Takako Morimoto

Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells

β‐Amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development. However, the functional role of APP during early development remains unclear. In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells. Cells were prepared from 13‐day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters. After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin‐positive proliferative cells, i.e. neural stem cells. To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast. Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition. The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino‐terminal region of sAPP. Then, we examined the effect of the amino‐terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino‐terminal region of sAPP is responsible for the biological activity. Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia.

ATP (10–100 μm), but not glutamate (100 μm), stimulated the release of plasminogen from microglia in a concentration‐dependent manner during a 10 min stimulation. However, neither ATP (100 μm) nor glutamate (100 μm) stimulated the release of NO. A one hour pretreatment with BAPTA‐AM (200 μm), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 μm). The Ca2+ ionophore A23187 induced plasminogen release in a concentration‐dependent manner (0.3 μm to 10 μm). ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration‐dependent manner which was very similar to the ATP‐evoked plasminogen release, whereas glutamate (100 μm) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μm) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). The ATP‐evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2‐Methylthio ATP was active (7 out of 7 cells), but α,β‐methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 μm) was shown not to inhibit the ATP‐evoked increase in [Ca2+]i (20 out of 20 cells). 2′‐ and 3′‐O‐(4‐Benzoylbenzoyl)‐adenosine 5′‐triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long‐lasting increase in [Ca2+]i even at 1 μm, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′‐triphosphate‐2′, 3′‐dialdehyde (oxidized ATP, 100 μm), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 μm). These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.

INVOLVEMENT OF AMYLOID PRECURSOR PROTEIN IN FUNCTIONAL SYNAPSE FORMATION IN CULTURED HIPPOCAMPAL NEURONS

Amyloid precursor protein (APP) is known to be widely expressed in neuronal cells, and enriched in the central and peripheral synaptic sites. Although it has been proposed that APP functions in synaptogenesis, no direct evidence has yet been reported. In this study we investigated the involvement of APP in functional synapse formation by monitoring spontaneous oscillations of intracellular Ca2+ concentration ([Ca2+]i) in cultured hippocampal neurons. As more and more neurons form synapses with each other during the culture period, increasing numbers of neuronal cells show synchronized spontaneous oscillations of [Ca2+]i. The number of neurons that showed synchronized spontaneous oscillations of [Ca2+]i was significantly lower when cultured in the presence of monoclonal antibody 22C11 against the N‐terminal portion of APP. Moreover, incubation with excess amounts of the secretory form of APP or the N‐terminal fragment of APP also inhibited the increase in number of neurons with synchronized spontaneous oscillations of [Ca2+]i. The addition of monoclonal antibody 22C11 or secretory form of APP did not, however, affect MAP‐2‐positive neurite outgrowth. These findings suggest that APP play a role in functional synapse formation during CNS development. J. Neurosci. Res. 51:185–195, 1998. © 1998 Wiley‐Liss, Inc.

Mg2+ Block of Drosophila NMDA Receptors Is Required for Long-Term Memory Formation and CREB-Dependent Gene Expression

NMDA receptor (NMDAR) channels allow Ca(2+) influx only during correlated activation of both pre- and postsynaptic cells; a Mg(2+) block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg(2+) block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg(2+) block, we found that Mg(2+) block mutants are defective for LTM formation but not associative learning. We demonstrate that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg(2+) block defective NMDARs. Furthermore, we show that genetic and pharmacological reduction of Mg(2+) block significantly increases expression of a CREB repressor isoform. Our results suggest that Mg(2+) block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction.

Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures

In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4‐[2‐(phenylsulphonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. PEPA dose‐dependently potentiated AMPA‐induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell‐to‐cell variation in rat hippocampal cultures. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. Reverse transcriptase‐polymerase chain reaction analysis suggested that GluR2‐flip, GluR1‐flip, GluR2‐flop, and GluR1‐flop were abundantly expressed (in this rank order) in the cultures used. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice‐variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. These results suggest that the potentiation by PEPA alone reveals cell‐to‐cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

The effect of a secreted form of β-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones

The effects of secreted forms of β‐amyloid‐precursor proteins (APPSs) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day‐cultured hippocampal cells, but not in 7 to 13 day‐cultured cells. Eighty‐one percent of neurones which were immunocytochemically positive for microtubule‐associated protein 2 responded to APP695S with an increase in [Ca2+]i. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol‐1,4,5‐trisphosphate (IP3) in a concentration‐dependent manner from 100 to 500 ng ml−1. In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPS, but not by carboxy terminal peptides. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3‐dependent mechanism that changes according to the stage of development.

Overexpression of synaptotagmin modulates short-term synaptic plasticity at developing neuromuscular junctions

The level of synaptotagmin I or II in developing spinal neurons was increased by injection of synaptotagmin messenger RNA into early blastomeres of Xenopus embryos. The effect of overexpression of synaptotagmin on synaptic function was assayed in Xenopus nerve-muscle cultures within two days after injection. At neuromuscular synapses made by synaptotagmin-overexpressing neurons, the frequency of miniature postsynaptic currents was markedly reduced, while their mean amplitude was unchanged, as compared to those of control neurons in the same culture. The amplitude of evoked postsynaptic currents elicited by low-frequency test stimuli was not affected by overexpression. However, synapses made by synaptotagmin-overexpressing neurons exhibited significantly higher paired-pulse facilitation and reduced tetanus-induced depression of the synaptic response, and there was also an increased number of synaptic vesicles at regions 100-300 nm from the plasmalemma at such synapses. These results show that synaptotagmins can exert an inhibitory action on the spontaneous exocytosis of synaptic vesicles. The effects on short-term plasticity suggest that synaptotagmin may facilitate vesicular supply for the evoked release during higher frequency transmission.

A Group of Segmental Premotor Interneurons Regulates the Speed of Axial Locomotion in Drosophila Larvae

BACKGROUND Animals control the speed of motion to meet behavioral demands. Yet, the underlying neuronal mechanisms remain poorly understood. Here we show that a class of segmentally arrayed local interneurons (period-positive median segmental interneurons, or PMSIs) regulates the speed of peristaltic locomotion in Drosophila larvae. RESULTS PMSIs formed glutamatergic synapses on motor neurons and, when optogenetically activated, inhibited motor activity, indicating that they are inhibitory premotor interneurons. Calcium imaging showed that PMSIs are rhythmically active during peristalsis with a short time delay in relation to motor neurons. Optogenetic silencing of these neurons elongated the duration of motor bursting and greatly reduced the speed of larval locomotion. CONCLUSIONS Our results suggest that PMSIs control the speed of axial locomotion by limiting, via inhibition, the duration of motor outputs in each segment. Similar mechanisms are found in the regulation of mammalian limb locomotion, suggesting that common strategies may be used to control the speed of animal movements in a diversity of species.

Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca2+ mobilization from the endoplasmic reticulum (ER) and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca2+ release. Here these issues are addressed in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. We report that neuronal excitation acts with ER Ca2+ stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca2+ provided by different presynaptic ER Ca2+ release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca2+ in the terminal, while translocation to AZs requires Ca2+ microdomains generated by IP3Rs.

Secreted form of β-amyloid precursor protein activates protein kinase C and phospholipase Cγ1 in cultured embryonic rat neocortical cells

Abstract The secreted form of β-amyloid precursor protein (sAPP) has been reported to exert various biological activities in cultured neurons. The signal transduction mechanisms underlying these physiological functions of sAPP remain unclear. We now report that treatment of neural cells with the secreted form of APP695 (sAPP695) leads to dose- and time-dependent increase in phosphorylation of the endogenous substrates with a molecular mass of 80, 57 and 43 kDa. Pretreatment of cells with protein kinase C (PKC) inhibitor H-7 reduced phosphorylation of the 80- and 43-kDa proteins in a dose-dependent manner. The effect of sAPP695 on the phosphorylation is mimicked by phorbol 12-myristate-13-acetate (PMA). Downregulation of PKC by prolonged treatment of cells with PMA abolished sAPP695-enhanced phosphorylation of the 80- and 43-kDa proteins, indicating PKC is involved in the sAPP695-enhanced phosphorylation of these proteins in the cells. We also suggest that the 80- and 43-kDa proteins phosphorylated by sAPP695-stimulation are the major PKC substrates myristoylated alanine-rich C-kinase substrate and growth-associated protein-43. Furthermore, we demonstrate that tyrosine phosphorylation of phospholipase Cγ1 and formation of inositol 1,4,5-trisphosphate were increased by sAPP695-stimulation. These observations suggest that sAPP695 induces the activation of the signaling pathways through a stimulation of phosphoinositide–PKC cascade.