Serine phosphorylation of the secreted extracellular domain of APP.

The phosphorylation status of full-length APP (FL-APP) and secreted APP (s-APP) was investigated in stably transfected cells. 32P incorporation was detected in the mature full-length APP both in the absence and presence of phorbol ester. Surprisingly, 32P-phosphate was incorporated in the secreted ectodomain, and this was stable to treatment of the [32P]-phospho-s-APP with a large excess of PNGase F, suggesting that N-linked oligosaccharide sites do not account for phosphate incorporation.

Regulated cleavage of Alzheimer beta-amyloid precursor protein in the absence of the cytoplasmic tail.

Alzheimer beta-amyloid precursor protein can be phosphorylated on residues Thr654, Ser655 and Thr668 on its cytoplasmic domain. Proteolytic cleavage of the amyloid precursor protein and release of the amyloid precursor protein ectodomain into the medium of cultured cells can be activated by phorbol esters which stimulate protein kinase C. In the present study, using mutated amyloid precursor protein, we show that phosphorylation of cytoplasmic residues is not required for the phorbol ester-activated cleavage and release of the amyloid precursor protein ectodomain.

Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures.

Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane.

Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum.

Despite physiological evidence that cholecystokinin (CCK) is an excitatory neurotransmitter in the brain, little is known about its mechanism of action. CCK immunoreactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phosphorylation of a dopamine- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) in striatal neurons.

Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy.

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence.

Protein phosphorylation inhibits production of Alzheimer amyloid beta/A4 peptide.

The major component of amyloid plaque cores and cerebrovascular amyloid deposits found in Alzheimer disease is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence suggests that abnormalities in beta/A4 peptide production or beta/A4 peptide aggregation may underlie cerebral amyloidosis. In the present study, treatment of cells with phorbol dibutyrate, which activates protein kinase C, and/or okadaic acid, which inhibits protein phosphatases 1 and 2A, reduced beta/A4 peptide production by 50-80%.

Protein phosphorylation regulates relative utilization of processing pathways for Alzheimer beta/A4 amyloid precursor protein.

The Alzheimer amyloid precursor protein (APP) is a phosphoprotein, and the phosphorylation state of APP at Ser655 can be regulated by protein kinase C, calcium/calmodulin-dependent protein kinase II, and okadaic acid-sensitive protein phosphatases. Other enzymes may also play a role at Ser655 of APP and, perhaps, at other residues. Signal transduction via protein phosphorylation regulates APP metabolism.

Rapid binding of synapsin I to F- and G-actin. A study using fluorescence resonance energy transfer.

Synapsin I is a nerve terminal phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation-dependent manner. By using fluorescence resonance energy transfer between purified components labeled with fluorescent probes, we now show that the binding of synapsin I to actin is a rapid phenomenon. Binding of synapsin I to actin can also be demonstrated when synaptic vesicles are present in the medium and appears to be modulated by ionic strength and synapsin I phosphorylation.

Comparison of the immunocytochemical localization of DARPP-32 and I-1 in the amygdala and hippocampus of the rhesus monkey.

Dopamine and adenosine 3':5'-monophosphate (cAMP) regulated phosphoprotein of M(r) 32 kDa (DARPP-32) and phosphatase inhibitor 1 (I-1) have been associated with intracellular signal transduction processes and share several biochemical features. Localization of each phosphoprotein in distinct neural structures will aid investigation of their physiologic properties and help identify their unique roles in the nervous system. We have compared the distribution of the two phosphoproteins in the amygdala and hippocampus of the rhesus monkey with the aid of immunocytochemical procedures.

Calcitonin gene-related peptide potentiates synaptic responses at developing neuromuscular junction.

Protein phosphorylation is important in synaptic transmission and plasticity. At the neuromuscular junction, phosphorylation of acetylcholine (ACh) receptor-channels increases the rate of agonist-induced channel desensitization. In contrast, potentiation of ACh channel activity through protein phosphorylation has not been described.

Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration.

In the brains of individuals with Alzheimer disease, senile plaques containing aggregates of beta-amyloid peptide, derived from the beta-amyloid precursor protein (APP), are seen in association with degenerating nerve terminals. It is not known whether the degenerating nerve terminals cause the formation of these aggregates or whether beta-amyloid peptide in the aggregates causes nerve-terminal degeneration. In the present study of rat brain, degeneration either of local neurons or of nerve terminals caused decreased levels of a neuron-enriched isoform of APP, increased levels of a glia-enriched isoform of APP, and increased levels of potentially amyloidogenic, as well as nonamyloidogenic, COOH-terminal fragments of APP.

Nitric oxide/cGMP pathway stimulates phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, in the substantia nigra.

DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000, has been shown to be phosphorylated on threonine-34, both in vitro with high efficiency by cAMP-dependent and cGMP-dependent protein kinases and in vivo by dopamine acting through cAMP-dependent protein kinase. In the present study, we investigated the nitric oxide (NO)/cGMP pathway for its ability to regulate the state of phosphorylation of DARPP-32 in slices of rat substantia nigra. DARPP-32 was phosphorylated on threonine-34 in these slices by sodium nitroprusside (SNP), an NO donor.

Dopamine D1 agonist SKF 38393 increases the state of phosphorylation of ARPP-21 in substantia nigra.

ARPP-21 is a cyclic AMP-regulated phosphoprotein (M(r) = 21,000) that has a distribution in brain similar to that of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) = 32,000). It is enriched in the medium-sized spiny neurons in the striatum and in the striatonigral nerve terminals in the pars reticulata of the substantia nigra. The present study shows that dopamine D1 agonist SKF 38393 increases the state of phosphorylation of ARPP-21 by 26% in nigral slices and that pretreatment of the slices with D1 antagonist SCH 23390 blocks this effect.

Identification of a functional silencer element involved in neuron-specific expression of the synapsin I gene.

We have identified a functional silencer element (positions -231 to -211) in the human synapsin I gene that selectively represses its transcription in nonneuronal cells. Transfection experiments using synapsin I-luciferase constructs show that site-specific mutations or deletion of this silencer sequence results in expression of the reporter gene in nonneuronal cells. Moreover, the silencer element is capable of conferring repression on a heterologous promoter in nonneuronal cells.

Synaptic vesicle phosphoproteins and regulation of synaptic function.

Complex brain functions, such as learning and memory, are believed to involve changes in the efficiency of communication between nerve cells. Therefore, the elucidation of the molecular mechanisms that regulate synaptic transmission, the process of intercellular communication, is an essential step toward understanding nervous system function. Several proteins associated with synaptic vesicles, the organelles that store neurotransmitters, are targets for protein phosphorylation and dephosphorylation.

Calcium-dependent serine phosphorylation of synaptophysin.

The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca(2+)-dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K(+)-induced depolarization of slices and synaptosomes prelabeled with 32P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca2+ in the incubation medium.

Proteolytic processing of human amyloid beta protein precursor in insect cells. Major carboxyl-terminal fragment is identical to its human counterpart.

The predominant component of amyloid plaques of Alzheimer's disease is the amyloid beta protein (A beta), a 39-42-amino-acid peptide derived by proteolysis of a family of precursors known as amyloid precursor proteins (APP). In mammalian brain and in cultured mammalian cells, the release of APP amino-terminal fragments into the extracellular medium occurs by a proteolytic cleavage within the A beta domain, thereby precluding amyloidogenesis. Infection of Sf9 insect cells with baculovirus vectors containing APP cDNAs results in high levels of APP expression.

Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells.

The Alzheimer beta/A4 amyloid precursor protein (APP) can be proteolytically processed by at least two separate pathways in PC12 cells: chloroquine-insensitive secretory cleavage and chloroquine-sensitive intracellular degradation, presumably in the endosomal/lysosomal system. To further investigate the possibility of APP processing in the endosomal/lysosomal system, we have examined whether APP is present in clathrin-coated vesicles (CCVs), which mediate the transport of many proteins to the endosomal compartment. Using a procedure derived from established protocols for the purification of CCVs from mammalian organs, we obtained from PC12 cells highly purified CCVs that displayed the same morphological features as described for CCVs purified from other sources.

ARPP-39, a membrane-associated substrate for cyclic AMP-dependent protein kinase present in neostriatal neurons.

This study describes a cyclic AMP-regulated phosphoprotein that displays a distinct cellular and regional distribution in rat brain. The protein is found only in neostriatal regions, where it is enriched in nerve cells and not in afferent or efferent axons or in glial cells. On subcellular fractionation, it appears tightly associated with particulate components, possibly the synaptic plasma membrane fraction.

Bidirectional regulation of Na+,K(+)-ATPase activity by dopamine and an alpha-adrenergic agonist.

Catecholamines have pronounced effects on the renal handling of sodium and water, dopamine-promoting sodium and water excretion, and norepinephrine-promoting sodium and water retention. In the present study, using isolated permeabilized renal tubule cells and intact rats, we have shown that these effects can be attributed to opposing actions of these transmitters on renal tubular Na+,K(+)-ATPase activity. The ability of each of these catecholamines to regulate Na+,K(+)-ATPase activity is affected by the concentration of Na+ as well as by the absence or presence of the opposing catecholamine.

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.

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.

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.

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.