ABSTRACT: 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.
