Autophagy and mitophagy-related extracellular mitochondrial dysfunction of cerebrospinal fluid cells in patients with hemorrhagic moyamoya disease

Abstract

We aimed to investigate whether mitochondrial dysfunction in extracellular cerebrospinal fluid (CSF), which is associated with autophagy and mitophagy, might be involved in neurological outcomes in adult patients with hemorrhagic moyamoya disease (MMD) whose pathogenesis related to poor outcomes is not well-known. CSF samples were collected from 43 adult MMD patients and analyzed according to outcomes at 3 months. Fluorescence-activated cell sorter analysis (FACS) and the JC-1 red/green ratio were used to assess mitochondrial cells and intact mitochondrial membrane potential (MMP). We performed quantitative real-time polymerase chain reaction and Western blotting analyses of autophagy and mitophagy-related markers, including HIF1α, ATG5, pBECN1, BECN1, BAX, BNIP3L, DAPK1, and PINK1. Finally, FACS analysis with specific fluorescence-conjugated antibodies was performed to evaluate the potential cellular origin of CSF mitochondrial cells. Twenty-seven females (62.8%) with a mean age of 47.4 ± 9.7 years were included in the study. Among 43 patients with hemorrhagic MMD, 23 (53.5%) had poor outcomes. The difference in MMP was evident between the two groups (2.4 ± 0.2 in patients with poor outcome vs. 3.5 ± 0.4 in patients with good outcome; p = 0.02). A significantly higher expression (2^-ΔCt) of HIF1α, ATG5, DAPK1 followed by BAX and BNIP3L mRNA and protein was also observed in poor-outcome patients compared to those with good outcomes. Higher percentage of vWF-positive mitochondria, suggesting endothelial cell origins, was observed in patients with good outcome compared with those with poor outcome (25.0 ± 1.4% in patients with good outcome vs. 17.5 ± 1.5% in those with poor outcome; p < 0.01). We observed the association between increased mitochondrial dysfunction concomitant with autophagy and mitophagy in CSF cells and neurological outcomes in adult patients with hemorrhagic MMD. Further prospective multicenter studies are needed to determine whether it has a diagnostic value for risk prediction.