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Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

Alirezaei M, Kiosses WB, Flynn CT, Brady NR, Fox HS - PLoS ONE (2008)

Bottom Line: We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival.Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons.Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

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Western blot analysis of autophagy related proteins.(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.
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pone-0002906-g004: Western blot analysis of autophagy related proteins.(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.

Mentions: We further investigated the observed impairment of neuronal autophagy by SIV-infected microglia supernatant by Western blot detection of different parameters of autophagic activity. The conversion of LC-I to LC3-II is correlated with the number of AV, and can be assessed by immunoblotting [10], [32], [33]. A second approach to examine potential impairment of autophagy is measuring the protein level of the conjugate Atg12-Atg5 that drives the elongation step of autophagy [8]. A third alternative technique for identifying the inhibition of autophagy is measuring p62 protein upregulation [16], [34]. Its level therefore can be used as an index of autophagic degradative capacity. We used all three of these methods to examine the potential impairment of autophagy induced by the SIV-infected microglia supernatant. Similar to three dimensional imaging results (Figures 2 and 3), Western blot detection of LC3 revealed a time-dependent decrease in LC3-II levels, indicating that neuronal AV numbers were significantly decreased in the presence of the SIV-infected microglia supernatant in a time-dependent manner (Figure 4A). Detection of the Atg12-Atg5 conjugate, revealed a time-dependent decrease in conjugate formation (Figure 4A), evidencing inhibition to autophagy. Examination of p62 protein levels revealed a significant increase after 24 hr exposure to the microglia supernatant (Figure 4A). Importantly, rapamycin increased both LC3-II and Atg5-Atg12 conjugate levels, in both control cells and cells exposed to SIV-infected microglia supernatant (Figure 4A), consistent with impairment of autophagy. Moreover, rapamycin decreased p62 levels in neurons treated with SIV-infected microglia supernatant to control levels. The optical density of bands was measured using imageJ software and the associated graphs were presented (Figures 4B–4D). Control supernatant from uninfected monkeys did not affect the LC3-II level for 3 and 24 hr exposure time (data not shown).


Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

Alirezaei M, Kiosses WB, Flynn CT, Brady NR, Fox HS - PLoS ONE (2008)

Western blot analysis of autophagy related proteins.(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483417&req=5

pone-0002906-g004: Western blot analysis of autophagy related proteins.(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.
Mentions: We further investigated the observed impairment of neuronal autophagy by SIV-infected microglia supernatant by Western blot detection of different parameters of autophagic activity. The conversion of LC-I to LC3-II is correlated with the number of AV, and can be assessed by immunoblotting [10], [32], [33]. A second approach to examine potential impairment of autophagy is measuring the protein level of the conjugate Atg12-Atg5 that drives the elongation step of autophagy [8]. A third alternative technique for identifying the inhibition of autophagy is measuring p62 protein upregulation [16], [34]. Its level therefore can be used as an index of autophagic degradative capacity. We used all three of these methods to examine the potential impairment of autophagy induced by the SIV-infected microglia supernatant. Similar to three dimensional imaging results (Figures 2 and 3), Western blot detection of LC3 revealed a time-dependent decrease in LC3-II levels, indicating that neuronal AV numbers were significantly decreased in the presence of the SIV-infected microglia supernatant in a time-dependent manner (Figure 4A). Detection of the Atg12-Atg5 conjugate, revealed a time-dependent decrease in conjugate formation (Figure 4A), evidencing inhibition to autophagy. Examination of p62 protein levels revealed a significant increase after 24 hr exposure to the microglia supernatant (Figure 4A). Importantly, rapamycin increased both LC3-II and Atg5-Atg12 conjugate levels, in both control cells and cells exposed to SIV-infected microglia supernatant (Figure 4A), consistent with impairment of autophagy. Moreover, rapamycin decreased p62 levels in neurons treated with SIV-infected microglia supernatant to control levels. The optical density of bands was measured using imageJ software and the associated graphs were presented (Figures 4B–4D). Control supernatant from uninfected monkeys did not affect the LC3-II level for 3 and 24 hr exposure time (data not shown).

Bottom Line: We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival.Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons.Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

Show MeSH
Related in: MedlinePlus