Specific Extracellular Vesicles, Generated and Operating at Synapses, Contribute to Neuronal Effects and Signaling.

In all cell types, small EVs, very abundant extracellular vesicles, are generated and accumulated within MVB endocytic cisternae. Upon MVB fusion and exocytosis with the plasma membrane, the EVs are released to the extracellular space. In the central nervous system, the release of neuronal EVs was believed to occur only from the surface of the body and dendrites. About 15 years ago, MVB cisternae and EVs were shown to exist and function at synaptic boutons, the terminals' pre- and post-synaptic structures essential for canonical neurotransmitter release. Recent studies have revealed that synaptic EVs are peculiar in many respects and heterogeneous with respect to other neuronal EVs. The distribution of synaptic EVs and the effect of their specific molecules are found at critical sites of their distribution. The role of synaptic EVs could consist of the modulation of canonical neurotransmitter release or a distinct, non-canonical form of neurotransmission. Additional roles of synaptic EVs are still not completely known. In the future, additional investigations will clarify the role of synaptic EVs in pathology, concerning, for example, circuits, trans-synaptic transmission, diagnosis and the therapy of diseases.

Autism Spectrum Disorder: Brain Areas Involved, Neurobiological Mechanisms, Diagnoses and Therapies.

Autism spectrum disorder (ASD), affecting over 2% of the pre-school children population, includes an important fraction of the conditions accounting for the heterogeneity of autism. The disease was discovered 75 years ago, and the present review, based on critical evaluations of the recognized ASD studies from the beginning of 1990, has been further developed by the comparative analyses of the research and clinical reports, which have grown progressively in recent years up to late 2023. The tools necessary for the identification of the ASD disease and its related clinical pathologies are genetic and epigenetic mutations affected by the specific interaction with transcription factors and chromatin remodeling processes occurring within specific complexes of brain neurons. Most often, the ensuing effects induce the inhibition/excitation of synaptic structures sustained primarily, at dendritic fibers, by alterations of flat and spine response sites. These effects are relevant because synapses, established by specific interactions of neurons with glial cells, operate as early and key targets of ASD. The pathology of children is often suspected by parents and communities and then confirmed by ensuing experiences. The final diagnoses of children and mature patients are then completed by the combination of neuropsychological (cognitive) tests and electro-/magneto-encephalography studies developed in specialized centers. ASD comorbidities, induced by processes such as anxieties, depressions, hyperactivities, and sleep defects, interact with and reinforce other brain diseases, especially schizophrenia. Advanced therapies, prescribed to children and adult patients for the control of ASD symptoms and disease, are based on the combination of well-known brain drugs with classical tools of neurologic and psychiatric practice. Overall, this review reports and discusses the advanced knowledge about the biological and medical properties of ASD.

Role of Senescent Astrocytes in Health and Disease.

For many decades after their discovery, astrocytes, the abundant glial cells of the brain, were believed to work as a glue, supporting the structure and metabolic functions of neurons. A revolution that started over 30 years ago revealed many additional functions of these cells, including neurogenesis, gliosecretion, glutamate homeostasis, assembly and function of synapses, neuronal metabolism with energy production, and others. These properties have been confirmed, limited however, to proliferating astrocytes. During their aging or following severe brain stress lesions, proliferating astrocytes are converted into their no-longer-proliferating, senescent forms, similar in their morphology but profoundly modified in their functions. The changed specificity of senescent astrocytes is largely due to their altered gene expression. The ensuing effects include downregulation of many properties typical of proliferating astrocytes, and upregulation of many others, concerned with neuroinflammation, release of pro-inflammatory cytokines, dysfunction of synapses, etc., specific to their senescence program. The ensuing decrease in neuronal support and protection by astrocytes induces the development, in vulnerable brain regions, of neuronal toxicity together with cognitive decline. Similar changes, ultimately reinforced by astrocyte aging, are also induced by traumatic events and molecules involved in dynamic processes. Senescent astrocytes play critical roles in the development of many severe brain diseases. The first demonstration, obtained for Alzheimer's disease less than 10 years ago, contributed to the elimination of the previously predominant neuro-centric amyloid hypothesis. The initial astrocyte effects, operating a considerable time before the appearance of known Alzheimer's symptoms evolve with the severity of the disease up to their proliferation during the final outcome. Involvement of astrocytes in other neurodegenerative diseases and cancer is now intensely investigated.

Cancer Stem Cells and Their Vesicles, Together with Other Stem and Non-Stem Cells, Govern Critical Cancer Processes: Perspectives for Medical Development.

Stem cells, identified several decades ago, started to attract interest at the end of the nineties when families of mesenchymal stem cells (MSCs), concentrated in the stroma of most organs, were found to participate in the therapy of many diseases. In cancer, however, stem cells of high importance are specific to another family, the cancer stem cells (CSCs). This comprehensive review is focused on the role and the mechanisms of CSCs and of their specific extracellular vesicles (EVs), which are composed of both exosomes and ectosomes. Compared to non-stem (normal) cancer cells, CSCs exist in small populations that are preferentially distributed to the niches, such as minor specific tissue sites corresponding to the stroma of non-cancer tissues. At niches and marginal sites of other cancer masses, the tissue exhibits peculiar properties that are typical of the tumor microenvironment (TME) of cancers. The extracellular matrix (ECM) includes components different from non-cancer tissues. CSCs and their EVs, in addition to effects analogous to those of MSCs/EVs, participate in processes of key importance, specific to cancer: generation of distinct cell subtypes, proliferation, differentiation, progression, formation of metastases, immune and therapy resistance, cancer relapse. Many of these, and other, effects require CSC cooperation with surrounding cells, especially MSCs. Filtered non-cancer cells, especially macrophages and fibroblasts, contribute to collaborative cancer transition/integration processes. Therapy developments are mentioned as ongoing preclinical initiatives. The preliminary state of clinical medicine is presented in terms of both industrial development and future treatments. The latter will be administered to specific patients together with known drugs, with the aim of eradicating their tumor growth and metastases.

Neurotrophin Trk Receptors: New Targets for Cancer Therapy.

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.

Gene Expression in the Physiology and Pathology of Neurons.

The expression of genes is the first process governing the molecular and structural specificity of the various types of cells, initiated by their transcription into the corresponding pre-mRNA [...].

Alternative Splicing by NOVA Factors: From Gene Expression to Cell Physiology and Pathology.

NOVA1 and NOVA2, the two members of the NOVA family of alternative splicing factors, bind YCAY clusters of pre-mRNAs and assemble spliceosomes to induce the maintenance/removal of introns and exons, thus governing the development of mRNAs. Members of other splicing families operate analogously. Activity of NOVAs accounts for up to 700 alternative splicing events per cell, taking place both in the nucleus (co-transcription of mRNAs) and in the cytoplasm. Brain neurons express high levels of NOVAs, with NOVA1 predominant in cerebellum and spinal cord, NOVA2 in the cortex. Among brain physiological processes NOVAs play critical roles in axon pathfinding and spreading, structure and function of synapses, as well as the regulation of surface receptors and voltage-gated channels. In pathology, NOVAs contribute to neurodegenerative diseases and epilepsy. In vessel endothelial cells, NOVA2 is essential for angiogenesis, while in adipocytes, NOVA1 contributes to regulation of thermogenesis and obesity. In many cancers NOVA1 and also NOVA2, by interacting with specific miRNAs and by additional mechanisms, activate oncogenic roles promoting cell proliferation, colony formation, migration, and invasion. In conclusion, NOVAs regulate cell functions of physiological and pathological nature. Single cell identification and distinction, and new therapies addressed to NOVA targets might be developed in the near future.

Alzheimer's disease: Key developments support promising perspectives for therapy.

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 Aß 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. Aß 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.

News about the Role of the Transcription Factor REST in Neurons: From Physiology to Pathology.

RE-1 silencing transcription factor (REST) (known also as NRSF) is a well-known transcription repressor whose strong decrease induces the distinction of neurons with respect to the other cells. Such distinction depends on the marked increased/decreased expression of specific genes, accompanied by parallel changes of the corresponding proteins. Many properties of REST had been identified in the past. Here we report those identified during the last 5 years. Among physiological discoveries are hundreds of genes governed directly/indirectly by REST, the mechanisms of its neuron/fibroblast conversions, and the cooperations with numerous distinct factors induced at the epigenetic level and essential for REST specific functions. New effects induced in neurons during brain diseases depend on the localization of REST, in the nucleus, where functions and toxicity occur, and in the cytoplasm. The effects of REST, including cell aggression or protection, are variable in neurodegenerative diseases in view of the distinct mechanisms of their pathology. Moreover, cooperations are among the mechanisms that govern the severity of brain cancers, glioblastomas, and medulloblastomas. Interestingly, the role in cancers is relevant also for therapeutic perspectives affecting the REST cooperations. In conclusion, part of the new REST knowledge in physiology and pathology appears promising for future developments in research and brain diseases.

REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability.

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.

Neurotrophin receptors in the pathogenesis, diagnosis and therapy of neurodegenerative diseases.

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.

Neurite outgrowth induced by NGF or L1CAM via activation of the TrkA receptor is sustained also by the exocytosis of enlargeosomes.

NGF binding to its protein kinase receptor TrkA is known to induce neurite outgrowth and neural cell differentiation. The plasma membrane expansion, necessary for the process, was shown to be contributed by the VAMP7-dependent exocytosis of endocytic vesicles. Working with wild-type PC12 (wtPC12), a cell model widely used to investigate NGF-induced neurite outgrowth, we found that a few hours of treatment with the neurotrophin (and to a lower extent with basic FGF and EGF) induces the appearance of enlargeosome vesicles competent for VAMP4-dependent exocytosis abundant in high REST-PC12 clones. Both the neurite length assay and the immunocytochemistry of enlargeosomes exocytosis revealed that activation of TrkA is induced not only by NGF, but also by the L1 adhesion protein, L1CAM, whose soluble construct binds the receptor with submicromolar affinity. In the intact wtPC12, the L1CAM construct induced autophosphorylation and internalization of TrkA followed by the activation of the PI3K, MEK, and PKCγ signaling cascades, analogous to the responses induced by NGF. Down-regulation of either VAMP7 or VAMP4 revealed the coparticipation of the two corresponding vesicles to the outgrowth responses induced by NGF and L1CAM. Finally, mixing experiments of wtPC12 cells rich in TrkA with high REST PC12 cells transfected with L1CAM documented the transactivation of the receptor by the adhesion protein surface-exposed in adjacent cells. In view of the known inhomogeneous surface distribution of both L1CAM and TrkA in various neural cells including neurons, their transcellular binding could be restricted to discrete sites, governing local signaling events distinct from those induced by soluble messengers.

Pharmacology of the cell/matrix form of adhesion.

Cell adhesions are heterogeneous processes including two main forms, CAM and cell/matrix forms. Both these forms induce the interaction among cells and with the extracellular matrix, and the generation of intracellular signals. The signaling of the two adhesion forms include, at the cell surface, involvement of distinct integrins, necessary for intracellular cascade activation. I will focus on the cell/integrin form based on two specific integrins, α5ß1 (the most important) and αvß3, activated by the preferential binding of fibronectin, a unique extracellular matrix protein. Such binding induces local assembly of stratified adhesion complexes containing protein kinases, that trigger the intracellular signaling cascades (Akt, ERK and others); proteins that sustain mechanical processes; and proteins associated with the cytoskeleton. In view of its role in several diseases, from cancers to the eye macular-degeneration; from brain neurodegeneration to fibroses, the pharmacological interest for the cell/integrin adhesion has grown, and presumably will further grow in the near future. The agents identified and developed for therapy include antibodies, many peptides and chemical drugs against α5ß1 integrin; drugs against fibronectin and metalloproteinases 2/9, responsible of the latter enzyme proteolysis; anti-kinase and anti-cascade drugs, some of which targeted to the activation of transcription factors and/or their transfer to the nucleus, with repression or activation of gene expression. A new perspective, based on the investigation of both animal models and human patients, includes factors active on the cell/matrix and CAM adhesions, considered separately or coordinately in distinct therapeutic approaches, integrated or not with classical chemotherapic treatments.

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

L1CAM and its cell-surface mutants: new mechanisms and effects relevant to the physiology and pathology of neural cells.

The L1 syndrome, a genetic disease that affects 1/30 000 newborn males, is sustained by numerous missense mutations of L1 cell adhesion molecule (L1CAM), an adhesion surface protein active also in transmembrane signaling, essential for the development and function of neurons. To investigate the cell biology of L1CAM, we employed a high RE1-silencing transcription (factor) clone of the pheochromocytoma PC12 line, defective in L1CAM expression and neurite outgrowth. The clone was transfected with wild-type L1CAM and four missense, disease-inducing point mutants encoding proteins distributed to the cell surface. The mutant-expressing cells, defective in adhesion to extracellular matrix proteins and in migration, exhibited unchanged proliferation. The nerve growth factor (NGF)-induced neurite outgrowth was re-established in defective clone cells transfected with the wild-type and the H210Q and I219T L1CAMs mutants, but not in the others. The stimulated outgrowth was confirmed in a second defective PC12 clone over-expressing the NGF receptor TrkA, treated with NGF and/or a recombinant L1CAM chimera. These results revealed a new function of L1CAM, a positive, robust and dose-dependent modulation of the TrkA receptor activated spontaneously or by NGF. The variable effects observed with the different L1CAM mutants suggest that this function contributes to the marked heterogeneity of symptoms and severity observed in the patients affected by the L1 syndrome.

A signaling loop of REST, TSC2 and ß-catenin governs proliferation and function of PC12 neural cells.

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 ß-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of ß-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-ß-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 ß-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-ß-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

A new form of neurite outgrowth sustained by the exocytosis of enlargeosomes expressed under the control of REST.

In neurons and neurosecretory (nerve) cells, neurite outgrowth requires surface enlargement sustained by exocytosis of specific but poorly characterized vesicles. A canonical, relatively slow form of outgrowth is known to require the v-SNARE Ti-VAMP. Recently, we have identified a new, rapid form, triggered by activation of Rac1 and sustained by the exocytosis of enlargeosomes (v-SNARE: VAMP4). By parallel study of various pheochromocytoma PC12 cell clones exhibiting either a single or both forms of outgrowth, we show that expression of enlargeosomes, their exocytosis at growth cones and their form of neurite outgrowth are positively governed by the RE-1 silencing transcription factor (REST), a repressor of many nerve cell-specific genes. Using a high REST/enlargeosome-rich PC12 clone transfected with TrkA, we found (i) that nerve growth factor (NGF) can increase the expression of both REST and the enlargeosome maker, Ahnak; and (ii) that outgrowth triggered by NGF, independent from the form triggered by Rac1 and supported mostly by exocytic, Ti-VAMP-positive organelles distinct from enlargeosomes, occurs at slow or fast rates depending on the strength of the TrkA signaling. These results confirm the duality of the outgrowth forms sustained by the two types of exocytic vesicles, reveal their distinct properties and identify new aspects of the REST impact in nerve cell specificity/function.

Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes.

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.

Dual REST-dependence of L1CAM: from gene expression to alternative splicing governed by Nova2 in neural cells.

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.