HIV-1 Clade B and C Isolates Exhibit Differential Replication: Relevance to Macrophage-Mediated Neurotoxicity

HIV-associated neurocognitive disorders (HAND) continue to be a consequence of HIV-1 infection among clade B-infected individuals. In contrast, the incidence of severe neurological impairment is lower among clade C-infected patients in regions of Sub-Saharan Africa and India. Biological aspects such as replication, cytopathicity, inflammatory response, and neurotoxicity unique to each clade influence neuropathogenicity and ultimately affect the clinical outcome of the disease. We hypothesize that productive infection by clade C isolates leads to macrophagemediated neurotoxicity, although to a lesser extent than clade B isolates. Using a panel of primary isolates of clades B and C we demonstrated that clade B has higher replication efficiency in monocyte-derived macrophages (MDM) through reverse transcriptase activity assay and HIV-1 p24 antigen ELISA. To test the neurotoxicity of clades B and C, we used an in vitro neurotoxicity model. Conditioned medium from clade B-infected MDM was neurotoxic to rat and human neuron cultures. In contrast, clade C isolates mediated neurotoxicity when a higher initial viral titer was used for MDM infection. Furthermore, neurotoxicity mediated by isolates of both clades correlated with virus replication in MDM. Together, these results suggest that in comparison to clade B, primary isolates of clade C have slower replication kinetics in primary MDM, leading to lower levels of macrophage-mediated neurotoxicity. Elucidating the differences in replication and macrophage-mediated neurotoxicity between isolates of HIV-1 clades B and C will provide important insights needed to clarify the disparity seen in HAND incidence

Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in HepG2 hepatocellular carcinoma cells

AbstractApoptosis and autophagy are crucial mechanisms regulating cell death, and the relationship between apoptosis and autophagy in the liver has yet to be thoroughly explored. TIGAR (TP53-induced glycolysis and apoptosis regulator), which is a p53-inducible gene, functions in the suppression of ROS (reactive oxygen species) and protects U2OS cells from undergoing cell death. In this study, silencing TIGAR by RNAi (RNA interference) in HepG2 cells down-regulated both TIGAR mRNA (∼75%) and protein levels (∼80%) and led to the inhibition of cell growth (P<0.01) by apoptosis (P<0.001) and autophagy. We demonstrated that TIGAR can increase ROS levels in HepG2 cells. The down-regulation of TIGAR led to the induction of LC-3 II (specific autophagic marker), the formation of the autophagosome, and increased Beclin-1 expression. 3-MA (3-Methyladenine), an inhibitor of autophagic sequestration blocker, inhibited TIGAR siRNA-enhanced autophagy, as indicated by the decrease in LC-3 II levels. Consequently, these data provide the first evidence that targeted silencing of TIGAR induces apoptotic and autophagic cell death in HepG2 cells, and our data raise hope for the future successful application of TIGAR siRNA in patients with hepatocellular carcinoma (HCC)

Interferon-α regulates glutaminase 1 promoter through STAT1 phosphorylation: relevance to HIV-1 associated neurocognitive disorders.

HIV-1 associated neurocognitive disorders (HAND) develop during progressive HIV-1 infection and affect up to 50% of infected individuals. Activated microglia and macrophages are critical cell populations that are involved in the pathogenesis of HAND, which is specifically related to the production and release of various soluble neurotoxic factors including glutamate. In the central nervous system (CNS), glutamate is typically derived from glutamine by mitochondrial enzyme glutaminase. Our previous study has shown that glutaminase is upregulated in HIV-1 infected monocyte-derived-macrophages (MDM) and microglia. However, how HIV-1 leads to glutaminase upregulation, or how glutaminase expression is regulated in general, remains unclear. In this study, using a dual-luciferase reporter assay system, we demonstrated that interferon (IFN) α specifically activated the glutaminase 1 (GLS1) promoter. Furthermore, IFN-α treatment increased signal transducer and activator of transcription 1 (STAT1) phosphorylation and glutaminase mRNA and protein levels. IFN-α stimulation of GLS1 promoter activity correlated to STAT1 phosphorylation and was reduced by fludarabine, a chemical that inhibits STAT1 phosphorylation. Interestingly, STAT1 was found to directly bind to the GLS1 promoter in MDM, an effect that was dependent on STAT1 phosphorylation and significantly enhanced by IFN-α treatment. More importantly, HIV-1 infection increased STAT1 phosphorylation and STAT1 binding to the GLS1 promoter, which was associated with increased glutamate levels. The clinical relevance of these findings was further corroborated with investigation of post-mortem brain tissues. The glutaminase C (GAC, one isoform of GLS1) mRNA levels in HIV associated-dementia (HAD) individuals correlate with STAT1 (

Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms: relevance to excitotoxic neurodegeneration.

BACKGROUND: Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis, which converts glutamine into glutamate. Glutamate is the major neurotransmitter of excitatory synapses, executing important physiological functions in the central nervous system. There are two isoforms of glutaminase 1, KGA and GAC, both of which are generated through alternative splicing from the same gene. KGA and GAC both transcribe 1-14 exons in the N-terminal, but each has its unique C-terminal in the coding sequence. We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection. Furthermore, glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis, Alzheimer\u27s disease, and hepatic encephalopathy. Core enzyme structure of KGA and GAC has been published recently. However, how other coding sequences affect their functional enzyme activity remains unclear. METHODS: We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids (AAs). Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate. RESULTS: When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal, KGA and GAC retained enzyme activity comparable to the full length proteins. In contrast, deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC. Consistently, when both N- and C-terminal of the KGA and GAC were removed, creating a truncated protein that expressed the central 219 AA - 550 AA, the protein retained enzyme activity. Furthermore, expression of the core 219 AA - 550 AA coding sequence in cells increased extracellular glutamate concentrations to levels comparable to those of full-length KGA and GAC expressions, suggesting that the core enzyme activity of the protein lies within the central 219 AA - 550 AA. Full-length KGA and GAC retained enzyme activities when kept at 4 °C. In contrast, 219 AA - 550 AA truncated protein lost glutaminase activities more readily compared with full-length KGA and GAC, suggesting that the N-terminal and C-terminal coding regions are required for the stability KGA and GAC. CONCLUSIONS: Glutaminase isoforms KGA and GAC have similar efficacy to catalyze the conversion of glutamine to glutamate. The core enzyme activity of glutaminase 1 protein is within the central 219 AA - 550 AA. The N-terminal and C-terminal coding regions of KGA and GAC help maintain the long-term activities of the enzymes

Characterization Of Induced Neural Progenitors From Skin Fibroblasts By A Novel Combination Of Defined Factors

Recent reports have demonstrated that somatic cells can be directly converted to other differentiated cell types through ectopic expression of sets of transcription factors, directly avoiding the transition through a pluripotent state. Our previous experiments generated induced neural progenitor-like cells (iNPCs) by a novel combination of five transcription factors (Sox2, Brn2, TLX, Bmi1 and c-Myc). Here we demonstrated that the iNPCs not only possess NPC-specific marker genes, but also have qualities of primary brain-derived NPCs (WT-NPCs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. Importantly, the mature neurons derived from iNPCs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. These results suggest that directly reprogrammed iNPCs closely resemble WT-NPCs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (iNCs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders

Characterization of induced neural progenitors from skin fibroblasts by a novel combination of defined factors.

Recent reports have demonstrated that somatic cells can be directly converted to other differentiated cell types through ectopic expression of sets of transcription factors, directly avoiding the transition through a pluripotent state. Our previous experiments generated induced neural progenitor-like cells (iNPCs) by a novel combination of five transcription factors (Sox2, Brn2, TLX, Bmi1 and c-Myc). Here we demonstrated that the iNPCs not only possess NPC-specific marker genes, but also have qualities of primary brain-derived NPCs (WT-NPCs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. Importantly, the mature neurons derived from iNPCs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. These results suggest that directly reprogrammed iNPCs closely resemble WT-NPCs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (iNCs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders

Direct Conversion of Mouse Astrocytes Into Neural Progenitor Cells and Specific Lineages of Neurons

Background: Cell replacement therapy has been envisioned as a promising treatment for neurodegenerative diseases. Due to the ethical concerns of ESCs-derived neural progenitor cells (NPCs) and tumorigenic potential of iPSCs, reprogramming of somatic cells directly into multipotent NPCs has emerged as a preferred approach for cell transplantation. Methods: Mouse astrocytes were reprogrammed into NPCs by the overexpression of transcription factors (TFs) Foxg1, Sox2, and Brn2. The generation of subtypes of neurons was directed by the force expression of cell-type specific TFs Lhx8 or Foxa2/Lmx1a. Results: Astrocyte-derived induced NPCs (AiNPCs) share high similarities, including the expression of NPC-specific genes, DNA methylation patterns, the ability to proliferate and differentiate, with the wild type NPCs. The AiNPCs are committed to the forebrain identity and predominantly differentiated into glutamatergic and GABAergic neuronal subtypes. Interestingly, additional overexpression of TFs Lhx8 and Foxa2/Lmx1a in AiNPCs promoted cholinergic and dopaminergic neuronal differentiation, respectively. Conclusions: Our studies suggest that astrocytes can be converted into AiNPCs and lineage-committed AiNPCs can acquire differentiation potential of other lineages through forced expression of specific TFs. Understanding the impact of the TF sets on the reprogramming and differentiation into specific lineages of neurons will provide valuable strategies for astrocyte-based cell therapy in neurodegenerative diseases

Selective Generation of Dopaminergic Precursors from Mouse Fibroblasts by Direct Lineage Conversion.

Degeneration of midbrain dopaminergic (DA) neurons is a key pathological event of Parkinson\u27s disease (PD). Limited adult dopaminergic neurogenesis has led to novel therapeutic strategies such as transplantation of dopaminergic precursors (DPs). However, this strategy is currently restrained by a lack of cell source, the tendency for the DPs to become a glial-restricted state, and the tumor formation after transplantation. Here, we demonstrate the direct conversion of mouse fibroblasts into induced DPs (iDPs) by ectopic expression of Brn2, Sox2 and Foxa2. Besides expression with neural progenitor markers and midbrain genes including Corin, Otx2 and Lmx1a, the iDPs were restricted to dopaminergic neuronal lineage upon differentiation. After transplantation into MPTP-lesioned mice, iDPs differentiated into DA neurons, functionally alleviated the motor deficits, and reduced the loss of striatal DA neuronal axonal termini. Importantly, no iDPs-derived astrocytes and neoplasia were detected in mouse brains after transplantation. We propose that the iDPs from direct reprogramming provides a safe and efficient cell source for PD treatment

Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

Association of genetic polymorphisms in the interleukin-10 promoter with risk of prostate cancer in Chinese

<p>Abstract</p> <p>Background</p> <p>Recent studies identified an increased risk of prostate cancer (PCa) in Caucasian men harboring polymorphisms of genes involved in innate immunity and inflammation. This study was designed to assess whether single nucleotide polymorphisms in the IL-10 promoter play a role in predisposing individuals to PCa in a Chinese population.</p> <p>Methods</p> <p>We genotyped three SNPs of the <it>IL-10 </it>promoter (-1082A/G, -819T/C and -592A/C) using polymerase chain reaction-restriction fragment length polymorphism analysis in 262 subjects with PCa and 270 age-matched healthy controls. Odds ratio and 95% confidence interval were determined by logistic regression for the associations between IL-10 genotypes and haplotypes with the risk of PCa and advanced PCa grade.</p> <p>Results</p> <p>No significant differences in allele frequency or genotype distribution were observed for any of the <it>IL-10 </it>SNPs between PCa patients and control subjects. Significantly higher frequencies of -1082G, -819C and -592C allele and GCC haplotype were observed, however, in early stage patients in comparison to advanced PCa patients (for -1082 G, 13.9% vs 6.1%, OR = 2.48, <it>P </it>= 0.005; for -819 C 40.3% vs 30.8%, OR = 1.51, <it>P </it>= 0.043; for -512C, 40.3% vs 30.8%, OR = 1.51, <it>P </it>= 0.043; and for haplotype GCC 11.1%vs 5.1%, OR = 2.66, P = 0.008, respectively).</p> <p>Conclusions</p> <p>Our results identify that <it>IL-10 </it>promoter polymorphisms might not be a risk factor for PCa in Chinese cohorts, but rather incidence of polymorphisms associates with PCa grade, suggesting that IL-10 expression may impact PCa progression.</p