Roles of Extracellular Vesicles in Opioid Addiction: Potential Applications

Extracellular Vesicles (EVs) are lipid-bilayer membranous vesicles that facilitate intercellular communication via their secretion. EVs contain a variety of cargoes that reflect the intracellular environment of their host cells, and these cargoes can induce functional changes in recipient cells. A wide body of previous research has demonstrated that EVs play a role in a diverse range of disease pathologies as well as regular function and have emerged as promising vehicles for therapeutics and drug-delivery systems. Unsurprisingly, some work has recently been published implicating EVs in drug addiction pathways and therapeutics. Given the pressing scope of the opioid misuse and abuse in the U.S., it is necessary to consider the role of EVs in the development of opioid dependence and tolerance, as well as their role in potential therapeutics. The current review seeks to identify work investigating the role of EVs in opioid addiction and identify gaps and future directions in the literature.https://digitalcommons.unmc.edu/surp2020/1024/thumbnail.jp

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure

MicroRNA-142 reduces monoamine oxidase A expression and activity in neuronal cells by downregulating SIRT1

Aberrant expression of microRNAs (miRs) has been implicated in the pathogenesis of several neurodegenerative disorders. In HIV-associated neurocognitive disorders (HAND), miR-142 was found to be upregulated in neurons and myeloid cells in the brain. We investigated the downstream effects of chronic miR-142 upregulation in neuronal cells by comparing gene expression in stable clones of the human neuroblastoma cell line BE(2)M17 expressing miR-142 to controls. Microarray analysis revealed that miR-142 expression led to a reduction in monoamine oxidase (MAO) A mRNA, which was validated by qRT-PCR. In addition to the mRNA, the MAOA protein level and enzyme activity were also reduced. Examination of primary human neurons revealed that miR-142 expression indeed resulted in a downregulation of MAOA protein level. Although MAOA is not a direct target of miR-142, SIRT1, a key transcriptional upregulator of MAOA is, thus miR-142 downregulation of MAOA expression is indirect. MiR-142 induced decrease in MAOA expression and activity may contribute to the changes in dopaminergic neurotransmission reported in HAND

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

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure

Upregulation of cathepsin D in the caudate nucleus of primates with experimental parkinsonism

<p>Abstract</p> <p>Background</p> <p>In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease.</p> <p>Results</p> <p>Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. <it>In vitro </it>overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis.</p> <p>Conclusions</p> <p>Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our <it>in vitro </it>studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.</p

Integrated Systems Analysis of Mixed Neuroglial Cultures Proteome Post Oxycodone Exposure

Opioid abuse has become a major public health crisis that affects millions of individuals across the globe. This widespread abuse of prescription opioids and dramatic increase in the availability of illicit opioids have created what is known as the opioid epidemic. Pregnant women are a particularly vulnerable group since they are prescribed for opioids such as morphine, buprenorphine, and methadone, all of which have been shown to cross the placenta and potentially impact the developing fetus. Limited information exists regarding the effect of oxycodone (oxy) on synaptic alterations. To fill this knowledge gap, we employed an integrated system approach to identify proteomic signatures and pathways impacted on mixed neuroglial cultures treated with oxy for 24 h. Differentially expressed proteins were mapped onto global canonical pathways using ingenuity pathway analysis (IPA), identifying enriched pathways associated with ephrin signaling, semaphorin signaling, synaptic long-term depression, endocannabinoid signaling, and opioid signaling. Further analysis by ClueGO identified that the dominant category of differentially expressed protein functions was associated with GDP binding. Since opioid receptors are G-protein coupled receptors (GPCRs), these data indicate that oxy exposure perturbs key pathways associated with synaptic function

Methamphetamine Induces the Release of Proadhesive Extracellular Vesicles and Promotes Syncytia Formation: A Potential Role in HIV-1 Neuropathogenesis

Despite the success of combinational antiretroviral therapy (cART), the high pervasiveness of human immunodeficiency virus-1 (HIV)-associated neurocognitive disorders (HAND) poses a significant challenge for society. Methamphetamine (meth) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. Intriguingly, HIV-infected individuals who are meth users have a comparatively higher rate of neuropsychological impairment and exhibit a higher viral load in the brain than infected individuals who do not abuse meth. Effectively, all cell types secrete nano-sized lipid membrane vesicles, referred to as extracellular vesicles (EVs) that can function as intercellular communication to modulate the physiology and pathology of the cells. This study shows that meth treatments on chronically HIV-infected promonocytic U1 cells induce the release of EVs that promote cellular clustering and syncytia formation, a phenomenon that facilitates HIV pathogenesis. Our analysis also revealed that meth exposure increased intercellular adhesion molecule-1 (ICAM-1) and HIV-Nef protein expression in both large (10 K) and small (100 K) EVs. Further, when meth EVs are applied to uninfected naïve monocyte-derived macrophages (MDMs), we saw a significant increase in cell clustering and syncytia formation. Furthermore, treatment of MDMs with antibodies against ICAM-1 and its receptor, lymphocyte function-associated antigen 1 (LFA1), substantially blocked syncytia formation, and consequently reduced the number of multinucleated cells. In summary, our findings reveal that meth exacerbates HIV pathogenesis in the brain through release of proadhesive EVs, promoting syncytia formation and thereby aiding in the progression of HIV infection in uninfected cells

Effect of Combined Methamphetamine and Oxycodone Use on the Synaptic Proteome in an In Vitro Model of Polysubstance Use

Polysubstance use (PSU) generally involves the simultaneous use of an opioid along with a stimulant. In recent years, this problem has escalated into a nationwide epidemic. Understanding the mechanisms and effects underlying the interaction between these drugs is essential for the development of treatments for those suffering from addiction. Currently, the effect of PSU on synapses-critical points of contact between neurons-remains poorly understood. Using an in vitro model of primary neurons, we examined the combined effects of the psychostimulant methamphetamine (METH) and the prescription opioid oxycodone (oxy) on the synaptic proteome using quantitative mass-spectrometry-based proteomics. A further ClueGO analysis and Ingenuity Pathway Analysis (IPA) indicated the dysregulation of several molecular functions, biological processes, and pathways associated with neural plasticity and structural development. We identified one key synaptic protein, Striatin-1, which plays a vital role in many of these processes and functions, to be downregulated following METH+oxy treatment. This downregulation of Striatin-1 was further validated by Western blot. Overall, the present study indicates several damaging effects of the combined use of METH and oxy on neural function and warrants further detailed investigation into mechanisms contributing to synaptic dysfunction

Identification of YWHAH as a Novel Brain-Derived Extracellular Vesicle Marker Post Long-Term Midazolam Exposure during Early Development

Recently, the long-term use of sedative agents in the neonatal intensive care unit (NICU) has raised concerns about neurodevelopmental outcomes in exposed neonates. Midazolam (MDZ), a common neonatal sedative in the NICU, has been suggested to increase learning disturbances and cognitive impairment in children. However, molecular mechanisms contributing to such outcomes with long-term MDZ use during the early stages of life remain unclear. In this study, we for the first time elucidate the role of brain-derived extracellular vesicles (BDEVs), including mining the BDEV proteome post long-term MDZ exposure during early development. Employing our previously established rodent model system that mimics the exposure of MDZ in the NICU using an increasing dosage regimen, we isolated BDEVs from postnatal 21-days-old control and MDZ groups using a differential sucrose density gradient. BDEVs from the control and MDZ groups were then characterized using a ZetaView nanoparticle tracking analyzer and transmission electron microscopy analysis. Next, using RT-qPCR, we examined the expression of key ESCRT-related genes involved in EV biogenesis. Lastly, using quantitative mass spectrometry-based proteomics, we mined the BDEV protein cargo that revealed key differentially expressed proteins and associated molecular pathways to be altered post long-term MDZ exposure. Our study characterized the proteome in BDEV cargo from long-term MDZ exposure at early development. Importantly, we identified and validated the expression of YWHAH as a potential target for further characterization of its downstream mechanism and a potential biomarker for the early onset of neurodevelopment and neurodegenerative diseases. Overall, the present study demonstrated long-term exposure to MDZ at early development stages could influence BDEV protein cargo, which potentially impact neural functions and behavior at later stages of development