Maja Mustapic

Low neural exosomal levels of cellular survival factors in Alzheimer's disease.

Transcription factors that mediate neuronal defenses against diverse stresses were quantified in plasma neural‐derived exosomes of Alzheimers disease or frontotemporal dementia patients and matched controls. Exosomal levels of low‐density lipoprotein receptor‐related protein 6, heat‐shock factor‐1, and repressor element 1‐silencing transcription factor all were significantly lower in Alzheimers disease patients than controls (P < 0.0001). In frontotemporal dementia, the only significant difference was higher levels of repressor element 1‐silencing transcription factor than in controls. Exosomal transcription factors were diminished 2–10 years before clinical diagnosis of Alzheimers disease. Low exosomal levels of survival proteins may explain decreased neuronal resistance to Alzheimers disease neurotoxic proteins.

RNA in extracellular vesicles.

Cells release a range of membrane‐enclosed extracellular vesicles (EVs) into the environment. Among them, exosomes and microvesicles (collectively measuring 40–1000 nm in diameter) carry proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been proposed to serve as intercellular mediators of communication. EVs transport cellular materials in many physiologic processes, including differentiation, stem cell homeostasis, immune responses, and neuronal signaling. EVs are also increasingly recognized as having a direct role in pathologies such as cancer and neurodegeneration. Accordingly, EVs have been the focus of intense investigation as biomarkers of disease, prognostic indicators, and even therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs (messenger RNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The rising attention to EV‐resident RNAs as biomarkers stems from the fact that RNAs can be detected at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we discuss EV RNAs in cancer and neurodegeneration, two major age‐associated disease processes. WIREs RNA 2017, 8:e1413. doi: 10.1002/wrna.1413

Cargo proteins of plasma astrocyte-derived exosomes in Alzheimer's disease.

Efficient intercellular transfer of RNAs, proteins, and lipids as protected exosomal cargo has been demonstrated in the CNS, but distinct physiologic and pathologic roles have not been well defined for this pathway. The capacity to isolate immunochemically human plasma neuron‐derived exosomes (NDEs), containing neuronspecific cargo, has permitted characterization of CNS‐derived exosomes in living humans. Constituents of the amyloid β‐peptide (Aβ)42‐generating system now are examined in 2 distinct sets of human neural cells by quantification in astrocyte‐derived exosomes (ADEs) and NDEs, enriched separately from plasmas of patients with Alzheimers disease (AD) or frontotemporal dementia (FTD) andmatched cognitively normal controls. ADE levels of β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE‐1), γ‐secretase, soluble Aβ42, soluble amyloid precursor protein (sAPP)β, sAPPα, glial‐derived neurotrophic factor (GDNF), P‐T181‐tau, and P‐S396‐tau were significantly (3‐ to 20‐fold) higher than levels in NDEs for patients and controls. BACE‐1 levels also were a mean of 7‐fold higher in ADEs than in NDEs from cultured rat type‐specific neural cells. Levels of BACE‐1 and sAPPβ were significantly higher and of GDNF significantly lower in ADEs of patients with AD than in those of controls, but not significantly different in patients with FTD than in controls. Abundant proteins of the Aβ42 peptide‐generating system in ADEs may sustain levels in neurons. ADE cargo proteins may be useful for studies of mechanisms of cellular interactions and effects of BACE‐1 inhibitors in AD.—Goetzl, E. J., Mustapic, M., Kapogiannis, D., Eitan, E., Lobach, I. V., Goetzl, L., Schwartz, J. B., Miller, B. L. Cargo proteins of plasma astrocyte‐derived exosomes in Alzheimers disease. FASEB J. 30, 3853–3859 (2016) www.fasebj.org

Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes

Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimers and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum, or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EVs enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner.

Exosomal biomarkers of brain insulin resistance associated with regional atrophy in Alzheimer's disease.

Brain insulin resistance (IR), which depends on insulin‐receptor‐substrate‐1 (IRS‐1) phosphorylation, is characteristic of Alzheimers disease (AD). Previously, we demonstrated higher pSer312‐IRS‐1 (ineffective insulin signaling) and lower p‐panTyr‐IRS‐1 (effective insulin signaling) in neural origin‐enriched plasma exosomes of AD patients vs. controls. Here, we hypothesized that these exosomal biomarkers associate with brain atrophy in AD. We studied 24 subjects with biomarker‐supported probable AD (low CSF Aβ42). Exosomes were isolated from plasma, enriched for neural origin using immunoprecipitation for L1CAM, and measured for pSer312‐ and p‐panTyr‐IRS‐1 phosphotypes. MPRAGE images were segmented by brain tissue type and voxel‐based morphometry (VBM) analysis for gray matter against pSer312‐ and p‐panTyr‐IRS‐1 was conducted. Given the regionally variable brain expression of IRS‐1, we used the Allen Brain Atlas to make spatial comparisons between VBM results and IRS‐1 expression. Brain volume was positively associated with P‐panTyr‐IRS‐1 and negatively associated with pSer312‐IRS‐1 in a strikingly similar regional pattern (bilateral parietal‐occipital junction, R middle temporal gyrus). This volumetric association pattern was spatially correlated with Allen Human Brain atlas normal brain IRS‐1 expression. Exosomal biomarkers of brain IR are thus associated with atrophy in AD as could be expected by their pathophysiological roles and do so in a pattern that reflects regional IRS‐1 expression. Furthermore, neural‐origin plasma exosomes may recover molecular signals from specific brain regions. Hum Brain Mapp 38:1933–1940, 2017.

Extracellular vesicle-associated Aβ mediates trans-neuronal bioenergetic and Ca 2+ -handling deficits in Alzheimer’s disease models

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder in which aggregation-prone neurotoxic amyloid β-peptide (Aβ) accumulates in the brain. Extracellular vesicles (EVs), including exosomes, are small 50–150 nm membrane vesicles that have recently been implicated in the prion-like spread of self-aggregating proteins. Here we report that EVs isolated from AD patient cerebrospinal fluid and plasma, from the plasma of two AD mouse models, and from the medium of neural cells expressing familial AD presenilin 1 mutations, destabilize neuronal Ca2+ homeostasis, impair mitochondrial function, and sensitize neurons to excitotoxicity. EVs contain a relatively low amount of Aβ but have an increased Aβ42/ Aβ40 ratio; the majority of Aβ is located on the surface of the EVs. Impairment of lysosome function results in increased generation of EVs with elevated Aβ42 levels. EVs may mediate transcellular spread of pathogenic Aβ species that impair neuronal Ca2+ handling and mitochondrial function, and may thereby render neurons vulnerable to excitotoxicity.

Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease

Plasma endothelial cell–derived exosomes (EDEs) and platelet‐derived exosomes (PDEs) were precipitated and enriched separately by immunospecific absorption procedures for analyses of cargo proteins relevant to atherosclerosis. EDEs had usual exosome size and marker protein content, and significantly higher levels than PDEs of the endothelial proteins vascular cell adhesion molecule‐1 (VCAM‐1) and endothelial nitric oxide synthase, whereas PDEs had significantly higher levels of platelet glycoprotein VI. EDE levels of VCAM‐1, von Willebrand factor, platelet‐derived growth factor (PDGF)‐BB, angiopoietin‐1, and lysyl oxidase‐2 and the cerebrovascular‐selective proteins glucose transporter 1, permeability‐glycoprotein, and large neutral amino acid transporter 1 were significantly higher for 18 patients with cerebrovascular disease (CeVD) than for 18 age‐ and gender‐matched control subjects. PDE levels of PDGF‐AA, platelet glycoprotein VI, integrin‐linked kinase‐1, high mobility group box‐1 protein, chemokine CXCL4, and thrombospondin‐1 were significantly higher in patients with CeVD than in control subjects, but differences were less with greater overlaps than for EDE proteins. EDE levels of Yes‐associated protein (YAP) were higher and of P(S127)‐YAP lower in patients with CeVD than in control subjects, consistent with heightened activity of this mechanical force–sensitive system in atherosclerosis. Elevated EDE and PDE levels of atherosclerosis‐promoting proteins in CeVD justify clinical studies of their potential value as biomarkers.—Goetzl, E. J., Schwartz, J. B., Mustapic, M., Lobach, I. V., Daneman, R., Abner, E. L., Jicha, G. A. Altered cargo proteins of human plasma endothelial cell–derived exosomes in atherosclerotic cerebrovascular disease. FASEB J. 31, 3689–3694 (2017). www.fasebj.org

Detection of Aggregation-Competent Tau in Neuron-Derived Extracellular Vesicles

Progressive cerebral accumulation of tau aggregates is a defining feature of Alzheimer’s disease (AD). A popular theory that seeks to explain the apparent spread of neurofibrillary tangle pathology proposes that aggregated tau is passed from neuron to neuron. Such a templated seeding process requires that the transferred tau contains the microtubule binding repeat domains that are necessary for aggregation. While it is not clear how a protein such as tau can move from cell to cell, previous reports have suggested that this may involve extracellular vesicles (EVs). Thus, measurement of tau in EVs may both provide insights on the molecular pathology of AD and facilitate biomarker development. Here, we report the use of sensitive immunoassays specific for full-length (FL) tau and mid-region tau, which we applied to analyze EVs from human induced pluripotent stem cell (iPSC)-derived neuron (iN) conditioned media, cerebrospinal fluid (CSF), and plasma. In each case, most tau was free-floating with a small component inside EVs. The majority of free-floating tau detected by the mid-region assay was not detected by our FL assays, indicating that most free-floating tau is truncated. Inside EVs, the mid-region assay also detected more tau than the FL assay, but the ratio of FL-positive to mid-region-positive tau was higher inside exosomes than in free solution. These studies demonstrate the presence of minute amounts of free-floating and exosome-contained FL tau in human biofluids. Given the potential for FL tau to aggregate, we conclude that further investigation of these pools of extracellular tau and how they change during disease is merited.

In a randomized trial in prostate cancer patients, dietary protein restriction modifies markers of leptin and insulin signaling in plasma extracellular vesicles

Obesity, metabolic syndrome, and hyperleptinemia are associated with aging and age‐associated diseases including prostate cancer. One experimental approach to inhibit tumor growth is to reduce dietary protein intake and hence levels of circulating amino acids. Dietary protein restriction (PR) increases insulin sensitivity and suppresses prostate cancer cell tumor growth in animal models, providing a rationale for clinical trials. We sought to demonstrate that biomarkers derived from plasma extracellular vesicles (EVs) reflect systemic leptin and insulin signaling and respond to dietary interventions. We studied plasma samples from men with prostate cancer awaiting prostatectomy who participated in a randomized trial of one month of PR or control diet. We found increased levels of leptin receptor in the PR group in total plasma EVs and in a subpopulation of plasma EVs expressing the neuronal marker L1CAM. Protein restriction also shifted the phosphorylation status of the insulin receptor signal transducer protein IRS1 in L1CAM+ EVs in a manner suggestive of improved insulin sensitivity. Dietary PR modifies indicators of leptin and insulin signaling in circulating EVs. These findings are consistent with improved insulin and leptin sensitivity in response to PR and open a new window for following physiologic responses to dietary interventions in humans.

Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease

Exosomes derived from chondroitin sulfate proteoglycan (CSPG) 4 type neural precursor cells (CSPG4Es) were purified from human plasma by sequential immunoabsorption with anti‐CSPG4 and anti‐platelet growth factor receptor α mAb to characterize the potential in vivo roles of CSPG4 cells in neuronal repair. Hepatocyte growth factor, fibroblast growth factors (FGFs)‐2 and ‐13, and type 1 insulin‐like growth factor (IGF‐1), which enhance neuronal survival and functions, were quantified in CSPG4E extracts. For CSPG4Es of 24 healthy control subjects, mean levels of hepatocyte growth factor, FGF‐13, and IGF‐1, but not FGF‐2, were significantly higher by up to 7‐fold than in their neuronal‐derived exosomes, and mean levels of all 4 growth factors were significantly higher by up to 8‐fold than in their astrocyte‐derived exosomes. Mean CSPG4E levels of all growth factors were significantly lower in patients with mild Alzheimer disease (AD) (n = 24) than in age‐ and sex‐matched cognitively normal control subjects (n = 24). Mean CSPG4E levels of all growth factors were also significantly lower in 15 patients at the stage of moderate dementia from AD (AD2) and at their preclinical stage 3 to 8 yr earlier (AD1), with no differences between values at stages AD1 and AD2. Current findings suggest that CSPG4 cells export in exosomes higher levels of neurotrophic factors than neurons or astrocytes and that CSPG4E neurotrophic factors are diminished early in AD, with no significant progression of decreases later in the course.—Goetzl, E. J., Nogueras‐Ortiz, C., Mustapic, M., Mullins, R. J., Abner, E. L., Schwartz, J. B., Kapogiannis, D. Deficient neurotrophic factors of CSPG4‐type neural cell exosomes in Alzheimer disease. FASEB J. 33, 231–238 (2019). www.fasebj.org