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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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Confocal imaging and three-dimensional model reconstruction of neuron transfected with GFP-LC3.(A–J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A–D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A–D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K–O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M–O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N–O).
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pone-0002906-g001: Confocal imaging and three-dimensional model reconstruction of neuron transfected with GFP-LC3.(A–J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A–D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A–D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K–O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M–O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N–O).

Mentions: Although there are indications that autophagy is active in developing primary neuronal cultures [3], [29], techniques for detecting autophagy in neurons have to date been qualitative. As a first objective we established a method to quantitatively determine the number of AV in primary neuronal cultures, both in the soma and processes. Primary neuronal cultures were transfected with GFP-LC3 to allow visualization of AV. Conditions of low neuronal transfection efficiency (<2%) were used in order to observe the soma and processes of single GFP-LC3 expressing neurons within the matrix of other neurons. To quantify the spatial and temporal distribution of AV we acquired confocal z-stacks for transfected neurons (Figures 1A–1J, 0.2 µm step size, approximately 150 images acquired per cell). From these data sets we were able to determine the 3D structure of the soma and processes, as well as the number and distribution of GFP-LC3-labelled AV (Figures 1K–1O). This imaging approach allowed for the quantitative investigation of AV content and localization in different conditions in order to understand their potential changes in our in vitro model of SIV microglia supernatant inducing neuronal cell death.


Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

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

Confocal imaging and three-dimensional model reconstruction of neuron transfected with GFP-LC3.(A–J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A–D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A–D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K–O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M–O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N–O).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483417&req=5

pone-0002906-g001: Confocal imaging and three-dimensional model reconstruction of neuron transfected with GFP-LC3.(A–J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A–D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A–D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K–O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M–O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N–O).
Mentions: Although there are indications that autophagy is active in developing primary neuronal cultures [3], [29], techniques for detecting autophagy in neurons have to date been qualitative. As a first objective we established a method to quantitatively determine the number of AV in primary neuronal cultures, both in the soma and processes. Primary neuronal cultures were transfected with GFP-LC3 to allow visualization of AV. Conditions of low neuronal transfection efficiency (<2%) were used in order to observe the soma and processes of single GFP-LC3 expressing neurons within the matrix of other neurons. To quantify the spatial and temporal distribution of AV we acquired confocal z-stacks for transfected neurons (Figures 1A–1J, 0.2 µm step size, approximately 150 images acquired per cell). From these data sets we were able to determine the 3D structure of the soma and processes, as well as the number and distribution of GFP-LC3-labelled AV (Figures 1K–1O). This imaging approach allowed for the quantitative investigation of AV content and localization in different conditions in order to understand their potential changes in our in vitro model of SIV microglia supernatant inducing neuronal cell death.

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