these fish with the mnx1:mKO2 motor neuron line to selectively use this reporter to detect dying neurons in the spinal cord. UV ablation of motor neurons in these double-labeled fish showed the same consistent morphological changes of somal degeneration and axonal blebbing as described earlier (compare Figure 4 and Figure 2; Supplementary Video 2). Moreover, within several minutes after ablation we detected the activation and accumulation of AnnexinV at the neuron (Figure 4B, yellow channel), firstly at the targeted soma site and then progressively along the axon toward the more distal parts of the neuron. As the neuron degenerated, AnnexinV-labeled fragments of neuronal debris were observed around the ablation site (Figures 4D–F).


In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.

Morsch M, Radford R, Lee A, Don EK, Badrock AP, Hall TE, Cole NJ, Chung R - Front Cell Neurosci (2015)

UV ablation activates annexinV-mediated neuronal apoptosis. UV ablation of a single neuron in a transgenic fish expressing the neuronal marker mnx1:mKO2 as well as the apoptotic marker AnnexinV (ubiq:secAnnexinV-mVenus). Shortly, after UV ablation of the soma of the neuron (A; circle), phosphatidylserine (PS) gets switched to the outer leaflet of the plasma membrane and trapped by the AnnexinV marker indicated by the yellow fluorescence (B,C, arrowheads). Throughout the time-course of neuronal degeneration, AnnexinV puncta (yellow fluorescence) were present within the degenerating cell body (C,D) as well as along the axon, progressing anterogradely from the site of ablation (D–F). Scale bars = 20 μm. Supplementary Video 2 shows the time-lapse video of this process.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: UV ablation activates annexinV-mediated neuronal apoptosis. UV ablation of a single neuron in a transgenic fish expressing the neuronal marker mnx1:mKO2 as well as the apoptotic marker AnnexinV (ubiq:secAnnexinV-mVenus). Shortly, after UV ablation of the soma of the neuron (A; circle), phosphatidylserine (PS) gets switched to the outer leaflet of the plasma membrane and trapped by the AnnexinV marker indicated by the yellow fluorescence (B,C, arrowheads). Throughout the time-course of neuronal degeneration, AnnexinV puncta (yellow fluorescence) were present within the degenerating cell body (C,D) as well as along the axon, progressing anterogradely from the site of ablation (D–F). Scale bars = 20 μm. Supplementary Video 2 shows the time-lapse video of this process.
Mentions: When cells undergo stress or death, they release “eat-me” signals that mediate the rapid recognition and engulfment of these dying neurons and neuronal debris to avoid a spread of inflammation. Eat-me signals, such as the phospholipid phosphatidylserine (PS) act as crucial detection signals for the recognition and ultimately the efficient digestion of these cells (Davalos et al., 2005; Takahashi et al., 2005; Ravichandran, 2011; Brown and Neher, 2014). We utilized AnnexinV, a protein that binds to PS lipids exposed on apoptotic cells (Vermes et al., 1995), to visualize this apoptotic/phagocytic signaling process in vivo. We generated a stable transgenic line (ubiq:secAnnexinV-mVenus; see Materials and Methods) and crossed these fish with the mnx1:mKO2 motor neuron line to selectively use this reporter to detect dying neurons in the spinal cord. UV ablation of motor neurons in these double-labeled fish showed the same consistent morphological changes of somal degeneration and axonal blebbing as described earlier (compare Figure 4 and Figure 2; Supplementary Video 2). Moreover, within several minutes after ablation we detected the activation and accumulation of AnnexinV at the neuron (Figure 4B, yellow channel), firstly at the targeted soma site and then progressively along the axon toward the more distal parts of the neuron. As the neuron degenerated, AnnexinV-labeled fragments of neuronal debris were observed around the ablation site (Figures 4D–F).

Bottom Line: In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord.This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation.This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

View Article: PubMed Central - PubMed

Affiliation: Motor Neuron Disease Research Group, Faculty of Medicine and Health Sciences, Macquarie University Sydney, NSW, Australia.

ABSTRACT
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

No MeSH data available.


Related in: MedlinePlus