Subcellular mechanisms of presenilin-mediated enhancement of calcium signaling.

Mutations in presenilin-1 (PS1), the leading cause of early-onset, autosomal-dominant familial Alzheimer's disease (FAD), enhance calcium signaling mediated by inositol 1,4,5-trisphosphate (IP3). To elucidate the subcellular mechanisms underlying this enhancement, we used high resolution line-scanning confocal microscopy to image elementary calcium release events ("puffs") in Xenopus oocytes expressing wild-type or mutant PS1. Here we report that mutant PS1-rendered puffs more sensitive to IP3 and increased both the magnitude and the rate of calcium release during each event. These effects were not attributable to quantitative changes in the levels of IP3 receptors or their distribution on the ER, but were instead associated with an abnormal elevation of ER calcium stores. Together, our results suggest that the effects of mutant PS1 on calcium signaling are manifested predominantly at the level of the regulation of calcium stores rather than via perturbations in the numbers or activity of IP3-activated calcium release channels.

Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling.

DsRed, a recently cloned red fluorescent protein, has attracted great interest as an expression tracer and fusion partner for multicolor imaging. We report that three-photon excitation (lambda <760 nm) rapidly changes the fluorescence of DsRed from red to green when viewed subsequently by conventional (one-photon) epifluorescence. Mechanistically, three-photon excitation (lambda <760 nm) selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction of fluorescence resonance energy transfer (FRET). The "greening" effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 h without affecting cell viability. This technique allows individual cells, organelles, and fusion proteins to be optically marked and has potential utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population. We describe optimal parameters to induce the color change of DsRed, and demonstrate applications that show the potential of this optical highlighter.

Calsenilin reverses presenilin-mediated enhancement of calcium signaling.

Most cases of autosomal-dominant familial Alzheimer's disease are linked to mutations in the presenilin genes (PS1 and PS2). In addition to modulating beta-amyloid production, presenilin mutations also produce highly specific and selective alterations in intracellular calcium signaling. Although the molecular mechanisms underlying these changes are not known, one candidate molecular mediator is calsenilin, a recently identified calcium-binding protein that associates with the C terminus of both PS1 and PS2. In this study, we investigated the effects of calsenilin on calcium signaling in Xenopus oocytes expressing either wild-type or mutant PS1. In this system, mutant PS1 potentiated the amplitude of calcium signals evoked by inositol 1,4,5-trisphosphate and also accelerated their rates of decay. We report that calsenilin coexpression reverses both of these potentially pathogenic effects. Notably, expression of calsenilin alone had no discernable effects on calcium signaling, suggesting that calsenilin modulates these signals by a mechanism independent of simple calcium buffering. Our findings further suggest that the effects of presenilin mutations on calcium signaling are likely mediated through the C-terminal domain, a region that has also been implicated in the modulation of beta-amyloid production and cell death.

Capacitative calcium entry deficits and elevated luminal calcium content in mutant presenilin-1 knockin mice.

Dysregulation of calcium signaling has been causally implicated in brain aging and Alzheimer's disease. Mutations in the presenilin genes (PS1, PS2), the leading cause of autosomal dominant familial Alzheimer's disease (FAD), cause highly specific alterations in intracellular calcium signaling pathways that may contribute to the neurodegenerative and pathological lesions of the disease. To elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations.

Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders.

Endoplasmic reticulum (ER) is a multifaceted organelle that regulates protein synthesis and trafficking, cellular responses to stress, and intracellular Ca2+ levels. In neurons, it is distributed between the cellular compartments that regulate plasticity and survival, which include axons, dendrites, growth cones and synaptic terminals. Intriguing communication networks between ER, mitochondria and plasma membrane are being revealed that provide mechanisms for the precise regulation of temporal and spatial aspects of Ca2+ signaling. Alterations in Ca2+ homeostasis in ER contribute to neuronal apoptosis and excitotoxicity, and are being linked to the pathogenesis of several different neurodegenerative disorders, including Alzheimer's disease and stroke.

Regional hypomyelination and dysplasia in transgenic mice with astrocyte-directed expression of interferon-gamma.

Interferon-gamma (IFN-gamma), traditionally associated with a variety of physiological and pathological processes of the immune system, manifests an array of biological effects on cells of the nervous system. Clinical and in vitro studies support a key role for IFN-gamma in the pathogenesis of immune-mediated demyelinating disorders such as multiple sclerosis (MS). To investigate the role of this cytokine within the central nervous system (CNS), transgenic mice were derived in which IFN-gamma transgene expression was selectively targeted to astrocytes, a potentially important cellular source of this cytokine. Here we report that astrocyte-directed expression of IFN-gamma results in regional hypomyelination and selective disruption of brain histogenesis, which included severe cerebellar and hippocampal dysplasia. Transgenic mice were markedly ataxic and the majority died prior to reaching sexual maturity. This study demonstrates that astrocyte-directed expression of IFN-gamma profoundly affects the differentiation and morphogenesis of the brain and provides additional evidence that this cytokine has deleterious consequences on myelin-producing cells, independent of the cellular source.

Presenilin-2 mutations modulate amplitude and kinetics of inositol 1, 4,5-trisphosphate-mediated calcium signals.

Mutations in the two presenilin genes (PS1, PS2) account for the majority of early-onset familial Alzheimer's disease (FAD) cases. Converging evidence from a variety of experimental systems, including fibroblasts from FAD patients and transgenic animals, indicates that PS1 mutations modulate intracellular calcium signaling pathways. Despite the potential relevance of these changes to the pathogenesis of FAD, a comparable effect for PS2 has not yet been demonstrated experimentally. We examined the effects of wild-type PS2, and both of the identified FAD mutations in PS2, on intracellular calcium signaling in Xenopus oocytes. Inositol 1,4, 5-trisphosphate (IP(3))-evoked calcium signals were significantly potentiated in cells expressing either of the PS2 mutations relative to wild-type PS2-expressing cells and controls. Decay rates of calcium signals were also significantly accelerated in mutant PS2-expressing cells in a manner dependent upon IP(3) concentration. The finding that mutations in both PS1 and PS2 modulate intracellular calcium signaling suggests that these disturbances may represent a common pathogenic mechanism of presenilin-associated FAD.

Alzheimer's presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes.

Perturbations in intracellular Ca2+ signaling may represent one mechanism underlying Alzheimer's disease (AD). The presenilin-1 gene (PS1), associated with the majority of early onset familial AD cases, has been implicated in this signaling pathway. Here we used the Xenopus oocyte expression system to investigate in greater detail the role of PS1 in intracellular Ca2+ signaling pathways. Treatment of cells expressing wild-type PS1 with a cell surface receptor agonist to stimulate the phosphoinositide second messenger pathway evoked Ca2+-activated Cl- currents that were significantly potentiated relative to controls. To determine which elements of the signal transduction pathway are responsible for the potentiation, we used photolysis of caged inositol 1,4,5-trisphosphate (IP3) and fluorescent Ca2+ imaging to demonstrate that PS1 potentiates IP3-mediated release of Ca2+ from internal stores. We show that an AD-linked mutation produces a potentiation in Ca2+ signaling that is significantly greater than that observed for wild-type PS1 and that cannot be attributed to differences in protein expression levels. Our findings support a role for PS1 in modulating IP3-mediated Ca2+ liberation and suggest that one pathophysiological mechanism by which PS1 mutations contribute to AD neurodegeneration may involve perturbations of this function.

Herpes simplex virus infections and Alzheimer's disease. Implications for drug treatment and immunotherapy.

Interest in the possible role of herpes simplex virus type 1 (HSV1) as a cofactor in the pathogenesis of Alzheimer's disease (AD) has re-emerged following the detection of viral DNA sequences in the central nervous system (CNS). Evidence from 2 independent laboratories indicates that HSV1 may interact with a host-specific factor, the apolipoprotein E epsilon 4 allele, to further augment the risk for AD. In this review, we consider the arguments implicating HSV1 in the pathogenesis of AD. Although further studies are required to confirm a role for HSV1 in AD and to elucidate its underlying molecular basis, implicating a virus in the pathogenesis of this insidious disease clearly offers novel potential treatments.

Presenilin-1 immunoreactivity is localized intracellularly in Alzheimer's disease brain, but not detected in amyloid plaques.

The identification of the cellular and subcellular regions of the Alzheimer's disease brain to which the presenilin-1 (PS-1) protein localizes is expected to contribute to an understanding of its pathophysiological role. Toward this end, we have derived an affinity-purified antibody to a synthetic PS-1 peptide. In this report, we demonstrate that this antibody, called SW2, specifically recognizes full-length, 47-kDa PS-1 protein from rat primary cortical neurons, from a human neuronal cell line, and from human brain extracts on Western immunoblots. Immunohistochemical analysis of postmortem brain tissue from control and Alzheimer's disease patients using this SW2 antibody indicates an intracellular localization of PS-1 immunoreactivity with prominent perinuclear characteristics in neurons, with staining also detected in neuritic processes. Despite various treatments of the tissue sections, no PS-1 immunoreactivity was observed in neuritic plaques, the hallmark pathological lesions of Alzheimer's disease. In addition, confocal microscopic analysis of immunostained cultured primary neurons revealed a prominent perinuclear pattern of PS-1 immunoreactivity consistent with vesicular localization, as well as punctate staining in neuritic processes.