Francesca Navone

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide‐containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal‐containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well‐characterized membrane protein of small vesicles in neurons and endocrine cells.

Synaptophysin immunoreactivity and small clear vesicles in neuroendocrine cells and related tumours

Synaptophysin (protein p38) immunoreactivity has been detected immunohistochemically in neuroendocrine cells of the human adrenal medulla, carotid body, skin, pituitary, thyroid, lung, pancreas and gastrointestinal mucosa as well as in 87 out of 93 neuroendocrine tumours investigated, including pheochromocytomas, chromaffin and non-chromaffin paragangliomas, ganglioneuromas, pituitary adenomas, thyroid medullary carcinomas, parathyroid adenomas, lung carcinoids and neuroendocrine carcinomas, pancreatic and gut endocrine tumours and cutaneous merkelomas. Parallel ultrastructural investigation of synaptophysin-reactive cells and tumours revealed the presence, in addition to dense-cored, secretory granules, of a population of pleomorphic, small, clear vesicles resembling synaptic vesicles of nerve terminals as well as the synaptophysin immunoreactive vesicles already described in rat adrenal medullary and pituitary cells. Synaptophysin immunoreactivity showed several differences in its distribution among tumour and non-tumour endocrine cells when compared to chromogranin A immunoreactivity, a well known marker of the core of endocrine granules. Synaptophysin represents a reliable general marker of neuroendocrine cells and tumours, which may be useful in diagnostic histopathology.

A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum

VAPB (vesicle‐associated membrane protein‐associated protein B) is an endoplasmic reticulum (ER)‐resident tail‐anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S‐VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell‐free systems and in cultured mammalian cells were used to investigate P56S‐VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post‐translationally into ER membranes in a manner indistinguishable from the wild‐type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild‐type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a ~30 nm‐thick electron‐dense cytosolic layer. Our results demonstrate that the ALS‐linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons.—Fasana, E., Fossati, M., Ruggiano, A., Brambillasca, S., Hoogenraad, C. C., Navone, F., Francolini, M., Borgese, N. A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum. FASEB J. 24, 1419–1430 (2010). www.fasebj.org

Regulated secretory pathways of neurons and their relation to the regulated secretory pathway of endocrine cells.

Neurons are highly specialized secretory cells that can secrete in a regulated way at least two types of neurotransmitter substances: small nonpeptide molecules and peptides.’** Evidence is accumulating that the release of these two classes of substances involves two types of secretory vesicles. One type, the small synaptic vesicle (SSV), is the “typical” synaptic vesicle, ie., a small (40-60 nm in diameter) vesicle which, after standard fixation conditions, exhibits by electron microscopy a clear core. The other type, the large dense-core vesicle (LDCV), is a larger vesicle (diameter greater than 60 nm and variable from neuron to neuron) that has an electron-dense core.”‘-’ Small synaptic vesicles are thought to contain classical neurotransmitters only, whereas increasing evidence indicates that peptide neurotransmitters are stored in LDCVs.2.’ Large dense-core vesicles may also contain nonpeptide molecules such as amines? The segregation of different types of neurotransmitters in two classes of vesicles is related to the different mechanisms involved in their biosynthesis. All the machinery necessary to synthesize and load classical neurotransmitters into vesicles is present in nerve terminals, where SSVs undergo a continued local exo-endocytotic recycling. Thus, at each of these cycles, SSVs can be refilled with classical neurotransmitter^?^ In contrast, peptides can be synthetized, loaded, and concentrated into granules only in perikarya, which are therefore the only sites at which LDCVs can be assembled?*6 Recently we have found that the synaptic-vesicle-associated protein synapsin I, a protein thought to play an important regulatory role in the control of neurotransmitter release,’ is selectively associated with the surface of SSVs.8 These findings have indicated that, even though some features may be common to regulated exocytosis of all secretory organelles of all cell types:.9 neuronal secretion via SSVs might be characterized by some special type of regulation. In fact, evidence is accumulating

Restructured endoplasmic reticulum generated by mutant amyotrophic lateral sclerosis-linked VAPB is cleared by the proteasome.

Summary VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56S-VAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein.

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

Abstract: The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only ∼2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron‐specific kinesin is up‐regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule‐based transport during the maturation of neuronal cells in vivo.

Expression of KIF3C kinesin during neural development and in vitro neuronal differentiation

KIF3A, KIF3B and KIF3C are kinesin‐related motor subunits of the KIF3 family that associate to form the kinesin‐II motor complex in which KIF3C and KIF3B are alternative partners of KIF3A. We have analysed the expression of Kif3 mRNAs during prenatal murine development. Kif3c transcripts are detectable from embryonic day 12.5 and persist throughout development both in the CNS and in some peripheral ganglia. Comparison of the expression patterns of the Kif3 genes revealed that Kif3c and Kif3a mRNAs colocalize in the CNS, while only Kif3a is also present outside the CNS. In contrast, Kif3b is detectable in several non‐neural tissues. We have also performed immunocytochemical analyses of the developing rat brain and have found the presence of the KIF3C protein in selected brain regions and in several fibre systems. Using neuroblastoma cells as an in vitro model for neuronal differentiation, we found that retinoic acid stimulated the expression of the three Kif3 and the kinesin‐associated protein genes, although with different time courses. The selective expression of Kif3c in the nervous system during embryonic development and its up‐regulation during neuroblastoma differentiation suggest a role for this motor during maturation of neuronal cells.

Amyotrophic lateral sclerosis-linked mutant VAPB inclusions do not interfere with protein degradation pathways or intracellular transport in a cultured cell model.

VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3δ, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3δ or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.

Immunocytochemistry as a tool in the study of neurotransmitter actions

Abstract Protein phosphorylation mediated by second messengers appears to be an important general mechanism by which neurotransmitters accomplish some of their effects on target neurons. In recent years several protein kinases and a variety of phosphoproteins have been identified in mammalian brain. However, the physiological function of the large majority of protein phosphorylation reactions occurring in nerve cells is still poorly understood. This review summarizes how immunocytochemistry can be important in relating biochemical information to specific cellular functions.