Circulating GLAST+ EVs are increased in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder, hallmarked by the gradual deterioration of motor neurons, culminating in muscle weakness and fatal paralysis. The exact etiology of ALS remains elusive, and there is a critical need for reliable biomarkers to aid in diagnosis and monitoring of disease progression. Extracellular vesicles (EVs) have emerged as promising candidates for biomarker discovery in neurodegenerative diseases such as ALS, giving access to pathologically relevant tissues otherwise typically challenging or invasive to sample. Indeed, EVs can derive by many cell types within the central nervous system, cross the blood-brain barrier and reach the blood, where they can be easily measured. One of the central mechanisms implicated in ALS pathology is glutamate excitotoxicity, which involves excessive glutamate accumulation due to impaired uptake by astrocytes and other glial cells, leading to neuronal damage. GLAST is a key glutamate transporter responsible for maintaining extracellular gluta-mate levels, and its dysregulation is thought to contribute significantly to ALS development and associated neuropathogenesis. Here, we applied a quick and validated method, to evaluate GLAST⁺ EVs in ALS patients' plasma and age-matched healthy controls. We found an increase in GLAST⁺ EVs that holds promise for uncovering novel diagnostic and therapeutic avenues in ALS research.

Keywords: amyotrophic lateral sclerosis; biomarker; extracellular vesicles; flow cytometry; glutamate transporter.

FIGURE 1

Absolute counts of (A) leukocytes-(leuko), (B) endothelial-endo and (C) platelets-derived EVs in ALS (n = 61) compared to HC (n = 30).

FIGURE 2

Gating strategy. The custom kit from BD incorporates an APC-emitting lipophilic cationic dye (LCD), which permeates double-layer structures assisted by membrane potential. This dye effectively stains EVs and cells, both dotated of a lipidic membrane. Additionally, Phalloidin-FITC selectively binds to the cytoskeleton protein actin, specifically targeting EVs/cells with damged membrane. To exclude platelets from the analysis, anti-CD41-FITC antibody was used. EVs were then characterized based on their LCD positivity and smaller size as measured by forward scatter (FSC) (EVs area). Within intact EVs region (defined as LCD+/phalloidin-), GLAST⁺ EVs were identified. To verify the specificity of staining, an isotype-PE antibody served as a negative control. Lastly, samples were treated with Triton X-100 solution and re-acquired in order to confirm the specificity of LCD staining for EVs. Samples were acquired using FACSymphony A5 and data were analyzed using FACSDiva software.

FIGURE 3

GLAST⁺ EVs counts in ALS patients (n = 61) and HC (n = 30). Mann-Whitney test was used, ****p < 0.0001.

FIGURE 4

Gating strategy. The custom kit from BD incorporates an APC-emitting lipophilic cationic dye (LCD), which permeates double-layer structures assisted by membrane potential. This dye effectively stains EVs and cells, both dotated of a lipidic membrane. Additionally, Phalloidin-FITC selectively binds to the cytoskeleton protein actin, specifically targeting EVs/cells with damged membrane. To exclude platelets from the analysis, anti-CD41-FITC antibody was used. EVs were then characterized based on their LCD positivity and smaller size as measured by forward scatter (FSC) (EVs area). Within intact EVs region (defined as LCD+/phalloidin-), GLAST⁺ EVs were identified. Finally, tetraspanin positive events were identified according to the expression of CD63 and CD81 in BV605 and BV711, respectively.