Regino Perez-Polo

Role of nerve growth factor in oxidant homeostasis: glutathione metabolism.

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin‐like NGF‐responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ‐glutamylcysteine synthetase (GCS) activity, the rate‐limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6‐phosphate dehydroge‐nase by 50% after 2 days. Treatment with NGF and cyclo‐heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6‐phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide‐induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl‐buthionine‐(S,R)‐sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl‐buthionine‐(S,R)‐sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6‐phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain‐derived neurotrophic factor, neurotro‐phin‐3, epidermal growth factor, insulin‐like growth factor‐I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro‐blastoma SK‐N‐SH‐SY5Y and the human melanoma A‐875 in serum‐free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF‐responsive cell lines, SK‐N‐SH‐SY5Y, A‐875, and the CNS C6 rat glioma subclone 2BD.

IL-1 Receptor Antagonist Prevents Apoptosis and Caspase-3 Activation after Spinal Cord Injury

One of the consequences of cytokine-orchestrated inflammation after CNS trauma is apoptosis. Our hypothesis is that cell death in the spinal cord after injury results in part from increased synthesis and release of IL-1beta. Using a ribonuclease protection assay, we demonstrated that there is increased transient expression of IL-1beta mRNA and, by using IL-1beta protein ELISA assay, that there are increased IL-1beta protein levels in the contused rat spinal cord, initially localized to the impact region of the spinal cord (segment T8). Using an ELISA cell death assay, we showed that there is apoptosis in the spinal cord 72 h after injury, a finding that was confirmed by measuring caspase-3 activity, which also significantly increased at the site of injury 72 h after trauma. Treatment of the contused spinal cord at the site of injury with the IL-1 receptor antagonist (rmIL-lra, 750 ng/mL) for 72 h using an osmotic minipump completely abolished the increases in contusion-induced apoptosis and caspase-3 activity.

The role of eNOS, iNOS, and NF-κB in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.

Challenges in the Development of Rodent Models of Mild Traumatic Brain Injury

Approximately 75% of traumatic brain injuries (TBI) are classified mild (mTBI). Despite the high frequency of mTBI, it is the least well studied. The prevalence of mTBI among service personnel returning from Operations Iraqi Freedom (OIF) and Enduring Freedom (OEF) and the recent reports of an association between repeated mTBI and the early onset of Alzheimers and other types of dementias in retired athletes has focused much attention on mTBI. The study of mTBI requires the development and validation of experimental models and one of the most basic requirements for an experimental model is that it replicates important features of the injury or disease in humans. mTBI in humans is associated with acute symptoms such as loss of consciousness and pre- and/or posttraumatic amnesia. In addition, many mTBI patients experience long-term effects of mTBI, including deficits in speed of information processing, attention and concentration, memory acquisition, retention and retrieval, and reasoning and decision-making. Although methods for the diagnosis and evaluation of the acute and chronic effects of mTBI in humans are well established, the same is not the case for rodents, the most widely used animal for TBI studies. Despite the magnitude of the difficulties associated with adapting these methods for experimental mTBI research, they must be surmounted. The identification and testing of treatments for mTBI depends of the development, characterization and validation of reproducible, clinically relevant models of mTBI.

The transcription factor Yin Yang 1 is an activator of BACE1 expression

The β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) is a prerequisite for the generation of β‐amyloid peptides, the principle constituents of senile plaques in the brains of patients with Alzheimers disease (AD). BACE1 expression and enzymatic activity are increased in the AD brain, but the regulatory mechanisms of BACE1 expression are largely unknown. Here we show that Yin Yang 1 (YY1), a highly conserved and multifunctional transcription factor, binds to its putative recognition sequence within the BACE1 promoter and stimulates BACE1 promoter activity in rat pheochromocytoma 12 (PC12) cells, rat primary neurones and astrocytes. In rat brain YY1 and BACE1 are widely expressed by neurons, but there was only a minor proportion of neurones that co‐expressed YY1 and BACE1, suggesting that YY1 is not required for constitutive neuronal BACE1 expression. Resting astrocytes in the untreated rat brain did not display either YY1 or BACE1 immunoreactivity. When chronically activated, however, astrocytes expressed both YY1 and BACE1 proteins, indicating that YY1 is important for the stimulated BACE1 expression by reactive astrocytes. This is further emphasized by the expression of YY1 and BACE1 by reactive astrocytes in proximity to β‐amyloid plaques in the AD brain. Our observations suggest that interfering with expression, translocation or binding of YY1 to its BACE1 promoter‐specific sequence may have therapeutic potential for treating patients with AD.

Altered NGF protein levels in different brain areas after immunolesion

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low‐affinity NGF receptor (p75NGFR) in the adult rat brain. Intraventricular injection of 192 IgG‐saporin, made by coupling the monoclonal antibody to p75NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75NGFR‐bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG‐saporin produced significant long‐lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75NGFR‐immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF‐like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial‐derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues.

Regulation of Antioxidant Enzyme Expression by NGF

The rapid decreases in viability seen in H2O2-treated PC12 cells reflect enhanced susceptibility of neural cell types to oxidant injury. The dose-response relationship between NGF concentration and survival after H2O2 treatment resembles that for NGF effects on PC12 survival in serumless medium. Previously we have shown that NGF treatment enhances the activity of GSH-Px and catalase which catalyze the degradation of H2O2. Here in order to ascertain whether NGF stimulates transcription, affects mRNA stability, or acts post-transcriptionally, we measured catalase and GSH-Px mRNA half-lives. While both catalase and GSH-Px transcripts are stable with a relatively long half life and a gradual decay in mRNA levels, NGF had different effects on their stability. NGF had marked effects on catalase mRNA stability. The catalase gene has a 3′ flanking region with T-rich clusters and CA repeats known to be susceptible to regulation by destabilization or ubiquination. NGF maintained catalase mRNA levels of actinomycin D (ACT-D) treated PC12 cells at twice that of cells exposed to ACT-D alone, delaying the rate of decay for catalase mRNA for 24 h. The NGF induction of GSH-Px and catalase mRNA was inhibited by cycloheximide (CHX) treatment with a slight decrease in their mRNA levels due to prolonged exposure to CHX. When the CHX treatment was delayed relative to the NGF treatment there was no effect on NGF effects on catalase and GSH-Px. The GSH-Px gene has conserved sequences in the open reading frame and 3′ untranslated region which forms a stem-loop structure necessary for the incorporation of Se into this selenoprotein. While Se is important in stabilizing GSH-Px transcripts, it did not affect transcription rates or mRNA stability. These results are consistent with the hypothesis that NGF regulates catalase and GSH-Px expression via a primary effect on transcription factor pathways.

Delayed cell death signaling in traumatized central nervous system: Hypoxia

There are two different ways for cells to die: necrosis and apoptosis. Cell death has traditionally been described as necrotic or apoptotic based on morphological criteria. There are controversy about the respective roles of apoptosis and necrosis in cell death resulting from trauma to the central nervous system (CNS). An evaluation of work published since 1997 in which electron microscopy was applied to ascertain the role of apoptosis and necrosis in: spinal cord injury, stroke, and hypoxia/ischemia (H/I) showed evidence for necrosis and apoptosis based on DNA degradation, presence of histones in cytoplasm, and morphological evidence in spinal cord. In the aftermath of stroke, many of the biochemical markers for apoptosis were present but the morphological determinations suggested that necrosis is the major source of post-traumatic cell death. This was not the case in H/I where both biochemical assays and the morphological studies gave more consistent results in a manner similar to the spinal cord injury studies. After H/I, major factors affecting cell death outcomes are DNA damage and repair processes, expression of bcl-like gene products and inflammation-triggered cytokine production.

Proteomic study of amyloid beta (25–35) peptide exposure to neuronal cells: Impact on APE1/Ref-1's protein–protein interaction

The genotoxic, extracellular accumulation of amyloid β (Aβ) protein and subsequent neuronal cell death are associated with Alzheimers disease (AD). APE1/Ref‐1, the predominant apurinic/apyrimidinic (AP) endonuclease and essential in eukaryotic cells, plays a central role in the base excision repair (BER) pathway for repairing oxidized and alkylated bases and single‐strand breaks (SSBs) in DNA. APE1/Ref‐1 is also involved in the redox activation of several trans‐acting factors (TFs) in various cell types, but little is known about its role in neuronal functions. There is emerging evidence for APE1/Ref‐1s role in neuronal cells vulnerable in AD and other neurodegenerative disorders, as reflected in its nuclear accumulation in AD brains. An increase in APE1/Ref‐1 has been shown to enhance neuronal survival after oxidative stress. To address whether APE1/Ref‐1 level or its association with other proteins is responsible for this protective effect, we used 2‐D proteomic analyses and identified cytoskeleton elements (i.e., tropomodulin 3, tropomyosin alpha‐3 chain), enzymes involved in energy metabolism (i.e., pyruvate kinase M2, N‐acetyl transferase, sulfotransferase 1c), proteins involved in stress response (i.e., leucine‐rich and death domain, anti‐NGF30), and heterogeneous nuclear ribonucleoprotien‐H (hnRNP‐H) as being associated with APE1/Ref‐1 in Aβ(25–35)‐treated rat pheochromocytoma PC12 and human neuroblastoma SH‐SY5Y cell lines, two common neuronal precursor lines used in Aβ neurotoxicity studies. Because the levels of some of these proteins are affected in the brains of AD patients, our study suggests a neuroprotective role for APE1/Ref‐1 via its association with those proteins and modulating their cellular functions during Aβ‐mediated neurotoxicity.

Nerve Growth Factor and Oxidative Stress in the Nervous System

Nerve growth factor is a target-derived neurotrophic factor acting on sympathetic and neural crest-derived sensory neurons in the peripheral nervous system (PNS) and some populations of cholinergic neurons in the central nervous system (CNS; Levi-Montalcini and Hamburger, 1951; 1953; Levi-Montalcini, 1987; Hefti and Weiner, 1986; Thoenen and Barde, 1980). Nerve growth factor isolated from mouse submaxillary glands has a sedimentation coefficient of 7S which lacks biological activity (Varon et al.,1972; Stach and Shooter, 1980) and is made up of two alphas, one beta, and two gamma subunits (α2βγ2). The biologically active form is the β-subunit, a homodimer made up of two identical polypeptides. Each chain contains three intrachain disulfide bonds which are crucial for biological activity since the reduction of these bonds abolishes biological activity (Greene and Shooter, 1980; Perez-Polo et al., 1990; Fahnestock, 1991). There are significant homologies in the amino acid sequence of the α -and γ-subunits (Greene et al.,1969; Thomas et al., 1981; Evans and Richards, 1985). The γ-subunit of 7S NGF (γ-NGF) is an arginine- or lysine-specific trypsin-like serine proteinase in the kallikrein gene family (Thomas et al., 1981; Evans and Richards, 1985; Evans et al., 1987). The γ-NGF has been postulated to function during the processing of the β-NGF precursor and may participate in cellular migration or tissue remodeling. The γ-NGF can also cleave recombinant single chain urokinase-type plasminogen activators which might be involved in cellular migration by activating a proteinase cascade (Wolf et al.,1993).The α-subunit also belongs to the kallikrein gene family (Evans and Richards, 1985). Although α-NGF may protect β-NGF from proteolytic degradation or inhibit NGF biological activity via formation of the 7S complex, a definitive biological function for the α-subunit has not been established.