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Background Vascular abnormalities and endothelial dysfunction are part of the spectrum of autosomal dominant polycystic kidney disease (ADPKD). The mechanisms behind these manifestations, including potential effects on the endothelial surface layer (ESL) and glycocalyx integrity, remain unknown. Methods Forty-five ambulatory adult patients with ADPKD were enrolled in this prospective, observational, cross-sectional, single-centre study. Fifty-one healthy volunteers served as a control group. All participants underwent real-time microvascular perfusion measurements of the sublingual microcirculation using sidestream dark field imaging. After image acquisition, the perfused boundary region (PBR), an inverse parameter for red blood cell (RBC) penetration into the ESL, was automatically calculated. Microvascular perfusion was assessed by RBC filling and capillary density. Concentrations of circulating glycocalyx components were determined by enzyme-linked immunosorbent assay. Results ADPKD patients showed a significantly larger PBR compared with healthy controls (2.09 ± 0.23 µm versus 1.79 ± 0.25 µm; P Conclusions Dimensions and integrity of the ESL are impaired in ADPKD patients. Increased capillary density may be a compensatory mechanism for vascular dysfunction to ensure sufficient tissue perfusion and oxygenation.

Background The identification of new biomarkers in autosomal-dominant polycystic kidney disease (ADPKD) is crucial to improve and simplify prognostic assessment as a basis for patient selection for targeted therapies. Post-Hoc-Analyses of the TEMPO 3:4 study indicated that copeptin could be one of those biomarkers. Methods Copeptin was tested in serum samples from patients of the AD(H)PKD study. Serum copeptin levels were measured using a time-resolved amplified cryptate emission (TRACE) based assay. In total, we collected 711 values from 389 patients without tolvaptan treatment and a total of 243 values (of which 64 were pre-tolvaptan) from 94 patients on tolvaptan. These were associated to rapid progression and disease-causing gene variants and their predictive capacity tested and compared to the Mayo Classification. Results As expected, copeptin levels showed a significant negative correlation with eGFR. Measurements on tolvaptan showed significantly higher copeptin levels (9.871 pmol/l vs. 23.90 pmol/l at 90/30 mg; p Conclusion Copeptin levels are associated with kidney function and independently explained future eGFR slopes. As expected, treatment with tolvaptan strongly increases copeptin levels.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt

BACKGROUND: Imaging-based total kidney volume (TKV) and total liver volume (TLV) are major prognostic factors in autosomal dominant polycystic kidney disease (ADPKD) and end points for clinical trials. However, volumetry is time consuming and reader dependent in clinical practice. Our aim was to develop a fully automated method for joint kidney and liver segmentation in magnetic resonance imaging (MRI) and to evaluate its performance in a multisequence, multicenter setting. METHODS: The convolutional neural network was trained on a large multicenter dataset consisting of 992 MRI scans of 327 patients. Manual segmentation delivered ground-truth labels. The model’s performance was evaluated in a separate test dataset of 93 patients (350 MRI scans) as well as a heterogeneous external dataset of 831 MRI scans from 323 patients. RESULTS: The segmentation model yielded excellent performance, achieving a median per study Dice coefficient of 0.92–0.97 for the kidneys and 0.96 for the liver. Automatically computed TKV correlated highly with manual measurements (intraclass correlation coefficient [ICC]: 0.996–0.999) with low bias and high precision (−0.2%±4% for axial images and 0.5%±4% for coronal images). TLV estimation showed an ICC of 0.999 and bias/precision of −0.5%±3%. For the external dataset, the automated TKV demonstrated bias and precision of −1%±7%. CONCLUSIONS: Our deep learning model enabled accurate segmentation of kidneys and liver and objective assessment of TKV and TLV. Importantly, this approach was validated with axial and coronal MRI scans from 40 different scanners, making implementation in clinical routine care feasible. Clinical Trial registry name and registration number: The German ADPKD Tolvaptan Treatment Registry (AD[H]PKD), NCT02497521

Background Ketogenic dietary interventions (KDI) have been shown to be effective in animal models of polycystic kidney disease (PKD), but data from clinical trials are lacking. Methods Ten autosomal dominant PKD (ADPKD) patients with rapid disease progression were enrolled at visit V1 and initially maintained a carbohydrate-rich diet. At V2, patients entered one of the two KDI arms: a 3-day water fast (WF) or a 14-day ketogenic diet (KD). At V3, they resumed their normal diet for 3–6 weeks until V4. At each visit, magnetic resonance imaging kidney and liver volumetry was performed. Ketone bodies were evaluated to assess metabolic efficacy and questionnaires were used to determine feasibility. Results All participants [KD n = 5, WF n = 5; age 39.8 ± 11.6 years; estimated glomerular filtration rate 82 ± 23.5 mL/min/1.73 m2; total kidney volume (TKV) 2224 ± 1156 mL] were classified as Mayo Class 1C–1E. Acetone levels in breath and beta-hydroxybutyrate (BHB) blood levels increased in both study arms (V1 to V2 average acetone: 2.7 ± 1.2 p.p.m., V2 to V3: 22.8 ± 11.9 p.p.m., P = .0006; V1 to V2 average BHB: 0.22 ± 0.08 mmol/L, V2 to V3: 1.88 ± 0.93 mmol/L, P = .0008). Nine of 10 patients reached a ketogenic state and 9/10 evaluated KDIs as feasible. TKV did not change during this trial. However, we found a significant impact on total liver volume (ΔTLV V2 to V3: −7.7%, P = .01), mediated by changes in its non-cystic fraction. Conclusions RESET-PKD demonstrates that short-term KDIs potently induce ketogenesis and are feasible for ADPKD patients in daily life. While TLV quickly changed upon the onset of ketogenesis, changes in TKV may require longer-term interventions.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disease. Patients at high risk of severe disease progression should be identified early in order to intervene with supportive and therapeutic measures. However, the glomerular filtration rate (GFR) may remain within normal limits for decades until decline begins, making it a late indicator of rapid progression. Kidney volumetry is frequently used in clinical practice to allow for an assessment of disease severity. Due to limited prognostic accuracy, additional imaging markers are of high interest to improve outcome prediction in ADPKD, but data from clinical cohorts are still limited. In this study, we examined cyst fraction as one of these parameters in a cohort of 142 ADPKD patients. A subset of 61 patients received MRIs in two consecutive years to assess longitudinal changes. All MRIs were analyzed by segmentation and volumetry of the kidneys followed by determination of cyst fraction. As expected, both total kidney volume (TKV) and cyst fraction correlated with estimated GFR (eGFR), but cyst fraction showed a higher R2 in a univariate linear regression. Besides, only cyst fraction remained statistically significant in a multiple linear regression including both htTKV and cyst fraction to predict eGFR. Consequently, this study underlines the potential of cyst fraction in ADPKD and encourages prospective clinical trials examining its predictive value in combination with other biomarkers to predict future eGFR decline.

The coronavirus disease-19 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has taken the world by surprise. To date, a worldwide approved treatment remains lacking and hence in the context of rapid viral spread and the growing need for rapid action, drug repurposing has emerged as one of the frontline strategies in the battle against SARS-CoV2. Repurposed drugs currently being evaluated against COVID-19 either tackle the replication and spread of SARS-CoV2 or they aim at controlling hyper-inflammation and the rampaged immune response in severe disease. In both cases, the target for such drugs resides in the lungs, at least during the period where treatment could still provide substantial clinical benefit to the patient. Yet, most of these drugs are administered systemically, questioning the percentage of administered drug that actually reaches the lung and as a consequence, the distribution of the remainder of the dose to off target sites. Inhalation therapy should allow higher concentrations of the drug in the lungs and lower concentrations systemically, hence providing a stronger, more localized action, with reduced adverse effects. Therefore, the nano-reformulation of the repurposed drugs for inhalation is a promising approach for targeted drug delivery to lungs. In this review, we critically analyze, what nanomedicine could and ought to do in the battle against SARS-CoV2. We start by a brief description of SARS-CoV2 structure and pathogenicity and move on to discuss the current limitations of repurposed antiviral and immune-modulating drugs that are being clinically investigated against COVID-19. This account focuses on how nanomedicine could address limitations of current therapeutics, enhancing the efficacy, specificity and safety of such drugs. With the appearance of new variants of SARS-CoV2 and the potential implication on the efficacy of vaccines and diagnostics, the presence of an effective therapeutic solution is inevitable and could be potentially achieved via nano-reformulation. The presence of an inhaled nano-platform capable of delivering antiviral or immunomodulatory drugs should be available as part of the repertoire in the fight against current and future outbreaks., Graphical abstract Unlabelled Image

BACKGROUND Prediction of disease progression is one of the main challenges in the treatment of Autosomal-Dominant Polycystic Kidney Disease (ADPKD). In addition to already established biomarkers such as total kidney volume (TKV), further biomarkers that are feasible and easy to collect would be important to allow conclusions on prognosis as well as, in the best case, on long-term treatment response. Post-hoc analyses of the TEMPO 3:4 study showed that copeptin could serve as such a biomarker [1, 2]. In this study, we investigated copeptin over a multi-year course in participants of the AD(H)PKD study. METHOD Copeptin was measure from serum samples of 486 patients in the AD(H)PKD study at both initial and follow-up presentations. A total of 95 patients were on tolvaptan therapy at least at one of the measurement time points. We examined the measured values with respect to their distribution in different patient groups (e.g. age, Mayo class) and their changes on tolvaptan administration. Baseline eGFR values were developed using historic creatinine measurements up to 8000 days prior and 1500 days after copeptin measurements. For each patient with at least 5 creatinine measurements an eGFR slope was calculated. The models were developed using a robust linear modelling approach. Slopes were extracted to be the parameter associated to date and multiplied by 365 to retrieve an annual eGFR slope. eGFRs predicted for the specific dates at which copeptin has been measured were then calculated and used in subsequent modelling approaches. To develop a model predictive of eGFR, we used a stepwise regression approach, removing insignificant coefficients at each step. RESULTS As expected, copeptin was significantly lower in 391 tolvaptan naive patients than in 95 patients on therapy (9.95 pmol/L versus 19.78 pmol/L; P CONCLUSION Our data obtained from the real-life setting should again be reconciled with the results from the TEMPO 3:4 cohort. Our findings show that copeptin levels are both associated with kidney function and with future eGFR loss. Adding copeptin to the model of the Mayo Classification indeed improves the prediction of future eGFR. However, it is not clear yet whether this mild effect will have an impact in routine clinical care. Copeptin has the potential to facilitate prognostic assessment as a biomarker and thus could be helpful in deciding on therapy initiation and possibly more patient-specific dosing. Future analyses of outcome in relation to copeptin will provide further data and might enable implementation in clinical practice.

BACKGROUND AND AIMS Over the last years, there has been increasing evidence that defects in the energy metabolism of polycystic kidney disease (PKD) cyst lining cells, especially increased glucose dependency and defects in fatty acid oxidation, may underlie the pathogenesis of ADPKD, Rowe et al. (Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 2013; 19(4): 488–493). Based on these data, ketogenic dietary interventions have effectively been used in PKD animal models, Kipp et al. (A mild reduction of food intake slows disease progression in an orthologous mouse model of polycystic kidney disease. Am J Physiol Renal Physiol 2016;310(8):F726-F31), Torres et al. (Ketosis ameliorates renal cyst growth in polycystic kidney disease. Cell Metab 2019;30(6):1007–23 e5) and Warner et al. (Food restriction ameliorates the development of polycystic kidney disease. J Am Soc Nephrol 2016;27(5):1437–1447). As a first-in-human pilot study, the RESET-PKD study now strives to translate these promising results in the clinical setting. METHOD The present study enrolled 10 ADPKD patients with rapid disease progression. After a screening visit (V1), patients followed their usual carbohydrate-rich diet for up to 4 weeks. In a second visit (V2), patients chose to either perform a 3-day water fast (WF) or a 14-day ketogenic diet (KD) until a third study visit (V3), after which they returned to their normal high-carbohydrate diet for 3–6 weeks until a final visit (V4). At all visits, total kidney volume (TKV) and total liver volume (TLV) were monitored using MRI; anthropometric parameters, body composition and biochemical parameters (i.e. serum creatinine, complete blood cell count, glucose, β-hydroxybutyrate in fingerstick blood and acetone in breath) were measured. Ketone bodies were evaluated at all visits and in between. Feasibility was examined using respective questionnaires. Undesirable effects such as feelings of hunger and discomfort were documented in a study diary. RESULTS All participants (KD: n = 5, WF: n = 5; age: 39.8 ± 11.6 years; eGFR: 82 ± 23.5 mL/min; TKV: 2224 ± 1156 mL) were classified as Mayo Class 1C to 1E. Serum creatinine values were not altered by the ketogenic dietary interventions (V2: 1.17 ± 0.33 mg/dL versus V3: 1.20 ± 0.35 mg/dL, P = 0.826), but serum glucose levels decreased significantly (V2: 84 ± 3 mg/dL, V3: 70 ± 13 mg/dL, P = 0.004). BHB blood levels as well as acetone levels in breath increased in both study arms (V1 to V2 average acetone: 2.6 ± 1.18 ppm, V2 to V3: 22.8 ± 11.9 ppm, P CONCLUSION The RESET-PKD study demonstrates feasibility of short-term ketogenic interventions both regarding reliable attainment of ketosis and patient-reported feasibility of this intervention in everyday life. In this proof of principle study, short-term ketogenic interventions could not show a significant reduction in TKV. However, this endpoint is likely to require long-term exposure to ketogenesis. Future large-scale clinical trials examining such long-term dietary interventions—e.g. the KETO-ADPKD study—will be of great importance to further evaluate long-term feasibility and the therapeutic potential of ketogenic diets in ADPKD.

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are two rare genetic disorders that are caused by impairment of the mitochondrial enzyme sulfite oxidase. Sulfite oxidase is catalyzing the terminal reaction of cellular cysteine catabolism, the oxidation of sulfite to sulfate. Absence of sulfite oxidase leads to the accumulation of sulfite, which has been identified as a cellular toxin. However, the molecular pathways leading to the production of sulfite are still not completely understood. In order to identify novel treatment options for both disorders, the understanding of cellular cysteine catabolism - and its alterations upon loss of sulfite oxidase - is of utmost importance. Here we applied a new detection method of sulfite in cellular extracts to dissect the contribution of cytosolic and mitochondrial glutamate oxaloacetate transaminase (GOT) in the transformation of cysteine sulfinic acid to sulfite and pyruvate. We found that the cytosolic isoform GOT1 is primarily responsible for the production of sulfite. Moreover, loss of sulfite oxidase activity results in the accumulation of sulfite, H2S and persulfidated cysteine and glutathione, which is consistent with an increase of SQR protein levels. Surprisingly, none of the known H2S-producing pathways were found to be upregulated under conditions of sulfite toxicity suggesting an alternative route of sulfite-induced shift from oxidative to H2S dependent cysteine catabolism.

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are two rare genetic disorders that are caused by impairment of the mitochondrial enzyme sulfite oxidase. Sulfite oxidase is catalyzing the terminal reaction of cellular cysteine catabolism, the oxidation of sulfite to sulfate. Absence of sulfite oxidase leads to the accumulation of sulfite, which has been identified as a cellular toxin. However, the molecular pathways leading to the production of sulfite are still not completely understood. In order to identify novel treatment options for both disorders, the understanding of cellular cysteine catabolism – and its alterations upon loss of sulfite oxidase – is of utmost importance. Here we applied a new detection method of sulfite in cellular extracts to dissect the contribution of cytosolic and mitochondrial glutamate oxaloacetate transaminase (GOT) in the transformation of cysteine sulfinic acid to sulfite and pyruvate. We found that the cytosolic isoform GOT1 is primarily responsible for the production of sulfite. Moreover, loss of sulfite oxidase activity results in the accumulation of sulfite, H2S and persulfidated cysteine and glutathione, which is consistent with an increase of SQR protein levels. Surprisingly, none of the known H2S-producing pathways were found to be upregulated under conditions of sulfite toxicity suggesting an alternative route of sulfite-induced shift from oxidative to H2S dependent cysteine catabolism., Graphical abstract Alterations in cysteine catabolism following sulfite oxidase (SO) deficiency. Cysteine catabolism in wildtype and SO-deficient cells. Steps and enzymes that localize to mitochondria are depicted by the light blue background. The area outside refers to the cytosol. Cytosolic glutamate oxaloacetate transaminase 1 (GOT1) is responsible for cellular sulfite production, whereas mitochondrial GOT (GOT2), plays an important role in H2S production in SO deficiency. In absence of SO, sulfite (SO32−) and hydrogen sulfide (H2S) accumulate. SO deficiency furthermore further impacts cellular cysteine catabolism resulting in lower intracellular cysteine levels and downregulation of many involved enzymes (depicted in gray letters). Moderate increase in GOT2 and strong induction of sulfide:quinone oxidoreductase (SQR) suggest an increased flux of sulfur via the GOT2-MPST-SQR axis. Abbreviations: CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; CDO, cysteine dioxygenase; GOT, glutamate oxaloacetate transaminase; MPST, mercaptopyruvate sulfurtransferase; SQR, sulfide:quinone oxidoreductase; ETHE1, persulfide dioxygenase; TST, thiosulfate sulfurtransferase.Image 1, Highlights • Cellular sulfite detection enables investigation of cysteine catabolism in cell extracts. • Cytosolic glutamate oxaloacetate transaminase 1 is the primary source for cellular sulfite. • Deficiency of sulfite oxidase leads to accumulation of H2S and persulfidated small molecules. • Sulfite oxidase deficiency results in a downregulation of H2S synthesis and increased SQR expression.

BackgroundRapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.MethodsTo avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including loop-mediated isothermal amplification (LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).ResultsWhilst specificity of standard LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel LAMP assays with SHERLOCK.ConclusionIn summary, this approach reveals one-step multiplexed LAMP assays as a prime-option for the development of easy and cheap POC test kits.

BackgroundRapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.MethodsTo avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).ResultsWhilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.ConclusionIn summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.

OBJECTIVE Autosomal dominant polycystic kidney disease (ADPKD) is a chronic progressive disorder with a significant disease burden leading to end-stage renal disease in more than 75% of the affected individuals. Although prediction of disease progression is highly important, all currently available biomarkers-including height-adjusted total kidney volume (htTKV)-have important drawbacks in the everyday clinical setting. Thus, the purpose of this study was to evaluate T2 mapping as a source of easily obtainable and accurate biomarkers, which are needed for improved patient counseling and selection of targeted treatment options. MATERIALS AND METHODS A total of 139 ADPKD patients from The German ADPKD Tolvaptan Treatment Registry and 10 healthy controls underwent magnetic resonance imaging on a clinical 1.5-T system including acquisition of a Gradient-Echo-Spin-Echo T2 mapping sequence. The ADPKD patients were divided into 3 groups according to kidney cyst fraction (0%-35%, 36%-70%, >70%) as a surrogate marker for disease severity. The htTKV was calculated based on standard T2-weighted imaging. Mean T2 relaxation times of both kidneys (kidney-T2) as well as T2 relaxation times of the residual kidney parenchyma (parenchyma-T2) were measured on the T2 maps. RESULTS Calculation of parenchyma-T2 was 6- to 10-fold faster than determination of htTKV and kidney-T2 (0.78 ± 0.14 vs 4.78 ± 1.17 minutes, P < 0.001; 0.78 ± 0.14 vs 7.59 ± 1.57 minutes, P < 0.001). Parenchyma-T2 showed a similarly strong correlation to cyst fraction (r = 0.77, P < 0.001) as kidney-T2 (r = 0.76, P < 0.001), the strongest correlation to the serum-derived biomarker copeptin (r = 0.37, P < 0.001), and allowed for the most distinct separation of patient groups divided according to cyst fraction. In contrast, htTKV showed an only moderate correlation to cyst fraction (r = 0.48, P < 0.001). These observations were even more evident when considering only patients with preserved kidney function. CONCLUSIONS The rapidly assessable parenchyma-T2 shows a strong association with disease severity early in disease and is superior to htTKV when it comes to correlation with renal cyst fraction.

Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.

Mammalian sulfite oxidase (SO) is a dimeric enzyme consisting of a molybdenum cofactor- (Moco) and haem-containing domain and catalyses the oxidation of toxic sulfite to sulfate. Following sulfite oxidation, electrons are passed from Moco via the haem cofactor to cytochrome c, the terminal electron acceptor. In contrast, plant SO (PSO) lacks the haem domain and electrons shuttle from Moco to molecular oxygen. Given the high similarity between plant and mammalian SO Moco domains, factors that determine the reactivity of PSO towards oxygen, remained unknown. In the present study, we generated mammalian haem-deficient and truncated SO variants and demonstrated their oxygen reactivity by hydrogen peroxide formation and oxygen-consumption studies. We found that intramolecular electron transfer between Moco and haem showed an inverse correlation to SO oxygen reactivity. Haem-deficient SO variants exhibited oxygen-dependent sulfite oxidation similar to PSO, which was confirmed further using haem-deficient human SO in a cell-based assay. This finding suggests the possibility to use oxygen-reactive SO variants in sulfite detoxification, as the loss of SO activity is causing severe neurodegeneration. Therefore we evaluated the potential use of PEG attachment (PEGylation) as a modification method for future enzyme substitution therapies using oxygen-reactive SO variants, which might use blood-dissolved oxygen as the electron acceptor. PEGylation has been shown to increase the half-life of other therapeutic proteins. PEGylation resulted in the modification of up to eight surface-exposed lysine residues of SO, an increased conformational stability and similar kinetic properties compared with wild-type SO.

Molybdenum cofactor deficiency (MoCD) is a lethal autosomal recessive inborn error of metabolism with devastating neurologic manifestations. Currently, experimental treatment with cyclic pyranopterin monophosphate (cPMP) is available for patients with MoCD type A caused by a mutation in the MOCS-1 gene. Here we report the first case of an infant, prenatally diagnosed with MoCD type A, whom we started on treatment with cPMP 4 hours after birth. The most reliable method to evaluate neurologic functioning in early infancy is to assess the quality of general movements (GMs) and fidgety movements (FMs). After a brief period of seizures and cramped-synchronized GMs on the first day, our patient showed no further clinical signs of neurologic deterioration. Her quality of GMs was normal by the end of the first week. Rapid improvement of GM quality together with normal FMs at 3 months is highly predictive of normal neurologic outcome. We demonstrated that a daily cPMP dose of even 80 μg/kg in the first 12 days reduced the effects of neurodegenerative damage even when seizures and cramped-synchronized GMs were already present. We strongly recommend starting cPMP treatment as soon as possible after birth in infants diagnosed with MoCD type A.

Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurological damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of MoCD and sulfite oxidase deficiency (SOD). Recently, a first successful treatment of a human MoCD type A patient based on a substitution therapy with the molybdenum cofactor precursor cPMP has been reported, resulting in nearly complete normalization of MoCD biomarkers. Knowing the rapid progression of the disease symptoms in nontreated patients, an early diagnosis of MoCD as well as a sensitive method to monitor daily changes in SSC levels, a key marker of sulfite toxicity, is crucial for treatment outcome. Here, we describe a fast and sensitive method for the analysis of SSC in human urine samples using high performance liquid chromatography (HPLC). The analysis is based on precolumn derivatization with O-phthaldialdehyde (OPA) and separation on a C18 reverse phase column coupled to UV detection. The method was extended to human serum analysis and no interference with endogenous amino acids was found. Finally, SSC values from 45 pediatric urine, 75 adult urine, and 24 serum samples from control individuals as well as MoCD patients are reported. Our method represents a cost-effective technique for routine diagnosis of MoCD and SOD, and can be used also to monitor treatment efficiency in those sulfite toxicity disorders on a daily basis.