Varicella zoster virus infection of highly pure terminally differentiated human neurons

In vitro analyses of varicella zoster virus (VZV) reactivation from latency in human ganglia have been hampered by the inability to isolate virus by explantation or cocultivation techniques. Furthermore, attempts to study interaction of VZV with neurons in experimentally infected ganglion cells in vitro have been impaired by the presence of nonneuronal cells, which become productively infected and destroy the cultures. We have developed an in vitro model of VZV infection in which highly pure (>95 %) terminally differentiated human neurons derived from pluripotent stem cells were infected with VZV. At 2 weeks post-infection, infected neurons appeared healthy compared to VZV-infected human fetal lung fibroblasts (HFLs), which developed a cytopathic effect (CPE) within 1 week. Tissue culture medium from VZV-infected neurons did not produce a CPE in uninfected HFLs and did not contain PCR-amplifiable VZV DNA, but cocultivation of infected neurons with uninfected HFLs did produce a CPE. The nonproductively infected neurons contained multiple regions of the VZV genome, as well as transcripts and proteins corresponding to VZV immediate-early, early, and late genes. No markers of the apoptotic caspase cascade were detected in healthy-appearing VZV-infected neurons. VZV infection of highly pure terminally differentiated human neurons provides a unique in vitro system to study the VZV-neuronal relationship and the potential to investigate mechanisms of VZV reactivation

Dramatic Co-Activation of WWOX/WOX1 with CREB and NF-κB in Delayed Loss of Small Dorsal Root Ganglion Neurons upon Sciatic Nerve Transection in Rats

BACKGROUND:Tumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats. METHODOLOGY/PRINCIPAL FINDINGS:Sciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-kappaB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-kappaB (40-65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-kappaB. WOX1 directly activated NF-kappaB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons. CONCLUSIONS/SIGNIFICANCE:Rapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-kappaB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-kappaB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury

Retroviral expression of a kinase-defective IGF-I receptor suppresses growth and causes apoptosis of CHO and U87 cells in-vivo

BACKGROUND: Phosphatidylinositol-3,4,5-triphosphate (PtdInsP3) signaling is elevated in many tumors due to loss of the tumor suppressor PTEN, and leads to constitutive activation of Akt, a kinase involved in cell survival. Reintroduction of PTEN in cells suppresses transformation and tumorigenicity. While this approach works in-vitro, it may prove difficult to achieve in-vivo. In this study, we investigated whether inhibition of growth factor signaling would have the same effect as re-expression of PTEN. METHODS: Dominant negative IGF-I receptors were expressed in CHO and U87 cells by retroviral infection. Cell proliferation, transformation and tumor formation in athymic nude mice were assessed. RESULTS: Inhibition of IGF-IR signaling in a CHO cell model system by expression of a kinase-defective IGF-IR impairs proliferation, transformation and tumor growth. Reduction in tumor growth is associated with an increase in apoptosis in-vivo. The dominant-negative IGF-IRs also prevented growth of U87 PTEN-negative glioblastoma cells when injected into nude mice. Injection of an IGF-IR blocking antibody αIR3 into mice harboring parental U87 tumors inhibits tumor growth and increases apoptosis. CONCLUSION: Inhibition of an upstream growth factor signal prevents tumor growth of the U87 PTEN-deficient glioma to the same extent as re-introduction of PTEN. This result suggests that growth factor receptor inhibition may be an effective alternative therapy for PTEN-deficient tumors

Activation of adherent vascular neutrophils in the lung during acute endotoxemia

BACKGROUND: Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. METHODS: Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. RESULTS: Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein. CONCLUSION: These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells

Comparative study of classification algorithms using molecular descriptors in toxicological databases

The rational development of new drugs is a complex and expensive process, comprising several steps. Typically, it starts by screening databases of small organic molecules for chemical structures with potential of binding to a target receptor and prioritizing the most promising ones. Only a few of these will be selected for biological evaluation and further refinement through chemical synthesis. Despite the accumulated knowledge by pharmaceutical companies that continually improve the process of finding new drugs, a myriad of factors affect the activity of putative candidate molecules in vivo and the propensity for causing adverse and toxic effects is recognized as the major hurdle behind the current "target-rich, lead-poor" scenario. In this study we evaluate the use of several Machine Learning algorithms to find useful rules to the elucidation and prediction of toxicity using ID and 2D molecular descriptors. The results indicate that: i) Machine Learning algorithms can effectively use ID molecular descriptors to construct accurate and simple models; ii) extending the set of descriptors to include 2D descriptors improve the accuracy of the models

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases