Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese.

Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma-MS, we validated that this assay, termed here "manganese-extracting small molecule estimation route" (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.

Identification of Three Small Molecules That Can Selectively Influence Cellular Manganese Levels in a Mouse Striatal Cell Model.

Manganese (Mn) is a biologically essential metal, critical as a cofactor for numerous enzymes such a glutamine synthetase and kinases such as ataxia-telangiectasia mutated (ATM). Similar to other essential metals such as iron and zinc, proper levels of Mn need to be achieved while simultaneously being careful to avoid excess levels of Mn that can be neurotoxic. A lifetime of occupational exposure to Mn can often lead to a Parkinsonian condition, also known as "manganism", characterized by impaired gait, muscle spasms, and tremors. Despite the importance of its regulation, the mechanisms underlying the transport and homeostasis of Mn are poorly understood. Rather than taking a protein or gene-targeted approach, our lab recently took a high-throughput-screening approach to identify 41 small molecules that could significantly increase or decrease intracellular Mn in a neuronal cell model. Here, we report characterization of these small molecules, which we refer to as the "Mn toolbox". We adapted a Fura-2-based assay for measuring Mn concentration and for measuring relative concentrations of other divalent metals: nickel, copper, cobalt, and zinc. Of these 41 small molecules, we report here the identification of three that selectively influence cellular Mn but do not influence the other divalent metals tested. The patterns of activity across divalent metals and the discovery of Mn-selective small molecules has potential pharmacological and scientific utility.

Sigma-1 receptor ligands inhibit catecholamine secretion from adrenal chromaffin cells due to block of nicotinic acetylcholine receptors.

Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100 nM to 1 µM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈ 50% block at 100 nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.

Role of d-amino acid oxidase in the production of kynurenine pathway metabolites from d-tryptophan in mice.

The kynurenine pathway (KP), the major catabolic route of the essential amino acid l-tryptophan (l-TRP), contains several neuroactive compounds, including kynurenic acid, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN). The role of the d-enantiomer (d-TRP) in KP metabolism has received little attention so far. d-TRP can be converted to l-TRP by d-amino acid oxidase, and the same enzyme can produce d-kynurenine, a known bioprecursor of KYNA. To analyze these complex metabolic events systematically in vivo, we injected mice with d-TRP (300 mg/kg, i.p.) and examined KP metabolism in the absence or presence of the d-amino acid oxidase inhibitor 3-methylpyrazole-5-carboxylic acid (MPC; 100 mg/kg, i.p.,). After 90 min, newly formed l-TRP was recovered in plasma, liver, forebrain, and cerebellum, and MPC prevented its neosynthesis in all tissues. In the same animals, de novo production of d-kynurenine from d-TRP was also observed, but was much higher in the periphery than in the brain. d-TRP administration raised KYNA, 3-HK, and QUIN levels in all tissues examined, and KYNA production from d-TRP was significantly reduced after pre-treatment with MPC. These results indicate that catabolic routes other than those classically ascribed to l-TRP and l-kynurenine can account for the synthesis of KYNA, 3-HK and QUINin vivo. The essential amino acid l-tryptophan is catabolized via the kynurenine pathway (KP). We explored the role of the d-enantiomer in KP metabolism in mice in vivo. We report that d-tryptophan is metabolized in both brain and periphery and converted to KP metabolites, including d-kynurenine and l-kynurenine, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid. Pharmacological experiments confirm the involvement of d-amino acid oxidase in these processes. Our results indicate that this enzyme participates in the synthesis of KP metabolites from d-tryptophan.

Manganese Is Essential for Neuronal Health.

The understanding of manganese (Mn) biology, in particular its cellular regulation and role in neurological disease, is an area of expanding interest. Mn is an essential micronutrient that is required for the activity of a diverse set of enzymatic proteins (e.g., arginase and glutamine synthase). Although necessary for life, Mn is toxic in excess. Thus, maintaining appropriate levels of intracellular Mn is critical. Unlike other essential metals, cell-level homeostatic mechanisms of Mn have not been identified. In this review, we discuss common forms of Mn exposure, absorption, and transport via regulated uptake/exchange at the gut and blood-brain barrier and via biliary excretion. We present the current understanding of cellular uptake and efflux as well as subcellular storage and transport of Mn. In addition, we highlight the Mn-dependent and Mn-responsive pathways implicated in the growing evidence of its role in Parkinson's disease and Huntington's disease. We conclude with suggestions for future focuses of Mn health-related research.

A method for the determination of D-kynurenine in biological tissues.

D-kynurenine (D-KYN), a metabolite of D-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of D-KYN in biological tissues, we developed a novel assay based on the conversion of D-KYN to KYNA by purified D-amino acid oxidase (D-AAO). Samples were incubated with D-AAO under optimal conditions for measuring D-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for D-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other D-amino acids, including D-serine and D-aspartate, were present in the incubation mixture at 50-fold higher concentrations than D-KYN. Using this new method, D-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with D-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for D-KYN measurement will be instrumental for evaluating whether D-KYN should be considered for a role in physiology and pathology.

Patterns of developmental regression and associated clinical characteristics in SLC6A1-related disorder.

Introduction: SLC6A1-related disorder is a genetic neurodevelopmental disorder that is caused by loss of function variants in the SLC6A1 gene. Solute Carrier Family 6 Member 1 (SLC6A1) gene encodes for gamma-aminobutyric acid (GABA) transporter type 1 (GAT1), which is responsible for reuptake of GABA from the synaptic cleft. Tight regulation of GABA levels plays an important role in brain development by balancing inhibitory and excitatory neuronal signaling. Consequently, individuals with SLC6A1-related disorder can have manifestations such as developmental delay, epilepsy, autism spectrum disorder, and a subset have developmental regression. Methods: In this study, we identified patterns of developmental regression among a cohort of 24 patients with SLC6A1-related disorder and assessed for clinical characteristics associated with regression. We reviewed medical records of patients with SLC6A1-related disorder and divided subjects into two groups: 1) regression group and 2) control group. We described the patterns of developmental regression including whether there was a trigger prior to the regression, multiple episodes of regression, and whether or not skills were recovered. We assessed the relationship of clinical characteristics among the regression and control groups including demographic factors, seizures, developmental milestone acquisition, gastrointestinal problems, sleep problems, autism spectrum disorder, and behavioral problems. Results: Individuals with developmental regression had a loss of skills that were previously mastered in developmental domains including speech and language, motor, social, and adaptive skills. The mean age at regression was 2.7 years and most subjects had regression of language or motor skills triggered by seizures, infection, or spontaneously. Although there was no significant difference in clinical characteristics between the two groups, there was a higher prevalence of autism and severe language impairment in the regression group. Discussion: Future studies of a larger cohort of patients are required to make definitive conclusions. Developmental regression is often a sign of severe neurodevelopmental disability in genetic syndromes, but it is poorly understood in SLC6A1-related disorder. Understanding the patterns of developmental regression and the associated clinical characteristics in this rare disorder will be important to medical management, prognostication, and could impact the design of future clinical trials.

A disease concept model for STXBP1-related disorders.

OBJECTIVE: STXBP1-related disorders are rare genetic epilepsies and neurodevelopmental disorders, but the impact of symptoms across clinical domains is poorly understood. Disease concept models are formal frameworks to assess the lived experience of individuals and their families and provide a basis for generating outcome measures. METHODS: We conducted semistructured, qualitative interviews with 19 caregivers of 16 individuals with STXBP1-related disorders and 7 healthcare professionals. We systematically coded themes using NVivo software and grouped concepts into the domains of symptoms, symptom impact, and caregiver impact. We quantified the frequency of concepts throughout the lifespan and across clinical subgroups stratified by seizure history and developmental trajectories. RESULTS: Over 25 hours of interviews, we coded a total of 3626 references to 38 distinct concepts. In addition to well-recognized clinical features such as developmental delay (n = 240 references), behavior (n = 201), and seizures (n = 147), we identified previously underrepresented symptoms including gastrointestinal (n = 68) and respiratory symptoms (n = 24) and pain (n = 30). The most frequently referenced symptom impacts were autonomy (n = 96), socialization (n = 64), and schooling (n = 61). Emotional impact (n = 354), support (n = 200), and daily life & activities (n = 108) were highly cited caregiver impacts. We found that seizures were more commonly referenced in infancy than in other age groups, while behavior and socialization were more likely to be referred to in childhood. We found that caregivers of individuals with ongoing seizures were less likely to reference developmental delay, possibly due to the relatively high impact of seizures. SIGNIFICANCE: STXBP1-related disorders are complex conditions affecting a wide range of clinical and social domains. We comprehensively mapped symptoms and their impact on families to generate a comprehensive disease model as a foundation for clinical endpoints in future trials.

A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens.

Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess metal uptake can be toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. aureus through disruption of metal homeostasis. VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship, reactive oxygen species (ROS) generation assays, metal binding assays, and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of enzymes, generation of reactive oxygen species, or disruption of other cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple Gram-positive bacteria, leading to increased reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates VU0026921 as a probe to further decipher mechanisms required to maintain metal homeostasis in Gram-positive bacteria.IMPORTANCEStaphylococcus aureus is a leading agent of antibiotic-resistant bacterial infections in the world. S. aureus tightly controls metal homeostasis during infection, and disruption of metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of VU0026921 is modulated by metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in metal detoxification. This work defines a small-molecule probe for further defining the role of metal toxicity in S. aureus and validates future antibiotic development targeting metal toxicity pathways.

Small Molecule Modifiers of In Vitro Manganese Transport Alter Toxicity In Vivo.

Manganese (Mn) is essential for several species and daily requirements are commonly met by an adequate diet. Mn overload may cause motor and psychiatric disturbances and may arise from an impaired or not fully developed excretion system, transporter malfunction and/or exposure to excessive levels of Mn. Therefore, deciphering processes regulating neuronal Mn homeostasis is essential to understand the mechanisms of Mn neurotoxicity. In the present study, we selected two small molecules (with opposing effects on Mn transport) from a previous high throughput screen of 40,167 to test their effects on Mn toxicity parameters in vivo using Caenorhabditis elegans. We pre-exposed worms to VU0063088 and VU0026921 for 30 min followed by co-exposure for 1 h with Mn and evaluated Mn accumulation, dopaminergic (DAergic) degeneration and worm survival. Control worms were exposed to vehicle (DMSO) and saline only. In pdat-1::GFP worms, with GFP labeled DAergic neurons, we observed a decrease of Mn-induced DAergic degeneration in the presence of both small molecules. This effect was also observed in an smf-2 knockout strain. SMF-2 is a regulator of Mn transport in the worms and this strain accumulates higher Mn levels. We did not observe improved survival in the presence of small molecules. Our results suggest that both VU0063088 and VU0026921 may modulate Mn levels in the worms through a mechanism that does not require SMF-2 and induce protection against Mn neurotoxicity.