Therapeutic Potential of Suvorexant on Intergenerational Maternal Oxycodone Exposure

Background: Maternal opioid misuse is a rising public health concern. Our lab previously published findings that in-utero oxycodone exposure (IUO) has detrimental impacts that persist to the F2 generation, including abnormal genetic expression, increased anxiety, and a difference in phenotypic measurements. The estimated cost of hospital admissions for infants suffering from Neonatal Abstinence Syndrome was $316 million in 2012 and is still rising. This figure does not take into account long-term costs, nor does it consider lasting effects on the F2 generation. Thus, it is critical to find a way to mitigate the negative impacts of IUO. Suvorexant (suvo) is a dual hypocretin receptor antagonist that is FDA-approved for the treatment of insomnia. The hypocretin system is involved in the regulation of the sleep/wake cycle, feeding behavior, and notably, addiction. Our previous findings showed that Hcrtr1 is upregulated in both F1 and F2 IUO offspring. This project will test the therapeutic potential of suvorexant to attenuate the impacts of IUO. Hypothesis: The administration of suvorexant on F1 animals that have been subjected to IUO will result in the alleviation of developmental impairments in the F2 generation. Methods: Female Sprague Dawley rats in the F0 generation were orally gavaged with 15mg/kg oxycodone or equal volumes of saline. Dosing was maintained from mating until weaning at post-natal day 21 (P21). F1 animals were given ascending doses of suvorexant (3mg/kg P3-P6, 10mg/kg P7-P10, 30mg/kg P11-P21) or an equal volume of DMSO through subcutaneous injection. At P60, 2 females from each condition were mated with naïve breeders. Phenotypic measurements of the F2 generation including weight, head size circumference, and body length were taken at P3 and P14. At P21, 6-8 animals were sacrificed, and organs were collected. Social preference/novelty tests were conducted at P28 and P45. The next phase of this study will consist of molecular assays, imaging, and further behavior testing including an oxycodone self-administration study. Results: Our preliminary results showed that F2 IUO-Suvo animals exhibited significant differences in body weight, body length, and head size circumference at P7 and P14 compared to the control. Furthermore, in the social preference test, the IUO-Suvo animals had significantly more entries into both the toy and naïve chambers at P45, as well as significantly more contacts with both the toy and naïve animal at P28. Conclusions: Administering suvorexant in the F1 generation may mitigate physical and behavioral deficits in the F2 generation caused by intergenerational IUO.https://digitalcommons.unmc.edu/chri_forum/1066/thumbnail.jp

A Holistic Systems Approach to Characterize the Impact of Pre- and Post-natal Oxycodone Exposure on Neurodevelopment and Behavior

Background: Increased risk of oxycodone (oxy) dependency during pregnancy has been associated with altered behaviors and cognitive deficits in exposed offspring. However, a significant knowledge gap remains regarding the effect of in utero and postnatal exposure on neurodevelopment and subsequent behavioral outcomes. Methods: Using a preclinical rodent model that mimics oxy exposure in utero (IUO) and postnatally (PNO), we employed an integrative holistic systems biology approach encompassing proton magnetic resonance spectroscopy (1H-MRS), electrophysiology, RNA-sequencing, and Von Frey pain testing to elucidate molecular and behavioral changes in the exposed offspring during early neurodevelopment as well as adulthood. Results: 1H-MRS studies revealed significant changes in key brain metabolites in the exposed offspring that were corroborated with changes in synaptic currents. Transcriptomic analysis employing RNA-sequencing identified alterations in the expression of pivotal genes associated with synaptic transmission, neurodevelopment, mood disorders, and addiction in the treatment groups. Furthermore, Von Frey analysis revealed lower pain thresholds in both exposed groups. Conclusions: Given the increased use of opiates, understanding the persistent developmental effects of these drugs on children will delineate potential risks associated with opiate use beyond the direct effects in pregnant women

Morphology and virulence in C. albicans: Determining what role KRE5 gene has in the filamentation process of Candida albicans

Candida albicans is a human fungal pathogen that causes candidiasis, a potentially lethal systemic disease. The morphology of C. albicans is an important factor for pathogenesis, and the ability to transition between forms is essential for disease. A recent study in our lab identified a number of genes that are vital for the switch between yeast like form, including the glucosyltransferase gene KRE5. A number of pathways are known to regulate filamentation and KRE5 has a previously unknown role in one of these pathways. To determine which pathway it belongs to, 7 genes were overexpressed within in the kre5Δ/Δ mutant strain which are downstream components of the known filamentation-regulatory pathways. The overexpressed genes were cleaved from the plasmid backbone through digestion followed by transformation of these overexpressed gene in kre5Δ/Δ mutant strain. A colony PCR was run in order to determine if the overexpressed construct inserted into the genomic DNA in the right location. Positive transformants have been collected for each over expression construct, but colony PCR has not identified correct placement of the over expression construct. These transformants will then be tested in various types of media to determine if filamentation was restored in the mutant cells

MicroRNA cluster miR199a/214 are differentially expressed in female and male rats following nicotine self-administration

Previous research has established sex differences associated with nicotine intake, however a significant gap in knowledge remains regarding the molecular mechanisms that govern these differences at the transcriptional level. One critical regulator of transcription are microRNAs (miRNAs). miRNAs are a family of non-coding RNAs that regulate an array of important biological functions altered in several disease states, including neuroadaptive changes within the brain associated with drug dependence. We examined the prefrontal cortex (PFC) from male and female Sprague-Dawley rats following self-administration (22 days) of nicotine or yoked saline controls using next generation RNA-Sequencing (RNA-Seq) technology and found an array of miRNAs to be significantly and differentially regulated by nicotine self-administration. Of these, we found the expression of miR-199a and 214, which are expressed on the same cluster of chromosome 1, to be upregulated in the female rats exposed to nicotine; upregulation in this group was further validated by real time polymerase chain reaction (RTPCR). Bioinformatics analysis to assess common targets of miR-199/214 identified Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD)- dependent deacetylase that plays a role in apoptosis, neuron survival, and stress resistance. Using western-blot, we confirmed downregulation of SIRT1 and increased cleaved caspase 3 expression in the brains of nicotine-exposed female rats and no change in expression levels in the other groups. Collectively, our findings highlight a miR-199/214 regulatory network that, through SIRT1, may be associated with nicotine seeking in females which may serve as a potential therapeutic target for sex-specific treatment approaches

Role of Brain Derived Extracellular Vesicles in Decoding Sex Differences Associated with Nicotine Self-Administration

Smoking remains a significant health and economic concern in the United States. Furthermore, the emerging pattern of nicotine intake between sexes further adds a layer of complexity. Nicotine is a potent psychostimulant with a high addiction liability that can significantly alter brain function. However, the neurobiological mechanisms underlying nicotine’s impact on brain function and behavior remain unclear. Elucidation of these mechanisms is of high clinical importance and may lead to improved therapeutics for smoking cessation. To fill in this critical knowledge gap, our current study focused on identifying sex-specific brain-derived extracellular vesicles (BDEV) signatures in male and female rats post nicotine self-administration. Extracellular vesicles (EVs) are comprised of phospholipid nanovesicles such as apoptotic bodies, microvesicles (MVs), and exosomes based on their origin or size. EVs are garnering significant attention as molecules involved in cell–cell communication and thus regulating the pathophysiology of several diseases. Interestingly, females post nicotine self-administration, showed larger BDEV sizes, along with impaired EV biogenesis compared to males. Next, using quantitative mass spectrometry-based proteomics, we identified BDEV signatures, including distinct molecular pathways, impacted between males and females. In summary, this study has identified sex-specific changes in BDEV biogenesis, protein cargo signatures, and molecular pathways associated with long-term nicotine self-administration

Distinct Synaptic Vesicle Proteomic Signatures Associated with Pre- and Post-Natal Oxycodone-Exposure

The current opioid crisis, which has ravaged all segments of society, continues to pose a rising public health concern. Importantly, dependency on prescription opioids such as oxycodone (oxy) during and after pregnancy can significantly impact the overall brain development of the exposed offspring, especially at the synapse. A significant knowledge gap that remains is identifying distinct synaptic signatures associated with these exposed offspring. Accordingly, the overall goal of this current study was to identify distinct synaptic vesicle (SV) proteins as signatures for offspring exposed to oxy in utero (IUO) and postnatally (PNO). Using a preclinical animal model that imitates oxycodone exposure in utero (IUO) and postnatally (PNO), we used a quantitative mass spectrometry-based proteomics platform to examine changes in the synaptic vesicle proteome on post-natal day 14 (P14) IUO and PNO offspring. We identified MEGF8, associated with carpenter syndrome, to be downregulated in the IUO offspring while LAMTOR4, associated with the regulator complex involved in lysosomal signaling and trafficking, was found to be upregulated in the PNO groups, respectively. Their respective differential expression was further validated by Western blot. In summary, our current study shows exposure to oxy in utero and postnatally can impact the SV proteome in the exposed offspring and the identification of these distinct SV signatures could further pave the way to further elucidate their downstream mechanisms including developing them as potential therapeutic targets

Role of Brain Derived Extracellular Vesicles in Decoding Sex Differences Associated with Nicotine Self-Administration

Smoking remains a significant health and economic concern in the United States. Furthermore, the emerging pattern of nicotine intake between sexes further adds a layer of complexity. Nicotine is a potent psychostimulant with a high addiction liability that can significantly alter brain function. However, the neurobiological mechanisms underlying nicotine’s impact on brain function and behavior remain unclear. Elucidation of these mechanisms is of high clinical importance and may lead to improved therapeutics for smoking cessation. To fill in this critical knowledge gap, our current study focused on identifying sex-specific brain-derived extracellular vesicles (BDEV) signatures in male and female rats post nicotine self-administration. Extracellular vesicles (EVs) are comprised of phospholipid nanovesicles such as apoptotic bodies, microvesicles (MVs), and exosomes based on their origin or size. EVs are garnering significant attention as molecules involved in cell–cell communication and thus regulating the pathophysiology of several diseases. Interestingly, females post nicotine self-administration, showed larger BDEV sizes, along with impaired EV biogenesis compared to males. Next, using quantitative mass spectrometry-based proteomics, we identified BDEV signatures, including distinct molecular pathways, impacted between males and females. In summary, this study has identified sex-specific changes in BDEV biogenesis, protein cargo signatures, and molecular pathways associated with long-term nicotine self-administration