Inhibition of post-lanosterol biosynthesis by fentanyl: potential implications for Fetal Fentanyl Syndrome (FFS).

A recent study discovered a novel, complex developmental disability syndrome, most likely caused by maternal fentanyl use disorder. This Fetal Fentanyl Syndrome (FFS) is biochemically characterized by elevated 7-dehydrocholesterol (7-DHC) levels in neonates, raising the question if fentanyl inhibition of the dehydrocholesterol reductase 7 (DHCR7) enzyme is causal for the emergence of the pathophysiology and phenotypic features of FFS. To test this hypothesis, we undertook a series of experiments on Neuro2a cells, primary mouse neuronal and astrocytic cultures, and human dermal fibroblasts (HDFs) with DHCR7+/+ and DHCR7+/- genotype. Our results revealed that in vitro exposure to fentanyl disrupted sterol biosynthesis across all four in vitro models. The sterol biosynthesis disruption by fentanyl was complex, and encompassed the majority of post-lanosterol intermediates, including elevated 7-DHC and decreased desmosterol (DES) levels across all investigated models. The overall findings suggested that maternal fentanyl use in the context of an opioid use disorder leads to FFS in the developing fetus through a strong disruption of the whole post-lanosterol pathway that is more complex than a simple DHCR7 inhibition. In follow-up experiments we found that heterozygous DHCR7+/- HDFs were significantly more susceptible to the sterol biosynthesis inhibitory effects of fentanyl than wild-type DHCR7+/+ fibroblasts. These data suggest that DHCR7+/- heterozygosity of mother and/or developing child (and potentially other sterol biosynthesis genes), when combined with maternal fentanyl use disorder, might be a significant contributory factor to the emergence of FFS in the exposed offspring. In a broader context, we believe that evaluation of new and existing medications for their effects on sterol biosynthesis should be an essential consideration during drug safety determinations, especially in pregnancy.

Effects of Neonatal Hypoxic-Ischemic Injury on Brain Sterol Synthesis and Metabolism.

BACKGROUND: Neonatal hypoxic-ischemic brain injury (HIBI) results from disruptions to blood supply and oxygen in the perinatal brain. The goal of this study was to measure brain sterol metabolites and plasma oxysterols after injury in a neonatal HIBI mouse model to assess for potential therapeutic targets in the brain biochemistry as well as potential circulating diagnostic biomarkers. METHODS: Postnatal day 9 CD1-IGS mouse pups were randomized to HIBI induced by carotid artery ligation followed by 30 minutes at 8% oxygen or to sham surgery and normoxia. Brain tissue was collected for sterol analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Plasma was collected for oxysterol analysis by LC-MS/MS. RESULTS: There were minimal changes in brain sterol concentrations in the first 72 hours after HIBI. In severely injured brains, there was a significant increase in desmosterol, 7-DHC, 8-DHC, and cholesterol 24 hours after injury in the ipsilateral tissue. Lanosterol, 24-dehydrolathosterol, and 14-dehydrozymostenol decreased in plasma 24 hours after injury. Severe neonatal HIBI was associated with increased cholesterol and sterol precursors in the cortex at 24 hours after injury. CONCLUSIONS: Differences in plasma oxysterols were seen at 24 hours but were not present at 30 minutes after injury, suggesting that these sterol intermediates would be of little value as early diagnostic biomarkers.

Dysregulated cholesterol metabolism, aberrant excitability and altered cell cycle of astrocytes in fragile X syndrome.

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. While neuronal contribution to FXS has been extensively studied in both animal and human-based models of FXS, the roles of astrocytes, a type of glial cells in the brain, are largely unknown. Here, we generated a human-based FXS model via differentiation of astrocytes from human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) and characterized their development, function, and proteomic profiles. We identified shortened cell cycle, enhanced Ca2+ signaling, impaired sterol biosynthesis, and pervasive alterations in the proteome of FXS astrocytes. Our work identified astrocytic impairments that could contribute to the pathogenesis of FXS and highlight astrocytes as a novel therapeutic target for FXS treatment.

Medication effects on developmental sterol biosynthesis.

Cholesterol is essential for normal brain function and development. Genetic disruptions of sterol biosynthesis result in intellectual and developmental disabilities. Developing neurons synthesize their own cholesterol, and disruption of this process can occur by both genetic and chemical mechanisms. Many commonly prescribed medications interfere with sterol biosynthesis, including haloperidol, aripiprazole, cariprazine, fluoxetine, trazodone and amiodarone. When used during pregnancy, these compounds might have detrimental effects on the developing brain of the offspring. In particular, inhibition of dehydrocholesterol-reductase 7 (DHCR7), the last enzyme in the biosynthesis pathway, results in accumulation of the immediate cholesterol precursor, 7-dehydrocholesterol (7-DHC). 7-DHC is highly unstable, giving rise to toxic oxysterols; this is particularly pronounced in a mouse model when both the mother and the offspring carry the Dhcr7+/- genotype. Studies of human dermal fibroblasts from individuals who carry DCHR7+/- single allele mutations suggest that the same gene*medication interaction also occurs in humans. The public health relevance of these findings is high, as DHCR7-inhibitors can be considered teratogens, and are commonly used by pregnant women. In addition, sterol biosynthesis inhibiting medications should be used with caution in individuals with mutations in sterol biosynthesis genes. In an age of precision medicine, further research in this area could open opportunities to improve patient and fetal/infant safety by tailoring medication prescriptions according to patient genotype and life stage.

Individual and simultaneous treatment with antipsychotic aripiprazole and antidepressant trazodone inhibit sterol biosynthesis in the adult brain.

Polypharmacy, or the simultaneous use of multiple drugs to treat a single patient, is a common practice in psychiatry. Unfortunately, data on the health effects of commonly used combinations of medications are very limited. In this study, we therefore investigated the effects and interactions between two commonly prescribed psychotropic medications with sterol inhibiting side effects, trazodone (TRZ), an antidepressant, and aripiprazole (ARI), an antipsychotic. In vitro cell culture experiments revealed that both medications alone disrupted neuronal and astroglial sterol biosynthesis in dose-dependent manners. Furthermore, when ARI and TRZ were combined, exposure resulted in an additive 7-dehydrocholesterol (7-DHC) increase, as well as desmosterol (DES) and cholesterol decreases in both cell types. In adult mice, at baseline, we found that the three investigated sterols showed significant differences in distribution across the eight assessed brain regions. Furthermore, experimental mice treated with ARI or TRZ, or a combination of both medications for 8 days, showed strong sterol disruption across all brain regions. We show ARI or TRZ alone elevated 7-DHC and decreased DES levels in all brain regions, but with regional differences. However, the combined utilization of these two medications for 8 days did not lead to additive changes in sterol disturbances. Based on the complex roles of 7-DHC derived oxysterols, we conclude that individual and potentially simultaneous use of medications with sterol biosynthesis-inhibiting properties might have undesired side effects on the adult brain, with as yet unknown long-term consequences on mental or physical health.

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.

Maternal cariprazine exposure inhibits embryonic and postnatal brain cholesterol biosynthesis.

Cariprazine (CAR) is a strong inhibitor of the Dhcr7 enzyme, the last enzyme in the cholesterol biosynthesis pathway. We assessed the effects of CAR on maternally exposed Dhcr7+/- and wild-type mouse offspring, and tested the biochemical effects of CAR in human serum samples. Dhcr7+/- and wild-type time-pregnant mice were exposed to vehicle or 0.2 mg/kg CAR from E12 to E19. Levels of CAR, CAR metabolites, sterols, and oxysterols were measured in the brain of maternally exposed offspring at various time points using LC-MS/MS. Embryonic exposure to CAR significantly increased levels of 7-DHC in all organs of exposed embryos, with a particularly strong effect in the brain. Detectable levels of CAR and elevated 7-DHC were observed in the brain of newborn pups 14 days after drug exposure. In addition, CAR altered sterol metabolism in all animals analyzed, with the strongest effect on the brain of Dhcr7+/- pups born to Dhcr7+/- dams. Furthermore, CAR elevated toxic oxysterols in the brain of maternally exposed Dhcr7+/- offspring to levels approaching those seen in a mouse model of Smith-Lemli-Opitz syndrome. Finally, we observed that patients taking CAR have elevated 7-DHC in their serum. In summary, maternal DHCR7 heterozygosity, combined with offspring DHCR7 heterozygosity might represent a vulnerability factor to medications that interfere with sterol biosynthesis. Due to the conserved sterol biosynthesis between mice and humans, we suggest that the 1-3% of patient population with single-allele DHCR7 mutations might not be ideal candidates for CAR use, especially if they are nursing, pregnant or plan to become pregnant.

Maternal aripiprazole exposure interacts with 7-dehydrocholesterol reductase mutations and alters embryonic neurodevelopment.

Mutations in both copies in the gene encoding 7-dehydrocholesterol reductase (DHCR7) cause Smith-Lemli-Opitz Syndrome (SLOS), which is characterized by a toxic elevation in 7-dehydrocholesterol (7-DHC). Aripiprazole (ARI) exposure, independent of genetic mutations, also leads to elevation of 7-DHC. We investigated the combined effect of a single-copy Dhcr7+/- mutation and maternal ARI exposure on the developing offspring brain. We generated a time-pregnant mouse model where WT and Dhcr7+/- embryos were maternally exposed to ARI or vehicle (VEH) from E12 to E19 (5 mg/kg). Levels of cholesterol, its precursors, ARI and its metabolites were measured at P0. We found that ARI and its metabolites were transported across the placenta and reached the brain of offspring. Maternal ARI exposure led to decreased viability of embryos and increased 7-DHC levels, regardless of maternal or offspring Dhcr7 genotype. In addition, Dhcr7+/- pups were more vulnerable to maternal ARI exposure than their WT littermates, and maternal Dhcr7+/- genotype also exacerbated offspring response to ARI treatment. Finally, both 7-DHC levels and 7-DHC/cholesterol ratio is the highest in Dhcr7+/- pups from Dhcr7+/- mothers exposed to ARI, underscoring a potentially dangerous interaction between maternal genotype×embryonic genotype×treatment. Our findings have important clinical implications. SLOS patients should avoid drugs that increase 7-DHC levels such as ARI, trazodone and haloperidol. In addition, treatment with 7-DHC elevating substances might be potentially unsafe for the 1-1.5% of population with single-allele disruptions of the DHCR7 gene. Finally, prenatal and parental genetic testing for DHCR7 should be considered before prescribing sterol-interfering medications during pregnancy.

Desmosterolosis and desmosterol homeostasis in the developing mouse brain.

Cholesterol serves as a building material for cellular membranes and plays an important role in cellular metabolism. The brain relies on its own cholesterol biosynthesis, which starts during embryonic development. Cholesterol is synthesized from two immediate precursors, desmosterol and 7-dehydrocholesterol (7-DHC). Mutations in the DHCR24 enzyme, which converts desmosterol into cholesterol, lead to desmosterolosis, an autosomal recessive developmental disorder. In this study, we assessed the brain content of desmosterol, 7-DHC, and cholesterol from development to adulthood, and analyzed the biochemical, molecular, and anatomical consequences of Dhcr24 mutations on the sterol profile in a mouse model of desmosterolosis and heterozygous Dhcr24+/- carriers. Our HPLC-MS/MS studies revealed that by P0 desmosterol almost entirely replaced cholesterol in the Dhcr24-KO brain. The greatly elevated desmosterol levels were also present in the Dhcr24-Het brains irrespective of maternal genotype, persisting into adulthood. Furthermore, Dhcr24-KO mice brains showed complex changes in expression of lipid and sterol transcripts, nuclear receptors, and synaptic plasticity transcripts. Cultured Dhcr24-KO neurons showed increased arborization, which was also present in the Dhcr24-KO mouse brains. Finally, we observed a shared pathophysiological mechanism between the mouse models of desmosterolosis and Smith-Lemli-Opitz syndrome (a genetic disorder of conversion of 7-DHC to cholesterol).

Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

Dichlorophenyl piperazines, including a recently-approved atypical antipsychotic, are potent inhibitors of DHCR7, the last enzyme in cholesterol biosynthesis.

While antipsychotic medications provide important relief from debilitating psychotic symptoms, they also have significant adverse side effects, which might have relevant impact on human health. Several research studies, including ours, have shown that commonly used antipsychotics such as haloperidol and aripiprazole affect cholesterol biosynthesis at the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. This transformation is promoted by the enzyme DHCR7 and its inhibition causes increases in plasma and tissue levels of 7-DHC. The inhibition of this enzymatic step by mutations in the Dhcr7 gene leads to Smith-Lemli-Opitz syndrome, a devastating human condition that can be replicated in rats by small molecule inhibitors of DHCR7. The fact that two compounds, brexpiprazole and cariprazine, that were recently approved by the FDA have substructural elements in common with the DHCR7 inhibitor aripiprazole, prompted us to evaluate the effect of brexpiprazole and cariprazine on cholesterol biosynthesis. We report that cariprazine affects levels of 7-DHC and cholesterol in cell culture incubations at concentrations as low as 5 nM. Furthermore, a common metabolite of cariprazine and aripiprazole, 2,3-(dichlorophenyl) piperazine, inhibits DHCR7 activity at concentrations comparable to those of the potent teratogen AY9944. The cell culture experiments were corroborated in mice in studies showing that treatment with cariprazine elevated 7-DHC in brain and serum. The consequences of sterol inhibition by antipsychotics in the developing nervous system and the safety of their use during pregnancy remains to be established.

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

Smith-Lemli-Opitz syndrome is a recessive disorder caused by mutations in 7-dehydrocholesterol reductase (DHCR)7 with a heterozygous (HET) carrier frequency of 1-3%. A defective DHCR7 causes accumulation of 7-dehydrocholesterol (DHC), which is a highly oxidizable and toxic compound. Recent studies suggest that several antipsychotics, including the highly prescribed pharmaceuticals, aripiprazole (ARI) and trazodone (TRZ), increase 7-DHC levels in vitro and in humans. Our investigation was designed to compare the effects of ARI and TRZ on cholesterol (Chol) synthesis in fibroblasts from DHCR7+/- human carriers and controls (CTRs). Six matched pairs of fibroblasts were treated and their sterol profile analyzed by LC-MS. Significantly, upon treatment with ARI and TRZ, the total accumulation of 7-DHC was higher in DHCR7-HET cells than in CTR fibroblasts. The same set of experiments was repeated in the presence of 13C-lanosterol to determine residual Chol synthesis, revealing that ARI and TRZ strongly inhibit de novo Chol biosynthesis. The results suggest that DHCR7 carriers have increased vulnerability to both ARI and TRZ exposure compared with CTRs. Thus, the 1-3% of the population who are DHCR7 carriers may be more likely to sustain deleterious health consequences on exposure to compounds like ARI and TRZ that increase levels of 7-DHC, especially during brain development.

An altered peripheral IL6 response in major depressive disorder.

Major depressive disorder (MDD) is one of the most prevalent major psychiatric disorders with a lifetime prevalence of 17%. Recent evidence suggests MDD is not only a brain dysfunction, but a systemic disease affecting the whole body. Central and peripheral inflammatory changes seem to be a centerpiece of MDD pathology: a subset of patients show elevated blood cytokine and chemokine levels that partially normalize with symptom improvement over the course of anti-depressant treatment. As this inflammatory process in MDD is poorly understood, we hypothesized that the peripheral tissues of MDD patients will respond differently to inflammatory stimuli, resulting in an aberrant transcriptional response to elevated pro-inflammatory cytokines. To test this, we used MDD patient- and control-derived dermal fibroblast cultures to investigate their response to an acute treatment with IL6, IL1ß, TNFα, or vehicle. Following RNA isolation and subsequent cDNA synthesis, quantitative PCR was used to determine the relative expression level of several families of inflammation-responsive genes. Our results showed comparable expression of the tested genes between MDD patients and controls at baseline. In contrast, MDD patient fibroblasts had a diminished transcriptional response to IL6 in all the gene sets tested (oxidative stress response, mitochondrial function, and lipid metabolism). We also found a significant increase in baseline and IL6 stimulated transcript levels of the IL6 receptor gene. This IL6 receptor transcript increase in MDD fibroblasts was accompanied by an IL6 stimulated increase in induction of SOCS3, which dampens IL6 receptor signaling. Altogether our results demonstrate that there is an altered transcriptional response to IL6 in MDD, which may represent one of the molecular mechanisms contributing to disease pathophysiology. Ultimately we hope that these studies will lead to validation of novel MDD drug targets focused on normalizing the altered IL6 response in patients.

Inhibitors of 7-Dehydrocholesterol Reductase: Screening of a Collection of Pharmacologically Active Compounds in Neuro2a Cells.

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 µM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 µM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 µM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS.

Metabolic stress-induced microRNA and mRNA expression profiles of human fibroblasts.

Metabolic and oxidative stresses induce physiological adaptation processes, disrupting a finely tuned, coordinated network of gene expression. To better understand the interplay between the mRNA and miRNA transcriptomes, we examined how two distinct metabolic stressors alter the expression profile of human dermal fibroblasts. Primary fibroblast cultures were obtained from skin biopsies of 17 healthy subjects. Metabolic stress was evoked by growing subcultured cells in glucose deprived, galactose enriched (GAL) or lipid reduced, cholesterol deficient (RL) media, and compared to parallel-cultured fibroblasts grown in standard (STD) medium. This was followed by mRNA expression profiling and assessment of >1000 miRNAs levels across all three conditions. The miRNA expression levels were subsequently correlated to the mRNA expression profile. Metabolic stress by RL and GAL both produced significant, strongly correlated mRNA/miRNA changes. At the single gene level four miRNAs (miR-129-3p, miR-146b-5p, miR-543 and miR-550a) showed significant and comparable expression changes in both experimental conditions. These miRNAs appeared to have a significant physiological effect on the transcriptome, as nearly 10% of the predicted targets reported changes at mRNA level. The two distinct metabolic stressors induced comparable changes in the miRNome profile, suggesting a common defensive response of the fibroblasts to altered homeostasis. The differentially expressed miR-129-3p, miR-146b-5p, miR-543 and miR-550a regulated multiple genes (e.g. NGEF, NOVA1, PDE5A) with region- and age-specific transcription in the human brain, suggesting that deregulation of these miRNAs might have significant consequences on CNS function. The overall findings suggest that analysis of stress-induced responses of peripheral fibroblasts, obtained from patients with psychiatric disorders is a promising avenue for future research endeavors.

A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome.

We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD "ene" reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.

Chemical Inhibition of Sterol Biosynthesis.

Cholesterol is an essential molecule of life, and its synthesis can be inhibited by both genetic and nongenetic mechanisms. Hundreds of chemicals that we are exposed to in our daily lives can alter sterol biosynthesis. These also encompass various classes of FDA-approved medications, including (but not limited to) commonly used antipsychotic, antidepressant, antifungal, and cardiovascular medications. These medications can interfere with various enzymes of the post-lanosterol biosynthetic pathway, giving rise to complex biochemical changes throughout the body. The consequences of these short- and long-term homeostatic disruptions are mostly unknown. We performed a comprehensive review of the literature and built a catalogue of chemical agents capable of inhibiting post-lanosterol biosynthesis. This process identified significant gaps in existing knowledge, which fall into two main areas: mechanisms by which sterol biosynthesis is altered and consequences that arise from the inhibitions of the different steps in the sterol biosynthesis pathway. The outcome of our review also reinforced that sterol inhibition is an often-overlooked mechanism that can result in adverse consequences and that there is a need to develop new safety guidelines for the use of (novel and already approved) medications with sterol biosynthesis inhibiting side effects, especially during pregnancy.

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.

Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia-ischemia.

Neonatal hypoxic-ischemic brain injury (HIBI) results in part from excess reactive oxygen species and iron-dependent lipid peroxidation (i.e. ferroptosis). The vitamin D precursor 7-dehydrocholesterol (7-DHC) may inhibit iron-dependent lipid peroxidation. Primary neurons underwent oxygen and glucose deprivation (OGD) injury and treatment with 7-DHC-elevating medications such as cariprazine (CAR) or vehicle. Postnatal day 9 mice underwent sham surgery or carotid artery ligation and hypoxia and received intraperitoneal CAR. In neurons, CAR administration resulted in significantly increased cell survival compared to vehicle controls, whether administered 48 h prior to or 30 min after OGD, and was associated with increased 7-DHC. In the mouse model, malondialdehyde and infarct area significantly increased after HIBI in the vehicle group, which were attenuated by post-treatment with CAR and were negatively correlated with tissue 7-DHC concentrations. Elevating 7-DHC concentrations with CAR was associated with improved cellular and tissue viability after hypoxic-ischemic injury, suggesting a novel therapeutic avenue.

CPT2 Deficiency Modeled in Zebrafish: Abnormal Neural Development, Electrical Activity, Behavior, and Schizophrenia-Related Gene Expression.

Carnitine palmitoyltransferase 2 (CPT2) is an inner mitochondrial membrane protein of the carnitine shuttle and is involved in the beta-oxidation of long chain fatty acids. Beta-oxidation provides an alternative pathway of energy production during early development and starvation. CPT2 deficiency is a genetic disorder that we recently showed can be associated with schizophrenia. We hypothesize that CPT2 deficiency during early brain development causes transcriptional, structural, and functional abnormalities that may contribute to a CNS environment that is susceptible to the emergence of schizophrenia. To investigate the effect of CPT2 deficiency on early vertebrate development and brain function, CPT2 was knocked down in a zebrafish model system. CPT2 knockdown resulted in abnormal lipid utilization and deposition, reduction in body size, and abnormal brain development. Axonal projections, neurotransmitter synthesis, electrical hyperactivity, and swimming behavior were disrupted in CPT2 knockdown zebrafish. RT-qPCR analyses showed significant increases in the expression of schizophrenia-associated genes in CPT2 knockdown compared to control zebrafish. Taken together, these data demonstrate that zebrafish are a useful model for studying the importance of beta-oxidation for early vertebrate development and brain function. This study also presents novel findings linking CPT2 deficiency to the regulation of schizophrenia and neurodegenerative disease-associated genes.