Omega images at the active zone may be endocytotic rather than exocytotic: Implications for the vesicle hypothesis of transmitter release

A Ca2+-dependent synaptic vesicle-recycling pathway emanating from the plasma membrane adjacent to the dense body at the active zone has been demonstrated by blocking pinch-off of recycling membrane by using the Drosophila mutant, shibire. Exposure of wild-type Drosophila synapses to low Ca2+/high Mg2+ saline is shown here to block this active zone recycling pathway at the stage in which invaginations of the plasma membrane develop adjacent to the dense body. These observations, in combination with our previous demonstration that exposure to high Ca2+ causes “docked” vesicles to accumulate in the identical location where active zone endocytosis occurs, suggest the possibility that a vesicle-recycling pathway emanating from the active zone may exist that is stimulated by exposure to elevated Ca2+, thereby causing an increase in vesicle recycling, and is suppressed by exposure to low Ca2+ saline, thereby blocking newly forming vesicles at the invagination stage. The presence of a Ca2+-dependent endocytotic pathway at the active zone opens up the following possibilities: (i) electron microscopic omega-shaped images (and their equivalent, freeze fracture dimples) observed at the active zone adjacent to the dense body could represent endocytotic images (newly forming vesicles) rather than exocytotic images; (ii) vesicles observed attached to the plasma membrane adjacent to the dense body could represent newly formed vesicles rather than vesicles “docked” for release of transmitter.

Memory-enhancing effects of secreted forms of the β-amyloid precursor protein in normal and amnestic mice

When administered intracerebroventricularly to mice performing various learning tasks involving either short-term or long-term memory, secreted forms of the β-amyloid precursor protein (APPs751 and APPs695) have potent memory-enhancing effects and block learning deficits induced by scopolamine. The memory-enhancing effects of APPs were observed over a wide range of extremely low doses (0.05-5,000 pg intracerebroventricularly), blocked by anti-APPs antisera, and observed when APPs was administered either after the first training session in a visual discrimination or a lever-press learning task or before the acquisition trial in an object recognition task. APPs had no effect on motor performance or exploratory activity. APPs695 and APPs751 were equally effective in the object recognition task, suggesting that the memory-enhancing effect of APPs does not require the Kunitz protease inhibitor domain. These data suggest an important role for APPss on memory processes.

Inhibition of the electrostatic interaction between β-amyloid peptide and membranes prevents β-amyloid-induced toxicity

The accumulation of β-amyloid peptides (Aβ) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Aβ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Aβ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Aβ1-42 and the toxic fragment Aβ25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic β-sheet conformation by Aβ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Aβ1-40 and Aβ25-35 to negatively charged lipid vesicles and Aβ induced cell toxicity. These results suggest that Aβ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis

Amyloid β peptide (Aβ) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Aβ induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Aβ and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Aβ decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Aβ may be relevant to the chronic neurodegeneration observed in AD.

Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Cerebral deposition of the amyloid β protein (Aβ) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of Aβ (Aβ40) accounts for ≈90% of all Aβ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (Aβ42), accounting for only ≈10% of secreted Aβ, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of Aβ42 production is a prime therapeutic goal. The same protease, γ-secretase, is assumed to generate the C termini of both Aβ40 and Aβ42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit γ-secretase, on β-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various Aβ40- and Aβ42-specific antibodies, we demonstrate a much stronger inhibition of the γ-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the Aβ40 and Aβ42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general β-amyloid precursor protein metabolism, including Aβ40 production, but specifically block the generation of the pathogenic Aβ42 peptide.

Possible ecological risks of transgenic organism release when transgenes affect mating success: Sexual selection and the Trojan gene hypothesis

Widespread interest in producing transgenic organisms is balanced by concern over ecological hazards, such as species extinction if such organisms were to be released into nature. An ecological risk associated with the introduction of a transgenic organism is that the transgene, though rare, can spread in a natural population. An increase in transgene frequency is often assumed to be unlikely because transgenic organisms typically have some viability disadvantage. Reduced viability is assumed to be common because transgenic individuals are best viewed as macromutants that lack any history of selection that could reduce negative fitness effects. However, these arguments ignore the potential advantageous effects of transgenes on some aspect of fitness such as mating success. Here, we examine the risk to a natural population after release of a few transgenic individuals when the transgene trait simultaneously increases transgenic male mating success and lowers the viability of transgenic offspring. We obtained relevant life history data by using the small cyprinodont fish, Japanese medaka (Oryzias latipes) as a model. Our deterministic equations predict that a transgene introduced into a natural population by a small number of transgenic fish will spread as a result of enhanced mating advantage, but the reduced viability of offspring will cause eventual local extinction of both populations. Such risks should be evaluated with each new transgenic animal before release.

Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid

Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer’s disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid β (Aβ) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer’s disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Aβ in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Aβ as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an App-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Aβ is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid β (Aβ) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes Aβ deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of Aβ in the pathogenesis of the disease.

Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P0 promoter

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P0) produces an unstable transcript, while the known downstream promoter (P1) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P0 but retaining P1. We confirm the previous finding that P0 is dispensable for Xist expression in undifferentiated cells and that P1 can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P1 is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P0 does not represent the 5′ end of a distinct Xist isoform. Instead, P0 is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P1 originates from the DNA strand opposite Xist and represents the 3′ end of the antisense Tsix RNA. Thus, these data do not support the existence of a P0 promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer’s disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.

Specific intercellular binding of the β-amyloid precursor protein to the presenilins induces intercellular signaling: Its significance for Alzheimer’s disease

Genetic evidence has implicated three proteins, the β-amyloid precursor protein (β-APP) and the two homologous presenilins (PS-1 and PS-2), in the etiology of Alzheimer’s disease (AD). How these three proteins jointly contribute to AD, however, is not clear. Nor is any of their normal physiological functions known. Herein, we demonstrate, confirming a prediction made earlier, that β-APP and either PS-1 or PS-2 act as a specific membrane-bound ligand binding intercellularly with either of its two membrane receptors. This results in a cell–cell adhesion, after which rapid transient increases in protein tyrosine kinase activity and protein tyrosine phosphorylation occur coordinately inside one or both of the two adherent cells. The spectrum of proteins modified by tyrosine phosphorylation differs depending on whether PS-1 or PS-2 is involved in the specific intercellular binding to β-APP, which implies that PS-1 and PS-2 have distinct, rather than redundant, functions in normal physiology. The relevance of this intercellular interaction and signaling process to AD is discussed.

Amyloid-β peptide–Receptor for Advanced Glycation Endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: A proinflammatory pathway in Alzheimer disease

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Aβ) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Aβ, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor κB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Aβ deposits, and in cerebrospinal fluid from AD patients there was ≈5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Aβ-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Aβ, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Aβ on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation

Amyloid plaques in Alzheimer disease are primarily aggregates of Aβ peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Aβ peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the β-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3′,5′-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.