Oxysterols Present in Alzheimer’s Disease Braininduce Synaptoxicity by Activating Astrocytes: A Major Role for Lipocalin-2

Abstract

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.

Impaired astrocytic synaptic function by peripheral cholesterol metabolite 27-hydroxycholesterol

“…We propose that the downregulation of GLT-1 and GLAST due to high levels of 27-OH plays an important role in this aberrant phenotype. The impact of 27-OH on astrocytes, including their activation and induction of sterile inflammation, aligns with the stress signals identified for both neurons and astrocytes by our group Loera-Valencia et al, 2021a) and others (Staurenghi et al, 2021).…”

“…Similarly, RxRγ induces the transcription factor REST in 2D primary cultures treated with 27-OH (Merino-Serrais et al, 2019), but mRNA levels were like controls in our 3D co-cultures, and the dendrite growth of its neurons was indeed compromised, which suggests that early 27-OH induced alterations in RxRγ signaling are sufficient to induce neuronal and astrocytic effects observed in this study. Our results also suggest a potential link between astrocytic function and the cognitive deficits observed in AD since the impact of 27-OH on astrocytes, including their activation and induction of sterile inflammation, aligns with the stress signals identified for both neurons and astrocytes Loera-Valencia et al, 2021a;Staurenghi et al, 2021). These findings imply a broader role for oxysterols such as 27-OH in disrupting astrocyte synaptic function, with implications for cognitive function and neurodegeneration.…”

Up-regulation of LCN2 in the anterior cingulate cortex contributes to neural injury-induced chronic pain

“…Previous studies have reported that LCN2 protein is secreted by neurons (Jha et al, 2015;Nagase and Tohda, 2021) and glial cells (Bi et al, 2013;Jang et al, 2013;Staurenghi et al, 2021;Weng et al, 2021). To determine the cellular distribution of LCN2 protein in the ACC of mice, we detected LCN2 by immunofluorescence staining and found that it mainly co-localized with neurons, and to a small extent with microglia and astrocytes in the ACC of naïve mice (Figures 5A, B).…”

“…Lipocalin-2 protein is secreted from neurons (Jha et al, 2015;Nagase and Tohda, 2021) as well as glial cells (Bi et al, 2013;Jang et al, 2013;Staurenghi et al, 2021;Weng et al, 2021). In order to explore whether up-regulation of LCN2 in the ACC is involved in the generation of chronic pain, we next constructed generated a broad-spectrum virus expressing short hairpin RNAs (shRNAs) to knock down LCN2 expression.…”

“…Lipocalin-2 (LCN2), a 25-kDa secreted protein, also called 24p3 or neutrophil gelatinase associated lipid transporter (NGAL) (Kjeldsen et al, 1993;Flo et al, 2004), has been shown to play a crucial role in immune system diseases (Xu et al, 2015;Chen et al, 2020), metabolic diseases (Elkhidir et al, 2017;Moschen et al, 2017), central nervous system diseases (Ferreira et al, 2015;Jha et al, 2015;Suk, 2016), kidney damage (Viau et al, 2010;Ashraf et al, 2016), iron homeostasis (Xiao et al, 2016;Roemhild et al, 2021), and other diseases (Makhezer et al, 2019;Staurenghi et al, 2021). LCN2 is synthesized and secreted in response to inflammation, injury, or infection (Jha et al, 2015), and has been implicated in the regulation of a variety of biological/behavioral responses via its cell surface receptor (24p3R), including cognition and emotion by regulating neural plasticity (Ferreira et al, 2013;Jha et al, 2015).…”