Ward A. Pedersen

Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease

Alzheimers disease (AD) is an age-related disorder characterized by deposition of amyloid β-peptide (Aβ) and degeneration of neurons in brain regions such as the hippocampus, resulting in progressive cognitive dysfunction. The pathogenesis of AD is tightly linked to Aβ deposition and oxidative stress, but it remains unclear as to how these factors result in neuronal dysfunction and death. We report alterations in sphingolipid and cholesterol metabolism during normal brain aging and in the brains of AD patients that result in accumulation of long-chain ceramides and cholesterol. Membrane-associated oxidative stress occurs in association with the lipid alterations, and exposure of hippocampal neurons to Aβ induces membrane oxidative stress and the accumulation of ceramide species and cholesterol. Treatment of neurons with α-tocopherol or an inhibitor of sphingomyelin synthesis prevents accumulation of ceramides and cholesterol and protects them against death induced by Aβ. Our findings suggest a sequence of events in the pathogenesis of AD in which Aβ induces membrane-associated oxidative stress, resulting in perturbed ceramide and cholesterol metabolism which, in turn, triggers a neurodegenerative cascade that leads to clinical disease.

Folic Acid Deficiency and Homocysteine Impair DNA Repair in Hippocampal Neurons and Sensitize Them to Amyloid Toxicity in Experimental Models of Alzheimer's Disease

Recent epidemiological and clinical data suggest that persons with low folic acid levels and elevated homocysteine levels are at increased risk of Alzheimers disease (AD), but the underlying mechanism is unknown. We tested the hypothesis that impaired one-carbon metabolism resulting from folic acid deficiency and high homocysteine levels promotes accumulation of DNA damage and sensitizes neurons to amyloid β-peptide (Aβ) toxicity. Incubation of hippocampal cultures in folic acid-deficient medium or in the presence of methotrexate (an inhibitor of folic acid metabolism) or homocysteine induced cell death and rendered neurons vulnerable to death induced by Aβ. Methyl donor deficiency caused uracil misincorporation and DNA damage and greatly potentiated Aβ toxicity as the result of reduced repair of Aβ-induced oxidative modification of DNA bases. When maintained on a folic acid-deficient diet, amyloid precursor protein (APP) mutant transgenic mice, but not wild-type mice, exhibited increased cellular DNA damage and hippocampal neurodegeneration. Levels of Aβ were unchanged in the brains of folate-deficient APP mutant mice. Our data suggest that folic acid deficiency and homocysteine impair DNA repair in neurons, which sensitizes them to oxidative damage induced by Aβ.

Cellular and Molecular Mechanisms Underlying Perturbed Energy Metabolism and Neuronal Degeneration in Alzheimer's and Parkinson's Diseases

ABSTRACT: Synaptic degeneration and death of nerve cells are defining features of Alzheimers disease (AD) and Parkinsons disease (PD), the two most prevalent age‐related neurodegenerative disorders. In AD, neurons in the hippocampus and basal forebrain (brain regions that subserve learning and memory functions) are selectively vulnerable. In PD dopamine‐producing neurons in the substantia nigra‐striatum (brain regions that control body movements) selectively degenerate. Studies of postmortem brain tissue from AD and PD patients have provided evidence for increased levels of oxidative stress, mitochondrial dysfunction and impaired glucose uptake in vulnerable neuronal populations. Studies of animal and cell culture models of AD and PD suggest that increased levels of oxidative stress (membrane lipid peroxidation, in particular) may disrupt neuronal energy metabolism and ion homeostasis, by impairing the function of membrane ion‐motive ATPases and glucose and glutamate transporters. Such oxidative and metabolic compromise may thereby render neurons vulnerable to excitotoxicity and apoptosis. Studies of the pathogenic mechanisms of AD‐linked mutations in amyloid precursor protein (APP) and presenilins strongly support central roles for perturbed cellular calcium homeostasis and aberrant proteolytic processing of APP as pivotal events that lead to metabolic compromise in neurons. Specific molecular “players” in the neurodegenerative processes in AD and PD are being identified and include Par‐4 and caspases (bad guys) and neurotrophic factors and stress proteins (good guys). Interestingly, while studies continue to elucidate cellular and molecular events occurring in the brain in AD and PD, recent data suggest that both AD and PD can manifest systemic alterations in energy metabolism (e.g., increased insulin resistance and dysregulation of glucose metabolism). Emerging evidence that dietary restriction can forestall the development of AD and PD is consistent with a major “metabolic” component to these disorders, and provides optimism that these devastating brain disorders of aging may be largely preventable.

Neurodegenerative disorders and ischemic brain diseases

Degeneration and death of neurons is the fundamental process responsible for the clinical manifestations of many different neurological disorders of aging, incuding Alzheimers disease, Parkinsons disease and stroke. The death of neurons in such disorders involves apoptotic biochemical cascades involving upstream effectors (Par-4, p53 and pro-apoptotic Bcl-2 family members), mitochondrial alterations and caspase activation. Both genetic and environmental factors, and the aging process itself, contribute to intiation of such neuronal apoptosis. For example, mutations in the amyloid precursor protein and presenilin genes can cause Alzheimers disease, while head injury is a risk factor for both Alzheimers and Parkinsons diseases. At the cellular level, neuronal apoptosis in neurodegenerative disorders may be triggered by oxidative stress, metabolic compromise and disruption of calcium homeostasis. Neuroprotective (anti-apoptotic) signaling pathways involving neurotrophic factors, cytokines and “conditioning responses” can counteract the effects of aging and genetic predisposition in experimental models of neurodegenerative disorders. A better understanding of the molecular underpinnings of neuronal death is leading directly to novel preventative and therapeutic approaches to neurodegenerative disorders.

Presenilins, the Endoplasmic Reticulum, and Neuronal Apoptosis in Alzheimer's Disease

Abstract: Many cases of autosomal dominant inherited forms of early‐onset Alzheimers disease are caused by mutations in the genes encoding presenilin‐1 (PS‐1; chromosome 14) and presenilin‐2 (PS‐2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrities. PS‐1 and PS‐2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel‐12, which is involved in Notch signaling, and phenotypic similarities of PS‐1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid β‐peptide (Aβ1‐42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid β‐peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimers disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.

ALS-linked Cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis.

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Evidence that accumulation of ceramides and cholesterol esters mediates oxidative stress-induced death of motor neurons in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the spinal cord resulting in progressive paralysis and death. The pathogenic mechanism of ALS is unknown but may involve increased oxidative stress, overactivation of glutamate receptors, and apoptosis. We report abnormalities in sphingolipid and cholesterol metabolism in the spinal cords of ALS patients and in a transgenic mouse model (Cu/ZnSOD mutant mice), which manifest increased levels of sphingomyelin, ceramides, and cholesterol esters; in the Cu/ZnSOD mutant mice, these abnormalities precede the clinical phenotype. In ALS patients and Cu/Zn‐SOD mutant mice, increased oxidative stress occurs in association with the lipid alterations, and exposure of cultured motor neurons to oxidative stress increases the accumulation of sphingomyelin, ceramides, and cholesterol esters. Pharmacological inhibition of sphingolipid synthesis prevents accumulation of ceramides, sphingomyelin, and cholesterol esters and protects motor neurons against death induced by oxidative and excitotoxic insults. These findings suggest a pivotal role for altered sphingolipid metabolism in the pathogenesis of ALS.

Urocortin, but not urocortin II, protects cultured hippocampal neurons from oxidative and excitotoxic cell death via corticotropin-releasing hormone receptor type I

Urocortin and urocortin II are members of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. Two high-affinity G-protein-coupled receptors have been identified that bind CRH and/or urocortin I and II, designated CRHR1 and CRHR2, both of which are present in hippocampal regions of mammalian brain. The hippocampus plays an important role in regulating stress responses and is a brain region in which neurons are vulnerable during disease and stress conditions, including cerebral ischemia, Alzheimers disease, and anxiety disorders. Here we report that urocortin exerts a potent protective action in cultured rat hippocampal neurons with concentrations in the range of 0.5–5.0 pm, increasing the resistance of the cells to oxidative (amyloid β-peptide, 4-hydroxynonenal, ferrous sulfate) and excitotoxic (glutamate) insults. We observed that urocortin is 10-fold more potent than CRH in protecting hippocampal neurons from insult, whereas urocortin II is ineffective. RT-PCR and sequencing analyses revealed the presence of both CRHR1 and CRHR2 in the hippocampal cultures, with CRHR1 being expressed at much higher levels than CRHR2. Using subtype-selective CRH receptor antagonists, we provide evidence that the neuroprotective effect of exogenously added urocortin is mediated by CRHR1. Furthermore, we provide evidence that the signaling pathway that mediates the neuroprotective effect of urocortin involves cAMP-dependent protein kinase, protein kinase C, and mitogen-activated protein kinase. This is the first demonstration of a biological activity of urocortin in hippocampal neurons, suggesting a role for the peptide in adaptive responses of hippocampal neurons to potentially lethal oxidative and excitotoxic insults.

A mechanism for the neuroprotective effect of apolipoprotein E: isoform-specific modification by the lipid peroxidation product 4-hydroxynonenal.

Abstract: Inheritance of the apolipoprotein E (apoE) ε4 allele increases the risk for Alzheimers disease and may also influence the pathogenesis of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The influence of apoE genotype on disease susceptibility must ultimately be explained by the fact that apoE proteins differ in only two amino acids: apoE2 has two cysteine residues, apoE3 has one cysteine residue, and apoE4 has none. We previously reported increased protein modification by the lipid peroxidation product 4‐hydroxynonenal (HNE), which covalently binds to proteins on cysteine residues, in human ALS lumbar spinal cord. We now report increased levels of HNE‐modified apoE in lumbar spinal cord samples from mice expressing an ALS‐linked mutation in Cu/Zn‐superoxide dismutase relative to controls. Studies of interactions of pure apoE proteins with HNE showed that the isoforms differ in the amount of HNE they can bind, with the order E2 > E3 > E4. This correlated with the differential ability of apoE isoforms to protect against apoptosis induced by HNE in cultures of mouse spinal cord motor neurons and by the amyloid β‐peptide in cultures of rat hippocampal neurons. These data suggest that apoE plays a major role in detoxifying HNE, and the differential neuroprotective effect of its isoforms may help explain the relationship between apoE genotype and the susceptibility to neurodegenerative diseases.

The Lipid Peroxidation Product 4-Hydroxynonenal Impairs Glutamate and Glucose Transport and Choline Acetyltransferase Activity in NSC-19 Motor Neuron Cells

Both oxidative stress and excitotoxicity are implicated in the pathogenesis of a number of neurodegenerative disorders, such as amyotrophic lateral sclerosis. We previously reported increased modification of proteins by 4-hydroxynonenal (HNE), a product of membrane lipid peroxidation, in the spinal cords of patients with amyotrophic lateral sclerosis relative to controls. In the current study, we examined the functional consequences of protein modification by HNE in a cell line with a motor neuron phenotype, NSC-19. Treatment of NSC-19 cells with FeSO4, which catalyzes lipid peroxidation, or HNE induced concentration-dependent decreases in glucose and glutamate transport. Vitamin E and propyl gallate blocked the impairment of glucose and glutamate transport caused by FeSO4 in these cells, but not that caused by HNE, whereas glutathione blocked the effects of FeSO4 as well as HNE. Both FeSO4 and HNE caused an increase in the number of apoptotic nuclei in NSC-19 cultures, but this occurred subsequent to the impairment of glucose and glutamate transport. Reductions in choline acetyltransferase activity were also observed in FeSO4- or HNE-treated NSC-19 cells before induction of apoptosis. Our results suggest that, prior to cell death, oxidative stress and HNE down-regulate cholinergic markers and impair glucose and glutamate transport in motor neurons, the latter of which may lead to excitotoxic degeneration of the cells.