Neurobeachin, a promising target for use in the treatment of alcohol use disorder.

Hazardous, heavy drinking increases risk for developing alcohol use disorder (AUD), which affects ~7% of adult Americans. Thus, understanding the molecular mechanisms promoting risk for heavy drinking is essential to developing more effective AUD pharmacotherapies than those currently approved by the FDA. Using genome-wide bisulfate sequencing, we identified DNA methylation (DNAm) signals within the nucleus accumbens core (NAcC) that differentiate nonheavy and heavy ethanol-drinking rhesus macaques. One differentially DNAm region (D-DMR) located within the gene neurobeachin (NBEA), which promotes synaptic membrane protein trafficking, was hypermethylated in heavy drinking macaques. A parallel study identified a similar NBEA D-DMR in human NAcC that distinguished alcoholic and nonalcoholic individuals. To investigate the role of NBEA in heavy ethanol drinking, we engineered a viral vector carrying a short hairpin RNA (shRNA) to reduce the expression of NBEA. Using two murine models of ethanol consumption: 4 days of drinking-in-the-dark and 4 weeks of chronic intermittent access, the knockdown of NBEA expression did not alter average ethanol consumption in either model. However, it did lead to a significant increase in the ethanol preference ratio. Following withdrawal, whole-cell patch clamp electrophysiological experiments revealed that Nbea knockdown led to an increase in spontaneous excitatory postsynaptic current amplitude with no alteration in spontaneous inhibitory postsynaptic currents, suggesting a specific role of NBEA in trafficking of glutamatergic receptors. Together, our findings suggest that NBEA could be targeted to modulate the preference for alcohol use.

A Comparative Study of the Pharmacokinetics of Clozapine N-Oxide and Clozapine N-Oxide Hydrochloride Salt in Rhesus Macaques.

Translating chemogenetic techniques from nonhuman primates to potential clinical applications has been complicated in part due to in vivo conversion of the chemogenetic actuator, clozapine N-oxide (CNO), to its pharmacologically active parent compound, clozapine, a ligand with known side effects, including five boxed warnings from the Food and Drug Administration. Additionally, the limited solubility of CNO requires high concentrations of potentially toxic detergents such as dimethylsulfoxide (DMSO). To address these concerns, pharmacokinetic profiling of commercially available CNO in DMSO (CNO-DMSO, 10% v/v DMSO in saline) and a water-soluble salt preparation (CNO-HCl, saline) was conducted in rhesus macaques. A time course of blood plasma and cerebrospinal fluid (CSF) concentrations of CNO and clozapine was conducted (30-240 minutes post-administration) following a range of doses (3-10 mg/kg, i.m. and/or i.v.) of CNO-DMSO or CNO-HCl. CNO-HCl resulted in 6- to 7-fold higher plasma concentrations of CNO compared to CNO-DMSO, and relatively less clozapine (3%-5% clozapine/CNO in the CNO-DMSO group and 0.5%-1.5% clozapine/CNO in the CNO-HCl group). Both groups had large between-subjects variability, pointing to the necessity of performing individual CNO pharmacokinetic studies prior to further experimentation. The ratio of CNO measured in the CSF was between 2% and 6% of that measured in the plasma and did not differ across drug preparation, indicating that CSF concentrations may be approximated from plasma samples. In conclusion, CNO-HCl demonstrated improved bioavailability compared with CNO-DMSO with less conversion to clozapine. Further investigation is needed to determine if brain concentrations of clozapine following CNO-HCl administration are pharmacologically active at off-target monoaminergic receptor systems in the primate brain.

Hospital-Acquired Sacral Pressure Ulcer Complicated by a Spinal Epidural Abscess.

A spinal epidural abscess is a rare condition characterized by the accumulation of pus between the dura mater and vertebral column, often caused by hematogenous spread from a distant site or local spread from infection in nearby structures. The abscess leads to compression of the spinal cord and can result in neurological damage, including dysfunction or permanent neurological deficits. Treatment of spinal epidural abscesses should not be delayed and requires a combination of decompression by surgical drainage and antibiotic therapy. The authors present a rare case in which a spinal epidural abscess developed from a hospital-acquired pressure ulcer, further complicated by bacteremia.

Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Characterization of DREADD receptor expression and function in rhesus macaques trained to discriminate ethanol.

Circuit manipulation has been a staple technique in neuroscience to identify how the brain functions to control complex behaviors. Chemogenetics, including designer receptors exclusively activated by designer drugs (DREADDs), have proven to be a powerful tool for the reversible modulation of discrete brain circuitry without the need for implantable devices, thereby making them especially useful in awake and unrestrained animals. This study used a DREADD approach to query the role of the nucleus accumbens (NAc) in mediating the interoceptive effects of 1.0 g/kg ethanol (i.g.) in rhesus monkeys (n = 7) using a drug discrimination procedure. After training, stereotaxic surgery was performed to introduce an AAV carrying the human muscarinic 4 receptor DREADD (hM4Di) bilaterally into the NAc. The hypothesis was that decreasing the output of the NAc by activation of hM4Di with the DREADD actuator, clozapine-n-oxide (CNO), would potentiate the discriminative stimulus effect of ethanol (i.e., a leftward shift the ethanol dose discrimination curve). The results showed individual variability shifts of the ethanol dose-response determination under DREADD activation. Characterization of the expression and function of hM4Di with MRI, immunohistochemical, and electrophysiological techniques found the selectivity of NAc transduction was proportional to behavioral effect. Specifically, the proportion of hM4Di expression restricted to the NAc was associated with the potency of the discriminative stimulus effects of ethanol. Together, these experiments highlight the NAc in mediating the interoceptive effects of ethanol, provide a framework for validation of chemogenetic tools in primates, and underscore the importance of robust within-subjects examination of DREADD expression for interpretation of behavioral findings.

Modulation of Gpr39, a G-protein coupled receptor associated with alcohol use in non-human primates, curbs ethanol intake in mice.

Alcohol use disorder (AUD) is a chronic condition with devastating health and socioeconomic effects. Still, pharmacotherapies to treat AUD are scarce. In a prior study aimed at identifying novel AUD therapeutic targets, we investigated the DNA methylome of the nucleus accumbens core (NAcc) of rhesus macaques after chronic alcohol use. The G-protein coupled receptor 39 (GPR39) gene was hypermethylated and its expression downregulated in heavy alcohol drinking macaques. GPR39 encodes a Zn2+-binding metabotropic receptor known to modulate excitatory and inhibitory neurotransmission, the balance of which is altered in AUD. These prior findings suggest that a GPR39 agonist would reduce alcohol intake. Using a drinking-in-the-dark two bottle choice (DID-2BC) model, we showed that an acute 7.5 mg/kg dose of the GPR39 agonist, TC-G 1008, reduced ethanol intake in mice without affecting total fluid intake, locomotor activity or saccharin preference. Furthermore, repeated doses of the agonist prevented ethanol escalation in an intermittent access 2BC paradigm (IA-2BC). This effect was reversible, as ethanol escalation followed agonist "wash out". As observed during the DID-2BC study, a subsequent acute agonist challenge during the IA-2BC procedure reduced ethanol intake by ~47%. Finally, Gpr39 activation was associated with changes in Gpr39 and Bdnf expression, and in glutamate release in the NAcc. Together, our findings suggest that GPR39 is a promising target for the development of prevention and treatment therapies for AUD.

Stimulation-dependent induction of CD154 on a subset of CD4+ FoxP3+ T-regulatory cells.

CD40-ligand/CD154 is predominantly expressed on activated CD4 T cells and plays a central role in regulating CD4 T-cell-dependent responses. To define the relative abilities of CD4 T-cell functional subsets in the induction of CD154--specifically FoxP3- effector, versus FoxP3+ regulatory, CD4 T cells--multiple CD4 T cell preparations were isolated from B6 and B6.FoxP3-GFP mice and stimulated in vitro to examine the kinetics of stimulation-dependent CD154 expression. CD154 was induced in 40-60% of total CD4 T cells in various cell preparations. However, despite similar kinetics of CD154-induced expression, the average percentage of CD154 expression among CD4+ FoxP3+ T regulatory (Treg) cells was only about 4-9%. Such differential, stimulation-dependent CD154 induction by total CD4+ T cells versus CD4+ FoxP3+ Treg cells was consistent, despite multiple stimulation conditions utilizing a variety of cell preparations of different composition. Similar induction of CD154 occurred irrespective of whether the CD4+ FoxP3+ Treg cells were first sorted to 98% purity and stimulated in vitro alone, or stimulated as non-purified cells in the presence of CD4+ FoxP3- T effector cells, suggesting that CD154 induction by CD4+ FoxP3+ Treg cells is regulated by cell-intrinsic mechanisms. Differential CD154 induction may be a key factor in determining the distinguishable functions of FoxP3- T-effector, versus FoxP3+ Treg, CD4+ T cells.

Alternative translational reading frames as a novel source of epitopes for an expanded CD8 T-cell repertoire: use of a retroviral system to assess the translational requirements for CTL recognition and lysis.

CD8 T-cell responses constitute a key host defense mechanism against tumor cells and a variety of viral infections, including retroviral infections that lead to acquired immunodeficiency. However, both for tumor cells and for many viral infections, there can be a relative paucity of immunodominant protective CD8 T-cell responses. For retroviruses, their rapid and error-prone replication, coupled with initial CD8 T-cell immunoselection of epitope-variant, retroviral quasi-species, are major impediments to sustaining a protective CD8 T-cell response. To approach this limitation of functional CD8 T-cell epitopes, here we further characterize an underappreciated source of additional T-cell epitopes: cryptic determinants, in particular those encoded in unconventional, alternative reading frames (ARFs). By use of the CD8 T-cell epitope, SYNTGRFPPL, which we have defined as encoded by the +1NT ARF of the gag gene of the LP-BM5 retrovirus that causes murine AIDS, we further characterize the regulation of ARF-epitope expression. Specifically, we examine the translation initiation requirements for production of sufficient epitope for effector CD8 T-cell recognition. Such translation must arise from rare frame-shifting events, making it crucial to understand any other constraints on epitope production, and therefore on the ability of the anti-Kd/SYNTGRFPPL CD8 T cells to protect from LP-BM5 pathogenesis and retroviral load, as we have previously shown. The data herein demonstrate that ARF epitope production depends entirely on conventional AUG-initiated translation, and that the more proximal in-frame ARF AUG is most important. However, maximal epitope production for protective CD8 T-cell lytic function also requires synergy of this initiation codon with a counterpart conventional AUG codon upstream in the same ARF (ORF 2), and with the classic ORF 1 AUG that initiates conventional gag polyprotein translation. These results have implications for the design of ARF-epitope-based vaccines, both to counter retroviral pathogenesis, as well as potentially more broadly, including in tumor systems.