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Background and Importance: Fusiform vertebrobasilar aneurysms carry significant morbidity. Endovascular strategies are preferred; however, unsafe or unfeasible access can call for innovative strategies., Clinical Presentation: An octogenarian patient with an enlarging fusiform proximal basilar artery aneurysm causing a sixth nerve palsy was found to have multiple anatomic features that precluded a transradial or transfemoral endovascular approach. She was thus treated with direct microsurgical access of the V3 segment of the vertebral artery for subsequent coil embolization and flow diversion., Conclusion: This case introduces a novel combined microsurgical and endovascular strategy for treating a complex partially thrombosed fusiform basilar artery aneurysm. This approach should be reserved only for patients where conventional endovascular access is dangerous or unfeasible., (Copyright © Congress of Neurological Surgeons 2024. All rights reserved.)

Objective: Stereotactic techniques play an important role in neurosurgery. The development of a miniaturized cranial robot with an efficient workflow and accurate surgical execution is an important step in a broader application of these techniques. Herein, the authors describe their experience with the Medtronic Stealth Autoguide miniaturized cranial robot., Methods: A retrospective review of 75 cases from 2020 to 2022 was performed. The patients who had undergone surgery utilizing the Stealth Autoguide robot were analyzed for surgical indication and accuracy, operative time, and clinical outcome. The outcomes were defined as follows: for stereoelectroencephalography (SEEG), the electrode placement pattern that identified the seizure focus and did not require any revision or additional leads; for biopsy, the percentage of cases in which diagnostic tissue was obtained; and for laser interstitial thermal therapy (LITT), the percentage of cases in which laser fiber placement was adequate for ablation. Surgical complications were defined as any asymptomatic or symptomatic intracerebral hemorrhage, new neurological deficit, or need for electrode, laser fiber, or biopsy needle repositioning or revision., Results: The Stealth Autoguide robot was utilized in 75 on-label cases, including 40 SEEG cases for seizure focus localization, 19 LITT cases, and 16 stereotactic biopsy cases. The mean real target error (RTE) at the entry was 1.48 ± 0.84 mm for biopsy, 1.36 ± 0.89 mm for Visualase laser fiber placement, and 1.24 ± 0.72 mm for SEEG. The mean RTE at the target was 1.56 ± 0.95 mm for biopsy needle placement, 1.42 ± 0.93 mm for Visualase laser fiber placement, and 1.31 ± 0.87 mm for SEEG electrode placement. The surgical time for unilateral SEEG cases took an average 52 minutes (average 6.5 mins/lead, average 8 electrodes). Bilateral SEEG cases took an average 105 minutes (average 7.5 mins/lead, average 14 electrodes). In the SEEG population, there were no revised or unsuccessful seizure localizations. For biopsy, diagnostic tissue was obtained in 100% of cases. For LITT, fiber placement was adequate for ablation in 100% of cases. There were no cases of symptomatic or asymptomatic intracerebral hemorrhage, and no cases required repositioning or replacement of the laser fiber, electrode, or biopsy needle. One patient experienced transient cranial nerve III palsy following laser ablation that resolved in 10 weeks. A failure of communication between the robotic platform and the Stealth Autoguide as a station required the cancellation of 1 procedure., Conclusions: The Medtronic Stealth Autoguide robot system is versatile across biopsy, SEEG, and laser ablation indications. Setup and surgical execution are efficient with a high degree of accuracy and consistency.

Objective: We review the outcomes of open surgical treatment of middle cerebral artery aneurysms (MCAAs) at a single center, focusing on aneurysm obliteration rates and functional outcomes at the most recent follow-up. These findings can be used for future comparisons of surgical outcomes with MCAAs., Methods: We retrospectively reviewed cases from a prospectively maintained database of patients receiving open surgical treatment for ruptured or unruptured MCAAs between July 2014 and December 2022. We utilized patients' modified Rankin Scale (mRS) score and Glasgow Outcome Scale score as functional outcome measures. Means, standard deviations, medians, and interquartile ranges were calculated, and a student's t test or its nonparametric equivalent was used to compare subgroups., Results: One hundred fifty patients (114 women, 76%; mean age 55.0 ± 14.7 years) with a total of 156 MCAAs comprised 152 cases; 85 (56%) ruptured and 71 (46%) unruptured. Bypass was performed in 34 cases (22.4%); 18 ruptured (51.4%) and 16 unruptured (48.6%). Intraoperative rupture occurred in 5 (5%) ruptured and 1 (2%) unruptured cases. Onwe hundred forty-five patients (95.4%) had aneurysm obliteration with initial surgery, with 98.4% of patients having complete occlusion at 40.2± 65.5 weeks of follow-up. Intrahospital mortality occurred in 7 (6.9%) ruptured versus 1 (2.0%) unruptured case. Fifty-two (51.5%) of the ruptured compared to 43 (86%) unruptured patients were discharged home, with the remaining patients requiring inpatient rehabilitation or long-term hospitalization. The ruptured group had a mean hospital stay of 18.4 ± 10.5 days versus. 5.7 ± 6.0 days for unruptured. Length of stay, discharge mRS/ Glasgow Outcome Scale, and mRS at 4-6 weeks favored unruptured cases (P < 0.0001-0.0336). Mean change in mRS from presentation to last follow-up favored ruptured cases (-0.7 ± 1.2 vs. -0.04 ± 1.2, P = 0.0215)., Conclusions: Open surgery remains a safe and definitive treatment option for MCAAs in the endovascular era., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Objective: Cerebral revascularization of multiple territories traditionally requires multiple constructs, serial anastomoses, or a combination of direct and indirect approaches. A novel 3-vessel anastomosis technique allows for direct, simultaneous multiterritory cerebral revascularization using a single interposition graft. We herein present our experience with this approach., Methods: Retrospective review of perioperative data and outcomes for patients undergoing multiterritory cerebral revascularization using a 3-vessel anastomosis from 2019 to 2023., Results: Five patients met inclusion criteria (median age 53 years [range 12-73]). Three patients with complex middle cerebral artery aneurysms (1 ruptured) were treated with proximal ligation or partial/complete clip trapping and multiterritory external carotid artery-M2-M2 revascularization using a saphenous vein interposition graft. Two patients with moyamoya disease, prior strokes, and predominately bilateral anterior cerebral artery hypoperfusion were treated with proximal superficial temporal artery-A3-A3 revascularization using a radial artery or radial artery fascial flow-through free flap graft. No patients experienced significant surgery-related ischemia. Bypass patency was 100%. One patient had new strokes from vasospasm after subarachnoid hemorrhage. One patient required a revision surgery for subdural hematoma evacuation and radial artery fascial flow-through free flap debridement, without affecting bypass patency or neurologic outcome. On hospital discharge, median Glasgow Outcome Scale and modified Rankin Scale scores were 4 (range 3-5) and 2 (range 0-5), respectively. On follow-up, 1 patient died from medical complications of their presenting stroke; Glasgow Outcome Scale and modified Rankin Scale scores were otherwise stable or improved., Conclusion: The 3-vessel anastomosis technique can be considered for simultaneous revascularization of multiple intracranial territories., (Copyright © Congress of Neurological Surgeons 2023. All rights reserved.)

Vestibular schwannoma (VS) is an intracranial tumor arising from neoplastic Schwann cells and typically presenting with hearing loss. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. We conducted profiling of patients' plasma for 66 immune-related factors in patients with sporadic VS ( N > 170) and identified and validated candidate biomarkers associated with tumor size (S100B) and hearing (MCP-3). We further identified a nine-biomarker panel (TNR-R2, MIF, CD30, MCP-3, IL-2R, BLC, TWEAK, eotaxin, and S100B) with outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS, providing a unique diagnostic tool that may predict hearing change and tumor growth in VS patients, and may inform the timing of tumor resection to preserve hearing.

Background and Purpose: The treatment of complex intracranial aneurysms can be challenging with stand-alone open or endovascular techniques, particularly after rupture. A combined open and endovascular strategy can potentially limit the risk of extensive dissections with open-only techniques, and allow for aggressive definitive endovascular treatments with minimized downstream ischemic risk., Materials and Methods: Retrospective, single-institution review of consecutive patients undergoing combined open revascularization and endovascular embolization/occlusion for complex intracranial aneurysms from 1/2016 to 6/2022., Results: Ten patients (4 male [40%]; mean age 51.9 ± 8.7 years) underwent combined open revascularization and endovascular treatment of intracranial aneurysms. The majority of aneurysms, 9/10 (90%), were ruptured and 8/10 (80%) were fusiform in morphology. Aneurysms of the posterior circulation represented 8/10 (80%) of the cases (vertebral artery [VA] involving the posterior inferior cerebellar artery [PICA] origin, proximal PICA or anterior inferior cerebellar artery/PICA complex, or proximal posterior cerebral artery). Revascularization strategies included intracranial-to-intracranial (IC-IC; 7/10 [70%]) and extracranial-to-intracranial (EC-IC; 3/10 [30%]) constructs, with 100% postoperative patency. Initial endovascular procedures (consisting of aneurysm/vessel sacrifice in 9/10 patients) were performed early after surgery (0.7 ± 1.5 days). In one patient, secondary endovascular vessel sacrifice was performed after an initial sub-occlusive embolization. Treatment related strokes were diagnosed in 3/10 patients (30%), largely from involved or nearby perforators. All bypasses with follow-up were patent (median 14.0, range 4-72 months). Good outcomes (defined as a Glasgow Outcomes Scale ≥4 and modified Rankin Scale ≤2) occurred in 6/10 patients (60%)., Conclusion: A variety of complex aneurysms not amenable to stand-alone open or endovascular techniques can be successfully treated with combined open and endovascular approaches. Recognition and preservation of perforators is critical to treatment success., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rennert, Nguyen, Abedi, Atai, Carey, Tenser, Amar, Mack and Russin.)

Background: Spinal cord ischemia remains a devastating complication when treating patients with complex thoracoabdominal aortic aneurysms using fenestrated endovascular aortic repair. This approach is progressively deployed. However, to date, no strategy has been identified to reduce the feared risk of spinal cord ischemia., Objective: To introduce a novel bypass technique using a customized composite graft to create a direct extra-anatomic revascularization before fenestrated endovascular aortic repair in patients with high-risk of spinal cord ischemia., Methods: To demonstrate this novel concept, we present here a clinical case that reports the strategy of this novel concept in detail. An 83-year-old man with medical history of endovascular repair of an abdominal aortic aneurysm and thoracic aorta presented with a type IA endoleak, located along the posterior superior aspect of the aortic stent graft adjacent to the lumbar arteries. A multidisciplinary plan was developed, which included a novel bypass from the profunda femoris to the left L1 radicular artery before fenestrated endovascular aortic repair to prevent spinal cord ischemia., Results: The patient successfully receives the novel extra-anatomic revascularization bypass before fenestrated endovascular aortic repair. During the first implementation of this strategy, no intraoperative difficulties and postoperative complications were observed., Conclusion: This case demonstrates a novel surgical technique before fenestrated endovascular aortic repair for prevention of spinal cord ischemia. In addition, this concept provides a promising direction to not only complement the existing surgical techniques but also to generate more future innovations., (Copyright © Congress of Neurological Surgeons 2022. All rights reserved.)

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo., Competing Interests: Conflict of interest S. J. G is a paid consultant for Capricor, holds equity in Capricor, and is coinventor of intellectual property licensed by Capricor. S. J. T. is coinventor of intellectual property licensed by Capricor. C. G. is coinventor of intellectual property licensed by Capricor. N. A. A., J. N., M. C., A. Salehi, A. Sedgwick, S. K., S. S., and C. L. contributed to this work as employees of Capricor., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined., Methods: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock., Results: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy., Conclusion: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Extracellular vesicles (EVs) carry RNA, DNA, proteins, and lipids. Specifically, tumor-derived EVs have the potential to be utilized as disease-specific biomarkers. However, a lack of methods to isolate tumor-specific EVs has limited their use in clinical settings. Here we report a sensitive analytical microfluidic platform ( EV HB-Chip) that enables tumor-specific EV-RNA isolation within 3 h. Using the EV HB-Chip, we achieve 94% tumor-EV specificity, a limit of detection of 100 EVs per μL, and a 10-fold increase in tumor RNA enrichment in comparison to other methods. Our approach allows for the subsequent release of captured tumor EVs, enabling downstream characterization and functional studies. Processing serum and plasma samples from glioblastoma multiforme (GBM) patients, we can detect the mutant EGFRvIII mRNA. Moreover, using next-generation RNA sequencing, we identify genes specific to GBM as well as transcripts that are hallmarks for the four genetic subtypes of the disease.

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As 2 O 3 ) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As 2 O 3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As 2 O 3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As 2 O 3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As 2 O 3 , possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As 2 O 3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As 2 O 3 -treated MPA-si spheroids under our experimental conditions, there was an increase in As 2 O 3 -mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage., Methods: Using differential centrifugation, we isolated EVs from VS cell line HEI-193 and primary cultured human VS cells from patients with good hearing or poor hearing. The EVs were characterized using a Nanosight device and transmission electron microscopy and by extracting their RNA content. The EVs' effects on cultured murine spiral ganglion cells and organotypic cochlear cultures were studied using a transwell dual-culture system and by direct labeling of EVs with PKH-67 dye. EV-induced changes in cochlear cells were quantified using confocal immunohistochemistry. Transfection of VS cells with a green fluorescent protein-containing plasmid was confirmed with reverse transcription PCR., Results: Human VS cells, from patients with poor hearing, produced EVs that could damage both cultured murine cochlear sensory cells and neurons. In contrast, EVs derived from VS cells from patients with good hearing did not damage the cultured cochlear cells., Conclusions: This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.

Background: Cancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression., Methods: Comparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells., Results: Of the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells., Conclusions: The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904&#95;906delGAG/c.907&#95;909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA(+) protein family that localizes to the endoplasmic reticulum lumen and nuclear envelope. This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients: R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia., (© 2014 WILEY PERIODICALS, INC.)

Background: Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis., New Method: The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells., Results: We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement., Comparison With Existing Method: The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells., Conclusion: We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Glioma is the most common brain tumor. For the more aggressive form, glioblastoma, standard treatment includes surgical resection, irradiation with adjuvant temozolomide and, on recurrence, experimental chemotherapy. However, the survival of patients remains poor. There is a critical need for minimally invasive biomarkers for diagnosis and as measures of response to therapeutic interventions. Glioma shed extracellular vesicles (EVs), which invade the surrounding tissue and circulate within both the cerebrospinal fluid and the systemic circulation. These tumor-derived EVs and their content serve as an attractive source of biomarkers. In this review, we discuss the current state of the art of biomarkers for glioma with emphasis on their EV derivation.

Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin or its derivatives as a therapeutic for disease in which EVs play a role.

The isocitrate dehydrogenase 1 (IDH1) mutation occurs in high frequency in glioma and secondary glioblastoma (GBM). Mutated IDH1 produces the oncometabolite 2-hydroxyglutarate rather than α-ketoglutarate or isocitrate. The oncometabolite is considered to be the major cause of the association between the IDH1 mutation and gliomagenesis. On the other hand, the IDH1 mutation in GBM is associated with prolonged patient survival. This association is not well understood yet but IDH1 involvement in epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme is considered to be an important mechanism. However, it was shown recently that the IDH1 mutation and MGMT silencing are independent prognostic factors. Here, we hypothesize that the IDH1 mutation reduces the capacity to produce NADPH and thus reduces the capacity to scavenge reactive oxygen species that are generated during irradiation and chemotherapy. IDH1 activity is responsible for two-thirds of the NADPH production capacity in normal brain, whereas the IDH1 mutation reduces this capacity by almost 40%. Therefore, we hypothesize that the reduced NADPH production capacity due to the IDH1 mutation renders GBM cells more vulnerable to irradiation and chemotherapy thus prolonging survival of the patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Most cases of early onset DYT1 dystonia in humans are caused by a GAG deletion in the TOR1A gene leading to loss of a glutamic acid (ΔE) in the torsinA protein, which underlies a movement disorder associated with neuronal dysfunction without apparent neurodegeneration. Mutation/deletion of the gene (Dst) encoding dystonin in mice results in a dystonic movement disorder termed dystonia musculorum, which resembles aspects of dystonia in humans. While torsinA and dystonin proteins do not share modular domain architecture, they participate in a similar function by modulating a structural link between the nuclear envelope and the cytoskeleton in neuronal cells. We suggest that through a shared interaction with the nuclear envelope protein nesprin-3α, torsinA and the neuronal dystonin-a2 isoform comprise a bridge complex between the outer nuclear membrane and the cytoskeleton, which is critical for some aspects of neuronal development and function. Elucidation of the overlapping roles of torsinA and dystonin-a2 in nuclear/endoplasmic reticulum dynamics should provide insights into the cellular mechanisms underlying the dystonic phenotype.

The somatic IDH1(R132) mutation in the isocitrate dehydrogenase 1 gene occurs in high frequency in glioma and in lower frequency in acute myeloid leukemia and thyroid cancer but not in other types of cancer. The mutation causes reduced NADPH production capacity in glioblastoma by 40% and is associated with prolonged patient survival. NADPH is a major reducing compound in cells that is essential for detoxification and may be involved in resistance of glioblastoma to treatment. IDH has never been considered important in NADPH production. Therefore, the authors investigated NADPH-producing dehydrogenases using in silico analysis of human cancer gene expression microarray data sets and metabolic mapping of human and rodent tissues to determine the role of IDH in total NADPH production. Expression of most NADPH-producing dehydrogenase genes was not elevated in 34 cancer data sets except for IDH1 in glioma and thyroid cancer, indicating an association with the IDH1 mutation. IDH activity was the main provider of NADPH in human normal brain and glioblastoma, but its role was modest in NADPH production in rodent brain and other tissues. It is concluded that rodents are a poor model to study consequences of the IDH1(R132) mutation in glioblastoma.

Osteopontin (OPN) is a glycophosphoprotein with multiple intracellular and extracellular functions. In vitro, OPN enhances migration of mouse neutrophils and macrophages. In cancer, extracellular OPN facilitates migration of cancer cells via its RGD sequence. The present study was designed to investigate whether osteopontin is responsible for neutrophil and macrophage infiltration in human cancer and in particular in glioblastoma. We found that in vitro mouse neutrophil migration was RGD-dependent. In silico, we found that the OPN gene was one of the 5% most highly expressed genes in 20 out of 35 cancer microarray data sets in comparison with normal tissue in at least 30% of cancer patients. In some types of cancer, such as ovarian cancer, lung cancer and melanoma, the OPN gene was one of those with the highest expression levels in at least 90% of cancer patients. In glioblastoma, the most invasive type of brain tumours/glioma, but not in lower grades of glioma it was one of the 5% highest expressed genes in 90% of patients. In situ, we found increased protein levels of OPN in human glioblastoma versus normal human brain confirming in silico results. OPN protein expression was co-localized with neutrophils and macrophages. In conclusion, OPN in tumours not only induces migration of cancer cells but also of leucocytes.

Somatic mutations in the isocitrate dehydrogenase 1 gene (IDH1) occur at high frequency in gliomas and seem to be a prognostic factor for survival in glioblastoma patients. In our set of 98 glioblastoma patients, IDH1 ( R132 ) mutations were associated with improved survival of 1 year on average, after correcting for age and other variables with Cox proportional hazards models. Patients with IDH1 mutations were on average 17 years younger than patients without mutation. Mutated IDH1 has a gain of function to produce 2-hydroxyglutarate by NADPH-dependent reduction of alpha-ketoglutarate, but it is unknown whether NADPH production in gliomas is affected by IDH1 mutations. We assessed the effect of IDH1 (R132 ) mutations on IDH-mediated NADPH production in glioblastomas in situ. Metabolic mapping and image analysis was applied to 51 glioblastoma samples of which 16 carried an IDH1 (R132 ) mutation. NADP+-dependent IDH activity was determined in comparison with activity of NAD+-dependent IDH and all other NADPH-producing dehydrogenases, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and hexose-6-phosphate dehydrogenase. The occurrence of IDH1 mutations correlated with approx. twofold diminished NADP+-dependent IDH activity, whereas activity of NAD+-dependent IDH and the other NADP+-dependent dehydrogenases was not affected in situ in glioblastoma. The total NADPH production capacity in glioblastoma was provided for 65% by IDH activity and the occurrence of IDH1 (R132 ) mutation reduced this capacity by 38%. It is concluded that NADPH production is hampered in glioblastoma with IDH1 (R132 ) mutation. Moreover, mutated IDH1 consumes rather than produces NADPH, thus likely lowering NADPH levels even further. The low NADPH levels may sensitize glioblastoma to irradiation and chemotherapy, thus explaining the prolonged survival of patients with mutated glioblastoma.