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Drosophila Neuronal Injury Follows a Temporal Sequence of Cellular Events Leading to Degeneration at the Neuromuscular Junction.

Lincoln BL, Alabsi SH, Frendo N, Freund R, Keller LC - J Exp Neurosci (2015)

Bottom Line: At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components.Our data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism.Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Quinnipiac University, Hamden, CT, USA.

ABSTRACT
Neurodegenerative diseases affect millions of people worldwide, and as the global population ages, there is a critical need to improve our understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. Here, we use an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila melanogaster. We find that sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. Our data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies have implications for our understanding of the cellular and molecular events that occur in humans and will facilitate the identification of biomedically relevant targets for future treatments.

No MeSH data available.


Related in: MedlinePlus

Neuronal injury induces moderate-to-severe neurodegeneration at the NMJ of muscle 6/7. (A) Wild-type uninjured NMJs show the presynaptic active zone maker Brp stained with nc82 (green, bottom panel) in apposition to the postsynaptic marker Dlg (red, middle panel) throughout the entire NMJ. (B) Neuronal injury can induce moderate neurodegeneration (<10 boutons retracted) in which the majority of the NMJ has Brp (green, bottom panel) and Dlg (red, middle panel) in perfect apposition. However, some of the boutons stained with Dlg lack the presynaptic active zone marker Brp. (C) Neuronal injury can also induce severe neurodegeneration in which >10 boutons lack Brp staining (green, bottom panel), suggesting that the neuron has retracted from the muscle as shown by the remaining postsynaptic Dlg staining without any accompanying Brp (red, middle panel). (D) Quantification of neurodegeneration severity was measured as the number of boutons per NMJ that were retracted (averages: uninjured = 0.0714 [n = 140 NMJs]; injured = 4.44 [n = 237 NMJs]; P = 0.030). (E–F) Quantification of neurodegeneration frequency was measured as the average percentage of NMJs with any retractions (E) or with >1 and >3 boutons retracted (F) (averages: uninjured = 0.1429; injured = 10.33; P = 0.0018). Error bars represent SEM. Scale bar = 10 μm. Inset scale bar = 5 μm.
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f6-jen-suppl.2-2015-001: Neuronal injury induces moderate-to-severe neurodegeneration at the NMJ of muscle 6/7. (A) Wild-type uninjured NMJs show the presynaptic active zone maker Brp stained with nc82 (green, bottom panel) in apposition to the postsynaptic marker Dlg (red, middle panel) throughout the entire NMJ. (B) Neuronal injury can induce moderate neurodegeneration (<10 boutons retracted) in which the majority of the NMJ has Brp (green, bottom panel) and Dlg (red, middle panel) in perfect apposition. However, some of the boutons stained with Dlg lack the presynaptic active zone marker Brp. (C) Neuronal injury can also induce severe neurodegeneration in which >10 boutons lack Brp staining (green, bottom panel), suggesting that the neuron has retracted from the muscle as shown by the remaining postsynaptic Dlg staining without any accompanying Brp (red, middle panel). (D) Quantification of neurodegeneration severity was measured as the number of boutons per NMJ that were retracted (averages: uninjured = 0.0714 [n = 140 NMJs]; injured = 4.44 [n = 237 NMJs]; P = 0.030). (E–F) Quantification of neurodegeneration frequency was measured as the average percentage of NMJs with any retractions (E) or with >1 and >3 boutons retracted (F) (averages: uninjured = 0.1429; injured = 10.33; P = 0.0018). Error bars represent SEM. Scale bar = 10 μm. Inset scale bar = 5 μm.

Mentions: It has been previously reported in Drosophila that segmental nerve injury causes a significant loss of presynaptic vesicular glutamate transporter (VGlut) proteins at NMJ synapses.13 We wanted to examine if other presynaptic markers were lost after injury and to examine the extent of neurodegeneration using our previously established method for quantitatively investigating neurodegeneration at the Drosophila NMJ.2,17,19–23 In this assay, NMJs were stained for the presynaptic active zone marker Bruchpilot (Brp; stained with nc82 antibody) and the postsynaptic marker discs large (Dlg) at various time points after mechanical injury. Boutons clearly stained with Dlg but lacking nc82 immunoreactivity signify a neurodegenerative event. The NMJs from uninjured animals show the presynaptic marker Brp in perfect apposition with the postsynaptic marker Dlg (Fig. 6A). However, 24 hours post injury, NMJs exhibited various degrees of missing presynaptic Brp staining at the NMJ ranging from moderate neurodegeneration (<10 boutons retracted) to severe neurodegeneration (>10 boutons retracted; Fig. 6B and C). The highly compact muscle membrane folds that create the postsynaptic part of the NMJ degenerate more slowly then their presynaptic counterpart and thus persist beyond the neurodegenerative events that we are characterizing.19 The severity of neurodegeneration was quantified as the average number of boutons per NMJ exhibiting degeneration and was significantly higher in animals with neuronal injury (average = 4.44, n = 237 NMJs) compared to uninjured animals (average = 0.07, n = 140 NMJs; Fig. 6D; t-test: P = 0.030; significance remains with Wilcoxon–Mann–Whitney test: P = 0.0007). The frequency of neurodegeneration was quantified as the average percentage of NMJs per animal with degeneration and was determined to be significantly higher after neuronal injury in animals (average = 10.33, n = 25 animals) compared to in uninjured animals (average = 0.14, n = 14 animals; Fig. 6E; t-test: P = 0.0018; significance remains with Wilcoxon–Mann–Whitney test: P = 0.0016). To more fully understand the neurodegenerative frequency, we also measured the percentage of NMJs with degeneration of >1 bouton retracted (average = 11.20) and >3 boutons retracted (average = 4.49; Fig. 6F; t-test: P = 0.0286; significance remains with Wilcoxon–Mann–Whitney test: P = 0.0429). This neurodegenerative phenotype is less severe than what is seen by genetic modifications but is similar to previously published results examining the loss of presynaptic VGlut proteins at the NMJ after neuronal injury.2,13,17,19–23 Together, these data suggest a spatial and temporal sequence of cellular events originating at the site of axonal injury with immediate cytoskeletal defects inducing axonal transport dysfunction by six hours, followed by accumulations of ubiquitinated proteins by 12 hours and subsequent neurodegeneration at the NMJ by 24 hours (Supplementary Table 1).


Drosophila Neuronal Injury Follows a Temporal Sequence of Cellular Events Leading to Degeneration at the Neuromuscular Junction.

Lincoln BL, Alabsi SH, Frendo N, Freund R, Keller LC - J Exp Neurosci (2015)

Neuronal injury induces moderate-to-severe neurodegeneration at the NMJ of muscle 6/7. (A) Wild-type uninjured NMJs show the presynaptic active zone maker Brp stained with nc82 (green, bottom panel) in apposition to the postsynaptic marker Dlg (red, middle panel) throughout the entire NMJ. (B) Neuronal injury can induce moderate neurodegeneration (<10 boutons retracted) in which the majority of the NMJ has Brp (green, bottom panel) and Dlg (red, middle panel) in perfect apposition. However, some of the boutons stained with Dlg lack the presynaptic active zone marker Brp. (C) Neuronal injury can also induce severe neurodegeneration in which >10 boutons lack Brp staining (green, bottom panel), suggesting that the neuron has retracted from the muscle as shown by the remaining postsynaptic Dlg staining without any accompanying Brp (red, middle panel). (D) Quantification of neurodegeneration severity was measured as the number of boutons per NMJ that were retracted (averages: uninjured = 0.0714 [n = 140 NMJs]; injured = 4.44 [n = 237 NMJs]; P = 0.030). (E–F) Quantification of neurodegeneration frequency was measured as the average percentage of NMJs with any retractions (E) or with >1 and >3 boutons retracted (F) (averages: uninjured = 0.1429; injured = 10.33; P = 0.0018). Error bars represent SEM. Scale bar = 10 μm. Inset scale bar = 5 μm.
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Related In: Results  -  Collection

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f6-jen-suppl.2-2015-001: Neuronal injury induces moderate-to-severe neurodegeneration at the NMJ of muscle 6/7. (A) Wild-type uninjured NMJs show the presynaptic active zone maker Brp stained with nc82 (green, bottom panel) in apposition to the postsynaptic marker Dlg (red, middle panel) throughout the entire NMJ. (B) Neuronal injury can induce moderate neurodegeneration (<10 boutons retracted) in which the majority of the NMJ has Brp (green, bottom panel) and Dlg (red, middle panel) in perfect apposition. However, some of the boutons stained with Dlg lack the presynaptic active zone marker Brp. (C) Neuronal injury can also induce severe neurodegeneration in which >10 boutons lack Brp staining (green, bottom panel), suggesting that the neuron has retracted from the muscle as shown by the remaining postsynaptic Dlg staining without any accompanying Brp (red, middle panel). (D) Quantification of neurodegeneration severity was measured as the number of boutons per NMJ that were retracted (averages: uninjured = 0.0714 [n = 140 NMJs]; injured = 4.44 [n = 237 NMJs]; P = 0.030). (E–F) Quantification of neurodegeneration frequency was measured as the average percentage of NMJs with any retractions (E) or with >1 and >3 boutons retracted (F) (averages: uninjured = 0.1429; injured = 10.33; P = 0.0018). Error bars represent SEM. Scale bar = 10 μm. Inset scale bar = 5 μm.
Mentions: It has been previously reported in Drosophila that segmental nerve injury causes a significant loss of presynaptic vesicular glutamate transporter (VGlut) proteins at NMJ synapses.13 We wanted to examine if other presynaptic markers were lost after injury and to examine the extent of neurodegeneration using our previously established method for quantitatively investigating neurodegeneration at the Drosophila NMJ.2,17,19–23 In this assay, NMJs were stained for the presynaptic active zone marker Bruchpilot (Brp; stained with nc82 antibody) and the postsynaptic marker discs large (Dlg) at various time points after mechanical injury. Boutons clearly stained with Dlg but lacking nc82 immunoreactivity signify a neurodegenerative event. The NMJs from uninjured animals show the presynaptic marker Brp in perfect apposition with the postsynaptic marker Dlg (Fig. 6A). However, 24 hours post injury, NMJs exhibited various degrees of missing presynaptic Brp staining at the NMJ ranging from moderate neurodegeneration (<10 boutons retracted) to severe neurodegeneration (>10 boutons retracted; Fig. 6B and C). The highly compact muscle membrane folds that create the postsynaptic part of the NMJ degenerate more slowly then their presynaptic counterpart and thus persist beyond the neurodegenerative events that we are characterizing.19 The severity of neurodegeneration was quantified as the average number of boutons per NMJ exhibiting degeneration and was significantly higher in animals with neuronal injury (average = 4.44, n = 237 NMJs) compared to uninjured animals (average = 0.07, n = 140 NMJs; Fig. 6D; t-test: P = 0.030; significance remains with Wilcoxon–Mann–Whitney test: P = 0.0007). The frequency of neurodegeneration was quantified as the average percentage of NMJs per animal with degeneration and was determined to be significantly higher after neuronal injury in animals (average = 10.33, n = 25 animals) compared to in uninjured animals (average = 0.14, n = 14 animals; Fig. 6E; t-test: P = 0.0018; significance remains with Wilcoxon–Mann–Whitney test: P = 0.0016). To more fully understand the neurodegenerative frequency, we also measured the percentage of NMJs with degeneration of >1 bouton retracted (average = 11.20) and >3 boutons retracted (average = 4.49; Fig. 6F; t-test: P = 0.0286; significance remains with Wilcoxon–Mann–Whitney test: P = 0.0429). This neurodegenerative phenotype is less severe than what is seen by genetic modifications but is similar to previously published results examining the loss of presynaptic VGlut proteins at the NMJ after neuronal injury.2,13,17,19–23 Together, these data suggest a spatial and temporal sequence of cellular events originating at the site of axonal injury with immediate cytoskeletal defects inducing axonal transport dysfunction by six hours, followed by accumulations of ubiquitinated proteins by 12 hours and subsequent neurodegeneration at the NMJ by 24 hours (Supplementary Table 1).

Bottom Line: At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components.Our data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism.Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Quinnipiac University, Hamden, CT, USA.

ABSTRACT
Neurodegenerative diseases affect millions of people worldwide, and as the global population ages, there is a critical need to improve our understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. Here, we use an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila melanogaster. We find that sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. Our data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies have implications for our understanding of the cellular and molecular events that occur in humans and will facilitate the identification of biomedically relevant targets for future treatments.

No MeSH data available.


Related in: MedlinePlus