Alzheimer's A beta(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Alzheimer's disease (AD) is a neurodegenerative disorder involving the florid deposition of vascular and cerebral plaques composed chiefly of amyloid beta-peptide (A beta) derived from cleavage of the amyloid precursor protein (APP). Varying in length from 39 to 43 amino acids, A beta, particularly the longer A beta(42), is thought to play a significant role in AD pathogenesis. To better understand AD it is important to identify the subcellular organelles generating A beta. Studies using agents that disrupt endosomal/lysosomal function suggest that A beta is generated late in the secretory and endocytic pathways. However, much of what is known about A beta biosynthesis has been inferred by monitoring extracellular A beta levels since intracellular A beta is undetectable in most cell types. Consequently, the precise site or sites that generate A beta, or whether A beta(1-40) and A beta(1-42) are generated at the same point in the biosynthetic pathway, is not known. Using human NT2N neurons, we found that retention of APP in the endoplasmic reticulum/intermediate compartment (ER/IC) by three independent approaches eliminated production of intracellular A beta(1-40), but did not alter intracellular A beta(1-42) synthesis. These findings suggest that the ER/IC may be an important site for generating this highly amyloidogenic species of A beta.

A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor.

The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.

Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum.

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.

Posttranslational oligomerization and cooperative acid activation of mixed influenza hemagglutinin trimers.

The influenza virus hemagglutinin (HA) is a well-characterized integral membrane glycoprotein composed of three identical subunits. We have analyzed the formation of mixed trimers in cells expressing two different HA gene products. The results show efficient and essentially random assembly of functional hybrid trimers provided that the HAs are from the same HA subtype. Trimerization is thus a posttranslational event, and subunits are recruited randomly from a common pool of monomers in the endoplasmic reticulum. Mixed trimers were not observed between HAs derived from different subtypes, indicating that the trimerization event is sequence specific. Mixed trimers containing mutant subunits were, moreover, used to establish that the acid-induced conformational change involved in the membrane fusion activity of HA is a highly cooperative event.

Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus.

Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.

Detection of a novel intraneuronal pool of insoluble amyloid beta protein that accumulates with time in culture.

The amyloid-beta peptide (Abeta) is produced at several sites within cultured human NT2N neurons with Abeta1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since Abeta is found as insoluble deposits in senile plaques of the AD brain, and the Abeta peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that Abeta1-40, and particularly the more highly amyloidogenic Abeta1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable Abeta that accounted for over half of the total intracellular Abeta. Abeta1-42 was more abundant than Abeta1-40 in this pool, and most of the insoluble Abeta1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble Abeta were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-beta precursor protein. This insoluble intracellular pool of Abeta was exceptionally stable, and accumulated in NT2N neurons in a time-dependent manner, increasing 12-fold over a 7-wk period in culture. These novel findings suggest that Abeta amyloidogenesis may be initiated within living neurons rather than in the extracellular space. Thus, the data presented here require a reexamination of the prevailing view about the pathogenesis of Abeta deposition in the AD brain.

Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein.

The vesicular stomatitis virus glycoprotein (G protein) is an integral membrane protein which assembles into noncovalently associated trimers before transport from the endoplasmic reticulum. In this study we have examined the folding and oligomeric assembly of twelve mutant G proteins with alterations in the cytoplasmic, transmembrane, or ectodomains. Through the use of conformation-specific antibodies, we found that newly synthesized G protein folded into a conformation similar to the mature form within 1-3 min of synthesis and before trimer formation. Mutant proteins not capable of undergoing correct initial folding did not trimerize, were not transported, and were found in large aggregates. They had, as a rule, mutations in the ectodomain, including several with altered glycosylation patterns. In contrast, mutations in the cytoplasmic domain generally had little effect on folding and trimerization. These mutant proteins, whose ectodomains were identical to the wild-type by several assays, were either transported to the cell surface slowly or not at all. We concluded that while correct ectodomain folding and trimer formation are prerequisites for transport, they alone are not sufficient. The results suggest that the cytoplasmic domain of the wild-type protein may facilitate rapid, efficient transport from the ER, which can be easily affected or eliminated by tail mutations that do not detectably affect the ectodomain.

Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.

Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks.

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

Genetic acceleration of AIDS progression by a promoter variant of CCR5.

The CCR5 gene encodes a cell surface chemokine receptor molecule that serves as the principal coreceptor, with CD4, for macrophage-tropic (R5) strains of human immunodeficiency virus-type 1 (HIV-1). Genetic association analysis of five cohorts of people with acquired immunodeficiency syndrome (AIDS) revealed that infected individuals homozygous for a multisite haplotype of the CCR5 regulatory region containing the promoter allele, CCR5P1, progress to AIDS more rapidly than those with other CCR5 promoter genotypes, particularly in the early years after infection. Composite genetic epidemiologic analyses of genotypes bearing CCR5P1, CCR5-Delta32, CCR2-64I, and SDF1-3'A affirmed distinct regulatory influences for each gene on AIDS progression. An estimated 10 to 17 percent of patients who develop AIDS within 3.5 years of HIV-1 infection do so because they are homozygous for CCR5P1/P1, and 7 to 13 percent of all people carry this susceptible genotype. The cumulative and interactive influence of these AIDS restriction genes illustrates the multigenic nature of host factors limiting AIDS disease progression.

HIV-1 coreceptors and their inhibitors.

Entry of human immunodeficiency virus (HIV) into target cells is mediated by the viral Envelope glycoprotein (Env) and its coordinated interaction with a receptor (CD4) and a coreceptor (usually the chemokine receptors CCR5 or CXCR4). This review describes the identification of chemokine receptors as coreceptors for HIV-1 Env-mediated fusion, the determinants of chemokine receptor usage, and the impact of nonfunctional chemokine receptor alleles on HIV-1 resistance and disease progression. Due to the important role of chemokine receptors in HIV-1 entry, inhibitors of these coreceptors are good candidates for blocking entry and development of antiretroviral therapies. We discuss the different CCR5- and CXCR4-based antiretroviral drugs that have been developed thus far, highlighting the most promising drug candidates. Resistance to these coreceptor inhibitors as well as the impact of these drugs on clinical monitoring and treatment are also discussed.

HIV-1 entry and its inhibition.

Entry of HIV-1 virions into cells is a complex and dynamic process carried out by envelope (Env) glycoproteins on the surface of the virion that promote the thermodynamically unfavorable fusion of highly stable viral and target cell membranes. Insight gained from studies of the mechanism of viral entry allowed insight into the design of novel inhibitors of HIV-1 entry, several of which are now in clinical trials. This review highlights the mechanism by which viral and cellular proteins mediate entry of HIV-1 into permissive cells, with an emphasis on targeting this process in the design of novel therapies that target distinct steps of the entry process, including antagonizing receptor binding events and blocking conformational changes intimately involved in membrane fusion.

Intracellular APP processing and A beta production in Alzheimer disease.

Senile plaques composed of A beta peptides are a histopathological hallmark of Alzheimer disease (AD). A role for A beta in the etiology of AD has been argued from analysis of mutations associated with a subset of early-onset familial AD (FAD). Expression of autosomal dominant mutations in the genes for the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) in affected patients, cultured cells, or transgenic mice leads to increased production of total A beta or increased production of A beta ending at residue 42 (A beta42). Since A beta42 is the more amyloidogenic and toxic species in vitro and is the major component of amyloid senile plaques in vivo, overproduction of this peptide may play a crucial role in the pathogenesis of AD. Thus, an understanding of the production of A beta within the cell in normal and pathological conditions is critical to understanding early events in AD. Studies in cell culture have established that processing of APP to form A beta can occur at multiple locations within the cell and leads to the production of 2 pools of A beta: a secreted pool composed predominantly of A beta40 and a nonsecreted, intracellular pool composed preferentially of more amyloidogenic A beta42. The purpose of this review is to provide a summary of our current understanding of APP processing in the generation of the secreted and intracellular pools of A beta and to propose a model linking the intracellular pool to the formation of extracellular plaques and neuronal pathology in AD.

Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer's disease.

Accumulation of the amyloid-beta (A beta) peptide in the central nervous system (CNS) is considered by many to be the crucial pathological insult that ultimately leads to the development of Alzheimer's disease (AD). Regulating the production and/or aggregation of A beta could therefore be of considerable benefit to patients afflicted with AD. It has long been known that A beta is derived from the proteolytic processing of the amyloid precursor protein (APP) by two enzymatic activities, beta-secretase and gamma-secretase. Recent breakthroughs have led to the identification of the aspartyl protease BACE (beta-site APP-cleaving enzyme) as beta-secretase and the probable identification of the presenilin proteins as gamma-secretases. This review discusses what is know about BACE and the presenilins, focusing on their capacity as secretases, as well as the options for therapeutic advancement the careful characterization of these proteins will provide. These findings are presented in the context of the "amyloid cascade hypothesis" and its physiological relevance in AD pathogenesis.

Vaccinia virus serves as an efficient vector for expressing heterologous proteins in human NTera 2 neurons.

The human teratocarcinoma cell line NTera 2 (NT2) can be induced to differentiate into post-mitotic neurons possessing well-defined axonal and dendritic morphology. Highly enriched neurons (NT2-N cells) can be prepared in large numbers, thus combining many of the advantages of both primary and continuous cell culture systems. Unfortunately, it has proven difficult to express foreign genes in NT2-N cells. We examined whether vaccinia virus (VV) can express heterologous proteins in NT2-N cells and characterized the response of NT2-N cells to VV infection. NT2-N cells were infected with VV vectors expressing the envelope glycoprotein (gp160) from the human immunodeficiency type 1 virus (HIV 1). These vectors were chosen because VV-directed synthesis and post-translational processing of gp160 have been well characterized in many cell types. Approximately 85% of the neurons expressed gp160 which underwent native post-translational cleavage. The rate of gp160 synthesis was maximal at 5-48 hours postinfection, but was detectable for as long as 4 days. Surprisingly, NT2-N cells showed no VV-induced alterations in morphology, downregulation of host protein synthesis, or cytotoxicity, as measured by lactate dehydrogenase release. These results indicate that VV can serve as an efficient vector for introducing foreign genes in NT2-N cells without the cytotoxic effects often associated with VV infection. These properties, in conjunction with the advantages provided by NT2-N cells, provide new options for analyzing the cellular and molecular functions of human neurons.

Chemokine receptors as fusion cofactors for human immunodeficiency virus type 1 (HIV-1).

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.

Expression of multiple functional chemokine receptors and monocyte chemoattractant protein-1 in human neurons.

Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.

Safe use of the CXCR4 inhibitor ALX40-4C in humans.

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.