Characterization of alternative routes for processing of the Alzheimer beta/A4-amyloid precursor protein. Differential effects of phorbol esters and chloroquine.
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Alzheimer Disease↗
Biomedical subjects
P Greengard
Publications and source records attributed to P Greengard.
Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor.
Activation of protein kinase C by phorbol esters is known to accelerate the processing and secretion of the beta/A4 amyloid protein precursor. We have now examined various first messengers that increase protein kinase C activity of target cells for their ability to affect beta/A4 amyloid protein precursor metabolism. Acetylcholine and interleukin 1, which are altered in Alzheimer disease, were shown to increase processing of the beta/A4 amyloid protein precursor via the secretory cleavage pathway. Cholinergic agonists stimulated secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the M1 receptor, while interleukin 1 stimulated secretion in human endothelial and glioma cells.
Acetylcholine↗
Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.
Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.
Amino Acid Sequence↗
Immunocytochemical localization of DARPP-32, a dopamine and cyclic-AMP-regulated phosphoprotein, in the primate brain.
The localization of DARPP-32, a dopamine and cAMP-regulated phosphoprotein, has been studied in monkey brain by immunocytochemistry. This study indicates that DARPP-32 is enriched in neurons in regions receiving a dense dopamine input from the substantia nigra and ventral tegmental area. Thus, the majority of somata in the anterior olfactory area, nucleus accumbens, caudate nucleus, and putamen are immunoreactive for DARPP-32. In the caudate nucleus, immunoreactive spines receive asymmetric contacts from unlabeled axon terminals. Immunoreactive somata have diameters of 10-15 microns. In regions known to receive projections from these nuclei, immunoreactivity is confined to small puncta that represent axons and axon terminals. Regions in which immunoreactivity is present in puncta include the ventral pallidum, globus pallidus, and substantia nigra pars reticulata. Dopaminergic neurons themselves are not immunoreactive. Neurons containing moderate to weak immunoreactivity for DARPP-32 are observed in portions of the cerebral cortex, particularly in the temporal cortex (layer VI). DARPP-32-positive neurons are also present in the cerebellum, in the medial habenula, and in portions of the bed nucleus of the stria terminalis and amygdaloid complex. DARPP-32 immunoreactivity is also present in astrocytes in the subcortical white matter and in tanycytes in the arcuate nucleus and median eminence. DARPP-32 may be an effective marker for dopaminoceptive neurons in which the actions of dopamine on the D-1 dopamine receptor are mediated through cAMP and its associated protein kinase.
Animals↗
Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.
The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/calmodulin-dependent protein phosphatase). Activation of the Na+,K(+)-ATPase by either oxymetazoline or A23187 was associated with a greater than 2-fold increase in its affinity for Na+. The results provide a biochemical mechanism by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention.
Animals↗
Calcium-dependent transmitter secretion reconstituted in Xenopus oocytes: requirement for synaptophysin.
Calcium-dependent glutamate secretion was reconstituted in Xenopus oocytes by injecting the oocyte with total rat cerebellar messenger RNA (mRNA). Co-injection of total mRNA with antisense oligonucleotides to synaptophysin message decreased the expression of synaptophysin in the oocyte and reduced the calcium-dependent secretion. A similar effect on secretion was observed for oocytes injected with total mRNA together with an antibody to rat synaptophysin. These results indicate that synaptophysin is necessary for transmitter secretion and that the oocyte expression system may be useful for dissecting the molecular events associated with the secretory process.
Animals↗
Phosphorylation of the cystic fibrosis transmembrane conductance regulator.
Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.
Amino Acid Sequence↗
Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect.
Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle.
Actins↗
Effects of the neuronal phosphoprotein synapsin I on actin polymerization. II. Analytical interpretation of kinetic curves.
The general features of the kinetics of actin polymerization are investigated by mathematical models, with the aim of identifying the kinetically relevant parameters in the process and detecting and interpreting the alterations occurring in actin polymerization under various experimental conditions. Polymerization curves, obtained by following the increase in fluorescence of actin derivatized with N-(1-pyrenyl) iodoacetamide, are fitted using analytical equations derived from biochemical models of the actin polymerization process. Particular attention is given to the evaluation of the effects of the neuronal phosphoprotein synapsin I. The models obtained under various ionic conditions reveal that synapsin I interacts with actin in a very complex fashion, sharing some of the properties of classical nucleating proteins but displaying also actions not described previously for other actin-binding proteins. Synapsin I appears to bind G-actin with a very high stoichiometry (1:2-4), and the complex behaves as an F-actin nucleus, producing actin filaments under conditions where spontaneous polymerization is negligible. These actions of synapsin I are markedly affected by site-specific phosphorylation of the protein. An original transformation of the fluorescence data, which estimates the disappearance rate of actin monomer toward the critical concentration, is presented and shown to be of general usefulness for the study of actin-binding proteins.
Actins↗
Synapsin IIa: expression in insect cells, purification, and characterization.
Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.
Animals↗
Structure and function of the synapsins.
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Actins↗
Protein phosphorylation regulates secretion of Alzheimer beta/A4 amyloid precursor protein.
Extracellular deposition of the beta/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. beta/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP751, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the beta/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP695, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-beta/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.
Adrenal Gland Neoplasms↗
Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein.
The metabolic fate of the Alzheimer beta/A4 amyloid precursor protein (APP) includes intraamyloid proteolysis that leads to the production of secreted N-terminal and cell-associated C-terminal fragments. The cellular sites at which this processing occurs are not known. We have examined the route of APP processing in metabolically labeled PC12 cells. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein. In addition, recovery of a C-terminal fragment resulting from normal intraamyloid cleavage was significantly increased in the presence of chloroquine, suggesting that further degradation of the C-terminal fragment was inhibited. Chloroquine had virtually no effect on APP maturation (N- and O-glycosylation and tyrosine sulfation) or secretion. Treatment with either monensin (which inhibits distal Golgi function) or brefeldin A (which causes resorption of the Golgi into the endoplasmic reticulum and fusion of the trans-Golgi network with the endosomal system) prevented normal APP maturation and abolished APP secretion and recovery of C-terminal fragments, indicating that intact Golgi function is necessary for APP maturation and processing. Our results suggest that a substantial proportion of APP is degraded in an intracellular acidic compartment but that the coupled APP cleavage/secretion event occurs in a chloroquine-insensitive compartment. The observations are consistent with the existence of multiple cellular routes for the trafficking and proteolysis of APP.
Ammonium Chloride↗
Amyloidogenesis in Alzheimer's disease: some possible therapeutic opportunities.
Cerebral deposition of fibrils formed from the beta/A4 amyloid protein is an invariable feature of Alzheimer's disease. Evidence suggests that generation of such fibrils may be involved in the etiology of this disease, since mutations in the coding region of the beta/A4 amyloid precursor protein (APP) gene segregate with familial cerebral amyloidoses, including familial Alzheimer's disease. Transgenic models of cerebral amyloidosis have been produced, and some progress has been made in elucidating the cell biology of amyloidogenesis. For example, agents that alter protein phosphorylation are potent modulators of the expression and proteolytic processing of APP. Sam Gandy and Paul Greengard review these recent studies, and discuss those that may provide rational therapeutic opportunities.
Alzheimer Disease↗
Phosphorylation of Alzheimer amyloid precursor protein by protein kinase C.
The beta/A4 amyloid precursor protein is a membrane protein with one transmembrane domain. The accumulation and deposition of beta/A4 amyloid protein in Alzheimer's disease is thought to be brought about by altered processing of beta/A4 amyloid precursor protein. Activation of protein kinase C and/or inhibition of protein phosphatases 1 and 2A results in an increase in the proteolytic processing and secretion of beta/A4 amyloid precursor protein. These effects might result either from phosphorylation of beta/A4 amyloid precursor protein by protein kinase C or from phosphorylation of components of the beta/A4 amyloid precursor protein processing apparatus. We have previously reported phosphorylation by protein kinase C of a synthetic peptide corresponding to part of the cytoplasmic domain of beta/A4 amyloid precursor protein. However, it was not known whether beta/A4 amyloid precursor protein holoprotein was phosphorylated in its native conformation in the cell membrane. Using a PC12 (rat pheochromocytoma) semi-intact cell system, we now report that mature isoforms of beta/A4 amyloid precursor protein are phosphorylated by protein kinase C at Ser655. Five COOH-terminal fragments which are generated by processing of mature beta/A4 amyloid precursor protein were also phosphorylated by protein kinase C at Ser655. The results support the idea that the beta/A4 amyloid precursor protein haloprotein is a physiological substrate for protein kinase C. These observations should facilitate our understanding of the relationship between altered protein phosphorylation and beta/A4 amyloid production.
Adenosine Triphosphate↗
Developmental regulation of phosphoprotein gene expression in the caudate-putamen of rat: an in situ hybridization study.
The regional and cellular ontogeny of the mRNA encoding the dopamine- and cAMP-regulated phosphoprotein, DARPP-32, has been studied in rat striatum by quantitative in situ hybridization histochemistry. The mRNA for DARPP-32 exhibited a characteristic developmental profile. The hybridization signal was first visible on the day of birth, at which time DARPP-32 mRNA was concentrated in patches in the caudate-putamen. By the end of the first postnatal week, the majority of neurons in the caudate-putamen expressed the DARPP-32 message. Levels of mRNA per cell increased markedly during the second postnatal week, and peaked around the beginning of the third week. The adult level of DARPP-32 mRNA was lower than that observed at the apex of mRNA expression, on a per cell basis, while the proportion of neurons expressing detectable levels of message remained relatively constant. In the nucleus accumbens and olfactory tubercle, DARPP-32 mRNA development lagged somewhat behind that observed in the caudate-putamen, but was similar in other respects. A non-quantitative study employing an oligonucleotide probe complementary to the mRNA encoding another cAMP-regulated phosphoprotein, ARPP-21, revealed a similar developmental sequence to DARPP-32. The present results suggest that for DARPP-32 mRNA, genetic and, possibly, environmental factors play a role in determining the developmental patterns observed.
Aging↗
Antibodies to synaptophysin interfere with transmitter secretion at neuromuscular synapses.
The involvement of synaptophysin, a synaptic vesicle-specific protein, in transmitter release at neuromuscular synapses was studied by intracellular application of synaptophysin antibodies into presynaptic neurons. Polyclonal antibodies or their Fab fragments were loaded into spinal neurons by injection into one of the early blastomeres of Xenopus embryos 1 day prior to culturing or, alternatively, directly through a whole-cell recording pipette at the soma of cultured neurons. At synapses made by antibody-loaded neurons in culture, the spontaneous synaptic currents showed marked reduction in frequency without significant change in their mean amplitude. The impulse-evoked synaptic currents showed reduced amplitude and increased failure rate. These results suggest that interference with synaptophysin function by antibody binding inhibits transmitter secretion.
Animals↗
Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation.
The distribution of the synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on synaptic vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to synaptic vesicles at rest. The value of this ratio was intermediate in synaptic vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with synaptic vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release.
Animals↗