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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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Neuronal survival after exposure to SIV-infected microglia supernatant in the presence of different autophagy related drugs.(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments. (B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.
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pone-0002906-g006: Neuronal survival after exposure to SIV-infected microglia supernatant in the presence of different autophagy related drugs.(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments. (B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.

Mentions: Both quantitative confocal microscopy and Western Blot analysis indicated that SIV-infected microglia supernatant significantly inhibited neuronal autophagy activity and this affect was reversed by rapamycin. We then sought to determine whether these changes in autophagy were involved in neuronal survival. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we found that neuronal survival decreased significantly after 3 or 24 hr in the presence of the SIV-infected microglia supernatant (Figure 6A, 91.1±1.1% and 68.9±1.1% of 100% baseline, respectively). Rapamycin pretreatment conferred significant protection of the neurons against the neurotoxic effect of SIV-infected microglia supernatant after 24 hr (Figure 6A, 86.2±1.9%), although not recovering completely to the baseline level. Furthermore, pre-treatment with (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), a non-competitive antagonist of the NMDA receptor, also abrogated neuronal cell death, confirming the importance of the excitotoxic glutamate pathways (Figure 6A). Data obtained from the lactate dehydrogenase (LDH) release assay confirmed the neurotoxicity results of the MTT assay (data not shown). These data strongly indicate that impaired autophagy contributes to the neuronal damage, and enhancing autophagy with rapamycin can inhibit a significant portion of neurotoxic effect of SIV-infected microglia supernatant.


Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

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

Neuronal survival after exposure to SIV-infected microglia supernatant in the presence of different autophagy related drugs.(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments. (B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483417&req=5

pone-0002906-g006: Neuronal survival after exposure to SIV-infected microglia supernatant in the presence of different autophagy related drugs.(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments. (B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.
Mentions: Both quantitative confocal microscopy and Western Blot analysis indicated that SIV-infected microglia supernatant significantly inhibited neuronal autophagy activity and this affect was reversed by rapamycin. We then sought to determine whether these changes in autophagy were involved in neuronal survival. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we found that neuronal survival decreased significantly after 3 or 24 hr in the presence of the SIV-infected microglia supernatant (Figure 6A, 91.1±1.1% and 68.9±1.1% of 100% baseline, respectively). Rapamycin pretreatment conferred significant protection of the neurons against the neurotoxic effect of SIV-infected microglia supernatant after 24 hr (Figure 6A, 86.2±1.9%), although not recovering completely to the baseline level. Furthermore, pre-treatment with (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), a non-competitive antagonist of the NMDA receptor, also abrogated neuronal cell death, confirming the importance of the excitotoxic glutamate pathways (Figure 6A). Data obtained from the lactate dehydrogenase (LDH) release assay confirmed the neurotoxicity results of the MTT assay (data not shown). These data strongly indicate that impaired autophagy contributes to the neuronal damage, and enhancing autophagy with rapamycin can inhibit a significant portion of neurotoxic effect of SIV-infected microglia supernatant.

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