Neurons expel vesicles called exosomes when multivesicular bodies fuse with the plasma membrane and release these tiny packets. Exosomes rid cells of waste, carry messages between them, and can cross the blood-brain barrier. Scientists led by Edward Goetzl, University of California, San Francisco, and Dimitrios Kapogiannis, National Institute on Aging, Baltimore, have developed a method to capture neuron-derived exosomes in blood and are exploring their potential as disease markers. Previously they reported that exosome levels of pathogenic proteins, insulin-resistance markers, and cell-survival proteins distinguished people with Alzheimer’s from controls (Aug 2014 news; Fiandaca et al., 2014; Kapogiannis et al., 2014). In the July 7 Neurology, the group now reports differences in lysosomal proteins, as well. A small set distinguished patients from controls up to 10 years before clinical diagnosis, introducing them as new candidates for early disease detection, wrote the authors.
“It’s refreshing to see the biomarker search expand to emphasize another pathophysiology that is prominent in AD,” said Ralph Nixon, New York University School of Medicine, who was not involved in the study. “This could be the tip of the iceberg in terms of other exosomal markers that reflect endosomal, lysosomal, and autophagy pathology.”
Lysosomes are acidic vesicles inside cells that digest unwanted material. The lysosomal network in neurons clogs up and malfunctions in AD and other disorders (for a review, see Ihara et al., 2012). When lysosomes stop breaking down garbage, the cell has to get rid of it in other ways, one of which may be to eject it in exosomes (Alvarez-Erviti et al., 2011). Could the contents of neurally derived exosomes offer a window on lysosomal dysfunction?
To answer this question, Goetzl and colleagues conducted a two-part study. In a cross-sectional portion, they took blood from 26 patients with mild cognitive impairment (MCI), eight with AD dementia, and 16 with frontotemporal dementia to compare the lysosomal proteins in their neuron-derived exosomes to each other and those of age-matched controls. Patients with AD were diagnosed by either Dubois or NINCDS-ADRDA criteria, with Clinical Dementia Rating (CDR) global scores of at least 1.0 (Dubois et al., 2007; McKhann et al., 1984). MCI patients were diagnosed according to Petersen criteria and had CDR global scores of 0.5 (Petersen, 2004). The scientists examined another 20 AD patients longitudinally. These people gave blood twice: once when they were cognitively intact—one to 10 years before disease onset—and again once they were symptomatic.
From those blood samples, the researchers isolated neuronal exosomes using an antibody for L1CAM, a neural adhesion protein highly expressed in the central nervous system. They then measured the levels of lysosomal proteins inside, including total protein with a ubiquitin tag that targets waste to lysosomes; lysosome-associated membrane protein 1 (LAMP-1), the lysosomal aspartyl endoproteinase cathepsin D, and the chaperone heat-shock protein 70 (HSP70).
In this cross-sectional data, there was some overlap in protein levels between groups. Even so, average cathepsin D and total ubiquitinylated protein levels were twice as high in the AD group as in controls, whereas HSP70 was lower. AD patients had more LAMP-1 on average, but values between groups overlapped so much that protein levels did not distinguish AD patients. FTD followed the same trend, though to a lesser extent. The combination of cathepsin D, total ubiquitin, and HSP70 distinguished all AD patients from controls, and 96 percent of AD from FTD patients. Cathepsin D and HSP70 likewise distinguished all FTD patients from controls. Researchers who were not involved in this study interpreted this result cautiously. They noted that the error rate inherent in clinical diagnosis makes perfect distinction by a biomarker unlikely, and called for replication of the data.
In the longitudinal data, all four lysosomal proteins separated AD patients from controls. What’s more, levels started changing when the study participants were still cognitively normal, hinting that the underlying process happens years before disease onset.
The results suggest that neuronal lysosome proteins in exosomes could become useful biomarkers for preclinical AD, the authors believe. Goetzl suspects the proteins may be abnormal even earlier than 10 years before diagnosis. He and Kapogiannis have started mining data from 400 people in the Baltimore Longitudinal Study on Aging, who gave blood regularly 40 to 50 years before getting Alzheimer’s disease. He predicts a shift in lysosomal proteins in middle age.
The authors acknowledged the small sample size in this study. Goetzl said ongoing studies include observation of exosomal profiles in healthy aging, their ability to predict conversion from MCI to dementia, and correlation with imaging markers and cerebrospinal fluid proteins.
Kaj Blennow, University of Gothenburg, Sweden, noted that certain peripheral cells produce L1CAM, so the assay may extract peripheral exosomes as well (see full comment below). Goetzl conceded that the vesicles are likely not 100 percent neuronal; he estimated based on enrichment of other neuronal markers that 80 percent are.
Can these exosomal markers distinguish diseases that are each tied to lysosomal dysfunction? Nixon said this might be achieved by looking for additional lysosome markers in exosomes, or for disease-specific markers. Exosomal α-synuclein, Aβ, or p-tau could yield disease-specific profiles for Parkinson’s or Alzheimer’s, he said. —Gwyneth Dickey Zakaib
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