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Astrocytes export cholesterol to oligodendrocytes to regulate their survival and remyelination a Primary astrocytes treatment plan and collection of astrocyte-conditioned media (ACM). b Nrf2+ (yellow) astrocytes (Aldh1l1+; magenta), with Hoechst in blue, following treatment with CDDOTFEA or CDDOTFEA, then Luteolin, or CDDOTFEA then CS-6253. Scale bar, 50 µm. c Mean percentage of astrocytes expressing nuclear Nrf2 ± s.e.m. One-way ANOVA and Tukey’s multiple comparisons test; aP = 0.0286, bP = 0.0085, cP = 0.0178. ANOVA summary P = 0.0023, F = 6.994. n = 6 independent litters (no treatment, CDDO, CDDO/Luteolin, CDDO/CS-6253). d Astrocyte expression of genes involved in cholesterol and Nrf2 signalling following CDDOTFEA treatment, represented as Log2FC over no treatment condition ± s.e.m. One-tailed Wilcoxon test for 2−ΔΔCt, P value = 0.0156. n = 4 independent litters (Nfe2l2, Gclc, Nqo1, Hmgcs1, Fdps) and n = 3 independent litters (Mvk, Fdft1). e Astrocyte expression of genes after CDDOTFEA treatment followed by Luteolin treatment, represented as Log2FC over CDDOTFEA condition ± s.e.m. Kolmogorov–Smirnov tests on 2−ΔΔCt, Hmgcs1 P = 0.0286. n = 4 independent litters (Hmgcs1, Fdps) and n = 3 independent litters (Mvk, Fdft1). f Astrocyte expression of genes after CDDOTFEA treatment followed by CS-6253 treatment, represented as Log2FC over CDDOTFEA condition ± s.e.m. Kolmogorov–Smirnov tests on 2−ΔΔCt, Hmgcs1 P = 0.0476, Fdps P = 0.0476, Mvk P = 0.0286. n = 4 independent litters (Hmgcs1, Fdps) and n = 3 independent litters (Mvk, Fdft1). g ACM applied to oligodendrocytes to track the uptake of Bodipy-FL-C12 exported from astrocytes. h Percentage of TPPP/p25+ cells cholesterol which are Bodipy+ ± s.e.m. Kruskal–Wallis test and Dunn’s multiple comparisons test, aP = 0.0561, bP = 0.0360. n = 4 independent litters. i Oligodendrocytes (TPPP/p25+ Sox10+; magenta/cyan) in unconditioned astrocyte media (AST) or following exposure to ACM, and uptake of Bodipy-FL-C12 (yellow). Hoechst in blue. Scale bar, 50 µm. j Immature oligodendrocyte lineage cells (Sox10+ TPPP/p25-) were Bodipy negative (arrows). Scale bar, 50 µm. k Mean percentage of TPPP/p25+ cells which are Tunel+ ± s.e.m., normalised to oligodendrocyte (OL) media control. Kruskal–Wallis test and Dunn’s multiple comparisons test, P value = 0.0349, aP = 0.0083, bP = 0.0265, cP = 0.0265. n = 4 independent litters. l Apoptotic (Tunel+; yellow) TPPP/p25+ Sox10+ oligodendrocytes (magenta/cyan), in OL media or AST control, or following exposure to ACM. Scale bar, 50 µm. m Brain explants myelinated for 14 days in vitro (DIV), were demyelinated with LPC, then fixed at 5 days post-LPC (dpl) when remyelination is initiated. n Remyelination index ± s.e.m. for AST: slice culture media (SC) control, or following exposure to ACM. Two-tailed unpaired Student’s t-test with Welch’s correction, aP = 0.0048, t = 4.842; bP = 0.0125, t = 3.723. n = 3 mice/condition (AST:SC media, SC media: ACM), n = 4 mice/condition (CDDO, CDDO+Luteolin, CDDO + CS-6253), n = 3 mice/condition (AST:SC media, ACM no treatment). o Explants exposed to AST or ACM from astrocytes following no treatment or exposure to CDDOTFEA, CDDOTFEA then Luteolin, and CDDOTFEA then CS-6253 stained for myelin basic protein (MBP; magenta) and neurofilament (NF; green). Scale bar, 50 µm. p Explants myelinated for 14 DIV, were demyelinated with LPC, then treated with the ABCA1 inhibitor PSC833 or vehicle control from 2 to 7 dpl when remyelination is underway. q Explants treated with vehicle control or PSC833 (5 µM) and stained for MBP (magenta) and neurofilament (NF; green). Scale bar, 50 µm. r Remyelination index ± s.e.m. for vehicle or PSC833-treated brain explants. One-way ANOVA with Tukey´s multiple comparisons test, aP = 0.0052, bP = 0.0026, cP = 0.0007. ANOVA summary (F = 17.11 and P value = 0.0008). n = 3 mice/condition. Source data is provided with this paper. The images in 5a, g, m, p were created with Biorender.com.
Source publication
Failed regeneration of myelin around neuronal axons following central nervous system damage contributes to nerve dysfunction and clinical decline in various neurological conditions, for which there is an unmet therapeutic demand. Here, we show that interaction between glial cells – astrocytes and mature myelin-forming oligodendrocytes – is a determ...
Citations
... Astrocytes, the predominant glial cells in the CNS, play a crucial role in maintaining neural homeostasis, supporting synaptic plasticity and facilitating myelination. Originating from the neuroepithelium, astrocytes encase neuronal synapses, thus facilitating communication between neurons and oligodendrocytes (Molina-Gonzalez et al. 2023;Schober et al. 2022). Under physiological conditions, astrocytes actively promote myelination by releasing pro-myelination factors, transferring lipids, and signaling via gap junctions (Seiler et al. 2024;Traiffort et al. 2020). ...
Neuroinflammation is a key factor in the development of preterm white matter injury (PWMI), leading to glial cell dysfunction, arrest of oligodendrocyte maturation, and long-term neurological damage. As a potential therapeutic strategy, mesenchymal stem cells (MSCs) exhibit significant immunomodulatory and regenerative potential. Recent studies suggest that the primary mechanism of MSC action is their paracrine effects, particularly mediated by extracellular vesicles, with MSC-derived exosomes (MSC-Exos) being the key mediators. MSC-Exos, enriched with lipids, proteins, and nucleic acids, regulate neuroinflammation by modulating glial cell activity and influencing signaling pathways associated with inflammation and repair. Preclinical evidence has indicated that MSC-Exos can suppress the activation of microglia and astrocytes, promote oligodendrocyte maturation, and enhance myelination, highlighting their potential as a cell-free treatment for PWMI. However, there are a paucity of comprehensive reviews on how MSC-Exos regulate neuroinflammation in PWMI through specific signaling pathways. This review aims to summarize the key signaling pathways through which MSC-Exos modulate neuroinflammation in PWMI and discuss the challenges associated with the clinical application of MSC-Exos-based therapies.
Graphical Abstract
... Live imaging has shown limited mitochondrial dynamics in mature astrocytes in vivo compared to cultured cells, suggesting tightly regulated mitochondrial activity (Bergami and Motori, 2020). Astrocyte-oligodendrocyte interactions are critical for CNS remyelination, blood-brain barrier (BBB) integrity, and synaptogenesis Molina-Gonzalez et al., 2023) Understanding these interactions could inform therapeutic strategies for CNS diseases. The AD-, GBM-specific and common biological pathways identified in our pipeline are summarized in Figure 13. ...
Introduction
Alzheimer’s disease (AD) and glioblastoma (GBM) are severe neurological disorders that pose significant global healthcare challenges. Despite extensive research, the molecular mechanisms, particularly those involving mitochondrial dysfunction, remain poorly understood. A major limitation in current studies is the lack of cell-specific markers that effectively represent mitochondrial dynamics in AD and GBM.
Methods
In this study, we analyzed single-cell transcriptomic data using 10 machine learning algorithms to identify mitochondria-associated cell-specific markers. We validated these markers through the integration of gene expression and methylation data across diverse cell types. Our dataset comprised single-nucleus RNA sequencing (snRNA-seq) from AD patients, single-cell RNA sequencing (scRNA-seq) from GBM patients, and additional DNA methylation and transcriptomic data from the ROSMAP, ADNI, TCGA, and CGGA cohorts.
Results
Our analysis identified four significant cross-disease mitochondrial markers: EFHD1, SASH1, FAM110B, and SLC25A18 . These markers showed both shared and unique expression profiles in AD and GBM, suggesting a common mitochondrial mechanism contributing to both diseases. Additionally, oligodendrocytes and their interactions with astrocytes were implicated in disease progression, particularly through the APP signaling pathway. Key hub genes, such as HS6ST3 and TUBB2B , were identified across different cellular subpopulations, highlighting a cell-specific co-expression network linked to mitochondrial function.
... In WM, ApoE is lipidated to meet the lipid demands of oligodendrocytes, including cholesterol, phospholipids, sphingolipids, and glucosylceramides, which are essential for assembling and maintaining the multilayered membrane of the myelin sheath. These lipids act through lipidation, which occurs either within oligodendrocytes or via transport from astrocytes [18,23,24]. During aging, oligodendrocytes become less capable of synthesizing fatty acids (FAs) and lipids, leading to an increase in lipid transport from astrocytes to maintain myelin integrity, which are more vulnerable during pathological conditions [23,25,26]. ...
The ApoE ε4 allele (APOEε4) is a major genetic risk factor for sporadic Alzheimer's disease (AD) and is linked to demyelination and cognitive decline. However, its effects on the lipid transporters apolipoprotein E (ApoE) and fatty acid-binding protein 7 (Fabp7), which are crucial for the maintenance of myelin in white matter (WM) during the progression of AD remain underexplored. To evaluate the effects of APOEε4 on ApoE, Fabp7 and myelin in the WM of the frontal cortex (FC), we examined individuals carrying one ε4 allele that came to autopsy with a premortem clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI) and mild to moderate AD compared with non-carrier counterparts. ApoE, Fabp7 and Olig2 immunostaining was used to visualize cells, whereas myelin basic protein (MBP) immunocytochemistry and luxol fast blue (LFB) histochemistry of myelin in the WM of the FC were combined with quantitative morphometry. We observed increased numbers of ApoE-positive astrocytes in the WM of both NCI and MCI APOEε4 carriers compared with non-carriers, whereas Fabp7-positive cells were elevated only in AD. Conversely, Olig2 cell counts and MBP immunostaining decreased in MCI APOEε4 carriers compared to non-carriers, while LFB levels were higher in NCI APOEε4 carriers compared to non-carriers. Although no correlations were found between ApoE, Fabp7, and cognitive status, LFB measurements were positively correlated with perceptual speed, global cognition, and visuospatial scores in APOEε4 carriers across clinical groups. The present findings suggest that the ε4 allele compromises FC myelin homeostasis by disrupting the lipid transporters ApoE, Fabp7 and myelination early in the onset of AD. These data support targeting cellular components related to WM integrity as possible treatments for AD.
... LPC disrupts the integrity of astrocytes and thus causes astrocytopathy [40]. LPC significantly induced loss in primary astrocytes, enhanced apoptosis, and increased ROS levels in the culture consistent with previous reports [41]. Using the MTT assay, we determined the cytoprotective concentration of UA at different doses (0.0001 mM, 0.001 mM, 0.01 mM, 0.1 mM, and 1 mM). ...
Background
Multiple sclerosis (MS) is a chronic autoimmune condition that damages the myelin sheath of neurons in the central nervous system, resulting in compromised nerve transmission and motor impairment. The astrocytopathy is considered one of the prominent etiological factor in the pathophysiology of demyelination in MS. The expression level of ceramide synthase-2 (CS-2) is yet to be established in the pathophysiology of astrocytopathy although the derailed ceramide biosynthetic pathways is well demonstrated in the pathophysiology of demyelination. Therefore, in the present study, the expression level of CS-2 has been evaluated in lysophosphatidylcholine (LPC)-challenged primary astrocytes in the presence of anti-demyelinating agents such as Fingolimod, a clinically approved anti-demyelinating drug, and Ursolic Acid (UA), an experimentally accepted anti-demyelinating agent, in MS.
Methods and Results
LPC (150 µg/ml) caused astrocytopathy evident by decrease in percentage of viable astrocytes, increase in percentage of apoptotic astrocytes, increase in the level of reactive oxygen species (ROS), and increase in the level of activated astrocytes. Interestingly, LPC significantly increased the expression level of CS-2 in the primary culture of astrocytes where as fingolimod and UA (1, 10, and 100 µM) were able to attenuate the extent of LPC-induced astrocytopathy in the primary culture model of MS. Further, both the drugs drastically reduced the LPC-induced increase in the level of expression of CS-2 in the astrocytes.
Conclusions
These observations indicate the fact that CS-2 could be a potential target in the management of astrocytopathy and astrocytopathy-related neurological disorders including MS. In conclusion, CS-2-targeted drugs could be potential therapeutic options in the management of MS.
... Although the blood-brain-barrier essentially prevents the influx of cholesterol from the bloodstream, the adult brain is the most cholesterol-rich organ in the body (41), mainly owing to cholesterol biosynthesis in astrocytes which provide it to neighboring cells upon demand (42). The significance of cholesterol biosynthesis in astrocytes is crucial for re-myelination (43). In addition to its being an essential component of cellular membranes, cholesterol within the brain also serves as a precursor for various molecules such as steroid hormones. ...
A devastating genetic recessive neurodegenerative disorder, Vanishing White Matter Disease (VWMD), stems from mutations in eIF2B, a master regulator of mRNA translation initiation and mediator of cellular stress response. While astrocytes, the brain's essential support cells, are known to be central to VWMD pathology, the molecular mechanisms underlying their dysfunction remain poorly understood. Our study reveals that even a mild mutation in eIF2B5 profoundly disrupts astrocyte mRNA translation regulation upon cytokine-mediated activation, affecting nearly one-third of all expressed genes. Through innovative integration of RNA-seq and Ribo-seq analyses using primary cell cultures of astrocytes isolated from eIF2B5R132H/R132H mice, we discovered attempts to compensate for impaired protein production by increasing mRNA levels. However, this compensation proves insufficient to maintain critical cellular functions. Our comprehensive analysis uncovered significant disruptions in cellular energy production and protein synthesis machinery. We also predicted previously unknown defects in cholesterol biosynthesis within mutant astrocytes. Moreover, a meta-analysis of translation initiation scores pinpointed, for the first time, a short list of specific 'effector' gene candidates that may drive disease progression. This powerful combination of transcriptome and translatome illuminates the complex pathophysiology of VWMD and identifies promising new biomarkers and therapeutic target opportunities.
... Although oligodendrocytes predominantly synthesize cholesterol de novo, they also rely on astrocytes for cholesterol acquisition through apolipoprotein particles released into the extracellular space [13]. Studies have shown that when cholesterol synthesis is impaired in astrocytes, the survival and myelination of oligodendrocytes are adversely affected [60]. Furthermore, when cholesterol synthesis in astrocytes is compromised, oligodendrocytes can circumvent this defect by utilizing dietary cholesterol for myelin membrane synthesis [13]. ...
The brain is the organ with the highest cholesterol content in the body. Cholesterol in the brain plays a crucial role in maintaining the integrity of synapses and myelin sheaths to ensure normal brain function. Disruptions in cholesterol metabolism are closely associated with various central nervous system (CNS) diseases, including Alzheimer’s disease (AD), Huntington’s disease (HD), and multiple sclerosis (MS). In this review, we explore the synthesis, regulation, transport, and functional roles of cholesterol in the CNS. We discuss in detail the associations between cholesterol homeostasis imbalance and CNS diseases including AD, HD, and MS, highlighting the significant role of cholesterol metabolism abnormalities in the development of these diseases. Sterol regulatory element binding protein-2 (SREBP2) and liver X receptor (LXR) are two critical transcription factors that play central roles in cholesterol synthesis and reverse transport, respectively. Their cooperative interaction finely tunes the balance of brain cholesterol metabolism, presenting potential therapeutic value for preventing and treating CNS diseases. We particularly emphasize the alterations in SREBP2 and LXR under pathological conditions and their impacts on disease progression. This review summarizes current therapeutic agents targeting these two pathways, with the hope of broadening the perspectives of CNS drug developers and encouraging further study into SREBP2 and LXR-related therapies for CNS diseases.
... Peptides were analyzed by data-independent acquisition (DIA) mass spectrometry as described previously 88,89 . In summary, peptides were injected onto a nanoscale C18 reverse-phase chromatography system (UltiMate 3000 RSLC nano, Thermo Scientific) and electrosprayed into an Orbitrap Exploris 480 Mass Spectrometer (Thermo Fisher). ...
Cortical layer 5 (L5) intratelencephalic (IT) and pyramidal tract (PT) neurons are embedded in distinct information processing pathways. Their morphology, connectivity, electrophysiological properties, and role in behavior have been extensively analyzed. However, the molecular composition of their synapses remains largely uncharacterized. Here, we dissect the protein composition of the excitatory postsynaptic compartment of mouse L5 neurons in intact somatosensory circuits, using an optimized proximity biotinylation workflow with high spatial accuracy. We find distinct synaptic signatures of L5 IT and PT neurons that are defined by proteins regulating synaptic organization and transmission, including cell-surface proteins (CSPs), neurotransmitter receptors and ion channels. In addition, we find a differential vulnerability to disease, with a marked enrichment of autism risk genes in the synaptic signature of L5 IT neurons compared to PT neurons. These results align with human studies and suggest that the excitatory postsynaptic compartment of L5 IT neurons is susceptible in autism. Our approach is versatile and can be broadly applied to other neuron types to create a protein-based, synaptic atlas of cortical circuits.
... MS survey scan was followed by MS/MS DIA scan events using the following parameters: multiplex ions set to false, collision energy mode set to stepped, collision energy type set to normalized, HCD collision energies set to 25.5, 27 and 30, orbitrap resolution 30000, first mass 200, RF lens 40, AGC target set to custom, normalized AGC target 3000, maximum injection time 55 ms. proteomes examining the impact of MYC inhibition on B cells, peptides were analysed by data independent acquisition (DIA) as described previously[64][65][66] . 1.5 μg of peptides was injected onto a nanoscale C18 reverse-phase chromatography system (UltiMate 3000 RSLC nano, Thermo Scientific) and electrosprayed into an Orbitrap Exploris 480 Mass Spectrometer (Thermo Fisher). ...
Using high resolution quantitative mass spectrometry, we have explored how immune activation and the metabolic checkpoint kinase mTORC1 (mammalian target of rapamycin complex 1) regulate the proteome of B lymphocytes. B cell activation via the B cell receptor, CD40 and the IL-4 receptor induced considerable re-modelling of the B cell protein landscape, with a 5-fold increase in total cellular protein mass within 24 hours of activation. Analysis of copy numbers per cell of >7,500 proteins revealed increases in the metabolic machinery that supports B cell activation and nutrient and amino acid transporters that fuel B cell biosynthetic capacity. We reveal that mTORC1 controls activation-induced cell growth and B cell proteome remodelling and inhibiting mTORC1 impairs the expression of amino acid transporters that fuel B cell protein production. We also show that mTORC1 activity regulates the expression of the transcription factor MYC and the transferrin receptor CD71. Blocking MYC activity phenocopied mTORC1 inhibition in many ways including impaired CD71 expression, while limiting iron availability during B cell activation impaired B cell growth and protein synthesis. This work provides a detailed map of naïve and immune activated B cell proteomes and a greater understanding of the cellular machinery that direct B cell phenotypes. This work also provides new insights into the role of mTORC1, MYC and iron in regulating activation-induced proteome remodelling and protein production in B cells.
... Nevertheless, the mechanism involved is still elusive and might not be related (at least exclusively) to the role of Nrf2 in oligodendrocytes but to other cell types such as microglia and astrocytes. In this vein, it has been recently shown that during remyelination, astrocytes downregulate Nrf2 pathway, turning their metabolism towards cholesterol biosynthesis [141], which they export to oligodendrocytes to regulate their survival and remyelination. Indeed, these authors use a model that constitutively overexpresses Nrf2 in astrocytes (GFAP-Nrf2) and they show an impairment in remyelination [141]. ...
... In this vein, it has been recently shown that during remyelination, astrocytes downregulate Nrf2 pathway, turning their metabolism towards cholesterol biosynthesis [141], which they export to oligodendrocytes to regulate their survival and remyelination. Indeed, these authors use a model that constitutively overexpresses Nrf2 in astrocytes (GFAP-Nrf2) and they show an impairment in remyelination [141]. On the contrary, opposite results have been obtained in other studies, where the activation of Nrf2 in the astrocytes using GFAP-specific Keap1 deletion animals showed lower oligodendrocyte loss and demyelination [142]. ...
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial regulator of cellular defence mechanisms, essential for maintaining the brain’s health. Nrf2 supports mitochondrial function and protects against oxidative damage, which is vital for meeting the brain’s substantial energy and antioxidant demands. Furthermore, Nrf2 modulates glial inflammatory responses, playing a pivotal role in preventing neuroinflammation. This review explores these multifaceted functions of Nrf2 within the central nervous system, focusing on its activity across various brain cell types, including neurons, astrocytes, microglia, and oligodendrocytes. Due to the brain’s vulnerability to oxidative stress and metabolic challenges, Nrf2 is emerging as a key therapeutic target to enhance resilience against oxidative stress, inflammation, mitochondrial dysfunction, and demyelination, which are central to many neurodegenerative diseases.
... As almost sole producers of cholesterol in the CNS, astrocytes are integral for the biosynthesis of cell membranes in the brain and spinal cord. Astrocyte-derived cholesterol has also been reported to support the survival of oligodendrocytes and remyelination 326 , which may add to neuroprotection in the context of AD. Cholesterol is also an important trophic molecule for microglia, and evidence suggests that astrocytes expressing the AD-associated APOE4 allele are less competent at producing and secreting cholesterol. ...
Increasing evidence points to a pivotal role of immune processes in the pathogenesis of Alzheimer disease, which is the most prevalent neurodegenerative and dementia-causing disease of our time. Multiple lines of information provided by experimental, epidemiological, neuropathological and genetic studies suggest a pathological role for innate and adaptive immune activation in this disease. Here, we review the cell types and pathological mechanisms involved in disease development as well as the influence of genetics and lifestyle factors. Given the decade-long preclinical stage of Alzheimer disease, these mechanisms and their interactions are driving forces behind the spread and progression of the disease. The identification of treatment opportunities will require a precise understanding of the cells and mechanisms involved as well as a clear definition of their temporal and topographical nature. We will also discuss new therapeutic strategies for targeting neuroinflammation, which are now entering the clinic and showing promise for patients.
