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In all cells, generation and release of specific vesicles are the initial steps of back-and-forth intercellular communication. These processes are critical in normal physiology and pathophysiology. Vesicles have particular functions appropriate to their targets. When stimulated, they are released into the extracellular space. Four cytoplasmic membrane-bound structures generate their particular vesicles. Among these structures, multivesicular bodies (MVBs) can accumulate many small vesicles in their lumen; release occurs upon MVB exocytosis. Ectosomes are larger vesicles characterized by their responses and are generated directly and released independently from specific microdomains pre-established in the thickness of the plasma membrane. Most lysosomes do not generate vesicles. However, unique components of a minor form, the endo-lysosome, constitute the third class of structures that release a few vesicles by exocytosis with molecules and structures inducing changes in the extracellular environment. The autophagosome, the fourth structure, releases several heterogeneous vesicles by exocytosis with malformed bio-molecules, assembled structures, and damaged organelles. Interestingly, the frequent interaction of autophagosomes with MVBs and their exosomes contributes to the regulation and intensity of their action. The specificity and function of released vesicles depend on their membranes’ and luminal cargoes’ composition and dynamics. An ongoing investigation of the various vesicles reveals new properties regarding their generation, release, and resulting extracellular processes. The growth of information about structures and their vesicles progressively extends the knowledge base regarding cell communication and contributes to their clinical applications.

The fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes. Other exocytic vesicles, however, do not contain cargoes, and thus, their fusion is not followed by secretion. Therefore, two distinct processes of exocytosis exist, one secretory and the other non-secretory. The present review deals with the knowledge of non-secretory exocytosis developed during recent years. Among such developments are the dual generation of the exocytic vesicles, initially released either from the trans-Golgi network or by endocytosis; their traffic with activation of receptors, channels, pumps, and transporters; the identification of their tethering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes that govern membrane fusions; the growth of axons and the membrane repair. Examples of potential relevance of these processes for pathology and medicine are also reported. The developments presented here offer interesting chances for future progress in the field.

Mesenchymal stem cells (MSCs), the cells distributed in the stromas of the body, are known for various properties including replication, the potential of various differentiations, the immune-related processes including inflammation. About two decades ago, these cells were shown to play relevant roles in the therapy of numerous diseases, dependent on their immune regulation and their release of cytokines and growth factors, with ensuing activation of favorable enzymes and processes. Such discovery induced great increase of their investigation. Soon thereafter, however, it became clear that therapeutic actions of MSCs are risky, accompanied by serious drawbacks and defects. MSC therapy has been therefore reduced to a few diseases, replaced for the others by their extracellular vesicles, the MSC-EVs. The latter vesicles recapitulate most therapeutic actions of MSCs, with equal or even better efficacies and without the serious drawbacks of the parent cells. In addition, MSC-EVs are characterized by many advantages, among which are their heterogeneities dependent on the stromas of origin, the alleviation of cell aging, the regulation of immune responses and inflammation. Here we illustrate the MSC-EV therapeutic effects, largely mediated by specific miRNAs, covering various diseases and pathological processes occurring in the bones, heart and vessels, kidney, and brain. MSC-EVs operate also on the development of cancers and on COVID-19, where they alleviate the organ lesions induced by the virus. Therapy by MSC-EVs can be improved by combination of their innate potential to engineering processes inducing precise targeting and transfer of drugs. The unique properties of MSC-EVs explain their intense studies, carried out with extraordinary success. Although not yet developed to clinical practice, the perspectives for proximal future are encouraging.

This is a Commentary of a review about extracellular vesicles of immune cells published two years ago in Clinical and Experimental Immunology, a prestigious journal of the field. The aim is to establish whether, and to what extent, results in scientific area of the review have been extended and strengthened by innovative findings of considerable interest. The analysis of the recently published results has revealed that in various areas of the review developments have occurred. However, innovative findings have been only about the extracellular vesicles secreted by mesenchymal stem cells, usually indicated as MSC-EVs. Based on these findings, the Commentary has been focused on recent MSC-EVs findings presented in three Sections dealing with 1. recently appeared, relevant functions of the latter vesicles; 2. therapeutic processes developed according well known criteria, however innovative in many respects; and 3. protection of COVID-19 disease patients from organ lesions induced by the specific virus, SARS-CoV-2, during the disease. As everybody knows, the COVID-19 pandemic started at the end of 2019, thus after the publication of the aforementioned review. Data of Section 3 are therefore innovative, of great potential interest also at the clinical level, applied by translational medicine to various organs, from lung to brain, heart, kidney, immune and other cells. In view of its relevance, the author expects that research and medical use of MSC-EV, active at present, will be further developed, acquiring additional relevance in the near future.

In the last few years, exciting properties have emerged regarding the activation, signaling, mechanisms of action, and therapeutic targeting of the two types of neurotrophin receptors: the p75NTR with its intracellular and extracellular peptides, the Trks, their precursors and their complexes. This review summarizes these new developments, with particular focus on neurodegenerative diseases. Based on the evolving knowledge, innovative concepts have been formulated regarding the pathogenesis of these diseases, especially the Alzheimer's and two other, the Parkinson's and Huntington's diseases. The medical progresses include original procedures of diagnosis, started from studies in mice and now investigated for human application, based on innovative classes of receptor agonists and blockers. In parallel, comprehensive studies have been and are being carried out for the development of drugs. The relevance of these studies is based on the limitations of the therapies employed until recently, especially for the treatment of Alzheimer's patients. Starting from well known drugs, previously employed for non-neurodegenerative diseases, the ongoing progress has lead to the development of small molecules that cross rapidly the blood-brain barrier. Among these molecules the most promising are specific blockers of the p75NTR receptor. Additional drugs, that activate Trk receptors, were shown effective against synaptic loss and memory deficits. In the near future such approaches, coordinated with treatments with monoclonal antibodies and with developments in the microRNA field, are expected to improve the therapy of neurodegenerative diseases, and may be relevant also for other human disease conditions.

Not available.

Exosomes and ectosomes, two distinct types of extracellular vesicles generated by all types of cell, play key roles in intercellular communication. The formation of these vesicles depends on local microdomains assembled in endocytic membranes for exosomes and in the plasma membrane for ectosomes. These microdomains govern the accumulation of proteins and various types of RNA associated with their cytosolic surface, followed by membrane budding inward for exosome precursors and outward for ectosomes. A fraction of endocytic cisternae filled with vesicles — multivesicular bodies — are later destined to undergo regulated exocytosis, leading to the extracellular release of exosomes. In contrast, the regulated release of ectosomes follows promptly after their generation. These two types of vesicle differ in size — 50–150 nm for exosomes and 100–500 nm for ectosomes — and in the mechanisms of assembly, composition, and regulation of release, albeit only partially. For both exosomes and ectosomes, the surface and luminal cargoes are heterogeneous when comparing vesicles released by different cell types or by single cells in different functional states. Upon release, the two types of vesicle navigate through extracellular fluid for varying times and distances. Subsequently, they interact with recognized target cells and undergo fusion with endocytic or plasma membranes, followed by integration of vesicle membranes into their fusion membranes and discharge of luminal cargoes into the cytosol, resulting in changes to cellular physiology. After fusion, exosome/ectosome components can be reassembled in new vesicles that are then recycled to other cells, activating effector networks. Extracellular vesicles also play critical roles in brain and heart diseases and in cancer, and are useful as biomarkers and in the development of innovative therapeutic approaches.

In the last few years, exciting reports have emerged regarding the role of the two types of neurotrophin receptors, p75

In the last few years, exciting reports have emerged regarding the role of the two types of neurotrophin receptors, p75NTR and Trks, not only in neurons, where they were discovered, but also in non-neural cells and, especially, in numerous cancers, including breast, lung, colon-rectum, pancreas, prostate, glioblastoma, neuroblastoma, myeloma, and lymphoid tumors. Traditionally, p75NTR, activated by all neurotrophins and their precursors, is an inhibitor. In various cancers, however, activated p75NTR induces variable effects, from inhibition to stimulation of cell proliferation, dependent on their direct or coordinate/indirect mechanism(s) of action. TrkA, TrkB, and TrkC, activated by distinct neurotrophins, are high affinity stimulatory receptors. In cancers, activation of Trks, especially of TrkB, are stimulators of cell proliferation, aggressiveness, and metastases. In rare cancers, these processes are due not to receptor activation but to fusion or mutation of the encoding genes. A considerable panel of anti-Trk drugs, developed recently, has been investigated both in vitro and in living mice for their effects on cancer cells. Many such drugs protect from cancers by preventing cell proliferation and inducing apoptosis. At present, these drugs are under control by trials, to promote introduction in human therapy. Moreover, anti-Trk drugs have been employed also in combination with classical chemotherapeutic drugs. So far, studies in mice have been positive. The chemotherapeutic/anti-receptor combinations exhibited in fact increased potency and down-regulation of resistance, with no increase of side effects.

Alzheimer's is the neurodegenerative disease affecting the largest number of patients in the world. In spite of the intense research of the last decades, progress about its knowledge and therapy was limited. In particular, various cytotoxic processes remained debated, while the few drugs approved for therapy were of only marginal relevance. Recent studies have identified key aspects of the disease, such as the mechanisms governing the development of pathology. In order to operate the Aβ peptide, known as the key factor, requires a complex assembled by its high affinity binding to PrPc, a cell surface prion protein, and mGluR5, a metabotropic glutamate receptor. Aβ and its associates bind also phosphorylated tau transferred to the extracellular space, with final activation of intracellular cytotoxic signals. Pathology is further affected by factors (including genes, receptors and their agonists) and by glial cells governing (via vesicles, cytokines and enzymes) cell immunology, inflammation and oxidative stress. Concomitant to pathology studies, strong attempts have been made for the development of new, effective therapies. Critical for this are biomarkers, by which Alzheimer's patients are recognized even before appearance of their symptoms. The question was whether patients take advantage from drugs not yet approved. The latter, first identified in mice, were found effective also in men, however only before appearance or at early stage of the disease. In other words, the drugs not yet approved induce effective protection of patients still healthy or in a preliminary stage of the disease. In contrast, developed Alzheimer's disease is practically irreversible.

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na+ channels, thereby decreasing the neuronal Na+ current density. This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, re-establishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.

Summary PC12-27, a PC12 clone characterized by high levels of the transcription repressor REST and by very low mTORC2 activity, had been shown to be unresponsive to NGF, possibly because of its lack of the specific TrkA receptor. The neurotrophin receptor repressed by high REST in PC12-27 cells, however, is shown now to be not TrkA, which is normal, but p75NTR, whose expression is inhibited at the transcriptional level. When treated with NGF, the PC12-27 cells lacking p75NTR exhibited a defective TrkA autophosphorylation restricted, however, to the TrkA(Y490) site, and an impairment of the PI3K signaling cascade. This defect was sustained in part by a mTORC1-dependent feed-back inhibition that in wtPC12 cells appeared marginal. Transfection of p75NTR to a level and surface distribution analogous to wtPC12 did not modify various high REST-dependent properties of PC12-27 cells such as high β-catenin, low TSC2 and high proliferation rate. In contrast, the defective PI3K signaling cascade and its associated mTORC2 activity were largely rescued together with the NGF-induced neurite outgrowth response. These changes were not due to p75NTR alone but required its cooperation with TrkA. Our results demonstrate that, in PC12, high REST induces alterations of NGF signaling which, however, are indirect, dependent on the repression of p75NTR; and that the well-known potentiation by p75NTR of the TrkA signaling does not concern all the effects induced by NGF but primarily the PI3K cascade and its associated mTORC2, a complex known to play an important role in neural cell differentiation.

The Repressor Element-1 (RE-1) Silencing Transcription (REST) factor, which is highly expressed in stem cells and non-neural cells, with low expression in neurons and other neural cells, orchestrates neural differentiation and preserves the unique neural phenotype. REST also plays a role in proliferation, although its effect differs depending on the cell type. It acts as an oncogene in neural cells and tumors (medulloblastomas, neuroblastomas, glioblastomas) and as a tumor suppressor in carcinomas of the lung, breast, and colon. The mechanisms underlying this duality have started to emerge recently and new therapeutic approaches based on these findings are being developed. Here, we present the mechanisms proposed to account for the oncogenic and antioncogenic roles of REST and discuss the therapeutic perspective of recent advances, particularly for small-cell lung cancer.

The membrane of dense-core vesicles is present only in neural cells, where it is instrumental to the regulated discharge of important molecules such as the catecholamine neurotransmitters. The mechanism underlying the specificity of this membrane to certain cell types has so far been unclear. Studies of this problem have been carried out by employing the pheochromocytoma PC12 cell line and its clones defective of dense-core vesicles. REST, the transcription repressor expressed at high levels in non-neural and at very low levels in neural cells, was found to regulate the genes encoding almost all the proteins of both the core and the membrane of the dense-core vesicles, including the transporter for catecholamines and the SNAREs for their exocytosis. Moreover, REST appears to control the assembly of the vesicle membrane. The role of REST in the various steps of the expression and function of the dense-core vesicle membrane is critical during development and participates in the dynamic regulation of mature cell physiology.

In the past few years, proline-rich transmembrane protein (PRRT)2 has been identified as the causative gene for several paroxysmal neurological disorders. Recently, an important role of PRRT2 in synapse development and function has emerged. Knock down of the protein strongly impairs the formation of synaptic contacts and neurotransmitter release. At the nerve terminal, PRRT2 endows synaptic vesicle exocytosis with Ca2+ sensitivity by interacting with proteins of the fusion complex and with the Ca2+ sensors synaptotagmins (Syts). In the postsynaptic compartment, PRRT2 interacts with glutamate receptors. The study of PRRT2 and of its mutations may help in refining our knowledge of the process of synaptic transmission and elucidating the pathogenetic mechanisms leading to derangement of network function in paroxysmal disorders.

This short comment deals with the recent review by Emanuele Cocucci and Jacopo Meldolesi on "Ectosomes and exosomes: shedding the confusion between extracellular vesicles" [1]. The confusion and shedding issues that are re-considered here are the following: 1. The biogenesis of the two vesicles. Ectosomes are generated directly from the plasma membrane, the exosomes, accumulated within the multivesicular bodies, are released upon exocytosis of the latter organelles. 2. The dissolution/navigation of the two types of extracellular vesicles. 3. Their fusion with specific target cells. 4. Their accumulation in the serum and urine. The conclusion emphasizes the relevance of ectosomes and exosomes in physiological and also pathological processes. The physiological processes include the expansion of the functional cell space; the concentration of specific components in the ectosome/exosome membranes, distinct from the plasma and multivesicular body membranes; the concentration of content molecules (proteins, RNAs and also DNA sequences) with respect to the cytosol; the external fusions with target cells. Among the pathological processes are the diagnoses of cancers and of other diseases.

L1 cell adhesion molecule (L1CAM), an adhesion/signaling protein encoded by a gene target of the transcription repressor RE-1-Silencing Transcription factor (REST), is expressed in two alternatively spliced isoforms. The full-length isoform, typical of low-REST neural cells, plays key roles in survival/migration, outgrowth/fasciculation/regeneration of axons, synaptic plasticity; the isoform missing two mini-exons, abundant in a few high-REST non-neural cells, maintains some effect on migration and proliferation. To investigate whether and how L1CAM alternative splicing depends on REST we used neural cell models expressing low or high levels of REST (PC12, SH-SY5Y, differentiated NT2/D1 and primary neurons transduced or not with REST). The short isoform was found to rise when the low-REST levels of neural cells were experimentally increased, while the full-length isoform increased in high-REST cells when the repressor tone was attenuated. These results were due to Nova2, a neural cell-specific splicing factor shown here to be repressed by REST. REST control of L1CAM occurs therefore by two mechanisms, transcription and alternative splicing. The splicing mechanism, affecting not only L1CAM but all Nova2 targets (∼7% of brain-specific splicing, including the mRNAs of other adhesion and synaptic proteins) is expected to be critical during development and important also for the structure and function of mature neural cells.

Cultured astrocytes exhibit a flat/epitelioid phenotype much different from the star-like phenotype of tissue astrocytes. Upon exposure to treatments that affect the small GTPase Rho and/or its effector ROCK, however, flat astrocytes undergo stellation, with restructuring of cytoskeleton and outgrowth of processes with lamellipodia, assuming a phenotype closer to that exhibited in situ. The mechanisms of this change are known only in part. Using the ROCK blocker drug Y27632, which induces rapid (tens of min), dose-dependent and reversible stellations, we focused on two specific aspects of the process: its dependence on small GTPases and the large surface expansion of the cells. Contrary to previous reports, we found stellation to be governed by the small G protein Rac1, up to disappearance of the process when Rac1 was downregulated or blocked by a specific drug. In contrast cdc42, the other G-protein often involved in phenotype changes, appeared not involved. The surface expansion concomitant to cytoskeleton restructuring, also dependent on Rac1, was found to be at least partially sustained by the exocytosis of enlargeosomes, small vesicles distinct from classical cell organelles, which are abundant in astrocytes. Exhaustion of stellation induced by repeated administrations of Y27632 correlated with the decrease of the enlargeosome pool. A whole-cell process like stellation of cultured astrocytes might be irrelevant in the brain tissue. However, local restructuring of the cytoskeleton coordinate with surface expansion, occurring at critical cell sites and sustained by mechanisms analogous to those of stellation, might be of importance in both astrocyte physiology and pathology. © 2011 Wiley Periodicals, Inc.

The RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin–neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor β-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of β-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST–TSC2–β-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active β-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST–TSC2–β-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

Neurite outgrowth is a fundamental process in the differentiation of neurons. The first, seminal study documenting the generation of "appendages" (now known as filopodia and lamellipodia) on the "cones d'accroissement," the specialized growth cones at the tips of neurites, was reported by Cajal still in the XIXth century, investigating chicken neurons embryos stained by the Golgi's reazione nera. Since then, studies have continued using, in addition to brain tissues, powerful in vitro models, i.e. primary cultures of pyramidal neurons from the hippocampus and neurosecretory cell lines, in particular PC12 cells. These studies have documented that neuronal neurites, upon sprouting from the cell body, give rise to both axons and dendrites. The specificity of these differentiated neurites depends on the diffusion barrier established at the initial segment of the axon and on the specialized domains, spines and presynaptic boutons, assembled around complexes of scaffold proteins. The two main, coordinate mechanisms that support neurite outgrowth are (a) the rearrangement of the cytoskeleton and (b) the expansion of the plasma membrane due to the exo/endocytosis of specific vesicles, distinct from those filled with neurotransmitters (clear and dense-core vesicles). The latter process is the main task of this review. In axons the surface-expanding exocytoses are concentrated at the growth cones; in dendrites they may be more distributed along the shaft. At least two types of exocytic vesicles appear to be involved, the enlargeosomes, positive for VAMP4, during early phases of development, and Ti-VAMP-positive vesicles later on. Outgrowth studies, that are now intensely pursued, have already yielded results of great importance in brain cell biology and function, and are playing an increasing role in pathology and medicine.

A rapid drop of the transcription repressor REST/NRSF during precursor differentiation into nerve cells is known to release the repression of hundreds of specific genes and thus to orchestrate the acquisition of the specific phenotype. REST, however, is important not only for differentiation, but also for the maintenance of key properties in mature nerve cell. The PC12 line is uniquely favorable for studying REST because, in addition to the wild-type, low REST neurosecretory cells, it includes spontaneously defective clones lacking neurosecretion, where REST is as high as in non-nerve cells. In this article, we summarize our cell biologic studies of two nerve cell-specific processes dependent on REST, neurosecretion and neurite outgrowth. We demonstrate that, in wild-type PC12 transfected with REST constructs, expression of genes encoding proteins of dense-core and synaptic-like vesicles is decreased, though, to different extents, with chromogranins being the most and the SNAREs (except SNAP25) the least affected. Concomitantly, dense core-vesicles decrease markedly in size but can still be discharged by regulated exocytosis. When, in contrast, dominant-negative constructs of REST are transfected in high-REST PC12, and the main effector enzymes of REST, histone deacetylases, are blocked, dense-core vesicles reappear and are discharged upon stimulation. In high-REST PC12, also neurite outgrowth is inhibited by down regulation of the NGF receptor. Concomitantly, however, high REST induces the expression of proteins and of an exocytic organelle, the enlargeosome, which sustain a Rac1-dependent form of neurite outgrowth, unknown until now, operative in PC12, in neuroblastoma SH-SY5Y cells, and also in neurons.

The cytosolic protein alpha-synuclein is enriched at the pre-synaptic terminals of almost all types of neurons in the central nervous system. alpha-Synuclein overexpression and the expression of three different mutants have been shown to sustain the pathogenesis of selected forms of Parkinson's disease. The localization of the protein and the defects found in knocked out or transgenic animals suggest a role of alpha-synuclein in the regulation of synaptic efficiency. However, the precise function of the protein and the molecular mechanisms of its action are still unclear. At synapses the synaptic vesicle release cycle is a finely tuned process composed of sequential steps that require the interconnected participation of several proteins and cytoskeletal elements. Actin microfilaments are required for the regulation of synaptic vesicle mobilization between different functional pools, for their organization at the active zone and influence the exocytotic process. We recently identified actin as a possible target of alpha-synuclein function. Through its binding to actin and the regulation of actin dynamics, alpha-synuclein could participate in the tuning of the vesicle release process, thereby modulating synaptic function and plasticity.

The function of alpha-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, alpha-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that alpha-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type alpha-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant alpha-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant alpha-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the alpha-synuclein gene, electroporation of wild-type alpha-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P alpha-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, alpha-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.

Triggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (Kd = 3.8 μM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain. © 2009 International Society for Neurochemistry.

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.

The small vesicles shed from the surface of many cells upon stimulation, considered for a long time to be artefacts, are now recognized as specific structures that are distinct from the exosomes released upon exocytosis of multivesicular bodies. Recent reports indicate that shedding vesicles participate in important biological processes, such as the surface-membrane traffic and the horizontal transfer of protein and RNAs among neighboring cells, which are necessary for the rapid phenotype adjustments in a variety of conditions. In addition, shedding vesicles have important physiological and pathological roles: in coagulation, by mediating the coordinate contribution of platelets, macrophages and neutrophils; in inflammatory diseases, via the release of cytokines; and in tumor progression, facilitating the spreading and release of cancer cells to generate metastases.

The mechanism by which neurons and neurosecretory cells govern the expression and the exocytic discharge of their clear and dense-core vesicles had remained unclear until recently when studies in the neurosecretory cell model PC12 revealed these processes to be orchestrated by the transcriptional repressor neuron restrictive silencer factor (NRSF)/repressor element-1 silencing transcription factor (REST). In wild-type PC12 fully competent for neurosecretion, NRSF/REST is low. The genes of the proteins involved in neurosecretion [from the secretory to vesicle membrane and plasma membrane proteins, including the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of exocytosis] were all repressed by increases of NRSF/REST expression to various extents when the increase was only a fewfold but were completely or almost completely repressed when the increase was large, as in spontaneously defective PC12 clones. In the first case the dense-core vesicles were still competent for exocytosis but were smaller and less dense than in wild-type cells; in the second they were no longer visible but did reappear when the repression was attenuated by transfection of a dominant-negative construct of NRSF/REST combined with a secretory chromogranin or strengthened by treatment with a blocker of NRSF/REST-associated enzymes, the histone deacetylases.

We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli.The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media.Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules.The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.