Decoding the Synaptic Proteome with Long-Term Exposure to Midazolam during Early Development.

The intensive use of anesthetic and sedative agents in the neonatal intensive care unit (NICU) has raised controversial concerns about the potential neurodevelopmental risks. This study focused on midazolam (MDZ), a common benzodiazepine regularly used as a sedative on neonates in the NICU. Mounting evidence suggests a single exposure to MDZ during the neonatal period leads to learning disturbances. However, a knowledge gap that remains is how long-term exposure to MDZ during very early stages of life impacts synaptic alterations. Using a preclinical rodent model system, we mimicked a dose-escalation regimen on postnatal day 3 (P3) pups until day 21. Next, purified synaptosomes from P21 control and MDZ animals were subjected to quantitative mass-spectrometry-based proteomics, to identify potential proteomic signatures. Further analysis by ClueGO identified enrichment of proteins associated with actin-binding and protein depolymerization process. One potential hit identified was alpha adducin (ADD1), belonging to the family of cytoskeleton proteins, which was upregulated in the MDZ group and whose expression was further validated by Western blot. In summary, this study sheds new information on the long-term exposure of MDZ during the early stages of development impacts synaptic function, which could subsequently perturb neurobehavioral outcomes at later stages of life.

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.

Role of Extracellular Vesicles in Substance Abuse and HIV-Related Neurological Pathologies.

Extracellular vesicles (EVs) are a broad, heterogeneous class of membranous lipid-bilayer vesicles that facilitate intercellular communication throughout the body. As important carriers of various types of cargo, including proteins, lipids, DNA fragments, and a variety of small noncoding RNAs, including miRNAs, mRNAs, and siRNAs, EVs may play an important role in the development of addiction and other neurological pathologies, particularly those related to HIV. In this review, we summarize the findings of EV studies in the context of methamphetamine (METH), cocaine, nicotine, opioid, and alcohol use disorders, highlighting important EV cargoes that may contribute to addiction. Additionally, as HIV and substance abuse are often comorbid, we discuss the potential role of EVs in the intersection of substance abuse and HIV. Taken together, the studies presented in this comprehensive review shed light on the potential role of EVs in the exacerbation of substance use and HIV. As a subject of growing interest, EVs may continue to provide information about mechanisms and pathogenesis in substance use disorders and CNS pathologies, perhaps allowing for exploration into potential therapeutic options.

Chronic SIV and morphine treatment increases heat shock protein 5 expression at the synapse.

The abuse of opiates such as morphine in synergy with HIV infection accelerates neurocognitive impairments and neuropathology in the CNS of HIV-infected subjects, collectively referred to as HAND. To identify potential pathogenic markers associated with HIV and morphine in perturbing the synaptic architecture, we performed quantitative mass spectrometry proteomics on purified synaptosomes isolated from the caudate of two groups of rhesus macaques chronically infected with SIV differing by one regimen-morphine treatment. The upregulation of heat shock 70-kDa protein 5 in the SIV + morphine group points to increased cellular stress during SIV/morphine interaction thus leading to CNS dysfunction.

Impaired synaptic development in a maternal immune activation mouse model of neurodevelopmental disorders.

Both genetic and environmental factors are thought to contribute to neurodevelopmental and neuropsychiatric disorders with maternal immune activation (MIA) being a risk factor for both autism spectrum disorders and schizophrenia. Although MIA mouse offspring exhibit behavioral impairments, the synaptic alterations in vivo that mediate these behaviors are not known. Here we employed in vivo multiphoton imaging to determine that in the cortex of young MIA offspring there is a reduction in number and turnover rates of dendritic spines, sites of majority of excitatory synaptic inputs. Significantly, spine impairments persisted into adulthood and correlated with increased repetitive behavior, an ASD relevant behavioral phenotype. Structural analysis of synaptic inputs revealed a reorganization of presynaptic inputs with a larger proportion of spines being contacted by both excitatory and inhibitory presynaptic terminals. These structural impairments were accompanied by altered excitatory and inhibitory synaptic transmission. Finally, we report that a postnatal treatment of MIA offspring with the anti-inflammatory drug ibudilast, prevented both synaptic and behavioral impairments. Our results suggest that a possible altered inflammatory state associated with maternal immune activation results in impaired synaptic development that persists into adulthood but which can be prevented with early anti-inflammatory treatment.

Neurobehavioral Characterization of Perinatal Oxycodone-Exposed Offspring in Early Adolescence.

The opioid epidemic has received considerable attention, but the impact on perinatal opioid-exposed (POE) offspring remains underexplored. This study addresses the emerging public health challenge of understanding and treating POE children. We examined two scenarios using preclinical models: offspring exposed to oxycodone (OXY) in utero (IUO) and acute postnatal OXY (PNO). We hypothesized exposure to OXY during pregnancy primes offspring for neurodevelopmental deficits and severity of deficits is dependent on timing of exposure. Notable findings include reduced head size and brain weight in offspring. Molecular analyses revealed significantly lower levels of inflammasome-specific genes in the prefrontal cortex (PFC). Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) highlighted the enrichment of genes associated with mitochondrial and synapse dysfunction in POE offspring. Western blot analysis validated IPA predictions of mitochondrial dysfunction in PFC-derived synaptosomes. Behavioral studies identified significant social deficits in POE offspring. This study presents the first comparative analysis of acute PNO- and IUO-offspring during early adolescence finding acute PNO-offspring have considerably greater deficits. The striking difference in deficit severity in acute PNO-offspring suggests that exposure to opioids in late pregnancy pose the greatest risk for offspring well-being.

Role of the Gut Bacteria-Derived Metabolite Phenylacetylglutamine in Health and Diseases.

Over the past few decades, it has been well established that gut microbiota-derived metabolites can disrupt gut function, thus resulting in an array of diseases. Notably, phenylacetylglutamine (PAGln), a bacterial derived metabolite, has recently gained attention due to its role in the initiation and progression of cardiovascular and cerebrovascular diseases. This meta-organismal metabolite PAGln is a byproduct of amino acid acetylation of its precursor phenylacetic acid (PAA) from a range of dietary sources like egg, meat, dairy products, etc. The microbiota-dependent metabolism of phenylalanine produces PAA, which is a crucial intermediate that is catalyzed by diverse microbial catalytic pathways. PAA conjugates with glutamine and glycine in the liver and kidney to predominantly form phenylacetylglutamine in humans and phenylacetylglycine in rodents. PAGln is associated with thrombosis as it enhances platelet activation mediated through the GPCRs receptors α2A, α2B, and ß2 ADRs, thereby aggravating the pathological conditions. Clinical evidence suggests that elevated levels of PAGln are associated with pathology of cardiovascular, cerebrovascular, and neurological diseases. This Review further consolidates the microbial/biochemical synthesis of PAGln and discusses its role in the above pathophysiologies.

Developmental outcomes with perinatal exposure (DOPE) to prescription opioids.

Researchers have found considerable evidence in the past 20 years that perinatal opioid exposure leads to an increased risk of developmental disorders in offspring that persist into adulthood. The use of opioids to treat pain concerning pregnancy, delivery, and postpartum complications has been rising. As a result, communities have reported a 300-400 % increase in Neonatal Opioid Withdrawal Syndrome (NOWS). NOWS represents the initial stage of several behavioral, phenotypic, and synaptic deficits. This review article summarizes the Developmental Outcomes of Perinatal Exposure (DOPE) to prescription opioids. Moreover, we also seek to connect these findings to clinical research that describes DOPE at multiple stages of life. Since specific mechanisms that underlie DOPE remain unclear, this article aims to provide a framework for conceptualizing across all ages and highlight the implications they may have for longevity.

The 27th Scientific Conference of the Society on NeuroImmune Pharmacology: New Delhi, India, March 15-18, 2023.

The 27th Scientific Conference of the Society on Neuroimmune Pharmacology (SNIP) in New Delhi, India, on March 15-18, 2023 is a historic summit of experts from around the world. The four day conference provides insights into the latest and most advanced science in the intersecting areas of neuroscience, immunology, pharmacology, and its translational aspects, in particular, HIV and drug abuse. Abstracts are ordered in three major groups: (1) Symposium speakers (S1-S64), (2) Investigator Posters (I1-I18), and (3) Trainee Poster (T1-T28).

In utero and post-natal opioid exposure followed by mild traumatic brain injury contributes to cortical neuroinflammation, mitochondrial dysfunction, and behavioral deficits in juvenile rats.

Maternal opioid use poses a significant health concern not just to the expectant mother but also to the fetus. Notably, increasing numbers of children born suffering from neonatal opioid withdrawal syndrome (NOWS) further compounds the crisis. While epidemiological research has shown the heightened risk factors associated with NOWS, little research has investigated what molecular mechanisms underly the vulnerabilities these children carry throughout development and into later life. To understand the implications of in utero and post-natal opioid exposure on the developing brain, we sought to assess the response to one of the most common pediatric injuries: minor traumatic brain injury (mTBI). Using a rat model of in utero and post-natal oxycodone (IUO) exposure and a low force weight drop model of mTBI, we show that not only neonatal opioid exposure significantly affects neuroinflammation, brain metabolites, synaptic proteome, mitochondrial function, and altered behavior in juvenile rats, but also, in conjunction with mTBI these aberrations are further exacerbated. Specifically, we observed long term metabolic dysregulation, neuroinflammation, alterations in synaptic mitochondria, and impaired behavior were impacted severely by mTBI. Our research highlights the specific vulnerability caused by IUO exposure to a secondary stressor such as later life brain injury. In summary, we present a comprehensive study to highlight the damaging effects of prenatal opioid abuse in conjunction with mild brain injury on the developing brain.

An Integrated Systems Approach to Decode the Impact of Adolescent Nicotine Exposure in Utero and Postnatally Oxycodone Exposed Offspring.

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found alterations of the blood-brain barrier (B.B.B.) and synaptic proteins. RT-qPCR further validated immune dysfunction in the central nervous system (CNS) consistent with compromised B.B.B. Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning PNO & IUO, a predictor of greater addiction risk. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal.

Impact of Adolescent Nicotine Exposure in Pre- and Post-natal Oxycodone Exposed Offspring.

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found synaptic protein alterations, especially upregulation of synaptophysin in IUO-withdrawal animals. RT-qPCR further validated immune dysfunction in the central nervous system (CNS). Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning IUO animals. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal. Graphical Abstract.

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.

Novel RNA-Seq Signatures Post-Methamphetamine and Oxycodone Use in a Model of HIV-Associated Neurocognitive Disorders.

In the 21st century, the effects of HIV-associated neurocognitive disorders (HAND) have been significantly reduced in individuals due to the development of antiretroviral therapies (ARTs). However, the growing epidemic of polysubstance use (PSU) has led to concern for the effects of PSU on HIV-seropositive individuals. To effectively treat individuals affected by HAND, it is critical to understand the biological mechanisms affected by PSU, including the identification of novel markers. To fill this important knowledge gap, we used an in vivo HIV-1 Transgenic (HIV-1 Tg) animal model to investigate the effects of the combined use of chronic methamphetamine (METH) and oxycodone (oxy). A RNA-Seq analysis on the striatum-a brain region that is primarily targeted by both HIV and drugs of abuse-identified key differentially expressed markers post-METH and oxy exposure. Furthermore, ClueGO analysis and Ingenuity Pathway Analysis (IPA) revealed crucial molecular and biological functions associated with ATP-activated adenosine receptors, neuropeptide hormone activity, and the oxytocin signaling pathway to be altered between the different treatment groups. The current study further reveals the harmful effects of chronic PSU and HIV infection that can subsequently impact neurological outcomes in polysubstance users with HAND.

The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption.

Period (Per) genes are involved in regulation of the circadian clock and are thought to modulate several brain functions. We demonstrate that Per2(Brdm1) mutant mice, which have a deletion in the PAS domain of the Per2 protein, show alterations in the glutamatergic system. Lowered expression of the glutamate transporter Eaat1 is observed in these animals, leading to reduced uptake of glutamate by astrocytes. As a consequence, glutamate levels increase in the extracellular space of Per2(Brdm1) mutant mouse brains. This is accompanied by increased alcohol intake in these animals. In humans, variations of the PER2 gene are associated with regulation of alcohol consumption. Acamprosate, a drug used to prevent craving and relapse in alcoholic patients is thought to act by dampening a hyper-glutamatergic state. This drug reduced augmented glutamate levels and normalized increased alcohol consumption in Per2(Brdm1) mutant mice. Collectively, these data establish glutamate as a link between dysfunction of the circadian clock gene Per2 and enhanced alcohol intake.

HIV-1 Tat and cocaine impact astrocytic energy reservoirs and epigenetic regulation by influencing the LINC01133-hsa-miR-4726-5p-NDUFA9 axis.

Clinical research has proven that HIV-positive (HIV+) individuals with cocaine abuse show behavioral and neurocognitive disorders. Noncoding RNAs (ncRNAs), such as long ncRNAs (lncRNAs) and microRNAs (miRNAs), are known to regulate gene expression in the contexts of HIV infection and drug abuse. However, there are no specific lncRNA or miRNA biomarkers associated with HIV-1 Transactivator of transcription protein (Tat) and cocaine coexposure. In the central nervous system (CNS), astrocytes are the primary regulators of energy metabolism, and impairment of the astrocytic energy supply can trigger neurodegeneration. The aim of this study was to uncover the roles of lncRNAs and miRNAs in the regulation of messenger RNA (mRNA) targets affected by HIV infection and cocaine abuse. Integrative bioinformatics analysis revealed altered expression of 10 lncRNAs, 10 miRNAs, and 4 mRNA/gene targets in human primary astrocytes treated with cocaine and HIV-1 Tat. We assessed the alterations in the expression of two miRNAs, hsa-miR-2355 and hsa-miR-4726-5p; four lncRNAs, LINC01133, H19, HHIP-AS1, and NOP14-AS1; and four genes, NDUFA9, KYNU, HKDC1, and LIPG. The results revealed interactions in the LINC01133-hsa-miR-4726-5p-NDUFA9 axis that may eventually help us understand cocaine- and HIV-1 Tat-induced astrocyte dysfunction that may ultimately result in neurodegeneration.

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.

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.

Metabolomic analysis of the cerebrospinal fluid reveals changes in phospholipase expression in the CNS of SIV-infected macaques.

HIV infiltrates the CNS soon after an individual has become infected with the virus, and can cause dementia and encephalitis in late-stage disease. Here, a global metabolomics approach was used to find and identify metabolites differentially regulated in the cerebrospinal fluid (CSF) of rhesus macaques with SIV-induced CNS disease, as we hypothesized that this might provide biomarkers of virus-induced CNS damage. The screening platform used a non-targeted, mass-based metabolomics approach beginning with capillary reverse phase chromatography and electrospray ionization with accurate mass determination, followed by novel, nonlinear data alignment and online database screening to identify metabolites. CSF was compared before and after viral infection. Significant changes in the metabolome specific to SIV-induced encephalitis were observed. Metabolites that were increased during infection-induced encephalitis included carnitine, acyl-carnitines, fatty acids, and phospholipid molecules. The elevation in free fatty acids and lysophospholipids correlated with increased expression of specific phospholipases in the brains of animals with encephalitis. One of these, a phospholipase A2 isoenzyme, is capable of releasing a number of the fatty acids identified. It was expressed in different areas of the brain in conjunction with glial activation, rather than linked to regions of SIV infection and inflammation, indicating widespread alterations in infected brains. The identification of specific metabolites as well as mechanisms of their increase illustrates the potential of mass-based metabolomics to address problems in CNS biochemistry and neurovirology, as well as neurodegenerative diseases.

Generational Effects of Opioid Exposure.

The inheritance of substance abuse, including opioid abuse, may be influenced by genetic and non-genetic factors related to the environment, such as stress and socioeconomic status. These non-genetic influences on the heritability of a trait can be attributed to epigenetics. Epigenetic inheritance can result from modifications passed down from the mother, father, or both, resulting in either maternal, paternal, or parental epigenetic inheritance, respectively. These epigenetic modifications can be passed to the offspring to result in multigenerational, intergenerational, or transgenerational inheritance. Human and animal models of opioid exposure have shown generational effects that result in molecular, developmental, and behavioral alterations in future generations.