Search

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappaB (NF-kappaB) transcription factor. Bcl-X(L) is a potent regulator of apoptosis in the CNS and is highly expressed in the developing and adult brain. We identified three putative NF-kappaB DNA binding sequences clustered upstream of the brain-specific transcription start site in the upstream promoter region. Recombinant p50/p50 and NF-kappaB proteins from nuclear extracts bound to these sites as determined by electrophoretic mobility shift assay and biotin-oligonucleotide/streptavidin affinity assays. NF-kappaB overexpression, coupled with bcl-x promoter/reporter assays using a series of murine bcl-x promoter and deletion mutants, has identified the downstream 1.1 kb of the bcl-x promoter as necessary for basal promoter activity and induction by NF-kappaB. The mutagenic removal of NF-kappaB binding sites individually or in combination revealed altered response patterns to p49/p65 and p50/p65 overexpression. These results support the hypothesis that NF-kappaB can act to enhance Bcl-X(L) expression via highly selective interactions, where NF-kappaB binding and bcl-x promoter activation are dependent on both DNA binding site sequence and NF-kappaB subunit composition. Our data suggest that molecular events associated with NF-kappaB promote regulation of neuronal apoptosis in the developing or injured CNS.

A key component of the cognitive deficits associated with aging is the loss of function of cholinergic neurons in the basal forebrain due to neuronal losses and decreased cholinergic function of spared neurons. A model to mimic one aspect of this phenomenon is to kill cholinergic neurons selectively in the basal forebrain via administration of the immunotoxin IgG-192-saporin. Here we discuss apoptotic regulators, such as nerve growth factor, in age-associated changes present in the cholinergic system and the role of the NF-kappaB signaling system in cellular commitment to apoptosis. We also examine the age-associated decline in intrinsic response mechanisms, which may account for the age-associated reduction in recovery from both acute and chronic insults to the central nervous system.

There are age-associated cognitive and cholinergic deficits in the neurotrophin-dependent cholinergic basal forebrain neurons (CBFNs). There are also increases in the activity of the transcription factor NF-kappaB in the aged rodent brain that may reflect chronic enhancement of stress response signaling. We used partial immunolesions (PIL) to CBFN to examine the role of endogenous NGF on choline acetyltransferase (ChAT) activity and NGF-mediated NF-kappaB alteration after cholinergic deafferentation. We injected 192 IgG-saporin, an immunotoxin selectively taken up by neurotrophin receptor p75(NTR)-bearing neurons, into lateral ventricles, followed by infusions of anti-NGF to assess NF-kappaB, ChAT and NGF responses to PIL after anti-NGF infusion. Treatment with anti-NGF decreased ChAT activity by 17-34% in the cortex, hippocampus, and olfactory bulb and PIL decreased ChAT activity by 47-73%. Changes in AChE activity levels paralleled those observed for ChAT after PIL. NGF protein levels in the olfactory bulb, but not the cortex or hippocampus, increased significantly after PIL treatment. Infusion of anti-NGF abolished the PIL-induced eight-fold NGF increase in CNS. NF-kappaB binding activity to the IgG-kappaB and ChAT specific NF-kappaB consensus sequences, increased in the cortex but not hippocampus after PIL followed by anti-NGF infusion. It is likely that immunolesion-induced changes in ambient NGF levels may perturb NF-kappaB activity.

Conditions associated with systemic infection, such as endotoxinemia, are known to increase the levels of pro-inflammatory cytokines such as interleukin (IL)-1 in the central nervous system. Systemic infection has been shown to be a common preexisting condition in patients with stroke. To examine a possible consequence of systemic infection, we used a novel electrochemical technique, which combines measurement of cerebral blood flow with measurement of superoxide anion concentrations, to examine the effect of pretreatment of pial vasculature with a proinflammatory cytokine, IL-1 beta, on cerebral blood flow and superoxide anion concentration in a rat model of middle cerebral artery occlusion and reperfusion. In addition, neutrophil recruitment was measured using an immunohistochemical technique. Our results indicate that exposure of pial and cerebral vasculature to IL-1 beta significantly accelerates recruitment of neutrophils, reduces cerebral blood flow, and increases superoxide anion concentration at the pial surface during reperfusion. These results support the idea that prior exposure of brain vasculature to IL-1 beta results in acceleration of cerebrovascular injury by accelerating recruitment of neutrophils, which secrete superoxide anion, during reperfusion. This finding has possible implications for the treatment of stroke with reperfusion agents in patients with preexisting infections., (Copyright 2000 Wiley-Liss, Inc.)

Oxidative stress has been linked to neuronal cell death resulting from either acute insults due to ischemia, trauma, excitotoxicity, or chronic neurodegenerative diseases. Cholinergic basal forebrain neurons (CBFNs) compete for nerve growth factor (NGF) synthesized in the hippocampus and cortex via retrograde transport. NGF affects CBFN survival and cholinergic function via activation of the NF-kappaB transcription factor and this signaling pathway appears to be impaired in aged rats. Here, we demonstrate that activation of NF-kappaB in basal forebrain primary culture via treatment with hydrogen peroxide or TNF-alpha is predominantly restricted to CBFNs, and that NF-kappaB activation appears to mostly affect p65 translocation to the nucleus, but not the p50 subunit. These results are consistent with NF-kappaB activation being a part of recovery processes after acute oxidative stress. Since p50 or p49 (also called p52) binding to promoter sites does not stimulate transcription - both p50 and p49 lack an activating domain - and p65 does contain an activating domain and thus can act as a transcription enhancer, differential translocation of different NF-kappaB dimers can act as repressors of constitutive activity or enhancers. These results are in agreement with the hypothesis that p50/p65 is the active trans-activating species of NF-kappaB, as compared to p50/p50 homodimers which bind to NF-kappaB binding sites but do not trans-activate promoters. Our results also suggest that selective activation of different NF-kappaB dimer species may have regulatory significance in neuronal responses to acute or chronic insults to CNS. Thus, increased p65 translocation could have enhancing effects while increased p50 translocation could have a repressor role. Manipulation of the types of NF-kappaB species being translocated could provide a basis for therapeutic strategies.

Cholinergic basal forebrain neurons (CBFNs) retrogradely transport neurotrophins released in the hippocampus and cortex as part of a general response to injury in a process that is impaired in the aged rodent and can be spared by the exogenous addition of pharmacological doses of nerve growth factor (NGF). This observation suggests that components of stress response signal transduction pathways in the aged CNS can be exogenously activated. The extent and mechanism of the endogenous stimulation of NGF in response to injury can be mimicked via treatment with 192 IgG-saporin of rat CNS, an immunolesion model. Here we report on the use of a conditioning lesion paradigm to determine if repeated partial immunolesions have a conditioning effect on the immunolesion-induced increases in NGF protein or decreases in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity. We report that chronic repeated immunolesions, as used here, were not as effective as a one time equivalent immunolesion in terms of induced NGF protein increases or decreasing ChAT and AChE activity in the hippocampus and cortex. Thus, chronic lesions resulting in cholinergic impairment typical of the aged CNS may differ from acute toxic models as a result of desensitization due to a conditioning effect of chronic subthreshold lesioning events in the CNS.

Gene therapy using cationic liposomes containing cDNA is a relatively new approach with great potential; however, little is known about the mechanisms of dermal gene transfer, its biodistribution, systemic transfection, and cellular uptake. This study identifies mechanisms, transfection rates, and biodistribution of liposomal gene transfers in the skin of thermally injured rats using cDNA gene constructs coding for insulin-like growth factor-I (IGF-I) and Lac Z. Male Sprague-Dawley rats (350 to 375 g) were given a 60% total body surface area full-thickness scald burn that was followed by weekly subcutaneous injections of normal saline (control, n = 10), liposomes plus 0.2 microg Lac Z cDNA construct driven by a cytomegalovirus (CMV) promoter (vehicle, n = 10), or liposomes containing 2.2 microg cDNA coding for IGF-I plus 0.2 microg Lac Z cDNA construct driven by a CMV promoter (IGF-I cDNA, n = 10). Gene transfection was determined by histochemical and luminescent beta-galactosidase assays of blood, skin, liver, spleen, and kidney. Transcription of IGF-I cDNA to IGF-I mRNA was determined in skin cells by Northern blot analyses. Levels of IGF-I protein in blood, skin, liver, spleen, and kidney were measured by radioimmunoassay. The biological activity of the translated IGF-I was evaluated by the mitogenic activity in dermal cells and the rate of re-epithelization. Gene transfection was observed only in skin cells. The expression of IGF-I mRNA increased in skin cells of burned rats receiving liposomes containing the IGF-I cDNA construct compared with liposomes without the construct or normal saline. IGF-I protein levels in the skin of rats receiving the IGF-I cDNA was 176 +/- 4 ng/ml compared with 105 +/- 6 ng/ml for liposomes alone or 90 +/-3 ng/ml for saline (p < 0.05). The translated IGF-I protein was found biologically active in the skin by increasing skin cell proliferation and accelerating re-epithelization 33 days after thermal injury (p < 0.05). No systemic transfection could be detected. Skin cells transfected with liposomes encapsulating the IGF-I cDNA constructs increased the expression of IGF-I mRNA transcript and the expression of a biologically active IGF-I protein. Liposomes containing the cDNA coding for IGF-I present an effective approach to gene therapy in the skin.

Rat pheochromocytoma (PC12) cells exhibit apoptotic cell death when deprived of serum and can be rescued by nerve growth factor (NGF). We characterized AP-1 DNA binding activity in PC12 cells after serum deprivation in the presence or absence of NGF or other neurotrophic agents. There was a decline in AP-1 DNA binding activity concomitant with apoptosis in PC12 cells after serum deprivation. Treatment of serum-deprived PC12 with NGF induced persistent AP-1 binding activity that was blocked by the Trk receptor inhibitor K252a. PC12 cells treated with dibutyryl cyclic AMP or insulin also displayed increased AP-1 DNA binding activity. While NGF somewhat increased c-Fos and c-Jun protein levels transiently, it had a more robust and persistent stimulatory effect on Jun B protein levels. AP-1 transcriptional activity increased after NGF, dibutyryl cAMP, or insulin treatment under serum free conditions. Curcumin, which inhibits AP-1 activity, blocked the NGF-mediated rescue. These results would suggest that the rescue of serum-deprived PC12 cells from apoptosis requires increasing endogenous levels of specific Fos/Jun components of AP-1.

In humans, cerebral hypoxia is a common component of severe brain insults, including trauma, stroke, and perinatal asphyxia. Oxidative stress and free radicals incidental to cerebral hypoxia are implicated in damaging macromolecules, leading to collapse of cellular homeostasis and cell death. Neuronal DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Here, we measured hypoxia-induced damage and repair in nuclear and mitochondrial DNA in rat hippocampus and cortex. Two highly sensitive quantitative polymerase chain reaction (QPCR) assays were used to measure DNA damage. One assay measures the integrity of the entire mitochondrial genome and the other the integrity of nuclear DNA. The latter is a novel assay, developed in our laboratory, which utilizes the high copy number of short interspersed DNA elements (SINEs) residing in introns and untranslated regions of mammalian genes. A unique feature of the SINE-mediated QPCR is its ability to amplify simultaneously long random segments of DNA. Consequently, the SINE assay offers sufficient sensitivity for detecting DNA damage at levels that are compatible with the cellular capacity for DNA repair, and are likely to be consistent with cellular survival and therefore adequate for studying the DNA damage response in the brain. In rats, we found that exposure to an atmosphere of 4% oxygen for 30 min resulted in induction of DNA damage in nuclear and to a greater extent, in mitochondrial DNA. Following a 3-hr recovery period in ambient air, dissimilar repair kinetics for nuclear and mitochondrial DNA were measured., (Copyright 1999 Wiley-Liss, Inc.)

Hypothesis: Cholesterol-containing cationic liposomes alone modulate the acute-phase response and cytokine expression in thermally injured rats and are an effective delivery system for gene therapy in trauma., Setting: Laboratory., Intervention: Fifty-six adult male Sprague-Dawley rats with a full-thickness scald burn covering 60% of total body surface area were randomly divided into 2 groups to receive either intravenous injections of cholesterol-containing cationic liposomes or saline (control)., Main Outcome Measures: Body weights, muscle and liver dry-wet weights, serum levels of constitutive hepatic proteins, acute-phase protein levels, and cytokine levels were determined at 1, 2, 5, and 7 days after thermal injury., Results: Rats receiving cholesterol-containing cationic liposomes had less body weight loss, increased serum transferrin levels, and decreased serum alpha1-acid glycoprotein levels when compared with controls (P<.05). Serum interleukin 1beta and tumor necrosis factor alpha levels were decreased in rats receiving liposomes at 1 and 2 days after burn compared with controls (P<.05)., Conclusions: These results suggest that cholesterol-containing cationic liposomes alone may have a beneficial effect in modulating the hypermetabolic response after burn injury by decreasing type 1 acute-phase proteins and the expression of the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha. Therefore, cholesterol-containing cationic liposomes appear to be suitable as a delivery system for gene therapy in trauma.

Hypothesis: Multiple subcutaneous injections of cholesterol-containing cationic liposomes encapsulating the complementary DNA (cDNA) gene for insulinlike growth factor I (IGF-I) increase the rate of transfected skin cells and result in increased IGF-I protein levels in the skin with subsequent improvement in wound healing when compared with a single injection., Setting: Laboratory., Intervention: Twenty-four adult male Sprague-Dawley rats (350-375 g) received a full-thickness scald burn on 60% of their body surface. These rats were randomly divided to receive either 1 injection of liposomes containing 2.2 microg-cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct, or 2 injections of liposomes containing 2.2 microg cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct., Main Outcome Measures: Transfection rates and IGF-I protein levels in the skin and physiological responses to the IGF-I gene therapy, evaluated from changes in body weight, protein content in serum and liver, and the rate of burn wound healing., Results: There was a significant decrease in transfection rate and IGF-I protein expression distal from the injection site in animals receiving 1 injection, as compared with a consistent increase in rats receiving multiple injections. Multiple injections improved the response to thermal trauma by increasing the extent of the healed burn wound 33 days after thermal injury (single injection, 31% +/- 1% vs multiple injections, 38% +/- 2%), total serum protein (single injection, 52 +/- 0.5 g/L vs multiple injections, 55 +/- 0.6 g/L), and total liver protein (single injection, 82.0 +/- 0.3 mg/mL vs multiple injections, 91.0 +/- 3.8 mg/mL), P<.05., Conclusions: Gene transfer rates can be increased by multiple injections of liposomes encapsulating IGF-I cDNA constructs. Increased transfer results in greater IGF-I protein skin concentrations, accelerated wound healing, and increased serum and liver protein concentrations. The clinical relevance of these findings is that liposomal gene constructs should be applied in well-defined distances to improve gene transfer in the skin, and thus clinical outcome after thermal injury.

We have shown recently in the pheochromocytoma PC-12 cell line, that the activation of the high-affinity receptor for nerve growth factor (NGF), tyrosine kinase receptor (TrkA), results in increased secretion of the amyloid precursor protein (APP) into the culture medium. In order to reveal through which TrkA-associated signaling pathway the secretory APP processing is mediated, signaling cascades activated by TrkA stimulation were selectively inhibited under conditions of selective TrkA stimulation via non-NGF mechanisms and APP secretion into the culture medium was followed by Western analysis. Our data demonstrate, that activation of mitogen activated protein (MAP) kinase alone is sufficient to promote APP secretion, whereas inhibition of MAP kinase will reduce APP secretion only when phospholipase Cgamma or phosphatidylinositol 3-kinase are additionally inhibited. This suggests that pharmacological manipulations activating the MAP kinase pathway may result in increased secretory APP processing.

Inflammation and the associated release of inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) may be a component of neurodegenerative diseases associated with aging or chronic HIV-1 infection. Most of the neurons that are affected under these conditions require a constant supply of trophic factors such as nerve growth factor (NGF) for survival. NGF acts via binding to a specific tyrosine kinase receptor (TrkA). NGF also binds to the common neurotrophin receptor (p75(NTR)), a member of the TNFalpha receptor (TNFR-I) superfamily, whose function may be to modulate apoptosis via the release of ceramide and the activation of the transcription factor nuclear factor kappa B (NFkappaB). The similarity between p75(NTR) and TNFR-I signal transduction pathways suggests that one of the mechanisms by which TNFalpha affects neuronal survival is by impacting upon these pathways that normally promote NGF support of neurons. Here we show that arachidonic acid (AA), a signaling lipid potentially associated with TNFR-I signal cascade, induces apoptosis in PC12 cells through inhibition of both protein kinase C zeta (PKCzeta) and NFkappaB activity. We also show that apoptosis induced by AA cannot be prevented by NGF. These data support the idea that PKCzeta and NFkappaB are both essential signaling elements for mediating NGF-promoted rescue from apoptosis. Our results also suggest that AA, an inflammatory signal lipid induced by TNFalpha via binding to TNFR-I, may reduce neuronal survival by inhibiting elements of the signal cascade induced by NGF., (Copyright 1999 Wiley-Liss, Inc.)

Nitric Oxide (NO) mediates a series of physiological processes including regulation of vascular tone, macrophage-mediated cytotoxicity, platelet aggregation, learning and long-term potentiation, neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in the pathophysiology of focal cerebral ischemia. In the present study NOS activities were evaluated in cerebellum and cerebral cortex of ischemic and post-ischemic reperfused rats using an experimental model of partial cerebral ischemia; moreover, the effects of L-N(G)Nitroarginine (NA, nonselective NOS inhibitor) or 7-Nitroindazole (7-NI, selective neuronal NOS inhibitor) administration were assayed on percentage survival of ischemic rats. An increase of NOS activity in the cerebellum and in cerebral cortex of ischemic and post-ischemic reperfused rats was observed. NA administration failed to induce neuroprotective effects, by increasing percentage of mortality of treated ischemic rats with respect to control group. In contrast, the treatment with the selective neuronal NOS inhibitor, 7-NI, induced a significant neuroprotective effect.

Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for beta-galactosidase, liposomes plus cDNA for IGF-I and beta-galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for beta-galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). Local IGF-I protein concentrations in the skin were higher when compared to liposomes containing only the lacZ gene (P < 0.05) Transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. Liposomes containing the IGF-I gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.

We have systematically investigated the therapeutic potential of cationic liposome-mediated neurotrophic gene transfer for treatment of CNS injury. Following determination of optimal transfection conditions, we examined the effects of dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) liposome-mediated NGF cDNA transfection in injured and uninjured primary septo-hippocampal cell cultures and rat brains. In in vitro studies, we detected an increase of NGF mRNA in cultures 1 day after transfection. Subsequent ELISA and PC12 cell biological assays confirmed that cultured cells secreted soluble active NGF into the media from day 2 after gene transfection. Further experiments showed that such NGF gene transfection reduced the loss of chol- ine acetyltransferase (ChAT) activity in cultures following calcium-dependent depolarization injury. In in vivo studies, following intraventricular injections of NGF cDNA complexed with DC-Chol liposomes, ELISA detected nine- to 12-fold increases of NGF in rat CSF. Further studies showed that liposome/NGF cDNA complexes could attenuate the loss of cholinergic neuronal immunostaining in the rat septum after traumatic brain injury (TBI). Since deficits in cholinergic neurotransmission are a major consequence of TBI, our studies demonstrate for the first time that DC-Chol liposome-mediated NGF gene transfection may have therapeutic potential for treatment of brain injury.

Ischemic insults to the brain result in a time-dependent increase in neuronal death that is responsible for some of the functional deficits associated with stroke. Our working hypothesis is that ischemia results in a prompt depletion of high energy phosphate species resulting in decreased pH and glutathione levels in brain in a temporal and spatial pattern that disrupts nerve growth factor homeostasis and increases neuronal apoptosis. Here we show hemispheric depletion of active phosphate species after ischemia. Also, we observed that the striatum is an early target for oxidative stress that is followed by energy metabolic impairment and altered neurotrophin levels that were detected by noninvasive magnetic resonance imaging (MRI) measurements of cytotoxicity and conventional biochemical determinations of apoptosis, glutathione, and nerve growth factor (NGF) protein levels in a pattern distinct from that observed in the hippocampus. Furthermore, early assessment of intracellular pH by 31P-magnetic resonance spectroscopy (31P-MRS) was a predictor of later infarct development as determined by MRI. We also show that pretreatment with pharmacological doses of NGF did not have overall significant beneficial consequences on irreversible ischemia in an intraluminal unilateral irreversible model of stroke in rat brain.

Cerebral ischemia and the aftermath of reperfusion form a hypoxic/hyperoxic sequence of events that can trigger oxidative stress response cascades in neurons of the central nervous system. After transient ischemia there is an increase in intracellular Ca2+ release, extracellular glutamate, reactive oxygen species (ROS) and nitric oxide, genotoxic events that stimulate DNA repair. Increased oxidative stress and interrupted blood flow in ischemia, like DNA repair, also deplete cellular ATP and commit neurons to apoptosis. We report that levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) decreased significantly in the hippocampus but not other brain areas after 6 h of reperfusion following an induced ischemic insult. This specific inhibition of APE/Ref-1 expression may affect the extent of apoptosis after ischemia.

Chronic oxidative stress has been hypothesized to be a major contributor to the aging process. The continued exposure to reactive oxygen species (ROS) generated by oxidative metabolism or environmental sources can damage critical cellular structures and be responsible for some age-related pathology. The exposure of rodents to 100% oxygen, isobaric hyperoxia, increases ambient ROS levels and significantly increases apoptosis in brain. The deleterious effects of ROS also include increased lipid peroxidation, protein oxidation, and DNA damage. Although differences in the relative amounts of oxidative stress in young and old brains have been observed, the mechanisms responsible for impaired aging-associated DNA repair processes have not been characterized. We measured DNA levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) protein by Western blot analysis in the brains of young (3-month) and old (30-month) male rats exposed to isobaric hyperoxia. Given that APE/Ref-1 is the rate-limiting enzyme in the repair pathway of apurinic/apyrimidinic sites generated in DNA by oxidative damage, we assumed that APE/Ref-1 protein levels were a good reflection of ongoing DNA base excision repair. Isobaric hyperoxia stimulated APE/Ref-1 expression in the hippocampus and basal forebrain of young rats experiencing 100% oxygen for 6 hr, while aged rats showed no significant changes in APE/Ref-1 protein levels in all brain areas at any time tested (0-48 hr) after hyperoxia. Differences in the stress-induced levels of expression of DNA repair enzymes may contribute to apoptotic increases and pathology associated with the aging process.

The increased expression and/or abnormal processing of the amyloid precursor protein (APP) is associated with the formation of amyloid plaques and cerebrovascular amyloid deposits, which are one of the major morphological hallmarks of Alzheimer's disease (AD). Among the processes regulating APP metabolism, the proteolytic cleavage of APP into amyloidogenic or nonamyloidogenic fragments is of special interest. The cleavage of the APP by the alpha-secretase within the beta-amyloid sequence generates nonamyloidogenic C-terminal APP fragments and soluble APPs alpha, which has neurotrophic and neuroprotective activities. Proteolytic processing of APP by beta-secretase, on the other hand, exposes the N-terminus of beta-amyloid, which is liberated after gamma-secretase cleavage at the variable amyloid C-terminus. The resulting 39-43 amino acid beta-amyloid may be neurotoxic and disrupt neuronal connectivity after its accumulation in senile plaques. In this review, we discuss evidence derived from in vitro experiments, suggesting that the stimulation of protein kinase C (PKC)-coupled M1/M3 muscarinic acetylcholine receptors increases the nonamyloidogenic, secretory pathway of APP processing. It has also been shown in animal models that under conditions of reduced M1/M3 muscarinic acetylcholine receptor stimulation the secretory pathway of APP processing is inhibited and that constitutive upregulation of M1/M3-associated PKC increases APP secretion. Thus, the cortical cholinergic hypoactivity characteristic of AD may inhibit the nonamyloidogenic APP processing pathway and lead to increased beta-amyloid generation. It has been shown in vitro that nerve growth factor (NGF)-associated signaling also influences the expression and catabolism of APP. Recent experiments with NGF-responsive cells revealed a specific role for the high-affinity NGF receptor, TrkA, in the increases in secretory APP processing and a role for the low-affinity neurotrophin receptor, p75NTR, in the transcriptional regulation of APP. Therefore, treatments with NGF could ameliorate cortical cholinergic dysfunction in AD. These findings may influence the design of therapeutic strategies aimed at stimulating cholinergic function and at increasing nonamyloidogenic APP processing without elevating APP expression.

Apoptosis in the central nervous system (in contrast to necrosis) is an endogenous cell suicide mechanism triggered in response to biological factors and genotoxic stimuli often resulting from oxidative stress. Excessive neural apoptosis may result in longterm brain dysfunction. A significant proportion of prematurely born infants are exposed to high oxygen and nutritional regimens deficient in antioxidant precursors. Such infants frequently display cognitive deficits when studied in later childhood. Studies in cell culture have characterized a close relationship between oxidative stress, glutathione availability and cell death. Here, we assessed this relationship in rat brain, as a model approximation of the situation that occurs in human infants. Two day old rats were exposed to an atmosphere of 95% oxygen and treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. Control groups consisted of rat-pups kept in air, air plus BSO, or oxygen alone. At the end of 5 days of treatment, brains were harvested, dissected and nerve growth factor protein (NGF), glutathione, and extent of apoptosis were measured. Hyperoxia induced a decrease in NGF protein while BSO induced a decrease in glutathione concentrations. Animals treated with both hyperoxia and BSO had a dramatic increase in the extent of brain apoptosis detected. We conclude from these studies that the brains of animals exposed to both oxidative stress and limited antioxidant protection are liable to pro-apoptotic changes. Increased cell death via apoptosis reflecting changes in neurotrophin and glutathione homeostasis may represent the mechanism responsible for the induction of the longterm cognitive deficits observed in some preterm infants.

In the present study, we compare the effects of cholinergic deafferentation of the hippocampus, cortex, and olfactory bulb of young and aged rats on nerve growth factor (NGF) protein levels in these areas. We also describe glial responses to intraventricular injections of the immunotoxin, 192 IgG-saporin in the aged. Choline acetyltransferase (ChAT) activity was dramatically decreased in the basal forebrain and target areas of the cholinergic basal forebrain neurons (CBFNs) in the young immunolesioned rats and to a lesser extent in their aged counterparts. After total immunolesion, NGF protein levels significantly increased in the hippocampus, cortex, and olfactory bulb of the young rats but not of the aged rats, except for small increases in the olfactory bulb after two weeks. After immunolesion NGF protein levels in the basal forebrain increased in young rats and less so in the aged rats. The total immunolesions had no effects on NGF and BDNF mRNA levels in the hippocampus and cortex. Two weeks after injection of the immunotoxin, the profiles of AChE- and p75NTR-positive cells significantly decreased in medial septum, vertical and horizontal limbs of diagonal band and nucleus basalis of Meynert. There was also an increase in microglia while but not astrocytes in the subnuclei of basal forebrain. In conclusion, 192 IgG-saporin was effective in producing cholinergic lesions in both young and aged rat brains, the lesion-induced NGF response was partially extinguished in the aged rat brains and immunolesions induced a microglial response in aged brain.

It has been hypothesized that the main olfactory bulb, with its relatively rich source of neurotrophins, may provide trophic support for neurons that project to the bulb. We monitored expression of the common, low affinity receptor for neurotrophins, p75NGFR, in the olfactory bulb and basal forebrain of unilaterally bulbectomized and sham-treated rats, 1-16 weeks post-surgery, using the monoclonal antibody MAb192. An induction of p75NGFR-immunoreactivity was observed in both the glomerular and olfactory nerve layers of the right, contralateral main olfactory bulb of lesioned animals. The naturally occurring regeneration taking place in the olfactory neuroepithelium is known to be altered by olfactory bulbectomy, with subsequent changes in the sensory input to the remaining bulb. These changes in expression of p75NGFR in the olfactory bulb support the hypothesis we have developed in previous papers, that changes in the extent of the peripheral input from the olfactory neuroepithelium to the main olfactory bulb regulate p75NGFR expression in both the glomerular and the olfactory nerve layers. Expression of p75NGFR in the basal forebrain of bulbectomized animals was found to be no different than sham-treated controls and does not support the hypothesis that the olfactory bulb provides trophic support to this region of the central nervous system.

There are deficits in cholinergic basal forebrain neurons (CBFNs) in the aged brain and patients suffering Alzheimer's disease associated with a partial loss of the CBFNs. To mimic this partial loss and assess its long term effects on residual cholinergic activity and resultant target-derived nerve growth factor (NGF) levels, we produced a partial immunolesion to CBFNs with 192 IgG-saporin, an immunotoxin selectively taken up by p75NTR-bearing neurons. We measured two cholinergic markers, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and NGF protein levels at 10 days, 1, 6 and 12 months postlesion. There were no significant changes in the cholinergic markers and the NGF protein levels in the sham-treated animal controls during the one year experiment. Ten days after 192 IgG-saporin treatment, ChAT activity decreased to 35-50% of controls in the olfactory bulb, hippocampus, and cortex. There was a minor but significant recovery of ChAT activity one year after the immunolesion in the hippocampus. Changes in AChE activity mirrored the ChAT changes but were less robust. There were transient increases in NGF protein levels in the hippocampus and cortex that returned to basal levels at 6 months and 12 months postlesion, respectively. In summary, partial immunolesions resulted in partial region-specific and time-dependent recoveries of cholinergic activity in the target areas of the basal forebrain after a partial elimination of CBFNs and a return to basal levels of NGF protein consistent with the hypothesis that the remaining CBFNs compensated for losses of ChAT and NGF due to changes in cholinergic innervation of basal forebrain target areas., (Copyright 1998 Elsevier Science B.V.)

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 kappaB.

The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.

The present paper reports the effects of GSH depletion (diethylmaleate induced) on partial cerebral ischemia and reperfusion for 7 and 20 days. Our results confirm that there is a paradoxical protective effect of the GSH-depletor and suggest an improved neuronal trophism induced by diethylmaleate treatment.

The cholinergic neurons of the basal forebrain (CNBF) are the major source of cholinergic innervation of the cortex and hippocampus. In Alzheimer's disease and aged brain, there are severe losses of cholinergic neurons in the nucleus basalis of Meynert, leading to a reduction of cortical cholinergic activity which correlates with the severity of cognitive deficits. While there is evidence that aged central nervous system (CNS) displays impaired stress response signaling, pharmacologic treatments with neurotrophic factors appear to ameliorate these age-associated cholinergic deficits. To mimic these cholinergic deficits in experimental animals and study the acute effects of nerve growth factor (NGF), we induced a partial lesion of CBFNs by the intracerebroventricular (i.c.v.) injection of the cholinergic immunotoxin 192IgG-saporin, in groups of 3- and 30-month-old rats. The lesion was followed 14 days later by i.c.v. administration of NGF, known to restore partial immunolesion-induced cholinergic deficits in rat CNS, and all rats were killed 2 days after the NGF treatment. Here we report the effects of partial immunolesions on the levels of choline acetyltransferase (ChAT) activity and NGF receptor mRNA levels in the basal forebrain of 3- and 30-month-old rats. Because of their presence in the promoters of the NGF, NGF receptors, and ChAT genes, we also measured DNA-binding activity of the transcription factors NFB and AP-1 in the cortex and hippocampus. We discuss these findings in the context of endogenous NGF-mediated signal transduction mechanisms and conclude that we have evidence for age-associated decreases in endogenous NGF responses to partial deafferentation of the basal forebrain cholinergic projections.

Brain-derived neurotrophic factor (BDNF) has therapeutic potential for treatment of the injured central nervous system. BDNF induces both differentiation and survival of neurons by binding to trkB receptors. This interaction stimulates the intrinsic tyrosine kinase activity of trkB, initiating a signal cascade involving the phosphorylation of intracellular protein on tyrosine, serine, and threonine residues. The purpose of this investigation was to examine the effects of cationic lipid-mediated gene transfection of BDNF on phosphatidylinositol 3 (PI3)-kinase activity in primary septo-hippocampal cell cultures. Thirty-six hours after BDNF gene transfection in the primary CNS cell culture, PI3-kinase activity was significantly increased. The increased PI3-kinase activity was inhibited by wortmannin, a selective and irreversible inhibitor of PI3-kinase. In addition, wortmannin blocked neurofilament increases induced by BDNF gene transfection. This result suggests a possible role of PI3-kinase activation in neuroprotective effects produced by BDNF gene transfection.

Ageing of the central nervous system (CNS) is characterized by a progressive apoptotic loss of neurons that may be in part due to impaired neurotrophin signaling mediated by such elements as the transcription factor nuclear factor kappa B (NFkappaB). To support this hypothesis, we inhibited nuclear translocation of NFkappaB in vivo by injecting a proteasome inhibitor (PSI) directly in the CNS lateral ventricle of rats and then measured fragmented DNA in various CNS areas as an index of ongoing apoptosis. Our results show that after PSI injection there was a significant inhibition of NFkappaB activity in vivo that resulted in the appearance of fragmented (apoptotic) DNA in the CNS of rats. These results suggest that alteration of NFkappaB may cause apoptotic cell death in the rat CNS during ageing.

In the present study the pheochromocytoma cell line (PC-12) was used as a model system to determine the role of the two neurotrophin receptors in the regulation of amyloid precursor protein (APP) secretion by nerve growth factor (NGF). To stimulate TrkA and/or p75NTR signaling in PC-12 cells, we used NGF, brain-derived neurotrophic factor (BDNF), and NGF in the presence of an excess of BDNF or the monoclonal antibody 192IgG, to block p75NTR binding to NGF. Our results demonstrate that NGF stimulates APP secretion in a dose dependent fashion with maximum effects at 10 ng/ml, known to saturate high-affinity NGF binding sites. Treatment of PC-12 cells with varying concentrations of BDNF, 1-1,000 ng/ml, did not alter APP secretion, suggesting that binding to p75NTR alone is not sufficient to affect APP secretion. When blocking NGF binding to p75NTR with BDNF or 192IgG, on the other hand, NGF effects on APP secretion were abolished. These findings suggest that in cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on APP secretion. Our data are also consistent with a proposed function of the p75NTR in receptor recruitment and "presentation" of NGF to receptors.

The proteolytic cleavage of the amyloid precursor protein (APP) has been shown to be modulated through specific muscarinic receptor activation in vitro in both transfected cell lines and native brain slices, whereas a demonstration of receptor-mediated control of APP processing under in vivo conditions is still lacking. To simulate alterations in muscarinic receptor stimulation in vivo, we have (i) specifically reduced the cortical cholinergic innervation in rats using partial immunolesions with 192IgG-saporin, and (ii) restored cholinergic function in lesioned rats by transplantation of nerve growth factor producing fibroblasts. While total APP levels in cortical homogenates were unaffected by cholinergic deafferentation, we observed a significant reduction in the abundance of secreted APP and a concomitant increase in membrane-bound APP. These changes were reversed in immunolesioned rats with nerve growth factor-producing fibroblasts. There was a strong positive correlation between the ratio of secreted APP to membrane-bound APP and the activity of choline acetyltransferase and M1 muscarinic acetylcholine receptor density (measured by [3H]pirenzepine binding) in experimental groups. Additionally, we observed a transient decrease in the ratio of cortical APP transcripts containing the Kunitz protease inhibitor domain (APP 770 and APP 751) versus APP 695 in rats with cholinergic hypoactivity. The data presented suggest that cortical APP processing is under basal forebrain cholinergic control, presumably mediated through M1 muscarinic acetylcholine receptors on cholinoceptive cortical target cells.

Nerve growth factor is a well-characterized neurotrophin essential for the development and maintenance of certain central and peripheral neurons. As many neurons affected by aging depend for their survival on a constant supply of neurotrophins, nerve growth factor has been proposed as a possible treatment to prevent aging-associated neurodegeneration. There is evidence that nerve growth factor also plays a role in the immune system and modulates certain aspects of endocrine function. Here we have determined the effects of prolonged peripheral (intraperitoneal) treatment with nerve growth factor on body weight, blood pressure, and serum corticosterone levels in the rat. Our data indicate that intraperitoneally-injected nerve growth factor can affect body weight gain in rats. This effect may not be mediated by nerve growth factor-induced increases in serum corticosterone levels, as exogenous administration of corticosterone did not result in a similar body weight loss. These results show that, as previously reported for intracerebroventricular treatment with nerve growth factor, intraperitoneally-injected nerve growth factor also reduces body weight gain in rats. The data also suggest that exogenous delivery of nerve growth factor as part of therapeutic regimens is likely to have several effects.

It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG-saporin and chronic pharmacological treatment of sham-treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham-treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham-treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion-induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.

Excessive activation of glutamate receptors via the N-methyl-D-aspartate (NMDA) subtype appears to play a role in the sequence of cellular events which lead to irreversible ischemic damage to neurons. Furthermore, NMDA receptor activation induces a stimulation of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine (PA) biosynthesis. In order to better understand the role of PA we have measured ODC activity and the effect of methionine sulfoximine (MSO), a molecule able to stimulate ODC, on a model of transient cerebral ischemia. There was a significant increase in ODC activity in the rat cerebral cortex during post-ischemic reperfusion. The treatment with MSO induced a significant decrease in cerebral glutamine synthetase activity accompanied by a marked increase in ODC activity. In MSO-pretreated rats there was a significant decrease in the survival rate when compared to untreated ischemic rats.

Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H2O2 treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H2O2-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H2O2 but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H2O2 exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.

Cholinergic basal forebrain neurons are the major source of cortical cholinergic innervation. The number of these neurons is regulated by the availability of nerve growth factor (NGF) during development while in adulthood their cholinergic activity is modulated by NGF. In previous studies we have shown that cholinergic immunolesions of basal forebrain neurons increase local immediate early gene expression and NGF synthesis in the regions of degeneration. In this study we identify the cellular source of c-Jun and NGF expression using dual immunolabeling of c-Jun and NGF in combination with neuronal and glial markers. We demonstrate that both c-Jun and NGF are exclusively expressed in reactive astrocytes but not in microglia or in GABAergic basal forebrain neurons. These observations support the hypothesis that reactive astrocytes synthesize neurotrophic substances in vivo in response to neuronal degeneration in the basal forebrain.

Many of the anabolic effects of growth hormone (GH) act through insulin-like growth factor-I (IGF-I). Systemic administration of GH in combination with IGF-I has been shown to stimulate wound reepithelialization, however, it causes hyperglycemia or hypoglycemia, respectively. We hypothesize that very low doses of IGF-I in a liposome form will have the same positive wound-healing effect when administered locally as the higher doses of GH plus IGF-I given systemically. To test this hypothesis, rats were given a 40% TBSA full-thickness scald injury and received either placebo IGF-I (5.0 mg/kg/day, subcutaneously), IGF-I liposome (0.9 microgram/kg/week, subcutaneously), or a combination of GH and IGF-I, or IGF-I liposomes for 8 weeks. GH in combination with IGF-I showed a significant increase in postburn weight. Wound reepithelialization, measured by computerized planimetry as percentage original wound area, was significantly increased in rats receiving GH plus IGF-I, GH plus IGF-I liposomes, and IGF-I liposomes when compared to sham, or IGF-I (p < 0.05). Results indicate that small doses of IGF-I, delivered in the form of liposomes, are equally effective in increasing burn wound reepithelialization as the higher doses of GH plus IGF-I, or GH plus IGF-I liposomes.

In rat brain dynamic susceptibility contrast magnetic resonance (MR) images, vessels visible on the same scan plane as the brain tissue were used to measure the characteristics of the input function of the MR contrast agent gadopentetate dimeglumine. MR images were acquired 30 and 60 minutes after intravenous injections of 3 mg/kg and 15 mg/kg NG-Nitro-L-arginine methyl ester (L-NAME) (n = 9). The time of arrival (TOA) and the mean transit time corrected for TOA of the input function were increased by 3 mg/kg or 15 mg/kg L-NAME. The area of the input function was increased by 15 mg/kg L-NAME. In two animals, similar modifications of the input function induced by 20 mg/kg L-NAME were reversed by infusion of sodium nitroprusside. In two other animals, MABP was increased by phenylephrine to a similar extent as in L-NAME experiments, but did not induce the same modifications of the input function, showing that the action of L-NAME on the input function was not simply caused by an effect on MABP. These results show that the input function can be significantly altered by manipulations widely used in cerebrovascular studies. These input function changes have important implications for calculation of cerebral blood flow.

Age-related cognitive deficits are often associated with loss of cholinergic activity within the neurotrophin-dependent cholinergic neurons that project from the basal forebrain to the hippocampus. The cause of reduced cholinergic function is unknown, but alterations in transcription factor-signaling pathways causing altered gene expression may cause decreased specific tissue function, resulting in loss of cholinergic activity. We measured transcription factor Nuclear Factor kappa B by electrophoretic mobility shift assay and Western analysis in young and aged rat brain tissues and report that basal levels of Nuclear Factor kappa B DNA-binding activity increase in the hippocampus and basal forebrain with age to significantly higher levels at 30 months of age. This age-associated increase in binding activity is associated with increased translocation of p65 to the nucleus. These data show an age-associated alteration in Nuclear Factor kappa B signal transduction pathways that may contribute to age-associated decreases in specific tissue function.

PC12 cells are known to undergo programmed cell death (apoptosis) when they are deprived of serum. Nerve growth factor (NGF) rescues PC12 cells from serum deprivation-induced apoptosis. In the present study, we examined the effects of retinoic acid (RA), a classic morphogen, on apoptosis in PC12 cells after serum deprivation and NGF-mediated rescue. In naive PC12 cells, all trans-RA treatment induced cell death in the presence of NGF. RA also abolished the protective effects of dibutyryl cyclic AMP or insulin under serum-free conditions. The death process was accompanied by nuclear condensation and DNA fragmentation, typical of apoptosis. In addition, RA also increased the extent of apoptosis in PC12 cells after serum deprivation. Cycloheximide, an inhibitor of protein synthesis, did not abolish the effects of RA on serum-deprived PC12 cells. RA also decreased thymidine incorporation and proliferation in NGF-treated PC12 cells. Furthermore, although the total DNA binding activity of the AP-1 transcription factor was not changed after RA treatment, RA decreased a specific AP-1 transcriptional activity. It is surprising that differentiated PC12 cells resisted the toxic effects of RA. These data suggest that RA might function as an endogenous inducer of apoptosis during neural differentiation by a mechanism distinct from that of serum deprivation.

The mechanism(s) underlying nerve growth factor (NGF)-mediated rescue of neurons from apoptosis is poorly understood, although it is well established that the high-affinity NGF receptor (TrkA) plays a pivotal role in mediating NGF effects. The report that the low-affinity NGF receptor (p75NGFR) can induce apoptosis prompted us to analyze the role played by a putative p75NGFR-associated signal-transduction element, the transcription factor nuclear factor kappa B (NFkappaB), in the modulation of apoptosis in PC12 cells. Here, we report that inhibition of NFkappaB function results in apoptosis of rat PC12 cells, a neuroblast-like cell line model of NGF-responsive neural tissues. Furthermore, NGF did not protect PC12 cells from cell death induced by the inhibition of NFkappaB. These results indicate that NFkappaB function is essential to maintain PC12 cell survival and to permit NGF-mediated rescue, consistent with the idea that signaling elements potentially associated with both TrkA- and p75NGFR are involved in the regulation of apoptosis.

Plasticity in the central nervous system after cerebral ischemia is a controversial issue; focal cerebral ischemia produces an area of infarction that is surrounded by neurons that may respond to nearby damage by creating new synapses. In the present study the expression of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein, synaptophysin, was investigated by immunocytochemical techniques in the CA1 sector of hippocampus and in cerebellum of rats made ischemic by bilateral clamping of common carotid arteries and reperfused for 7 and 30 days. In addition, ischemia-induced behavioral alterations were also evaluated after 7 and 30 days of reperfusion. The present study demonstrates a decreased postsynaptic MAP2 immunoreactivity, representative of neuronal loss, particularly in CA1 sector of hippocampus and in cerebellum of ischemic rats reperfused for 7 days. After 30 days of reperfusion, MAP2 immunostaining was similar to control. In the same brain sections an increased presynaptic synaptophysin immunoreactivity has been observed only after 30 days of reperfusion. These data suggest compensatory regenerative changes associated with synaptic remodelling and are supported by behavioral recovery observed under the same experimental conditions.

During aging there is a progressive loss of neuronal function in the basal forebrain that results in cognitive impairment and cholinergic deficits. While altered neurotrophin (NT)-mediated signal transduction may account for some age-associated deficits, there are differences in the extent of NT responsiveness among different laboratory rat strains. Here we measured nerve growth factor (NGF) protein levels and fragmented DNA in the CNS, and basal and NGF-stimulated activity levels of the hypothalamus-pituitary-adrenocortical axis (HPAA) in 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. Our results show that while there is no age-associated differences in NGF protein levels, in aged Fischer-344/Brown Norway rats, there are increases in levels of immunoreactive fragmented DNA in the CNS and in adrenocortical responses to the peripheral administration of NGF. These data contribute to the characterization of the Fischer-344/Brown Norway F1 hybrid rat and provide baseline values useful for future studies on aged CNS.

We examined the effect of cationic lipid-mediated gene transfection of nerve growth factor (NGF) in primary septo-hippocampal cell cultures. Rat NGF cDNA was subcloned into a pUC19-based plasmid containing a CMV promoter. Two days after NGF gene transfection in primary cell cultures, ELISA confirmed increases in NGF protein secretion from transfected cells. To study the biological effect of cationic lipid-mediated NGF gene transfection, we analyzed the amount of neurofilament protein from NGF-transfected cell cultures. Western blot and immunohistochemical analyses detected significant increases in the phosphorylated form of neurofilament proteins in the cultures after cationic lipid-mediated NGF cDNA transfection. Cationic lipid-mediated NGF cDNA transfection did rot cause significant changes in the total amount of neurofilament protein. Our studies suggest that cationic lipid-mediated NGF gene transfection can increase neurofilament phosphorylation but not total neurofilament protein.

We studied the effects of endotoxin from Escherichia coli (E. coli) on Ca2+ channel activity in PC12 cells using the cell-attached patch clamp technique. Endotoxin (1-100 ng/ml) decreased channel availability (n x Po) to about one third of control values, an effect that required 3.5 +/- 1 min (mean +/- SD; n = 13) to reach steady state. The biophysical properties of the channel, including slope conductance (22 pS; 40 mM Ba2+), voltage dependence of n x Po, and open times (tau 1 = 0.78 ms, tau 2 = 8.9 ms) for the two open states at 0 mV, were not altered. The effect of endotoxin was blocked by polymyxin-B, indicating involvement of the lipid-A moiety of lipopolysaccharide, and by the tyrosine kinase (tk) inhibitor, tyrphostin. The effect of endotoxin was mimicked by 8-bromo-cGMP (100 microM), and was blocked by the inhibitor of cGMP-dependent protein kinase (PKG), H-8, suggesting involvement of the cGMP/PKG pathway. The effect of endotoxin also was blocked by the nitric oxide (NO) synthase inhibitor, NG-monomethyl-L-arginine monoacetate, suggesting involvement of nitric oxide synthase (NOS). The rapidity of the effect of endotoxin on Ca2+ channel activity suggested that constitutive NOS (cNOS) was involved, in accordance with our finding that endotoxin-induced transcriptional induction of NOS, as measured by nitrite production, required > 6 hr. We conclude that early signaling events by endotoxin in PC12 cells involve tk, cNOS, cGMP/PKG, and Ca2+ channels.

Prenatal alcohol exposure (FAE) has been associated with multiple anomalies, including a selective developmental delay of sympathetic innervation in lymphoid organs. Sympathetic neurons require nerve growth factor (NGF) for their development and maintenance, and recent evidence has suggested that alcohol impairs the synthesis and/or biological activity of NGF in selected central and peripheral neurons. Thus, the present study examined the hypothesis that NGF administration to FAE rats during early postnatal development would reverse some of the peripheral sympathetic deficits. Neonate rats, FAE and the corresponding control cohorts, received daily treatments of NGF or cytochrome C (0.3 mg/kg; s.c.) for various time intervals, and were killed 24 hr or 10 days after the last treatment. The measured parameters included norepinephrine (NE) concentrations in the spleen and heart, which receive nor-adrenergic innervation from the coeliac ganglion and the superior cervical ganglion (SCG), respectively. In addition, we measured the activity of pineal N-acetyltransferase (NAT), the rate-limiting enzyme of melatonin biosynthesis, which depends on sympathetic innervation from the SCG. The data show that chronic, but not acute, NGF treatments reversed the FAE-related deficits in splenic NE concentrations as well as in pineal NAT activity in a time- and age-dependent manner. Sympathetic neurons play an important role in immune modulation. Thus, the altered splenic NE levels and pineal NAT activity may play a role in immune deficits associated with exposure to alcohol in utero.

Both intrinsic signals, such as serum and neurotrophic factor deprivation, and extrinsic events or agents, such as oxidative stress and glucose deprivation, can induce cell death in pheochromocytoma (PC12) cells. Also, treatment with nerve growth factor (NGF) reduces cell death due to the treatments mentioned. Serumless-induced cell death, as a model of apoptosis, has been intensively investigated in PC12 cells. In the present study, we investigated the molecular components of H2O2-induced cell death and compared it with serumless-induced cell death. Exposure of PC12 cells to intermediate concentrations of H2O2 (100 microM) induced nuclear condensation and DNA fragmentation, indicating that there is an apoptotic component in H2O2-induced cell death. Since transcription factors have been shown to play an essential role in the control of cellular proliferation, differentiation, and survival, we measured changes in the DNA binding activities of the transcription factors activator protein-1 (AP-1), nuclear factor kappa B (NF-kappa B), and octamer-binding protein (Oct) by electrophoretic mobility shift assay (EMSA) after H2O2 treatment and serum deprivation, both in the absence and presence of exogenous NGF in PC12 cells. AP-1 DNA binding activity transiently increased during apoptosis due to serum deprivation, and NGF treatment further stimulated AP-1 DNA binding activity in a more persistent fashion. NF-kappa B DNA binding activity only increased slightly after serum deprivation, and NGF treatment of PC12 cells decreased NF-kappa B binding activity in the late stages of serum deprivation. Oct DNA binding activity decreased after serum deprivation, while NGF had an opposite effect. AP-1 DNA binding activity also transiently increased after H2O2 treatment, as did NF-kappa B DNA binding activity. Our results suggest that AP-1 is likely to be a common component of signaling pathways associated with both the induction or suppression of apoptosis induced by intrinsic or extrinsic stimuli.

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 micrograms 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.

Neurons of the mammalian CNS differ in their vulnerability to various disease processes and other insults, particularly in their response to total anoxia/ischemia. In this study we have tested the histological and behavioral modifications induced by experimental conditions of partial cerebral ischemia in the rats. The specific morphological and histological alterations, observed in our experimental conditions of reversible partial cerebral ischemia, confirm the selective vulnerability of certain neuronal populations to ischemic injury and are also evidenced by behavioral modifications which may mirror the functional impairment observed in humans after a transitory ischemic attack.