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We previously reported that a-disintegrin and metalloproteinase (ADAM)17 is a key protease regulating myelin formation. We now describe a role for ADAM17 during the Wallerian degeneration (WD) process. Unexpectedly, we observed that glial ADAM17, by regulating p75NTRprocessing, cell autonomously promotes remyelination, while neuronal ADAM17 is dispensable. Accordingly, p75NTRabnormally accumulates specifically when ADAM17 is maximally expressed leading to a downregulation of tissue plasminogen activator (tPA) expression, excessive fibrin accumulation over time, and delayed remyelination. Mutant mice also present impaired macrophage recruitment and defective nerve conduction velocity (NCV). Thus, ADAM17 expressed in Schwann cells, controls the whole WD process, and its absence hampers effective nerve repair. Collectively, we describe a previously uncharacterized role for glial ADAM17 during nerve regeneration. Based on the results of our study, we posit that, unlike development, glial ADAM17 promotes remyelination through the regulation of p75NTR-mediated fibrinolysis.SIGNIFICANCE STATEMENTThe α-secretase a-disintegrin and metalloproteinase (ADAM)17, although relevant for developmental PNS myelination, has never been investigated in Wallerian degeneration (WD). We now unravel a new mechanism of action for this protease and show that ADAM17 cleaves p75NTR, regulates fibrin clearance, and eventually fine-tunes remyelination. The results presented in this study provide important insights into the complex regulation of remyelination following nerve injury, identifying in ADAM17 and p75NTRa new signaling axis implicated in these events. Modulation of this pathway could have important implications in promoting nerve remyelination, an often-inefficient process, with the aim of restoring a functional axo-glial unit.

Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases.

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.

The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a and Otx2); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development.

Background The increasing use of gold nanoparticles (AuNPs) in the field of neuroscience instilled hope for their rapid translation to the clinical practice. AuNPs can be engineered to carry therapeutics or diagnostics in the diseased brain, possibly providing greater cell specificity and low toxicity. Although there is a general enthusiasm for these tools, we are in early stages of their development. Overall, their brain penetrance, stability and cell specificity are critical issues that must be addressed to drive AuNPs to the clinic. Results We studied the kinetic, distribution and stability of PEG-coated AuNPs in mice receiving a single injection into the cisterna magna of the 4th ventricle. AuNPs were conjugated with the fluorescent tag Cy5.5 (Cy5.5-AuNPs) to track their in vivo distribution. Fluorescence levels from such particles were detected in mice for weeks. In situ analysis of brains by immunofluorescence and electron microscopy revealed that Cy5.5-AuNPs penetrated the brain parenchyma, spreading in the CNS parenchyma beneath the 4th ventricle. Cy5.5-AuNPs were preferentially found in neurons, although a subset of resting microglia also entrapped these particles. Conclusions Our results suggest that the ICM route for delivering gold particles allows the targeting of neurons. This approach might be pursued to carry therapeutics or diagnostics inside a diseased brain with a surgical procedure that is largely used in gene therapy approaches. Furthermore, this approach could be used for radiotherapy, enhancing the agent’s efficacy to kill brain cancer cells. Electronic supplementary material The online version of this article (10.1186/s12951-019-0481-3) contains supplementary material, which is available to authorized users.

Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell-extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α-secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effect on myelin formation. Collectively, our data indicate that the neuronal α-secretase ADAM17 is required for proper CNS myelination. Further, our studies confirm that secretases are important post-translational regulators of myelination although the mechanisms controlling CNS and peripheral nervous system (PNS) myelination are distinct.

Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous “eat me” signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases. Extracellular vesicles (EVs) are a physiological way of intercellular communication. We show here that it is possible to deliver EVs engineered to contain different molecules for therapeutic purposes. Using this strategy, we have successfully delivered an anti-inflammatory cytokine to treat a mouse model of multiple sclerosis.

Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2β1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin., Author summary Myelin is formed by the wrapping of glial cell membranes around axons and is required for the fast conduction of nerve impulses and to support axons. In the peripheral nervous system, myelin is produced by Schwann cells. Peripheral myelin defects cause debilitating diseases, whose molecular pathogeneses are only partially understood. Here, we reveal for the first time how 2 crucial extracellular modulators of myelin formation, neuregulin 1 type III (Nrg1III) and laminin α2β1γ1 (Lm211), work together in the peripheral nervous system. Although Lm211 was believed to promote myelination, we show that it can also inhibit myelin formation by suppressing the activity of Nrg1III, limiting the activation of its downstream signaling cascade. These results help to explain why certain inherited neuropathies are characterized by hypermyelination and redundant myelin sheaths.

Growing evidence indicates that alterations within the peripheral nervous system (PNS) are involved at an early stage in the amyotrophic lateral sclerosis (ALS) pathogenetic cascade. In this study, magnetic resonance imaging (MRI), neurophysiologic analyses, and histologic analyses were used to monitor the extent of PNS damage in the hSOD-1 ALS rat model. The imaging signature of the disease was defined using in vivo MRI of the sciatic nerve. Initial abnormalities were detected in the nerves by an increase in T2 relaxation time before the onset of clinical disease; diffusion MRI showed a progressive increase in mean and radial diffusivity and reduction of fractional anisotropy at advanced stages of disease. Histologic analysis demonstrated early impairment of the blood-nerve barrier followed by acute axonal degeneration associated with endoneurial edema and macrophage response in motor nerve compartments. Progressive axonal degeneration and motor nerve fiber loss correlated with MRI and neurophysiologic changes. These functional and morphologic investigations of the PNS might be applied in following disease progression in preclinical therapeutic studies. This study establishes the PNS signature in this rat ALS model (shedding new light into pathogenesis) and provides a rationale for translating into future systematic MRI studies of PNS involvement in patients with ALS.

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 study investigates if alpha-lipoic acid is neuroprotective against chemotherapy induced neurotoxicity, if mitochondrial damage plays a critical role in toxic neurodegenerative cascade, and if neuroprotective effects of alpha-lipoic acid depend on mitochondria protection. We used an in vitro model of chemotherapy induced peripheral neuropathy that closely mimic the in vivo condition by exposing primary cultures of dorsal root ganglion (DRG) sensory neurons to paclitaxel and cisplatin, two widely used and highly effective chemotherapeutic drugs. This approach allowed investigating the efficacy of alpha-lipoic acid in preventing axonal damage and apoptosis and the function and ultrastructural morphology of mitochondria after exposure to toxic agents and alpha-lipoic acid. Our results demonstrate that both cisplatin and paclitaxel cause early mitochondrial impairment with loss of membrane potential and induction of autophagic vacuoles in neurons. Alpha-lipoic acid exerts neuroprotective effects against chemotherapy induced neurotoxicity in sensory neurons: it rescues the mitochondrial toxicity and induces the expression of frataxin, an essential mitochondrial protein with anti-oxidant and chaperone properties. In conclusion mitochondrial toxicity is an early common event both in paclitaxel and cisplatin induced neurotoxicity. Alpha-lipoic acid protects sensory neurons through its anti-oxidant and mitochondrial regulatory functions, possibly inducing the expression of frataxin. These findings suggest that alpha-lipoic acid might reduce the risk of developing peripheral nerve toxicity in patients undergoing chemotherapy and encourage further confirmatory clinical trials.

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.

Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca2+concentration ([Ca2+]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 [Ca2+]irange, no matter whether induced by photolysis of the caged Ca2+compound o-nitrophenyl EGTA or by the Ca2+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 [Ca2+]idependent. 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.

Together with its adaptor protein, the adaptor protein of 12 kDa also known as KARAP and TYROBP (DAP12), triggering receptor expressed in myeloid cells 2 (TREM2) is a stimulatory membrane receptor of the immunoglobulin/lectin-like superfamily, well known in myeloid cells. In humans, however, loss-of-function mutations of TREM2/DAP12 leave myeloid cells unaffected but induce an autosomal recessive disease characterized, together with bone cysts, by a spectrum of pathological lesions in the cortex, thalamus and basal ganglia with clinical symptoms of progressive dementia (polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy). Nothing was known about the role of TREM2/DAP12 in brain cell biology and physiology. By confocal immunocytochemistry we demonstrate that, in both human and mouse cerebral cortex, TREM2/DAP12, strongly expressed by microglia, is also present in a fraction of neurons but not in astrocytes and oligodendrocytes. In contrast, in the hippocampal cortex TREM2-expressing neurons are rare. Both in neurons and microglia the receptor appears to be located mostly intracellularly in a discrete compartment(s) partially coinciding with (or adjacent to) the Golgi complex/trans-Golgi network. Four nerve cell lines were identified as expressing the intracellular receptor system. In living human microglia CHME-5 and glioblastoma T98G cells, activation of TREM2 by its specific antibody induced [Ca2+]i responses, documenting its surface expression and functioning. Surface expression of TREM2, low in resting CHME-5 and T98G cells, increases significantly and transiently (60 min) when cells are stimulated by ionomycin, as revealed by both surface biotinylation and surface immunolabeling, Our results provide the first information about the expression, distribution (mostly intracellular) and functioning of TREM2/DAP12 system in nerve cells, a necessary step in the understanding of the cellular mechanisms affected in polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

Electrophysiological studies in some secretory and non-secretory cells have identified an extensive form of calcium-induced exocytosis that is rapid (hundreds of milliseconds), insensitive to tetanus toxin and distinct from regulated secretion. We have now identified a marker of the process, desmoyokin-AHNAK, in a clonal derivative of the neuronal cell line, PC12. In resting cells, desmoyokin-AHNAK is localized within the lumen of specific vesicles, but appears on the cell surface during stimulation. Desmoyokin-AHNAK-positive vesicles exist in a variety of cells and tissues and are distinct from the endoplasmic reticulum, Golgi, trans-Golgi, endosomes and lysosomes, and from Glut4 and constitutive secretion vesicles. They seem to be involved in two models of plasmalemma enlargement: differentiation and membrane repair. We therefore propose that these vesicles should be called 'enlargosomes'.

Neurosecretion competence is intended as the ability of neurosecretory cells to express dense and clear vesicles discharged by regulated exocytosis (neurotransmitter release). Such a property, which so far has never been studied independently, is investigated here by a heterotypic cell fusion approach, using a clone of rat pheochromocytoma PC12 cells totally incompetent for neurosecretion that still largely maintains its typical molecular and cellular phenotype. When fused with wild-type partners of various species (rat, human) and specialization (PC12, neuroblastoma SH-SY5Y, HeLa), the defective cells reacquire their competence as revealed by the expression of their secretion-specific proteins. Fused wild-type cells therefore appear able to complement defective cells by providing them with factor(s) inducing the reactivation of their secretory program. The mechanism of action of these factors may consist not in a coordinate unblocking of transcription but in the prevention of a rapid post-transcriptional degradation of the mRNAs for secretion-specific genes.

The REST/NRSF transcriptional repressor prevents cultured astrocytes from forming DCVs, and its variable expression in human brain cortex astrocytes may account for their functional heterogeneity., Astrocytes are the brain nonnerve cells that are competent for gliosecretion, i.e., for expression and regulated exocytosis of clear and dense-core vesicles (DCVs). We investigated whether expression of astrocyte DCVs is governed by RE-1–silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), the transcription repressor that orchestrates nerve cell differentiation. Rat astrocyte cultures exhibited high levels of REST and expressed neither DCVs nor their markers (granins, peptides, and membrane proteins). Transfection of a dominant-negative construct of REST induced the appearance of DCVs filled with secretogranin 2 and neuropeptide Y (NPY) and distinct from other organelles. Total internal reflection fluorescence analysis revealed NPY–monomeric red fluorescent protein–labeled DCVs to undergo Ca2+-dependent exocytosis, which was largely prevented by botulinum toxin B. In the I–II layers of the human temporal brain cortex, all neurons and microglia exhibited the expected inappreciable and high levels of REST, respectively. In contrast, astrocyte REST was variable, going from inappreciable to high, and accompanied by a variable expression of DCVs. In conclusion, astrocyte DCV expression and gliosecretion are governed by REST. The variable in situ REST levels may contribute to the well-known structural/functional heterogeneity of astrocytes.

Taking advantage of a fluorescent Ca 2+ indicator selectively targeted to the trans -Golgi lumen, we here demonstrate that its Ca 2+ homeostatic mechanisms are distinct from those of the other Golgi subcompartments: ( i ) Ca 2+ uptake depends exclusively on the activity of the secretory pathway Ca 2+ ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA) is excluded; ( ii ) IP 3 generated by receptor stimulation causes Ca 2+ uptake rather than release; ( iii ) Ca 2+ release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans -Golgi Ca 2+ content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.

Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to probe the endoplasmic reticulum in the cell body and dendrites of rat cerebellar Purkinje neurons. Two markers, previously investigated in non-nerve cells, the membrane protein p91 (calnexin) and the lumenal protein BiP, were found to be highly expressed and widely distributed to the various endoplasmic reticulum sections of Purkinje neurons, from the cell body to dendrites and dendritic spines. An antibody (denominated anti-rough-surfaced endoplasmic reticulum), which recognized two membrane proteins, p14 and p40, revealed a similar immunogold labeling pattern. However, centrifugation results consistent with a widespread distribution were obtained for p14 only, while p40 was concentrated in the rough microsome-enriched subfractions. The areas enriched in the inositol 1,4,5-triphosphate receptor and thus presumably specialized in Ca2+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and p91, and were positive for the anti-rough-surfaced endoplasmic reticulum antibody (presumably via the p14 antigen). Additional antibodies, that yielded inadequate immunocytochemical signals, were employed only by Western blotting of the microsomal subfractions, while the ryanodine receptor was studied by specific binding. The latter receptor and the Ca2+ ATPase, known in other species to be concentrated in Purkinje neurons, exhibited bimodal distributions with a peak in the light and another in the heavy subfractions. A similar distribution was also observed with another lumenal protein, protein disulfide isomerase. Taken as a whole, the results that we have obtained suggest the existence in the endoplasmic reticulum of Purkinje neurons of two levels of organization; the first identified by widespread, probably general markers (BiP, p91, possibly p14 and others), the second by specialization markers, such as the inositol 1,4,5-triphosphate receptor and, possibly, p40, which appear restricted to areas where specific functions appear to be localized.

Various aspects of the rapidly exchanging intracellular Ca2+ stores of neurons and nerve cells are reviewed: their multiplicity, with separate sensitivity to either the second messenger, inositol 1,4,5-trisphosphate, or ryanodine-caffeine (the latter stores are probably activated via Ca(2+)-induced Ca2+ release); their control of the plasma membrane Ca2+ permeability, via the activation of a peculiar type of cation channels; their ability to sustain localized heterogeneities of the [Ca2+]i that could be of physiological key-importance. Finally, the molecular composition of these stores is discussed. They are shown (by high resolution immunocytochemistry and subcellular fractionation) to express: i) a Ca2+ ATPase responsible for the accumulation of the cation; ii) Ca2+ binding protein(s) of low affinity and high capacity to keep Ca2+ stored; and iii) a Ca2+ channel, activated by either one of the mechanisms mentioned above, to release Ca2+ to the cytosol. Results obtained in Purkinje neurons document the heterogeneity of the stores and the strategical distribution of the corresponding organelles (calciosomes; specialized portions of the ER) within the cell body, dendrites and dendritic spines.

The neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the dominant-negative construct induced the rescue of many vesicle/exocytosis genes but not of those of chromogranins. Small dense-core vesicles, exocytized upon stimulation, were rescued when the construct-transfected defective cells were transfected also with chromograninA or treated with trichostatinA, a blocker of histone deacetylases. Our results identify REST, working by direct and indirect mechanisms, as the factor governing the maintenance of the neurosecretory process and the properties of dense-core vesicles in PC12 cells.

Enlargeosomes are cytoplasmic organelles discharged by regulated exocytosis, identified by immunofluorescence of their membrane marker, desmoyokin/Ahnak, but never revealed at the ultrastructural level. Among the numerous enlargeosome-positive cells, the richest and most extensively characterized are those of a PC12 clone, PC12-27, defective of classical neurosecretion. By using ultrastructural immunoperoxidase labeling of formaldehyde-fixed, Triton-X-100-permeabilized PC12-27 cells, we have now identified the enlargeosomes as small vesicles scattered in the proximity of, but never docked to, the plasma membrane. Upon stimulation, these vesicles undergo exocytosis [rapid after the Ca(2+) ionophore, ionomycin, much slower after either the phorbol ester, phorbol myristate acetate (PMA), or ATP, working through a P2Y receptor], with appearance in the plasma membrane of typical desmoyokin/Ahnak (d/A)-positive, Omega-shaped and open profiles evolving into flat patches. Postexocytic removal of the exocytized d/A-positive membrane occurs by two processes: generation of endocytic vesicles, predominant after ionomycin and ATP 100-500 microM; and shedding of membrane-bound cytoplasmic bodies, predominant after PMA and 1 mM ATP, containing little or no trace of endoplasmic reticulum, Golgi, endo/lysosomes and also of a plasma membrane marker. Depending on the stimulation, therefore, the cell-surface expansion by enlargeosome exocytosis is not always recycled but can induce release of specific membranes, possibly important in the pericellular environment.

The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the variations organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

Two cerebral cortex areas (frontal and occipital) and the retina of rats varying in age from 0.4 to 30 months were investigated for the expression levels and distribution of two cytosolic high affinity Ca2+ binding proteins, calbindin-28 and calretinin, and of two presynaptic protein markers. Of these latter proteins, one is integral (synaptophysin) the other peripheral (synapsin I) to the synaptic vesicle membranes. In the cortex areas, no significant changes of the markers were observed, except for a drop of calretinin from 0.4 to 2 months, probably related to a stage of neuronal development. In the retina, calbindin-28 decreased progressively during ageing (−40% at 30 months) while calretinin remained unchanged. Concomitantly, the two synaptic vesicle proteins dropped, synaptophysin >50% and synapsin I >85%. The role of these changes in sustaining the functional alterations previously described in the retina of aged animals remains to be investigated.

The expression of two cytosolic, high affinity Ca(2+)-binding proteins, calbindin-28 and calretinin, has been investigated in the cerebellum and hippocampus of young and old rats (from 12 days to 30 months) by combining immunofluorescence and Western blotting. Three markers, calreticulin (the major Ca2+ binding protein within the lumen of the endoplasmic reticulum), MAP-2 (a microtubule binding protein concentrated in neuronal dendrites) and synaptophysin (an integral protein of synaptic vesicles), were studied in parallel. In the cerebellar cortex a rise from 12 to 60 days was observed with calbindin-28 and, especially, calretinin, concentrated in the Purkinje and granule neurons, respectively. The level of expression of the two proteins subsequently remained high and the distribution was unchanged, even in the cerebellum of old animals. A completely different pattern was observed in the hippocampus. Here calretinin, present especially in fibres and interneurons, was abundant in the young, decreased in the adult and reached low values in the old rats. Calbindin-28 accumulated during growth, especially in a subpopulation of CA1 pyramidal cells and in the mossy fibres of CA3, then declined, although irregularly, during ageing. These changes of the two proteins were more marked in the dorsal and central parts than in the ventral part of the hippocampus. In the same brain areas the levels of expression of the three additional markers and their distribution within neurons and synapses were unchanged by ageing.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryosection immunofluorescence and immunogold labeling with antibodies against specific markers were used in rat vas deferens smooth muscle fibers to reveal the molecular arrangement of the endomembrane system (referred to variously in the text as ER or sarcoplasmic reticulum [SR]; S-ER or ER/SR) known to participate in the control of Ca2+ homeostasis. The lumenal ER chaperon, immunoglobulin binding protein (BiP), as well as protein disulfide isomerase, and calreticulin, a Ca2+ binding protein expressed by most eukaryotic cells, appeared to be evenly distributed throughout the entire system (i.e., within [a] the nuclear envelope and the few rough-surfaced cisternae clustered near the nucleus; [b] single elements scattered around in the contractile cytoplasm; and [c] numerous, heterogeneous, mainly smooth-surfaced elements concentrated in the peripheral cytoplasm, part of which is in close apposition to the plasmalemma). All other structures, including nuclei, mitochondria, Golgi complex, and surface caveolae were unlabeled. An even distribution throughout the endomembrane system appeared also for the proteins recognized by anti-ER membrane antibodies. In contrast, calsequestrin (the protein that in striated muscles is believed to be the main actor of the rapidly exchanging Ca2+ storage within the lumen of the sarcoplasmic reticulum) was found preferentially clustered at discrete lumenal sites, most often within peripheral smooth-surfaced elements of moderate electron density. Within these elements dual labeling revealed intermixing of calsequestrin with the other lumenal ER proteins. Moreover, the calsequestrin-rich elements were enriched also in the receptor for inositol 1,4,5-trisphosphate, the second messenger that induces Ca2+ release from intracellular stores. These results document the previously hypothesized molecular heterogeneity of the smooth muscle endomembrane system, particularly in relation to the rapid storage and release of Ca2+.

Rat cerebellum microsomes were subfractionated on isopycnic linear sucrose (20-42%)-density gradients. The distribution of endoplasmic reticulum (ER) markers (RNA, signal-sequence receptor alpha, calnexin, calreticulin, the immunoglobulin-binding protein Bip) and markers of intracellular rapidly exchanging Ca2+ stores [Ca2+ channels sensitive to either Ins(1,4,5)P3 or ryanodine) was investigated biochemically and immunologically. The comparison indicates that: (a) vesicles bearing the InsP3 receptor were separated from those bearing the ryanodine receptor; (b) ER markers, i.e. Bip, calnexin, signal-sequence receptor alpha, RNA, did not sediment as either InsP3 or ryanodine receptors did; (c) calreticulin, an intralumenal low-affinity high-capacity Ca(2+)-binding protein, had a widespread distribution, similar to that of Bip and calnexin, and was present in Purkinje, granule, Golgi and stellate neurons, as indicated by immunofluorescent labelling of cerebellum cortex cryosections. The present results show that the ER is not a homogeneous entity, and that Ca2+ stores are heterogeneous insofar as InsP3 receptors and ryanodine receptors are segregated, either to discrete intracellular organelles or to specialized ER subcompartments.

Stacks of regularly spaced, flat, smooth-surfaced endoplasmic reticulum cisternae frequently observed in both the cell body and dendrites of cerebellar Purkinje neurons, were previously shown by immunocytochemistry to be highly enriched in receptors for the second messenger, inositol 1,4,5-trisphosphate. Morphometric analyses have been carried out on randomly selected thin section images of rat Purkinje neurons to reveal the tridimensional organization of these structures. Individual stacked cisternae (on the average approximately 3.5 per stack) were shown to be separated from each other by a 23.5 nm space occupied by perpendicular bridges, approximately 20 nm in diameter, most probably composed by two apposed receptor homotetramer molecules, inserted into the parallel membranes in their hydrophobic domains. In the stacked membranes the density of the bridges was approximately 500 microns -2, corresponding to approximately 15% of the surface area. The lateral distribution of bridges was not random, but revealed regular distances that might correspond to unoccupied receptor slots. In each stack, the external cisternae were often in direct lumenal continuity with conventional elements of the endoplasmic reticulum, whereas the internal cisternae were not. Since continuities between stacked cisternae were never observed, the results indicate that the internal cisternae are at least transitorily discrete, i.e. they are not in permanent lumenal continuity with the rest of the endoplasmic reticulum. To our knowledge this is the first demonstration of a physical subcompartmentalization of the latter endomembrane system in a non-mitotic cells. A model for the biogenesis of cisternal stacks, based on the head-to-head binding and lateral interaction of the inositol 1,4,5-trisphosphate receptor molecules in the plane of the interacting membranes, is proposed and critically discussed.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.

To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.

The microsomal fraction of chicken cerebellum expresses a large amount of Ca(2+)-ATPase (105 kDa), which is phosphorylated by ATP in the presence of Ca2+. The Ca(2+)-ATPase activity is highly sensitive to temperature and to the presence of detergents. This ATPase has kinetic properties similar to those of chicken skeletal-muscle sarcoplasmic reticulum, as (i) it is activated by low (microM) and inhibited by high (mM) Ca2+ concentrations, (ii) it shows biphasic activation with ATP and (iii) it is inhibited by vanadate. However, the vanadate-sensitivity is at least 10 times greater than that observed in chicken skeletal or cardiac sarcoplasmic-reticulum Ca(2+)-ATPases. Thus, despite cross-reacting with antibodies against the cardiac and skeletal isoforms, the cerebellar microsomal Ca(2+)-ATPase appears to be distinct from both muscle enzymes. The Ca(2+)-ATPase is concentrated in, but not exclusive to, Purkinje neurons. In Purkinje neurons the Ca(2+)-ATPase appears to be expressed throughout the cell body, the dendritic tree (and the spines) and the axons. At the electron-microscope level the Ca(2+)-ATPase is found in smooth and rough endoplasmic-reticulum cisternae as well as in other, yet unidentified, smooth-surfaced structures.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.