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. © 2011 Wiley Periodicals, Inc

Overall Lack of Regulated Secretion in a PC12 Variant Cell Clone

Abstract A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944-953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and α-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-β-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription-polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretory proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation

Nanotopography and microconfinement impact on primary hippocampal astrocyte morphology, cytoskeleton and spontaneous calcium wave signalling

Astrocytes' organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation

Micropatterning of substrates for the culture of cell networks by stencil-assisted additive nanofabrication

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown

Extracellular Vesicles of Mesenchymal Stem Cells: Therapeutic Properties Discovered with Extraordinary Success

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

The Dysfunctional Mechanisms Throwing Tics: Structural and Functional Changes in Tourette Syndrome

Tourette Syndrome (TS) is a high-incidence multifactorial neuropsychiatric disorder characterized by motor and vocal tics co-occurring with several diverse comorbidities, including obsessive-compulsive disorder and attention-deficit hyperactivity disorder. The origin of TS is multifactorial, with strong genetic, perinatal, and immunological influences. Although almost all neurotransmettitorial systems have been implicated in TS pathophysiology, a comprehensive neurophysiological model explaining the dynamics of expression and inhibition of tics is still lacking. The genesis and maintenance of motor and non-motor aspects of TS are thought to arise from functional and/or structural modifications of the basal ganglia and related circuitry. This complex wiring involves several cortical and subcortical structures whose concerted activity controls the selection of the most appropriate reflexive and habitual motor, cognitive and emotional actions. Importantly, striatal circuits exhibit bidirectional forms of synaptic plasticity that differ in many respects from hippocampal and neocortical plasticity, including sensitivity to metaplastic molecules such as dopamine. Here, we review the available evidence about structural and functional anomalies in neural circuits which have been found in TS patients. Finally, considering what is known in the field of striatal plasticity, we discuss the role of exuberant plasticity in TS, including the prospect of future pharmacological and neuromodulation avenues

Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca(2+) concentration ([Ca(2+)](i)) rises. Cell biology of enlargeosomes is still largely unknown. By combining immunocytochemistry (marker desmoyokin-Ahnak, d/A) to capacitance electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that 1) the two responses, cell surface enlargement and d/A surface appearance, occur with similar kinetics and in the same low micromolar [Ca(2+)](i) range, no matter whether induced by photolysis of the caged Ca(2+) compound o-nitrophenyl EGTA or by the Ca(2+) ionophore ionomycin. Thus, enlargeosomes seem to account, at least in large part, for the exocytic processes triggered by the two stimulations. 2. The enlargeosome membranes are resistant to nonionic detergents but distinct from other resistant membranes, rich in caveolin, Thy1, and/or flotillin1. 3. Cell cholesterol depletion, which affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated exocytosis. 4. The postexocytic cell surface decline is [Ca(2+)](i) dependent. 5. Exocytized d/A-rich membranes are endocytized and trafficked along an intracellular pathway by nonacidic organelles, distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes and suggest their participation, in addition to cell differentiation and repair, for which evidence already exists, to other physiological and pathological processes

Nanotopography and Microconfinement Impact on Primary Hippocampal Astrocyte Morphology, Cytoskeleton and Spontaneous Calcium Wave Signalling

Astrocytes’ organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation

Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown