Impact of hippocampal neuronal ablation on neurogenesis and cognition in the aged brain.

Neuronal loss is the most common and critical feature of a spectrum of brain traumas and neurodegenerative disorders such as Alzheimer's disease (AD). The capacity to generate new neurons in the central nervous system diminishes early during brain development and is restricted mainly to two brain areas in the mature brain: subventricular zone and subgranular zone. Extensive research on the impact of brain injury on endogenous neurogenesis and cognition has been conducted primarily using young animals, when neurogenesis is most active. However, a critical question remains to elucidate the effect of brain injury on endogenous neurogenesis and cognition in older animals, which is far more relevant for age-related neurodegenerative disorders such as AD. Therefore, we examined the impact of neuronal loss on endogenous neurogenesis in aged animals using CaM/Tet-DTA mice, a transgenic model of hippocampal cell loss. Additionally, we investigated whether the upregulation of adult neurogenesis could mitigate cognitive deficits following substantial hippocampal neuronal loss. Our findings demonstrate that aged CaM/Tet-DTA mice that sustain severe neuronal loss exhibit an upregulation of endogenous neurogenesis. However, despite this significant upregulation, neurogenesis alone is not able to mitigate the cognitive deficits observed. Our studies suggest that the aged brain has the capacity to stimulate neurogenesis post-injury; however, multiple therapeutic approaches, including upregulation of endogenous neurogenesis, will be necessary to recover brain function after severe neurodegeneration.

Long term changes in phospho-APP and tau aggregation in the 3xTg-AD mice following cerebral ischemia.

The risk of Alzheimer's disease increases following cerebral hypoperfusion. We studied the long-term interaction between low blood flow to the brain and Alzheimer's disease by inducing a transient global ischemic insult in aged 3xTg-AD mice and determining the effects on AD pathology 3-months post injury. We found that global ischemia does not increase the levels of amyloid-ß in these mice. However, the injury did lead to enhanced phosphorylation of the amyloid precursor protein (APP) at the Thr668 site in both the 3xTg-AD mice and wild-type controls. Furthermore, we found an increase in insoluble total tau 3-months post-injury. Together these findings further elucidate the long-term impact of cerebral hypoperfusion on Alzheimer's disease.

Amyloid-beta expression in retrosplenial cortex of triple transgenic mice: relationship to cholinergic axonal afferents from medial septum.

Triple transgenic (3xTg-AD) mice harboring the presenilin 1, amyloid precursor protein, and tau transgenes (Oddo et al., 2003b) display prominent levels of amyloid-beta (Abeta) immunoreactivity in forebrain regions. The Abeta immunoreactivity is first seen intracellularly in neurons and later as extracellular plaque deposits. The present study examined Abeta immunoreactivity that occurs in layer III of the granular division of retrosplenial cortex (RSg). This pattern of Abeta immunoreactivity in layer III of RSg develops relatively late, and is seen in animals older than 14 months. The appearance of the Abeta immunoreactivity is similar to an axonal terminal field and thus may offer a unique opportunity to study the relationship between afferent projections and the formation of Abeta deposits. Axonal tract tracing techniques demonstrated that the pattern of axon terminal labeling in layer III of RSg, following placement of DiI in medial septum, is remarkably similar to the pattern of cholinergic axons in RSg, as detected by acetylcholinesterase histochemical staining, choline acetyltransferase immunoreactivity, or p75 receptor immunoreactivity; this pattern also is strikingly similar to the band of Abeta immunoreactivity. In animals sustaining early damage to the medial septal nucleus (prior to the advent of Abeta immunoreactivity), the band of Abeta in layer III of RSg does not develop; the corresponding band of cholinergic markers also is eliminated. In older animals (after the appearance of the Abeta immunoreactivity) damage to cholinergic afferents by electrolytic lesions, immunotoxin lesions, or cutting the cingulate bundle, result in a rapid loss of the cholinergic markers and a slower reduction of Abeta immunoreactivity. These results suggest that the septal cholinergic axonal projections transport Abeta or amyloid precursor protein (APP) to layer III of RSg.

Role of calcium in the pathogenesis of Alzheimer's disease and transgenic models.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the elderly that is characterized by memory loss. Neuropathologically, the AD brain is marked by an increased AP burden, hyperphosphorylated tau aggregates, synaptic loss, and inflammatory responses. Disturbances in calcium homeostasis are also one of the earliest molecular changes that occur in AD patients, alongside alterations in calcium-dependent enzymes in the post-mortem brain. The sum of these studies suggests that calcium dyshomeostasis is an integral part of the pathology, either influencing AP production, mediating its effects or both. Increasing evidence from in vitro studies demonstrates that the AP peptide could modulate a number of ion channels increasing calcium influx, including voltage-gated calcium and potassium channels, the NMDA receptor, the nicotinic receptor, as well as forming its own calcium-conducting pores. In vivo evidence has shown that A3 impairs both LTP and cognition, whereas all of these ion channels cluster at the synapse and underlie synaptic transmission and hence cognition. Here we consider the evidence that AP causes cognitive deficits through altering calcium homeostasis at the synapse, thus impairing synaptic transmission and LTP. Furthermore, this disruption appearr to occur without overt or extensive neuronal loss, as it is observed in transgenic mouse models of AD, but may contribute to the synaptic loss, which is an early event that correlates best with cognitive decline.

Modeling behavioral and neuronal symptoms of Alzheimer's disease in mice: a role for intraneuronal amyloid.

The amyloid Abeta-peptide (Abeta) is suspected to play a critical role in the cascade leading to AD as the pathogen that causes neuronal and synaptic dysfunction and, eventually, cell death. Therefore, it has been the subject of a huge number of clinical and basic research studies on this disease. Abeta is typically found aggregated in extracellular amyloid plaques that occur in specific brain regions enriched in nAChRs in Alzheimer's disease (AD) and Down syndrome (DS) brains. Advances in the genetics of its familiar and sporadic forms, together with those in gene transfer technology, have provided valuable animal models that complement the traditional cholinergic approaches, although modeling the neuronal and behavioral deficits of AD in these models has been challenging. More recently, emerging evidence indicates that intraneuronal accumulation of Abeta may also contribute to the cascade of neurodegenerative events and strongly suggest that it is an early, pathological biomarker for the onset of AD and associated cognitive and other behavioral deficits. The present review covers these studies in humans, in in vitro and in transgenic models, also providing more evidence that adult 3xTg-AD mice harboring PS1M146V, APPSwe, tauP301L transgenes, and mimicking many critical hallmarks of AD, show cognitive deficits and other behavioral alterations at ages when overt neuropathology is not yet observed, but when intraneuronal Abeta, synaptic and cholinergic deficits can already be described.

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.

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.

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.

Fibril formation and neurotoxicity by a herpes simplex virus glycoprotein B fragment with homology to the Alzheimer's A beta peptide.

Despite significant progress in the elucidation of the genetic basis of early-onset familial Alzheimer's disease (AD), the etiology of sporadic cases remains elusive. Although certain genetic loci play a role in conferring susceptibility in some sporadic AD cases, it is likely that the etiology is multifactorial; hence, the majority of cases cannot be attributed to genetic factors alone, indicating that environmental factors may modulate the onset and/or progression of the disease. Head injury and infectious agents are environmental factors that have been periodically implicated, but no plausible mechanisms have been clearly identified. With regard to infectious agents, speculation has often centered on the neurotropic herpes viruses, with herpes simplex virus 1 (HSV1) considered a likely candidate. We report that an internal sequence of HSV1 glycoprotein B (gB) is homologous to the carboxyl-terminal region of the A beta peptide that accumulates in diffuse and neuritic plaques in AD. Synthetic peptides were generated and the biophysical and biological properties of the viral peptide compared to those of A beta. Here we show that this gB fragment forms beta-pleated sheets, self-assembles into fibrils that are thioflavin-positive and ultrastructurally indistinguishable from A beta, accelerates the formation of A beta fibrils in vitro, and is toxic to primary cortical neurons at doses comparable to those of A beta. These findings suggest a possible role for this infectious agent in the pathophysiology of sporadic cases of AD.

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.

Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Abeta stabilization.

The brains of individuals with Alzheimer's disease (AD) are characterized by extracellular deposition of beta-amyloid protein (Abeta), intracellular neurofibrillary tangles, and loss of neurons. To study molecular markers associated with dying cells in the AD brain, in situ DNA labeling techniques were used to visualize cells with DNA fragmentation. We observed that intracellular accumulation of apolipoprotein E (apoE) is correlated with the detection of intracellular Abeta-like immunoreactivity within the same cytoplasmic granules, suggesting that uptake of lipids may have stabilized the hydrophobic Abeta protein within the cell. These apoE-containing neurons also exhibit high expression of a cell surface receptor, gp330, which is known to bind apoE. Cells containing significant nuclear DNA fragmentation express the highest level of cell surface gp330. Extracellular deposition of Abeta is detected only upon neuronal cell death, initially as halos of Abeta immunoreactivity around individual dying neurons, and subsequently as Abeta plaques containing numerous neuronal cell ghosts. Based on our in situ analysis of nuclear DNA fragmentation, we conclude that neuronal cell death likely occurs before the extracellular deposition of Abeta in AD brains.

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.

Extracellular deposition of beta-amyloid upon p53-dependent neuronal cell death in transgenic mice.

The finding that intracellular expression of the beta-amyloid protein (Abeta) under a neuron-specific promoter led progressively to degeneration and death of neurons in the brains of transgenic mice provides a unique opportunity to utilize this animal model to both understand the mechanism that underlies neuronal cell death and define the complexity of events which may ensue. We observed a correlation between Abeta accumulation in selective neurons and activation of p53, a protein that has been implicated in the induction of apoptosis. Histological and immunohistochemical evaluations of adjacent brain sections suggest that expression of p53 is accompanied by nuclear DNA fragmentation. In certain regions with marked neuronal cell death, extracellular deposition of A(beta) became evident, together with the local activation of astrocytes. Interestingly, the neuritic structures underlying the Abeta deposits showed altered synaptophysin immunoreactivity and morphologic evidence for damage. This transgenic mouse model suggests that intracellular generation of the Abeta protein not only leads to the death of the neuron but may also functionally impair neighboring neurons as well. It further offers a mechanism whereby neuritic plaques may be derived.

Injury induces presenilin-1 gene expression in mouse brain.

Information regarding the genetic factors and environmental conditions that influence presenilin-1 (PS-1) gene expression is essential for the elucidation of its pathophysiological role in Alzheimer's disease (AD). Previous in situ hybridization studies have demonstrated that neurons are the predominant cell type expressing PS-1 in the mammalian central nervous system (CNS) under physiological conditions. In this study, we examined the consequences of an experimentally induced focal injury on PS-1 gene expression in the mouse CNS. Physical lesions to white matter regions produced a robust increase in PS-1 gene expression in non-neuronal cells immediately surrounding the site of injury. These findings underscore the epidemiological evidence that implicate head injury as a risk factor for AD and suggest a possible role for PS-1 in this capacity.

Widespread neuronal expression of the presenilin-1 early-onset Alzheimer's disease gene in the murine brain.

Mutations in the presenilin-1 (S182) gene have been genetically linked to early-onset Alzheimer's disease. To clarify the underlying molecular mechanism through which presenilin-1 is involved in the pathogenesis of this neurodegenerative disorder, the regional and cellular transcription profile of this gene was characterized in primary cells isolated from the murine brain by Northern blot hybridization using digoxigenin-labeled riboprobes. Our results indicate that presenilin-1 mRNA transcripts are widely distributed throughout the adult mouse brain. Furthermore, immunohistochemical labeling of hybridized sections indicates that expression was predominantly localized to neuronal cells. Neurons in the hippocampus and cerebral cortex, which are severely compromised in Alzheimer's disease, showed prominent expression of presenilin-1. In contrast, white matter areas and endothelial cells do not appear to express presenilin-1 to detectable levels. presenilin-1 transcripts, however, are also present less frequently in certain nonneuronal cell populations such as ependymal cells in the choroid plexus. Analysis of primary cells isolated from murine brain supported the results obtained by in situ hybridization and showed that cultured primary neurons and astrocytes express presenilin-1. Overall, it appears that the pattern of presenilin-1 gene expression parallels that previously described for the amyloid precursor protein.