Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 are involved in both excitotoxin-induced neurodegeneration and regeneration.

Intrahippocamal injections of kainic acid (KA) significantly increase the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in the ipsilateral hippocampus at 2-4 h and 21-45 days post-administration, suggesting the possible involvement of these chemokines in both neurodegenerative and regenerative processes. To examine the possible role of these chemokines on neuronal cell death, hippocampal neurons were incubated with either MCP-1 or MIP-2 in vitro and examined to assess the effects on neuronal cell viability. These treatments resulted in significant neuronal apoptosis that could be abrogated by prior treatment with the caspase-1 inhibitor, Z-VAD-FMK, the caspase-3 inhibitor, Z-DEVD-FMK, the Galphai inhibitor, pertussis toxin, or the MAO-B inhibitor, (-)deprenyl. Furthermore, this chemokine apoptotic effect could also be observed in vivo as intrahippocampal injections of MCP-1 or MIP-2 resulted in the apoptosis of hippocampal neurons, thus supporting a direct role of these chemokines in neuronal death. In contrast, immunohistological analysis of kainic acid lesions on days 21-45 revealed significant expression of MCP-1 and MIP-2 associated with reactive astrocytes and macrophages, respectively, with no apoptotic populations being observed. These results suggested that these chemokines might also mediate distinct biological effects on local microenvironmental cell populations at various stages post truama and during cellular repair. To address this possibility, astrocyte were cultured in the presence or absence of these chemokines and examined by microarray analysis for effects on astrocytes gene expression. A number of genes encoding proteins associated with inflammation, cellular signaling, differentiation, and repair were directly modulated by chemokine treatment. More specifically, the RNA and protein expression of the neurotrophic factor, basic fibroblast growth factor (bFGF), was found to be significantly increased upon culture with MCP-1 and MIP-2. Conditioned media derived from chemokine-stimulated astrocytes also facilitated bFGF-dependent neuronal cell differentiation and promoted survival of H19-7 neurons in vitro, suggesting a possible role for chemokine-activated astrocytes as a source of trophic support. Taken together, these data support possible autocrine and paracrine roles for MCP-1 and MIP-2 in both the "death and life" of hippocampal neurons following CNS injury.

Nerve growth factor uses Ras/ERK and phosphatidylinositol 3-kinase cascades to up-regulate the N-methyl-D-aspartate receptor 1 promoter.

We reported previously that nerve growth factor (NGF) up-regulates activity of the N-methyl-D-aspartate receptor 1 (NR1) promoter. We have explored the pathways and nuclear targets of NGF signaling in regulating the NR1 promoter. PD98059 and wortmannin, but not rapamycin, significantly attenuated NGF-induced transcriptional activity from an NR1 promoter-luciferase construct. Coexpressing constitutively active forms of Ras, Raf, or MAPK/ERK kinase 1 (MEK1) increased promoter activity dramatically. The MEK1-induced increase was largely prevented by mutations of the tandem GC boxes in the promoter. Promoter activity was also increased significantly by coexpressed GC box-binding proteins (Sp1, 3, or 4) in nonstimulated PC12 cells. Either an extracellular signal-regulated kinase-1 (ERK1)- or Sp1-specific antibody coprecipitated Sp1 with ERKs, and the coprecipitation was enhanced significantly by NGF treatment of PC12 cells. ERK2 also incorporated radioactivity of [gamma(32)P]ATP into recombinant Sp1. However, ERK2-treated Sp1 and PC12 nuclear extracts or nuclear extracts from NGF-treated cells exhibited reduced binding to the promoter or a consensus GC box. Our results suggest that NGF utilizes both the Ras/ERK and phosphatidylinositol 3-kinase pathways to up-regulate NR1 promoter activity and that Sp1 is a novel substrate of NGF-activated ERKs. NGF-increased NR1 promoter activity may involve a complicated mechanism of Sp1 phosphorylation and possible transcription factor exchange.

Expression of mutant amyloid precursor proteins decreases adhesion and delays differentiation of Hep-1 cells.

The amyloid precursor protein (APP) is a type I integral membrane protein and is processed to generate several intra-cellular and secreted fragments. The physiological role of APP and its processed fragments is unclear. Several mutations have been discovered in APP, which are causative of early-onset, familial, neurological disease, including Alzheimer's disease (FAD). These mutations alter the processing of APP and lead to excess production and extra-cellular deposition of A-beta peptide (Abeta). We have examined the role of APP in a cell culture model of endothelial cell function. The endothelial cell line, Hep-1, was stably transfected with wild-type (wt) and FAD mutant forms of APP (mAPP). Secretion of sAPPalpha was reduced in cell lines over-expressing mAPP when these cells were grown on several different substrates. Levels of secreted Abeta were increased as measured by ELISA in the mutant cell lines. Cell adhesion to laminin-, fibronectin-, collagen I-, and collagen IV-coated culture flasks was reduced in all mAPP-expressing cell lines, while in lines over-expressing wt-APP, adhesiveness was slightly increased. Cell lines over-expressing mAPP differentiated more slowly into capillary network-like structures on Matrigel than those expressing wt-APP. No differences were detected among all cell lines in a migration/invasion assay. The results suggest that APP may have a role in cell adhesiveness and maturation of endothelial cells into capillary-like networks. The reduction in adhesion and differentiation in mutant cell lines may be due to reduced amounts of sAPPalpha released into the culture media or toxic effects of increased extracellular Abeta.

Characterization of the neurotrophic interaction between nerve growth factor and secreted alpha-amyloid precursor protein.

The expression and secretion of amyloid precursor protein (beta APP) is increased in rat cerebral cortices that have been denervated by subcortical lesions of the nucleus basalis of Meynert. The physiological role of the secreted beta APP in response to this injury has not been established. We have previously shown that secreted beta APP produced by alpha-secretase activity (sAPP(alpha)) potentiates the neuritogenic activity of nerve growth factor (NGF) in vitro on naive PC12 cells. In this investigation, we have further characterized the neurotrophic interaction of NGF and sAPP(alpha) using differentiated PC12 cells and rat primary cortical neurons. NGF required the expression of beta APP to maintain a neuronal phenotype. Reduction of endogenous beta APP expression by introduction of antisense oligonucleotides in the presence of NGF resulted in loss of neurites from differentiated PC12 cells but no apparent cell death. Addition of exogenous sAPP(alpha) (60--200 pM) potentiated the protective activity of NGF in serum-deprived differentiated PC12 cells as determined by retention of neurites and cell viability. In addition, exogenous sAPP(alpha) increased neuron viability in both short-term (3 days) cortical neuron cultures grown in the absence of serum and in long-term (9 days) cultures grown with serum. Disruption of the insulin signaling pathway by reduction of IRS-1 expression inhibited the ability of sAPP(alpha) to potentiate neurotrophic activity. These observations suggest that sAPP(alpha) acts as an injury-induced neurotrophic factor that interacts with NGF to enhance neuronal viability using the insulin signaling pathway.

Death of PC12 cells and hippocampal neurons induced by adenoviral-mediated FAD human amyloid precursor protein gene expression.

We used adenoviral-mediated gene transfer of human amyloid precursor proteins (h-APPs) to evaluate the role of various h-APPs in causing neuronal cell death. We were able to infect PC12 cells with very high efficiency because approximately 90% of the cells were cytochemically positive for beta-galactosidase activity when an adenoviral vector containing LacZ cDNA was used to infect cells. Cells infected with adenovirus containing h-APP cDNA showed high-level transcription and expression of h-APP as measured by reverse transcriptase-polymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17-and 24-fold in cultures infected with recombinant adenovirus containing wild-type mutant and 13- and 17-fold with V642F mutant. No elevation in h-APP was seen in cultures infected with antisense h-APP or null adenovirus. H-APP levels were maximal 3 days after infection. Overexpression of V642F mutant h-APP in PC12 cells and hippocampal neurons resulted in about a twofold increase in death compared with overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral mediated gene transfer in cell culture studies and suggest that overexpression of a familial Alzheimer's disease mutant APP may be toxic to neuronal cells.

Effects of PS1 deficiency on membrane protein trafficking in neurons.

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

Synergistic activation of the N-methyl-D-aspartate receptor subunit 1 promoter by myocyte enhancer factor 2C and Sp1.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays important roles in neuronal development, plasticity, and cell death. NMDA receptor subunit 1 (NR1) is an essential subunit of the NMDA receptor and is developmentally expressed in postnatal neurons of the central nervous system. Here we identify on the NR1 promoter a binding site for myocyte enhancer factor 2C (MEF2C), a developmentally expressed neuron/muscle transcription factor found in cerebrocortical neurons, and study its regulation of the NR1 gene. Co-expression of MEF2C and Sp1 cDNAs in primary neurons or cell lines synergistically activates the NR1 promoter. Disruption of the MEF2 site or the MEF2C DNA binding domain moderately reduces this synergism. Mutation of the Sp1 sites or the activation domains of Sp1 protein strongly reduces the synergism. Results of yeast two-hybrid and co-immunoprecipitation experiments reveal a physical interaction between MEF2C and Sp1 proteins. The MEF2C DNA binding domain is sufficient for this interaction. Dominant-negative MEF2C interferes with expression of NR1 mRNA in neuronally differentiated P19 cells. Growth factors, including epidermal growth factor and basic fibroblast growth factor, can up-regulate NR1 promoter activity in stably transfected PC12 cells, even in the absence of the MEF2 site, but the Sp1 sites are necessary for this growth factor regulation, suggesting that Sp1 sites may mediate these effects.

Single-stranded DNA-binding proteins and neuron-restrictive silencer factor participate in cell-specific transcriptional control of the NMDAR1 gene.

Our previous studies revealed that a proximal region of the N-methyl-D-aspartate receptor 1 (NMDAR1) promoter is important for cell-type-specific expression. We have now explored the contributions of several regulatory elements to this specificity. Deletion of the neuron-restrictive silencer element partially relieved the suppression of promoter activity in C6 glioma and HeLa cells. An overlapping G(C/G)G/tandem Sp1-containing region crucial for both basal and nerve growth factor (NGF)-regulated promoter activity specifically bound nuclear proteins on its purine-rich sense strand. A faster migrating complex, single-stranded binding protein complex 1 (SBPC1), was highly enriched in HeLa cells, whereas a slower migrating complex, SBPC2, was enriched in PC12 cells. A high ratio of 2/1 complex correlated with a high level of promoter activity. NGF treatment of PC12 cells reduced SBPC1 but increased SBPC2. Competition experiments showed that the SBPC1 binding required a dG4 sequence and the SBPC2 needed a core of TG3A plus a 5'-flanking sequence. Single-stranded DNA encompassing TG3A and/or dG4 specifically suppressed cotransfected NMDAR1 promoter activity. UV cross-linking studies indicated that a 31.5-kDa protein mainly formed SBPC1, whereas SBPC2 contained several larger proteins. Our results suggest that neuron-restrictive silencer factor and single-stranded DNA-binding proteins may both play a role in cell-type specificity of the NMDAR1 gene, and the latter may also be involved in basal and NGF-regulated activity.

Processing of Alzheimer's amyloid precursor protein during H2O2-induced apoptosis in human neuronal cells.

The processing of Alzheimer's amyloid precursor protein was studied by Western blotting during H2O2 induced apoptosis in cultures of human neuroblastoma cells. A new 5.5 kDa fragment putatively containing intact A beta was detected and found to be highly associated with apoptosis. The results suggest a possible vicious cycle involving H2O2, A beta and apoptosis which may contribute to the neuronal death mechanism in Alzheimer's Disease.

Expression of mutant amyloid precursor proteins induces apoptosis in PC12 cells.

The cause of neuronal loss in Alzheimer disease is unknown. We investigated the effects on survival of PC12 cells expressing A692G, E693Q, and V717F mutant amyloid precursor proteins (APP). Differentiated cells expressing mutant APPs exhibited somal shrinkage, followed by cell detachment from the plates. Increased levels of oligonucleosome-sized DNA ladders and TUNEL-positive nuclei were observed, and electron microscopy revealed extensive plasma membrane blebbing, margination of condensed chromatin, and well-preserved organelles in these transfectants. The levels of TUNEL-positive cells, analyzed by a flow-cytometric method, were increased by four- to sevenfold in mutant APP transfectants, but less than twofold in wild-type APP transfectants relative to untransfected cells. Our results provide evidence that expression of mutant APPs in differentiated PC12 cells induces cell death via an apoptotic pathway.

Nerve growth factor up-regulates the N-methyl-D-aspartate receptor subunit 1 promoter in PC12 cells.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays important roles in synaptic plasticity, the induction of long term potentiation, and excitotoxicity. Mechanisms governing the regulation of expression of its subunit genes remain largely unknown. The promoter of the essential subunit of the NMDA receptor heteromer, NMDAR1, contains DNA binding elements recognized by the nerve growth factor-inducible/early growth reaction factor (NGFI/Egr) family of transcription factors that are rapidly induced by neurotrophins, such as nerve growth factor (NGF). This study examined the effect of NGF on the activity of the N-methyl-D-aspartate receptor subunit 1 (NMDAR1) promoter/luciferase reporter constructs in PC12 cells, which contain the high affinity TrkA receptor for NGF and the low affinity p75(NTR) receptor for neurotrophins. NGF up-regulated the activity of the NMDAR1 promoter by 3-4-fold in a time- and dose-dependent manner. 5' deletional analysis of the promoter indicated that the responsive element(s) resides in the proximal region containing GSG and Sp1 sites. Mutational analysis of these sites revealed that both were important for NGF regulation. Transient expression of Egr-1 increased activity of the wild type promoter but failed to increase activity of a GSG mutant promoter. Other neurotrophins did not activate the promoter, while K-252a inhibited the action of NGF. These results suggest that the NGF effect is mediated by the high affinity NGF receptor, Trk A and that neurotrophin binding to the low affinity neurotrophin receptor, p75(NTR), alone does not affect the promoter activity. Our results suggest that NGF is able to up-regulate the activity of the NMDAR1 promoter and may play a role in controlling the expression levels of NMDA receptors.

Participation of presenilin 2 in apoptosis: enhanced basal activity conferred by an Alzheimer mutation.

Overexpression of the familial Alzheimer's disease gene Presenilin 2 (PS2) in nerve growth factor-differentiated PC12 cells increased apoptosis induced by trophic factor withdrawal or beta-amyloid. Transfection of antisense PS2 conferred protection against apoptosis induced by trophic withdrawal in nerve growth factor-differentiated or amyloid precursor protein-expressing PC12 cells. The apoptotic cell death induced by PS2 protein was sensitive to pertussis toxin, suggesting that heterotrimeric GTP-binding proteins are involved. A PS2 mutation associated with familial Alzheimer's disease was found to generate a molecule with enhanced basal apoptotic activity. This gain of function might accelerate the process of neurodegeneration that occurs in Alzheimer's disease, leading to the earlier age of onset characteristic of familial Alzheimer's disease.

Neurodegeneration in Alzheimer disease. Is apoptosis involved?

Alzheimer disease (AD), the most common dementia of the elderly, results from a significant loss of neuronal cells in brain regions important in memory and cognition. Several lines of evidence suggest that the A beta peptide is directly responsible for some of this neuronal cell death. We review recent evidence from in vitro toxicity and immunohistochemical studies that suggest some of the cell loss in AD is the result of apoptosis.

Laminin alpha 2 is a component of brain capillary basement membrane: reduced expression in dystrophic dy mice.

In the present study we demonstrate low level expression of the laminin alpha 2 chain in brain and localize the alpha 2 protein to the capillary basement membrane. While in peripheral basement membranes the laminin alpha 1 and alpha 2 chains have an almost mutually exclusive distribution, the present results suggest both alpha 1 and alpha 2 in the cerebral capillary basement membrane. Towards elucidating the function of alpha 2 in brain, we have performed ultrastructural analysis of the capillary basement membrane in dystrophic dy mice, which show a 70-90% and > 95% reduction of alpha 2 messenger RNA compared to heterozygous and wild-type mice, respectively, and show a nearly total absence of the alpha 2 protein by immunofluorescence. In contrast to the muscle and Schwann cell basement membrane, where alpha 2 deficiency causes structural basement membrane abnormalities, the present results show that the lack of the alpha 2 subunit in the cerebral capillary basement membrane is not detrimental to its structure. This observation might be explained by the fact that the cerebral capillary basement membrane expresses both alpha chains and therefore exhibits structural redundancy.

Changes in gene transcription during a beta-mediated cell death.

The a beta peptide induces cell death in neurons grown in cell culture. Previous studies have shown that the mechanism of a beta-mediated cell death of central nervous system neurons appears to be via apoptosis. Apoptosis is an active process that involves both gene transcription and translation. Using semi-quantitative polymerase chain reaction, we have analyzed the levels of a variety of transcripts in primary neuronal cultures treated with a beta that are likely to play important roles in apoptosis. Following addition of 10 microM a beta 1-42 the immediate early response gene, c-fos, shows a rapid and sustained increase in transcript level while c-jun levels increase at a slower rate. Bcl-2 and its homologues, bcl-X and bax, also increase in amount with bcl-2 and bcl-X increasing more rapidly than bax. These data provide support indicating that a beta-mediated cell death in central nervous system neurons is an active process similar to that seen in apoptosis.

Studies on TGF-beta 1 gene expression in the intima of the human aorta in regions with high and low probability of developing atherosclerotic lesions.

Certain regions of the human aorta are at greater risk for early and more severe atherosclerotic lesions development than others. Cornhill and coworkers (Cornhill FJ et al.: Arteriosclerosis 5:415, 1985) created maps for the probability of developing atherosclerosis defining the high-probability region (HPR) in the dorsal descending thoracic aorta and the low-probability region (LPR) in the ventral descending thoracic aorta. Our study examines the hypothesis that transforming growth factor beta -1 (TGF-beta 1), a well-known suppressor of growth and function in many human cell lines, is one of the inhibitors of human atherogenesis. The present experiment analyzes the expression of mRNA for TGF-beta 1 in both the HPR and the LPR of aortas from young (age 17 to 25 y) males of black (n = 8) and white (n = 7) race. The level of TGF-beta 1 gene expression was assessed in the aortic intima in both the HPR and the LPR, using National Institutes of Health Image 1.47, an Apple Macintosh application capable of digital image processing, analysis, and morphometric measurement. There was significantly lower (P = 0.002, alpha = 0.05) TGF-beta 1 gene expression in the HPR than in the LPR in the 22- to 25-y age group. There was no significant difference in the 17- to 21-y age group and between the HPR and the LPR in the entire study group.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional analysis of the proximal 5'-flanking region of the N-methyl-D-aspartate receptor subunit gene, NMDAR1.

The NMDAR1 receptor subunit is a common subunit of N-methyl-D-aspartate receptors. We have previously characterized 3 kilobases (kb) of 5'-flanking sequence of the NMDAR1 gene and now report on the ability of this region to direct transcription of a reporter gene and on its interaction with nuclear proteins. The sequence 356 base pairs (bp) 5' of the first nucleotide of codon 1 was sufficient to express a luciferase reporter gene in rat PC12 pheochromocytoma cells. Additional sequences upstream of nucleotide -356 influenced the activity approximately 2-fold. A labeled 112-bp fragment (position -356 to -245) formed six complexes (C1A and -B, C2A and -B, and C3A and -B), grouped as three double bands, with nuclear extracts from PC12 cells. Competition with Sp1 oligonucleotides abolished formation of C2A and -B and C3A and -B complexes. Sp1 antibody recognized the C3A complex in supershift experiments. Prior immunoprecipitation of nuclear extracts with Sp1 antibody abolished formation CA2 and -B and C3A and -B complexes. Purified Sp1 protein alone did not form a C3A complex but potentiated its formation when PC12 nuclear extract was added. A GC-rich sequence in this fragment was protected from DNase I digestion by nuclear extract. These results suggest that a 356-bp sequence comprises the NMDAR1 basal promoter, and that NMDAR1 gene expression may be regulated by Sp1-like nuclear factors.

Altered processing of a mutant amyloid precursor protein in neuronal and endothelial cells.

Altered proteolysis of the amyloid precursor protein (APP) may play an important role in Alzheimer disease (AD). To better understand the role of mutant APP in the pathogenesis of the disease, we stably overexpressed the mutant APP717F approximately twofold vs. the endogenous wild-type gene in several cell types. The processing of APP was examined by Western blot analysis and immunoprecipitation. We observed distinctive patterns of APP metabolites among various cell lines. Neuronal and endothelial cells expressing mutant APP717F generated higher levels of large, potentially amyloidogenic carboxyl terminal fragments, which were enhanced upon treatment of the cells with leupeptin. These results suggest that mutations in the APP gene shift the protein processing towards the amyloidogenic pathway in neuronal and endothelial cells possibly involving the endosomal-lysosomal system.

Cloning and analysis of the 5' flanking sequence of the rat N-methyl-D-aspartate receptor 1 (NMDAR1) gene.

We cloned and analyzed a 3.8 kb EcoRI fragment of the rat NMDAR1 gene. It contains 3 kb of promoter/enhancer region, exon 1 and a portion of intron 1. Two major transcription start sites were identified at -276 and -238 from the first nucleotide in codon 1. One GSG and two SP1 motifs, but no TATA/CAAT boxes, exist in the region proximal to the transcription start sites. Our results suggest that NMDAR1 has the characteristics of a housekeeping gene and may be regulated by immediate-early genes.

A splice variant of the N-methyl-D-aspartate (NMDAR1) receptor.

A splice variant of the NMDA receptor (NMDAR1) was discovered containing a deletion of 37 amino acids near the carboxyl tail and has been designated NMDAR1b. The 111 nucleotides corresponding to the deleted amino acid sequence were found in a separate exon bounded by consensus intron/exon junction sequences in rat genomic DNA. A partial restriction map of genomic DNA bounding this region placed the deleted exon approximately 600 base pairs (bp) downstream of the upstream exon. RT/PCR analysis of RNA from different brain regions showed that the deletion variant is more abundantly expressed in the brain stem and cerebellum while the full-length form is expressed more abundantly in the olfactory bulb, striatum, hippocampus, and cortex. Northern analysis of poly(A)+ RNA from different brain regions with probes specific for the deleted exon (i.e., full-length form) and for the splice junction (deletion form) indicated approximately 4.4 kb transcripts. The probe for the deleted exon hybridized to transcripts in olfactory bulb, cortex, striatum, and hippocampus while the splice junction probe hybridized most strongly to transcripts in cerebellum. The results suggest an interesting rostral to caudal shift in the expression of splice variants of the NMDAR1 which may signify important functional differences in native forms of NMDA receptors.