Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration

Inactivation of glycogen synthase kinase-3β (GSK3β) by S9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y216, on GSK3β is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y216 phosphorylation on GSK3β in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y216, GSK3β activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3β and adenoviral-mediated transduction of dominant negative GSK3β constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S9 phosphorylation and inactivation of GSK3β but did not affect Y216 phosphorylation, suggesting that S9 phosphorylation is sufficient to override GSK3β activation by Y216 phosphorylation. Under the conditions examined, increased Y216 phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y216 phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y216-phosphorylated GSK3β selectively increased within the nucleus. In rats, Y216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y216 phosphorylation of GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.

Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

Hepatocyte gene therapy in a large animal: A neonatal bovine model of citrullinemia

The development of gene-replacement therapy for inborn errors of metabolism has been hindered by the limited number of suitable large-animal models of these diseases and by inadequate methods of assessing the efficacy of treatment. Such methods should provide sensitive detection of expression in vivo and should be unaffected by concurrent pharmacologic and dietary regimens. We present the results of studies in a neonatal bovine model of citrullinemia, an inborn error of urea-cycle metabolism characterized by deficiency of argininosuccinate synthetase and consequent life-threatening hyperammonemia. Measurements of the flux of nitrogen from orally administered 15NH4 to [15N]urea were used to determine urea-cycle activity in vivo. In control animals, these isotopic measurements proved to be unaffected by pharmacologic treatments. Systemic administration of a first-generation E1-deleted adenoviral vector expressing human argininosuccinate synthetase resulted in transduction of hepatocytes and partial correction of the enzyme defect. The isotopic method showed significant restoration of urea synthesis. Moreover, the calves showed clinical improvement and normalization of plasma glutamine levels after treatment. The results show the clinical efficacy of treating a large-animal model of an inborn error of hepatocyte metabolism in conjunction with a method for sensitively measuring correction in vivo. These studies will be applicable to human trials of the treatment of this disorder and other related urea-cycle disorders.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Contributions of cell kill and posttreatment tumor growth rates to the repopulation of intracerebral 9L tumors after chemotherapy: An MRI study

The drought of progress in clinical brain tumor therapy provides an impetus for developing new treatments as well as methods for testing therapeutics in animal models. The inability of traditional assays to simultaneously measure tumor size, location, growth kinetics, and cell kill achieved by a treatment complicates the interpretation of therapy experiments in animal models. To address these issues, tumor volume measurements obtained from serial magnetic resonance images were used to noninvasively estimate cell kill values in individual rats with intracerebral 9L tumors after treatment with 0.5, 1, or 2 × LD10 doses of 1,3-bis(2-chloroethyl)-1-nitrosourea. The calculated cell kill values were consistently lower than those reported using traditional assays. A dose-dependent increase in 9L tumor doubling time after treatment was observed that significantly contributed to the time required for surviving cells to repopulate the tumor mass. This study reveals that increases in animal survival are not exclusively attributable to the fraction of tumor cells killed but rather are a function of the cell kill and repopulation kinetics, both of which vary with treatment dose.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

NF-κB activation provides the potential link between inflammation and hyperplasia in the arthritic joint

The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-κB in animal models of RA. We demonstrate that in vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.

Unbalanced expression of the different subunits of elongation factor 1 in diabetic skeletal muscle

In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1α. With Northern blot analysis, EF-1α expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1β or -γ were noted. We observed similar results in animal models of Type 1 diabetes. EF-1α expression, but not EF-1β or -γ expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1α mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state.

The role of antimicrobial peptides in animal defenses

It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.

Chronic morphine induces visible changes in the morphology of mesolimbic dopamine neurons.

The mesolimbic dopamine system, which arises in the ventral tegmental area (VTA), is an important neural substrate for opiate reinforcement and addiction. Chronic exposure to opiates is known to produce biochemical adaptations in this brain region. We now show that these adaptations are associated with structural changes in VTA dopamine neurons. Individual VTA neurons in paraformaldehyde-fixed brain sections from control or morphine-treated rats were injected with the fluorescent dye Lucifer yellow. The identity of the injected cells as dopaminergic or nondopaminergic was determined by immunohistochemical labeling of the sections for tyrosine hydroxylase. Chronic morphine treatment resulted in a mean approximately 25% reduction in the area and perimeter of VTA dopamine neurons. This reduction in cell size was prevented by concomitant treatment of rats with naltrexone, an opioid receptor antagonist, as well as by intra-VTA infusion of brain-derived neurotrophic factor. In contrast, chronic morphine treatment did not alter the size of nondopaminergic neurons in the VTA, nor did it affect the total number of dopaminergic neurons in this brain region. The results of these studies provide direct evidence for structural alterations in VTA dopamine neurons as a consequence of chronic opiate exposure, which could contribute to changes in mesolimbic dopamine function associated with addiction.

Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease.

Huntington disease is a dominantly inherited, untreatable neurological disorder featuring a progressive loss of striatal output neurons that results in dyskinesia, cognitive decline, and, ultimately, death. Neurotrophic factors have recently been shown to be protective in several animal models of neurodegenerative disease, raising the possibility that such substances might also sustain the survival of compromised striatal output neurons. We determined whether intracerebral administration of brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, or ciliary neurotrophic factor could protect striatal output neurons in a rodent model of Huntington disease. Whereas treatment with brain-derived neurotrophic factor, nerve growth factor, or neurotrophin-3 provided no protection of striatal output neurons from death induced by intrastriatal injection of quinolinic acid, an N-methyl-D-aspartate glutamate receptor agonist, treatment with ciliary neurotrophic factor afforded marked protection against this neurodegenerative insult.