Extracellular Vesicles as EVocators in Brain Tumors
Xandra O. Breakefield, PhD1
Glioma tumors release extracellular vesicles (EVs) in support of tumor progression. These EVs range in size from 100 nm to 1 µm and contain RNAs and proteins that can act as directives to cells in the tumor microenvironment. For example, tumor-derived EVs steer the transcriptome of neighboring microglia towards a more immune suppressive and tumor growth-promoting phenotype. This has been demonstrated by comparing microglia isolated from normal brains and from the brains of mice bearing syngeneic gliomas, with the latter microglia separated by FACS into those that have taken up fluorescently labeled tumor-derived EVs and those that have not. Work is also underway to try to harness these infiltrative packets of information as therapeutic vehicles to carry pro-drug activating enzymes/RNA which can deliver therapeutic drugs on site within the brain, thus avoiding the blood-brain barrier (BBB). Glioma cells have been genetically modified using lentivirus vectors to express RNA/proteins for cytosine deaminase::uracil phosphoribosyltransferase that are incorporated into EVs. These therapeutic EVs are taken up by non-modified tumor cells, with both tumor cell types subsequently being killed upon exposure to the prodrug, 5-fluorouracil. Thus although tumor-derived EVs are normally supportive for cancer, it may be possible to take advantage of their infiltrative and communicative properties and to arm them to act as therapeutic vehicles.
Coauthors: Sybren L. N. Maas, MD1, Erik R. Abels, MSc1, Joseph El Khoury, MD2, Kristina P. Friis, PhD1, Justin Hall1 and Marieke L. Broekman, PhD, MD1
1 Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA
2 Department of Medicine, Harvard Medical School and Division of Infectious Disease, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, MA, USA
Diagnosing Traumatic Brain Injury on a Chip via Circulating Exosomes
Mild traumatic brain injury (mTBI) occurs in 2 million people annually. Most mTBI patients recover within one year, but 10% of mild cases result in a long-term disability. Although much is known about molecular changes in the brain following injury, access to biomarkers is lacking. Recently, brain-derived exosomes, which cross the blood-brain barrier and circulate following injury, have shown potential as a biomarker of brain recovery. Unfortunately, due to exosomes’ small size (30nm-200nm) and the scarcity of brain-derived exosomes, it has proven challenging to use these biomarkers to improve treatment. While microfluidics have been used successfully to precisely sort cells, scaling these approaches to exosomes has been limited by the low throughput and susceptibility to clogging of nanofluidics.
We solve this problem by developing a new approach to nanofluidic sorting of brain-derived exosomes, wherein millions of nanofluidic devices are incorporated onto a microchip platform and operated in parallel, increasing throughput by a million fold and eliminating susceptibility to clogging. Using our device, we isolate brain derived exosomes from V>10 mL serum and plasma in less than 15 minutes (> 6 hours using conventional methods), based on expression of brain-specific markers (GluR2). We incorporate our device with molecular analysis to profile exosomal nucleic acid cargo (miRNA). To validate clinical utility, we measured the exosomal RNA signature of healthy subjects and subjects with mTBI in two murine models and pilot clinical samples, and generated a predictive panel for post-concussion syndrome
Coauthors: Jina Ko1, Matt Hemphill1, Dave Meaney1
1 Department of Bioengineering, University of Pennsylvania, PA, USA
2 Department of Electrical and Systems Engineering, University of Pennsylvania, PA, USA
Secretion of Exosomes, a Neuroprotective Mechanism in Neurodegenerative Disorders
Efrat Levy, PhD1,2
Dysfunctional neuronal endosomes are an early characteristic of neurodegenerative diseases, including Down syndrome (DS) and Alzheimer’s disease. We hypothesized that endocytosed material is released by neuronal endosomal multivesicular bodies (MVB) into the extracellular space via exosomes to relieve the neurons of accumulated toxic endosomal content. We examined the level of extracellular vesicles (EV) and content in the brain of DS patients and a mouse model of the disease [Ts[Rb(12.1716)]2Cje] (Ts2Cje). In samples of frontal cortex of DS patients without amyloid pathology we found higher EV levels compared to age-matched diploid controls. Higher EV levels were also found in brain extracellular space of Ts2Cje mice compared to littermate controls. Examination of EV secretion into the media of cultured human fibroblasts showed that DS fibroblasts with endosomal abnormalities secrete higher levels of EV compared to age-matched 2N fibroblasts, revealing that higher levels of EV in the brain are due to enhanced secretion. Mass spectrometry identified significantly higher levels of exosomal markers in Ts2Cje EV compared to controls. mRNA and protein expression levels of the tetraspanin CD63, enriched in the membrane of MVB, were higher in DS brain homogenates and DS fibroblasts. To identify the mechanism for enhanced exosome secretion, we silenced CD63 expression and found diminished exosome release and impaired endosomes in DS fibroblasts. These data suggest that induction of CD63 expression enhances exosome release in DS brains, alleviating neuronal endosomal abnormalities, underscoring the regulation of exosome release as a therapeutic target for neurodegenerative disorders with endosomal pathology.
Coauthors: Rocío Pérez-González, PhD2, Sebastien A. Gauthier, PhD2
1 New York University Langone Medical Center, New York, NY, USA
2 Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA
Prediction of Conversion from Mild Cognitive Impairment to Dementia with Neuronally-Derived Blood Exosome Protein Profile
Charisse N. Winston, MS, PhD1
Levels of Alzheimer's disease (AD)-related proteins in plasma neuronal derived exosomes (NDEs) were quantified to identify biomarkers for prediction and staging of mild cognitive impairment (MCI) and AD
Plasma exosomes were extracted, precipitated, and enriched for neuronal source by anti-L1CAM antibody absorption. NDEs were characterized by size (Nanosight) and shape (TEM) and extracted NDE protein biomarkers were quantified by ELISAs. Plasma NDE cargo was injected into normal mice, and results were characterized by immunohistochemistry to determine pathogenic potential
Plasma NDE levels of P-T181-tau, P-S396-tau, and Aβ1-42 were significantly higher, whereas those of neurogranin (NRGN) and the repressor element 1-silencing transcription factor (REST) were significantly lower in AD and MCI converting to AD (ADC) patients compared to cognitively normal control subjects (CNC) and stable MCI patients. Mice injected with plasma NDEs from ADC patients displayed increased P-tau (PHF-1 antibody)-positive cells in the CA1 region of the hippocampus compared to plasma NDEs from CNC and stable MCI patients.
Abnormal plasma NDE levels of P-tau, Aβ1-42, NRGN, and REST accurately predict conversion of MCI to AD dementia. Plasma NDEs from demented patients seeded tau aggregation and induced AD-like neuropathology in normal mouse CNS.
Coauthors: Edward J. Goetzl, MD2, Jonny Akers, PhD1, Bob S. Carter, MD, PhD1, Edward Rockenstein, PhD1, Douglas R. Galasko, MD1, Eliezer Masliah, MD1 and Robert A. Rissman, PhD1
1 University of California, San Diego, La Jolla, CA, USA
2 University of California San Francisco, San Francisco, CA, USA
Exosome Pathway as a New Therapeutic Target of Alzheimer’s Disease
Tsuneya Ikezu, MD, PhD, Boston University School of Medicine, Boston, MA, USA
The neurofibrillary tangle is a pathological hallmark of Alzheimer’s disease and primarily consists of hyper-phosphorylated tau protein (pTau). pTau first appears in the entorhinal cortex in the pre-symptomatic stage, then gradually disseminates to the hippocampal region around the onset of clinical symptoms of AD. Halting this tau spread in the asympomatic stage is a promising therapeutic approach for AD. The exosome is a small vesicle of 50-100 nm in diameter, enriched in ceramide, and is suggested to contain neuropathogenic proteins, such as tau protein. Our recent study has shown that microglia transduce tau aggregates into nearby neuronal cells via exosomal secretion, and that inhibition of the exosome synthesis or secretory pathway reduces tau dissemination. pTau was also identified in exosomes in the cerebrospinal fluid and plasma in Alzheimer’s disease patients, suggesting the potential value of exosomes as biomarkers of Alzheimer’s disease. These results demonstrate that exosome secretion from microglia play a significant role in propagation of tau protein, and that targeting this pathway may be beneficial in ameliorating disease progression.
Biomarkers in Urinary Exosomes for Neurological Diseases
Andrew B. West, PhD, University of Alabama at Birmingham, Birmingham, AL, USA
Exosomes, often referred to as ‘liquid biopsies’, encapsulate a pool of protein in biofluids that may be used to discover new biomarkers useful for disease prediction, progression, and response to therapies. Urine provides a non-invasively derived source of exosomes conveniently obtained from clinical populations that can be stored indefinitely. Urinary exosomes include robust concentrations of many proteins and their post-translational modifications, linked to neurological diseases. Our whole proteome scans of clinical populations reveal a subset of urinary exosome proteins that are stable over time and vary little between subjects, as well as proteins that are extremely variable over time and are often idiosyncratic. Within this spectrum lies a potentially rich source of proteins for biomarker studies. As a proof-of-principle, we identify a panel of urinary exosome proteins that can differentiate Parkinson disease cases from controls. In addition, analysis of the phosphorylation status of a single protein in urinary exosomes, LRRK2, demonstrates successful prediction of disease onset for carriers positive for the most common genetic cause of neurodegeneration. We propose that urinary exosomes may provide a rich source of proteins useful for understanding and treating CNS disorders.
Coauthors: James A. Mobley, PhD, Kyle B. Fraser, PhD, Shijie Wang, BS
University of Alabama at Birmingham, Birmingham, AL, USA