Amyotrophic lateral sclerosis (ALS) is a terminalneurodegenerative disease. 
Clinical and molecular observations suggest that ALS pathology originates at a 
single site and spreads in an organized and prion-like manner, possibly driven 
by extracellular vesicles. Extracellular vesicles (EVs) transfer cargo molecules 
associated with ALS pathogenesis, such as misfolded and aggregated proteins and 
dysregulated microRNAs (miRNAs). However, it is poorly understood whether 
altered levels of circulating extracellular vesicles or their cargo components 
reflect pathological signatures of the disease. In this study, we used 
immuno-affinity-based microfluidic technology, electron microscopy, and 
NanoString miRNA profiling to isolate and characterize extracellular vesicles 
and their miRNA cargo from frontal cortex, spinal cord, and serum of sporadic 
ALS (n = 15) and healthy control (n = 16) participants. We found larger 
extracellular vesicles in ALS spinal cord versus controls and smaller sized 
vesicles in ALS serum. However, there were no changes in the number of 
extracellular vesicles between cases and controls across any tissues. 
Characterization of extracellular vesicle-derived miRNA cargo in ALS compared to 
controls identified significantly altered miRNA levels in all tissues; miRNAs 
were reduced in ALS frontal cortex and spinal cord and increased in serum. Two 
miRNAs were dysregulated in all three tissues: miR-342-3p was increased in ALS, 
and miR-1254 was reduced in ALS. Additional miRNAs overlapping across two 
tissues included miR-587, miR-298, miR-4443, and miR-450a-2-3p. Predicted 
targets and pathways associated with the dysregulated miRNAs across the ALS 
tissues were associated with common biological pathways altered in 
neurodegeneration, including axon guidance and long-term potentiation. A 
predicted target of one identified miRNA (N-deacetylase and N-sulfotransferase 
4; NDST4) was likewise dysregulated in an in vitro model of ALS, verifying 
potential biological relevance. Together, these findings demonstrate that 
circulating extracellular vesicle miRNA cargo mirror those of the central 
nervous system disease state in ALS, and thereby offer insight into possible 
pathogenic factors and diagnostic opportunities.