Role of endoplasmic reticulum, endosomal-lysosomal compartments, and microtubules in amyloid precursor protein metabolism of human neurons

A wide interest in amyloid precursor protein (APP) metabolism stems from the fact that increased amounts of amyloid beta peptide (Abeta), arising through proteolytic processing of APP, likely play a significant role in Alzheimer's disease. As Alzheimer's disease pathology is limited almost exclusively to the human species, we established human primary neuron cultures to address the possibility of distinctive APP processing in human CNS neurons. In the present study, we investigate the role of organelles and protein trafficking in APP metabolism. Using brefeldin A, we failed to detect APP processing into Abeta in the endoplasmic reticulum. Monensin and the lysomotropic agents, NH4Cl and chloroquine, revealed a bypass pH-dependent secretory pathway in a compartment between the endoplasmic reticulum and the medial Golgi, resulting in the secretion of full-length APP. Colchicine treatment resulting in the loss of neurites inhibited processing of APP through the secretory, but not the endosomal-lysosomal, pathway of APP metabolism. The serine protease inhibitor, leupeptin, indicates a role for lysosomes in APP, Abeta, and APP C-terminal fragment turnover. These results demonstrate that the regulation of APP metabolism in human neurons differs considerably from those reported in rodent CNS primary neuron cultures or continuously dividing cell types.     

Pathogenic A beta induces the expression and activation of matrix metalloproteinase-2 in human cerebrovascular smooth muscle cells

Cerebral amyloid angiopathy (CAA) is a major pathological feature of Alzheimer's disease and related disorders. Human cerebrovascular smooth muscle (HCSM) cells, which are intimately associated with CAA, have been used as an in vitro model system to investigate pathologic interactions with amyloid beta protein (A beta). Previously we have shown that pathogenic forms of A beta induce several pathologic responses in HCSM cells including fibril assembly at the cell surface, increase in the levels of A beta precursor, and apoptotic cell death. Here we show that pathogenic A beta stimulates the expression and activation of matrix metalloproteinase-2 (MMP-2). Furthermore, we demonstrate that the increase in MMP-2 activation is largely caused by increased expression of membrane type-1 (MT1)-MMP expression, the primary MMP-2 activator. Finally, treatment with MMP-2 inhibitors resulted in increased HCSM cell viability in the presence of pathogenic A beta. Our findings suggest that increased expression and activation of MMP-2 may contribute to HCSM cell death in response to pathogenic A beta. In addition, these activities may also contribute to loss of vessel wall integrity in CAA resulting in hemorrhagic stroke. Therefore, further understanding into the role of MMPs in HCSM cell degeneration may facilitate designing therapeutic strategies to treat CAA found in AD and related disorders.     

Overexpression of the neuritotrophic cytokine S100beta precedes the appearance of neuritic beta-amyloid plaques in APPV717F mice

Homozygous APPV717F transgenic mice overexpress a human beta-amyloid precursor protein (betaAPP) minigene encoding a familial Alzheimer's disease mutation. These mice develop Alzheimer-type neuritic beta-amyloid plaques surrounded by astrocytes. S100beta is an astrocyte-derived cytokine that promotes neurite growth and promotes excessive expression of betaAPP. S100beta overexpression in Alzheimer's disease correlates with the proliferation of betaAPP-immunoreactive neurites in beta-amyloid plaques. We found age-related increases in tissue levels of both betaAPP and S100beta mRNA in transgenic mice. Neuronal betaAPP overexpression was found in cell somas in young mice, whereas older mice showed betaAPP overexpression in dystrophic neurites in plaques. These age-related changes were accompanied by progressive increases in S100beta expression, as determined by S100beta load (percent immunoreactive area). These increases were evident as early as 1 and 2 months of age, months before the appearance of beta-amyloid deposits in these mice. Such precocious astrocyte activation and S100beta overexpression are similar to our earlier findings in Down's syndrome. Accelerated age-related overexpression of S100beta may interact with age-associated overexpression of mutant betaAPP in transgenic mice to promote development of Alzheimer-like neuropathological changes.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Sodium channel beta4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington's disease transgenic mice

Sodium channel beta4 is a very recently identified auxiliary subunit of the voltage-gated sodium channels. To find the primarily affected gene in Huntington's disease (HD) pathogenesis, we profiled HD transgenic mice using a high-density oligonucleotide array and identified beta4 as an expressed sequence tag (EST) that was significantly down-regulated in the striatum of HD model mice and patients. Reduction in beta4 started at a presymptomatic stage in HD mice, whereas other voltage-gated ion channel subunits were decreased later. In contrast, spinal cord neurons, which generate only negligible levels of expanded polyglutamine aggregates, maintained normal levels of beta4 expression even at the symptomatic stage. Overexpression of beta4 induced neurite outgrowth in Neuro2a cells, and caused a thickening of dendrites and increased density of dendritic spines in hippocampal primary neurons, indicating that beta4 modulates neurite outgrowth activities. These results suggest that down-regulation of beta4 may lead to abnormalities of sodium channel and neurite degeneration in the striatum of HD transgenic mice and patients with HD.     

Androgens regulate neprilysin expression: role in reducing beta-amyloid levels

Age-related testosterone depletion in men is a risk factor for Alzheimer's disease. Prior studies suggest that androgens affect Alzheimer's disease risk by regulating accumulation of β-amyloid protein (Aβ) by an undefined mechanism. In this study, we investigated the role of the Aβ-catabolizing enzyme neprilysin (NEP) in this process. First, we observed that androgens positively regulate neural expression of NEP in adult male rats. Next, we investigated androgen regulatory effects on both NEP expression and Aβ levels using cultured hippocampal neurons and neuronally differentiated rat pheochromocytoma cell 12 with or without androgen receptor (AR). Dihydrotestosterone (DHT) induced a time-dependent increase in NEP expression. DHT also significantly decreased levels of Aβ in AR-expressing cells transfected with amyloid precursor protein, but did not affect levels of either full-length or non-amyloidogenic, soluble amyloid precursor protein. Importantly, the DHT induced decrease of Aβ was blocked by pharmacological inhibition of NEP. The DHT-mediated increase in NEP expression and decrease in Aβ levels were (i) not observed in rat pheochromocytoma cell 12 lacking AR and (ii) blocked in AR-expressing cells by the antagonists, cyproterone acetate and flutamide. Together, these findings suggest that androgen regulation of Aβ involves an AR-dependent mechanism requiring up-regulation of the Aβ catabolizing enzyme NEP.     

Increased Expression of β-Amyloid Protein Precursor and Microtubule-Associated Protein τ During the Differentiation of Murine Embryonal Carcinoma Cells

Expression of the genes encoding the beta/A4 amyloid protein precursor (APP) and microtubule-associated protein tau was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of tau mRNA. Moreover, subsequent decreases in the levels of APP and tau mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that greater than 85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and tau protein in development of Alzheimer's disease in cholinergic neurons.     

Amyloid precursor protein cross-linking stimulates beta amyloid production and pro-inflammatory cytokine release in monocytic lineage cells

Beta amyloid peptide-containing neuritic plaques are a defining feature of Alzheimer's disease pathology. Beta amyloid are 38-43 residue peptides derived by proteolytic cleavage of amyloid precursor protein. Although much attention has focused on the proteolytic events leading to beta amyloid generation, the function of amyloid precursor protein remains poorly described. Previously, we reported that amyloid precursor protein functions as a pro-inflammatory receptor on monocytic lineage cells and defined a role for amyloid precursor protein in adhesion by demonstrating that beta(1) integrin-mediated pro-inflammatory activation of monocytes is amyloid precursor protein dependent. We demonstrated that antibody-induced cross-linking of amyloid precursor protein in human THP-1 monocytes and primary mouse microglia stimulates a tyrosine kinase-based pro-inflammatory signaling response leading to acquisition of a reactive phenotype. Here, we have identified pro-inflammatory mediators released upon amyloid precursor protein-dependent activation of monocytes and microglia. We show that amyloid precursor protein cross-linking stimulated tyrosine kinase-dependent increases in pro-inflammatory cytokine release and a tyrosine kinase-independent increase in beta amyloid 1-42 generation. These data provide much needed insight into the function of amyloid precursor protein and provide potential therapeutic targets to limit inflammatory changes associated with the progression of Alzheimer's disease.     

Mutant presenilin 1 increases the levels of Alzheimer amyloid beta-peptide Abeta42 in late compartments of the constitutive secretory pathway

Mutations in the presenilin 1 (PS1) gene are associated with autosomal dominant, early-onset, familial Alzheimer's disease and result in increased release of the hyperaggregatable 42-amino acid form of the amyloid beta-peptide (A(beta)42). To determine which subcellular compartments are potential source(s) of released Abeta42, we compared the levels and spatial segregation of intracellular A(beta)40 and A(beta)42 peptides between N2a neuroblastoma cells doubly transfected with the "Swedish" familial Alzheimer's disease-linked amyloid precursor protein variant and either wild-type PS1 (PS1(wt)) or familial Alzheimer's disease-linked delta9 mutant PS1 (PS1delta9). As expected, PS1delta9-expressing cells had dramatically higher levels of intracellular Abeta42 than did cells expressing PS1wt. However, the highest levels of A(beta)42 colocalized not with endoplasmic reticulum or Golgi markers but with rab8, a marker for trans-Golgi network (TGN)-to-plasma membrane (PM) transport vesicles. We show that PS1 mutants are capable of causing accumulation of A(beta)42 in late compartments of the secretory pathway, generating there a readily releasable source of A(beta)42. Our findings indicate that PS1 "bioactivity" localizes to the vicinity of the TGN and/or PM and reconcile the apparent discrepancy between the preponderant concentration of PS1 protein in proximal compartments of the secretory pathway and the recent findings that PS1 "bioactivity" can control gamma-secretase-like processing of another transmembrane substrate, Notch, at or near the PM.     

A beta oligomers - a decade of discovery

Converging lines of evidence suggest that progressive accumulation of the amyloid beta-protein (A beta) plays a central role in the genesis of Alzheimer's disease, but it was long assumed that A beta had to be assembled into extracellular amyloid fibrils to exert its cytotoxic effects. Over the past decade, data have emerged from the use of synthetic A beta peptides, cell culture models, beta-amyloid precursor protein transgenic mice and human brain to suggest that pre-fibrillar, diffusible assemblies of A beta are also deleterious. Although the precise molecular identity of these soluble toxins remains unsettled, accumulating evidence suggests that soluble forms of A beta are indeed the proximate effectors of synapse loss and neuronal injury. Here we review recent progress in understanding the role of soluble oligomers in Alzheimer's disease.     

Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of beta-amyloid peptide (Abeta), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Abeta on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT-PCR and specific radioimmunoassay demonstrated that aggregated Abeta(25-35), at concentrations up to 10(-4) m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Abeta(25-35) was prevented by Congo red. Measurement of the number of living cells revealed that Abeta(25-35) induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Abeta(25-35)-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Abeta-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.     

Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia

Activated microglia surrounding amyloid beta-containing senile plaques synthesize interleukin-1, an inflammatory cytokine that has been postulated to contribute to Alzheimer's disease pathology. Studies have demonstrated that amyloid beta treatment causes increased cytokine release in microglia and related cell cultures. The present work evaluates the specificity of this cellular response by comparing the effects of amyloid beta to that of amylin, another amyloidotic peptide. Both lipopolysaccharide-treated THP-1 monocytes and mouse microglia showed significant increases in mature interleukin-1beta release 48 h following amyloid beta or human amylin treatment, whereas nonfibrillar rat amylin had no effect on interleukin-1beta production by THP-1 cells. Lipopolysaccharide-stimulated THP-1 cells treated with amyloid beta or amylin also showed increased release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokines interleukin-8 and macrophage inflammatory protein-1alpha and -1beta. THP-1 cells incubated with fibrillar amyloid beta or amylin in the absence of lipopolysaccharide also showed significant increases of both interleukin-1beta and tumor necrosis factor-alpha mRNA. Furthermore, treatment of THP-1 cells with amyloid fibrils resulted in an elevated expression of the immediate-early genes c-fos and junB. These studies provide further evidence that fibrillar amyloid peptides can induce signal transduction pathways that initiate an inflammatory response that is likely to contribute to Alzheimer's disease pathology.     

Non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease: old and new mechanisms of action

Alzheimer's disease (AD) is characterized by cerebral deposits of beta-amyloid (A beta) peptides and neurofibrillary tangles (NFT) which are surrounded by inflammatory cells. Epidemiological studies have shown that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. It has been postulated that some NSAIDs target pathological hallmarks of AD by interacting with several pathways, including the inhibition of cyclooxygenases (COX) and activation of the peroxisome proliferator-activated receptor gamma. A variety of experimental studies indicate that a subset of NSAIDs such as ibuprofen, flurbiprofen, indomethacin and sulindac also possess A beta-lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin. While COX inhibition occurs at low concentrations in vitro (nM-low microm range), the A beta-lowering activity is observed at high concentrations (< or = 50 microm). Nonetheless, studies with flurbiprofen or ibuprofen in AD transgenic mice show that the effects on A beta levels or deposition are attained at plasma levels similar to those achieved in humans at therapeutic dosage. Still, it remains to be assessed whether adequate concentrations are reached in the brain. This is a crucial aspect that will allow defining the dose-window and the length of treatment in future clinical trials. Here, we will discuss how the combination of anti-amyloidogenic and anti-inflammatory activities of certain NSAIDs may produce a profile potentially relevant to their clinical use as disease-modifying agents for the treatment of AD.     

Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer's disease

Increased levels of a 40-42 amino-acid peptide called the amyloid beta protein (A beta) and evidence of oxidative damage are early neuropathological markers of Alzheimer's disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from A beta-mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimer's amyloidosis and monitored over time the effects of administering melatonin on brain levels of A beta, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time-dependent elevation of beta-amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD.     

Macrophage colony stimulatory factor and interferon-gamma trigger distinct mechanisms for augmentation of beta-amyloid-induced microglia-mediated neurotoxicity

Dysregulated stimulation of microglia, the resident macrophages in the brain, can lead to excessive induction of inflammatory agents and subsequently damage to neurons. Fibrillar beta-amyloid peptide (fA beta), a major component of senile plaques in Alzheimer's disease (AD) brain, is known to induce microglial-mediated neurotoxicity under certain conditions. Microglial 'priming' by macrophage colony stimulatory factor (MCSF) or interferon-gamma (IFN gamma) appears to be required for this fA beta-induced microglia mediated neurotoxicity in vitro. We report here that while both MCSF and IFN gamma induce microglial-mediated fA beta neurotoxicity, their mechanisms of toxicity differ. The enhancement of neurotoxicity by IFN gamma or MCSF is not due to enhanced A beta ingestion by microglia or to the direct effect of proinflammatory cytokine production. The neurotoxicity resulting from IFN gamma/fA beta treatment was blocked by pretreatment with nitric oxide synthase inhibitor L-N-5-(1-iminoethyl) ornithine hydrochloride (L-NIO), consistent with a role for nitric oxide in the IFN gamma-mediated toxicity mechanism. In contrast, no induction of nitric oxide production was detected for microglia treated with MCSF/fA beta. Furthermore, inhibiting the generation of reactive oxygen species (ROS) using the specific NADPH oxidase inhibitor apocynin reversed fA beta/MCSF-induced neurotoxicity while L-NIO had little effect. As MCSF is endogenously expressed within the brain, and both its level and that of the MCSF receptor are dramatically increased in the AD brain, the neurotoxicity resulting from ROS release by fA beta/MCSF coactivated microglia may be a more appropriate model for assessing fA beta-induced microglial-mediated neuropathology in AD.     

Inverse and distinct modulation of tau-dependent neurodegeneration by presenilin 1 and amyloid-beta in cultured cortical neurons: evidence that tau phosphorylation is the limiting factor in amyloid-beta-induced cell death

Alzheimer's disease (AD) is characterized by massive neuron loss in distinct brain regions, extracellular accumulations of the amyloid precursor protein-fragment amyloid-beta (A beta) and intracellular tau fibrils containing hyperphosphorylated tau. Experimental evidence suggests a relation between presenilin (PS) mutations, A beta formation, and tau phosphorylation in triggering cell death; however, how A beta and PS affect tau-dependent degeneration is unknown. Using herpes simplex virus 1-mediated gene-transfer of fluorescent-tagged tau constructs in primary cortical neurons, we demonstrate that tau expression exerts a neurotoxic effect that is increased with a construct mimicking disease-like hyperphosphorylation [pseudohyperphosphorylated (PHP) tau]. Live imaging revealed that PHP tau expression is associated with increased perikarya suggesting the development of a 'ballooned' phenotype as a specific feature of tau-mediated cell death. Transgenic expression of PS1 suppressed tau-induced neurodegeneration. In contrast, A beta amplified degeneration in the presence of wt tau but not of PHP tau. The data indicate that PS1 and A beta inversely modulate tau-dependent neurodegeneration at distinct steps. They indicate that the mode by which PHP tau causes neurotoxicity is downstream of A beta and that tau phosphorylation is the limiting factor in A beta-induced cell death. Suppression of tau expression or inhibition of tau phosphorylation at disease-relevant sites may provide an effective therapeutic strategy to prevent neurodegeneration in Alzheimer's disease.