Central Nervous System (CNS)-Derived Extracellular Vesicles (EVs) as Novel Biomarkers for Environmental Exposure and Disease Progression in ALS

Abstract

BACKGROUND AND AIM: Metal exposure is linked to amyotrophic lateral sclerosis (ALS) but biomarkers of central nervous system (CNS) metal burden are lacking. Here, we tested the use of blood-isolated CNS-derived extracellular vesicles (EVs) as biomarkers of metal exposure and disease progression in ALS. Specifically, we examined whether: 1) cultured brain cells can eliminate metals via EV release, 2) metal levels in ALS patient blood-isolated CNS-EVs predict metal load in their brain and spinal cord, and 3) differences in CNS-EV metal levels are associated with ALS disease progression (ongoing). METHODS: Metal levels were measured by ICP-MS. Cultured astrocytes were treated with 2.5µM Mn or As for 3 or 7 days. Released EVs were isolated by ultracentrifugation. In a pilot of N = 7 National ALS Registry patients, we measured metal levels in blood, CNS-EVs, spinal cord, and cortex to assess their correlations. CNS-EVs of neuronal (L1CAM) and astrocyte (GLAST) origin were enriched via immunoprecipitation. In 97 ALS patients at two disease stages, GLAST-EVs were immunopurified from blood and analyzed for Cu, Pb, Hg, Al, Mn, Cd, and Fe levels. Linear models are being employed to examine associations between changes in CNS-EV metal levels and ALS Functional Rating Scale progression. RESULTS:Mn and As levels in treated astrocyte EVs increased over time, whereas these metals were not detected in control cells. Circulating GLAST-EV metal load was overall more predictive of spinal cord and cortex metal levels than total blood or L1CAM-EV metal levels. Hg and Cd were not detected in ALS patient GLAST-EV samples, while geometric means of Cu, Pb, Al, Mn, and Fe decreased between the two timepoints. CONCLUSIONS:Preliminary evidence suggests that peripheral GLAST-EVs metal levels may reflect internal CNS metal exposure and that metal regulation via CNS-EVs weakens over time. The association of GLAST-EVs with ALS disease progression deserves further exploration. KEYWORDS: Heavy metals, Exposures, Biomarkers of exposure, Neurodegenerative outcomes, ALS, Extracellular vesicles