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Abeta42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila.

Ling D, Song HJ, Garza D, Neufeld TP, Salvaterra PM - PLoS ONE (2009)

Bottom Line: Abeta(1-42)-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition.Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42) expression.Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42) and Abeta(1-40), and reveal an Abeta(1-42)-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuroscience, Beckman Research Institute of the City of Hope, Duarte, California, United States of America.

ABSTRACT
The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Abeta(1-42)) is believed to play a causative role in the development of AD. Here we expressed human Abeta(1-42) and amyloid beta 40 (Abeta(1-40)) in Drosophila neurons. Abeta(1-42) but not Abeta(1-40) causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Abeta(1-42)-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42) expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42) and Abeta(1-40), and reveal an Abeta(1-42)-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

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Aβ1–40 and Aβ1–42 have differential neurotoxicity.(A–B) Aβ1–42 but not Aβ1–40 expression decreases fly lifespan (A) and climbing ability (B) (lifespan assay, N = 953, 633 and 965 for three parallel cohorts of control, Aβ1–40 and Aβ1–42 flies respectively; data are the mean±SEM; climbing assay, N = 160 for all three cohorts). Note that survival rates correlate well with climbing ability in control and Aβ1–40 flies. However, 88% of Aβ1–42 flies at 16 days survive with only 5% maintaining active climbing ability. Aβ1–42 flies thus have accelerated neurological deficits that precede animal death. (C) Levels of Aβ transcripts in fly heads are significantly higher for Aβ1–40 relative to Aβ1–42 (data are the mean+SEM, N = 3 for each group, two-tailed P value by student's t test). (D–E) Cytosolic GFP fluorescence exhibits an even distribution in Aβ1–40 flies (16-day-old adult, D) in contrast to an extensive accumulation of punctate structures in an age- and region-matched Aβ1–42 sample (E). GFP fluorescence in the Aβ1–42 sample is decreased in cytosol (arrowheads) but especially bright in puncta (arrows). Some neuronal somas appear abnormally large (stars). Cellular boundaries also appear to be indistinct (arrowheads). Note that cytosolic GFP expression is independent of the expression of Aβ1–40 or Aβ1–42 thus the fluorescent puncta are not likely to be the structure of Aβ1–42 aggregation. (F) An age-dependent increase of fluorescent puncta in Aβ1–42-targeted neurons (data are mean+SEM, two-tailed P values by student's t test, n = 9 for each group). Scale bars = 5 µm.
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pone-0004201-g001: Aβ1–40 and Aβ1–42 have differential neurotoxicity.(A–B) Aβ1–42 but not Aβ1–40 expression decreases fly lifespan (A) and climbing ability (B) (lifespan assay, N = 953, 633 and 965 for three parallel cohorts of control, Aβ1–40 and Aβ1–42 flies respectively; data are the mean±SEM; climbing assay, N = 160 for all three cohorts). Note that survival rates correlate well with climbing ability in control and Aβ1–40 flies. However, 88% of Aβ1–42 flies at 16 days survive with only 5% maintaining active climbing ability. Aβ1–42 flies thus have accelerated neurological deficits that precede animal death. (C) Levels of Aβ transcripts in fly heads are significantly higher for Aβ1–40 relative to Aβ1–42 (data are the mean+SEM, N = 3 for each group, two-tailed P value by student's t test). (D–E) Cytosolic GFP fluorescence exhibits an even distribution in Aβ1–40 flies (16-day-old adult, D) in contrast to an extensive accumulation of punctate structures in an age- and region-matched Aβ1–42 sample (E). GFP fluorescence in the Aβ1–42 sample is decreased in cytosol (arrowheads) but especially bright in puncta (arrows). Some neuronal somas appear abnormally large (stars). Cellular boundaries also appear to be indistinct (arrowheads). Note that cytosolic GFP expression is independent of the expression of Aβ1–40 or Aβ1–42 thus the fluorescent puncta are not likely to be the structure of Aβ1–42 aggregation. (F) An age-dependent increase of fluorescent puncta in Aβ1–42-targeted neurons (data are mean+SEM, two-tailed P values by student's t test, n = 9 for each group). Scale bars = 5 µm.

Mentions: Human Aβ1–40 or Aβ1–42 transgene is expressed in subtypes of Drosophila neurons where soluble GFP is also expressed as a cytosolic reporter that is independent of Aβ expression. GFP labels somas and neuropil of targeted neurons; while Aβ1–42 immunostaining is primarily limited to neuronal somas (Fig. S1). When expression is limited to cholinergic neurons, Aβ1–42 results in a 38.1% of decrease in mean lifespan relative to control (log-rank P<0.0001, Fig. 1A) suggesting a significant Aβ1–42 neurotoxicity. In contrast, Aβ1–40 expression does not shorten fly lifespan. Locomotor activity of Aβ1–42 flies shows an accelerated decrease compared with Aβ1–40 or control flies (Fig. 1B). Similar results were obtained for Aβ1–40 or Aβ1–42 expression limited to GABAergic (and glutamate motor) neurons (not shown). Relative expression levels of Aβ transgenes measured by reverse transcription quantitative PCR (RT-qPCR) show that Aβ1–40 expression is significantly higher than Aβ1–42 (Fig. 1C), thus ruling out the possibility that Aβ1–42-specific neurotoxicity is associated with a higher level of the transgene expression. Cytosolic GFP fluorescence in control (not shown) or Aβ1–40 flies (Fig. 1D) shows relatively homogeneous distribution in neurons. In contrast, region and age-matched Aβ1–42 samples exhibit numerous punctate structures with high GFP fluorescence (fluorescent puncta) relative to the surrounding cytosol with lower GFP fluorescence (Fig. 1E). These puncta show a significantly age-dependent increase (Fig. 1F) that has a negative correlation with animal climbing ability.


Abeta42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila.

Ling D, Song HJ, Garza D, Neufeld TP, Salvaterra PM - PLoS ONE (2009)

Aβ1–40 and Aβ1–42 have differential neurotoxicity.(A–B) Aβ1–42 but not Aβ1–40 expression decreases fly lifespan (A) and climbing ability (B) (lifespan assay, N = 953, 633 and 965 for three parallel cohorts of control, Aβ1–40 and Aβ1–42 flies respectively; data are the mean±SEM; climbing assay, N = 160 for all three cohorts). Note that survival rates correlate well with climbing ability in control and Aβ1–40 flies. However, 88% of Aβ1–42 flies at 16 days survive with only 5% maintaining active climbing ability. Aβ1–42 flies thus have accelerated neurological deficits that precede animal death. (C) Levels of Aβ transcripts in fly heads are significantly higher for Aβ1–40 relative to Aβ1–42 (data are the mean+SEM, N = 3 for each group, two-tailed P value by student's t test). (D–E) Cytosolic GFP fluorescence exhibits an even distribution in Aβ1–40 flies (16-day-old adult, D) in contrast to an extensive accumulation of punctate structures in an age- and region-matched Aβ1–42 sample (E). GFP fluorescence in the Aβ1–42 sample is decreased in cytosol (arrowheads) but especially bright in puncta (arrows). Some neuronal somas appear abnormally large (stars). Cellular boundaries also appear to be indistinct (arrowheads). Note that cytosolic GFP expression is independent of the expression of Aβ1–40 or Aβ1–42 thus the fluorescent puncta are not likely to be the structure of Aβ1–42 aggregation. (F) An age-dependent increase of fluorescent puncta in Aβ1–42-targeted neurons (data are mean+SEM, two-tailed P values by student's t test, n = 9 for each group). Scale bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2626277&req=5

pone-0004201-g001: Aβ1–40 and Aβ1–42 have differential neurotoxicity.(A–B) Aβ1–42 but not Aβ1–40 expression decreases fly lifespan (A) and climbing ability (B) (lifespan assay, N = 953, 633 and 965 for three parallel cohorts of control, Aβ1–40 and Aβ1–42 flies respectively; data are the mean±SEM; climbing assay, N = 160 for all three cohorts). Note that survival rates correlate well with climbing ability in control and Aβ1–40 flies. However, 88% of Aβ1–42 flies at 16 days survive with only 5% maintaining active climbing ability. Aβ1–42 flies thus have accelerated neurological deficits that precede animal death. (C) Levels of Aβ transcripts in fly heads are significantly higher for Aβ1–40 relative to Aβ1–42 (data are the mean+SEM, N = 3 for each group, two-tailed P value by student's t test). (D–E) Cytosolic GFP fluorescence exhibits an even distribution in Aβ1–40 flies (16-day-old adult, D) in contrast to an extensive accumulation of punctate structures in an age- and region-matched Aβ1–42 sample (E). GFP fluorescence in the Aβ1–42 sample is decreased in cytosol (arrowheads) but especially bright in puncta (arrows). Some neuronal somas appear abnormally large (stars). Cellular boundaries also appear to be indistinct (arrowheads). Note that cytosolic GFP expression is independent of the expression of Aβ1–40 or Aβ1–42 thus the fluorescent puncta are not likely to be the structure of Aβ1–42 aggregation. (F) An age-dependent increase of fluorescent puncta in Aβ1–42-targeted neurons (data are mean+SEM, two-tailed P values by student's t test, n = 9 for each group). Scale bars = 5 µm.
Mentions: Human Aβ1–40 or Aβ1–42 transgene is expressed in subtypes of Drosophila neurons where soluble GFP is also expressed as a cytosolic reporter that is independent of Aβ expression. GFP labels somas and neuropil of targeted neurons; while Aβ1–42 immunostaining is primarily limited to neuronal somas (Fig. S1). When expression is limited to cholinergic neurons, Aβ1–42 results in a 38.1% of decrease in mean lifespan relative to control (log-rank P<0.0001, Fig. 1A) suggesting a significant Aβ1–42 neurotoxicity. In contrast, Aβ1–40 expression does not shorten fly lifespan. Locomotor activity of Aβ1–42 flies shows an accelerated decrease compared with Aβ1–40 or control flies (Fig. 1B). Similar results were obtained for Aβ1–40 or Aβ1–42 expression limited to GABAergic (and glutamate motor) neurons (not shown). Relative expression levels of Aβ transgenes measured by reverse transcription quantitative PCR (RT-qPCR) show that Aβ1–40 expression is significantly higher than Aβ1–42 (Fig. 1C), thus ruling out the possibility that Aβ1–42-specific neurotoxicity is associated with a higher level of the transgene expression. Cytosolic GFP fluorescence in control (not shown) or Aβ1–40 flies (Fig. 1D) shows relatively homogeneous distribution in neurons. In contrast, region and age-matched Aβ1–42 samples exhibit numerous punctate structures with high GFP fluorescence (fluorescent puncta) relative to the surrounding cytosol with lower GFP fluorescence (Fig. 1E). These puncta show a significantly age-dependent increase (Fig. 1F) that has a negative correlation with animal climbing ability.

Bottom Line: Abeta(1-42)-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition.Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42) expression.Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42) and Abeta(1-40), and reveal an Abeta(1-42)-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuroscience, Beckman Research Institute of the City of Hope, Duarte, California, United States of America.

ABSTRACT
The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Abeta(1-42)) is believed to play a causative role in the development of AD. Here we expressed human Abeta(1-42) and amyloid beta 40 (Abeta(1-40)) in Drosophila neurons. Abeta(1-42) but not Abeta(1-40) causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Abeta(1-42)-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42) expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42) and Abeta(1-40), and reveal an Abeta(1-42)-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

Show MeSH
Related in: MedlinePlus