John W. Kusiak

Participation of Presenilin 2 in Apoptosis: Enhanced Basal Activity Conferred by an Alzheimer Mutation

Overexpression of the familial Alzheimers disease gene Presenilin 2 (PS2) in nerve growth factor-differentiated PC12 cells increased apoptosis induced by trophic factor withdrawal or β-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 Alzheimers 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 Alzheimers disease, leading to the earlier age of onset characteristic of familial Alzheimers disease.

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.

Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimers Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

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. J. Neurosci. Res. 47:253–263, 1997.

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 p75NTR 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, p75NTR, 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.

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.

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 dG4sequence 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.

Red cell perturbations by amyloid β-protein

Abstract Amyloid β-protein (Aβ) accumulation in brain is thought to be important in causing the neuropathology of Alzheimers disease (AD). Aβ interactions with both neurons and microglial cells play key roles in AD. Since vascular deposition of Aβ is also implicated in AD, the interaction of red cells with these toxic aggregates gains importance. However, the effects of Aβ interactions with red blood cells are less well understood. Synthetic amyloid β-protein (1–40) was labeled with biotin and preincubated at 37 °C for 4, 14 and 72 h to produce fibrils. Flow cytometry was used to study the binding of these fibrils to red cells. The amyloid fibrils had a high affinity for the red cell with increased binding for the larger fibrils produced by longer preincubation. Bovine serum albumin (BSA) did not reverse the binding, but actually resulted in a more efficient binding of the Aβ fibrils to the red cells. The interaction of Aβ with red cells increased the mean cell volume and caused the cells to become more spherical. This effect was greater for the longer fibrils. At the same time the interaction of Aβ with red cells produced an increase in their fluorescence measured after 16-h incubation at 37 °C. This increase in fluorescence is attributed to the formation of fluorescent heme degradation products. The effect of prior hemoglobin oxidation, catalase inhibition and glutathione peroxidase inhibition indicated that the amyloid-induced oxidative damage to the red cell involved hydrogen peroxide-induced heme degradation. These results suggest that amyloid interactions with the red cell may contribute to the pathology of AD.

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 ≈90% of the cells were cytochemically positive for β‐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 Alzheimers disease mutant APP may be toxic to neuronal cells. J. Neurosci. Res. 55:629–642, 1999

Characterization of the neurotrophic interaction between nerve growth factor and secreted α-amyloid precursor protein†

The expression and secretion of amyloid precursor protein (βAPP) 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 βAPP in response to this injury has not been established. We have previously shown that secreted βAPP produced by α‐secretase activity (sAPPα) 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 sAPPα using differentiated PC12 cells and rat primary cortical neurons. NGF required the expression of βAPP to maintain a neuronal phenotype. Reduction of endogenous βAPP 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 sAPPα (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 sAPPα 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 sAPPα to potentiate neurotrophic activity. These observations suggest that sAPPα acts as an injury‐induced neurotrophic factor that interacts with NGF to enhance neuronal viability using the insulin signaling pathway. J. Neurosci. Res. 63:410–420, 2001.