Relationship between miR-335 and sterol levels after in vitro hypoxia-ischemia of primary brain cells

Background/Purpose: Cholesterol homeostasis is vital for synaptogenesis and myelination during fetal and neonatal brain development. Both brain and plasma sterol levels have been shown to be associated with outcomes in adult stroke, and animal studies have suggested brain and plasma sterol changes in neonatal hypoxic-ischemic brain injury. MicroRNA-335 (miR-335) has been associated with brain sterol metabolism and our preliminary data showed that sterol metabolism is dysregulated in the brain after hypoxic-ischemic injury in a mouse model. This study sought to describe the effect of oxygen-glucose deprivation in vitro on individual brain cell populations and to validate the associations between sterol levels and miR-335 levels. Lastly, we assessed the feasibility of transfecting miR-335 mimics or miRNA antagonists (antagomiRs) in these brain cell cultures to set up future experiments to alter miR-335 levels for neuroprotection. Methods: Primary cells were isolated from embryonic day 18 CD1 mouse brains. Cells were maintained in DMEM for mixed glial culture and Neurobasal media with B27 for neurons. Once astrocytes were confluent, microglia were separated from the mixed glial culture by shaking to provide pure cell populations. Each of the three cell types underwent oxygen and glucose deprivation (OGD: glucose/pyruvate/B27-free media, 1% O2, 5% CO2) for 4 hours followed by replacement of glucose/pyruvate/B27 and resumption of normoxia. At 24 hours after OGD, cells (n=12 wells OGD and 12 normoxia) were washed, counted, and sterols analyzed by LC-MS/MS, normalized to the number of cells/well. Additional cells underwent measurement of miR-335 expression using quantitative PCR and/or transfection with miR-335 mimic or antagomiR. Results: Although lanosterol is increased after OGD in neurons, desmosterol and cholesterol levels were decreased. In microglia and astrocytes, cholesterol levels were lower than in neurons but increased after OGD. MiR-335 expression in neurons and astrocytes were inverse to cholesterol level changes, though this association was not seen in microglia. Lastly, red fluorescent protein-labelled miR-335 mimic was visualized in both neurons and astrocytes after transfection and miR-335 expression changes were seen after transfection in astrocytes. Conclusions: Sterol levels are altered after OGD and may be associated with OGD changes. Transfection of miR-335 mimic and antagomiR is feasible and future studies using these tools will allow for better understanding of the effects of neonatal HIBI on sterol levels. This approach could allow for identification of targets to aid in developing therapeutics.https://digitalcommons.unmc.edu/chri_forum/1017/thumbnail.jp

Evaluation of brain cholesterol metabolism after neonatal hypoxic-ischemic brain injury

Introduction Neonatal hypoxic-ischemic brain injury (HIBI), resulting from impaired cerebral blood flow and oxygen delivery to the brain, affects at least 1.5 per 1,000 live births each year in the United States. Altered levels of cholesterol and cholesterol metabolites have been identified in brain tissue and in serum after adult brain injury such as traumatic brain injury and stroke. We hypothesized that there would be temporal and brain region-specific alterations in cholesterol and sterol precursors after neonatal HIBI. Methods Postnatal day 9 CD1 mouse pups were anesthetized with isoflurane and randomized to HIBI induced by carotid artery ligation or controls receiving sham surgery consisting of dissection without ligation (n=24/group). Pups were allowed to recover after surgery and then placed in a hypoxia chamber at 8% oxygen for HIBI or 21% for controls for 30 minutes. Each group was further divided into three sub-groups (n=8/group) for blood and brain tissue collection at 30 minutes, 24 hours, or 72 hours after injury. Brain tissue was dissected into four regions: cortex, cerebellum, striatum/thalamus, and hippocampus. For each region, protein was quantified by BCA assay, interleukin-6 (IL-6) levels were measured by ELISA as a marker of injury severity, and liquid chromatography mass spectrometry was performed to evaluate for the following sterols: cholesterol, desmosterol, 7-dehydrocholesterol, 8-dehydrocholesterol, and lanosterol. Levels were compared between HIBI and control groups at each time point and neuroanatomical region. Additionally, the four samples in each group demonstrating the most severe injury, as defined by IL-6 levels, were stratified as “severe injury”. Differences were analyzed with a two-sided Mann-Whitney test. Results When assessing the entire cohort, no statistically significant differences were seen between HIBI and controls with respect to temporal or regional differences for any of the sterols measured. In animals with severe HIBI, however, cholesterol, 7-dehydrocholesterol, 8-dehydrocholesterol, and desmosterol were higher in the cortex at 24 hours after injury compared to controls. Additionally, desmosterol was also elevated in the cerebellum but decreased in the striatum 24 hours after injury in the severely injured animals. Conclusion Severe neonatal encephalopathy appears to be associated with alterations in cortical brain sterol levels, peaking around 24 hours after injury. Further defining the aberrations in brain metabolism in infants with HIBI could provide opportunities for not only diagnostic biomarkers but also the development of targeted therapies.https://digitalcommons.unmc.edu/chri_forum/1003/thumbnail.jp

Engineering Neurotoxin-Functionalized Exosomes for Targeted Delivery to the Peripheral Nervous System

The administration of therapeutics to peripheral nerve tissue is challenging due to the complexities of peripheral neuroanatomy and the limitations imposed by the blood–nerve barrier (BNB). Therefore, there is a pressing need to enhance delivery effectiveness and implement targeted delivery methods. Recently, erythrocyte-derived exosomes (Exos) have gained widespread attention as biocompatible vehicles for therapeutics in clinical applications. However, engineering targeted Exos for the peripheral nervous system (PNS) is still challenging. This study aims to develop a targeted Exo delivery system specifically designed for presynaptic terminals of peripheral nerve tissue. The clostridium neurotoxin, tetanus toxin-C fragment (TTC), was tethered to the surface of red blood cell (RBC)-derived Exos via a facile and efficient bio-orthogonal click chemistry method without a catalyst. Additionally, Cyanine5 (Cy5), a reactive fluorescent tag, was also conjugated to track Exo movement in both in vitro and in vivo models. Subsequently, Neuro-2a, a mouse neuronal cell line, was treated with dye-labeled Exos with/without TTC in vitro, and the results indicated that TTC-Exos exhibited more efficient accumulation along the soma and axonal circumference, compared to their unmodified counterparts. Further investigation, using a mouse model, revealed that within 72 h of intramuscular administration, engineered TTC-Exos were successfully transported into the neuromuscular junction and sciatic nerve tissues. These results indicated that TTC played a crucial role in the Exo delivery system, improving the affinity to peripheral nerves. These promising results underscore the potential of using targeted Exo carriers to deliver therapeutics for treating peripheral neuropathies

The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts

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Vulnerability of DHCR7+/- mutation carriers to aripiprazole and trazodone exposure

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Plasma Retinol Concentrations and Dietary Intakes of Mother-Infant Sets in Singleton versus Twin Pregnancy

Vitamin A (retinol) is essential for normal fetal development, but the recommendation for maternal dietary intake (Retinol Activity Equivalent, RAE) does not differ for singleton vs. twin pregnancy, despite the limited evaluation of retinol status. Therefore, this study aimed to evaluate plasma retinol concentrations and deficiency status in mother-infant sets from singleton vs. twin pregnancies as well as maternal RAE intake. A total of 21 mother-infant sets were included (14 singleton, 7 twin). The HPLC and LC-MS/HS evaluated the plasma retinol concentration, and data were analyzed using the Mann-Whitney U test. Plasma retinol was significantly lower in twin vs. singleton pregnancies in both maternal (192.2 vs. 312.1 vs. mcg/L, p = 0.002) and umbilical cord (UC) samples (102.5 vs. 154.4 vs. mcg/L, p = 0.002). The prevalence of serum-defined vitamin A deficiency (VAD) \u3c200.6 mcg/L was higher in twins vs. singletons for both maternal (57% vs. 7%, p = 0.031) and UC samples (100% vs. 0%, p \u3c 0.001), despite a similar RAE intake (2178 vs. 1862 mcg/day, p = 0.603). Twin pregnancies demonstrated a higher likelihood of vitamin A deficiency in mothers, with an odds ratio of 17.3 (95% CI: 1.4 to 216.6). This study suggests twin pregnancy may be associated with VAD deficiency. Further research is needed to determine optimal maternal dietary recommendations during twin gestation

Ubiquitous Aberration in Cholesterol Metabolism Across Pancreatic Ductal Adenocarcinoma

Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens

Retinol and Pro-Vitamin A Carotenoid Nutritional Status during Pregnancy Is Associated with Newborn Hearing Screen Results

The prenatal period is critical for auditory development; thus, prenatal influences on auditory development may significantly impact long-term hearing ability. While previous studies identified a protective effect of carotenoids on adult hearing, the impact of these nutrients on hearing outcomes in neonates is not well understood. The purpose of this study is to investigate the relationship between maternal and umbilical cord plasma retinol and carotenoid concentrations and abnormal newborn hearing screen (NHS) results. Mother-infant dyads (n = 546) were enrolled at delivery. Plasma samples were analyzed using HPLC and LC-MS/MS. NHS results were obtained from medical records. Statistical analysis utilized Mann-Whitney U tests and logistic regression models, with p ≤ 0.05 considered statistically significant. Abnormal NHS results were observed in 8.5% of infants. Higher median cord retinol (187.4 vs. 162.2 μg/L, p = 0.01), maternal trans-β-carotene (206.1 vs. 149.4 μg/L, p = 0.02), maternal cis-β-carotene (15.9 vs. 11.2 μg/L, p = 0.02), and cord trans-β-carotene (15.5 vs. 8.0 μg/L, p = 0.04) were associated with abnormal NHS. Significant associations between natural log-transformed retinol and β-carotene concentrations and abnormal NHS results remained after adjustment for smoking status, maternal age, and corrected gestational age. Further studies should investigate if congenital metabolic deficiencies, pesticide contamination of carotenoid-rich foods, maternal hypothyroidism, or other variables mediate this relationshi

Visualizing Cholesterol in the Brain by On-Tissue Derivatization and Quantitative Mass Spectrometry Imaging.

Despite being a critical molecule in the brain, mass spectrometry imaging (MSI) of cholesterol has been under-reported compared to other lipids due to the difficulty in ionizing the sterol molecule. In the present work, we have employed an on-tissue enzyme-assisted derivatization strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific structures of the mouse brain, in a model of Niemann-Pick type C1 disease, and during brain development. MSI revealed that in the adult mouse, cholesterol is the highest in the pons and medulla and how its distribution changes during development. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience

Subcellular localization of sterol biosynthesis enzymes

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