Polysialic acid and activity-dependent synapse remodeling

Polysialic acid (PSA) is a large carbohydrate added post-translationally to the extracellular domain of the Neural Cell Adhesion Molecule (NCAM) that influences its adhesive and other functional properties. PSA-NCAM is widely distributed in the developing nervous system where it promotes dynamic cell interactions, like those responsible for axonal growth, terminal sprouting and target innervation. Its expression becomes restricted in the adult nervous system where it is thought to contribute to various forms of neuronal and glial plasticity. We here review evidence, obtained mainly from hypothalamic neuroendocrine centers and the olfactory system, that it intervenes in structural synaptic plasticity and accompanying neuronal-glial transformations, making possible the formation and elimination of synapses that occur under particular physiological conditions.     

Modeling activity-dependent synapse restructuring

The spread of electrical activity in a dendritic tree is shaped, in part, by its morphology. Conversely, experimental evidence is growing that electrical and chemical activity can slowly shape the morphology of the dendrite. In this theoretical study, the dendritic spines are dynamic elements, with biophysical properties that change in response to patterns of electrical activity. Recent experiments and diagrammatic models suggest that activity-dependent processes can regulate structural modifications in dendritic spines as well as their distribution along the dendrite. This study considers how local changes in spine structure (minutes to hours) can influence patterns of electrical activity along the dendrite; and how electrical activity due to synaptic events and excitable membrane dynamics can, over time, influence the morphology of the dendrite. The model presents a slow subsystem for structural synaptic plasticity associated with long-term potentiation. A perturbation problem evolves naturally when the spine stem shortens, since the ratio of spine stem resistance to input resistance is small. Hence, the difference between the spine head and dendritic potentials become negligible. This paper presents an asymptotic expansion of head potential in terms of dendritic potential. This leads to a reduced model for post-synaptic restructuring that captures the dynamics of the full model in a briefer computation period when the spines are well connected to the dendrite.     

Pre- and postsynaptic mechanisms in Hebbian activity-dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down-regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity-dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity-dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output.     

An activity-dependent determinant of synapse elimination in the mammalian brain

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Developmental remodeling of the retinogeniculate synapse

Anatomical rearrangement of retinogeniculate connections contributes to the refinement of synaptic circuits in the developing visual system, but the underlying changes in synaptic function are unclear. Here, we study such changes in mouse brain slices. Each geniculate cell receives a surprisingly large number of retinal inputs (>20) well after eye-specific zones are formed. All but one to three of these inputs are eliminated over a 3-week period spanning eye opening. Remaining inputs are strengthened approximately 50-fold, in part through an increase in quantal size, but primarily through an increase in the number of release sites. Changes in release probability do not contribute significantly. Thus, a redistribution of release sites from many inputs to few inputs at this late developmental stage contributes to the precise receptive fields of thalamic relay neurons.     

Novel mouse model reveals distinct activity-dependent and -independent contributions to synapse development

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.     

NCAM promotes assembly and activity-dependent remodeling of the postsynaptic signaling complex

The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. We show that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the N-methyl-d-aspartate (NMDA) receptor and Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) in a manner not firmly or directly linked to PSD95 and alpha-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM-spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM-spectrin-associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIalpha. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIalpha to synapses after NMDA receptor activation, which is a critical process in NMDA receptor-dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM.     

Phosphorylation reactions in activity-dependent synapse modification at the neuromuscular junction during development

We have studied developmental activity-dependent synapse diminution in both an in vitro tissue culture chamber system and at the intact rodent neuromuscular junction (nmj). In both types of preparations, pre- and postsynaptic alterations in synapse structure and function are produced by manipulations of thrombin (Thr) and protein kinase C (PKC) activity. An opposing postsynaptic effect of PKC and protein kinase A (PKA) action on the acetycholine receptor (AChR) can be shown in vitro with PKA stabilizing and PKC destabilizing the nmj synapses. In vivo studies of normal junctional maturation show that changes in axonal inputs and postsynaptic receptor cluster morphology occur, to a substantial degree, independently of one another. Presynaptic actions of PKA are involved in the activity dependent synapse modulation that can be demonstrated in vitro. Late in the elimination process, (>12 days in vivo) the process becomes independent of PKC, implying that diverse, redundant mechanisms are involved in this important developmental process.     

Mechanisms involved in activity-dependent synapse formation in mammalian central nervous system cell cultures

Differences in neuronal activity produced by electrical stimulation lead to competition between synapses from sensory afferents converging on a common spinal cord neuron. Studies were performed on neurons dissociated from the mouse spinal cord and grown in culture dishes with three compartments. Synaptic efficacy from stimulated afferents was increased compared with unstimulated convergents, and the number of functional connections was increased by stimulation compared with control cultures. Blocking NMDA channel activation with 100 microM APV in medium containing 1.8 mM calcium inhibited this synaptic plasticity, but plasticity was not blocked by APV in medium in which the calcium concentration was elevated to 3 mM. These experiments support the view that electrical activity differentially influences processes that cause a persistent decrease in synaptic efficacy or lead to synapse elimination and those that increase synaptic strength or lead to synapse augmentation. We interpret our results in terms of a model in which these antagonistic mechanisms are both regulated via changes in calcium levels and second messengers that are modulated by electrical activity. A significant portion of the activity-related calcium influx relevant to synaptic plasticity passes through the NMDA channel, but other sources of calcium are involved. In particular, competitive elimination of synapses appears to occur during blockade of NMDA channels if the extracellular concentration of calcium is elevated moderately. The outcome of competition between the two calcium-dependent but antagonistic processes may depend either on their differential sensitivity to intracellular calcium concentration or separate specificities to NMDA and non-NMDA receptor-linked mechanisms.     

Polysialic acid facilitates tumor invasion by glioma cells

Polysialic acid (PSA) is thought to attenuate neural cell adhesion molecule (NCAM) adhesion, thereby facilitating neural cell migration and regeneration. Although the expression of PSA has been shown to correlate with the progression of certain tumors such as small cell lung carcinoma, there have been no studies to determine the roles of PSA in gliomas, the most common type of primary brain tumor in humans. In this study, we first revealed that among patients with glioma, PSA was detected more frequently in diffuse astrocytoma cells, which spread extensively. To determine directly the role of PSA in glioma cell invasion, we transfected C6 glioma cells with polysialyltransferases to express PSA. In those transfected cells, PSA is attached mainly to NCAM-140, whereas the mock-transfected C6 cells express equivalent amounts of PSA-free NCAM-140. Both PSA negative and positive C6 cell lines exhibited almost identical growth rates measured in vitro. However, PSA positive C6 cells exhibited increased invasion to the corpus callosum, where the mock-transfected C6 glioma cells rarely invaded when inoculated into the brain. By contrast, the invasion to the corpus callosum by both the mock-transfected and PSA positive C6 cells was observed in NCAM-deficient mice. These results combined indicate that PSA facilitates tumor invasion of glioma in the brain, and that NCAM-NCAM interaction is likely attenuated in the PSA-mediated tumor invasion.     

Polysialic acid influences specific pathfinding by avian motoneurons.

The influence of polysialic acid (PSA) on the neural cell adhesion molecule on motoneuron outgrowth and pathway formation was investigated by determining its temporal and spatial pattern of expression and by the effect that its removal had on motoneuron projection patterns. Motoneurons first expressed PSA as their growth cones began to segregate into motoneuron pool-specific groups in the plexus region; furthermore, PSA levels differed between motoneurons projecting to different targets. When PSA was removed during the period of axonal segregation in the plexus region projection errors were common. However, later removal during the process of muscle nerve formation did not result in projection errors. These results suggest that PSA modulates interactions between motoneuron axons and guidance molecules in the plexus region during axonal pathfinding.     

Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment

The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Polysialic acids

• 1.1. Polysialic acids are linear homopolymers of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminated neuraminic acid (KDN) residues joined by α 2,8, α 2–9 or α2,8/α2,9 ketosidic linkages.
• 2.2. They occur in glycoproteins of embryonic neural membranes (playing a role of neural cell adhesion molecules), in non-neural tissues (postnatal kidney), tumours, (neuroectodermal tumours), fish eggs and in the capsule of certain bacteria such as Neisseria meningitidis group B.
• 3.3. These polymers are synthesized through reactions which involve (a) the synthesis of sialic acid; (b) its activation to a cytidine monophosphate sugar nucleotide and (c) the polymerization of the different residues by a polysialyl-transferase complex.
• 4.4. Polysialic acids are involved in organogenesis and in cell growth. In several tissues they act as oneodevelopmental antigens, and in bacteria are also virulent determinants.

     

Polysialic acid limits choline acetyltransferase activity induced by brain-derived neurotrophic factor

Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine, is known to be activated by brain derived neurotrophic factor (BDNF). We found that the specific removal of the carbohydrate polysialic acid (PSA) significantly increased BDNF-induced ChAT-activity in embryonic septal neurons. Using a p75 neurotrophin receptor (p75(NTR)) function-blocking antibody and K252a, a-pan tropomyosin related kinase (Trk) inhibitor, we demonstrate that BDNF-induced ChAT activity requires the stimulation of p75(NTR) and TrkB. PSA removal drastically increased radioactive iodinated ([(125)I])BDNF's maximal binding capacity (Bmax), derived from concentrations of [(125)I]BDNF ranging from 1 pM to 3.2 nM. In the presence of unlabeled nerve growth factor to prevent the binding of [(125)I]BDNF to p75(NTR) sites, the impact of PSA removal on the binding capacity of [(125)I]BDNF was greatly reduced. In conclusion, PSA limits BDNF-induced ChAT activity and BDNF-receptor interactions. BDNF-induced ChAT activity is TrkB and p75(NTR) dependent, and upon PSA removal the additional binding of BDNF to its receptors, especially p75(NTR), likely contributes to the maximal ChAT activity observed. In vivo, the ontogenetic loss of PSA in the postnatal period may allow more interactions between BDNF and its receptors to increase ChAT activity and assure the proper development of the cholinergic septal neurons.     

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.     

The thrombin receptor mediates functional activity-dependent neuromuscular synapse reduction via protein kinase C activation in vitro

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system.     

Experience-dependent retinogeniculate synapse remodeling is abnormal in MeCP2-deficient mice

Mutations in MECP2 underlie the neurodevelopmental disorder Rett syndrome (RTT). One hallmark of RTT is relatively normal development followed by a later onset of symptoms. Growing evidence suggests an etiology of disrupted synaptic function, yet it is unclear how these abnormalities explain the clinical presentation of RTT. Here we investigate synapse maturation in Mecp2-deficient mice at a circuit with distinct developmental phases: the retinogeniculate synapse. We find that synapse development in mutants is comparable to that of wild-type littermates between postnatal days 9 and 21, indicating that initial phases of synapse formation, elimination, and strengthening are not significantly affected by MeCP2 absence. However, during the subsequent experience-dependent phase of synapse remodeling, the circuit becomes abnormal in mutants as retinal innervation of relay neurons increases and retinal inputs fail to strengthen further. Moreover, synaptic plasticity in response to visual deprivation is disrupted in mutants. These results suggest a crucial role for Mecp2 in experience-dependent refinement of synaptic circuits.     

The role of the ubiquitin proteasome system in synapse remodeling and neurodegenerative diseases

The ubiquitin proteasome system is a potent regulatory mechanism used to control protein stability in numerous cellular processes, including neural development. Many neurodegenerative diseases are featured by the accumulation of UPS-associated proteins, suggesting the UPS dysfunction may be crucial for pathogenesis. Recent experiments have highlighted the UPS as a key player during synaptic development. Here we summarize recent discoveries centered on the role of the UPS in synapse remodeling and draw attention to the potential link between the synaptic UPS dysfunction and the pathology of neurodegenerative diseases.