Guinea-pig primary cell cultures provide a model to study expression and amyloidogenic processing of endogenous amyloid precursor protein

Until now guinea-pigs have been rarely used to investigate formation and deposition of Alzheimer's disease-associated amyloid beta peptides despite the sequence identity of human and guinea-pig amyloid beta peptides being known, and the overall similarity of human and guinea-pig amyloid precursor protein. We now describe a primary cell culture system of mixed fetal guinea-pig brain cells, which we have applied to characterize endogenous amyloid precursor protein processing and amyloid beta formation. These cell cultures were established at embryonic day 24 of guinea-pigs after comparison of selected stages of guinea-pig ontogenetic development with the known ontogeny of rats, and were characterized by immunocytochemical detection of neuronal and glial marker proteins. Amyloid precursor protein expression, processing and amyloid beta formation increased in parallel with cellular maturation during cultivation and reached a stable phase after approximately 14 days in vitro therefore providing a suitable time for analysis. Aged cultures display strong neuronal amyloid precursor protein immunoreactivity and an altered profile of amyloid precursor protein isoform messenger RNA expression due to glial proliferation as single neurons were shown to retain their typical pattern of amyloid precursor protein expression. We show that amyloid precursor protein in guinea-pig cells is processed by different protease activities which most likely represent alpha- and beta-secretase, leading to the generation of soluble amyloid precursor protein derivatives. Furthermore, endogenous amyloid precursor protein processing leads to production of substantial amounts of amyloid beta-peptides which accumulate in conditioned culture medium. Amyloid beta was readily detectable by western blot analysis and was shown to consist of approximately 80-90% amyloid beta(1-40). We suggest that primary guinea-pig cell cultures provide a valuable tool in amyloid research that resembles amyloid precursor protein processing under physiological concentrations and, therefore, the situation in humans more closely than current rodent models. It should be especially useful in screening experiments for secretase inhibiting compounds.     

A neural cell adhesion molecule-derived peptide reduces neuropathological signs and cognitive impairment induced by Abeta25-35

By means of i.c.v. administration of preaggregated oligomeric beta-amyloid (Abeta)25-35 peptide it was possible in rats to generate neuropathological signs related to those of early stages of Alzheimer's disease (AD). Abeta25-35-administration induced the deposition of endogenously produced amyloid protein. Furthermore, quantitative immunohistochemistry demonstrated time-related statistically significant increases in amyloid immunoreactivity, tau phosphorylation, microglial activation, and astrocytosis, and stereological investigations demonstrated statistically significant increased neuronal cell death and brain atrophy in response to Abeta25-35. Finally, the Abeta25-35-administration led to a reduced short-term memory as determined by the social recognition test. A synthetic peptide termed FGL derived from the neural cell adhesion molecule (NCAM) was able to prevent or, if already manifest, strongly reduce all investigated signs of Abeta25-35-induced neuropathology and cognitive impairment. The FGL peptide was recently demonstrated to be able to cross the blood-brain-barrier. Accordingly, we found that the beneficial effects of FGL were achieved not only by intracisternal, but also by intranasal and s.c. administration of the peptide. Furthermore, FGL-treatment was shown to inhibit the activity of GSK3beta, a kinase implicated in signaling regulating cell survival, tau phosphorylation and the processing of the amyloid precursor protein (APP). Thus, the peptide induced a statistically significant increase in the fraction of GSK3beta phosphorylated on the Ser9-position, a posttranslational modification known to inhibit the activity of the kinase. Hence, the mode of action of FGL with respect to the preventive and curative effects on Abeta25-35-induced neuropathological manifestations and cognitive impairment involves the modulation of intracellular signal-transduction mediated through GSK3beta.     

Cytoskeletal protein pathology and the formation of beta-amyloid fibers in Alzheimer's disease

Discovery of the abnormally phosphorylated tau in paired helical filaments, its accumulation preceding the formation of the tangles and the in vitro microtubule assembly defect suggest that an abnormality in the protein phosphorylation/dephosphorylation system is involved in the pathogenesis of Alzheimer cytoskeletal pathology. The levels of mRNA for the beta-amyloid precursor protein (beta APP) in the brain suggest that only a small deficiency in the processing of the precursor would be sufficient to account for the accumulation of beta-amyloid in Alzheimer brain. Identification of reticuloendothelial system cells responsible for the production/processing of beta-amyloid will help to elucidate the pathogenesis of the brain amyloidosis. The disproportionate accumulation of paired helical filaments and amyloid within the same affected brain and from disease to disease raises the possibility of different etiologies for each of these lesions coexisting in Alzheimer's disease.     

Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.     

The role of the protein glycosylation state in the control of cellular transport of the amyloid beta precursor protein

The amyloid beta precursor protein can exist as both a membrane-bound and a secreted protein, with the former having the potential to generate the amyloid beta peptide present in the neuritic plaques which are characteristic of Alzheimer's disease. In this study, we have used a clone of the AtT20 mouse pituitary cell line which expresses high levels of the amyloid beta precursor protein to characterize the glycosylation state of the secreted and membrane-bound forms of the protein and to examine the role of post-translational modifications in protein processing. Lectin blot analysis of immunoprecipitated amyloid beta precursor protein demonstrated that the soluble form of the protein contains significant amounts of sialic acid, with the lectin staining being reduced in the particulate cellular fractions. Treatment of the cells with mannosidase inhibitors to interfere with the formation of complex-type N-linked glycans resulted in a decrease in secreted amyloid beta precursor protein and an increase in the level of the cellular form of the protein. The increase in amyloid beta precursor protein levels in the cellular fraction was accompanied by an increase in perinuclear staining. Furthermore, cells overexpressing the alpha2,6(N)-sialyltransferase enzyme also demonstrated an increase in amyloid beta precursor protein secretion. These results suggest that the presence of terminal sialic acid residues on complex-type N-glycans may be required for the optimal transport of the amyloid beta precursor protein from the Golgi to the cell membrane with the subsequent cleavage to generate the secreted form of the protein.     

Secretory pathway of beta/A4 amyloid protein precursor in familial Alzheimer's disease with Val717 to Ile mutation.

To determine whether the secretory pathway of beta/A4 amyloid protein precursor (APP) was altered in familial Alzheimer's disease (FAD) with a mutation of Val717 to Ile, cerebrospinal fluid was studied by Western blotting. The ratio of the density of the bands labeled with the antibody against the amino-terminal part of beta/A4 protein to that with the antibody against amino-terminal part of beta/A4 protein to that with the antibody against amino-terminal part of APP was not decreased. The present result suggests that the secretory pathway is not altered by the mutation in such a way that amyloidogenic full-length beta/A4 protein is generated.     

Carboxyl-terminal fragments of beta-amyloid precursor protein bind to microtubules and the associated protein tau.

Alzheimer's disease is a neurodegenerative disorder characterized by protein depositions in intracellular and extracellular spaces in the brain. The intraneuronal deposits are formed by neurofibrillary tangles composed mainly of abnormally phosphorylated tau, a microtubule-associated protein, whereas the major constituent of the amyloid deposited extracellularly in the brain parenchyma and vessel walls is amyloid beta-protein (A beta). The proteolytic processing of the beta-amyloid precursor protein (beta PP) results in the generation of a complex set of carboxyl-terminal peptides that contain A beta. In this study, we have used fusion proteins containing carboxyl-terminal fragments of beta PP to investigate the association of beta PP with cellular components. We demonstrate that specific domains within the carboxyl end of beta PP contain binding sites for cytoskeletal components; one, within residues 1 to 28 of A beta, binds directly to tubulin, and the second one, within sequences carboxyl-terminal to A beta, binds tau and tubulin. We propose that the two neuropathological hallmarks of Alzheimer's disease, A beta deposition and neurofibrillary tangles, represent the residual of a disrupted beta PP-tubulin-tau complex.     

The chick embryo appears as a natural model for research in beta-amyloid precursor protein processing

This study reveals that the chick embryo has active the machinery for the production and degradation of the amyloid beta peptide characteristic of Alzheimer's disease. We cloned the principal beta-amyloid precursor protein isoforms in the chick embryo and observed that they are highly homologous to the human sequences and identical at the C-terminal sequence, including the amyloid beta domain. Mammals such as rat or mouse, more commonly used as animal models of human diseases, have a distinct amyloid beta sequence. The distribution of beta-amyloid precursor protein isoforms in the chick embryo revealed that, as in humans, their expression is ubiquitous and the prototype beta-amyloid precursor protein-695 predominated in the nervous system. We also found that the chick embryo expresses the genes for the main proteolytic proteases implicated in the production of amyloid beta, including BACE-1, BACE-2, presenilin-1, presenilin-2 and nicastrin, as well as the amyloid beta-degrading enzyme neprilysin, or ADAM-17, a protease implicated in the non-amyloidogenic processing of beta-amyloid precursor protein. We have also found that between amyloid beta40 and amyloid beta42, this latter seems to be the major amyloid beta peptide produced during chick embryogenesis. The chick embryo appears as a suitable natural model to study cell biology and developmental function of beta-amyloid precursor protein and a potential assay system for drugs that regulate beta-amyloid precursor protein processing.     

Ser(2194) is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A

Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193-2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatography-mass spectrometry. The elution time of the phosphopeptide determined by negative-ion electrospray ionization-mass spectrometry corresponded with the elution time of the majority of (32)P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization-tandem mass spectrometry revealed that Ser(2194) was the major phosphorylated residue on the phosphopeptide GpSPPSLASSSASQLSAPSLK. Substitution for Ser(2194) with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser(2194) and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser(2194) may be an important mechanism for modulating NS5A biological functions.     

Amyloid precursor protein associates independently and collaboratively with PTB and PDZ domains of mint on vesicles and at cell membrane.

The mint family consists of evolutionarily conserved adapter proteins from Caenorhabditis elegans to mammalian neurons. Three mammalian isoforms, mint1, 2, and 3, are extensively diverted in their N-terminal halves and, in striking contrast, are highly homologous to each other in their C-terminal halves containing phosphotyrosine-binding (PTB) and PSD-95/DLG-A/ZO-1 (PDZ) domains that work as protein-protein interaction modules. Biochemical and genetic analyses revealed that mint1 and LIN-10, a homolog in C. elegans, comprise macromolecular complexes in the presynaptic and postsynaptic terminals, thereby bringing synaptic vesicles to the exocytotic transmitter release site and localizing receptors and ion channels in the specific membrane domains. Amyloid precursor protein is one of the targets of the PTB domain of mint and this interaction modulates its proteolytic procedures ending up with amyloid beta peptide production, but its molecular mechanism is unclear. We show by an in situ hybridization technique that mint3, a ubiquitous isoform, is expressed both in polar cells like neurons, and in non-polar cells, such as glia and ependymal cells, in the mouse brain. In addition, a considerable amount of a human homolog mint3 (approximately 70 kDa) was expressed in a human epithelial cell line. Subcellularly, mint3 is specifically enriched in vesicles in the cytoplasm, cell membrane, and Golgi complex as reserves. A series of deletions or site-directed mutations revealed that mint3 double recognizes an amyloid precursor protein-containing macromolecular complex via the PTB and PDZb domains independently and cooperatively, not only in the cytoplasmic transporting vesicles but even after amyloid precursor protein was targeted and/or inserted to the specific cell membrane domains.From these results we suggest that mint3 links amyloid precursor protein to other components, thereby regulating its transport, endocytosis, and metabolism. Abnormal metabolism of amyloid precursor protein causes an early-onset type of Alzheimer's disease but its molecular mechanism is incompletely understood. The present findings give morphological evidence and a molecular framework of how mint interacts with amyloid precursor protein and modifies its processing on the secretory pathway.     

Phosphorylated protein kinases associated with neuronal and glial tau deposits in argyrophilic grain disease

Tau phosphorylation was examined in argyrophilic grain disease (AGD) by using the phosphospecific tau antibodies Thr181, Ser202, Ser214, Ser 396 and Ser422, and antibodies to non-phosphorylated and phosphorylated mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK), stress-activated kinase (SAPK), c-Jun N-terminal kinase (JNK), p38 kinase (p-38), alpha-calcium/calmodulin-dependent kinase II (alphaCaM kinase II), and glycogen synthase kinase-3 (GSK-3), all of which regulate phosphorylation at specific sites of tau. This is the first study in which the role of protein kinases in tau phosphorylation has been examined in AGD. Hyperphosphorylated tau accumulated in grains and pre-tangles in the hippocampus, dentate gyrus, entorhinal and trans-entorhinal cortices, and amygdala in all cases. Ballooned neurons in the amygdala, entorhinal, insular and cingulate cortex, and claustrum contained alphaB-crystallyn and phosphorylated neurofilament epitopes. Some astrocytes and scattered oligodendrocytes containing coiled bodies were recognized with anti-tau antibodies. A few tangles were observed in the entorhinal cortex and hippocampus corresponding to Alzheimer's disease (AD) stages I-III of Braak and Braak. None of the present cases was associated with progressive supranuclear palsy or with alpha-synuclein pathology. Two bands of phospho-tau of 64 and 68 kDa were observed in Western blots of sarkosyl-insoluble fractions enriched with abnormal filaments in AGD, a pattern that contrasts with the 4-band pattern obtained in AD. No modifications in the expression of non-phosphorylated MEK-1, ERK2 and GSK-3alpha/beta, as revealed by immunohistochemistry, were seen in AGD, but sarkosyl-insoluble fractions were particularly enriched in JNK-1 and alphaCaM kinase II. Increased expression of the phosphorylated (P) forms of MAPK/ERK, SAPK/JNK, p38 and GSK-3beta was found in grains and tau-containing cells in AGD. MAPK/ERK-P immunoreactivity was observed in pre-tangles and, diffusely, in the cytoplasm of ballooned neurons, but not in grains. Strong SAPK/JNK-P and P38-P, and moderate GSK-3b-P immunoreactivities selectively occured in grains, in neurons with pre-tangles and in the peripheral region of the cytoplasm of ballooned neurons. MAPK/ERK-P, SAPK/JNK-P, p38-P and GSK-3beta-P were expressed in tau-containing astrocytes and in oligodendrocytes with coiled bodies. Western blots revealed kinase expression in sarkosyl-insoluble fractions but none of the phospho-kinase antibodies recognized hyper-phosphorylated tau protein. These findings indicate complex, specific profiles of tau phosphorylation and concomitant activation of precise kinases that have the capacity to phosphorylate tau at specific sites in AGD. These kinases co-localize abnormal tau in selected structures and cells, including neurons with pre-tangles, ballooned neurons, astrocytes and oligodendrocytes. Most of these kinases are involved in cell death and cell survival in certain experimental paradigms. However, double-labeling studies with the method of in situ end-labeling of nuclear DNA fragmentation and cleaved (active) caspase-3 immunohistochemistry show no expression of apoptosis and death markers in cells bearing phosphorylated kinases.     

Cyclic AMP-dependent protein kinase A and protein kinase C phosphorylate α4β2 nicotinic receptor subunits at distinct stages of receptor formation and maturation

Neuronal nicotinic receptor α4 subunits associated with nicotinic α4β2 receptors are phosphorylated by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), but the stages of receptor formation during which phosphorylation occurs and the functional consequences of kinase activation are unknown. SH-EP1 cells transfected with DNAs coding for human α4 and/or β2 subunits were incubated with ³²Pi, and PKA or PKC was activated by forskolin or phorbol 12,13-dibutyrate, respectively. Immunoprecipitation and immunoblotting of proteins from cells expressing α4β2 receptors or only α4 subunits were used to identify free α4 subunits, and α4 subunits present in immature α4β2 complexes and mature α4β2 pentamers containing complex carbohydrates. In the absence of kinase activation, phosphorylation of α4 subunits associated with mature pentamers was three times higher than subunits associated with immature complexes. PKA and PKC activation increased phosphorylation of free α4 subunits on different serine residues; only PKC activation phosphorylated subunits associated with mature α4β2 receptors. Activation of both PKA and PKC increased the density of membrane-associated receptors, but only PKC activation increased peak membrane currents. PKA and PKC activation also phosphorylated β2 subunits associated with mature α4β2 receptors. Results indicate that activation of PKA and PKC leads to the phosphorylation α4β2 receptors at different stages of receptor formation and maturation and has differential effects on the expression and function of human α4β2 receptors.     

Overexpression of amyloid precursor protein reduces epsilon protein kinase C levels

Alzheimer’s disease (AD) is characterized by extracellular deposits of amyloid beta peptide (Aβ), a peptide that is generated upon proteolytic cleavage of amyloid precursor protein (APP). The events leading to the development of AD and their sequence are not yet fully understood. Protein kinase C (PKC) has been suggested to have a significant role in controlling neuronal degeneration and in the aberrant signal transduction taking place in AD. Several studies document a deficit in PKC levels and activity in brains of AD patients when compared with those of normal controls. Such a decrease in PKC could have serious implications since certain PKC isozymes were shown to drive the APP proteolytic cleavage into a non-amyloidogenic pathway. Reduced levels of distinct PKC isozymes could thus contribute to driving APP processing toward an amyloidogenic pathway.     

Amyloid precursor protein cytoplasmic domain antagonizes reelin neurite outgrowth inhibition of hippocampal neurons

The function of the amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD) remains unknown. Among the proteins that interact with the APP cytoplasmic domain in vitro and in heterologous systems is Disabled-1, a signaling molecule of the reelin pathway. The physiological consequence of this interaction is unknown. Here we used an in vitro model of hippocampal neurons grown on a reelin substrate that inhibits neurite outgrowth. Our results show that an excess of APP cytoplasmic domain internalized by a cell permeable peptide, is able to antagonize the neurite outgrowth inhibition of reelin. The APP cytoplasmic domain binds Disabled-1 and retains it in the cytoplasm, preventing it from reaching the plasma membrane and sequesters tyrosine phosphorylated Disabled-1, both of which disrupt reelin signaling. In the context of AD, increased formation of APP cytoplasmic domain in the cytosol released after cleavage of the A beta peptide, could then inhibit reelin signaling pathway in the hippocampus and thus influence synaptic plasticity.     

Aberrant glycosylation modulates phosphorylation of tau by protein kinase A and dephosphorylation of tau by protein phosphatase 2A and 5

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in Alzheimer's disease (AD). We recently found that the aberrant tau glycosylation precedes tau hyperphosphorylation in AD brain. In the present study, we developed assays to determine phosphorylation and dephosphorylation of tau at specific phosphorylation sites by using glycosylated tau purified from AD brain as a substrate. We then studied the effects of the aberrant glycosylation on phosphorylation and dephosphorylation of tau at each specific phosphorylation site. We found that deglycosylation of the aberrantly glycosylated tau decreased the subsequent phosphorylation of tau at Ser214, Ser262, and Ser356 in vitro by protein kinase A. On the other hand, deglycosylation of tau positively modulated the subsequent dephosphorylation by protein phosphatase 2A and protein phosphatase 5 in vitro at the phosphorylation sites Ser198, Ser199, and Ser202.Our results suggest that the aberrant glycosylation may modulate tau protein at a substrate level so that it is easier to be phosphorylated and more difficult to be dephosphorylated at some phosphorylation sites in AD brain. The combined impact of this modulation may be to make tau more susceptible to becoming abnormally hyperphosphorylated.     

Glycogen synthase kinase-3β–mediated CCAAT/enhancer-binding protein delta phosphorylation in astrocytes promot es migration and activation of microglia/macrophages

Alzheimer's disease is neuropathologically characterized by the accumulation of amyloid-β protein into senile plaques that are sites of chronic inflammation involving reactive microglia, astrocytes, and proinflammatory molecules, such as interleukin-1β and tumor necrosis factor-α. The human CCAAT/enhancer-binding protein (CEBP) delta (CEBPD) is known to be induced in many inflammation-related diseases. In Alzheimer's disease, this protein is responsive to amyloid-β and proinflammatory cytokines in astrocytes. However, the functional role of CEBPD in astrocytes remains largely unclear. In this study, we show that CEBPD is upregulated by interleukin-1β through the mitogen-activated protein kinase p38 (MAPKp38) signaling pathway and phosphorylated by glycogen synthase kinase (GSK)-3β at Ser167 in astrocytes. CEBPD in astrocytes is associated with microglia activation and migration in amyloid precursor protein transgenic mice (AppTg) mice. We further identified that the monocyte chemotactic protein-1, a chemoattractive factor, and migration factors matrix metalloproteinase-1 and -3 are responsive to GSK3β-mediated CEBPD Ser167 phosphorylation. Our results revealed the novel regulation of LiCl on astrocytes and that GSK3β-mediated CEBPD phosphorylation in astrocytes plays an important role in the activation of microglia.     

Phosphorylation at the hydrophobic site of protein kinase C Apl II is increased during intermediate term facilitation

In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans-phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase.     

Pro-inflammatory complement activation by the A beta peptide of Alzheimer's disease is biologically significant and can be blocked by vaccinia virus complement control protein

The amyloid plaque is the hallmark of Alzheimer's disease (AD). The transmembrane domain and a portion of the C-terminus (A beta) of the amyloid precursor protein, are known to form the nucleus of the amyloid plaque. It has been demonstrated recently, using in vitro assays, that the A beta peptide can activate both the classical (antibody-independent) and alternate pathways of complement activation. The proposed complement activation is due to the binding of A beta to the complement components C1q and C3, respectively, which initiate formation of the proinflammatory C5a and C5b-9 membrane attack complex. In this report, we have investigated the in vitro findings for the likely complement-dependent proinflammatory properties of the Alzheimer's disease A beta peptide. We have performed experiments using congenic C5-deficient and C5-sufficient mice injected with synthetic A beta and recombinant polypeptide (C-100) containing A beta. Injection of C-100 into C5-sufficient mice induced a clear increase in the number of polymorphonuclear cells (neutrophils) at the site of injection due to complement activation and the subsequent release of proinflammatory chemtoactic factors. In sharp contrast, the C5-deficient mice did not show any increase in cellular influx. The vaccinia virus complement control protein, an inhibitor of both the classical and alternate pathway can down-regulate the biologically significant activation of complement by A beta, as demonstrated by an in vitro immunassay. The therapeutic down-regulation of A beta-caused complement activation could greatly alleviate the progression of some of the chronic neurodegeneration characteristic of Alzheimer's disease.     

Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.