渡邉 知佳

Niemann-Pick disease type C1 (NPC1) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants of the NPC1 gene that encodes a protein essential for lysosomal cholesterol transport. A deficiency in NPC1 results in the accumulation of unesterified cholesterol and sphingolipids, leading to neurological, psychiatric, and hepatic manifestations from infancy to adulthood. The currently approved treatment is palliative. Although the efficacy of gene therapy has been demonstrated in murine models, reliable biomarkers for evaluating the treatment effects remain unknown. We evaluated adeno-associated virus (AAV) vector-mediated NPC1 gene therapy in Npc1 homo-knockout (Npc1-/-) mice, focusing on blood-based biomarkers. An AAV vector carrying human NPC1 under a cytomegalovirus promoter (AAV-hNPC1) was administered intraperitoneally on days 6-8 after birth at varying vector doses and analyzed at multiple time points: 1.8 × 1011 vector genomes/mouse analyzed at 7 weeks (Low/7w) and 1.0 × 1012 vector genomes/mouse at 4 weeks (High/4w) and 9 weeks (High/9w). hNPC1 is expressed in the brain and liver, and a degree of neuronal cell survival is observed. High-dose AAV treatment improves body weight and rotarod performance. Plasma N-palmitoyl-O-phosphocholine-serine (PPCS) and lysosphingomyelin (lyso-SM) levels were significantly elevated in Npc1-/- mice. PPCS increased with disease progression but was significantly decreased after later points of high-dose AAV treatment (saline-treated Npc1-/- mice: 12.88 ± 3.53 ng/mL, AAV-treated Npc1-/- mice: 7.87 ± 1.67 ng/mL, p = 0.0008). Lyso-SM and oxysterols showed limited changes after therapy. Vector genome analysis revealed higher and more sustained levels in the brain than in the liver, which is consistent with rapid hepatocyte proliferation-reducing vector persistence. These findings demonstrate that systemic AAV-hNPC1 therapy ameliorates motor and neurological deficits but has a limited impact on several cholesterol-related biomarkers. PPCS has been suggested as a sensitive biomarker of therapeutic response and warrants further evaluations in preclinical and clinical NPC1 gene therapy trials.

BACKGROUND: Primary coenzyme Q10 (CoQ10) deficiency is a group of mitochondrial disorders caused by pathogenic variants of genes involved in CoQ10 biosynthesis. Although some patients respond to oral CoQ10 supplementation, the pathophysiology remains poorly understood. Ferroptosis, a form of iron-dependent cell death driven by lipid peroxidation, is suppressed by reduced CoQ10via ferroptosis suppressor protein 1 (FSP1). However, its involvement in primary CoQ10 deficiency has not yet been studied using patient-derived cells. CASES AND RESULTS: We reported six patients from three families and investigated ferroptosis susceptibility in fibroblasts from three representative patients: one with COQ2 variants and two with COQ4 variants. Fibroblasts with COQ2 variants showed increased vulnerability to ferroptosis inducers, plasma membrane lipid peroxidation. In contrast, fibroblasts with COQ4 variants exhibited only mild changes. Notably, susceptibility to ferroptosis remained unchanged after increasing intracellular CoQ10 levels. Despite this persistent ferroptosis sensitivity in vitro, the COQ2 patient exhibited significant clinical improvement following CoQ10 supplementation. These findings suggest that ferroptosis may contribute to cellular vulnerability in primary CoQ10 deficiency but may not be the primary driver of renal and neurological symptoms. CONCLUSIONS: Our results highlight a complex interplay between CoQ10 biosynthesis, ferroptosis defense, and therapeutic response, warranting further investigation of subcellular CoQ10 distribution and ferroptosis-related mechanisms.

Mitochondrial electron transport chain (ETC) complexes partition between free complexes and quaternary assemblies known as supercomplexes (SCs). However, the physiological requirement for SCs and the mechanisms regulating their formation remain controversial. Here, we show that genetic perturbations in mammalian ETC complex III (CIII) biogenesis stimulate the formation of a specialized extra-large SC (SC-XL) with a structure of I2+III2, resolved at 3.7 Å by cryoelectron microscopy (cryo-EM). SC-XL formation increases mitochondrial cristae density, reduces CIII reactive oxygen species (ROS), and sustains normal respiration despite a 70% reduction in CIII activity, effectively rescuing CIII deficiency. Consequently, inhibiting SC-XL formation in CIII mutants using the Uqcrc1DEL:E258-D260 contact site mutation leads to respiratory decompensation. Lastly, SC-XL formation promotes fatty acid oxidation (FAO) and protects against ischemic heart failure in mice. Our study uncovers an unexpected plasticity in the mammalian ETC, where structural adaptations mitigate intrinsic perturbations, and suggests that manipulating SC-XL formation is a potential therapeutic strategy for mitochondrial dysfunction.

Nicotinamide adenine dinucleotide (NAD) is a critical cofactor in mitochondrial energy production. The NADH/NAD+ ratio, reflecting the balance between NADH (reduced) and NAD+ (oxidized), is a key marker for the severity of mitochondrial diseases. We recently developed a streamlined LC-MS/MS method for the precise measurement of NADH and NAD+. Utilizing this technique, we quantified NADH and NAD+ levels in fibroblasts derived from pediatric patients and in a Leigh syndrome mouse model in which mitochondrial respiratory chain complex I subunit Ndufs4 is knocked out (KO). In patient-derived fibroblasts, NAD+ levels did not differ significantly from those of healthy controls (p = 0.79); however, NADH levels were significantly elevated (p = 0.04), indicating increased NADH reductive stress. This increase, observed despite comparable total NAD(H) levels between the groups, was attributed to elevated NADH levels. Similarly, in the mouse model, NADH levels were significantly increased in the KO group (p = 0.002), further suggesting that NADH elevation drives reductive stress. This precise method for NADH measurement is expected to outperform conventional assays, such as those for lactate, providing a simpler and more reliable means of assessing disease progression.