Sphingolipid desaturase DEGS1 is essential for mitochondria-associated membrane integrity.

Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental disorders to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase Δ4-dihydroceramide desaturase 1 (DEGS1) acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18) (OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane-resident (MAM-resident) enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multiomics, and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: (a) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; (b) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; (c) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; and (d) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAM function.

A novel mutation in the GFAP gene expands the phenotype of Alexander disease.

BACKGROUND: Alexander disease, an autosomal dominant leukodystrophy, is caused by missense mutations in GFAP. Although mostly diagnosed in children, associated with severe leukoencephalopathy, milder adult forms also exist. METHODS: A family affected by adult-onset spastic paraplegia underwent neurological examination and cerebral MRI. Two patients were sequenced by whole exome sequencing (WES). A candidate variant was functionally tested in an astrocytoma cell line. RESULTS: The novel variant in GFAP (Glial Fibrillary Acidic Protein) N-terminal head domain (p.Gly18Val) cosegregated in multiple relatives (LOD score: 2.7). All patients, even those with the mildest forms, showed characteristic signal changes or atrophy in the brainstem and spinal cord MRIs, and abnormal MRS. In vitro, this variant did not cause significant protein aggregation, in contrast to most Alexander disease mutations characterised so far. However, cell area analysis showed larger size, a feature previously described in patients and mouse models. CONCLUSION: We suggest that this variant causes variable expressivity and an attenuated phenotype of Alexander disease type II, probably associated with alternative pathogenic mechanisms, that is, astrocyte enlargement. GFAP analysis should be considered in adult-onset neurological presentations with pyramidal and bulbar symptoms, in particular when characteristic findings, such as the tadpole sign, are present in MRI. WES is a powerful tool to diagnose atypical cases.

Aberrant regulation of the GSK-3ß/NRF2 axis unveils a novel therapy for adrenoleukodystrophy.

The nuclear factor erythroid 2-like 2 (NRF2) is the master regulator of endogenous antioxidant responses. Oxidative damage is a shared and early-appearing feature in X-linked adrenoleukodystrophy (X-ALD) patients and the mouse model (Abcd1 null mouse). This rare neurometabolic disease is caused by the loss of function of the peroxisomal transporter ABCD1, leading to an accumulation of very long-chain fatty acids and the induction of reactive oxygen species of mitochondrial origin. Here, we identify an impaired NRF2 response caused by aberrant activity of GSK-3ß. We find that GSK-3ß inhibitors can significantly reactivate the blunted NRF2 response in patients' fibroblasts. In the mouse models (Abcd1- and Abcd1-/Abcd2-/- mice), oral administration of dimethyl fumarate (DMF/BG12/Tecfidera), an NRF2 activator in use for multiple sclerosis, normalized (i) mitochondrial depletion, (ii) bioenergetic failure, (iii) oxidative damage, and (iv) inflammation, highlighting an intricate cross-talk governing energetic and redox homeostasis in X-ALD Importantly, DMF halted axonal degeneration and locomotor disability suggesting that therapies activating NRF2 hold therapeutic potential for X-ALD and other axonopathies with impaired GSK-3ß/NRF2 axis.

Oxidative stress and mitochondrial dynamics malfunction are linked in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a fatal hypomyelinating disorder characterized by early impairment of motor development, nystagmus, choreoathetotic movements, ataxia and progressive spasticity. PMD is caused by variations in the proteolipid protein gene PLP1, which encodes the two major myelin proteins of the central nervous system, PLP and its spliced isoform DM20, in oligodendrocytes. Large duplications including the entire PLP1 gene are the most frequent causative mutation leading to the classical form of PMD. The Plp1 overexpressing mouse model (PLP-tg66/66 ) develops a phenotype very similar to human PMD, with early and severe motor dysfunction and a dramatic decrease in lifespan. The sequence of cellular events that cause neurodegeneration and ultimately death is poorly understood. In this work, we analyzed patient-derived fibroblasts and spinal cords of the PLP-tg66/66 mouse model, and identified redox imbalance, with altered antioxidant defense and oxidative damage to several enzymes involved in ATP production, such as glycolytic enzymes, creatine kinase and mitochondrial proteins from the Krebs cycle and oxidative phosphorylation. We also evidenced malfunction of the mitochondria compartment with increased ROS production and depolarization in PMD patient's fibroblasts, which was prevented by the antioxidant N-acetyl-cysteine. Finally, we uncovered an impairment of mitochondrial dynamics in patient's fibroblasts which may help explain the ultrastructural abnormalities of mitochondria morphology detected in spinal cords from PLP-tg66/66 mice. Altogether, these results underscore the link between redox and metabolic homeostasis in myelin diseases, provide insight into the pathophysiology of PMD, and may bear implications for tailored pharmacological intervention.

Altered glycolipid and glycerophospholipid signaling drive inflammatory cascades in adrenomyeloneuropathy.

X-linked adrenomyeloneuropathy (AMN) is an inherited neurometabolic disorder caused by malfunction of the ABCD1 gene, characterized by slowly progressing spastic paraplegia affecting corticospinal tracts, and adrenal insufficiency. AMN is the most common phenotypic manifestation of adrenoleukodystrophy (X-ALD). In some cases, an inflammatory cerebral demyelination occurs associated to poor prognosis in cerebral AMN (cAMN). Though ABCD1 codes for a peroxisomal transporter of very long-chain fatty acids, the molecular mechanisms that govern disease onset and progression, or its transformation to a cerebral, inflammatory demyelinating form, remain largely unknown. Here we used an integrated -omics approach to identify novel biomarkers and altered network dynamic characteristic of, and possibly driving, the disease. We combined an untargeted metabolome assay of plasma and peripheral blood mononuclear cells (PBMC) of AMN patients, which used liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-Q-TOF), with a functional genomics analysis of spinal cords of Abcd1(-) mouse. The results uncovered altered nodes in lipid-driven proinflammatory cascades, such as glycosphingolipid and glycerophospholipid synthesis, governed by the ß-1,4-galactosyltransferase (B4GALT6), the phospholipase 2γ (PLA2G4C) and the choline/ethanolamine phosphotransferase (CEPT1) enzymes. Confirmatory investigations revealed a non-classic, inflammatory profile, consisting on the one hand of raised plasma levels of several eicosanoids derived from arachidonic acid through PLA2G4C activity, together with also the proinflammatory cytokines IL6, IL8, MCP-1 and tumor necrosis factor-α. In contrast, we detected a more protective, Th2-shifted response in PBMC. Thus, our findings illustrate a previously unreported connection between ABCD1 dysfunction, glyco- and glycerolipid-driven inflammatory signaling and a fine-tuned inflammatory response underlying a disease considered non-inflammatory.

Dietary intake of green tea polyphenols regulates insulin sensitivity with an increase in AMP-activated protein kinase α content and changes in mitochondrial respiratory complexes.

SCOPE: The intake of food rich in polyphenols is related to a lower incidence in almost all chronic degenerative diseases. However, relatively little is known about the molecular mechanisms involved in its antioxidant properties. The aim of this study was to determine whether the mechanism of action of polyphenols could be related to a modulation in energy uptake and metabolism, and further induced mitochondrial changes. METHODS AND RESULTS: For this purpose, male C57BL6 mice were fed during 3 months with a tea-based beverage rich in polyphenols. Insulin sensitivity, tissue oxidative damage biomarkers, as well as energy-related signaling pathways were determined to evaluate its mechanism of action. As a result, a tissue- and protein-specific subtle reduction in oxidative damage was observed. Skeletal muscle showed mitochondrial changes in respiratory complexes and an increase in AMP-activated protein kinase α levels, suggesting reduced energy availability. These changes were also associated with adipose tissue cellular metabolism. This was confirmed by a decline in the potential of energy uptake, evidenced by a diminished intestinal and systemic absorption of carbohydrates together with an inhibition of insulin sensitivity. CONCLUSIONS: Our results suggest that the mechanisms of action of green tea polyphenols may be related to their ability to modulate energy uptake leading to mitochondrial adaptations possibly responsible for the changes in protein oxidative damage.

Oxidative stress modulates mitochondrial failure and cyclophilin D function in X-linked adrenoleukodystrophy.

A common process associated with oxidative stress and severe mitochondrial impairment is the opening of the mitochondrial permeability transition pore, as described in many neurodegenerative diseases. Thus, inhibition of mitochondrial permeability transition pore opening represents a potential target for inhibiting mitochondrial-driven cell death. Among the mitochondrial permeability transition pore components, cyclophilin D is the most studied and has been found increased under pathological conditions. Here, we have used in vitro and in vivo models of X-linked adrenoleukodystrophy to investigate the relationship between the mitochondrial permeability transition pore opening and redox homeostasis. X-linked adrenoleukodystrophy is a neurodegenerative condition caused by loss of function of the peroxisomal ABCD1 transporter, in which oxidative stress plays a pivotal role. In this study, we provide evidence of impaired mitochondrial metabolism in a peroxisomal disease, as fibroblasts in patients with X-linked adrenoleukodystrophy cannot survive when forced to rely on mitochondrial energy production, i.e. on incubation in galactose. Oxidative stress induced under galactose conditions leads to mitochondrial damage in the form of mitochondrial inner membrane potential dissipation, ATP drop and necrotic cell death, together with increased levels of oxidative modifications in cyclophilin D protein. Moreover, we show increased expression levels of cyclophilin D in the affected zones of brains in patients with adrenomyeloneuropathy, in spinal cord of a mouse model of X-linked adrenoleukodystrophy (Abcd1-null mice) and in fibroblasts from patients with X-linked adrenoleukodystrophy. Notably, treatment with antioxidants rescues mitochondrial damage markers in fibroblasts from patients with X-linked adrenoleukodystrophy, including cyclophilin D oxidative modifications, and reverses cyclophilin D induction in vitro and in vivo. These findings provide mechanistic insight into the beneficial effects of antioxidants in neurodegenerative and non-neurodegenerative cyclophilin D-dependent disorders.

Antioxidants halt axonal degeneration in a mouse model of X-adrenoleukodystrophy.

OBJECTIVE: Axonal degeneration is a main contributor to disability in progressive neurodegenerative diseases in which oxidative stress is often identified as a pathogenic factor. We aim to demonstrate that antioxidants are able to improve axonal degeneration and locomotor deficits in a mouse model of X-adrenoleukodystrophy (X-ALD). METHODS: X-ALD is a lethal disease caused by loss of function of the ABCD1 peroxisomal transporter of very long chain fatty acids (VLCFA). The mouse model for X-ALD exhibits a late onset neurological phenotype with locomotor disability and axonal degeneration in spinal cord resembling the most common phenotype of the disease, adrenomyeloneuropathy (X-AMN). Recently, we identified oxidative damage as an early event in life, and the excess of VLCFA as a generator of radical oxygen species (ROS) and oxidative damage to proteins in X-ALD. RESULTS: Here, we prove the capability of the antioxidants N-acetyl-cysteine, α-lipoic acid, and α-tocopherol to scavenge VLCFA-dependent ROS generation in vitro. Furthermore, in a preclinical setting, the cocktail of the 3 compounds reversed: (1) oxidative stress and lesions to proteins, (2) immunohistological signs of axonal degeneration, and (3) locomotor impairment in bar cross and treadmill tests. INTERPRETATION: We have established a direct link between oxidative stress and axonal damage in a mouse model of neurodegenerative disease. This conceptual proof of oxidative stress as a major disease-driving factor in X-AMN warrants translation into clinical trials for X-AMN, and invites assessment of antioxidant strategies in axonopathies in which oxidative damage might be a contributing factor.

Oxidative damage compromises energy metabolism in the axonal degeneration mouse model of X-adrenoleukodystrophy.

AIMS: Chronic metabolic impairment and oxidative stress are associated with the pathogenesis of axonal dysfunction in a growing number of neurodegenerative conditions. To investigate the intertwining of both noxious factors, we have chosen the mouse model of adrenoleukodystrophy (X-ALD), which exhibits axonal degeneration in spinal cords and motor disability. The disease is caused by loss of function of the ABCD1 transporter, involved in the import and degradation of very long-chain fatty acids (VLCFA) in peroxisomes. Oxidative stress due to VLCFA excess appears early in the neurodegenerative cascade. RESULTS: In this study, we demonstrate by redox proteomics that oxidative damage to proteins specifically affects five key enzymes of glycolysis and TCA (Tricarboxylic acid) cycle in spinal cords of Abcd1(-) mice and pyruvate kinase in human X-ALD fibroblasts. We also show that NADH and ATP levels are significantly diminished in these samples, together with decrease of pyruvate kinase activities and GSH levels, and increase of NADPH. INNOVATION: Treating Abcd1(-) mice with the antioxidants N-acetylcysteine and α-lipoic acid (LA) prevents protein oxidation; preserves NADH, NADPH, ATP, and GSH levels; and normalizes pyruvate kinase activity, which implies that oxidative stress provoked by VLCFA results in bioenergetic dysfunction, at a presymptomatic stage. CONCLUSION: Our results provide mechanistic insight into the beneficial effects of antioxidants and enhance the rationale for translation into clinical trials for X-adrenoleukodystrophy.