Synaptic vesicle endocytosis: the races,places, and molecular faces

The classical experiments on synaptic vesicle recycling in the 1970s by Heuser and Reese, Ceccarelli, and their colleagues raised opposing theories regarding the speed, mechanisms, and locations of membrane retrieval at the synapse. The Heuser and Reese experiments supported a model in which synaptic vesicle recycling is mediated by the formation of coated vesicles, is relatively slow, and occurs distally from active zones, the sites of neurotransmitter release. Because heavy levels of stimulation were needed to visualize the coated vesicles, Ceccarelli’s experiments argued that synaptic vesicle recycling does not require the formation of coated vesicles, is relatively fast, and occurs directly at the active zone in a “kiss-and-run” reversal of exocytosis under more physiological conditions. For the next thirty years, these models have provided the foundation for studies of the rates, locations, and molecular elements involved in synaptic vesicle endocytosis. Here, we describe the evidence supporting each model and argue that the coated vesicle pathway is the most predominant physiological mechanism for recycling synaptic vesicles.     

Cellular mechanisms of connexin32 mutations associated with CNS manifestations

Both oligodendrocytes and myelinating Schwann cells express the gap junction protein connexin32 (Cx32). Mutations in the gene encoding Cx32 (GJB1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX). Although most CMTX patients do not have clinical central nervous system (CNS) manifestations, subclinical evidence of CNS dysfunction is common. We investigated the cellular effects of a subgroup of GJB1/Cx32 mutations that have been reported to cause clinical CNS dysfunction. We hypothesized that these mutants have dominant-negative effects on other connexins expressed by oligodendrocytes, specifically Cx45. We expressed these and other Cx32 mutants in communication-incompetent as well as Cx45-expressing HeLa cells, and analyzed the transfected cells by immunocytochemistry and immunoblotting. In communication-incompetent cells, the mutants associated with CNS phenotypes failed to reach the cell membrane and were instead retained in the endoplasmic reticulum (A39V, T55I) or Golgi apparatus (M93V, R164Q, R183H), although rare gap junction plaques were found in cells expressing M93V or R183H. In HeLa cells stably expressing Cx45, these Cx32 mutants showed a similar expression pattern, and did not alter the pattern of Cx45 expression. These results indicate that Cx32 mutants that are associated with a CNS phenotype do not interact with Cx45, but may instead have other toxic effects in oligodendrocytes.     

Decreased levels of brain-derived neurotrophic factor in serum of chronic schizophrenic patients

Neurotrophic factors regulate neuronal development as well as synaptic plasticity, and their impairment is often implicated as a cause of schizophrenia. Among various neurotrophic molecules, brain-derived neurotrophic factor (BDNF) levels have been found to be increased in the corticolimbic regions of patients’ brains. In the present study, we assessed peripheral BDNF levels in whole blood as well as in the serum of two independent groups of schizophrenic patients (n=34 in each group) and healthy volunteers (n=35 and n=27, respectively). BDNF protein levels in fresh serum and blood of the patients and volunteers were measured using a two-site enzyme immunoassay and correlated with the number and decay of platelets. In addition to the studies of patients and volunteers, neuroleptic effects on BDNF levels were assessed by administering haloperidol to adult rats for 2 weeks or 5 months. The major findings were as follows: BDNF levels were significantly reduced in the serum of schizophrenic patients (P<0.005, Mann–Whitney U-test) but not in their whole blood. Antipsychotic dose did not correlate with serum BDNF levels. Moreover, chronic administration of haloperidol failed to decrease serum BDNF levels in adult rats. Abnormal levels of BDNF are evident not only in the brain of schizophrenic patients, but also in their peripheral blood. The BDNF reduction in serum but not in whole blood suggests a potential deficit in neurotrophic factor release in patients with schizophrenia.     

Lipid Modification during Epidermal Cell Differentiation

As keratinocytes differentiate into corneocytes of the stratum corneum or epidermal permeability barrier, their lipids are modified so as to fulfill totally different functions. Recent experimentation has clarified the molecular mechanisms by which lipids of membrane origin are targeted to specialized lamellar bodies, where metabolic retailoring makes them suitable for use in the water-impermeable intercellular lamellae. In this latter structure the modified lipids are bound covalently to specialized proteins in a way that encourages the formation of lipid bilayers alternating with lipid monolayers. Only now are potential clues to the molecular regulation of this dramatic lipid transformation becoming apparent.     

Opioid receptors and their interacting proteins

The opioid receptors are among the most highly studied members of the family of G protein-coupled receptors. As for many other members of this family, recent studies have indicated that they do not exist in isolation but are able to interact with a substantial range of other polypeptides. Such interactions can alter the effectiveness of agonist-mediated cell signalling, determine the signals generated, alter the intracellular trafficking routes of the receptors and potentially determine cellular localization by providing a scaffold to link the receptors to the cytoskeletal network. Although virtually all studies on these interactions to date have employed expression into simple heterologous cell lines, the availability of knock-out mouse lines and the capacity to knock-down levels of opioid receptor-interacting proteins using techniques such as siRNA suggest that information on the functional consequences of such protein-protein interactions in a physiological setting will soon be forthcoming.     

Decreased plasma membrane targeting of NMDA-NR1 receptor subunit in dendrites of medial nucleus tractus solitarius neurons in rats self-administering morphine

Opioid abuse is associated with repeated administration and escalation of dose that can result in profound adaptations in homeostatic processes. Potential cellular mechanisms and neural sites mediating opiate-dependent adaptations may involve NMDA-dependent synaptic plasticity within brain areas participating in behaviors related to consumption of natural reinforcers, as well as affective-autonomic integration, notably the medial nucleus tractus solitarius (mNTS). NMDA-dependent synaptic plasticity may be mediated by changes in the intracellular and surface targeting of NMDA receptors, particularly in postsynaptic sites including spines or small distal dendrites. High-resolution immunogold electron microscopic immunocytochemistry combined with morphometry were used to measure changes in targeting of the NMDA-NR1 (NR1) receptor subunit between intracellular and plasmalemmal sites in dendrites of neurons of the intermediate mNTS of rats self-administering escalating doses of morphine (EMSA). In control and EMSA rats, the density of plasmalemmal and cytosolic gold particles was inversely related to profile size. Collapsed across all NR1-labeled dendrites, rats self-administering morphine had a lower number of plasmalemmal gold particles per unit surface area (7.1 +/- 0.8 vs. 14.4 +/- 1 per 100 microm), but had a higher number of intracellular gold particles per unit cross-sectional area (169 +/- 6.1 vs. 148 +/- 5.1 per 100 microm2) compared to saline self-administering rats. Morphometric analysis showed that the decrease in plasma membrane labeling of NR1 was most robust in small dendritic profiles (<1 microm), where there was a reciprocal increase in the density of intracellular particles. These results indicate that the plasmalemmal distribution of the essential NR1 subunits in distal sites may prominently contribute to NMDA receptor-dependent modulation of neural circuitry regulating homeostatic processes, and targeting of these proteins can be prominently affected by morphine self-administration.     

Ultrastructural localization of high-affinity choline transporter in the rat neuromuscular junction: enrichment on synaptic vesicles

In cholinergic neurons, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake system is thought to be the rate-limiting step in acetylcholine (ACh) synthesis. The system is highly regulated by neuronal activity; the choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity choline transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and choline uptake.     

The unfolded protein response in protein aggregating diseases

For many genetic diseases, clinical phenotypes arise through the dysfunction of the gene products encoded by mutant genes. Effective treatment entails providing a source of the gene product in the diet or circulation, as has been achieved for type I diabetes and hemophilia, or in cases of enzyme deficiency by supplementation with metabolites synthesized by the defective protein, as in adrenoleukodystrophy. However, a growing list of diseases do not appear to be amenable to such treatment strategies. In these instances, defective gene products acquire novel properties that disrupt normal cell function, even in the presence of proteins encoded by the normal allele. One class of such diseases, collectively termed “conformational diseases,” is composed of clinically unrelated disorders that share a common pathophysiology because the mutant proteins cannot adopt stable three-dimensional conformations. These mutant proteins aggregate in various subcellular compartments and may even cause cell death. Some of these diseases are associated with inclusion bodies containing the aggregating proteins whereas others do not exhibit such pathology; however, all appear to activate cell stress signaling pathways. Herein, we highlight one such disorder, Pelizaeus-Merzbacher disease, that disrupts formation of whiter matter in the brain. Accumulation of the mutant protein in oligodendrocytes activates the unfolded protein response. The well-characterized genetics and large number of animal models available for Pelizaeus-Merzbacher disease enables this disease to serve as an important model for conformational diseases, both in terms of defining molecular components of the unfolded protein response signaling pathway as well as testing therapeutic approaches to ameliorate disease.