Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice

Background

Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in PANK2, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.

Objective, Methods, Results and Conclusions

Cellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.     

The p53-target gene puma drives neutrophil-mediated protection against lethal bacterial sepsis

Disruption of p53/Puma-mediated apoptosis protects against lethality due to DNA damage. Here we demonstrate the unexpected requirement of the pro-apoptotic p53-target gene Puma to mount a successful innate immune response to bacterial sepsis. Puma^−/− mice rapidly died when challenged with bacteria. While the immune response in Puma^−/− mice was unchanged in cell migration, phagocytosis and bacterial killing, sites of infection accumulated large abscesses and sepsis was progressive. Blocking p53/Puma-induced apoptosis during infection caused resistance to ROS-induced cell death in the CD49d+ neutrophil subpopulation, resulting in insufficient immune resolution. This study identifies a biological role for p53/Puma apoptosis in optimizing neutrophil lifespan so as to ensure the proper clearance of bacteria and exposes a counter-balance between the innate immune response to infection and survival from DNA damage.     

Novelties in Stevia (Compositae: Eupatorieae)

Twenty-four new taxa (16 species, 8 varieties) are described in the genusStevia from North and Central America. Three new combinations are made. Brief discussions concerning the relationships of the new taxa are provided.     

Roles for lysophospholipid S1P receptors in multiple sclerosis

Sphingosine 1-phosphate (S1P) signaling in the treatment of multiple sclerosis (MS) has been highlighted by the efficacy of FTY720 (fingolimod), which upon phosphorylation can modulate S1P receptor activities. FTY720 has become the first oral treatment for relapsing MS that was approved by the FDA in September 2010. Phosphorylated FTY720 modulates four of the five known S1P receptors (S1P(1), S1P(3), S1P(4), and S1P(5)) at high affinity. Studies in human MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have revealed that FTY720 exposure alters lymphocyte trafficking via sequestration of auto-aggressive lymphocytes within lymphoid organs, representing the current understanding of its mechanism of action. These effects primarily involve S1P(1), which is thought to attenuate inflammatory insults in the central nervous system (CNS). In addition, FTY720's actions may involve direct effects on S1P receptor-mediated signaling in CNS cells, based upon the known expression of S1P receptors in CNS cell types relevant to MS, access to the CNS through the blood-brain barrier (BBB), and in vitro studies. These data implicate lysophospholipid signaling--via S1P(1) and perhaps other lysophospholipid receptors--in therapeutic approaches to MS and potentially other diseases with immunological and/or neurological components.     

Blockade of sphingosine 1-phosphate receptor 2 signaling attenuates streptozotocin-induced apoptosis of pancreatic β-cells

Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that acts through five cognate G protein-coupled receptors (S1P₁-S1P₅) and regulates many critical biological processes. Recent studies indicated that S1P at nanomolar concentrations significantly reduces cytokine-induced apoptosis of pancreatic β-cells in which genes for S1P₁-S1P₄ are co-expressed. However, the S1P receptor subtype(s) involved in this effect remains to be clarified. In this study, we investigated the potential role of S1P₂ in streptozotocin (STZ)-induced apoptosis of pancreatic β-cells and progression of diabetes. S1P₂-deficient (S1P₂^-/-) mice displayed a greater survive ability, lower blood glucose levels, and smaller numbers of TUNEL-positive apoptotic β-cells to administration of a high dose of STZ than wild-type (WT) mice. S1P₂^-/- mice showed higher insulin/glucose ratios (an index of relative insulin deficiency) and larger insulin-positive islet areas to administration of a low dose of STZ than WT mice. Moreover, administration of JTE-013, a S1P₂-specific antagonist, to WT mice ameliorated STZ-induced blood glucose elevation and reduced the incidence of diabetes. Our findings indicate that blockade of S1P₂ signaling attenuates STZ-induced apoptosis of pancreatic β-cells and decreases the incidence of diabetes.     

Lysophospholipids in the nervous system

This piece offers perspectives on the emerging roles of lysophospholipids, which include lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), for the biology and pathophysiology of the nervous system. It reflects opinions generated during a meeting sponsored by the National Institute on Drug Abuse (NIDA) entitled "Targeted Lipidomics: Signaling Lipids and Drugs of Abuse" held in Washington, D.C., 15-17 April 2004, organized by Dr. Rao Rapaka. Lysophospholipids represent one class of lipids that has many important actions mediated by G protein-coupled receptors. While influencing a large number of biologically important systems, this discussion will focus on the nervous system, including areas of future research.     

Emerging medicinal roles for lysophospholipid signaling

The two lysophospholipids (LPs) lysophosphatidic acid and sphingosine 1-phosphate (S1P) regulate diverse biological processes. Over the past decade, it has become clear that medically relevant LP activities are mediated by specific G protein-coupled receptors, implicating them in the etiology of a growing number of disorders. A new class of LP agonists shows promise for drug therapy: the experimental drug FTY720 is phosphorylated in vivo to produce a potent S1P receptor agonist (FTY720-P) and is currently in Phase III clinical trials for kidney transplantation and Phase II for multiple sclerosis. Recent genetic and pharmacological studies on LP signaling in animal disease models have identified new areas in which interventions in LP signaling might provide novel therapeutic approaches for the treatment of human diseases.     

Autotaxin, a synthetic enzyme of lysophosphatidic acid (LPA), mediates the induction of nerve-injured neuropathic pain

The extracellular signal-regulated kinase (ERK) cascade has been shown to be a key modulator of pain processing in the central nucleus of the amygdala (CeA) in mice. ERK is activated in the CeA during persistent inflammatory pain and this activation is both necessary and sufficient to induce peripheral tactile hypersensitivity. Interestingly, biochemical studies show that inflammation-induced ERK activation in the CeA only occurs in the right, but not the left hemisphere. This inflammation-induced ERK activation in the right CeA is independent of the side of peripheral inflammation, suggesting that there is a dominant role of the right hemisphere in the modulation of pain by ERK activation in the CeA. However, the functional significance of this biochemical lateralization has yet to be determined. In the present study, we tested the hypothesis that modulation of pain by ERK signaling in the CeA is functionally lateralized. We acutely blocked ERK activation in the CeA by infusing the MEK inhibitor U0126 into the right or the left hemisphere and then measured the behavioral effects on inflammation-induced mechanical hypersensitivity in mice. Our results show that blockade of ERK activation in the right, but not the left CeA, decreases inflammation-induced peripheral hypersensitivity independent of the side of peripheral injury. These findings demonstrate that modulation of pain by ERK signaling in the CeA is functionally lateralized to the right hemisphere, suggesting a dominant role of the right amygdala in pain processing.     

Preclinical models for neuroblastoma: establishing a baseline for treatment

Background

Preclinical models of pediatric cancers are essential for testing newchemotherapeutic combinations for clinical trials. The most widely usedgenetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse.This neuroblastoma-prone mouse recapitulates many of the features of humanneuroblastoma. Limitations of this model include the low frequency of bonemarrow metastasis, the lack of information on whether the gene expressionpatterns in this system parallels human neuroblastomas, the relatively slowrate of tumor formation and variability in tumor penetrance on differentgenetic backgrounds. As an alternative, preclinical studies are frequentlyperformed using human cell lines xenografted into immunocompromised mice,either as flank implant or orthtotopically. Drawbacks of this system includethe use of cell lines that have been in culture for years, the inappropriatemicroenvironment of the flank or difficult, time consuming surgery fororthotopic transplants and the absence of an intact immune system.

Principal Findings

Here we characterize and optimize both systems to increase their utility forpreclinical studies. We show that TH-MYCN mice develop tumors in theparaspinal ganglia, but not in the adrenal, with cellular and geneexpression patterns similar to human NB. In addition, we present a newultrasound guided, minimally invasive orthotopic xenograft method. Thisinjection technique is rapid, provides accurate targeting of the injectedcells and leads to efficient engraftment. We also demonstrate that tumorscan be detected, monitored and quantified prior to visualization usingultrasound, MRI and bioluminescence. Finally we develop and test a“standard of care” chemotherapy regimen. This protocol, which isbased on current treatments for neuroblastoma, provides a baseline forcomparison of new therapeutic agents.

Significance

The studies suggest that use of both the TH-NMYC model of neuroblastoma andthe orthotopic xenograft model provide the optimal combination for testingnew chemotherapies for this devastating childhood cancer.     

Nucleotide sequence of a cDNA clone of the horse (Equus caballus) DRA gene

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60100. Clone pL33EA is available upon request.