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N K Robakis

Publications and source records attributed to N K Robakis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Presenilin-1-immunoreactive neurons are preserved in late-onset Alzheimer's disease.

Recent studies have suggested that missense mutations in the presenilin-1 gene are causally related to the majority of familial early-onset Alzheimer's disease (AD). To examine the possible involvement of presenilin-1 in late-onset sporadic AD, a quantitative analysis of its distribution in the cerebral cortex of nondemented and AD patients was performed using immunocytochemistry. Stereological analyses revealed that AD brains showed a marked neuronal loss in the CA1 field of the hippocampus and hilus of the dentate gyrus, subiculum, and entorhinal cortex. In these areas, however, the fraction of neurofibrillary tangle (NFT)-free neurons showing presenilin-1 immunoreactivity was increased compared with nondemented controls. In contrast, cortical areas, which displayed no neuronal loss, did not show any significant increase in the fraction of presenilin-1-positive neurons. Moreover, presenilin-1 immunoreactivity was reduced in NFT-containing neurons. Thus, in AD, the fraction of NFT-free neurons that contained presenilin-1 varied from 0.48 to 0.77, whereas the fraction of NFT-containing neurons that were presenilin-1 positive varied from 0.1 to 0.24. Together, these observations indicate that presenilin-1 may have a neuroprotective role and that in AD low cellular expression of this protein may be associated with increased neuronal loss and NFT formation.

Age of Onset↗
PubMed1996

Identification and neuron specific expression of the S182/presenilin I protein in human and rodent brains.

Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I). Currently, the PS-I gene product has not been identified and its function remains unknown. Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa. This antigen was also present in immortalized human and mouse neuronal cell cultures. Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction. In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites. Remarkably the relative partition among these three compartments varied dramatically. A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya. In most of the cerebrum, PS-I staining in axons was very weak or undetectable. By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity. Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites. These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function. The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells.

Animals↗
PubMed1996

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Acetylcholine↗
PubMed1996

Characterization of appican, the chondroitin sulfate proteoglycan form of the Alzheimer amyloid precursor protein.

In this report we focus on the characterization of appican, the chondroitin sulfate proteoglycan form of amyloid precursor protein (APP), and the role that it and other proteoglycans may play in AD. Appican is expressed by certain transformed cell lines of neural origin, namely C6 cells and N2a neuroblastomas. It is detected in both human and rat brain and in primary cultures is expressed by astrocytes, but not neurons. The core protein of appican has been shown to be an alternatively spliced isoform of APP, lacking exon 15 of the APP gene, originally identified in leukocytes (L-APP). Splicing out of exon 15 results in the joining of exons 14 and 16, and formation of an Asp-Xaa-Ser-Gly consensus sequence for chondroitin sulfate chain attachment to serine 619 of L-APP, which lies 16 amino acids upstream of the A beta peptide sequence. Mutation of this serine residue to an alanine prevented chondroitin sulfate chain addition to the core protein. Levels of appican expression could be regulated by growth conditions independently of APP, suggesting that these molecules may serve distinct physiological roles within the cell. Morphological changes were also observed in both astrocytic and transformed cell cultures, that appeared to reflect changes in levels of appican expression. Preliminary data suggest that appican may be a strong cell adhesion molecule. Transfected C6 glioma cells overexpressing appican remained attached to tissue culture dishes markedly better than either C6 cells over-expressing exon-15 containing APP or WT C6 cells. Appican-enriched extracellular matrix (ECM) was also observed to serve as a much better substrate for attachment of N2a neuroblastomas, pheocromocytoma PC12 cells and primary astrocytes compared to APP enriched ECM.

Alzheimer Disease↗
PubMed1996

Intracellular cyclic AMP inhibits constitutive and phorbol ester-stimulated secretory cleavage of amyloid precursor protein.

alpha-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid beta peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP751 to examine whether the alpha-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the alpha-secretase cleavage of APP751. At 1 microM, forskolin inhibited secretion of NXII by approximately 50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the alpha-secretase cleavage of APP751. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.

Amyloid beta-Protein Precursor↗
PubMed1995

Heat-shock induces abnormalities in the cellular distribution of amyloid precursor protein (APP) and APP fusion proteins.

The heat shock or stress response may play a role in the pathogenesis of Alzheimer's disease. We conducted experiments to visualize microscopically the distribution of wild type amyloid precursor protein (APP) and the behavior of an APP deletion mutant under stress. This was achieved by heat-shock treatment of cells expressing fusion recombinant APP proteins tagged with secreted placental alkaline phosphatase (SEAP). The fusion proteins were cleaved and secreted in a manner similar to wild type APP in unstressed control cells. SEAP activity was detected by cytochemical methods within the cytoplasm in less than 10% of transfected unstressed cells. Heat shocked cells showed a striking difference from the control cells in that over 90% of the stressed cells displayed strong intracytoplasmic SEAP activity occurring with Golgi-like pattern and/or membranous distribution. The effects of heat shock were not due to a peculiar behavior of the clones and depended on the APP portion of the constructs. This study shows miscompartmentalization of APP under stress. Such cellular changes may bear important implications in the processing of APP.

Alkaline Phosphatase↗
PubMed1995

The Alzheimer amyloid precursor proteoglycan (appican) is present in brain and is produced by astrocytes but not by neurons in primary neural cultures.

Recent studies showed that the Alzheimer amyloid precursor (APP) occurs as the core protein of a chondroitin sulfate proteoglycan (appican) in C6 glioma cells. In the present study we show that appican is present in both human and rat brain tissue. Cortical rat brain cell cultures were used to identify appican-producing cells. Soluble secreted and cell-associated appican was produced by mixed glial cultures but not by primary neuronal cultures. Among the three major glial cell types, astrocytes produced high levels of appican, while oligodendrocytes failed to produce any. Only low levels of this molecule were occasionally detected in microglial cultures. Expression of appican in astrocyte cultures was regulated by the composition of the growth media. N2a neuroblastoma cells also produced appican; however, treatment with dibutyryl cAMP which promotes neuronal differentiation in these cells inhibited its production without inhibiting synthesis of APP. In contrast to the restricted expression of appican, APP was present in all cultures, and its production was independent of appican synthesis. Neuronal cultures produced mainly APP695 while glial cultures produced the Kunitz type protease inhibitor containing APP. The astrocyte-specific expression of appican suggests a function distinct from the function of APP. Brain appicans may play a role in the development of Alzheimer disease neuropathology.

Amyloid beta-Protein Precursor↗
PubMed1995

The chondroitin sulfate attachment site of appican is formed by splicing out exon 15 of the amyloid precursor gene.

Appicans are secreted and cell-associated chondroitin sulfate proteoglycans containing Alzheimer amyloid precursor (APP) as their core protein. Appicans are found in brain tissue, and in cell cultures their expression depends on both cell type and growth conditions. Here we report that the core protein of appicans derives from an APP mRNA lacking exon 15. Splicing out of this exon creates a new consensus sequence for the attachment of a chondroitin sulfate chain in the resulting APP product. Transfection of C6 glioma or 293 kidney fibroblast cells with APP cDNAs containing exon 15 produced no appican, while transfection with an APP cDNA lacking this exon induced high levels of appican production. Polymerase chain reactions indicated that appican-producing cells contained an APP mRNA species without exon 15, whereas cells without this mRNA produced no appican. Site-directed mutagenesis combined with immunoreactivity experiments showed that the chondroitin sulfate chain is attached to a serine residue 16 amino acids upstream of the amino terminus of the A beta sequence of APP. The attachment of a glycosaminoglycan chain close to the A beta sequence of APP may affect the proteolytic processing of APP and production of A beta. The proteoglycan nature of APP suggests that addition of the chondroitin sulfate glycosaminoglycan is important for the implementation of the biological function of these proteins.

Alternative Splicing↗
PubMed1995

Evidence that secretase cleavage of cell surface Alzheimer amyloid precursor occurs after normal endocytic internalization.

Three different treatments (methylamine, colchicine, and 18 degrees C temperature block), known to disrupt normal endocytic internalization, significantly reduced the secretory cleavage of cell surface-derived Alzheimer amyloid precursor (APP) in non-transfected C6 cell cultures. Conversely, treatments with methylamine or colchicine had no significant effect on the secretion of total APP. Treatment of these cells with the lysosomotropic amine chloroquine resulted in a significant increase in the levels of both cell surface full-length APP and cell surface-derived secreted nexin II (NXII). Immunofluorescence analysis of C6 glioma cells transfected with APP751 indicated that under normal conditions, cell surface APP was internalized, and within 30 minutes was localized in discrete intracellular vesicles. These vesicles contained the endocytic tracer Texas red-conjugated ovalbumin and probably represented late endosomes or lysosomes. However, treatment of the transfected C6 cultures with methylamine or colchicine prevented localization of cell surface APP in intracellular vesicles, suggesting that these treatments altered the normal intracellular trafficking of cell surface-derived APP. Both the biochemical and immunofluorescence data are compatible with the suggestion that inhibition of normal endocytic internalization reduces the secretory cleavage of cell surface APP. Furthermore, our results suggest that following internalization, cell surface APP is cleaved by secretase(s) and secreted or routed to the lysosomes where it is degraded.

Alzheimer Disease↗
PubMed1995

Full-length and truncated Alzheimer amyloid precursors in chromaffin granules: solubilization of membrane amyloid precursor is mediated by an enzymatic mechanism.

The amyloid beta peptide (A beta) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact A beta sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37 degrees C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+, and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of A beta from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing A beta. The detection of soluble APP with intact A beta sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions.

Adrenal Medulla↗
PubMed1995

Expression of the chondroitin sulfate proteoglycans of amyloid precursor (appican) and amyloid precursor-like protein 2.

The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific antibodies suggested that a CS chain is attached within or proximal to the A beta sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of approximately 100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗
PubMed1995

Molecular cloning and characterization of the mouse dopamine D3 receptor gene: an additional intron and an mRNA variant.

The intron-exon organization for the murine dopamine D3 receptor gene was determined. A novel intron of approximately 1 kb was identified in both rat and mouse D3 receptor genes. This intron (termed intron 4) is situated between coding nucleotides 723 and 724, resulting in a split of former exon 4 (containing nucleotides 527-801) into two separate exons (exon 4 and exon 5). Thus, the coding regions of the D2 and D3 receptor genes contain an identical number of exons (seven exons) and share a very similar gene structure. Reverse transcription-PCR experiments revealed a short form of mouse D3 mRNA (D3Short) that lacks the first 63 nucleotides from exon 6, and results from a splicing event occurring within this exon. However, this mRNA variant was not found in either rat or human brain. No dopamine D3 receptor mRNA variants were found deriving from the alternative splicing of exon 5, although its counterpart, exon 6 in the D2 receptor gene, is spliced out to produce the D2Short mRNA. These data suggest that, although the intron-exon organizations of the D2 and D3 receptor genes are similar, the encoded transcripts may be processed differently.

Alternative Splicing↗
PubMed1994

Solubilization of full-length amyloid precursor proteins from PC12 cell membranes.

The amyloid beta protein (A beta) of Alzheimer disease (AD) is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane proteins. Production of A beta from a transmembrane precursor predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Here we show that incubation of a membrane fraction of PC12 cells at 37 degrees C results in the solubilization of an APP species which migrates on SDS-PAGE as full-length APP. The release of this full-length APP was pH-dependent with a peak of activity of pH 9.0. At this pH about 19% of the membrane APP was released from the active subcellular fraction. Under the same conditions other transmembrane proteins remained insoluble. Very little APP was solubilized at 4 degrees C. APP solubilization was specifically inhibited by the serine protease inhibitors aprotinin and pefabloc. Other protease inhibitors, including leupeptin and alpha 1-antitrypsin, had no effect. Several metal cations, including Ca++ and Zn++, also inhibited release of soluble full-length APP. Low levels of full-length APP were also detected in both the soluble fraction of PC12 cell extracts and in the media of PC12 cell cultures. These data suggest the involvement of a serine protease in the solubilization of membrane, full-length APP. The release of this APP could provide a soluble substrate for the proteolytic enzymes involved in the production of A beta.

Amyloid↗
PubMed1994

Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene.

Glutamate dehydrogenase, an enzyme central to glutamate metabolism, is deficient in patients with heterogeneous neurological disorders characterized by multiple system atrophy. There is evidence for multiplicity of human glutamate dehydrogenase, which may account for the heterogeneity of the above disorders. However, only one mRNA that is encoded by an intron-containing gene (GLUD1) is presently known. Because blindness due to neuroretinal degeneration can occur in rare forms of multiple system atrophy, we searched for retina-specific GLUD mRNA(s) by screening a lambda gt10 library derived from human retina. A novel cDNA encoded by an X chromosome-linked intronless gene, designated GLUD2, was isolated and characterized. Reverse transcription-polymerase chain reaction analysis of human tissues revealed that the novel cDNA is expressed in human retina, testis, and, at lower levels, brain. In vitro translation of mRNAs derived from GLUD1 and GLUD2 genes generated proteins with distinct electrophoretic characteristics. The retinal cDNA was expressed in the baculovirus heterologous system, producing a protein capable of catalyzing the oxidative deamination of glutamate. The mobility of the expressed protein on SDS-polyacrylamide gel electrophoresis and its catalytic properties were very similar to those of the naturally occurring human brain glutamate dehydrogenases. The novel gene will be useful for understanding the biology of human neural and testicular tissues and in the study of X-linked neurodegenerative disorders.

Amino Acid Sequence↗
PubMed1994

Study of the phorbol ester effect on Alzheimer amyloid precursor processing: sequence requirements and involvement of a cholera toxin sensitive protein.

Phorbol esters (PDBu) stimulate alpha-secretase cleavage and secretion of the Alzheimer amyloid precursor protein (APP). To determine whether any cytoplasmic residues or sequence motifs mediate the PDBu effect on APP processing, this region of APP was altered by point mutations or deletions. To differentiate the mutated APP from the endogenous APP, the APP751 ectodomain between amino acids 1 and 647 was replaced by a human secreted alkaline phosphatase derivative (SEAP). The resultant fusion protein (SEAP-APP751) was cleaved by alpha-secretase at the same site as full-length APP, and its secretion was stimulated by PDBu at a level similar to APP751. However, PDBu-stimulated secretion of the SEAP-APP751 fusion protein reached its maximum level after 30 min of treatment, while secretion of APP751 reached its maximum after 60 min, suggesting that the APP ectodomain affects the kinetics of APP secretion. Mutation of the cytoplasmic serines to alanines had no effect on the PDBu-stimulated secretion of the SEAP-APP, indicating that protein kinase C (PKC) phosphorylation of the cytoplasmic domain of APP is not important for stimulation of APP secretion. Similarly, deletion of the cytoplasmic domain between amino acids 719 and 751 had no effect on the PDBu-stimulated secretion. However, deletion of amino acids 707-751 resulted in a significant increase in the secretory cleavage of the SEAP-APP707 delta C construct, suggesting that the sequence 707-719 is important for the regulated secretion of APP. Cholera toxin, but not pertussis toxin, reduced the PDBu-induced secretion of APP by more than two-fold, suggesting that the PDBu response may be modulated by a cholera toxin sensitive heterotrimeric G-protein.

Alzheimer Disease↗
PubMed1994

Non-amyloidogenic cleavage of the beta-amyloid precursor protein by an integral membrane metalloendopeptidase.

The beta-amyloid precursor protein (beta-APP) is a membrane spanning glycoprotein. The small beta-protein domain within the precursor is presumed to be the source of amyloid found in plaques characteristic of Alzheimer's disease. The amino terminus of beta-APP is released from cells by cleavages that produce both potentially amyloidogenic and nonamyloidogenic fragments of the carboxyl terminus. We developed a cell free system that imposes specificity and co-localization to characterize the proteolytic activity that cleaves the precursor within the beta-protein domain. A reporter protein containing the carboxyl-terminal 105 amino acids of beta-APP provided a specific substrate for cleavage at Lys16 of the beta-protein. The protease inhibitor profile and solubility characteristics of the activity demonstrate the cleavage is produced by an integral membrane metalloendopeptidase.

Alkaline Phosphatase↗