The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF‐hand Ca²⁺‐binding motifs in their N‐terminal domains. Both proteins and their C‐terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H⁺. Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca²⁺ on the external side of the inner mitochondrial membrane, where the Ca²⁺‐binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca²⁺ through a mechanism independent of Ca²⁺ entry into mitochondria, and suggest a novel mechanism of Ca²⁺ regulation of the aspartate/malate shuttle.

Ca²⁺signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca²⁺entry in mitochondria via the Ca²⁺uniporter followed by Ca²⁺activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca²⁺signals via a mechanism that does not require the entry of Ca²⁺in mitochondria, a mechanism depending on the activity of Ca²⁺-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P_icarriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca²⁺in the intermembrane space and function together with the Ca²⁺uniporter in decoding the Ca²⁺signal into a mitochondrial response.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium (www.cebiond.org), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer’s, Parkinson’s, and Huntington’s diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

Purpose To assess the efficacy of a risk-adapted treatment policy for patients with stage I seminoma by using universally accepted risk criteria. Patients and Methods Between 1999 and 2003, 314 patients with clinical stage I seminoma after orchiectomy were prospectively included. One hundred patients (31.8%) presented no risk factors and were managed with surveillance. In contrast, 131 patients (41.7%) had tumors larger than 4 cm, 33 patients (10.5%) had rete testis involvement, and 50 patients (15.9%) had both risk factors. All the latter received two courses of adjuvant carboplatin. Results Chemotherapy was well tolerated, as only 17 patients (7.9%) presented grade 3 to 4 toxicity. Relapses were observed in six patients (6.0%) on surveillance and in seven patients (3.3%) treated with carboplatin (0.8% of tumors larger than 4 cm, 9.1% of those involving the rete testis, and 6.0% of patients with both risk criteria). All were located at the retroperitoneum, except for one at the spermatic cord. Median tumor size was 25 mm (range, 11 to 70 mm), and median time to relapse was 9 months (range, 4 to 28 months). All patients were rendered disease-free with chemotherapy (etoposide plus cisplatin). Median follow-up was 34 months (range, 12 to 72 months). The actuarial 5-year disease-free survival rate was 93.4% for patients on surveillance and 96.2% for patients treated with adjuvant chemotherapy. Overall 5-year survival was 100%. Conclusion Adjuvant carboplatin is effective in reducing the relapse rate in patients with stage I seminoma and risk factors. A risk-adapted strategy is safe and feasible and should be considered an alternative to systematic approaches, such as irradiation, chemotherapy, or surveillance.

Significance: Aerobic glycolysis is a metabolic adaptation that helps cells in a tumor meet high anabolic demands. The M2 isoform of pyruvate kinase (PKM2) is associated with aerobic glycolysis in cancer cells. Aerobic glycolysis also accounts for most of the Glc metabolized in retinas. We find that photoreceptors (PRs) in retinas, like cancer cells in tumors, express PKM2. We also found very little expression of pyruvate kinase (PK) in Müller glia. We present metabolic flux analyses that show a metabolic relationship between PRs and Müller cells (MCs) that is different from the relationship between some neurons and astrocytes in brain. To compensate for PK deficiency and aspartate/glutamate carrier 1 deficiencies, MCs can fuel their mitochondria with lactate and aspartate produced by PRs.