Marcos Barbosa

The TNF-α/NF-κB signaling pathway has a key role in methamphetamine–induced blood–brain barrier dysfunction

Methamphetamine (METH) is a psychostimulant that causes neurologic and psychiatric abnormalities. Recent studies have suggested that its neurotoxicity may also result from its ability to compromise the blood–brain barrier (BBB). Herein, we show that METH rapidly increased the vesicular transport across endothelial cells (ECs), followed by an increase of paracellular transport. Moreover, METH triggered the release of tumor necrosis factor-alpha (TNF-α), and the blockade of this cytokine or the inhibition of nuclear factor-kappa B (NF-κB) pathway prevented endothelial dysfunction. Since astrocytes have a crucial role in modulating BBB function, we further showed that conditioned medium obtained from astrocytes previously exposed to METH had a negative impact on barrier properties also via TNF-α/NF-κB pathway. Animal studies corroborated the in vitro results. Overall, we show that METH directly interferes with EC properties or indirectly via astrocytes through the release of TNF-α and subsequent activation of NF-κB pathway culminating in barrier dysfunction.

Amplified and Homozygously Deleted Genes in Glioblastoma: Impact on Gene Expression Levels

Background Glioblastoma multiforme (GBM) displays multiple amplicons and homozygous deletions that involve relevant pathogenic genes and other genes whose role remains unknown. Methodology Single-nucleotide polymorphism (SNP)-arrays were used to determine the frequency of recurrent amplicons and homozygous deletions in GBM (n = 46), and to evaluate the impact of copy number alterations (CNA) on mRNA levels of the genes involved. Principal Findings Recurrent amplicons were detected for chromosomes 7 (50%), 12 (22%), 1 (11%), 4 (9%), 11 (4%), and 17 (4%), whereas homozygous deletions involved chromosomes 9p21 (52%) and 10q (22%). Most genes that displayed a high correlation between DNA CNA and mRNA levels were coded in the amplified chromosomes. For some amplicons the impact of DNA CNA on mRNA expression was restricted to a single gene (e.g., EGFR at 7p11.2), while for others it involved multiple genes (e.g., 11 and 5 genes at 12q14.1–q15 and 4q12, respectively). Despite homozygous del(9p21) and del(10q23.31) included multiple genes, association between these DNA CNA and RNA expression was restricted to the MTAP gene. Conclusions Overall, our results showed a high frequency of amplicons and homozygous deletions in GBM with variable impact on the expression of the genes involved, and they contributed to the identification of other potentially relevant genes.

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.

Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.

Tophaceous gout of the lumbar spine mimicking a spinal meningioma

PurposeAlthough gout is a common metabolic disorder, it usually affects distal joints of the appendicular skeleton. Axial spine involvement is rare, with only 131 cases reported in the literature. The authors report a rare case of lumbar spinal gout mimicking a spinal meningioma.MethodsA 77-year-old man with a history of gout presented with chronic low back pain and progressive paraparesis. Imaging revealed a lumbar spine compressive mass lesion with a dural tail signal. The differential diagnosis was thought to be straightforward favoring a spinal meningioma. Tophaceous gout was never considered. The presence of a dural tail associated with the lesion is an interesting detail of this case, that strongly misguided it and to the best of our knowledge it is the first one reported in the literature.ResultsThe patient underwent surgery and intra-operative findings were surprisingly different from those expected, revealing a chalky white mass lesion firmly adherent and compressing the dural sac. It was completely excised, leaving the dura intact. Histopathology confirmed the diagnosis of tophaceous gout. The patient was sent to physical therapy and had a complete remission of pain and neurological deficit, regaining his walking capacity.ConclusionAlthough spinal gout is rare, it should be considered in the differential diagnosis for patients presenting with symptoms of spinal stenosis, a suspicion of neoplastic lesion of the spine, and a previous history of gout. Early diagnosis can ensure proper and timely medical management, perhaps avoiding neurological compromise and the need for surgery.

Prognostic stratification of adult primary glioblastoma multiforme patients based on their tumor gene amplification profiles

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management.

Nucleolin is expressed in patient-derived samples and glioblastoma cells, enabling improved intracellular drug delivery and cytotoxicity

ABSTRACT One of the major challenges in Glioblastoma (GBM) therapy relates with the existence of glioma stem‐like cells (GSCs), known to be chemo‐ and radio‐resistant. GSCs and non‐stem GBM cells have the ability to interchange, emphasizing the importance of identifying common molecular targets among those cell sub‐populations. Nucleolin overexpression has been recently associated with breast cancer sub‐populations with different stem‐like phenotype. The goal of this work was to evaluate the potential of cell surface nucleolin as a target in GBM cells. Different levels of nucleolin expression resulted in a 3.4‐fold higher association of liposomes targeting nucleolin (functionalized with the nucleolin‐binding F3 peptide) in U87, relative to GBM11 glioblastoma cells. Moreover, nucleolin was suggested as a potential marker in OCT4‐, NANOG‐positive GSC, and in the corresponding non‐stem GBM cells, as well as in SOX2‐positive GSC. Doxorubicin delivered by liposomes targeting nucleolin enabled a level of cytotoxicity that was 2.5‐ or 4.6‐fold higher compared to the non‐targeted counterparts. Importantly, an overexpression of nucleolin was also observed in cells of patient‐derived samples, as compared with normal brain. Overall, these results suggested nucleolin as a therapeutic target in GBM. HIGHLIGHTSNucleolin is overexpressed in GSC sub‐populations and non‐stem glioblastoma cells;Nucleolin enables intracellular targeted drug delivery in glioblastoma cells;Nucleolin is overexpressed in glioblastoma patient‐derived samples.