Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Regulation of erythrocyte survival by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK), an energy-sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca(2+) activity ([Ca(2+)](i)) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca(2+)](i) (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence-activated cell sorting (FACS) analysis. Glucose removal increased [Ca(2+)](i), decreased cell volume, and increased PS exposure. The AMPK-inhibitor compound C (20 microM) did not significantly modify eryptosis under glucose-replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca(2+)](i) by Ca(2+) ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM). As compared with erythrocytes from wild-type littermates (ampk(+/+)), erythrocytes from AMPKalpha1-deficient mice (ampk(-/-)) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk(-/-) mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes.

Delineating MT-ATP6-associated disease: From isolated neuropathy to early onset neurodegeneration.

OBJECTIVE: To delineate the phenotypic and genotypic spectrum in carriers of mitochondrial MT-ATP6 mutations in a large international cohort. METHODS: We analyzed in detail the clinical, genetical, and neuroimaging data from 132 mutation carriers from national registries and local databases from Europe, USA, Japan, and China. RESULTS: We identified 113 clinically affected and 19 asymptomatic individuals with a known pathogenic MT-ATP6 mutation. The most frequent mutations were m.8993 T > G (53/132, 40%), m.8993 T > C (30/132, 23%), m.9176 T > C (30/132, 23%), and m.9185 T > C (12/132, 9%). The degree of heteroplasmy was high both in affected (mean 95%, range 20%-100%) and unaffected individuals (mean 73%, range 20%-100%). Age at onset ranged from prenatal to the age of 75 years, but almost half of the patients (49/103, 48%) became symptomatic before their first birthday. In 28 deceased patients, the median age of death was 14 months. The most frequent symptoms were ataxia (81%), cognitive dysfunction (49%), neuropathy (48%), seizures (37%), and retinopathy (14%). A diagnosis of Leigh syndrome was made in 55% of patients, whereas the classic syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP) was rare (8%). CONCLUSIONS: In this currently largest series of patients with mitochondrial MT-ATP6 mutations, the phenotypic spectrum ranged from asymptomatic to early onset multisystemic neurodegeneration. The degree of mutation heteroplasmy did not reliably predict disease severity. Leigh syndrome was found in more than half of the patients, whereas classic NARP syndrome was rare. Oligosymptomatic presentations were rather frequent in adult-onset patients, indicating the need to include MT-ATP6 mutations in the differential diagnosis of both ataxias and neuropathies.

Modulation of suicidal erythrocyte cation channels by an AMPA antagonist.

In neurons alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are heteromeric cation channels composed of different sub-units, including GluA1-GluA4. When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels. In erythrocytes, activation of Ca(2+)-permeable cation channels triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with subsequent exposure of phosphatidylserine at the cell surface. Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose). The present study explored whether GluA1 is expressed in human erythrocytes and whether pharmacological AMPA receptor inhibition modifies Ca(2+) entry and suicidal death of human erythrocytes. GluA1 protein abundance was determined by confocal microscopy, phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+) concentration from Fluo3 fluorescence and channel activity by whole-cell patch-clamp recordings. As a result, GluA1 is indeed expressed in the erythrocyte cell membrane. The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion. The present study reveals a novel action of AMPA receptor antagonists and raises the possibility that GluA1 or a pharmacologically related protein participates in the regulation of Ca(2+) entry into and suicidal death of human erythrocytes.

Caffeine inhibits suicidal erythrocyte death.

Eryptosis, the suicidal death of erythrocytes, is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by several stress conditions including isotonic cell shrinkage (Cl(-) removal) and energy depletion (glucose removal). Both are effective through an increase in the cytosolic Ca(2+) concentration. Phosphatidylserine-exposing erythrocytes are cleared from circulating blood. Enhanced eryptosis thus leads to anemia. Accordingly, drugs interfering with eryptosis may prove useful in the treatment of anemia. The present study explored, whether caffeine interferes with eryptosis. Erythrocyte phosphatidylserine exposure was estimated from annexin V-binding, cell volume from forward scatter and cytosolic Ca(2+) activity from Fluo3 fluorescence. Under control conditions, eryptosis affected less than 5% of the erythrocytes and was not significantly modified by the presence of caffeine (50-500 microM). Glucose depletion (for 48 hours) significantly increased Fluo3 fluorescence and annexin V-binding and decreased forward scatter, effects partially reversed by caffeine (500 microM). Low Cl(-) solution (Cl(-) exchanged by gluconate for 48 hours) similarly increased annexin V-binding and decreased forward scatter, effects again reversed by caffeine (50-500 microM). In conclusion, caffeine inhibits Ca(2+) entry following glucose depletion and thus counteracts eryptosis during isotonic cell shrinkage and energy depletion.