Manganese Overexposure Alters Neurogranin Expression and Causes Behavioral Deficits in Larval Zebrafish.

Manganese (Mn), a cofactor for various enzyme classes, is an essential trace metal for all organisms. However, overexposure to Mn causes neurotoxicity. Here, we evaluated the effects of exposure to Mn chloride (MnCl2) on viability, morphology, synapse function (based on neurogranin expression) and behavior of zebrafish larvae. MnCl2 exposure from 2.5 h post fertilization led to reduced survival (60%) at 5 days post fertilization. Phenotypical changes affected body length, eye and olfactory organ size, and visual background adaptation. This was accompanied by a decrease in both the fluorescence intensity of neurogranin immunostaining and expression levels of the neurogranin-encoding genes nrgna and nrgnb, suggesting the presence of synaptic alterations. Furthermore, overexposure to MnCl2 resulted in larvae exhibiting postural defects, reduction in motor activity and impaired preference for light environments. Following the removal of MnCl2 from the fish water, zebrafish larvae recovered their pigmentation pattern and normalized their locomotor behavior, indicating that some aspects of Mn neurotoxicity are reversible. In summary, our results demonstrate that Mn overexposure leads to pronounced morphological alterations, changes in neurogranin expression and behavioral impairments in zebrafish larvae.

The incidence of movement disorder increases with age and contrasts with subtle and limited neuroimaging abnormalities in argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy.

Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity.

Manganese is an essential metal, but elevated brain Mn concentrations produce a parkinsonian-like movement disorder in adults and fine motor, attentional, cognitive, and intellectual deficits in children. Human Mn neurotoxicity occurs owing to elevated exposure from occupational or environmental sources, defective excretion (e.g., due to cirrhosis), or loss-of-function mutations in the Mn transporters solute carrier family 30 member 10 or solute carrier family 39 member 14. Animal models are essential to study Mn neurotoxicity, but in order to be translationally relevant, such models should utilize environmentally relevant Mn exposure regimens that reproduce changes in brain Mn concentrations and neurological function evident in human patients. Here, we provide guidelines for Mn exposure in mice, rats, nematodes, and zebrafish so that brain Mn concentrations and neurobehavioral sequelae remain directly relatable to the human phenotype.

Imaging of changes in copper trafficking and redistribution in a mouse model of Niemann-Pick C disease using positron emission tomography.

Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder resulting from mutations in the NPC1 (95% of cases) or NPC2 genes. Disturbance of copper homeostasis has been reported in NPC1 disease. In this study we have used whole-body positron emission tomography (PET) and brain electronic autoradiography with copper-64 (64Cu), in the form of the copper(II) bis(thiosemicarbazonato) complex 64Cu-GTSM, to image short-term changes in copper trafficking after intravenous injection in a transgenic mouse model of NPC1 disease. 64Cu-GTSM is taken up in all tissues and dissociates rapidly inside cells, allowing monitoring of the subsequent efflux and redistribution of 64Cu from all tissues. Significantly enhanced retention of 64Cu radioactivity was observed in brain, lungs and blood at 15 h post-injection in symptomatic Npc1-/- transgenic mice compared to wildtype controls. The enhanced retention of 64Cu in brain was confirmed by electronic autoradiography, particularly in the midbrain, thalamus, medulla and pons regions. Positron emission tomography imaging with 64Cu in selected chemical forms could be a useful diagnostic and research tool for the management and understanding of NPC1 disease.

Novel founder intronic variant in SLC39A14 in two families causing Manganism and potential treatment strategies.

Congenital disorders of manganese metabolism are rare occurrences in children, and medical management of these disorders is complex and challenging. Homozygous exonic mutations in the manganese transporter SLC39A14 have recently been associated with a pediatric-onset neurodegenerative disorder characterized by brain manganese accumulation and clinical signs of manganese neurotoxicity, including parkinsonism-dystonia. We performed whole exome sequencing on DNA samples from two unrelated female children from the United Arab Emirates with progressive movement disorder and brain mineralization, identified a novel homozygous intronic mutation in SLC39A14 in both children, and demonstrated that the mutation leads to aberrant splicing. Both children had consistently elevated serum manganese levels and were diagnosed with SLC39A14-associated manganism. Over a four-year period, we utilized a multidisciplinary management approach for Patient 1 combining decreased manganese dietary intake and chelation with symptomatic management of dystonia. Our treatment strategy appeared to slow disease progression, but did not lead to a cure or reversal of already established deficits. Clinicians should consider testing for noncoding mutations in the diagnosis of congenital disorders of manganese metabolism and utilizing multidisciplinary approaches in the management of these disorders.

Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 Protein as a Manganese Transporter.

Manganese homeostasis involves coordinated regulation of specific proteins involved in manganese influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in manganese detoxification/efflux, by evaluating their ability to reduce manganese toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and the zinc-specific transporter hZnT1 showed that residue Asn(43), which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique manganese mobilization activity; residues Cys(52) and Leu(242) in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the His → Asn reversion mutant in hZnT1 conferred manganese transport activity and loss of zinc transport activity. These results provide important information about manganese detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a manganese transporter.

Syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia caused by mutations in SLC30A10, a manganese transporter in man.

Environmental manganese (Mn) toxicity causes an extrapyramidal, parkinsonian-type movement disorder with characteristic magnetic resonance images of Mn accumulation in the basal ganglia. We have recently reported a suspected autosomal recessively inherited syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia in cases without environmental Mn exposure. Whole-genome mapping of two consanguineous families identified SLC30A10 as the affected gene in this inherited type of hypermanganesemia. This gene was subsequently sequenced in eight families, and homozygous sequence changes were identified in all affected individuals. The function of the wild-type protein and the effect of sequence changes were studied in the manganese-sensitive yeast strain Δpmr1. Expressing human wild-type SLC30A10 in the Δpmr1 yeast strain rescued growth in high Mn conditions, confirming its role in Mn transport. The presence of missense (c.266T>C [p.Leu89Pro]) and nonsense (c.585del [p.Thr196Profs(∗)17]) mutations in SLC30A10 failed to restore Mn resistance. Previously, SLC30A10 had been presumed to be a zinc transporter. However, this work has confirmed that SLC30A10 functions as a Mn transporter in humans that, when defective, causes Mn accumulation in liver and brain. This is an important step toward understanding Mn transport and its role in neurodegenerative processes.

Hyperphosphatasia with mental retardation syndrome 3: Cerebrospinal fluid abnormalities and correction with pyridoxine and Folinic acid.

Glycosylphosphatidylinositol anchored proteins (GPI-APs) represent a class of molecules attached to the external leaflet of the plasma membrane by the GPI anchor where they play important roles in numerous cellular processes including neurogenesis, cell adhesion, immune response and signalling. Within the group of GPI anchor defects, six present with the clinical phenotype of Hyperphosphatasia with Mental Retardation Syndrome (HPMRS, Mabry Syndrome) characterized by moderate to severe intellectual disability, dysmorphic features, hypotonia, seizures and persistent hyperphosphatasia. We report the case of a 5-year-old female with global developmental delay associated with precocious puberty and persistently raised plasma alkaline phosphatase. Targeted next generation sequencing analysis of the HPMRS genes identified novel compound heterozygous variants in the PGAP2 gene (c.103del p.(Leu35Serfs*90)and c.134A > Gp.(His45Arg)) consistent with the diagnosis of HPMRS type 3. Cerebrospinal fluid (CSF) neurotransmitter analysis showed low levels of pyridoxal phosphate and 5-methyltetrahydrofolate and raised homovanillic acid. Supplementation with pyridoxine and folinic acid led to normalization of biochemical abnormalities. The patient continues to make developmental progress with significant improvement in speech and fine motor skills. Our reported case expands the clinical spectrum of HPMRS3 in which multisystem involvement is being increasingly recognized. Furthermore, it shows that miss-targeting GPI-APs and the effect on normal cellular function could provide a physiopathologic explanation for the CSF biochemical abnormalities with management implications for a group of disorders that currently has no treatment that can lead possibly to improved clinical outcomes.

Dystonia with brain manganese accumulation resulting from SLC30A10 mutations: a new treatable disorder.

BACKGROUND: The first gene causing early-onset generalized dystonia with brain manganese accumulation has recently been identified. Mutations in the SLC30A10 gene, encoding a manganese transporter, cause a syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia. METHODS: We present 10-year longitudinal clinical features, MRI data, and treatment response to chelation therapy of the originally described patient with a proven homozygous mutation in SLC30A10. RESULTS: The patient presented with early-onset generalized dystonia and mild hyperbilirubinemia accompanied by elevated whole-blood manganese levels. T1-sequences in MRI showed hyperintensities in the basal ganglia and cerebellum, characteristic of manganese deposition. Treatment with intravenous disodium calcium edetate led to clinical improvement and reduction of hyperintensities in brain imaging. CONCLUSIONS: We wish to highlight this rare disorder, which, together with Wilson's disease, is the only potentially treatable inherited metal storage disorder to date, that otherwise can be fatal as a result of complications of cirrhosis. © 2012 Movement Disorder Society.

Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish.

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.

Genotypic and phenotypic spectrum of pyridoxine-dependent epilepsy (ALDH7A1 deficiency).

Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signaling.

Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia.

Hepatic cirrhosis, dystonia, polycythaemia and hypermanganesaemia--a new metabolic disorder.

We report a new constellation of clinical features consisting of hypermanganesaemia, liver cirrhosis, an extrapyramidal motor disorder and polycythaemia in a 12 year-old girl born to consanguineous parents. Blood manganese levels were >3000 nmol/L (normal range <320 nmol/L) and MRI revealed signal abnormalities of the basal ganglia consistent with manganese deposition. An older brother with the same phenotype died at 18 years, suggesting a potentially lethal, autosomal recessive disease. This disorder is probably caused by a defect of manganese metabolism with the accumulation of manganese in the liver and the basal ganglia similar to the copper accumulation in Wilson disease. In order to assess the genetic basis of this syndrome we investigated two candidate genes: ATP2C2 and ATP2A3 encoding the manganese-transporting calcium-ATPases, SPCA2 and SERCA3, respectively. Genotyping of the patient and the family for microsatellite markers surrounding ATP2C2 and ATP2A3 excluded these genes. The patient was found to be heterozygous for both gene loci. Despite the unknown pathophysiology, we were able to develop a successful treatment regime. Chelation therapy with disodium calcium edetate combined with iron supplementation is the treatment of choice, lowering blood manganese levels significantly and improving clinical symptoms.

Sterols and oxysterols in plasma from Smith-Lemli-Opitz syndrome patients.

Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (Δ7-sterol reductase, DHCR7, EC 1.3.1.21) leading to a build-up of the cholesterol precursor 7-dehydrocholesterol (7-DHC) in tissues and blood plasma. Although the underling enzyme deficiency associated with SLOS is clear there are likely to be multiple mechanisms responsible for SLOS pathology. In an effort to learn more of the aetiology of SLOS we have analysed plasma from SLOS patients to search for metabolites derived from 7-DHC which may be responsible for some of the pathology. We have identified a novel hydroxy-8-dehydrocholesterol, which is either 24- or 25-hydroxy-8-dehydrocholesterol and also the known metabolites 26-hydroxy-8-dehydrocholesterol, 4-hydroxy-7-dehydrocholesterol, 3ß,5α-dihydroxycholest-7-en-6-one and 7α,8α-epoxycholesterol. None of these metabolites are detected in control plasma at quantifiable levels (0.5ng/mL).

Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry.

BACKGROUND: Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots. METHODS: Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively. RESULTS: Levels of most AA and GAA increased significantly with increasing hematocrit (p<0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p<0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p<0.0001). CONCLUSIONS: Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.

Hypermanganesemia with Dystonia, Polycythemia and Cirrhosis (HMDPC) due to mutation in the SLC30A10 gene.

Manganese (Mn) is an essential element for metabolic pathways but it can be toxic when present in excessive amounts in the body. Hypermanganesemia along with dystonia, polycythemia, characteristic MRI brain findings in the basal ganglia, and chronic liver disease are the hallmarks of an inherited Mn transporter defect due to mutations in the SLC30A10 gene. We are reporting three siblings who presented with features of dystonia, polycythemia, MRI brain showing basal ganglia hyperintensity on T1 weighted images and chronic liver disease. Blood Mn levels were markedly elevated in the affected patients. Mutation analysis of DNA samples of the affected children confirmed a homozygous missense mutation in SLC30A10. Chelation therapy with intravenous disodium calcium edetate was started in two siblings and led to a marked decrease in whole blood Mn. Oral Penicillamine was later added to the therapy which further improved blood Mn levels. This is a rare disorder and is one of the potentially treatable inherited metal storage disorders. It can be fatal if left untreated. Penicillamine may be an effective alternative to disodium calcium edetate.

Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism-dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.

Rapid analysis of total plasma homocysteine by tandem mass spectrometry.

Elevated plasma homocysteine levels may be an independent risk factor for premature vascular disease. Early detection and population screening are warranted to recognise hyperhomocysteinemia and initiate homocysteine lowering therapy. Current methods for homocysteine analysis are time consuming, labor intensive and/or expensive. We developed a sensitive and fast method for homocysteine analysis based on tandem mass spectrometry that avoids the need for derivatization and preanalytical chromatography.