Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities.

Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism.

PEN Receptor GPR83 in Anxiety-Like Behaviors: Differential Regulation in Global vs Amygdalar Knockdown.

Anxiety disorders are prevalent across the United States and result in a large personal and societal burden. Currently, numerous therapeutic and pharmaceutical treatment options exist. However, drugs to classical receptor targets have shown limited efficacy and often come with unpleasant side effects, highlighting the need to identify novel targets involved in the etiology and treatment of anxiety disorders. GPR83, a recently deorphanized receptor activated by the abundant neuropeptide PEN, has also been identified as a glucocorticoid regulated receptor (and named GIR) suggesting that this receptor may be involved in stress-responses that underlie anxiety. Consistent with this, GPR83 null mice have been found to be resistant to stress-induced anxiety. However, studies examining the role of GPR83 within specific brain regions or potential sex differences have been lacking. In this study, we investigate anxiety-related behaviors in male and female mice with global knockout and following local GPR83 knockdown in female mice. We find that a global knockdown of GPR83 has minimal impact on anxiety-like behaviors in female mice and a decrease in anxiety-related behaviors in male mice. In contrast, a local GPR83 knockdown in the basolateral amygdala leads to more anxiety-related behaviors in female mice. Local GPR83 knockdown in the central amygdala or nucleus accumbens (NAc) showed no significant effect on anxiety-related behaviors. Finally, dexamethasone administration leads to a significant decrease in receptor expression in the amygdala and NAc of female mice. Together, our studies uncover a significant, but divergent role for GPR83 in different brain regions in the regulation of anxiety-related behaviors, which is furthermore dependent on sex.

Insights into the Neurobiology of Craving in Opioid Use Disorder.

PURPOSE OF REVIEW: Opioids remain the most potent form of pain relief currently available, yet have a high abuse liability. Here we discuss underlying neurobiological changes in Opioid Use Disorder (OUD) that likely contribute to drug craving, which in turn drives continued drug use and relapse. RECENT FINDINGS: Craving has emerged as a strong indicator in drug-seeking and relapse. Studies have demonstrated a number of allostatic changes in circuitry that facilitate learning of drug-stimuli relationships, thereby augmenting cue-triggered drug use and relapse. SUMMARY: This review will focus on key neurobiological changes in underlying circuitry observed during the initial and continued exposure to opioids that result in an increase in neural-reactivity to drug-related intrinsic and extrinsic drug cues, and to enhanced learning of drug-context correlations. This sensitized learning state may be an indication of the underlying framework that drives craving and ultimately, motivates increased salience of drug cues and drives drug-seeking.

The role of the neuropeptide PEN receptor, GPR83, in the reward pathway: Relationship to sex-differences.

GPR83, the receptor for the neuropeptide PEN, exhibits high expression in the nucleus accumbens of the human and rodent brain, suggesting that it plays a role in modulating the mesolimbic reward pathway. However, the cell-type specific expression of GPR83, its functional impact in the reward pathway, and in drug reward-learning has not been fully explored. Using GPR83/eGFP mice, we show high GPR83 expression on cholinergic interneurons in the nucleus accumbens and moderate expression on ventral tegmental area dopamine neurons. In GPR83 knockout mice, baseline dopamine release in the nucleus accumbens is enhanced which disrupts the ratio of tonic vs phasic release. Additionally, GPR83 knockout leads to changes in the expression of dopamine-related genes. Using the morphine conditioned place preference model, we identify sex differences in morphine reward-learning, show that GPR83 is upregulated in the nucleus accumbens following morphine conditioned place preference, and show that shRNA-mediated knockdown of GPR83 in the nucleus accumbens leads to attenuation morphine reward. Together, these findings detect GPR83 expression in the reward-pathway, and show its involvement in dopamine release and morphine reward-learning.

The Contribution of the Descending Pain Modulatory Pathway in Opioid Tolerance.

Opioids remain among the most effective pain-relieving therapeutics. However, their long-term use is limited due to the development of tolerance and potential for addiction. For many years, researchers have explored the underlying mechanisms that lead to this decreased effectiveness of opioids after repeated use, and numerous theories have been proposed to explain these changes. The most widely studied theories involve alterations in receptor trafficking and intracellular signaling. Other possible mechanisms include the recruitment of new structural neuronal and microglia networks. While many of these theories have been developed using molecular and cellular techniques, more recent behavioral data also supports these findings. In this review, we focus on the mechanisms that underlie tolerance within the descending pain modulatory pathway, including alterations in intracellular signaling, neural-glial interactions, and neurotransmission following opioid exposure. Developing a better understanding of the relationship between these various mechanisms, within different parts of this pathway, is vital for the identification of more efficacious, novel therapeutics to treat chronic pain.

Targeting the Recently Deorphanized Receptor GPR83 for the Treatment of Immunological, Neuroendocrine and Neuropsychiatric Disorders.

G-protein coupled receptors (GPCRs) are a superfamily of receptors responsible for initiation of a myriad of intracellular signaling cascades. Currently, GPCRs represent approximately 34% of marketed pharmaceuticals, a large portion of which have no known endogenous ligand. These orphan GPCRs represent a large pool of novel targets for drug development. Very recently, the neuropeptide PEN, derived from the proteolytic processing of the precursor proSAAS, has been identified as a selective, high-affinity endogenous ligand for the orphan receptor, GPR83. GPR83 is highly expressed in the brain, spleen and thymus, indicating that this receptor may be a target to treat neurological and immune disorders. In the brain GPR83 is expressed in regions involved in the reward pathway, stress/anxiety responses, learning and memory and metabolism. However, the cell type specific expression of GPR83 in these regions has only recently begun to be characterized. In the immune system, GPR83 expression is regulated by Foxp3 in T-regulatory cells that are involved in autoimmune responses. Moreover, in the brain this receptor is regulated by interactions with other GPCRs, such as the recently deorphanized receptor, GPR171, and other hypothalamic receptors such as MC4R and GHSR. The following review will summarize the properties of GPR83 and highlight its known and potential significance in health and disease, as well as its promise as a novel target for drug development.

The Role of Phosphodiesterase-2 in Psychiatric and Neurodegenerative Disorders.

Cyclic nucleotide PDEs are a super-family of enzymes responsible for regulating intracellular levels of the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Through their catalysis, PDEs are able to exert tight regulation over these important intracellular signaling cascades. Previously, PDEs have been implicated in learning and memory, as well as in mood disorders, such as anxiety and depression. PDE2 is of special interest due to its high level of expression in the forebrain, specifically in the isocortex, entorhinal cortex, striatum, hippocampus, amygdala, and medial habenula. Many of these brain regions are considered participants of the limbic system, which is known as the emotional regulatory center of the brain, and is important for modulating emotion and long-term memory. Therefore, PDE2s coincidental expression in these areas suggests an important role for PDE2 in these behaviors, and researchers are continuing to uncover the complex connections. It was shown that PDE2 inhibitors have pro-cognitive effects in tests of memory, including the object recognition test. PDE2 inhibitors are also protective against cognitive deficits in various models of cognitive impairment. Additionally, PDE2 inhibitors are protective against many different forms of stress-induced anxiety-like and depression-like behaviors. Currently, there is a great need for novel therapeutics for the treatment of mood and cognitive disorders, especially anxiety and depression, and other neurodegenerative diseases, such as Alzheimer's disease, and PDE2 is emerging as a viable target for future drug development for many of these diseases.

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

Cyclic GMP-mediated memory enhancement in the object recognition test by inhibitors of phosphodiesterase-2 in mice.

RATIONALE AND OBJECTIVES: Cyclic nucleotide phosphodiesterase-2 (PDE2) is a potential therapeutic target for the treatment of cognitive dysfunction. Using the object recognition test (ORT), this study assessed the effects of two PDE2 inhibitors, Bay 60-7550 and ND7001, on learning and memory, and examined underlying mechanisms. METHODS: To assess the role of PDE2 inhibition on phases of memory, Bay 60-7550 (3 mg/kg) was administered: 30 min prior to training; 0, 1, or 3 h after training; or 30 min prior to recall testing. To assess cyclic nucleotide involvement in PDE2 inhibitor-enhanced memory consolidation, either the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg; intraperitoneal (IP)), soluble guanylyl cyclase inhibitor 1H-[-1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 20 mg/kg; IP), protein kinase G inhibitor KT5823 (2.5 µg; intracerebroventricular (ICV)), or protein kinase A inhibitor H89 (1 µg; ICV) was administered 30 min prior to the PDE2 inhibitor Bay 60-7550 (3 mg/kg) or ND7001 (3 mg/kg). Changes in the phosphorylation of 3'5'-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) at Ser-133 and vasodilator-stimulated phosphoprotein (VASP) at Ser-239 were determined to confirm activation of cAMP and 3'5'-cyclic guanosine monophosphate (cGMP) signaling. RESULTS: Bay 60-7550 (3 mg/kg) enhanced memory of mice in the ORT when given 30 min prior to training, immediately after training, or 30 min prior to recall. Inhibitors of the cGMP pathway blocked the memory-enhancing effects of both Bay 60-7550 (3 mg/kg) and ND7001 (3 mg/kg) on early consolidation processes. Bay 60-7550 (3 mg/kg) enhanced phosphorylation of CREB and VASP, both targets of cGMP-dependent protein kinase (PKG). CONCLUSIONS: These results confirm a potential of PDE2, or components of its signaling pathway, as a therapeutic target for drug discovery focused on restoring memory function.