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Research article
Oxysterols Present in Alzheimer’s Disease Braininduce Synaptoxicity by Activating Astrocytes: A Major Role for Lipocalin-2
Erica Staurenghi
Department of Clinical and Biological Sciences, University of Turin, Turin, Italy
Corresponding Author
ORCiD: https://orcid.org/0000-0001-5035-7401
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Among Alzheimer’s disease (AD) brain hallmarks, the presence of reactive astrocytes was demonstrated to correlate with neuronal loss and cognitive deficits. Evidence indeed supports the role of reactive astrocytes as mediators of changes in neurons, including synapses. However, the complexity and the outcomes of astrocyte reactivity are far from being completely elucidated.
Another key role in AD pathogenesis is played by alterations in brain cholesterol metabolism. Oxysterols (cholesterol oxidation products) are crucial for brain cholesterol homeostasis, and we previously demonstrated that changes in the brain levels of various oxysterols correlate with AD progression. Moreover, oxysterols have been shown to contribute to various pathological mechanisms involved in AD pathogenesis.
In order to deepen the role of oxysterols in AD, we investigated whether they could contribute to astrocyte reactivity, and consequently impact on neuronal health.
Methods: Mouse primary astrocyte cultures were used to test the effect of two oxysterol mixtures, that represent the oxysterol composition respectively of mild or severe AD brains, on astrocyte morphology, markers of reactivity, and secretion profile. Co-culture experiments were performed to investigate the impact of oxysterol-treated astrocytes on neurons. Neuronal cultures were exposed to astrocyte conditioned media (ACM) deprived of lipocalin-2 (Lcn2) to investigate the contribution of this mediator to synaptotoxicity.
Results: Results showed that oxysterols induce a clear morphological change in astrocytes, accompanied by the upregulation of some reactive astrocyte markers, including Lcn2. Moreover, ACM analysis revealed a significant increase in the release of Lcn2, cytokines, and chemokines in response to oxysterols. A significant reduction of postsynaptic density protein 95 (PSD95) and a concurrent increase in cleaved caspase-3 protein levels have been demonstrated in neurons co-cultured with oxysterol-treated astrocytes, pointing out that mediators released by astrocytes have an impact on neurons. Among these mediators, Lcn2 has been demonstrated to play a major role on synapses, affecting neurite morphology and decreasing dendritic spine density.
Conclusions: These data demonstrated that oxysterols present in the AD brain promote astrocyte reactivity, determining the release of several mediators that affect neuronal health and synapses. Lcn2 has been shown to exert a key role in mediating the synaptotoxic effect of oxysterol-treated astrocytes.
Keywords
Oxysterols, Astrocytes, Astrocyte reactivity, Lipocalin-2, Neurons, Synapses, Synaptotoxicity, Alzheimer’s disease
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- SupplementalFig4.tif
Supplemental Fig. 4 Oxysterol mixtures do not directly cause neuron death. Neuronal viability was assessed in neurons treated with the Early or Late AD oxysterol mixture (10 M) for 24h. The bar graph shows the proportion of lactate dehydrogenase (LDH) released into medium relative to total LDH in lysed cells, normalized to values for control media. Data are expressed as mean values ± SD from three different experiments (n=12, one-way ANOVA).
- SupplementalFig3.tif
Oxysterol analyses in astrocyte culture media. Astrocyte cultures were treated for 1, 3, 12 or 24h with the Early or Late AD oxysterol mixtures (10 M). Astrocyte conditioned media was collected and the amounts of the seven oxysterols present in the mixtures were determined by gas chromatography-mass spectrometry (GC-MS). The graph shows the amounts of oxysterol in media expressed as percentage of the original oxysterol concentrations present in each mixture. Data shown are averages of the measurements obtained from treatments with Early and Late AD mixtures. Data are expressed as mean values ± SD from two different experiments (n=6).
- SupplementalFig2.tif
Validation of lipocalin-2 (Lcn2) silencing efficiency. Astrocyte cultures were transfected for 6h with Lcn2 or scrambled siRNA, then the medium was changed and astrocytes were incubated with fresh medium for 36h. Transient Lcn2 gene knockdown was evaluated by real-time RT-PCR. Data were normalized to the corresponding -actin levels. Data are expressed as mean values ± SD of three different experiments (n=9, one-way ANOVA). ****P<0.0001 vs control.
- SupplementalFig1.tif
Evaluation of astrocyte culture purity. Astrocytic cultures were analysed by (A) immunocytochemistry and (B) Western blotting using antibodies against glial fibrillary acidic protein (GFAP, astrocytic marker) and ionized calcium binding adaptor molecule 1 (Iba1, microglial marker). Cells were imaged using an LSM800 confocal microscope (Zeiss, 40X objective; scale bar: 100 m).
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Research article
Oxysterols Present in Alzheimer’s Disease Braininduce Synaptoxicity by Activating Astrocytes: A Major Role for Lipocalin-2
Erica Staurenghi
Department of Clinical and Biological Sciences, University of Turin, Turin, Italy
Corresponding Author
ORCiD: https://orcid.org/0000-0001-5035-7401
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 14 Aug, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Neurology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Among Alzheimer’s disease (AD) brain hallmarks, the presence of reactive astrocytes was demonstrated to correlate with neuronal loss and cognitive deficits. Evidence indeed supports the role of reactive astrocytes as mediators of changes in neurons, including synapses. However, the complexity and the outcomes of astrocyte reactivity are far from being completely elucidated.
Another key role in AD pathogenesis is played by alterations in brain cholesterol metabolism. Oxysterols (cholesterol oxidation products) are crucial for brain cholesterol homeostasis, and we previously demonstrated that changes in the brain levels of various oxysterols correlate with AD progression. Moreover, oxysterols have been shown to contribute to various pathological mechanisms involved in AD pathogenesis.
In order to deepen the role of oxysterols in AD, we investigated whether they could contribute to astrocyte reactivity, and consequently impact on neuronal health.
Methods: Mouse primary astrocyte cultures were used to test the effect of two oxysterol mixtures, that represent the oxysterol composition respectively of mild or severe AD brains, on astrocyte morphology, markers of reactivity, and secretion profile. Co-culture experiments were performed to investigate the impact of oxysterol-treated astrocytes on neurons. Neuronal cultures were exposed to astrocyte conditioned media (ACM) deprived of lipocalin-2 (Lcn2) to investigate the contribution of this mediator to synaptotoxicity.
Results: Results showed that oxysterols induce a clear morphological change in astrocytes, accompanied by the upregulation of some reactive astrocyte markers, including Lcn2. Moreover, ACM analysis revealed a significant increase in the release of Lcn2, cytokines, and chemokines in response to oxysterols. A significant reduction of postsynaptic density protein 95 (PSD95) and a concurrent increase in cleaved caspase-3 protein levels have been demonstrated in neurons co-cultured with oxysterol-treated astrocytes, pointing out that mediators released by astrocytes have an impact on neurons. Among these mediators, Lcn2 has been demonstrated to play a major role on synapses, affecting neurite morphology and decreasing dendritic spine density.
Conclusions: These data demonstrated that oxysterols present in the AD brain promote astrocyte reactivity, determining the release of several mediators that affect neuronal health and synapses. Lcn2 has been shown to exert a key role in mediating the synaptotoxic effect of oxysterol-treated astrocytes.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.