First evidence of overlaps between HIV-Associated Dementia (HAD) and non-viral neurodegenerative diseases: proteomic analysis of the frontal cortex from HIV+ patients with and without dementia

<p>Abstract</p> <p>Background</p> <p>The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed.</p> <p>Result</p> <p>Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings.</p> <p>Conclusion</p> <p>These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.</p

Oncogenic B-RAFV600E Signaling Induces the T-Box3 Transcriptional Repressor to Repress E-Cadherin and Enhance Melanoma Cell Invasion

Approximately 50% of melanomas require oncogenic B-RAFV600E signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAFV600E regulates genes associated with epithelial–mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte–melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAFV600E induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas

Monitoring oncogenic B-RAF-induced senescence in melanocytes

The B-RAF kinase is a downstream effector of the RAS family of proto-oncogenes and is constitutively activated in the majority of human melanomas. The common oncogenic B-RAF(V600E) mutant cooperates with additional genetic lesions to transform immortal murine and human cells. In primary cells, however, B-RAF(V600E) triggers a rapid cell cycle arrest that is phenotypically indistinguishable from cellular senescence. Here we describe the analyses of B-RAF-induced senescence in primary human melanocytes using recombinant lentiviruses.14 page(s

The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of d-Alanine▿ †

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development

The Relative contributions of the p53 and pRb pathways in oncogene-induced melanocyte senescence

Oncogene-induced senescence acts as a barrier against tumour formation and has been implicated as the mechanism preventing the transformation of benign melanocytic lesions that frequently harbour oncogenic B-RAF or N-RAS mutations. In the present study we systematically assessed the relative importance of the tumour suppressor proteins p53, p21(Waf1), pRb and p16(INK4a) in mediating oncogene-induced senescence in human melanocytes. We now show that oncogenic N-RAS induced senescence in melanocytes is associated with DNA damage, a potent DNA damage response and the activation of both the p16(INK4a)/pRb and p53/p21(Waf1) tumour suppressor pathways. Surprisingly neither the pharmacological inhibition of the DNA damage response pathway nor silencing of p53 expression had any detectable impact on oncogene-induced senescence in human melanocytes. Our data indicate that the pRb pathway is the dominant effector of senescence in these cells, as its specific inactivation delays the onset of senescence and weakens oncogene-induced proliferative arrest. Furthermore, we show that although both p16(INK4a) and p21(Waf1) are upregulated in response to N-RAS(Q61K), the activities of these CDK inhibitors are clearly distinct and only the loss of p16(INK4a) weakens senescence. We propose that the ability of p16(INK4a) to inhibit the cyclin D-dependent kinases and DNA replication, functions not shared by p21(Waf1), contribute to its role in senescence. Thus, in melanocytes with oncogenic signalling only p16(INK4a) can fully engage the pRb pathway to alter chromatin structure and silence the genes that are required for proliferation.15 page(s

Absence of distinguishing senescence traits in human melanocytic nevi

Cellular senescence permanently restricts the replicative capacity of cells in response to various stress signals, including aberrant activation of oncogenes. The presence of predictive senescence markers in human premalignant lesions suggests that senescence may function as a genuine tumor suppressor. These markers are not exclusive to the senescence program, however, and it is possible that their expression in vivo does not discriminate irreversible from reversible forms of proliferative arrest. In this study, we aimed to clarify whether human nevus cells can be distinguished from primary and transformed melanocytes by examining the expression of eight senescence markers, including those previously purported to define nevi as senescent tumors. Specifically, we analyzed effectors of senescence, including p16 INK4a, p53, and DNA damage (γ-H2AX), as well as predictive markers of senescence including Ki67, PML, senescence-associated β-galactosidase, heterochromatic foci (H3K9Me, 4'-6-diamidino-2-phenylindole), and nuclear size. We found that these commonly accepted senescence markers do not in fact distinguish nevi from precursor/normal and transformed/malignant melanocytes. We conclude that on the basis of current evidence it cannot be reasonably inferred that nevi are permanently growth arrested via senescence.9 page(s

A parallel genome-wide mRNA and microRNA profiling of the frontal cortex of HIV patients with and without HIV-associated dementia shows the role of axon guidance and downstream pathways in HIV-mediated neurodegeneration

Background: HIV-associated dementia (HAD) is the most common dementia type in young adults less than 40 years of age. Although the neurotoxins, oxidative/metabolic stress and impaired activity of neurotrophic factors are believed to be underlying reasons for the development of HAD, the genomic basis, which ultimately defines the virus-host interaction and leads to neurologic manifestation of HIV disease is lacking. Therefore, identifying HIV fingerprints on the host gene machinery and its regulation by microRNA holds a great promise and potential for improving our understanding of HAD pathogenesis, its diagnosis and therapy. Results: A parallel profiling of mRNA and miRNA of the frontal cortex autopsies from HIV positive patients with and without dementia was performed using Illumina Human-6 BeadChip and Affymetrix version 1.0 miRNA array, respectively. The gene ontology and pathway analysis of the two data sets showed high concordance between miRNA and mRNAs, revealing significant interference with the host axon guidance and its downstream signalling pathways in HAD brains. Moreover, the differentially expressed (DE) miRNAs identified in this study, in particular miR-137, 153 and 218, based on which most correlations were built cumulatively targeted neurodegeneration related pathways, implying their future potential in diagnosis, prognosis and possible therapies for HIV-mediated and possibly other neurodegenerative diseases. Furthermore, this relationship between DE miRNAs and DE mRNAs was also reflected in correlation analysis using Bayesian networks by splitting-averaging strategy (SA-BNs), which revealed 195 statistically significant correlated miRNA-mRNA pairs according to Pearson’s correlation test (P<0.05). Conclusions: Our study provides the first evidence on unambiguous support for intrinsic functional relationship between mRNA and miRNA in the context of HIV-mediated neurodegeneration, which shows that neurologic manifestation in HIV patients possibly occurs through the interference with the host axon guidance and its downstream signalling pathways. These data provide an excellent avenue for the development of new generation of diagnostic/prognostic biomarkers and therapeutic intervention strategies for HIV-associated neurodegeneration