ABSTRACT
Mutations in the gene encoding the transient receptor potential vanilloid member 4 (TRPV4), a Ca2+ permeable nonselective cation channel, cause TRPV4-related disorders. TRPV4 is widely expressed in the brain; however, the pathogenesis underlying TRPV4-mediated Ca2+ deregulation in neurodevelopment remains unresolved and an effective therapeutic strategy remains to be established. These issues were addressed by isolating mutant dental pulp stem cells from a tooth donated by a child diagnosed with metatropic dysplasia with neurodevelopmental comorbidities caused by a gain-of-function TRPV4 mutation, c.1855C > T (p.L619F). The mutation was repaired using CRISPR/Cas9 to generate corrected isogenic stem cells. These stem cells were differentiated into dopaminergic neurons and the pharmacological effects of folic acid were examined. In mutant neurons, constitutively elevated cytosolic Ca2+ augmented AKT-mediated α-synuclein (α-syn) induction, resulting in mitochondrial Ca2+ accumulation and dysfunction. The TRPV4 antagonist, AKT inhibitor, or α-syn knockdown, normalizes the mitochondrial Ca2+ levels in mutant neurons, suggesting the importance of mutant TRPV4/Ca2+/AKT-induced α-syn in mitochondrial Ca2+ accumulation. Folic acid was effective in normalizing mitochondrial Ca2+ levels via the transcriptional repression of α-syn and improving mitochondrial reactive oxygen species levels, adenosine triphosphate synthesis, and neurite outgrowth of mutant neurons. This study provides new insights into the neuropathological mechanisms underlying TRPV4-related disorders and related therapeutic strategies.
ABSTRACT
Melatonin entrainment of suprachiasmatic nucleus-regulating circadian rhythms is mediated by MT1 and MT2 receptors. Melatonin also has neuroprotective and mitochondrial activating effects, suggesting it may affect neurodevelopment. We studied melatonin's pharmacological effects on autism spectrum disorder (ASD) neuropathology. Deciduous tooth-derived stem cells from children with ASD were used to model neurodevelopmental defects and differentiated into dopaminergic neurons (ASD-DNs) with or without melatonin. Without melatonin, ASD-DNs had reduced neurite outgrowth, mitochondrial dysfunction, lower mitochondrial Ca2+ levels, and Ca2+ accumulation in the endoplasmic reticulum (ER) compared to control DNs from typically developing children-derived stem cells. Melatonin enhanced IP3-dependent Ca2+ release from ER to mitochondria, improving mitochondrial function and neurite outgrowth in ASD-DNs. Luzindole, an MT1/MT2 antagonist, blocked these effects. Thus, melatonin supplementation may improve dopaminergic system development in ASD by modulating mitochondrial Ca2+ homeostasis via MT1/MT2 receptors.
ABSTRACT
Mitochondrial fission factor (MFF) is an adapter that targets dynamin-related protein 1 from the cytosol to the mitochondria for fission. Loss-of-function MFF mutations cause encephalopathy due to defective mitochondrial and peroxisomal fission 2 (EMPF2). To elucidate the molecular mechanisms that were involved, we analyzed the functional effects of MFF depletion in deciduous teeth-derived dental pulp stem cells differentiating into dopaminergic neurons (DNs). When treated with MFF-targeting small interfering RNA, DNs showed impaired neurite outgrowth and reduced mitochondrial signals in neurites harboring elongated mitochondria. MFF silencing also caused mitochondrial Ca2+ accumulation through accelerated Ca2+ influx from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor. Mitochondrial Ca2+ overload led DNs to produce excessive reactive oxygen species (ROS), and downregulated peroxisome proliferator-activated receptor-gamma co-activator-1 alpha (PGC-1α). MFF was co-immunoprecipitated with voltage-dependent anion channel 1, an essential component of the ER-mitochondrial Ca2+ transport system. Folic acid supplementation normalized ROS levels, PGC-1α mediated mitochondrial biogenesis, and neurite outgrowth in MFF depleted DNs, without affecting their mitochondrial morphology or Ca2+ levels. We propose that MFF negatively regulates the mitochondrial Ca2+ influx from the ER. MFF-insufficiency recapitulated the EMPF2 neuropathology with increased oxidative stress and suppressed mitochondrial biogenesis. ROS and mitochondrial biogenesis might be potential therapeutic targets for EMPF2.
