Steven W. Barger

β-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer's disease

Abstract Recent findings link altered processing of β-amyloid precursor protein (βAPP) to disruption of neuronal Ca 2+ homeostasis and an excitotoxic mechanism of cell death in Alzheimers disease. A major pathway of βAPP metabolism results in the release of secreted forms of βAPP, APP s s. These secreted forms are released in response to electrical activity and can modulate neuronal responses to glutamate, suggesting roles in developmental and synaptic plasticity. βAPP is upregulated in response to neural injury and APP s s can protect neurons against excitotoxic or ischemic insults by stabilizing the intracellular Ca 2+ concentration [Ca 2+ ] i . An alternative βAPP processing pathway liberates intact β - amyloid peptide, which can form aggregates that disrupt Ca 2+ homeostasis and render neurons vulnerable to metabolic or excitotoxic insults. Genetic abnormalities (e.g. certain βAPP mutations or Down syndrome) and age-related changes in brain metabolism (e.g. reduced energy availability or increased oxidative stress) may favor accumulation of [Ca 2+ i -destabilizing β - amyloid peptide and diminish the release of [Ca 2+ ] i -stabilizing, neuroprotective APP s s.

CALCIUM, FREE RADICALS, AND EXCITOTOXIC NEURONAL DEATH IN PRIMARY CELL CULTURE

Publisher Summary Elucidation of cellular and molecular mechanisms of excitotoxic neuronal injury have been facilitated by the development of several cell biological technologies—namely, (1) neural cell culture, (2) quantitative assays of neuronal injury and death, (3) fluorescence imaging of intracellular free calcium levels ([Ca 2+ ] i ), (4) evaluation of mitochondrial function, (5) quantification of reactive oxygen species, (6) assessment of cytoskeletal alterations elicited by excitotoxic/metabolic insults, and (7) detection of “stress response” proteins. This chapter presents protocols for each of these technical approaches, as applied to embryonic rat and human brain neurons in dissociated cell culture. One strategy employed to study neuronal death is to elucidate the mechanisms that normally protect neurons from adverse environmental conditions. These studies have shown that many cellular signaling mechanisms exist that are designed to protect neurons against adverse environmental conditions such as metabolic and excitotoxic insults. Many of the neuroprotective strategies acquired during evolution involve systems that control calcium and free radical metabolism.

S100β protects hippocampal neurons from damage induced by glucose deprivation

Abstract S100β is a calcium-binding protein elevated in Downs syndrome and Alzheimers disease. Previous studies have demonstrated that S100β is trophic for several neuronal populations. We tested the influence of S100β on hippocampal neurons. The initial response included a rapid increase in [Ca2+]i similar to that elicited by S100β in other populations. S100β also substantially decreased cell death and loss of mitochondrial function resulting from glucose deprivation. Therefore, S100β exerts a neuroprotective influence on CNS neurons, suggesting that its elevation in neurological disorders may be a compensatory response.

Role of glutathione redox cycle in TNF-α-mediated endothelial cell dysfunction

Modulation of the glutathione redox cycle may influence tumor necrosis factor-α (TNF)-mediated disturbances of endothelial integrity. To test this hypothesis, normal endothelial cells or cells with either increased or decreased glutathione levels were exposed to 100 ng (500 U) TNF/ml. Increased glutathione levels were achieved by exposure to 0.2 mM N-acetyl-L-cysteine (NAC) and decreased glutathione levels by exposure to 25 μM buthionine sulfoximine (BSO). Several components of the glutathione redox cycle as well as markers of endothelial integrity, such as cytoplasmic free calcium and transendothelial albumin transfer, were measured in the treated cells. Exposure to TNF for 3 and 6 h decreased total glutathione levels, which was followed by an increase at later time points. Moreover, treatment with TNF resulted in an increase in the ratio of oxidized to reduced glutathione, intracellular free calcium, albumin transfer across endothelial monolayers and lipid hydroperoxides. However, an increase in lipid hydroperoxides was seen only when endothelial cell cultures were supplemented with iron. BSO treatment increased susceptibility of endothelial cells to TNF-mediated metabolic disturbances. On the other hand, NAC partially protected against TNF-induced injury to endothelial monolayers. Our results demonstrate the important role of the glutathione redox cycle in TNF-mediated disturbances of the vascular endothelium and indicate that modulation of glutathione levels may potentiate the injurious effects of this inflammatory cytokine.

Staurosporine, K-252a, and K-252b Stabilize Calcium Homeostasis and Promote Survival of CNS Neurons in the Absence of Glucose

Abstract: Staurosporine, K‐252a, and the 9‐carboxylic related compound K‐2525 are low‐molecular‐weight alkaloids from microbial origin that at high concentrations are kinase inhibitors and can antagonize the effects of neuronal growth factors. Paradoxically, we have found that very low concentrations of these agents (10 fM‐10 nM) prolong the survival of hippocampal, septal, and cortical neurons deprived of glucose. These agents did not prevent the depletion of ATP caused by glucose deprivation. The large elevation of intracellular calcium levels that normally mediates glucose deprivation‐induced damage was attenuated by Staurosporine, K‐252a, and K‐252b. Western blot analysis using antiphosphotyrosine antibody showed that Staurosporine and the K‐252 compounds (10–100 pM) stimulated tyrosine phosphorylation of several different proteins. The tyrosine kinase inhibitor genistein significantly reduced the protective effect of Staurosporine and the K‐252 compounds, indicating that tyrosine phosphorylation was required for neuroprotection by these compounds. Taken together, the data demonstrate that low concentrations of Staurosporine and the K‐252 compounds can stabilize calcium homeostasis, possibly by a mechanism involving activation of receptor tyrosine kinase transduction pathways.

Participation of Gene Expression in the Protection against Amyloid β‐Peptide Toxicity by the β‐Amyloid Precursor Proteina

The amyloid β‐peptide (Aβ) is a toxic derivative of the β‐amyloid precursor protein. Alternative processing of this precursor also yields large soluble forms (APPSs) which are secreted from many cell types. These APPSs have neuritogenic and neuroprotective activities; indeed, APPSs can protect primary neurons from the toxicity of Aβ itself. To begin to explore the regulation of gene expression by APPS, we have focused on the NF‐κB transcription factor family. NF‐κB is induced by conditions of stress, including cellular oxidation. We report that NF‐κB can also be induced by APPS. Furthermore, we effected direct activation of NF‐κB through disinhibition using antisense oligonucleotide technology. This means of activating NF‐κB resulted in protection of neuroblastoma cells from the toxicity of a calcium ionophore and protection of primary hippocampal neurons from the toxicity of Aβ. Together, these data suggest that NF‐κB may exist as a common agent inducing a neuroprotective pattern of gene expression in response to either trophic cytokines or stress itself.

β-Amyloid Precursor Protein Mismetabolism and Loss of Calcium Homeostasis in Alzheimer's Diseasea

The suspected involvement of the β‐amyloid precursor protein (βAPP) in the etiology of Alzheimers disease (AD) has been strengthened by recent genetic evidence, but pursuit of the mechanisms involved will initially require basic cell biology approaches. Several studies have concentrated on toxic activities of β‐amyloid peptide (βAP) itself, illuminating its contributions to excitotoxicity and calcium‐mediated degeneration in general. We now know that generation of βAP from βAPP also compromises the production of an important set of trophic factors: the secreted forms of βAPP (APPS), which may act—ironically—by conferring protection from calcium‐mediated insults. Therefore, conditions which contribute to the formation of βAP (possibly including ischemia) not only produce an agent which exacerbates calcium‐mediated cell death, but also reduce the levels of one of the few factors able to rescue calcium homeostasis. The implications of these postulates and their relationship to the process of aging are discussed.

Programmed Cell Life: Neuroprotective Signal Transduction and Ischemic Brain Injury

Abstract Neurons (and other cell types) perceive and respond to environmental adversity by activating inter- and intracellular signaling pathways that bolster various molecular defense systems. Two major life-promoting programs neurons possess result in stabilization of cellular calcium homeostasis and enhancement of antioxidant defense systems. Different signal transduction systems can mediate neuroprotection and promote cell life. Neurotrophic factor signaling involves receptor tyrosine kinases and a cascade of intermediate kinase interactions that lead to increased or decreased expression of proteins involved in calcium and free radical metabolism. For example, basic fibroblast growth factor suppresses the production of an N-methyl-D-aspartate receptor protein, increases the expression of the calcium-binding protein calbindin, and induces the production of the antioxidant enzymes superoxide dismutase and glutathione reductase. Another pro-life signaling pathway uses cyclic guanosine monophosphate as a messenger; this latter pathway mediates the excitoprotective action of secreted forms of β-amyloid precursor protein. In some cases, the same intercellular signal may promote the survival of one kind of cell while killing another cell type. For example, tumor necrosis factor kills oligodendrocytes, protects hippocampal neurons against metabolic/excitotoxic insults, and protects astrocytes against acidotic injury. A detailed understanding of the intricate signaling mechanisms that mediate neuroprotection will provide opportunities for preventative and therapeutic intervention in both acute and chronic neurodegenerative disorders.

βAPP Metabolites, Radicals, Calcium, and Neurodegeneration: Novel Neuroprotective Strategies

Many biochemical and molecular alterations in the brains of victims of Alzheimer’s disease (AD) and other age-associated neurodegenerative disorders have been described. Unfortunately, it remains unclear which of the alterations contribute to the neuronal damage, which represent compensatory and cytoprotective responses to ongoing cell injury, and which are mere remnants of damaged cells in general. The present article describes studies that have been performed in our laboratories to help define roles for two major metabolites of the β-amyloid precursor protein (βAPP) in the pathogenesis of AD. In addition, we discuss findings concerning age-related alterations in brain metabolism (e.g., reduced glucose availability), and cellular signaling systems regulating neuronal plasticity and survival (e.g., neurotrophic factors), that are likely to impact on the biological activities of βAPP metabolites. Cellular systems regulating metabolism of calcium and reactive oxygen species (ROS) appear to be critical targets of both neurodegenerative and neuroprotective pathways. We therefore highlight the variety of both natural and synthetic compounds that can stabilize calcium homeostasis and ROS metabolism, and which may thus prove effective in reducing neuronal injury in a variety of neurodegenerative disorders. We emphasize βAPP in this chapter, not because it is the only determinant of AD, but rather because increasing data suggest it plays a pivotal role in many cases. This article is not intended to be a comprehensive review of the literature, and we refer the reader to review articles that provide more in-depth analyses of our current understanding of the molecular and cellular pathophysiology of AD1–4.