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.

Toxoplasma gondii, an intracellular protozoan parasite, is a major cause of opportunistic infectious disease affecting the brain and has been linked to an increased incidence of schizophrenia. In murine hosts, infection with T. gondii stimulates tryptophan degradation along the kynurenine pathway (KP), which contains several neuroactive metabolites, including 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN) and kynurenic acid (KYNA). As these endogenous compounds may provide a mechanistic connection between T. gondii and the pathophysiology of schizophrenia, we measured KP metabolites in both brain and periphery of T. gondii-treated C57BL/6 mice 8 and 28 days post-infection. Infected mice showed early decreases in the levels of tryptophan in brain and serum, but not in the liver. These reductions were associated with elevated levels of kynurenine, KYNA, 3-HK and QUIN in the brain. In quantitative terms, the most significant increases in these KP metabolites were observed in the brain at 28 days post-infection. Notably, the anti-parasitic drugs pyrimethamine and sulfadiazine, a standard treatment of toxoplasmosis, significantly reduced 3-HK and KYNA levels in the brain of infected mice when applied between 28 and 56 days post-infection. In summary, T. gondii infection, probably by activating microglia and astrocytes, enhances the production of KP metabolites in the brain. However, during the first two months after infection, the KP changes in these mice do not reliably duplicate abnormalities seen in the brain of individuals with schizophrenia.

The kynurenine pathway (KP), the major catabolic route of the essential amino acid L-tryptophan (L-TRP), contains several neuroactive compounds, including kynurenic acid (KYNA), 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 (D-AAO), 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-AAO 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 QUIN in vivo.

Huntington’s disease (HD) is caused by a mutation in the huntingtin gene (HTT), resulting in profound striatal neurodegeneration through an unknown mechanism. Perturbations in the urea cycle have been reported in HD models and in HD patient blood and brain. In neurons, arginase is a central urea cycle enzyme, and the metal manganese (Mn) is an essential cofactor. Deficient biological responses to Mn, and reduced Mn accumulation have been observed in HD striatal mouse and cell models. Here we report in vivo and ex vivo evidence of a urea cycle metabolic phenotype in a prodromal HD mouse model. Further, either in vivo or in vitro Mn supplementation reverses the urea-cycle pathology by restoring arginase activity. We show that Arginase 2 (ARG2) is the arginase enzyme present in these mouse brain models, with ARG2 protein levels directly increased by Mn exposure. ARG2 protein is not reduced in the prodromal stage, though enzyme activity is reduced, indicating that altered Mn bioavailability as a cofactor leads to the deficient enzymatic activity. These data support a hypothesis that mutant HTT leads to a selective deficiency of neuronal Mn at an early disease stage, contributing to HD striatal urea-cycle pathophysiology through an effect on arginase activity.

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. IMPORTANCE Staphylococcus 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.

Background and ObjectivesPathogenic variants at the voltage-gated sodium channel gene,SCN8A, are associated with a wide spectrum of clinical disease outcomes. A critical challenge for neurologists is to determine whether patients carry gain-of-function (GOF) or loss-of-function (LOF) variants to guide treatment decisions, yet in vitro studies to infer channel function are often not feasible in the clinic. In this study, we develop a predictive modeling approach to classify variants based on clinical features present at initial diagnosis.MethodsWe performed an exhaustive search for individuals deemed to carry SCN8A GOF and LOF variants by means of in vitro studies in heterologous cell systems, or because the variant was classified as truncating, and recorded clinical features. This resulted in a total of 69 LOF variants: 34 missense and 35 truncating variants, including 9 nonsense, 13 frameshift, 6 splice site, 6 indels, and 1 large deletion. We then assembled a truth set of variants with known functional effects, excluding individuals carrying variants at other loci associated with epilepsy. We then trained a predictive model based on random forest using this truth set of 45 LOF variants and 45 GOF variants randomly selected from a set of variants tested by in vitro methods.ResultsPhenotypic categories assigned to individuals correlated strongly with GOF or LOF variants. All patients with GOF variants experienced early-onset seizures (mean age at onset = 4.5 ± 3.1 months) while only 64.4% patients with LOF variants had seizures, most of which were late-onset absence seizures (mean age at onset = 40.0 ± 38.1 months). With high accuracy (95.4%), our model including 5 key clinical features classified individuals with GOF and LOF variants into 2 distinct cohorts differing in age at seizure onset, development of seizures, seizure type, intellectual disability, and developmental and epileptic encephalopathy.DiscussionThe results support the hypothesis that patients withSCN8AGOF and LOF variants represent distinct clinical phenotypes. The clinical model developed in this study has great utility because it provides a rapid and highly accurate platform for predicting the functional class of patient variants duringSCN8Adiagnosis, which can aid in initial treatment decisions and improve prognosis.

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

IntroductionSLC6A1-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.MethodsIn 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.ResultsIndividuals 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.DiscussionFuture 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.