James G. Begley

Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid β-peptide : Role of the lipid peroxidation product 4-hydroxynonenal

Abstract: Deposits of amyloid β‐peptide (Aβ), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimers disease. Synapse loss is closely correlated with cognitive impairments in Alzheimers disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that Aβ causes oxyradical‐mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. Aβ induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to Aβ and decreased further with time. The lipid peroxidation product 4‐hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented Aβ‐induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of Aβ. FeSO4 (an initiator of lipid peroxidation), Aβ, and 4‐hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration‐dependent decreases in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. Aβ also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that Aβ induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4‐hydroxynonenal appears to be a mediator of these actions of Aβ. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimers disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.

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.

Increased Activity-Regulating and Neuroprotective Efficacy of α-Secretase-Derived Secreted Amyloid Precursor Protein Conferred by a C-Terminal Heparin-Binding Domain

Abstract: Proteolytic cleavage of β‐amyloid precursor protein (βAPP) by α‐secretase results in release of one secreted form (sAPP) of APP (sAPPα), whereas cleavage by β‐secretase releases a C‐terminally truncated sAPP (sAPPβ) plus amyloid β‐peptide (Aβ). βAPP mutations linked to some inherited forms of Alzheimers disease may alter its processing such that levels of sAPPα are reduced and levels of sAPPβ increased. sAPPαs may play important roles in neuronal plasticity and survival, whereas Aβ can be neurotoxic. sAPPα was ∼100‐fold more potent than sAPPβ in protecting hippocampal neurons against excitotoxicity, Aβ toxicity, and glucose deprivation. Whole‐cell patch clamp and calcium imaging analyses showed that sAPPβ was less effective than sAPPα in suppressing synaptic activity, activating K+ channels, and attenuating calcium responses to glutamate. Using various truncated sAPPα and sAPPβ APP695 products generated by eukaryotic and prokaryotic expression systems, and synthetic sAPP peptides, the activity of sAPPα was localized to amino acids 591–612 at the C‐terminus. Heparinases greatly reduced the actions of sAPPαs, indicating a role for a heparin‐binding domain at the C‐terminus of sAPPα in receptor activation. These findings indicate that alternative processing of βAPP has profound effects on the bioactivity of the resultant sAPP products and suggest that reduced levels of sAPPα could contribute to neuronal degeneration in Alzhiemers disease.

Evidence for Synaptic Apoptosis

Increasing evidence indicates that neurons die by apoptosis, an active form of cell death involving a relatively stereotyped series of biochemical changes that culminate in nuclear fragmentation, in many different developmental and pathophysiological settings. In contrast to most other cell types, neurons have elaborate morphologies with complex neuritic arbors that often extend great distances from the cell body. Neuronal death signals are likely to be activated at remote synaptic sites and, indeed, overactivation of glutamate receptors and underactivation of trophic factor receptors are implicated in neurodegenerative disorders. We now report that biochemical changes consistent with apoptosis are engaged locally in synapses. Exposure of cortical synaptosomes to staurosporine and Fe2+ resulted in loss of membrane phospholipid asymmetry, caspase activation, and mitochondrial alterations (membrane depolarization, calcium overload, and oxyradical accumulation) characteristic of apoptosis. The caspase inhibitor zVAD-fmk prevented mitochondrial membrane depolarization in synaptosomes. Studies of the effects of cytosolic extracts from synaptosomes exposed to apoptotic insults, on isolated nuclei, showed that signals capable of inducing nuclear apoptosis are generated locally in synapses. Exposure of cultured hippocampal neurons to staurosporine and glutamate resulted in caspase activation and mitochondrial membrane depolarization in dendrites, and zVAD-fmk prevented the membrane depolarization. Glutamate-induced increases in caspase activity were first observed in dendrites and later in the cell body, and focal application of glutamate to individual dendrites resulted in local activation of caspases. Collectively, the data demonstrate that apoptotic biochemical cascades can be activated locally in synapses and dendrites and suggest a role for such local apoptotic signals in synapse loss and neuronal death in neurodegenerative disorders that involve excessive activation of glutamate receptors.

Anti-apoptotic Role of Telomerase in Pheochromocytoma Cells

Telomerase is a protein-RNA enzyme complex that adds a six-base DNA sequence (TTAGGG) to the ends of chromosomes and thereby prevents their shortening. Reduced telomerase activity is associated with cell differentiation and accelerated cellular senescence, whereas increased telomerase activity is associated with cell transformation and immortalization. Because many types of cancer have been associated with reduced apoptosis, whereas cell differentiation and senescence have been associated with increased apoptosis, we tested the hypothesis that telomerase activity is mechanistically involved in the regulation of apoptosis. Levels of telomerase activity in cultured pheochromocytoma cells decreased prior to cell death in cells undergoing apoptosis. Treatment of cells with the oligodeoxynucleotide TTAGGG or with 3,3′-diethyloxadicarbocyanine, agents that inhibit telomerase activity in a concentration-dependent manner, significantly enhanced mitochondrial dysfunction and apoptosis induced by staurosporine, Fe2+ (an oxidative insult), and amyloid β-peptide (a cytotoxic peptide linked to neuronal apoptosis in Alzheimer’s disease). Overexpression of Bcl-2 and the caspase inhibitor zVAD-fmk protected cells against apoptosis in the presence of telomerase inhibitors, suggesting a site of action of telomerase prior to caspase activation and mitochondrial dysfunction. Telomerase activity decreased in cells during the process of nerve growth factor-induced differentiation, and such differentiated cells exhibited increased sensitivity to apoptosis. Our data establish a role for telomerase in suppressing apoptotic signaling cascades and suggest a mechanism whereby telomerase may suppress cellular senescence and promote tumor formation.

Bcl-2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β-Peptide

Abstract: The bcl‐2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl‐2s potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl‐2 on membrane lipid peroxidation. Using hydrogen peroxide (H2O2) and amyloid β‐peptide (Aβ) as lipoperoxidation initiators, we determined the loss of EPR‐detectable paramagnetism of nitroxyl stearate (NS) spin labels 5‐NS and 12‐NS. In intact cell preparations and postnuclear membrane fractions, Aβ and H2O2 induced significant loss of 5‐NS and 12‐NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl‐2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl‐2‐expressing cells, both fractions contained Bcl‐2 protein. 5‐NS and 12‐NS signals were significantly decreased following Aβ and H2O2 exposure in control PC12 mitochondrial membranes, and Bcl‐2 largely prevented these effects. Plasma membrane preparations containing Bcl‐2 were also resistant to radical‐induced loss of spin label. Collectively, our data suggest that Bcl‐2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by Aβ and H2O2, further highlighting the important role of lipid peroxidation in apoptosis.

4‐Hydroxynonenal, a Lipid Peroxidation Product, Rapidly Accumulates Following Traumatic Spinal Cord Injury and Inhibits Glutamate Uptake

Abstract: Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4‐hydroxynonenal (HNE), which cross‐links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein‐bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein‐bound HNE increased in content in the damaged spinal cord at early times following injury (1–24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein‐bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose‐dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms.

17β-Estradiol attenuates oxidative impairment of synaptic Na+/K+-ATPase activity, glucose transport, and glutamate transport induced by amyloid β-peptide and iron

Synapse loss, deposits of amyloid β‐peptide (Aβ), impaired energy metabolism, and cognitive deficits are defining features of Alzheimers disease (AD). Estrogen replacement therapy reduces the risk of developing AD in postmenopausal women. Because synapses are likely sites for initiation of neurodegenerative cascades in AD, we tested the hypothesis that estrogens act directly on synapses to suppress oxidative impairment of membrane transport systems. Exposure of rat cortical synaptosomes to Aβ25‐35 (Aβ) and FeSO4 induced membrane lipid peroxidation and impaired the function of the plasma membrane Na+/K+‐ATPase, glutamate transporter, and glucose transporter. Pretreatment of synaptosomes with 17β‐estradiol or estriol largely prevented impairment of Na+/K+‐ATPase activity, glutamate transport, and glucose transport; other steroids were relatively ineffective. 17β‐Estradiol suppressed membrane lipid peroxidation induced by Aβ and FeSO4, but did not prevent impairment of membrane transport systems by 4‐hydroxynonenal (a toxic lipid peroxidation product), suggesting that an antioxidant property of 17β‐estradiol was responsible for its protective effects. By suppressing membrane lipid peroxidation in synaptic membranes, estrogens may prevent impairment of transport systems that maintain ion homeostasis and energy metabolism, and thereby forestall excitotoxic synaptic degeneration and neuronal loss in disorders such as AD and ischemic stroke. J. Neurosci. Res. 50:522–530, 1997.

Altered calcium homeostasis and mitochondrial dysfunction in cortical synaptic compartments of presenilin-1 mutant mice

Abstract : Alzheimers disease is characterized by amyloid β‐peptide deposition, synapse loss, and neuronal death, which are correlated with cognitive impairments. Mutations in the presenilin‐1 gene on chromosome 14 are causally linked to many cases of early‐onset inherited Alzheimers disease. We report that synaptosomes prepared from transgenic mice harboring presenilin‐1 mutations exhibit enhanced elevations of cytoplasmic calcium levels following exposure to depolarizing agents, amyloid β‐peptide, and a mitochondrial toxin compared with synaptosomes from nontransgenic mice and mice overexpressing wild‐type presenilin‐1. Mitochondrial dysfunction and caspase activation following exposures to amyloid β‐peptide and metabolic insults were exacerbated in synaptosomes from presenilin‐1 mutant mice. Agents that buffer cytoplasmic calcium or that prevent calcium release from the endoplasmic reticulum protected synaptosomes against the adverse effect of presenilin‐1 mutations on mitochondrial function. Abnormal synaptic calcium homeostasis and mitochondrial dysfunction may contribute to the pathogenic mechanism of presenilin‐1 mutations.

Aβ25-35 induces rapid lysis of red blood cells: Contrast with aβ1-42 and examination of underlying mechanisms

Abstract Amyloid β-peptide (Aβ) is produced by many different cell types and circulates in blood and cerebrospinal fluid in a soluble form. In Alzheimers disease (AD), Aβ forms insoluble fibrillar aggregates that accumulate in association with cells of the brain parenchyma and vasculature. Both full-length Aβ (Aβ1–40/42) and the Aβ25–35 fragment can damage and kill neurons by a mechanism that may involve oxidative stress and disruption of calcium homeostasis. Circulating blood cells are exposed to soluble Aβ1–40/42 and may also be exposed to Aβ aggregates associated with the luminal surfaces of cerebral microvessels. We therefore examined the effects of Aβ25–35 and Aβ1–42 on human red blood cells (RBCs) and report that Aβ25–35, in contrast to Aβ1–42, induces rapid (10–60 min) lysis of RBCs. The mechanism of RBC lysis by Aβ25–35 involved ion channel formation and calcium influx, but did not involve oxidative stress because antioxidants did not prevent cell lysis. In contrast, Aβ1–42 induced a delayed (4–24 h) damage to RBCs which was attenuated by antioxidants. The damaging effects of both Aβ25–35 and Aβ1–42 towards RBCs were completely prevented by Congo red indicating a requirement for peptide fibril formation. Aβ1–42 induced membrane lipid peroxidation in RBC, and basal levels of lipid peroxidation in RBCs from AD patients were significantly greater than in age-matched controls, suggesting a possible role for Aβ1–42 in previously reported alterations in RBCs from AD patients.