Nancy A. Muma

Huntington's disease is a progressive and lethal neurodegenerative disease caused by an increased CAG repeat mutation in exon 1 of the huntingtin gene (mutant huntingtin). Current drug treatments provide only limited symptomatic relief without impacting disease progression. Previous studies in our lab and others identified the abnormal binding of mutant huntingtin protein with calmodulin, a key regulator of calcium signaling. Disrupting the abnormal binding of mutant huntingtin to calmodulin reduces perturbations caused by mutant huntingtin in cell and mouse models of Huntington's disease and importantly normalizes receptor-stimulated calcium release. Using a series of high-throughput in vitro and cell-based screening assays, we identified numerous small-molecule hits that disrupt the binding of mutant huntingtin to calmodulin and demonstrate protective effects. Iterative optimization of one hit resulted in nontoxic, selective compounds that are protective against mutant huntingtin cytotoxicity and normalized receptor-stimulated intracellular calcium release in PC12 cell models of Huntington's disease. Importantly, the compounds do not work by reducing the levels of mutant huntingtin, allowing this strategy to complement future molecular approaches to reduce mutant huntingtin expression. Our novel scaffold will serve as a prototype for further drug development in Huntington's disease. These studies indicate that the development of small-molecule compounds that disrupt the binding of mutant huntingtin to calmodulin is a promising approach for the advancement of therapeutics to treat Huntington's disease.

Serotonin 1A receptors (5-HT1ARs) are implicated in the control of mood, cognition, and memory and in various neuropsychiatric disorders such as depression and anxiety. As such, understanding the regulation of 5-HT1ARs will inform the development of better treatment approaches. We previously demonstrated 5-HT1ARs are SUMOylated by SUMO1 in the rat brain. Agonist stimulation increased SUMOylation and was further enhanced when combined with 17β-estradiol-3-benzoate (EB), which are treatments that cause the transient and prolonged desensitization of 5-HT1AR signaling, respectively. In the current study, we identified the protein inhibitor of activated STAT (PIAS)xα as the enzyme that facilitates SUMOylation, and SENP2 as the protein that catalyzes the deSUMOylation of 5-HT1ARs. We demonstrated that PIASxα significantly increased in the membrane fraction of rats co-treated with EB and an agonist, compared to either the EB-treated or vehicle-treated groups. The acute treatment with an agonist alone shifted the location of SENP2 from the membrane to the cytoplasmic fraction, but it has little effect on PIASxα. Hence, two separate mechanisms regulate SUMOylation and the activity of 5-HT1ARs by an agonist and EB. The effects of EB on 5-HT1AR SUMOylation and signaling may be related to the higher incidence of mood disorders in women during times with large fluctuations in estrogens. Targeting the SUMOylation of 5-HT1ARs could have important clinical relevance for the therapy for several neuropsychiatric disorders in which 5-HT1ARs are implicated.

The aberrant protein–protein interaction between calmodulin and mutant huntingtin protein in Huntington’s disease patients has been found to contribute to Huntington’s disease progression. A high-throughput screen for small molecules capable of disrupting this interaction revealed a sultam series as potent small-molecule disruptors. Diversification of the sultam scaffold afforded a set of 24 analogs or further evaluation. Several structure–activity trends within the analog set were found, most notably a negligible effect of absolute stereochemistry and a strong beneficial correlation with electron-withdrawing aromatic substituents. The most promising analogs were profiled for off-target effects at relevant kinases and, ultimately, one candidate molecule was evaluated for neuroprotection in a neuronal cell model of Huntington’s disease.

Finasteride (FIN) is the prototypical inhibitor of steroid 5α-reductase (5αR), the enzyme that catalyzes the rate-limiting step of the conversion of progesterone and testosterone into their main neuroactive metabolites. FIN is clinically approved for the treatment of benign prostatic hyperplasia and male baldness; while often well-tolerated, FIN has also been shown to cause or exacerbate psychological problems in vulnerable subjects. Evidence on the psychological effects of FIN, however, remains controversial, in view of inconsistent clinical reports. Here, we tested the effects of FIN in a battery of tests aimed at capturing complementary aspects of mood regulation and stress reactivity in rats. FIN reduced exploratory, incentive, prosocial, and risk-taking behavior; furthermore, it decreased stress coping, as revealed by increased immobility in the forced-swim test (FST). This last effect was also observed in female and orchiectomized male rats, suggesting that the mechanism of action of FIN does not primarily reflect changes in gonadal steroids. The effects of FIN on FST responses were associated with a dramatic decrease in corticotropin release hormone (CRH) mRNA and adrenocorticotropic hormone (ACTH) levels. These results suggest that FIN impairs stress reactivity and reduces behavioral activation and impulsive behavior by altering the function of the hypothalamus–pituitary–adrenal (HPA) axis.

Transglutaminases (TGs) and especially TG2 play important roles in neurotransmitter and receptor signaling pathways. Three different mechanisms by which TG2 interacts with neurotransmitter and receptor signaling systems will be discussed in this review. The first way in which TG2 interacts with receptor signaling is via its function as a guanine nucleotide binding protein (G-protein) coupling to G-protein coupled receptors (GPCRs) to activate down-stream signaling pathways. TG2 can exist in a least two conformations, a closed GTP-bound conformation and an open calcium-bound conformation. In the closed GTP-bound conformation, TG2 is capable of functioning as a G-protein for GPCRs. In the open calcium-bound conformation, TG2 catalyzes a transamidation reaction cross-linking proteins or catalyzing the covalent binding of a mono- or polyamine to a protein. The second mechanism is regulation of the transamidation reaction catalyzed by TG2 via receptor stimulation which can increase local calcium concentrations and thereby increase transamidation reactions. The third way in which TG2 plays a role in neurotransmitter and receptor signaling systems is via its use of monoamine neurotransmitters as a substrate. Monoamine neurotransmitters including serotonin can be substrates for transamidation to a protein often a small G-protein (also known as a small GTPase) resulting in activation of the small G-protein. The transamidation of a monoamine neurotransmitter or serotonin has been designated as monoaminylation or more specifically serotonylation, respectively. Other proteins are also targets for monoaminylation such as fibronectin and cytoskeletal proteins. These receptor and neurotransmitter-regulated reactions by TG2 play roles in physiological and key pathophysiological processes.

Although serotonin was discovered over 65 years ago, it has been only within the past decade that serotonin was found to be involved in a covalent post-translational modification to proteins. The enzyme transglutaminase catalyzes the transamidation of serotonin to a protein-bound glutamine residue; the amino group of serotonin is covalently bound to the gamma carboxamide of glutamine. The term serotonylation is used to describe this transamidation reaction to serotonin. Not only can serotonin be a substrate for transamidation to proteins but also other monoamine neurotransmitters are substrates including histamine, dopamine, and noradrenaline. The term monoaminylation has been coined to describe the transamidation of monoamines to protein substrates. Small G proteins have emerged as the most common substrate for monoaminylation and are activated by this post-translational modification. Fibronectin and cytoskeletal proteins are also substrates for monoaminylation. Serotonylation and monoaminylation are involved in a number of physiological functions, including platelet activation, insulin release, smooth muscle contraction, and regulation of membrane localization of the serotonin transporter. Stimulation of 5-HT_2A receptors increases serotonylation and activates the small G protein Rac1, which plays a role in dendritic spine regulation. Monoaminylation is implicated in pathophysiological processes as well such as diabetes and hypertension. The availability of monoamines for monoaminylation is altered by antidepressants that target serotonin transporters, noradrenaline transporters, or the enzymatic degradation of monoamines as well as drugs of abuse such as cocaine and amphetamines. Further research on monoaminylation is needed to elucidate its physiological and pathophysiological roles and to explore monoaminylation as a novel target for drug therapy.

Hyperactivity of the hypothalamic-pituitary-adrenal axis is a consistent biological characteristic of depression, and response normalization coincides with clinical responsiveness to antidepressant medications. Desensitization of serotonin 1A receptor (5-HT_1AR) signaling in the hypothalamic paraventricular nucleus of the hypothalamus (PVN) follows selective serotonin reuptake inhibitor (SSRI) antidepressant treatment and contributes to the antidepressant response. Estradiol alone produces a partial desensitization of 5-HT_1AR signaling and synergizes with SSRIs to result in a complete and more rapid desensitization than with SSRIs alone as measured by a decrease in the oxytocin and adrenocorticotrophic hormone (ACTH) responses to 5-HT_1AR stimulation. G protein-coupled estrogen receptor 1 (GPER1) is necessary for estradiol-induced desensitization of 5-HT_1AR signaling, although the underlying mechanisms are still unclear. We now find that stimulation of GPER1 with the selective agonist G-1 and nonselective stimulation of estrogen receptors dramatically alter isoform expression of a key component of the 5-HT_1AR signaling pathway, RGSz1, a GTPase-activating protein selective for Gαz, the Gα subunit necessary for 5-HT_1AR-mediated hormone release. RGSz1 isoforms are differentially glycosylated, SUMOylated, and phosphorylated, and differentially distributed in subcellular organelles. High-molecular-weight RGSz1 is SUMOylated and glycosylated, localized to the detergent-resistant microdomain (DRM) of the cell membrane, and increased by estradiol and G-1 treatment. Because activated Gαz also localizes to the DRM, increased DRM-localized RGSz1 by estradiol and G-1 could reduce Gαz activity, functionally uncoupling 5-HT_1AR signaling. Peripheral G-1 treatment produced a partial reduction in oxytocin and ACTH responses to 5-HT_1AR stimulation similar to direct injections into the PVN. Together, these results identify GPER1 and RGSz1 as novel targets for the treatment of depression.