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Compromised lipid metabolism, mitochondria respiration and neuroprotective effects in iPSC-derived astrocytes from a Smith-Lemli-Opitz syndrome patient

ABSTRACT: Smith-Lemli-Opitz syndrome (SLOS) is a rare, autosomal recessive disorder characterized by congenital malformations, intellectual disability, and behavioral abnormalities. SLOS results from mutations in the DHCR7 gene, leading to impaired cholesterol biosynthesis due to dysregulation of 7-dehydrocholesterol reductase. Cholesterol plays crucial roles in neurophysiology, including synaptic formation and neurotransmitter receptor regulation, which likely contribute to neurological manifestations in SLOS patients. While astrocytes are the main cholesterol producing cells in the brain, their specific role in SLOS pathogenesis remains unclear. In this study, we utilized induced pluripotent stem cell (iPSC)-derived astrocytes from a SLOS patient with DHCR7 c.C278T mutation and the isogenic control. We found decreased lipid droplet formation in SLOS iPSC astrocytes compared to controls, accompanied with diminished efflux of cholesterol and apolipoprotein E. Lipidomics revealed reduced cholesterol and cholesterol esters, as well as altered profiles of other lipids in SLOS iPSC astrocytes. While RNA-sequencing identified various genes and pathways affected by the disease status, those related to mitochondria functions were top-ranked. Mitochondrial electron transport chain oxidative phosphorylation gene expression decreased in SLOS iPSC astrocytes, alongside impaired mitochondrial respiration. Furthermore, SLOS iPSC astrocytes less effectively mediated neuroprotection on iPSC neurons than control astrocytes in serum-starvation conditions. SLOS iPSC astrocytes also poorly contributed to synaptic networks when co-cultured with iPSC neurons. Overall, our findings provide mechanistic insights into how DHCR7 disruption impacts astrocyte function, contributing to SLOS neuropathology by dysregulating lipid metabolism, mitochondrial respiration, and impaired neuroprotection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE309707 | GEO | 2025/12/04

REPOSITORIES: GEO

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Project description:RELEVANCE: Smith-Lemli-Opitz syndrome (SLOS) is a human disease caused by mutations in the gene coding for the enzyme DHCR7 (7-dehydrocholesterol (7DHC) reductase), which catalyzes the final step of cholesterol biosynthesis. Accumulated 7DHC in tissues and body fluids of SLOS patients gives rise to numerous oxidation products (oxysterols) in situ, some of which are cytotoxic, and which may contribute to the pathophysiology of SLOS. The SLOS phenotype is broad, ranging from death in utero to viable individuals with malformations and malfunctions in numerous organ systems and tissues, including neurological and cognitive defects. The latter presentations suggest that impaired cholesterol synthesis, in particular the generation of toxic oxysterols, has a deleterious impact on the morphogenesis and viability of neurons in the CNS. In a rat model of SLOS (using a small molecule inhibitor of DHCR7), the loss of photoreceptors was also documented, distinct from the continuous viability of other retinal neurons and supporting cells, and this selective cell death was recapitulated in vitro using 661W cells (an immortalized line derived from mouse cones) incubated with purified 7DHC-derived, SLOS-associated oxysterols. Upon exposure to these compounds, cell viability assay results for 661W showed one to two orders of magnitude higher sensitivity with respect to efficacy and potency, and also an accelerated time frame for cell death, compared to retinal Mueller glia and retinal pigment epithelial cells. These findings inspired questions as to the molecular mechanisms underlying oxysterol-induced neuronal cell death. Therefore, we characterized differential gene expression associated with processes and pathways induced by oxysterol treatments, first, to provide insights regarding cell death and dysfunction, not only in SLOS, but also extended to other neurodegenerative diseases (including those affecting the retina), and second, to identify cellular protective responses, with the expectation that these findings would suggest possibilities for future prevention and treatment of neurological disease and damage. INTENT: Generate gene expression array profiles of 661W cells following exposure to either of two oxysterols. The oxysterols are employed at doses already determined to exert full cytotoxicity by 24 hours incubation time; however, for the purposes of the array, stop incubations for each oxysterol at time points preceding global cell death, to harvest still-intact RNA from a majority of cells that have maintained membrane integrity, and are also displaying microscopically observable indications of morphological response to the oxysterol treatments already known to precede the demise of the cells. Therefore, the samples are expected to manifest transcriptomes emblematic of gene expression changes in response to the oxysterols. These changes should fit patterns that correlate with pathways and processes associated with cellular damage and regulated cell death, or with cell survival/protective and repair mechanisms (with expected overlap of the two opposing scenarios). As a negative control, a separate set of replicates are incubated with cholesterol under conditions having no impact on cell viability (23 hours, at a (non-physiological) concentration intermediate between that of the two oxysterols). To identify differentially expressed genes, individual array probe set data for either oxysterol and for cholesterol are matched with those from cells incubated with a vehicle control (for 24 hours), for computing “-fold change” in expression and statistical significance of expression differences. EXPERIMENTAL WORKFLOW: 1) 661W cells were seeded in 100-mm cell culture-treated dishes at a density permitting proliferation to subconfluence, allowing cells to retain neurite-like extensions; includes overnight adaptation to a simplified incubation medium devoid of most growth factors and reagents supporting antioxidant activity. 2) Cells were exposed for a predetermined time period to a single concentration of: i) either of two different oxysterols (EPCD, an endoperoxide specific to SLOS, or 7-ketocholesterol); ii) cholesterol; or iii) vehicle control. 3) Total RNA was harvested, from each triplicate sample representing the above treatments, according to the RNeasy Plus minikit protocol (Qiagen). 4) Final sample preparation (amplification, labeling, fragmentation of cRNA) was carried out a core facility following Affymetrix specifications and protocols. 5) Hybridized chips were scanned to generate raw intensity data for further analysis.

Project description:Smith-Lemli-Opitz syndrome (SLOS) is a disorder of cholesterol production by the body. It is caused by changes in the DHCR7 gene, which is the blueprint for an enzyme called 7-dehydrocholesterol- delta7-reductase. This enzyme is necessary for the production of cholesterol by all cells in the body. People with SLOS often have malformations of major organs, slow growth, feeding difficulties and intellectual disability or learning problems. Because patients with SLOS cannot make enough cholesterol, it has been proposed that cholesterol supplementation (either with egg yolk or liquid suspensions of cholesterol) could help improve the symptoms of the disease. However, despite the widespread use of cholesterol supplementation, it is still not known whether it works or not. The study will try to provide an answer to this question by studying the disease and its progression while patients are receiving cholesterol. The clinical features of SLOS are thought to be related to low cholesterol and buildup of toxic cholesterol precursors (substances from which cholesterol is formed). But how exactly low cholesterol and toxic precursors contribute to the disease is poorly understood. This knowledge is critically important because it should help discover new therapeutic targets and develop treatments of the disease in the long run. The study will try to fill this gap with a comprehensive clinical and biochemical testing of the study participants over the course of several years. Last, a limitation of previous SLOS research studies has been their low number of participants. This is understandable because SLOS is a rare diseases and few research groups are working on it. However, in order to fully understand the disease, researchers need to study as many patients as possible. This study is unique in that it is run by a network of several highly specialized clinical research sites across the country. Having several sites involved increases the researcher's ability to recruit and study large number of patients, and centralize patients' information in a comprehensive SLOS clinical registry. This registry will be key to identify markers for diagnostic testing, screening and measuring outcomes in future studies of treatment. The purpose of this study is to learn as much as possible about Smith-Lemli-Opitz Syndrome (SLOS) by following a large group of affected children and adults over time. In this study, we will measure cholesterol and other similar chemicals in blood and urine, evaluate development and behavior, do limited medical evaluation, and carry out brain imaging studies.This study will help researchers: <ul> <li>learn more about what causes SLOS and how SLOS changes with age,</li> <li>note differences in features of SLOS among those affected,</li> <li>evaluate the effect of giving extra cholesterol in this condition, and</li> <li>develop ways to evaluate whether treatments developed in the future will be helpful.</li> </ul>

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Compromised lipid metabolism, mitochondria respiration and neuroprotective effects in iPSC-derived astrocytes from a Smith-Lemli-Opitz syndrome patient

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