Specific Binding of Alzheimer's Aß Peptides to Extracellular Vesicles.

Alzheimer's disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aß42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aß42 is detectable in the plasma, a phenomenon thought to result from Aß becoming more aggregated in the brain and less Aß42 and Aß40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse "cargos" of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aß42 and Aß40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aß and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aß42 and Aß40 bind to EVs in a manner that is sequence specific, saturable, and endothermic. In addition, Aß incubation with EVs overnight yielded larger amounts of bound Aß peptide that was fibrillar in structure. These findings point to a specific amyloid-EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.

A tale of two tumors: differential, but detrimental, effects of glioblastoma extracellular vesicles (EVs) on normal human brain cells.

Glioblastomas (GBMs) are dreadful brain tumors with abysmal survival outcomes. GBM EVs dramatically affect normal brain cells (largely astrocytes) constituting the tumor microenvironment (TME). EVs from different patient-derived GBM spheroids induced differential transcriptomic, secretomic, and proteomic effects on cultured astrocytes/brain tissue slices as GBM EV recipients. The net outcome of brain cell differential changes nonetheless converges on increased tumorigenicity. GBM spheroids and brain slices were derived from neurosurgical patient tissues following informed consent. Astrocytes were commercially obtained. EVs were isolated from conditioned culture media by ultrafiltration, ultraconcentration, and ultracentrifugation. EVs were characterized by nanoparticle tracking analysis, electron microscopy, biochemical markers, and proteomics. Astrocytes/brain tissues were treated with GBM EVs before downstream analyses. EVs from different GBMs induced brain cells to alter secretomes with pro-inflammatory or TME-modifying (proteolytic) effects. Astrocyte responses ranged from anti-viral gene/protein expression and cytokine release to altered extracellular signal-regulated protein kinase (ERK1/2) signaling pathways, and conditioned media from EV-treated cells increased GBM cell proliferation. Thus, astrocytes/brain slices treated with different GBM EVs underwent non-identical changes in various 'omics readouts and other assays, indicating "personalized" tumor-specific GBM EV effects on the TME. This raises concern regarding reliance on "model" systems as a sole basis for translational direction. Nonetheless, net downstream impacts from differential cellular and TME effects still led to increased tumorigenic capacities for the different GBMs.

High-Throughput Screening of Epigenetic Inhibitors in Meningiomas Identifies HDAC, G9a, and Jumonji-Domain Inhibition as Potential Therapies.

Background Epigenetics may predict treatment sensitivity and clinical course for patients with meningiomas more accurately than histopathology. Nonetheless, targeting epigenetic mechanisms is understudied for pharmacotherapeutic development for these tumors. The bio-molecular insights and potential therapeutic development of meningioma epigenetics led us to investigate epigenetic inhibition in meningiomas. Methods We screened a 43-tumor cohort using a 139-compound epigenetic inhibitor library to assess sensitivity of relevant meningioma subgroups to epigenetic inhibition. The cohort was composed of 5 cell lines and 38 tumors cultured directly from surgery; mean patient age was 56.6 years ± 13.9 standard deviation. Tumor categories: 38 primary tumors, 5 recurrent; 33 from females, 10 from males; 32 = grade 1; 10 = grade 2; 1 = grade 3. Results Consistent with our previous results, histone deacetylase inhibitors (HDACi) were the most efficacious class. Panobinostat significantly reduced cell viability in 36 of 43 tumors; 41 tumors had significant sensitivity to some HDACi. G9a inhibition and Jumonji-domain inhibition also significantly reduced cell viability across the cohort; tumors that lost sensitivity to panobinostat maintained sensitivity to either G9a or Jumonji-domain inhibition. Sensitivity to G9a and HDAC inhibition increased with tumor grade; tumor responses did not separate by gender. Few differences were found between recurrent and primary tumors, or between those with prior radiation versus those without. Conclusions Few efforts have investigated the efficacy of targeting epigenetic mechanisms to treat meningiomas, making the clinical utility of epigenetic inhibition largely unknown. Our results suggest that epigenetic inhibition is a targetable area for meningioma pharmacotherapy.

Immunopathology of Extracellular Vesicles in Macrophage and Glioma Cross-Talk.

Glioblastomas (GBM) are a devastating disease with extremely poor clinical outcomes. Resident (microglia) and infiltrating macrophages are a substantial component of the tumor environment. In GBM and other cancers, tumor-derived extracellular vesicles (EVs) suppress macrophage inflammatory responses, impairing their ability to identify and phagocytose cancerous tissues. Furthermore, these macrophages then begin to produce EVs that support tumor growth and migration. This cross-talk between macrophages/microglia and gliomas is a significant contributor to GBM pathophysiology. Here, we review the mechanisms through which GBM-derived EVs impair macrophage function, how subsequent macrophage-derived EVs support tumor growth, and the current therapeutic approaches to target GBM/macrophage EV crosstalk.

Rapid Activation of Neuroinflammation in Stroke: Plasma and Extracellular Vesicles Obtained on a Mobile Stroke Unit.

BACKGROUND: Neuroinflammation is ubiquitous in acute stroke and worsens outcome. However, the precise timing of the inflammatory response is unknown, hindering the design of acute anti-inflammatory therapeutic interventions. We sought to identify the onset of the neuroinflammatory cascade using a mobile stroke unit. METHODS: The study is a proof-of-concept, cohort investigation of ultra-early blood- and extracellular vesicle-derived markers of neuroinflammation and outcome in acute stroke. Blood was obtained, prehospital, on an mobile stroke unit. Outcomes were biomarker concentrations, modified Rankin Scale score, and National Institutes of Health Stroke Scale score. RESULTS: Forty-one adults were analyzed, including 15 patients treated on the mobile stroke unit between August 2021 and April 2022, and 26 healthy controls to establish biomarker reference levels. Median patient age was 74 (range, 36-97) years, 60% were female, and 80% White. Ten (67%) were diagnosed as stroke, with 8 (53%) confirmed and 2 likely transient ischemic attack or stroke averted by thrombolysis; 5 were stroke mimics. For strokes, median initial National Institutes of Health Stroke Scale score was 11 (range, 4-19) and 6 (75%) received tPA (tissue-type plasminogen activator). Blood was obtained a median of 58 (range, 36-133) minutes after symptom onset. Within 36 minutes after stroke, plasma IL-6 (interleukin-6), neurofilament light chain, UCH-L1 (ubiquitin C-terminal hydrolase L1), and GFAP (glial fibrillary acidic protein) were elevated by as much as 10 times normal. In EVs, MMP-9 (matrix metalloproteinase-9), CXCL4 (chemokine (C-X-C motif) ligand 4), CRP (C-reactive protein), IL-6, OPN (osteopontin), and PECAM1 (platelet and endothelial cell adhesion molecule 1) were elevated. Inflammatory markers increased rapidly in the first 2 hours and continued rising for 24 hours. CONCLUSIONS: The neuroinflammatory cascade was found to be activated within 36 to 133 minutes after stroke and progresses rapidly. This is earlier than observed previously in humans and suggests injury from neuroinflammation occurs faster than had been surmised. These findings could inform development of acute immunomodulatory stroke therapies and lead to new diagnostic tools and improved outcomes.

Phenotype and Neuronal Cytotoxic Function of Glioblastoma Extracellular Vesicles.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.

Glioblastoma Extracellular Vesicle-Specific Peptides Inhibit EV-Induced Neuronal Cytotoxicity.

WHO Grade 4 IDH-wild type astrocytoma (GBM) is the deadliest brain tumor with a poor prognosis. Meningioma (MMA) is a more common "benign" central nervous system tumor but with significant recurrence rates. There is an urgent need for brain tumor biomarkers for early diagnosis and effective treatment options. Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles that play essential functions in cell-to-cell communications among tumor cells. We aimed to identify epitopes of brain tumor EVs by phage peptide libraries. EVs from GBM plasma, MMA plasma, or brain tumor cell lines were used to screen phage-displayed random peptide libraries to identify high-affinity peptides. We purified EVs from three GBM plasma pools (23 patients), one MMA pool (10 patients), and four brain tumor cell lines. We identified a total of 21 high-affinity phage peptides (12 unique) specific to brain tumor EVs. The peptides shared high sequence homologies among those selected by the same EVs. Dose-response ELISA demonstrated that phage peptides were specific to brain tumor EVs compared to controls. Peptide affinity purification identified unique brain tumor EV subpopulations. Significantly, GBM EV peptides inhibit brain tumor EV-induced complement-dependent cytotoxicity (necrosis) in neurons. We conclude that phage display technology could identify specific peptides to isolate and characterize tumor EVs.

Targeting DNA Methyl Transferases with Decitabine in Cultured Meningiomas.

OBJECTIVE: Meningiomas are a common primary central nervous system tumor that lack a U.S. Food and Drug Administration-approved pharmacotherapy. Approximately 20%-35% of meningiomas are classified as higher grade with poor outcome, whereas patients with lower-grade meningiomas are known to have long-term neurologic deficits and reduced overall survival. Recent efforts to understand the epigenetic landscape of meningiomas have highlighted the importance of DNA methylation for predicting tumor outcomes and prognosis; therefore, inhibition of these pathways may present a viable therapy for these tumors. METHODS: In this study, we perform dose-response curves of decitabine, a DNA methyltransferase inhibitor, on patient-cultured tumors and meningioma cell lines. RESULTS: Thirty total samples were evaluated, including 24 patient-cultured tumors and 6 established meningioma cell lines. Meningiomas were found to have a significant reduction in cell viability after decitabine treatment in a dose dependent manner. The effect was primarily driven by 11 of the 30 tumors in our cohort, or 36.7%. Decitabine significantly reduced cell viability across all grades, tumors from different sexes, recurrent and primary tumors, as well as tumors without a history of previous radiation. Surprisingly, our single radiation-induced tumor did demonstrate greater viability after decitabine treatment. CONCLUSIONS: Our work has identified a potential drug candidate in decitabine for the treatment of meningiomas regardless of clinical subgroup. These data require further evaluation in preclinical models, and the conclusions based on clinical subgroups need to be evaluated in a larger cohort to achieve appropriate statistical power.

Higher Levels of IgG3 Antibodies in Serum, But Not in CSF, Distinguish Multiple Sclerosis From Other Neurological Disorders.

Increased intrathecal IgG and oligoclonal bands (OCB) are seminal features of multiple sclerosis (MS). Although no such differences in MS blood total IgG antibodies have been reported, serum OCB are a common and persistent finding in MS and have a systemic source. Recent studies showed that IgG3+ B cells and higher levels of serum IgG3 are linked to the development of MS. Additionally, intrathecal IgG synthesis in MS is associated with IgG3 heavy chain gene single nucleotide polymorphisms, and there is a strong relationship between susceptibility to MS and an IgG3 restriction fragment length polymorphism. These studies support the role of IgG3 in disease pathogenesis. Using multiple immunoassays, we investigated levels of total IgG, IgG1, and IgG3 in sera and CSF of 102 MS patients (19 paired CSF and sera), 76 patients with other neurological disorders (9 paired CSF and sera), and 13 healthy controls. We show that higher levels of total IgG and IgG3 antibodies were detected in MS serum, but not in CSF, which distinguishes MS from other inflammatory and non-inflammatory neurological disorders, with Receiver Operating Characteristic (ROC) Curves 0.79 for both IgG3 & total IgG. Our data support the notion that IgG3 antibodies may be a potential candidate for MS blood biomarker development.

Cytokine-Laden Extracellular Vesicles Predict Patient Prognosis after Cerebrovascular Accident.

BACKGROUND: A major contributor to disability after hemorrhagic stroke is secondary brain damage induced by the inflammatory response. Following stroke, global increases in numerous cytokines-many associated with worse outcomes-occur within the brain, cerebrospinal fluid, and peripheral blood. Extracellular vesicles (EVs) may traffic inflammatory cytokines from damaged tissue within the brain, as well as peripheral sources, across the blood-brain barrier, and they may be a critical component of post-stroke neuroinflammatory signaling. METHODS: We performed a comprehensive analysis of cytokine concentrations bound to plasma EV surfaces and/or sequestered within the vesicles themselves. These concentrations were correlated to patient acute neurological condition by the Glasgow Coma Scale (GCS) and to chronic, long-term outcome via the Glasgow Outcome Scale-Extended (GOS-E). RESULTS: Pro-inflammatory cytokines detected from plasma EVs were correlated to worse outcomes in hemorrhagic stroke patients. Anti-inflammatory cytokines detected within EVs were still correlated to poor outcomes despite their putative neuroprotective properties. Inflammatory cytokines macrophage-derived chemokine (MDC/CCL2), colony stimulating factor 1 (CSF1), interleukin 7 (IL7), and monokine induced by gamma interferon (MIG/CXCL9) were significantly correlated to both negative GCS and GOS-E when bound to plasma EV membranes. CONCLUSIONS: These findings correlate plasma-derived EV cytokine content with detrimental outcomes after stroke, highlighting the potential for EVs to provide cytokines with a means of long-range delivery of inflammatory signals that perpetuate neuroinflammation after stroke, thus hindering recovery.

High-Throughput Mechanistic Screening of Epigenetic Compounds for the Potential Treatment of Meningiomas.

BACKGROUND: Meningiomas are the most common primary central nervous system tumors. 20-30% of these tumors are considered high-grade and associated with poor prognosis and high recurrence rates. Despite the high occurrence of meningiomas, there are no FDA-approved compounds for the treatment of these tumors. METHODS: In this study, we screened patient-cultured meningiomas with an epigenetic compound library to identify targetable mechanisms for the potential treatment of these tumors. Meningioma cell cultures were generated directly from surgically resected patient tumors and were cultured on a neural matrix. Cells were treated with a library of compounds meant to target epigenetic functions. RESULTS: Although each tumor displayed a unique compound sensitivity profile, Panobinostat, LAQ824, and HC toxin were broadly effective across most tumors. These three compounds are broad-spectrum Histone Deacetylase (HDAC) inhibitors which target class I, IIa, and IIb HDACs. Panobinostat was identified as the most broadly effective compound, capable of significantly decreasing the average cell viability of the sample cohort, regardless of tumor grade, recurrence, radiation, and patient gender. CONCLUSIONS: These findings strongly suggest an important role of HDACs in meningioma biology and as a targetable mechanism. Additional validation studies are necessary to confirm these promising findings, as well to identify an ideal HDAC inhibitor candidate to develop for clinical use.

Making HSP90 Inhibitors Great Again? Unite for Better Cancer Immunotherapy.

HSP90 inhibitors are in numerous cancer clinical trials, but treatments often induce toxicity at effective dosages. In this issue of Cell Chemical Biology, Zavareh et al. (2020) serendipitously found that HSP90 inhibitors, at manageable doses, can reduce target cell expression of immune checkpoint molecules, potentially enabling improved anti-cancer immunotherapy.

Antibodies from Multiple Sclerosis Brain Identified Epstein-Barr Virus Nuclear Antigen 1 & 2 Epitopes which Are Recognized by Oligoclonal Bands.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), the etiology of which is poorly understood. The most common laboratory abnormality associated with MS is increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands (OCBs) in the brain and cerebrospinal fluid (CSF). However, the major antigenic targets of these antibody responses are unknown. The risk of MS is increased after infectious mononucleosis (IM) due to EBV infection, and MS patients have higher serum titers of anti-EBV antibodies than control populations. Our goal was to identify disease-relevant epitopes of IgG antibodies in MS; to do so, we screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. We identified two phage peptides that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays. Using a highly sensitive phage-mediated immuno-PCR assay, we determined specific bindings of the two EBV epitopes to IgG from CSF from 46 MS and 5 inflammatory control (IC) patients. MS CSF IgG have significantly higher bindings to EBNA1 epitope than to EBNA2 epitope, whereas EBNA1 and EBNA2 did not significantly differ in binding to IC CSF IgG. Further, the EBNA1 epitope was recognized by OCBs from multiple MS CSF as shown in blotting assays with samples separated by isoelectric focusing. The EBNA1 epitope is reactive to MS intrathecal antibodies corresponding to oligoclonal bands. This reinforces the potential role of EBV in the etiology of MS. Graphical abstract Antibodies purified from an MS brain plaque were panned by phage display peptide libraries to discern potential antigens. Phage displaying peptide sequences resembling Epstein-Barr Virus Nuclear Antigens 1 & 2 (EBNA1 & 2) epitopes were identified. Antibodies from sera and CSF from other MS patients also reacted to those epitopes.

Recombinant antibodies derived from laser captured single plasma cells of multiple sclerosis brain identified phage peptides which may be used as tools for characterizing intrathecal IgG response.

Oligoclonal bands and increased IgG antibody levels can be detected in the cerebrospinal fluid in vast majority of patients with Multiple Sclerosis (MS). However, the antigenic specificity of oligoclonal IgG has yet to be determined. Using laser capture microdissection, we isolated single CD38+ plasma cells from lesion areas in two autopsy MS brains, and generated three recombinant antibodies (rAbs) from clonally expanded plasma cells. Panning phage-displayed random peptide libraries was carried out to determine peptide antigen specificities of these MS brain rAbs. We identified 25 high affinity phage peptides from which 5 peptides are unique. Database searches revealed that they shared sequence homologies with Epstein-Barr nuclear antigens 4 and 6, as well as with other viral proteins. Significantly, these peptides were recognized by intrathecal IgG and oligoclonal IgG bands in other MS patients. Our results demonstrate that functional recombinant antibodies can be generated from clonally expanded plasma cells in MS brain lesions by laser capture microdissection, and that these MS brain rAbs have the potential for determining the targets of intrathecal IgG and oligoclonal bands.

Varicella-Zoster Virus infected human neurons are resistant to apoptosis.

Varicella-zoster virus (VZV) is a pathogenic human herpesvirus that causes varicella (chickenpox) as a primary infection following which it becomes latent in ganglionic neurons. Following viral reactivation many years later VZV causes herpes zoster (shingles) as well as a variety of other neurological syndromes. The molecular mechanisms of the conversion of the virus from a lytic to a latent state in ganglia are not well understood. In order to gain insights into the neuron-virus interaction, we studied virus-induced apoptosis in cultures of both highly pure terminally differentiated human neurons and human fetal lung fibroblasts (HFL). It was found that (a) VZV DNA did not accumulate in infected human neurons; (b) VZV transcripts were present at lower levels at all days studied post-infection in neurons; (c) Western blot analysis showed less VZV IE 63 and very little detectable VZV gE proteins in infected neurons compared with HFL; (d) lower levels of the apoptotic marker cleaved Caspase-3 protein were detected in VZV-infected neurons compared with HFL, and higher levels of the known anti-apoptotic proteins Bcl2, Bcl-XL and also the mitochondrial MT-CO2 protein were found in VZV-infected neurons compared with uninfected cells; and (e) both the MT-CO2 protein and VZV IE 63-encoded protein were detected in infected neurons by dual immunofluorescence. These findings showed that neurons are resistant to VZV-induced apoptosis, which may have relevance to the switching of VZV from a lytic to latent ganglionic neuronal infection.

Roles of Extracellular Vesicles in High-Grade Gliomas: Tiny Particles with Outsized Influence.

High-grade gliomas, particularly glioblastomas (grade IV), are devastating diseases with dismal prognoses; afflicted patients seldom live longer than 15 months, and their quality of life suffers immensely. Our current standard-of-care therapy has remained essentially unchanged for almost 15 years, with little new therapeutic progress. We desperately need a better biologic understanding of these complicated tumors in a complicated organ. One area of rejuvenated study relates to extracellular vesicles (EVs)-membrane-enclosed nano- or microsized particles that originate from the endosomal system or are shed from the plasma membrane. EVs contribute to tumor heterogeneity (including the maintenance of glioma stem cells or their differentiation), the impacts of hypoxia (angiogenesis and coagulopathies), interactions amid the tumor microenvironment (concerning the survival of astrocytes, neurons, endothelial cells, blood vessels, the blood-brain barrier, and the ensuing inflammation), and influences on the immune system (both stimulatory and suppressive). This article reviews glioma EVs and the ways that EVs manifest themselves as autocrine, paracrine, and endocrine factors in proximal and distal intra- and intercellular communications. The reader should note that there is much controversy, and indeed confusion, in the field over the exact roles for EVs in many biological processes, and we will engage some of these difficulties herein.

The Potential of Exosomes From Cow Milk for Oral Delivery.

Many pharmaceuticals must be administered intravenously due to their poor oral bioavailability. In addition to issues associated with sterility and inconvenience, the cost of repeated infusion over a 6-week course of therapy costs the health care system tens of billions of dollars per year. Attempts to improve oral bioavailability have traditionally focused on enhancing drug solubility and membrane permeability, and the use of synthetic nanoparticles has also been investigated. As an alternative strategy, some recent reports have clearly demonstrated that exosomes from cow milk are absorbed from the gastrointestinal tract in humans and could potentially be used for oral delivery of drugs that are traditionally administered intravenously. Our previous work has shown that antibodies are present in exosome preparations, and the current work with milk exosomes suggests that absorption from the gastrointestinal tract occurs via the "neonatal" Fc receptor, FcRn. Furthermore, our results demonstrate that milk exosomes are absorbed from the gut as intact particles that can be modified with ligands to promote retention in target tissues.