Karin Werrbach-Perez

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Abstract: Nerve growth factor (NGF) is a member of the neuro‐ trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per‐ oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time‐ and dose‐dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.

A human clonal cell line model of differentiating neurons

The human neuroblastoma cell line SK-N-SH and its thrice-subcloned derivative, SY-5Y, are nearly diploid cell lines with high levels of dopamine β-hydroxylase, a key enzyme in adrenergic neurotransmitter synthesis. The SY-5Y subclone also displays a neuroblast-like morphology in cell culture and in the undifferentiated state does not have electrically excitable membranes. In this study the response of the SY-5Y cells to the nerve growth factor protein, NGF, is described. After 5 days in culture in the presence of NGF, the SY-5Y cells extended neurites, ceased multiplying, and aggregated into clumps similar to those observed in primary cultures of sympathetic ganglia treated with NGF. Ultrastructural analysis of the induced processes showed them to be neurites. Whereas NGF treatment inhibited the incorporation of [ 3 H]thymidine into DNA and inhibited cellular proliferation, it stimulated incorporation of 3 H-amino acids into protein. Further analysis of these differentiated cells showed them to have developed electrically excitable membranes. Undifferentiated cells were selectively killed by 6-hydroxydopamine, whereas NGF treatment led to protection of the differentiated cells from 6-hydroxydopamine killing. Treatment of the SY-5Y clone with dibutyryl cyclic AMP induced neurite outgrowth and enhanced electrical excitability but did not lead to protection from 6-hydroxydopamine killing. The morphological response of the SY-5Y cells to dibutyryl cyclic AMP was quite different from that observed after NGF treatment. We propose the SY-5Y clone as a model for the study of the regulation of NGF-induced differentiation in neuronal cells.

Effect of retinoic acid on nerve growth factor receptors

SummaryRetinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of 125I-NGF to solubilized receptors. Analysis of 125I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.

Signal Transduction in Neuronal Death

Abstract: Apoptosis in the nervous system is a necessary event during the development of the nervous system and is also present after genotoxic events, be they chronic as in aging or more acute after trauma and ischemia. Apoptotic events reflect an interplay between intrinsic signaling events that rely on cytokines, neurotransmitters, and growth factors and responses to extrinsic events that increase levels of radical oxygen species. Both intrinsically and extrinsically driven signal‐transduction pathways act via transcription factors that regulate the coordinated timely expression of stress‐response genes as part of a decision‐making process that can commit cells to apoptosis or survival. Here we discuss the role of two transcription factors that participate in apoptosis in the nervous system: the activator protein AP‐1 and nuclear factor κB.

Acetyl-l-carnitine enhances the response of PC12 cells to nerve growth factor

We have demonstrated that treatment of rat pheochromocytoma (PC12) cells with acetyl-L-carnitine (ALCAR) stimulates the synthesis of nerve growth factor receptors (NGFR). ALCAR has also been reported to prevent some age-related impairments of the central nervous system (CNS). In particular, ALCAR reduces the loss of NGFR in the hippocampus and basal forebrain of aged rodents. On these bases, a study on the effect of NGF on the PC12 cells was carried out to ascertain whether ALCAR induction of NGFR resulted in an enhancement of NGF action. Treatment of PC12 cells for 6 days with ALCAR (10 mM) stimulated [125I]NGF PC12 cell uptake, consistent with increased NGFR levels. Also, neurite outgrowth elicited in PC12 cells by NGF (100 ng/ml) was greatly augmented by ALCAR pretreatment. When PC12 cells were treated with 10 mM ALCAR and then exposed to NGF (1 ng/ml), an NGF concentration that is insufficient to elicit neurite outgrowth under these conditions, there was an ALCAR effect on neurite outgrowth. The concentration of NGF necessary for survival of serum-deprived PC12 cells was 100-fold lower for ALCAR-treated cells as compared to controls. The minimal effective dose of ALCAR here was between 0.1 and 0.5 mM. This is similar to the reported minimal concentration of ALCAR that stimulates the synthesis of NGFR in these cells. The data here presented indicate that one mechanism by which ALCAR rescues aged neurons may be by increasing their responsiveness to neuronotrophic factors in the CNS.

Suppression of p140trkA Does Not Abolish Nerve Growth Factor-Mediated Rescue of Serum-Free PC12 Cells

Abstract: Programmed cell death, the intrinsic form of apoptosis, plays an integral role in those neurodegenerative events associated with age‐related neuropathology. Neurotrophins (NTs), such as nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and NT‐3, are required for survival of certain neurons, and thus their clinical use to counteract age‐ and pathology‐associated neurodegeneration has been suggested, although mechanistic descriptions for NT cell rescue from apoptosis are not definitive. Here we attempted to isolate the individual actions of high‐affinity tyrosine kinase (Trk) receptors and p75NGFR, the common low‐affinity NT receptor, in NT rescue of apoptotic PC12 cells. Our results showed that whereas inhibiting Trk receptor phosphorylation abolishes NGF rescue of serum‐deprived PC12 cells from apoptosis, TrkA suppression with antisense oligonucleotides did not. Also, although BDNF did not rescue naive serumless PC12 cells, which lack the BDNF‐specific TrkB receptor, it significantly increased survival of TrkA‐suppressed serum‐starved PC12 cells. These data confirm the hypothesis that binding of any NT to Trk‐free p75NGFR‐bearing cells blocks apoptosis but also suggest that if Trk receptors are expressed, prohibiting Trk phosphorylation also blocks NT‐mediated rescue from apoptosis.

Neurotrophin regulation of energy homeostasis in the central nervous system.

Our hypothesis is that one cause of neuronal cell death and shrinkage in the aged central nervous system is an inability of neurons to maintain oxidant homeostasis in the face of increased levels of reactive oxygen species, decreased endogenous antioxidants, and impaired energy metabolism associated with physiological senescence, Alzheimers, and Parkinsons diseases. Since treatment with nerve growth factor (NGF) reverses behavioral impairments in aged rats and stimulates cholinergic activity in the basal forebrain, while brain-derived neurotrophic factor appears to play a similar role in the striatum, we propose that neurotrophin-mediated cell-sparing reflects effects on oxidant homeostasis. Neurotrophins may play a similar cell-sparing role in hypoxic/ischemic injury to the nervous system, which also is mediated in part by reactive oxygen species. The degradation of one such species, H2O2, is catalyzed by catalase and glutathione peroxidase (GSH Px). The activity of the latter enzyme is dependent on glutathione reductase and the availability of NADPH for regeneration of reduced GSH. The GSH redox cycle is also regulated by enzymes of the hexose monophosphate shunt. NGF protects PC12 cells from H2O2 injury by stimulating the synthesis of antioxidant enzymes including catalase, GSH Px, glucose-6-phosphate dehydrogenase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. NGF also enhances recovery from the NAD+ losses occurring as a consequence of H2O2 treatment.

EFFECTS OF INTRAVENTRICULAR TRANSPLANTATION OF NGF-SECRETING CELLS ON CHOLINERGIC BASAL FOREBRAIN NEURONS AFTER PARTIAL IMMUNOLESION

The aim of the present study was to examine the effects of nerve growth factor on brain cholinergic function after a partial immunolesion to the rat cholinergic basal forebrain neurons (CBFNs) by 192 IgG‐saporin. Two weeks after intraventricular injections of 1.3 μg of 192 IgG‐saporin, about 50% of CBFNs were lost which was associated with 40–60% reductions of choline acetyltransferase (ChAT) and high‐affinity choline uptake (HACU) activities throughout the basal forebrain cholinergic system. Two groups of lesioned animals received intraventricular transplantations of mouse 3T3 fibroblasts retrovirally transfected with either the rat NGF gene (3T3NGF+) or the retrovirus alone (3T3NGF−) and were sacrificed eight weeks later. In vivo production of NGF by 3T3NGF+ cells was confirmed by NGF immunohistochemistry on the grafts and NGF immunoassay on cerebrospinal fluid (CSF) samples. Both ChAT and HACU activities returned to normal control levels in the basal forebrain and cortex after 3T3NGF+ transplants, whereas no recovery was observed in 3T3NGF− transplanted animals. There was a 25% increase in the size of remaining CBFNs and an increased staining intensity for NGF immunoreactivity in these cells after NGF treatments. Acetylcholinesterase (AChE) histochemistry revealed that the optical density of AChE‐positive fibers in the cerebral cortex and hippocampus were reduced by about 60% in immunolesioned rats which were completely restored by 3T3NGF+ grafts. In addition, decreases in growth‐associated protein (GAP)‐43 immunoreactivity after immunolesion and increases in synaptophysin immunoreactivity after 3T3NGF+ grafts were observed in the hippocampus. Our results further confirm the notion that transfected NGF‐secreting cells are useful in long‐term in vivo NGF treatment and NGF can upregulate CBFN function. They also highly suggest that NGF induces terminal sprouting from remaining CBFNs.

Stimulation of nerve growth factor receptors in PC12 by acetyl-l-carnitine

Acetyl-L-carnitine (ALCAR) prevents some deficits associated with aging in the central nervous system (CNS), such as the aged-related reduction of nerve growth factor (NGF) binding. The aim of this study was to ascertain whether ALCAR could affect the expression of an NGF receptor (p75NGFR). Treatment of PC12 cells with ALCAR increased equilibrium binding of 125I-NGF. ALCAR treatment also increased the amount of immunoprecipitable p75NGFR from PC12 cells. Lastly, the level of p75NGFR messenger RNA (mRNA) in PC12 was increased following ALCAR treatment. These results are in agreement with the hypothesis that there is a direct action of ALCAR on p75NGFR expression in aged rodent CNS.

Nerve growth factor effects on pyridine nucleotides after oxidant injury of rat pheochromocytoma cells

Neurotrophic factors regulate neuronal survival and neurite growth in development and following injury. Oxidative stress produced in neurons as a consequence of primary injury, or during reperfusion following ischemia, may contribute to cell death. Here, the effects of nerve growth factor (NGF) on the response to H2O2 injury were examined in the PC12 rat pheochromocytoma cell line. Specifically, the effect of NGF on cell viability after H2O2 injury was measured. Pretreatment with NGF enhanced survival after H2O2 treatment, as measured by Trypan blue dye exclusion, radiolabeled amino acid incorporation, tetrazolium salt reduction, or cytoplasmic enzyme release. One early event associated with H2O2 treatment was a rapid decrease in NAD+. Although initial decreases in NAD+ levels were similar in control and NGF-treated cells, the latter recovered more rapidly and extensively. The decline in total NAD observed after NGF treatment was almost equal in magnitude to the measured increase in NADP. Inhibition of poly(ADP-ribose) polymerase also enhanced viability following H2O2 injury. Treatment with both NGF and an inhibitor of this enzyme resulted in a greater reduction of H2O2 toxicity than was observed with either agent alone. These data suggest that NGF protection is multifactorial and that a significant component of the NGF effect is due to its regulatory role in the metabolism of pyridine nucleotides.