A knock-in rat model unravels acute and chronic renal toxicity in glutaric aciduria type I.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an autosomal recessive inborn error of metabolism caused by deficiency of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH). The principal clinical manifestation in GA-I patients is striatal injury most often triggered by catabolic stress. Early diagnosis by newborn screening programs improved survival and reduced striatal damage in GA-I patients. However, the clinical phenotype is still evolving in the aging patient population. Evaluation of long-term outcome in GA-I patients recently identified glomerular filtration rate (GFR) decline with increasing age. We recently created the first knock-in rat model for GA-I harboring the mutation p.R411W (c.1231 C>T), corresponding to the most frequent GCDH human mutation p.R402W. In this study, we evaluated the effect of an acute metabolic stress in form of high lysine diet (HLD) on young Gcdhki/ki rats. We further studied the chronic effect of GCDH deficiency on kidney function in a longitudinal study on a cohort of Gcdhki/ki rats by repetitive 68Ga-EDTA positron emission tomography (PET) renography, biochemical and histological analyses. In young Gcdhki/ki rats exposed to HLD, we observed a GFR decline and biochemical signs of a tubulopathy. Histological analyses revealed lipophilic vacuoles, thinning of apical brush border membranes and increased numbers of mitochondria in proximal tubular (PT) cells. HLD also altered OXPHOS activities and proteome in kidneys of Gcdhki/ki rats. In the longitudinal cohort, we showed a progressive GFR decline in Gcdhki/ki rats starting at young adult age and a decline of renal clearance. Histopathological analyses in aged Gcdhki/ki rats revealed tubular dilatation, protein accumulation in PT cells and mononuclear infiltrations. These observations confirm that GA-I leads to acute and chronic renal damage. This raises questions on indication for follow-up on kidney function in GA-I patients and possible therapeutic interventions to avoid renal damage.

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

TMEM70 deficiency: long-term outcome of 48 patients.

OBJECTIVES: TMEM70 deficiency is the most common nuclear-encoded defect affecting the ATP synthase. In this multicentre retrospective study we characterise the natural history of the disease, treatment and outcome in 48 patients with mutations in TMEM70. Eleven centers from eight European countries, Turkey and Israel participated. RESULTS: All 27 Roma and eight non-Roma patients were homozygous for the common mutation c.317-2A > G. Five patients were compound heterozygotes for the common mutation and mutations c.470 T > A, c.628A > C, c.118_119insGT or c.251delC. Six Arab Muslims and two Turkish patients were homozygous for mutations c.238C > T, c.316 + 1G > T, c.336 T > A, c.578_579delCA, c.535C > T, c.359delC. Age of onset was neonatal in 41 patients, infantile in six cases and two years in one child. The most frequent symptoms at onset were poor feeding, hypotonia, lethargy, respiratory and heart failure, accompanied by lactic acidosis, 3-methylglutaconic aciduria and hyperammonaemia. Symptoms further included: developmental delay (98%), hypotonia (95%), faltering growth (94%), short stature (89%), non-progressive cardiomyopathy (89%), microcephaly (71%), facial dysmorphism (66%), hypospadias (50% of the males), persistent pulmonary hypertension of the newborn (22%) and Wolff-Parkinson-White syndrome (13%). One or more acute metabolic crises occurred in 24 surviving children, frequently followed by developmental regression. Hyperammonaemic episodes responded well to infusion with glucose and lipid emulsion, and ammonia scavengers or haemodiafiltration. Ten-year survival was 63%, importantly for prognostication, no child died after the age of five years. CONCLUSION: TMEM70 deficiency is a panethnic, multisystemic disease with variable outcome depending mainly on adequate management of hyperammonaemic crises in the neonatal period and early childhood.

Alterations of oxidative phosphorylation complexes in astrocytomas.

The shift in cellular energy production from oxidative phosphorylation (OXPHOS) to glycolysis, even under aerobic conditions, called the Warburg effect, is a feature of most solid tumors. The activity levels of OXPHOS complexes and citrate synthase were determined in astrocytomas. A gradual decrease of citrate synthase and OXPHOS complexes was observed depending on tumor grade. In low-grade astrocytomas (WHO grade II), enzyme activities of citrate synthase, complex I, and complex V were comparable to those of normal brain tissue. A trend to reduced activities was observed for complexes II-IV. In glioblastoma (WHO grade IV), activities of citrate synthase and complexes I-IV were decreased by 56-92% as compared with normal brain. Immunohistochemical staining for porin revealed that the tumorpil of low-grade astrocytomas displays characteristics of the mitochondria-rich neuropil of normal brain tissue. In high-grade tumors (WHO grades III and IV), the tumorpil was characterized by severe morphologic alterations as well as loss of "pilem" structures. Specific alterations of OXPHOS complexes were observed in all astrocytic tumors by immunohistochemical analysis: 80% of astrocytomas exhibited severe deficiency of complex IV; complex I showed a gradual reduction in amount with increasing tumor grade, whereas complex II showed reduced levels only in high-grade (WHO grade IV) tumors (9/12); complexes III and V did not show significant alterations compared with normal brain tissue. OXPHOS defects were present not only in the cell bodies of tumor cells but also in the pilem structures, indicating that the ramifications/protuberances (tumorpil) in general originate from tumor cells.

Case Report and Review of the Literature: A New and a Recurrent Variant in the VARS2 Gene Are Associated With Isolated Lethal Hypertrophic Cardiomyopathy, Hyperlactatemia, and Pulmonary Hypertension in Early Infancy.

Mitochondriopathies represent a wide spectrum of miscellaneous disorders with multisystem involvement, which are caused by various genetic changes. The establishment of the diagnosis of mitochondriopathy is often challenging. Recently, several mutations of the VARS2 gene encoding the mitochondrial valyl-tRNA synthetase were associated with early onset encephalomyopathies or encephalocardiomyopathies with major clinical features such as hypotonia, developmental delay, brain MRI changes, epilepsy, hypertrophic cardiomyopathy, and plasma lactate elevation. However, the correlation between genotype and phenotype still remains unclear. In this paper we present a male Caucasian patient with a recurrent c.1168G>A (p.Ala390Thr) and a new missense biallelic variant c.2758T>C (p.Tyr920His) in the VARS2 gene which were detected by whole exome sequencing (WES). VARS2 protein was reduced in the patient's muscle. A resulting defect of oxidative phosphorylation (OXPHOS) was proven by enzymatic assay, western blotting and immunohistochemistry from a homogenate of skeletal muscle tissue. Clinical signs of our patient included hyperlactatemia, hypertrophic cardiomyopathy (HCM) and pulmonary hypertension, which led to early death at the age of 47 days without any other known accompanying signs. The finding of novel variants in the VARS2 gene expands the spectrum of known mutations and phenotype presentation. Based on our findings we recommend to consider possible mitochondriopathy and to include the analysis of the VARS2 gene in the genetic diagnostic algorithm in cases with early manifesting and rapidly progressing HCM with hyperlactatemia.

Low VDAC1 Expression Is Associated with an Aggressive Phenotype and Reduced Overall Patient Survival in Cholangiocellular Carcinoma.

Abstract: Cancer cells frequently exhibit dysfunctional oxidative phosphorylation (OXPHOS) and a concomitant increase in glycolytic flux. We investigated the expression of OXPHOS complex subunits and mitochondrial mass in 34 human cholangiocellular carcinomas (CCCs) and adjacent normal tissue by using tissue microarrays. In the tumor periphery, all OXPHOS complexes were reduced except complex I. In addition, significantly lower levels of complex IV were found at the tumor center (p < 0.0001). Mitochondrial mass, as indicated by VDAC1 expression, was significantly increased in CCCs compared to corresponding normal tissue (p < 0.0001). VDAC1 levels were inversely correlated with UICC (Union Internationale Contre le Cancer) cancer stage classification (p = 0.0065). Furthermore, significantly lower VDAC1 was present in patients with lymph node involvement (p = 0.02). Consistent with this, patients whose carcinomas expressed VDAC1 at low to moderate levels had significantly reduced survival compared to high expressors (p < 0.05). Therefore, low mitochondrial mass is associated with more aggressive CCC. These metabolic features are indicative of a Warburg phenotype in CCCs. This metabolic signature has potential therapeutic implications because tumors with low mitochondrial function may be targeted by metabolic therapies such as a high-fat, low-carbohydrate ketogenic diet.

A ketogenic diet supplemented with medium-chain triglycerides enhances the anti-tumor and anti-angiogenic efficacy of chemotherapy on neuroblastoma xenografts in a CD1-nu mouse model.

Neuroblastoma (NB) is a pediatric malignancy characterized by a marked reduction in aerobic energy metabolism. Recent preclinical data indicate that targeting this metabolic phenotype by a ketogenic diet (KD), especially in combination with calorie restriction, slows tumor growth and enhances metronomic cyclophosphamide (CP) therapy of NB xenografts. Because calorie restriction would be contraindicated in most cancer patients, the aim of the present study was to optimize the KD such that the tumors are sensitized to CP without the need of calorie restriction. In a NB xenograft model, metronomic CP was combined with KDs of different triglyceride compositions and fed to CD1-nu mice ad libitum. Metronomic CP in combination with a KD containing 8-carbon medium-chain triglycerides exerted a robust anti-tumor effect, suppressing growth and causing a significant reduction of tumor blood-vessel density and intratumoral hemorrhage, accompanied by activation of AMP-activated protein kinase in NB cells. Furthermore, the KDs caused a significant reduction in the serum levels of essential amino acids, but increased those of serine, glutamine and glycine. Our data suggest that targeting energy metabolism by a modified KD may be considered as part of a multimodal treatment regimen to improve the efficacy of classic anti-NB therapy.

Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model.

INTRODUCTION: Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer's oxidative phosphorylation system. METHODS: Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content). RESULTS: Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention. CONCLUSIONS: Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.