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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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In vitro analysis of the impact of SIV-infected microglia supernatant on autophagy in primary neurons.(A) Model of experimental design, as detailed in the Materials and Methods. (B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments. (C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.
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pone-0002906-g002: In vitro analysis of the impact of SIV-infected microglia supernatant on autophagy in primary neurons.(A) Model of experimental design, as detailed in the Materials and Methods. (B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments. (C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.

Mentions: Subsequently, to examine the role of autophagy in SIV mediated neuronal cell death, we treated GFP-LC3-transfected primary rat neurons, as described above, with supernatants from microglia/macrophages of SIV-infected or uninfected monkeys and determined the total number of AV. Separate monkey microglia cultures were derived from cultures from infected and uninfected animals, and the culture supernatants then subjected to ultrafiltration to isolate molecules less than 30 kDa for use as microglia-derived factors to treat primary neurons (Figure 2A). Under control conditions, the mean number of AV in primary control neurons was 119.3±7.4 (Figure 2B), likely representing the constitutive autophagy activity necessary for neuronal survival. In many cell types autophagy is induced as a starvation response, activated to provide sources of energy. This induction occurs through inhibition of the mammalian target of rapamycin (mTOR), which negatively regulates the autophagy pathway in cells, and can be mimicked by treatment with the mTOR inhibitor rapamycin [30]. However, perhaps given the priority of the brain in obtaining nutrients, autophagy is not strongly induced in CNS neurons as part of the starvation response [31]. We found that neurons treated for 24 hr with rapamycin exhibited a slight but significant increase in AV number (Figures 2B and 2C, 139.2±6.8), indicating that autophagy can be pharmacologically activated in our experimental model.


Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

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

In vitro analysis of the impact of SIV-infected microglia supernatant on autophagy in primary neurons.(A) Model of experimental design, as detailed in the Materials and Methods. (B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments. (C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483417&req=5

pone-0002906-g002: In vitro analysis of the impact of SIV-infected microglia supernatant on autophagy in primary neurons.(A) Model of experimental design, as detailed in the Materials and Methods. (B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments. (C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.
Mentions: Subsequently, to examine the role of autophagy in SIV mediated neuronal cell death, we treated GFP-LC3-transfected primary rat neurons, as described above, with supernatants from microglia/macrophages of SIV-infected or uninfected monkeys and determined the total number of AV. Separate monkey microglia cultures were derived from cultures from infected and uninfected animals, and the culture supernatants then subjected to ultrafiltration to isolate molecules less than 30 kDa for use as microglia-derived factors to treat primary neurons (Figure 2A). Under control conditions, the mean number of AV in primary control neurons was 119.3±7.4 (Figure 2B), likely representing the constitutive autophagy activity necessary for neuronal survival. In many cell types autophagy is induced as a starvation response, activated to provide sources of energy. This induction occurs through inhibition of the mammalian target of rapamycin (mTOR), which negatively regulates the autophagy pathway in cells, and can be mimicked by treatment with the mTOR inhibitor rapamycin [30]. However, perhaps given the priority of the brain in obtaining nutrients, autophagy is not strongly induced in CNS neurons as part of the starvation response [31]. We found that neurons treated for 24 hr with rapamycin exhibited a slight but significant increase in AV number (Figures 2B and 2C, 139.2±6.8), indicating that autophagy can be pharmacologically activated in our experimental model.

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