Molecular mechanisms underlying a unique intermediate phase of memory in aplysia

Short- and long-term synaptic facilitation induced by serotonin at Aplysia sensory-motor (SN-MN) synapses has been widely used as a cellular model of short- and long-term memory for sensitization. In recent years, a distinct intermediate phase of synaptic facilitation (ITF) has been described at SN-MN synapses. Here, we identify a novel intermediate phase of behavioral memory (ITM) for sensitization in Aplysia and demonstrate that it shares the temporal and mechanistic features of ITF in the intact CNS: (1) it declines completely prior to the onset of LTM, (2) its induction requires protein but not RNA synthesis, and (3) its expression requires the persistent activation of protein kinase A. Thus, in Aplysia, the same temporal and molecular characteristics that distinguish ITF from other phases of synaptic plasticity distinguish ITM from other phases of behavioral memory.     

Cell and molecular analysis of long-term sensitization in Aplysia

We have found that one cellular locus for the storage of the memory underlying short-term sensitization of the gill and siphon withdrawal reflex in Aplysia is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. These connections also participate in the storage of memory underlying long-term sensitization. In animals that have undergone long-term sensitization, the amplitudes of the monosynaptic connections are significantly larger (2.2x) than the ones in control animals. To study the mechanisms of onset and retention of long-term synaptic facilitation that underly long-term sensitization and the role of protein synthesis in long-term memory, we have developed two types of reduced preparations: the intact reflex isolated from the remainder of the animal, and a dissociated cell culture system in which the monosynaptic component (sensory neurons and motor neurons) of the neuronal circuit mediating the withdrawal reflex is reconstituted. We found that protein synthesis inhibitors, such as anisomycin or emetine, and RNA synthesis inhibitors, such as actinomycin D or alpha-amanitin, blocked long-term facilitation without interfering with short-term facilitation. These results suggest that the acquisition of long-term memory may require the expression of genes and the synthesis of proteins not needed for short-term memory.     

Postsynaptic regulation of long-term facilitation in Aplysia

Repeated exposure to serotonin (5-HT), an endogenous neurotransmitter that mediates behavioral sensitization in Aplysia[1-3], induces long-term facilitation (LTF) of the Aplysia sensorimotor synapse [4]. LTF, a prominent form of invertebrate synaptic plasticity, is believed to play a major role in long-term learning in Aplysia[5]. Until now, LTF has been thought to be due predominantly to cellular processes activated by 5-HT within the presynaptic sensory neuron [6]. Recent work indicates that LTF depends on the increased expression and release of a sensory neuron-specific neuropeptide, sensorin [7]. Sensorin released during LTF appears to bind to autoreceptors on the sensory neuron, thereby activating critical presynaptic signals, including mitogen-activated protein kinase (MAPK) [8, 9]. Here, we show that LTF depends on elevated postsynaptic Ca2+ and postsynaptic protein synthesis. Furthermore, we find that the increased expression of presynaptic sensorin resulting from 5-HT stimulation requires elevation of postsynaptic intracellular Ca2+. Our results represent perhaps the strongest evidence to date that the increased expression of a specific presynaptic neuropeptide during LTF is regulated by retrograde signals.     

Calreticulin modulates cell adhesiveness via regulation of vinculin expression

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)- binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell- substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.     

Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effects of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

Molecular approaches to the study of long-term sensitization in Aplysia californica

In order to gain insight into the molecular mechanisms underlying the establishment and maintenance of long-term sensitization in Aplysia new tools are being used to study the synaptic facilitation in the sensory-motor connection which mediates the gill-and-siphon withdrawal reflex. The supposition that long-term changes in neuronal properties share molecular pathways with other cells during development and differentiation points towards specific candidate genes as well as towards a general experimental strategy designed to find proteins which might mediate these changes. The unique properties of Aplysia cell biology may, in addition, provide a means to examine their specific roles in the triggering of the changes, as well as the nature of the changes themselves.     

Neuronal mechanisms contributing to long-term sensitization in Aplysia

1.) Cellular processes that contribute to the acquisition and expression of long-term sensitization have been examined in Aplysia. The tail-siphon withdrawal reflex was studied because the neural circuit for this reflex has been well characterized. Furthermore, the sensory neurons of this neural circuit exhibit cellular changes that accompany short-term sensitization. 2.) Repeated application of noxious stimuli to the animal produces a long-lasting enhancement of reflex withdrawal of the siphon when the animal is tested with a weak stimulus to the tail. These findings confirm the existence of long-term sensitization in Aplysia, first described by Pinkser et al. (1973). 3.) Biophysical correlates of long-term sensitization were examined in the first central relay of the tail-siphon reflex circuit, the sensory neurons that innervate the animal's tail. The net outward membrane currents of these cells reduced after 24 hours as a consequence of long-term sensitization training. 4.) The intracellular signal for the induction of these changes in membrane currents was examined by intracellular injection of cAMP into individual sensory neurons. This procedure mimics at least some of the effects of sensitization training at the single-cell level. cAMP induced a long-term reduction of membrane K+ currents 24 hours after the cells were injected with cAMP. The membrane currents reduced by cAMP were similar to those reduced by long-term sensitization training. 5.) Preliminary experiments indicate that neurotransmitters and agents that induce an evaluation of cAMP in the sensory neurons also alter the incorporation of labeled amino acids into specific proteins in the sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of calcium binding proteins calreticulin and calsequestrin during differentiation in the myogenic cell line L6

In this report we defined the structural and temporal limits within which calreticulin and calsequestrin participate in the muscle cell phenotype, in the L6 model myogenic system. Calreticulin and calsequestrin are two Ca²⁺ binding proteins thought to participate in intracellular Ca²⁺ homeostasis. We show that calsequestrin protein and mRNA were expressed when L6 cells were induced to differentiate, during which time the level of expression of calreticulin protein did not change appreciably. Calreticulin mRNA levels, however, were constant throughout L6 cell differentiation except for slight decline in the mRNA levels at the very late stages of L6 differentiation (day 11–12). We also show that the two Ca²⁺ binding proteins are coexpressed in differentiated L6 cells. Based on its mobility in SDS-PAGE, L6 rat skeletal muscle cells in culture expressed cardiac isoform of calsequestrin. In the mature rat skeletal muscle, calreticulin and calsequestrin were localized to sarcoplasmic reticulum (SR). Calreticulin, but not calsequestrin, staining was also observed in the perinuclear region. These data suggest that expression of calreticulin and calsequestrin may be under different control during myogenesis in rat L6 cells in culture. © 1996 Wiley-Liss, Inc.     

Calreticulin in T-lymphocytes. Identification of calreticulin in T-lymphocytes and demonstration that activation of T cells correlates with increased levels of calreticulin mRNA and protein.

Ca2+ is an essential second messenger for T cell activation, but the exact mechanisms of its action are poorly understood. The cytosolic Ca2+ concentration is significantly increased upon the stimulation of T cells with either mitogen, cross-linking antibodies, or their cognate ligands. In this study, expression of calreticulin, a major Ca(2+)-binding (storage), KDEL protein of the endoplasmic reticulum was examined in resting and concanavalin A (ConA)-stimulated mouse and human T-lymphocytes. Both resting, mouse and human lymphocytes contain very low levels of calreticulin mRNA and protein. Mouse splenocytes stimulated with ConA exhibited an induction in calreticulin mRNA which peaked by Day 4. A 5-fold increase in the immunoreactive calreticulin protein band was also observed in the cells during this period of stimulation. Similarly when human lymphocytes were cultured with ConA a significant increase in the levels of the calreticulin mRNA and protein was observed. The peak of calreticulin mRNA was observed at Day 1 rather than Day 4 as seen for the mouse. These results clearly demonstrate the presence of calreticulin, a Ca(2+)-binding protein originally characterized in muscle tissue, in activated T-lymphocytes. Furthermore, we show that expression of calreticulin correlates with T-lymphocyte activation. Our results suggest that calreticulin may be involved in the signaling pathway for the induction of Ca(2+)-dependent processes and may represent one regulatory mechanism operating in activation of T-lymphocytes.     

Overexpression of calreticulin fails to abolish its induction by perturbation of normal ER function.

Along with other endoplasmic reticulum (ER) Ca2+-binding proteins, notably the glucose-response proteins grp78 and grp94, expression of calreticulin is induced in response to perturbation of normal ER function. It has yet to be clearly defined how this stress is signaled from the ER to the nucleus in mammalian cells, particularly with regard to its initiation. Using a GFP-calreticulin fusion protein, we have generated and selected stably transfected HeLa cells that overexpress calreticulin to investigate whether the protein might be involved in signaling its own induction. Basal levels of endogenous calreticulin mRNA and protein were unaffected in these cells, indicating that overexpression alone does not induce a stress response. ER stress induced calreticulin expression in response to either thapsigargin or tunicamycin was equivalent in these cells to that seen in control, nontransfected cells, leading us to conclude that calreticulin is unlikely be involved in its own induction. Levels of the mRNA encoding the fusion protein were also increased by tunicamycin, but not thapsigargin, suggesting that, in agreement with our previous observations, inhibition of N-linked glycosylation may increase the stability of calreticulin mRNA. This indicates that in mammalian cells, there is more than one signaling pathway for the ER stress response.     

Protective role of calreticulin in HFE hemochromatosis

HFE gene mutations are associated with over 80% of cases of hereditary hemochromatosis (HH), an iron-overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. More recent studies have suggested a more complex function for this nonclassical MHC-I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte-like cell line. Gene expression was analyzed in HepG2 cells overexpressing wild-type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)-activated gene expression was analyzed in peripheral blood mononuclear cells from characterized HH patients. C282Y HFE down-regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y(+/+) patients calreticulin levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of hereditary hemochromatosis clinical manifestations. The data show that expression of C282Y HFE triggers a stress-protective response in HepG2 cells and suggest a role for calreticulin as a modifier of the clinical expression of HH.     

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.