Microglial activation induced by factor(s) contained in sera from Alzheimer-related ApoE genotypes

Several factors that increase the likelihood of developing Alzheimer's disease (AD) have already been identified. A correct evaluation of these may contribute to a better understanding of the etiology of the disease. The risk of developing AD definitely increases with (a) age, (b) head injuries, (c) family history of AD or Down syndrome, (d) sex (higher prevalence of AD in women), (e) vascular disease, (f) exposure to environmental toxins, (g) infectious processes, or (h) changes in immune function, and recent advances in molecular genetics have suggested that genetic predisposition (i) can be considered one of the most important risk factors in the development of AD. A significant increase in the number of amyloid plaques in AD patients with an apolipoprotein E4 (ApoE) allele has been observed and the results of several genetic studies indicate that the etiology of this neurodegenerative disease is associated with the presence of the allele E4 of ApoE. A potential source of damage in the AD brain is an altered response triggered by microglial activation, which is associated with amyloid plaques. It has become evident that a dysregulation of cytokine release appears within lesions of many types of brain disorders including infection, trauma, stroke, and neurodegenerative diseases. Many studies have shown that microglia secrete both cytokines and cytotoxins and since reactive microglia appears in nearly every type of brain damage, it is likely that their secreted products ultimately help to determine the rate of damaged brain tissue. In this study, in vitro cell cultures were established to investigate the effect of different concentrations of human sera (2.5% and 10%) with specific ApoE genotypes from Alzheimer's and non-Alzheimer's subjects on ameboid and flat microglial cells obtained from neonatal rat hippocampi. Results show that a modulation in the proliferation and activation of microglial cells was obtained and that AD sera, mainly in the ApoE 3/ 4 and 4/ 4 genotype contain factor(s) which are able to induce morphological changes, as measured by an increase in the ameboid cell type. In addition, major histocompatibility complex (MHC) class II antigen expression, as measured by flow cytometric analysis, and interleukin-1β (IL-1β) release as measured by enzyme linked immunoadsorbent assay (ELISA), in comparison with control groups and lipopolysaccharide (LPS)-treated cells, clearly demonstrate a direct effect of ApoE 3/ 4 and 4/ 4 and/ or an indirect effect mediated by the release of IL-1β on microglia activation. These results strongly suggest that primary in vitro microglial cell cultures can be used as a screening model to test human sera as well as the effect of new potential drugs aimed at down-regulating microglia activation. J. Neurosci. Res. 54:539–553, 1998. © 1998 Wiley-Liss, Inc.     

Neurons Induce the Activation of Microglial Cellsin Vitro

Although microglial cells are well known to become activated in the pathological brain, mechanisms underlying the microglial activation are not fully understood. In the present study, with an aim to elucidate whether neurons are involved in the microglial activation, we compared the morphology and the superoxide anion (O₂⁻)-generating activity of rat microglial cells in pure culture with those of cells cocultured with rat primary cortical neurons. Microglial cells in pure culture in serum-free Eagle's minimum essential medium on poly- -lysine-coated coverslips displayed ramified morphology and suppressed activity of O₂⁻generation. In contrast, microglial cells in neuron–microglia coculture under the same conditions as those for the pure culture displayed ameboid shape and upregulated activity of O₂⁻generation. Electron microscopic observation revealed that microglial cells in coculture were more abundant in Golgi apparatus and secretory granules than those in pure culture and that some of microglial cells in the vicinity of neurites exhibited membrane specialization reminiscent of a junctional apparatus with high electron density between a microglial soma and a neurite. Microglial cells in coculture tended to tie neurites in bundles by extending processes. Medium conditioned by neurons significantly enhanced O₂⁻generation by microglia, but microglial cells in contact with or in close apposition to cocultured neurons were much more intensely activated than those remote from the neurons. Furthermore, the membrane fraction of cortical neurons activated microglial cells, and this effect was abolished by treating the neuronal membrane with trypsin or neuraminidase. In conclusion, neuronal–microglial contact may be necessary to mediate microglial activation. The present findings suggest that the contact of microglia with damaged neurons in the brain is a plausible cause to activate microglia in the neuropathological pro cesses.     

Rat microglial interleukin-3

Interleukin-3 (IL-3, multi-CSF) is a growth factor for a variety of hematopoietic progenitor cells. Recently, microglial cells, the resident macrophages of the central nervous system (CNS) have been shown to proliferate in the presence of IL-3 both in vivo and in culture. Data obtained from cultured astrocytes gave rise to the hypothesis that astrocytes synthesize the microglial growth factor. This is the first report identifying rat microglial cells themselves as a source of IL-3. Culture media conditioned by isolated microglia enhanced microglial proliferation above fresh media controls. IL-3 polypeptide was detected in both conditioned media (CM) and in microglial cells by Western blotting and immunoprecipitation. Furthermore, anti-IL-3 antibodies were able to inhibit microglial proliferation induced by conditioned media. mRNA^IL-3 was present in single microglial cells as revealed by in situ hybridization. Total RNA prepared from purified microglia yielded a single PCR amplification product. Identity of the PCR product was confirmed by Southern blot hybridization using a cDNA^IL-3 probe and by DNA sequencing. Expression of mRNA^IL-3 was observed in both absence and presence of lipopolysaccharide, a bacterial endotoxin, that commonly induces expression of inflammatory cytokines and inhibits microglial proliferation. It is concluded that IL-3 expression in microglial cells is an early marker of inflammatory events in the brain preceding the expression of other cytokines and most likely ensuring the recruitment of enhanced numbers of immunocompetent cells at sites of lesion. In the light of weak immune reactions in the brain, it is hypothesized that the expression of a characteristic T cell feature in monocyte-derived microglia may be a partial compensation of T cell functions in brain lesions.     

Reduction of the microglial cell number in rat primary glial cell cultures by exogenous addition of dibutyryl cyclic adenosine monophosphate

The present work examined the effects induced by dibutyryl cyclic adenosine monophosphate (dB–cAMP) on microglial cells in primary glial cell cultures from newborn rats. Microglial cells were identified by OX42 immunohistochemistry and nucleoside diphosphatase histochemistry. Double staining for astrocytes was carried out by combination with glial fibrillary acidic protein immunolabeling. Addition of 0.25 mM dB–cAMP to the cultures decreased the microglial cell number about sixfold. The findings suggest that the effect of dB–cAMP on the microglial cells might be either a direct action of dB–cAMP on the microglial cells or an indirect effect mediated by the astroglial cells.     

Differential expression of chemokines and chemokine receptors during microglial activation and inhibition

Intrauterine infection produces an inflammatory response in the fetus characterized by increased inflammatory cytokines in the fetal brain and activation of brain microglial cells. Intrauterine infection can release bacterial cell wall products into the fetal circulation. Lipopolysaccharides (LPS) are derived from the cell walls of gram negative organisms. The degree of microglial cell activation may influence the extent of brain injury following an inflammatory stimulus. Chemokines, which are released by activated microglia, regulate the influx of inflammatory cells to the brain. Accordingly, therapeutic strategies that reduce the extent of chemokine expression in microglial cells may prove neuroprotective. Minocycline (MN), a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents and inhibits microglial cell activation. To determine if minocycline can reduce the production of chemokines and chemokine receptors in response to LPS, microglial-like BV-2 and HAPI cells were cultured in the presence or absence of 100 ng/ml of LPS. Enzyme-linked immunosorbent assay (ELISA) and semi-quantitative RT-PCR were used to examine changes in inflammatory chemokines (macrophage inflammatory protein-1 (MIP-1alpha), regulated upon activation, normal T cell expressed and secreted (RANTES), and inducible protein-10 (IP-10)) and chemokine receptor (C-C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3)) production, respectively. We found that in both cell lines chemokine release after 4-, 8-, and 16-h exposure to LPS was significantly higher compared to non-exposed cells for all the chemokines measured, P<0.001. Minocycline inhibited chemokine release of LPS-stimulated BV-2 cells. There was even greater inhibition (up to 50%) of mRNA expression after exposure to LPS (P<0.001). We conclude that endotoxin enhanced the expression of chemokines and chemokine receptors in microglial-like cell lines. Modulation of this expression was achieved with minocycline. Recognition of the mechanisms whereby minocycline exerts its anti-inflammatory effect on microglia may uncover specific targets for pharmacologic intervention.     

Secretion of apolipoprotein E by brain glia requires protein prenylation and is suppressed by statins

Apolipoprotein E (ApoE) genotype modulates the risk of Alzheimer's disease. ApoE has been shown essential for amyloid beta-peptide fibrillogenesis and deposition, a defining pathological feature of this disease. Because astrocytes and microglia represent the major source of extracellular apoE in brain, we investigated apoE secretion by glia. We determined that protein prenylation is required for apoE release from a continuous microglial cell line, primary mixed glia, and from organotypic hippocampal cultures. Using selective protein prenylation inhibitors, apoE secretion was found to require protein geranylgeranylation. This prenylation involved a protein critical to apoE secretion, not apoE proper. ApoE secretion could also be suppressed by inhibiting synthesis of mevalonate, the precursor to both types of protein prenylation, using hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins). Recent reports have described the beneficial effects of statins on the risk of dementia. Our finding that protein geranylgeranylation is required for apoE secretion in the brain parenchyma provides another contributing mechanism to explain the effective properties of statins against the development of dementia. In this model, statin-mediated inhibition of mevalonate synthesis, an essential reaction in forming geranylgeranyl lipid, would lower extracellular levels of parenchymal apoE. Because apoE has been found necessary for plaque development in transgenic models of Alzheimer's disease, suppressing apoE secretion by statins could reduce plaques and, in turn, improve cognitive function.     

Microglia express GABA(B) receptors to modulate interleukin release

gamma-Aminobutyric acid (GABA) can act as a neuroprotective agent besides its well-established role as the main inhibitory neurotransmitter in the CNS. Here we report that microglial cells express GABA(B) receptors indicating that these prominent immunocompetent cells in the brain are a target for GABA. Agonists of GABA(B) receptors triggered the induction of K(+) conductance in microglial cells from acute brain slices and in culture. Both subunits of GABA(B) receptors were identified in cultured microglia by Western blot analysis and immunocytochemistry, and were detected on a subpopulation of microglia in situ by immunohistochemistry. In response to facial nerve axotomy, we observed an increase in GABA(B) receptor expressing microglial cells in the facial nucleus. We activated microglial cells in culture with lipopolysaccharide (LPS) to induce the release of interleukin-6 and interleukin-12p40. This release activity was attenuated by simultaneous activation of the GABA(B) receptors indicating that GABA can modulate the microglial immune response.     

Microglial cell reaction in the gray and white matter in spinal cords from jimpy mice. An enzyme histochemical study at the light and electron microscope level

Jimpy is a genetic disorder which results in a severe hypomyelination in the central nervous system associated with a variety of astroglial and oligodendroglial abnormalities. In this study, we examined the morphology and distribution of microglial cells in spinal cord sections from jimpy and normal mice at 10–12 and 20–22 days postnatal using a specific microglial marker, the nucleoside diphosphatase staining. Compared to those of normal littermates, the spinal cords of jimpy mice showed an intense microglial cell reaction in white and gray matter, as revealed by quantitative analysis and light and electron microscope study. Microglial reactivity was apparent in all spinal cord areas, although it was more pronounced in white than in gray matter. The mean microglial densities in the jumpy white matter were about threefold (10–12 days) and fivefold (20–22 days) higher than in the normal, whereas in the gray matter, microglial density in jimpy was about 60% higher than in normal at both ages. Morphologically, microglial cells in the normal spinal cord showed a ramified appearance, similar in size and ramification pattern to those reported in other normal CNS areas. In contrast, microglial cells in the jimpy spinal cord showed a reactive morphology, characterized by a shortening and coarsening of their cell processes, swelling of their cell body and accumulation of lipid inclusions. Reactive microglial cells were found in close association with axons and oligodendroglial cells. The possible role of microglial cells in hypomyelination is discussed.     

Microglial activation in chronic neurodegenerative diseases: roles of apoptotic neurons and chronic stimulation

In chronic neurodegenerative diseases, microglial activation is an early sign that often precedes neuronal death. Increasing evidence indicates that in these chronic pathologies activated microglia sustain a local inflammatory response. Nonetheless, the potential detrimental or protective roles of such reaction remain to date not fully understood, mainly because of the lack of direct evidence of the functional properties acquired by microglia in the course of chronic diseases. Purified microglial cultures have been extensively used to investigate microglial functions associated with activation, but they are often criticized for some experimental constrains, including the abrupt addition of activators, the limited time of stimulation, and the absence of interactions with neurons or other elements of brain parenchyma. To limit these confounding factors, we developed in vitro models in which microglial cells were repeatedly challenged with lipopolysaccharide or co-cultured with healthy, apoptotic, or necrotic neuronal cells. We found that chronic stimulation and interaction with phosphatidylserine-expressing apoptotic cells induced microglial cells to release immunoregulatory and neuroprotective agents (prostaglandin E₂, transforming growth factor-β, and nerve growth factor), whereas the synthesis of pro-inflammatory molecules (tumor necrosis factor-α and nitric oxide) was inhibited. These findings suggest that signals that are relevant to chronic diseases lead to a progressive down-regulation of pro-inflammatory microglial functions and may help in understanding the atypical microglial activation that begins to be recognized in some chronic neuropathologies.     

Interferon-gamma differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages

During bacterial infections of the CNS, activated microglia could support leucocyte recruitment to the brain through the synthesis of cyto- and chemokines. In turn, invading leucocytes may feedback on microglial cells to influence their chemokine release pattern. Here, we analyzed the capacity of interferon-gamma (IFNgamma) to serve as such a leucocyte-to-microglia signal. Production of cyto- and chemokines was stimulated in mouse microglia cultures by treatments with lipopolysaccharide (LPS) from Gram-negative Escherichia coli or cell walls from Gram-positive Streptococcus pneumoniae (PCW). IFNgamma presence during the stimulation (0.1-100 ng/mL) modulated the patterns of LPS- and PCW-induced cyto- and chemokine release in a dose-dependent, potent and complex manner. While amounts of TNFalpha and IL-6 remained nearly unchanged, IFNgamma enhanced the production of IL-12, MCP-1 and RANTES, but attenuated that of KC, MIP-1alpha and MIP-2. Release modulation was obtained with IFNgamma preincubation (treatment of cells before LPS or PCW administration), coincubation and even delayed addition to an ongoing LPS or PCW stimulation. Together the changes observed for the microglial chemokine release under IFNgamma would shift the chemoattractive profile from favouring neutrophils to a preferential attraction of monocytes and T lymphocyte populations--as actually seen during the course of bacterial meningitis. The findings support the view of activated microglia as a major intrinsic source for an instant production of a variety of chemokines and suggest that leucocyte-derived IFNgamma could potentially regulate the microglial chemokine release pattern.     

Induction of immunoreactive interleukin-1β and tumor necrosis factor-α in the brains of rabies virus infected rats

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) are important cytokines in the development of brain inflammation during pathological process. During rabies virus infection, the level of these proinflammatory cytokines are enhanced in the brain. In the present study we determined the cellular localization of these two cytokines by immunocytochemistry in brains of rats infected with rabies virus, at different time-intervals of the disease (day 1, 3, 4, 5 and at final stage day 6 post-infection (p.i.)). Cellular identification of IL-1β (irIL-1β) and TNFα (irTNFα) immunopositive cells was studied using a polyclonal antibody against these cytokines and against glial fibrillary acidic protein (GFAP) to detect astrocytes and GSA-I-B4 isolectin to detect microglial cells and/or infiltrating macrophages. In brains of control and early infected rats, irIL-1β was only detected in fibers located in the hypothalamus, supraoptic and tractus optic nuclei and infundibular nucleus. From day 4 onwards until day 6 p.i., enhanced irIL-1β was found and identified either in activated ameboid and/or infiltrated macrophages (amygdala, thalamus, internal capsula, subtantia nigra, septal nuclei and around blood vessels), or in activated ramified cells (hypothalamus and periventricular nucleus, piriformis and cingulate cortex, hippocampus). IrTNFα was observed in the brains of rats at a final stage of disease (day 5 and 6 p.i.): in the hypothalamus, the amygdala, the internal capsula, the thalamus, the septal nuclei, the hippocampus, the habenular nuclei and around the blood vessels. Ir-TNFα was detected in round cells identified as ameboid microglia and/or infiltrated macrophages. A marked activation of microglial and astroglial cells was observed mainly in the hypothalamus, the thalamus and hippocampus and around the blood vessels, at day 4 p.i. and later, revealing a high central inflammatory reaction in brains of rabies virus infected rats. These results showed that IL-1β and TNFα are produced in the brain both by local microglial cells and infiltrating macrophages during rabies infection. Thus, these cytokines may play an important role in coordinating the dramatic inflammatory response associated with the rabies-encephalopathy as well as in the neural modification and alteration of brain functions.     

Microglia and neuroprotection: implications for Alzheimer's disease

The first part of this paper summarizes some of the key observations from experimental work in animals that support a role of microglia as neuroprotective cells after acute neuronal injury. These studies point towards an important role of neuronal–microglial crosstalk in the facilitation of neuroprotection. Conceptually, injured neurons are thought to generate rescue signals that trigger microglial activation and, in turn, activated microglia produce trophic or other factors that help damaged neurons recover from injury. Against this background, the second part of this paper summarizes recent work from postmortem studies conducted in humans that have revealed the occurrence of senescent, or dystrophic, microglial cells in the aged and Alzheimer's disease brain. These findings suggest that microglial cells become increasingly dysfunctional with advancing age and that a loss of microglial cell function may involve a loss of neuroprotective properties that could contribute to the development of aging-related neurodegeneration.     

Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-α

Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-α had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-α production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-α production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.     

Integrin Mac-1 and β-amyloid in microglial release of nitric oxide

The β-amyloid protein associated with Alzheimer's disease (AD) has been well characterized biochemically; however, its primary biological function and mode of action in AD has not been determined. We have shown previously that β-amyloid (β25–35), in combination with interferon-γ (IFN-γ), can induce nitric oxide release from cultured hippocampal microglial cells. In the present study, binding of β-amyloid with the leukocyte integrin Mac-1, a cell surface receptor on microglia, was studied by observing (1) inhibition of β-amyloid (β25–35)-mediated release of nitric oxide from cultured microglial cells following exposure to monoclonal antibodies against Mac-1 (anti-CD18 and anti-CD11b) and (2) competitive binding of fluorochrome-labeled β25–35 with anti-CD18 or anti-CD11b using fluorescent flow cytometry. Wt.3 (anti-CD18 antibody) and OX42 (anti-CD11b antibody) were as effective as opsonized zymosan at inducing the release of nitric oxide from microglia. Furthermore, Wt.3 and OX42 acted synergistically to induce maximum nitric oxide release. An interaction between β-amyloid and CD18 of Mac-1 was evidenced by the suppressive action of β25–35 on Wt.3-mediated release of nitric oxide and the synergistic action between OX42 and β25–35 in inducing nitric oxide release from microglia. The tissue culture study was supported by competitive binding assays of fluorochrome-labeled β25–35 and Mac-1 antibodies (Wt.3 or OX42). The majority of microglial cells (71%) did bind biotinylated β-amyloid in the presence of cytochalasin B, suggesting that β-amyloid binding to microglia is a receptor-mediated event. Furthermore, pre-exposure to Wt.3, but not OX42, significantly decreased binding of biotinylated β25–35 to microglia. These findings suggest that CD18 of Mac-1 may play a role in β-amyloid-mediated release of nitric oxide.     

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid β (Aβ) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt–Jakob disease, while Aβ is involved in the pathogenesis of Alzheimer's disease. Synthetic fragments of both proteins, PrP106–126 and β25–35 (β25–35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106–126 and β25–35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca²⁺]_i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca²⁺]_i concentration as observed after treatment with PrP106–126 and β25–35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106–126 and β25–35.     

Latex beads phagocytosis capacity and ecto-NAD glycohydrolase activity of rat brain microglia cells in vitro

Conditions are described which allow the preparation in vitro of pure (> 95%) microglial cell cultures isolated from newborn rat brain. Such ameboid cells cultivated in vitro can efficiently phagocytize opsonized latex beads and are capable of ingesting more (100–200 beads of 1.1 μm diameter per cell) and larger (6.4 μm) particles than other nerve cells, such as oligodendrocytes and astroglia. The microglial cells also show an important ecto-NAD⁺ glycohydrolase activity which is characteristic of phagocytic cells. We noted that the phagocytic capacity and ecto-NAD⁺ glycohydrolase of these cells were correlated and increased notably during the in vitro culture.Microglia cultivated in vitro appear to be a good model to study the activation of phagocytic properties in the central nervous system and corresponding modulation by natural or pharmacological immunomodulators.     

Induction of microglial nitric oxide synthesis by very low density lipoprotein

The effect of lipoproteins on adult rat CNS microglial nitric oxide (NO) production was studied. Incubation of cultured microglia with very low density lipoprotein (VLDL) for 48 h induced a 13-fold increase in NO production, compared with control. However, the induction of NO was negated when cells were incubated with VLDL and the NO synthase inhibitor N-methyl-L-arginine. High and low density lipoproteins (HDL and LDL) did not significantly induce NO synthesis in microglial cells. These results suggest that the lipoprotein environment in the brain can influence neurotoxic activity of microglia via production of NO. © 1996 Wiley-Liss, Inc.     

Apolipoprotein E expression in the cerebellum of normal and hypercholesterolemic rabbits

We have studied the expression of apolipoprotein E (ApoE) mRNA in the cerebella of control and experimental rabbits fed with a cholesterol-rich diet for 8 weeks. Cholesterol-treated rabbits show a dramatic increase in serum cholesterol levels; however, no significant variations in the expression level of cerebellar ApoE mRNA were found in comparison to control rabbits. In addition, no differences were observed between control and hypercholesterolemic rabbits in the in situ hybridization pattern of ApoE mRNA on cerebellar cortex sections. ApoE mRNA was localized in astroglial processes associated with Purkinje cell bodies and dendrites, granule cell clusters, blood vessels and nerve fibers of the white matter. No expression of ApoE mRNA was observed in Purkinje and granule cell neurons. Polarized light examination of cryostat cerebellar sections revealed the absence of cholesterol-rich microglia/macrophage cells induced by the hypercholesterolemia. In this way, neither reactive microglial cells nor perivascular phagocytes were found by ultrastructural analysis in hypercholesterolemic conditions. The pattern of glial fibrillary acidic protein of the astroglial cells of the cerebellar cortex as well as their nuclear size were unchanged following cholesterol treatment, indicating the absence of astroglial activation induced by hypercholesterolemia. Our results suggest that cerebellar ApoE does not contribute to the general cholesterol homeostasis outside of the brain and supports the view that this cerebellar ApoE is involved in paracrine and autocrine functions particularly related with synpase turnover and membrane remodelling of astroglial cells.     

Presenilin Is Essential for ApoE Secretion,a Novel Role of Presenilin Involved in Alzheimer's Disease Pathogenesis

Alzheimer''s disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-β (Aβ) protein into amyloid plaques in patient brains. Mutations in presenilin (PS) lead to abnormal generation of Aβ, which is the major cause of familial AD (FAD), and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin-deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear transport receptor. Moreover, the expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with the expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal control subjects. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion.SIGNIFICANCE STATEMENT Familial AD (FAD) typically results from mutations in the genes encoding amyloid precursor protein, presenilin 1 (PS1), or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased amyloid-β 42 (Aβ42)/Aβ40 ratio, which induce early amyloid deposition and FAD. On the other hand, apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) and contributes to AD pathogenesis because it has reduced Aβ clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms; however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis.