Guidance Molecules in Synapse Formation and Plasticity

A major goal of modern neuroscience research is to understand the cellular and molecular processes that control the formation, function, and remodeling of chemical synapses. In this article, we discuss the numerous studies that implicate molecules initially discovered for their functions in axon guidance as critical regulators of synapse formation and plasticity. Insights from these studies have helped elucidate basic principles of synaptogenesis, dendritic spine formation, and structural and functional synapse plasticity. In addition, they have revealed interesting dual roles for proteins and cellular mechanisms involved in both axon guidance and synaptogenesis. Much like the dual involvement of morphogens in early cell fate induction and axon guidance, many guidance-related molecules continue to play active roles in controlling the location, number, shape, and strength of neuronal synapses during development and throughout the lifetime of the organism. This article summarizes key findings that link axon guidance molecules to specific aspects of synapse formation and plasticity and discusses the emerging relationship between the molecular and cellular mechanisms that control both axon guidance and synaptogenesis.     

Neuronal Apoptosis Induced by Endoplasmic Reticulum Stress

Apoptosis is a conserved active cellular mechanism occurring under a range of physiological and pathological conditions. In the nervous system, apoptosis plays crucial roles in normal development and neuronal degenerating diseases. Various deleterious conditions, including accumulation of the mutant proteins in the endoplasmic reticulum (ER) and inhibition of ER to Golgi transport of proteins, may result in apoptosis. In this study, we examined the downstream events of apoptosis in differentiated PC 12 cells under ER stress induced by brefeldin A, an inhibitor of ER to Golgi protein transport. Activation of NF-B and degradation of I-B were observed within 2 hours, followed by up-regulation of GRP78 protein level in treated cells. Caspase-12 only appeared around 24 hours after brefeldin A treatment, coincident with cell nuclei fragmentation. These results suggest that neuronal apoptosis may be induced by ER stress through a NF-B and caspase related pathway.     

Highly Basic 30- and 32-Kilodalton Proteins Associated with Synapse Formation on Polylysine-Coated Beads in Enriched Neuronal Cell Cultures

Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.     

Microglial Response Factor (MRF)-1: Constitutive Expression in Ramified Microglia and Upregulation upon Neuronal Death Induced by Ischemia or Glutamate Exposure

We have isolated a new microglial gene, mrf-1, which is upregulated on microglia in response to apoptosis of granule neurons in cerebellar cell cultures. We examined whether or not upregulation of MRF-1 is observed in response to necrotic neuronal death both in vivo and in vitro. Though MRF-1 was detected on ramified/resting microglia in the brain of normal adult rats, activated microglia in the region of the brain where neuronal damage was induced by ischemia were strongly immunostained with anti-MRF-1 anti-body. In the in vitro system, we confirmed, with immunocytochemistry or RT-PCR, that MRF-1 or mrf-1 mRNA were constitutively expressed in ramified microglia at significant but lower levels than in amoeboid one. Moreover, by Northern blot, it was ascertained that expression level of mrf-1 mRNA on microglia was markedly upregulated in response to glutamate-induced death of granule cells in a cerebellar cell culture. These results indicate the following: 1) expression of mrf-1 in microglia may be markedly enhanced upon not only apoptotic but also necrotic neuronal death, and 2) MRF-1 is, thus, an useful marker for identifying all types of microglia in vivo and in vitro.     

Persistent Synapse Loss Induced by Repetitive LTD in Developing Rat Hippocampal Neurons

Synaptic pruning is a physiological event that eliminates excessive or inappropriate synapses to form proper synaptic connections during development of neurons. Appropriate synaptic pruning is required for normal neural development. However, the mechanism of synaptic pruning is not fully understood. Strength of synaptic activity under competitive circumstances is thought to act as a selective force for synaptic pruning. Long-term depression (LTD) is a synaptic plasticity showing persistent decreased synaptic efficacy, which is accompanied by morphological changes of dendritic spines including transient retraction. Repetitive induction of LTD has been shown to cause persistent loss of synapses in mature neurons. Here, we show that multiple, but not single, induction of LTD caused a persistent reduction in the number of dendritic synapses in cultured rat developing hippocampal neurons. When LTD was induced in 14 days in vitro cultures by application of (RS)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR) agonist, and repeated three times with a one day interval, there was a significant decrease in the number of dendritic synapses. This effect continued up to at least two weeks after the triple LTD induction. The persistent reduction in synapse number occurred in the proximal dendrites, but not the distal dendrites, and was prevented by simultaneous application of the group I/II mGluR antagonist (S)-a-methyl-4-carboxyphenylglycine (MCPG). In conclusion, we found that repetitive LTD induction in developing neurons elicits synaptic pruning and contributes to activity-dependent regulation of synapse number in rat hippocampal neurons.     

The Effect of Desflurane on Neuronal Communication at a Central Synapse

Although general anesthetics are thought to modify critical neuronal functions, their impact on neuronal communication has been poorly examined. We have investigated the effect induced by desflurane, a clinically used general anesthetic, on information transfer at the synapse between mossy fibers and granule cells of cerebellum, where this analysis can be carried out extensively. Mutual information values were assessed by measuring the variability of postsynaptic output in relationship to the variability of a given set of presynaptic inputs. Desflurane synchronized granule cell firing and reduced mutual information in response to physiologically relevant mossy fibers patterns. The decrease in spike variability was due to an increased postsynaptic membrane excitability, which made granule cells more prone to elicit action potentials, and to a strengthened synaptic inhibition, which markedly hampered membrane depolarization. These concomitant actions on granule cells firing indicate that desflurane re-shapes the transfer of information between neurons by providing a less informative neurotransmission rather than completely silencing neuronal activity.     

Local F-actin Network Links Synapse Formation and Axon Branching

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Postacute Effects of Kisspeptin‐10 on Neuronal Injury Induced by l‐Methionine in Rats

Apart from its effect on the regulation of reproductive function, recent studies indicate that kisspeptin may play roles in the antioxidant defense system. The antioxidant defense system and oxidative stress contribute to the etiology and pathogenesis of neuronal cell death after brain injury. We have investigated the postacute effect of kisspeptin‐10 on brain injury induced by l ‐methionine. DNA fragmentation, malondialdehyde (MDA), reduced glutathione levels, and superoxide dismutase (SOD) activities were analyzed. Our results showed that methionine treatment increases apoptotic cell death. Kisspeptin alone showed no side effect on apoptotic cell death. However, kisspeptin treatment reversed the proapoptotic effect of methionine associated with reduced MDA and increased glutathione levels. Furthermore, SOD activity was completely depleted in methionine‐treated animals. In conclusion, our results revealed that delayed kisspeptin‐10 treatment reduces neuronal cell death by activation of SOD activity.     

Characteristic Changes of Astrocyte and Microglia in Rat Striatum Induced by 3-NP and MCAO

Our previous studies had confirmed that both 3-NP and MCAO induced the behavioral defect as well as striatal neuronal injury and loss in experimental rats. This study aimed to examine different response forms of striatal astrocyte and microglia in 3-NP and MCAO rat models. The present results showed that the immunoreaction for GFAP was extremely weak in the lesioned core of striatum, but in the transition zone of 3-NP model and the penumbra zone of MCAO model, GFAP+ cells showed strong hypertrophic and proliferative changes. Statistical analysis for the number, size and integral optical density (IOD) of GFAP+ cells showed significant differences when compared with their controls and compared between the core and the transition zone or the penumbra zone, respectively, but no differences between the 3-NP and MCAO groups. However, Iba-1+ cells showed obvious hypertrophy and proliferation in the injured striatum in the 3-NP and the MCAO models, especially in the transition zone of 3-NP model and the penumbra zone of MCAO model. These Iba-1+ cells displayed two characteristic forms as branching cells with thick processes and amoeboid cells with thin processes. Statistical analysis showed that the number, size and IOD of Iba-1+ cells were significantly increased in the cores and the transition zone of 3-NP group and the penumbra zone of MCAO group than that of the controls, and the immune response of Iba-1 was stronger in the MCAO group than in the 3-NP group. The present results suggested that characteristic responses of astrocyte and microglia in the 3-NP and the MCAO models display their different effects on the pathological process of brain injury.     

Molecular Control of B Cell Activation and Immunological Synapse Formation

B cells form an essential part of the adaptive immune system by producing specific antibodies that can neutralize toxins and target infected or malignant cells for destruction. During B cell activation, a fundamental role is played by a specialized intercellular structure called the immunological synapse (IS). The IS serves as a platform for B cell recognition of foreign, often pathogenic, antigens on the surface of antigen‐presenting cells (APC). This recognition is elicited by highly specific B cell receptors (BCR) that subsequently trigger carefully orchestrated intracellular signaling cascades that lead to cell activation. Furthermore, antigen internalization, essential for full B cell activation and differentiation into antibody producing effector cells or memory cells, occurs in the IS. Recent developments especially in various imaging‐based methods have considerably advanced our understanding of the molecular control of B cell activation. Interestingly, the cellular cytoskeleton is emerging as a key player at several stages of B cell activation, including the initiation of receptor signaling. Here, we discuss the functions and molecular mechanisms of the IS and highlight the multifaceted role of the actin cytoskeleton in several aspects of B cell activation.      

MTM-6, a Phosphoinositide Phosphatase,is Required to Promote Synapse Formation in Caenorhabditis elegans

Forming the proper number of synapses is crucial for normal neuronal development. We found that loss of function of the phosphoinositide phosphatase mtm-6 results in a reduction in the number of synaptic puncta. The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action. MTM-6 shows relative specificity for EGL-20 over the other Wnt ligands. We suggest that the ability of MTM-6 to regulate EGL-20 secretion is a function of its expression pattern. We conclude that regulation of secretion of different Wnt ligands can use different components. Additionally, we present a novel neuronal function for MTM-6.     

Interleukin 10 in disseminated lupus erythematosus

A number of years ago several groups reported that Interleukin 10 (IL10) was vigorously overproduced in disseminated lupus erythematosus (reviewed in Llorente et al., 2000). This hyperproduction obeys two different patterns. In one pattern, IL10 serum concentrations become elevated during disease and evolve in parallel with them. In the other, the patients' blood mononucleated cells spontaneously release increased IL10 quantities; contrary to serum variations in the first pattern, this increased output is not correlated with disease spurts. Interestingly an increase of this type is frequently found in disease-free parents of these patients (Llorente et al., 1997; Gr?ndal et al., 1999). Some studies relate this IL10 hyperproduction to a genetic origin (Mehrian et al., 1998; Mok et al., 1998), while others seem to indicate an environmental cause (Gr?ndal et al., 1999). Together these findings suggest two different possible origins for lupus IL10 overproduction. The first would be due to intrinsic anomalous IL10 production by some immune cells, the second would result from high IL10 output by tissues damaged by the inflammatory process. Considering the properties of IL10, which activates B lymphocytes and inhibits T lymphocytes, overproduction in lupus could explain the immune anomalies of this disease, which is characterized by anti-self antibodies production and by deficient T responses. Interestingly several of these anomalies are found independently from disease outbreaks and, in the case of some, in relatives of patients. The part played by IL10 in lupus has been inferred from the murine NZB-W model, in which an anti-IL10 monoclonal antibody prevents development of the disease (Ishida et al., 1994); this antibody also prevents the appearance of human anti-ADN autoantibodies in immune-deficient mice restored with patients' lymphocytes. A direct demonstration of the role of IL10 in the etiology of lupus symptoms has recently been adduced in a pilot study bearing on six lupus patients, refractory to anti-inflammatory and immuno-suppressive treatments, who were treated for 3 weeks with a murine anti-IL10 monoclonal antibody (Llorente et al., 2000). This treatment brought about a rapid amelioration of the clinical picture, in particular of the cutaneous and articular symptoms, which was maintained for six months after this trial. This symptomatic amelioration was accompanied by a decrease of the biological signs of immune system hyperactivity and a partial amelioration of T lymphocyte function. The better clinical control of the disease allowed a significant decrease of corticotherapy. While this was an open study, without a control group and without randomisation, the rapid and important evolution of clinical and biological parameters towards normal strongly pleads for a favorable effect of the treatment. While confirming previous hypotheses about the rote of IL10 in lupus, this study leaves several points unexplained. First the clinical and biological amelioration in these patients treated with anti-IL10 monoclonal antibody occurred independently of circulating anti-ADN auto-antibodies, indicating that the role of this interleukin in the disease is more complex than could be thought from the initial experiments in mouse. This unexpected result does not exclude that the beneficial effect of the antibody could be mediated by its action on B lymphocytes, since it is well known that these cells play important parts in development auto-immune processes other than only the production of auto-antibodies. It is also possible that the negative effect of IL10 in this disease exerts on other targets than B lymphocytes, in particular on fibroblasts and endothelial cells. Indeed it should be recalled that, while IL10 has often been held as an anti-inflammatory cytokine, its biological properties are more ambiguous, since it can immuno-stimulate some cell types. Among other remaining unknowns, the reason why some IL10 overproducing individuals, belonging to the family of lupus patients, do not develop lupus, is not understood. This fact underlines the multi-factorial origin of this disease, which must stem from associated genetic and environmental causes. IL10 overproduction, while it apparently promotes the symptoms, is certainly not sufficient. The efficiency of anti-IL 10 monoclonal antibodies during lupus, which are well tolerated, points to their usefulness in the therapeutic arsenal, especially in forms that are refractory to conventional anti-inflammatory and immunosuppressive treatments. In order to be validated, this hypothesis must be submitted to more in depth, randomized, studies. Moreover humanized antibodies should be developed, which would make it possible to reiterate the treatment during further outbreaks. Once all these conditions are fulfilled, the place of anti-IL10 treatment in lupus and the benefice-risk ratio should be compared to these of therapeutic approaches currently used in refractory forms. If further studies confirm the efficiency of- and tolerance to- IL10 neutralizing antibodies in lupus, it is well possible that these antibodies will eventually equate, for this disease, the recent use of TNF antagonists in the treatment of rheumatoid arthritis.