Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. I. Specific association of bindin with gel-phase phospholipid vesicles.

Bindin is a 30,000-mol-wt protein of sea urchin sperm that is responsible for the specific adhesion of the sperm acrosomal process to the vitelline layer covering the egg plasma membrane during fertilization. Sulfated glycoconjugates are believed to be the egg surface receptors for bindin, but the mechanism by which bindin associates with the sperm acrosomal membrane is unknown. Here I report that bindin specifically associates with phospholipid vesicles in vitro. Interaction of the bindin polypeptide with liposomes was found to cause an increase in the density of the liposomes and induce the aggregation of the vesicles. A novel property of this association of bindin with membranes was that it required phospholipids in a gel phase. The interaction of bindin with liposomes was greatly reduced at temperatures above the phase transition temperature. The interaction of bindin with gel-phase vesicles appeared to be reversible, since the aggregated vesicles dissaggregated as the temperature was raised above the phase transition temperature. Association of bindin with the bilayer did not alter the accessibility of the polypeptide to cleavage by trypsin, which suggests that most of the polypeptide chain remains exposed at the surface of the membrane.

Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. II. Bindin induces the fusion of mixed-phase vesicles that contain phosphatidylcholine and phosphatidylserine in vitro.

Bindin from sea urchin sperm associates with gel-phase phospholipid bilayers (Glabe, C. G., 1985, J. Cell Biol., 100:794-799). Bindin also interacts with phospholipid vesicles containing both gel-phase and fluid-phase domains and thereby induces their aggregation. Association of bindin with vesicles containing gel-phase domains of dipalmitoylphosphatidylcholine (DPPC) and fluid-phase domains of brain phosphatidylserine (PS) was found to result in the fusion of the vesicles. After incubation with bindin, these mixed-phase vesicles were much larger as determined by gel filtration chromatography and electron microscopic observations of negatively stained samples. The average diameter of the vesicles after incubation was 190 +/- 109 nm compared with 39 +/- 20 nm for vesicles incubated in the absence of bindin. Resonance energy transfer studies also indicated that bindin induces the fusion of vesicle bilayers. Two fluorescent probes (NBD-PE and Rh-PE) were incorporated into the membrane of mixed-phase DPPC:PS vesicles at a density of 0.5 mol%, where efficient energy transfer occurs between the probes. The efficiency of energy transfer was proportional to the concentration of the fluorescence energy acceptor in the bilayer. The fluorescent vesicles were mixed with an excess of unlabeled target vesicles to quantify fusion. After bindin addition, there was a significant decrease in the efficiency of energy transfer compared with controls incubated in the absence of bindin. Although bindin induced the fusion of vesicles in the absence of calcium, the rate of fusion in the presence of 2 mM calcium was three-fourfold higher. In the presence of calcium, approximately half of the vesicles in the population had fused with another vesicle after incubation with bindin for 20 min. Bindin did not induce the fusion of gel-phase DPPC vesicles or mixed-phase vesicles of DPPC and dioleoylphosphatidylcholine, which suggests that the fusagenic activity of bindin requires specific phospholipids. Electron microscopic observations of DPPC:PS vesicles incubated in the presence of bindin suggest that the outer leaflets of bindin-aggregated vesicles are in close apposition. This is believed to be an important initial event for membrane fusion. These observations suggest that bindin may play a dual role in fertilization: Bindin mediates the attachment of sperm to glycoconjugate receptors of the egg surface and may also participate in the fusion of the sperm and egg plasma membranes.

Species-specific sperm adhesion in sea urchins. A quantitative investigation of bindin-mediated egg agglutination.

Bindin, a protein component of the acrosomal vesicle of sea urchin sperm, has been isolated from Arbacia punctulata and strongylocentrous purpuratus. Using this isolated bindin, we have devised a quantitative assay for bindin-mediated egg agglutination and compared the agglutination of bindin eggs from A. puntulata and S. purpuratus. Bindin- mediated agglutination is species -specific in both species, although a measurable degree of heterotypic interaction is observed. Homotypic bindin-egg interactions differ significantly from heterotypic interactions both in the extent of agglutination and the size of the resulting aggregates. We also provide direct evidence that bindin particles agglutinate eggs by adhering to the surfaces of adjacent eggs. Although the A. punctulata bindin preparation displays the same functional properties and consists of one major polypeptide of the same apparent molecular weight as S. purpuratus bindin, its morphology is very different. Unlike the spherical aggregates observed with S. purpuratus bindin, A punctulata bindin exists as lamellar vesicles and binds significant amounts of phospholipids and Triton X-100, suggesting that it may be tightly associated with the acrosomal membrane. Having defined a number of the basic parameters of bindin-mediated agglutination, we examined the effect of a number of saccharides and glycopeptides on bindin-mediated egg agglutination. Carbohydrate-containing components derived from the egg cell surface by proteolysis were found to inhibit bindin-mediated egg agglutination at low concentrations, but this inhibition is not species specific.

Isolation and characterization of the vitelline layer of sea urchin eggs.

The vitelline layers (VLs) of unfertilized sea urchin eggs were isolated by homogenization in a hypotonic medium containing Triton X-100 and EDTA. The surface topography of the VL is not changed by isolation. The thickness of the isolated VLs (300-400 A) is greater than that reported for VLs on intact eggs (100-200 A). Sperm adhere to the isolated VLs. When both internal and external VL surfaces are accessible to sperm, the sperm attach only to the external surface, suggesting that the external surface may carry sperm receptor proteins not present on the internal surface. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis shows that isolated VLs are composed of numerous proteins ranging from greater than 213,000 to 25,000 daltons. Lactoperoxidase-catalyzed 125I-iodination of unfertilized eggs labels two high molecular weight bands that stain faintly for carbohydrate. VLs are 90% protein and 3.5% carbohydrate. No predominance of a single amino acid or class of amino acids was found. Carbohydrate analysis yields fucose, mannose, galactose, glucose, xylose, glucosamine, galactosamine, and sialic acid. Controls for purity indicate that isolated VLs contain 2% protein of cytoplasmic origin and no more than 2.5% egg jelly.

Carbohydrate specificity of sea urchin sperm bindin: a cell surface lectin mediating sperm-egg adhesion.

We have examined the carbohydrate specificity of bindin, a sperm protein responsible for the adhesion of sea urchin sperm to eggs, by investigating the interaction of a number of polysaccharides and glycoconjugates with isolated bindin. Several of these polysaccharides inhibit the agglutination of eggs by bindin particles. An egg surface polysaccharide was found to be the most potent inhibitor of bindin-mediated egg agglutination. Fucoidin, a sulfated fucose heteropolysaccharide, was the next most potent inhibitor, followed by the egg jelly fucan, a sulfated fucose homopolysaccharide, and xylan, a beta(1 leads to 4) linked xylose polysaccharide. A wide variety of other polysaccharides and glycoconjugates were found to have no effect on egg agglutination. We also report that isolated bindin has a soluble lectinlike activity which is assayed by agglutination of erythrocytes. The bindin lectin activity is inhibited by the same polysaccharides that inhibit egg agglutination by particulate bindin. This suggests that the egg adhesion activity of bindin is directly related to its lectin activity. We have established that fucoidin binds specifically to bindin particles with a high apparent affinity (Kd = 5.5 X 10(-8) M). The other polysaccharides that inhibit egg agglutination also inhibit the binding of 125I-fucoidin to bindin particles, suggesting that they compete for the same site on bindin. The observation that polysaccharides of different composition and linkage type interact with bindin suggests that the critical structural features required for binding may reside at a higher level of organization. Together, these findings strengthen the hypothesis that sperm-egg adhesion in sea urchins is mediated by a lectin-polysaccharide type of interaction.

The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.

During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.

Characterization of the membrane-associating domain of the sperm adhesive protein, bindin.

Bindin is an adhesive protein that mediates the binding of sea urchin sperm to the egg during fertilization. Bindin selectively associates with gel-phase phospholipid vesicles in a peripheral fashion. Bindin interacts specifically with sulfated fucan on the egg's surface, and directly with the phospholipid bilayer of the sperm. Analysis of a series of deletion mutants of recombinant bindin was undertaken to define the membrane associating domain of bindin. Recombinant and sperm bindin display nearly identical binding kinetics to gel-phase phospholipids and have equivalent saturation points of approx. 250 lipid molecules per molecule of bindin. Deletion mutants of bindin which contain residues 75-130 retained specific membrane binding activity. Synthetic peptides corresponding to residues 69-130, and 92-130 also display gel-phase specific membrane association. This region is highly conserved within four different species of bindin molecules. Circular dichroism spectroscopy of synthetic peptides corresponding to residues 92-130 and 69-130 suggests that a distinct change in conformation takes place upon binding liposomes. Taken together, these data indicate that the membrane binding activity of bindin residues within this highly conserved region of the bindin molecule.

Specific recognition of sulfate esters by bindin, a sperm adhesion protein from sea urchins.

Bindin specifically binds to egg surface sulfated fucan polysaccharides and mediates the attachment of sperm to the egg during fertilization. Sulfate esters are critical for this interaction. We have examined the effect of different anionic groups on the relative binding affinities of a series of homologous anionic polymers for bindin to determine the extent to which other charged moieties can substitute for sulfate. We found that bindin displays a remarkable specificity for sulfate- or sulfonic acid-containing polymers. The relative affinities of poly(vinyl sulfate) and poly(styrenesulfonic acid) are four orders of magnitude higher than polymers containing phosphate esters or carboxyl groups. The bindin-mediated aggregation of sea urchin eggs was inhibited by the sulfated polymers but not the other anionic polymers. This high degree of selectivity for sulfated polymers is not observed for the binding of the polyanions to most other proteins and basic polypeptides. These results suggest that the binding is not due to the formation of simple salt bridges, and that all three non-ester oxygen atoms of the sulfate groups are involved in multiple bonding interactions with a complementary 'docking site' on the bindin polypeptide. The orientation of the polysaccharide sulfate oxygen atoms relative to the protein binding site may be an important determinant of the specificity of polysaccharide binding.

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions.

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.

Amyloid beta protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxyterminal fragments of the amyloid precursor protein.

A critical issue concerning Alzheimer's disease is its selectivity, which leads to cellular degeneration in certain brain areas but not in others, and whether this pathogenic selectivity involves products of the amyloid precursor protein (APP). Here, we show that the amyloid beta protein Abeta1-42 is accumulated gradually and is retained intact by field CA1, but not by other subdivisions, of organotypic hippocampal slice cultures. In contrast, the slightly shorter Abeta1-40 peptide was not sequestered selectively. Sequestration of Abeta1-42 was followed by the build-up of carboxyterminal fragments of the endogenous precursor protein that were identified by immunoprecipitation. Unlike the peptide uptake, this induction appeared to be stochastic at the cellular level. In addition, the APP fragments were distributed more broadly within the CA1 pyramidal neurons than the sequestered Abeta1-42, and they appeared to be localized to synaptic terminals in the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of the subfield CA3. Concentrations of synaptophysin, a presynaptic marker, decreased as the number of neurons producing amyloidogenic species increased. These results indicate that exogenous Abeta1-42 sets into motion a sequence that involves 1) selective uptake of the peptide by vulnerable cells at risk in Alzheimer's disease, 2) markedly enhanced production of amyloidogenic precursor material, and 3) slow deterioration of central synapses.

Complement activation by cross-linked truncated and chimeric full-length beta-amyloid.

The activation of complement by beta-amyloid (A beta) has been implicated in the local inflammatory response in Alzheimer's disease. To assess the structural parameters required for this activation, beta-sheet-containing fibrils of A beta1-28 were induced by low pH and then chemically cross-linked to constrain the beta-sheet conformation. Chimeric A beta peptides with a substituted C-terminal sequence derived from two different transmembrane proteins were also assessed for the ability to form fibrils rich in beta-sheet structure and to activate complement. Both the cross-linked A beta1-28 and the chimeric A beta peptides were strong activators of the classical complement pathway. These results suggest that the C-terminal residues (29-42) of A beta facilitate fibril assembly required for complement activation but do not contain the interaction sites required for complement activation, further supporting the hypothesis that C1q binds to the N-terminal hydrophilic domain of A beta, and that a fibrillar beta-sheet-rich conformation is required for effective binding and activation of C1.

Preferential adsorption, internalization and resistance to degradation of the major isoform of the Alzheimer's amyloid peptide, A beta 1-42, in differentiated PC12 cells.

A central question in Alzheimer's disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer A beta 1-42 form of amyloid in pathogenesis led us to characterize the interaction of A beta with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled A beta in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All A beta peptides examined absorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of A beta 1-42 is significantly greater than that of A beta 1-40 and A beta 1-28. The adsorption of A beta 1-42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized A beta 1-42 is also very different than shorter A beta peptides; a fraction of the internalized A beta 1-42 accumulates intracellularly and is resistant to degradation for at least 3 days while A beta 1-40 and shorter peptides are eliminated with a half life of about 1 h. A beta 1-42 does not appear to inhibit lysosomal hydrolases, since A beta 1-28 is degraded at the same rate in the presence or absence of A beta 1-42. The intracellular A beta 1-42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of A beta 1-42 compared to A beta 1-40 and A beta 1-28. These differences may be important for the preferential accumulation of the longer A beta 1-42 isoform and its association with AD pathogenesis.

Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP).

The secreted isoforms of the amyloid precursor protein (APP) that contain the Kunitz domain are also known as protease nexin 2 (PN2). Normal proteolytic processing of transmembrane APP, which results in the majority of soluble PN2, cleaves within the Alzheimer's A beta peptide, precluding A beta formation. Recent data indicate that soluble PN2 is internalized by cells via the low density lipoprotein receptor-related protein (LRP), which binds multiple ligands including apolipoprotein E (apoE) [23]. However, soluble PN2 cannot contribute to amyloid accumulation, so we examined whether the unprocessed, transmembrane form of APP751 containing the intact A beta sequence would form complexes with a PN2 ligand, EGF binding protein (EGFBP), and be internalized by LRP. We found that the addition of EGFBP to cells overexpressing APP751 induced the internalization of this amyloidogenic form of APP. The 39 kDa LRP receptor associated protein (RAP), an antagonist for LRP, blocked the internalization of APP751/PN2, suggesting a common LRP-mediated internalization pathway for both soluble and transmembrane APP751/PN2 after protease complex formation. Previous work has shown that internalization of transmembrane APP can lead to the formation of amyloidogenic carboxyl-terminal fragments and increased secretion of the Alzheimer's A beta peptide. Our data suggest the protease ligands for PN2 may play an important role in altering APP processing pathways to favor amyloid formation, and that LRP may be a point at which the apoE and amyloid processing pathways intersect.

In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity.

beta-Amyloid peptide forms the senile plaques of Alzheimer's disease and has been previously demonstrated to have both trophic and toxic effects on neurons in vitro. We report here that synthetic beta-amyloid peptide shows both aggregation and neurotoxicity after a 2-4 day incubation period, but is neurite-promoting and not toxic in its initially solubilized state. SDS-PAGE characterization shows that newly solubilized beta-amyloid is predominantly monomeric whereas incubated peptide has several high molecular weight species.

Beta-amyloid protein promotes neuritic branching in hippocampal cultures.

In the neuritic plaques of Alzheimer's disease, abnormal neuritic processes cluster around a core of beta-amyloid protein. Previous data have shown that beta 1-28, a peptide homologous to the first 28 amino acid residues of beta-amyloid protein, enhanced survival without affecting neuritic extension or branching in cultures of hippocampal neurons. In this paper we show that beta 1-42, a synthetic peptide which corresponds to the full 42 amino acid sequence of beta-amyloid protein, increased cell survival and also promoted the elongation of axon-like processes, raised the number of dendrite-like processes, and increased their arborization.

Transgenic mice as a model of pre-clinical Alzheimer's disease.

At diagnosis, Alzheimer's disease (AD) brains are extensively burdened with plaques and tangles and display a degree of synaptic failure most likely beyond therapeutic treatment. It is therefore crucial to identify early pathological events in the progression of the disease. While it is not currently feasible to identify and study early, pre-clinical stages of AD, transgenic (Tg) models offer a valuable tool in this regard. Here we investigated cognitive, structural and biochemical CNS alterations occurring in our newly developed McGill-Thyl-APP Tg mice (over-expressing the human amyloid precursor protein with the Swedish and Indiana mutations) prior to extracellular plaque deposition. Pre-plaque, 3-month old Tg mice already displayed cognitive deficits concomitant with reorganization of cortical cholinergic pre-synaptic terminals. Conformational specific antibodies revealed the early appearance of intracellular amyloid ß (Aß)-oligomers and fibrillar oligomers in pyramidal neurons of cerebral cortex and hippocampus. At the same age, the cortical levels of insulin degrading enzyme -a well established Aß-peptidase, were found to be significantly down-regulated. Our results suggest that, in the McGill-Thy1-APP Tg model, functional, structural and biochemical alterations are already present in the CNS at early, pre-plaque stages of the pathology. Accumulation of intraneuronal neurotoxic Aß-oligomers (possibly caused by a failure in the clearance machinery) is likely to be the culprit of such early, pre-plaque pathology. Similar neuronal alterations might occur prior to clinical diagnosis in AD, during a yet undefined 'latent' stage. A better understanding of such pre-clinical AD might yield novel therapeutic targets and or diagnostic tools.

The sequence of the Arbacia punctulata bindin cDNA and implications for the structural basis of species-specific sperm adhesion and fertilization.

Bindin is the major protein component of the acrosome granule of sea urchin sperm which mediates the species-specific adhesion of sperm to the egg surface during fertilization. Bindin isolated from both Arbacia punctulata and Strongylocentrotus purpuratus sperm demonstrate a distinct adhesive preference for eggs of the same species although a significant amount of cross-species reactivity is observed. Here we describe the isolation and sequence of A. punctulata bindin cDNA clones and a comparison of the predicted protein sequence with the sequence previously reported for S. purpuratus bindin (Gao et al., 1986, Proc. Natl. Acad. Sci., USA 83, 8634-8638). Bindins from these genera show substantial sequence similarity in both the mature bindin domain and the probindin precursor region. The most striking identity is a region of 42 conserved amino acids in the central part of the mature bindins. This conserved domain may be responsible for conserved functions of bindin. Regions flanking this conserved element on both the amino and carboxyl side are more highly divergent, suggesting that they are responsible for the species-specific properties of bindin. The mature A. punctulata sequence contains a putative transmembrane segment between residues 431 and 451 that is absent from S. purpuratus bindin. This structural element may account for the previous observation that isolated A. punctulata bindin uniquely forms multilamellar structures reminiscent of lipid bilayers and binds significant amounts of phospholipid and detergent. The structure of this hydrophobic segment also displays a number of similarities to viral fusion peptides.