Current Aspects on the Pathophysiology of Bone Metabolic Defects during Progression of Scoliosis in Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1), which is the most common phacomatoses, is an autosomal dominant disorder characterized by clinical presentations in various tissues and organs, such as the skin, eyes and nervous and skeletal systems. The musculoskeletal implications of NF1 include a variety of deformities, including scoliosis, kyphoscoliosis, spondylolistheses, congenital bony bowing, pseudarthrosis and bone dysplasia. Scoliosis is the most common skeletal problem, affecting 10–30% of NF1 patients. Although the pathophysiology of spinal deformities has not been elucidated yet, defects in bone metabolism have been implicated in the progression of scoliotic curves. Measurements of Bone Mineral Density (BMD) in the lumbar spine by using dual energy absorptiometry (DXA) and quantitative computer tomography (QCT) have demonstrated a marked reduction in Z-score and osteoporosis. Additionally, serum bone metabolic markers, such as vitamin D, calcium, phosphorus, osteocalcin and alkaline phosphatase, have been found to be abnormal. Intraoperative and histological vertebral analysis confirmed that alterations of the trabecular microarchitecture are associated with inadequate bone turnover, indicating generalized bone metabolic defects. At the molecular level, loss of function of neurofibromin dysregulates Ras and Transforming Growth factor-β1 (TGF-β1) signaling and leads to altered osteoclastic proliferation, osteoblastic activity and collagen production. Correlation between clinical characteristics and molecular pathways may provide targets for novel therapeutic approaches in NF1.

Improved Vascularization and Survival of White Compared to Brown Adipose Tissue Grafts in the Dorsal Skinfold Chamber

Fat grafting is a frequently applied procedure in plastic surgery for volume reconstruction. Moreover, the transplantation of white adipose tissue (WAT) and brown adipose tissue (BAT) increasingly gains interest in preclinical research for the treatment of obesity-related metabolic defects. Therefore, we herein directly compared the vascularization capacity and survival of WAT and BAT grafts. For this purpose, size-matched grafts isolated from the inguinal WAT pad and the interscapular BAT depot of C57BL/6N donor mice were syngeneically transplanted into the dorsal skinfold chamber of recipient animals. The vascularization and survival of the grafts were analyzed by means of intravital fluorescence microscopy, histology, and immunohistochemistry over an observation period of 14 days. WAT grafts showed an identical microvascular architecture and functional microvessel density as native WAT. In contrast, BAT grafts developed an erratic microvasculature with a significantly lower functional microvessel density when compared to native BAT. Accordingly, they also contained a markedly lower number of CD31-positive microvessels, which was associated with a massive loss of perilipin-positive adipocytes. These findings indicate that in contrast to WAT grafts, BAT grafts exhibit an impaired vascularization capacity and survival, which may be due to their higher metabolic demand. Hence, future studies should focus on the establishment of strategies to improve the engraftment of transplanted BAT.

Impaired ion homeostasis as a possible associate factor in mucopolysaccharidosis pathogenesis: transcriptomic, cellular and animal studies

Mucopolysaccharidoses (MPS) are a group of diseases caused by mutations resulting in deficiencies of lysosomal enzymes which lead to the accumulation of partially undegraded glycosaminoglycans (GAG). This phenomenon causes severe and chronic disturbances in the functioning of the organism, and leads to premature death. The metabolic defects affect also functions of the brain in most MPS types (except types IV, VI, and IX). The variety of symptoms, as well as the ineffectiveness of GAG-lowering therapies, question the early theory that GAG storage is the only cause of these diseases. As disorders of ion homeostasis increasingly turn out to be co-causes of the pathogenesis of various human diseases, the aim of this work was to determine the perturbations related to the maintenance of the ion balance at both the transcriptome and cellular levels in MPS. Transcriptomic studies, performed with fibroblasts derived from patients with all types/subtypes of MPS, showed extensive changes in the expression of genes involved in processes related to ion binding, transport and homeostasis. Detailed analysis of these data indicated specific changes in the expression of genes coding for proteins participating in the metabolism of Ca2+, Fe2+ and Zn2+. The results of tests carried out with the mouse MPS I model (Idua−/−) showed reductions in concentrations of these 3 ions in the liver and spleen. The results of these studies indicate for the first time ionic concentration disorders as possible factors influencing the course of MPS and show them as hypothetical, additional therapeutic targets for this rare disease.

AMPK modulation ameliorates dominant disease phenotypes of CTRP5 variant in retinal degeneration

Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder caused by a missense substitution in CTRP5. Distinctive clinical features include sub-retinal pigment epithelium (RPE) deposits, choroidal neovascularization, and RPE atrophy. In induced pluripotent stem cells-derived RPE from L-ORD patients (L-ORD-iRPE), we show that the dominant pathogenic CTRP5 variant leads to reduced CTRP5 secretion. In silico modeling suggests lower binding of mutant CTRP5 to adiponectin receptor 1 (ADIPOR1). Downstream of ADIPOR1 sustained activation of AMPK renders it insensitive to changes in AMP/ATP ratio resulting in defective lipid metabolism, reduced Neuroprotectin D1(NPD1) secretion, lower mitochondrial respiration, and reduced ATP production. These metabolic defects result in accumulation of sub-RPE deposits and leave L-ORD-iRPE susceptible to dedifferentiation. Gene augmentation of L-ORD-iRPE with WT CTRP5 or modulation of AMPK, by metformin, re-sensitize L-ORD-iRPE to changes in cellular energy status alleviating the disease cellular phenotypes. Our data suggests a mechanism for the dominant behavior of CTRP5 mutation and provides potential treatment strategies for L-ORD patients.

Quantification of Differential Metabolites in Dried Blood Spots Using Second-Tier Testing for SCADD/IBDD Disorders Based on Large-Scale Newborn Screening in a Chinese Population

Background: Although newborn screening (NBS) for metabolic defects using the marker butyl carnitine (C4) combined with the C4-to-acetylcarnitine ratio is adequate, the incorporation of novel parameters may improve differential testing for these disorders without compromising sensitivity.Methods: Analytical and clinical performance was evaluated by MS/MS using 237 initially positive neonatal samples between March 2019 and March 2020 at the Newborn Screening Center of Xuzhou Maternity and Child Health Care Hospital. Additionally, second-tier testing by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with the quantification of ethylmalonate (EMA) or isobutyryl-glycine (IBG) in dried blood spots (DBSs) was performed to reduce the false-positive rate.Results: We reviewed initial MS/MS data for DBSs from 469,730 neonates, and a second-tier test was performed using 237 samples that exceeded the C4 concentration cutoff value. Eleven variants of the ACADS gene were identified, with c.1031A>G (p.E344G) being the most common. Fifteen ACAD8 mutations were identified in seven patients, and Swiss modeling and amino acid conservation analyses were conducted for the novel variants. Based on a retrospective analysis of EMA and IBG, the application of second-tier tests before the release of neonatal screening results reduced referrals by over 91.89% and improved the positive predictive value (PPV) for short-chain acyl-CoA dehydrogenase deficiency/isobutyryl-CoA dehydrogenase deficiency (SCADD/IBDD) screening.Conclusion: A screening algorithm including EMA/IBG improves target differential testing for NBS and may eliminate unnecessary referrals while maintaining 100% sensitivity. Second-tier screening using UPLC-MS/MS as a rapid and convenient supplemental DNA sequencing method may be beneficial for differential detection.

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock

Biological clocks are fundamental to an organism′s health, controlling periodicity of behavior and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signaling, the generation of an ultradian oscillator, and its metabolic consequences.

TAMI-26. INVESTIGATING TUMOR MICROENVIRONMENT METABOLIC DEPENDENCIES IN GLIOBLASTOMA

Metabolic reprogramming is a hallmark of cancer. Malignant cells must acquire metabolic adaptations in response to a multitude of intrinsic and extrinsic factors to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Altered metabolism in GBM is becoming an increasingly promising area of research to identify novel therapeutic targets and biomarkers, as metabolic rewiring can occur across numerous genotypes. The unique features of the brain TME pose a difficult challenge when studying GBM and other primary brain cancers – currently, the availability of nutrients in the brain, as well as how they influence or are influenced by tumor metabolism, are not well understood. Our group has identified a subgroup of gliomas, hereafter termed TME-dependent, which can only form tumors in the brain TME. While genetically heterogeneous, these tumors share transcriptional identities linked to oligodendrocyte precursor cell (OPC) and neuronal lineages. Systematic molecular profiling of over 75 patient tumors and their matched cell culture and brain orthotopic xenograft derived models revealed that TME-dependent tumors display lipid metabolic signatures linked to signaling and interactions with surrounding neurons and glial cells. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity in fatty acid biosynthetic programs, indicating a potential brain-microenvironment specific metabolic dependency linked to transcriptional identity that can be targeted for therapy.

Mitochondrial Respiration Defects in Single-Ventricle Congenital Heart Disease

Background: Congenital heart disease (CHD) with single-ventricle (SV) physiology is now survivable with a three-stage surgical course ending with Fontan palliation. However, 10-year transplant-free survival remains at 39–50%, with ventricular dysfunction progressing to heart failure (HF) being a common sequela. For SV-CHD patients who develop HF, undergoing the surgical course would not be helpful and could even be detrimental. As HF risk cannot be predicted and metabolic defects have been observed in Ohia SV-CHD mice, we hypothesized that respiratory defects in peripheral blood mononuclear cells (PBMCs) may allow HF risk stratification in SV-CHD.Methods: SV-CHD (n = 20), biventricular CHD (BV-CHD; n = 16), or healthy control subjects (n = 22) were recruited, and PBMC oxygen consumption rate (OCR) was measured using the Seahorse Analyzer. Respiration was similarly measured in Ohia mouse heart tissue.Results: Post-Fontan SV-CHD patients with HF showed higher maximal respiratory capacity (p = 0.004) and respiratory reserve (p < 0.0001), parameters important for cell stress adaptation, while the opposite was found for those without HF (reserve p = 0.037; maximal p = 0.05). This was observed in comparison to BV-CHD or healthy controls. However, respiration did not differ between SV patients pre- and post-Fontan or between pre- or post-Fontan SV-CHD patients and BV-CHD. Reminiscent of these findings, heart tissue from Ohia mice with SV-CHD also showed higher OCR, while those without CHD showed lower OCR.Conclusion: Elevated mitochondrial respiration in PBMCs is correlated with HF in post-Fontan SV-CHD, suggesting that PBMC respiration may have utility for prognosticating HF risk in SV-CHD. Whether elevated respiration may reflect maladaptation to altered hemodynamics in SV-CHD warrants further investigation.

Chronic Kidney Disease-Induced Arterial Media Calcification in Rats Prevented by Tissue Non-Specific Alkaline Phosphatase Substrate Supplementation Rather Than Inhibition of the Enzyme

Patients with chronic kidney disease (CKD) suffer from arterial media calcification and a disturbed bone metabolism. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the calcification inhibitor pyrophosphate (PPi) into inorganic phosphate (Pi) and thereby stimulates arterial media calcification as well as physiological bone mineralization. This study investigates whether the TNAP inhibitor SBI-425, PPi or the combination of both inhibit arterial media calcification in an 0.75% adenine rat model of CKD. Treatments started with the induction of CKD, including (i) rats with normal renal function (control diet) treated with vehicle and CKD rats treated with either (ii) vehicle, (iii) 10 mg/kg/day SBI-425, (iv) 120 µmol/kg/day PPi and (v) 120 µmol/kg/day PPi and 10 mg/kg/day SBI-425. All CKD groups developed a stable chronic renal failure reflected by hyperphosphatemia, hypocalcemia and high serum creatinine levels. CKD induced arterial media calcification and bone metabolic defects. All treatments, except for SBI-425 alone, blocked CKD-related arterial media calcification. More important, SBI-425 alone and in combination with PPi increased osteoid area pointing to a less efficient bone mineralization. Clearly, potential side effects on bone mineralization will need to be assessed in any clinical trial aimed at modifying the Pi/PPi ratio in CKD patients who already suffer from a compromised bone status.