Gunilla Jacobsson

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle‐associated protein components characterized by an N‐terminal J‐domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT–PCR analysis demonstrated CSP mRNA expression in insulin‐producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin‐secreting cell lines. Punctate CSP‐like immunoreactivity (CSP‐LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP‐LI in close association with membranes of secretory granules of cells in the endo‐ and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS‐1 cells or intracellular administration of CSP antibodies into mouse ob/ob β‐cells did not affect voltage‐dependent Ca2+‐channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS‐1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage‐gated Ca2+‐channels.

Glutamate transporter mRNA and glutamate-like immunoreactivity in spinal motoneurones

Glutamate is the major excitatory neurotransmitter in the central nervous system. The release of glutamate is terminated by rapid uptake of glutamate into the presynaptic nerve terminals and into surrounding glial cells. Recently, a neuronal glutamate transporter was cloned from rabbit small intestine, thereby providing the possibility to study the distribution of cells that express glutamate transporter mRNA. Using oligonucleotide probes and in situ hybridization, glutamate transporter mRNA was demonstrated in large cell bodies, presumably motoneurones, in the thoracic spinal cord of the rabbit. Immunohistochemical analysis with rabbit polyclonal antibodies to glutamate showed immunoreactivity in the cytoplasm of large cell bodies in the ventral horn, presumably motoneurones, of the rat spinal cord. Glutamate-LI was in addition demonstrated in the motor end plate in hindlimb muscle of the rat, as visualized by double-labelling with mouse monoclonal antibodies to synaptophysin. Taken together, these data raise the possibility that glutamate has a function at the vertebrate neuromuscular junction.

Exocytotic proteins in enterochromaffin-like (ECL) cells of the rat stomach.

Abstract. Proteins participating in vesicular docking and fusion have been identified in the nervous system. Such proteins appear to be important for the molecular regulation of exocytosis also in non-neuronal cells. The enterochromaffin-like (ECL) cells of the gastric acid-secreting (oxyntic) mucosa secrete histamine and chromogranin A-derived peptides, such as pancreastatin. Using immunohistochemistry, we have examined whether the ECL cells of the rat stomach, identified with antibodies to histidine decarboxylase (HDC, the histamine-forming enzyme), express the same exocytotic proteins as neurons. The ECL cells displayed immunoreactivity for synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2 (VAMP-2), cysteine string protein (CSP), vesicular monoamine transporter-2 (VMAT-2), synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin, and Munc-18, but not for synaptotagmin I/II and VAMP-1. Synaptophysin and VMAT-2 could be detected not only in the ECL cells, but also in a population of HDC-negative cells. The demonstration of synaptotagmin III in only a limited number of ECL cells suggests the existence of a subpopulation of ECL cells. The results show that several exocytotic proteins, previously identified in neurons, are present in rat stomach ECL cells. Hence, proteins engaged in vesicular docking and in the fusion of granule/vesicle membrane with plasma membrane seem to exist in both neurons and endocrine cells.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

The thiol antioxidant N‐acetyl‐ l‐cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL‐2 and up‐regulation of the IL‐2 receptor. The 120‐kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen‐specific T cell clones (TCC). TCC were stimulated with anti‐CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down‐regulation of IL‐4, IL‐5 and IFN‐γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL‐4. Furthermore, they down‐regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 m m NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down‐regulation of IL‐4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL‐4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases.

VAMP‐1 and VAMP‐2 gene expression in rat spinal motoneurones: differential regulation after neuronal injury

Vesicle‐associated membrane protein (VAMP; synaptobrevin) is involved in the molecular regulation of transmitter release at the presynaptic plasma membrane. VAMP exists in two isoforms, VAMP‐1 and VAMP‐2, which are transcribed from two separate genes and differentially expressed in the nervous system. In situ hybridization was used to examine whether VAMP isoform mRNA expression may be altered by experimental manipulations. The effect of nerve injury on VAMP‐1 and VAMP‐2 mRNA levels in motoneurones of the rat lumbar spinal cord was compared with lesion‐induced changes in the expression of choline acetyl transferase (ChAT) and α‐calcitonin gene‐related peptide (α‐CGRP) mRNA. After unilateral sciatic nerve transection (axotomy), VAMP‐1 mRNA expression decreased significantly in parallel with a downregulation of ChAT mRNA in axotomized motoneurones compared with the corresponding motoneurones on the contralateral unlesioned side. There was a rapid decrease in VAMP‐1 and ChAT mRNA levels at 2 days after axotomy, and at 7 days there was a 65% decrease in VAMP‐1 mRNA and a 48% decrease in ChAT mRNA. VAMP‐1 mRNA levels continued to decrease at 14 and 21 days, while ChAT mRNA levels had returned to normal at this time. In contrast, VAMP‐2 and α‐CGRP mRNA levels were upregulated in axotomized motoneurones. A significant increase for both VAMP‐2 and α‐CGRP mRNA levels was present 2 days after axotomy, and a maximum was reached after 7 days for α‐CGRP mRNA (163%) and after 14 days for VAMP‐2 mRNA (587%). Immunohistochemical analysis did not reveal any detectable changes in VAMP‐1‐ or VAMP‐2‐like immunoreactivity in the motoneurone cell soma after axotomy. In the proximal end of the transected sciatic nerve, there was an increase in VAMP‐1‐ and VAMP‐2‐LI, which was most prominent at 2 days after lesion. The results show that, in axotomized spinal motoneurones, VAMP‐1 mRNA is downregulated and VAMP‐2 mRNA is upregulated, indicating differential regulation of the two separate VAMP genes and differential roles for the two VAMP isoforms in the regulation of exocytosis after nerve injury.

Differential subcellular localization of SNAP-25a and SNAP-25b RNA transcripts in spinal motoneurons and plasticity in expression after nerve injury

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in the molecular regulation of neurotransmitter release. SNAP-25 exists in two isoforms, which arise from alternative splicing of exon 5. In situ hybridization was used to examine whether SNAP-25 isoform mRNA expression may be altered by experimental manipulations. The effect of unilateral nerve injury on SNAP-25 mRNA levels was studied in motoneurons of the rat lumbar spinal cord. In all animals, SNAP-25a RNA transcripts were demonstrated in the nucleus of motoneurons, whereas SNAP-25b mRNA was present mainly in the cytoplasm. Cloning of the rat Snap gene intron spacing the alternative exon 5a and 5b sequences and generation of an intron-specific oligonucleotide probe used for in situ hybridization did not point to the presence of unspliced variants of SNAP-25b mRNA. After unilateral sciatic nerve transection (axotomy), SNAP-25a and SNAP-25b expression decreased in axotomized motoneurons compared with corresponding motoneurons on the unlesioned side. A significant decrease was demonstrated 2 days after axotomy, which reached a maximum after 7 days (62% for SNAP-25a and 67% for SNAP-25b), while levels had slightly recovered by 14 and 28 days. Ventral root avulsion also induced a decrease in levels of SNAP-25 RNA transcripts, suggesting that the axonal injury in itself was responsible for the down-regulation of Snap gene expression. This study shows that, in spinal motoneurons, SNAP-25a and SNAP-25b RNA transcripts have different subcellular localization and that levels of SNAP-25 RNA transcripts are down-regulated after axonal injury.

Estrogen Down-Regulates mRNA Encoding the Exocytotic Protein SNAP-25 in the Rat Pituitary Gland

Exocytosis is dependent on specific proteins that are located at the secretory granule membrane, in the cytoplasm or at the plasma membrane. The mRNA expression of synaptosomal‐associated protein of 25 kDa (SNAP‐25) isoforms SNAP‐25a and SNAP‐25b, vesicle associated membrane protein (VAMP) 2, mammalian homologue of unc‐18 (munc‐18) and Hrs‐2 was studied in the pituitary of ovariectomized rats after subcutaneous insertion of capsules containing estrogen or placebo using in situ hybridization. Estrogen treatment (0.25 mg estradiol) significantly decreased SNAP‐25a (32%; 10%) and SNAP‐25b (25%; 22%) mRNA levels in the anterior and intermediate lobes, respectively, whereas VAMP‐2, munc‐18 and Hrs‐2 mRNA levels remained unchanged. The results suggest that estrogen selectively regulates SNAP‐25 transcription in the pituitary gland, but leaves VAMP‐2, munc‐18 and Hrs‐2 mRNA levels unaffected.

Differential expression of SNAP-25a and SNAP-25b RNA transcripts in cranial nerve nuclei

Synaptosomal‐associated protein of 25 kDa (SNAP‐25) is involved in the molecular regulation of neurotransmitter release. SNAP‐25 exists in two isoforms, SNAP‐25a and SNAP‐25b, which arise from alternate splicing and which are differentially expressed throughout the nervous system. In situ hybridization was used to examine the presence and subcellular localization of SNAP‐25a and SNAP‐25b RNA transcript expression in motor and parasympathetic nuclei associated with cranial nerves. SNAP‐25a RNA transcripts were strongly expressed in the parasympathetic Edinger–Westphal nucleus and dorsal motor nucleus of the vagus nerve but weakly expressed in motor nuclei such as the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal and accessory nuclei and in motoneurons of mouse lumbar spinal cord. In contrast, SNAP‐25b RNA transcripts were not detectable in the Edinger–Westphal nucleus and dorsal motor nucleus of the vagus nerve but were strongly expressed in the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal, and accessory nuclei and in the motoneurons of mouse lumbar spinal cord. In the parasympathetic cranial nerve nuclei, displaying high levels of SNAP‐25a RNA transcripts, the labeling was cytoplasmic, whereas the labeling was nuclear in the cranial nerve motor nuclei, displaying lower levels of transcripts. In contrast, labeling of SNAP‐25b RNA transcripts was cytoplasmic in cranial nerve motor nuclei and not detectable in parasympathetic cranial nerve nuclei. Possible explanations for the region‐specific and differential subcellular localization of SNAP‐25a and SNAP‐25b RNA transcripts are discussed. J. Comp. Neurol. 411:591–600, 1999.

Isoform-specific exocytotic protein mRNA expression in hypothalamic magnocellular neurons: regulation after osmotic challenge.

Exocytosis is regulated by proteins which interact to promote docking and fusion of secretory granules with the plasma membrane. We have used in situ hybridization to study the mRNA expression for vesicle-associated membrane protein (VAMP) isoforms VAMP-1 and VAMP-2, synaptosomal-associated protein of 25-kDa (SNAP-25) isoforms SNAP-25a and SNAP-25b, mammalian homologue of unc-18 (munc-18) and Hrs-2 in neurosecretory neurons of the magnocellular paraventricular (PVN) and supraoptic (SON) nuclei of normal and osmotically challenged animals. In PVN and SON neurons of normal animals, strong labeling was demonstrated for VAMP-2 and SNAP-25a mRNA, whereas VAMP-1 or SNAP-25b mRNA could not be detected. Salt-loading (2% NaCl as drinking water), an animal model which increases the expression and secretion of hormones from hypothalamic magnocellular neurons, resulted in significantly increased mRNA levels for VAMP-2 (36%, 28%), munc-18 (74%, 68%) and SNAP-25a (59%, 77%) in the PVN and SON, respectively. There was no significant increase in Hrs-2 mRNA levels in the PVN, whereas a significant increase (22%) was observed in the SON. In the posterior pituitary, immunohistochemistry showed a marked decrease in numbers and intensity of vasopressin-immunoreactive (-IR) nerve endings after salt-loading. There were no obvious changes in numbers or intensity of VAMP-2-, munc-18-, Hrs-2- or SNAP-25-IR fibers. Large varicosities containing VAMP-2- and Hrs-2 immunocreactivity were seen in salt-loaded animals. The results show isoform-specific mRNA expression in neurosecretory neurons and an increased mRNA expression of proteins participating in the molecular regulation of exocytosis during an experimental situation characterized by increased secretion.

Hexokinase I Messenger RNA in the Rat Central Nervous System

Hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is the first enzyme required in the metabolism of glucose in the central nervous system and plays a major role in regulation of the cerebral glycolytic rate. The distribution of hexokinase I mRNA was examined throughout the central nervous system of the rat by use of oligonucleotide probes and in situ hybridization histochemistry. In the rhinencephalon, strong hexokinase I mRNA labeling was demonstrated in the glomerular, mitral, internal granular, and internal plexiform layers, whereas the olfactory nerve, external plexiform, and subependymal layers and ependyma were devoid of labeling. Within the telencephalon, strong labeling was present in all layers (with the exception of the molecular layer) of the cerebral cortex, in the septum, in CA1-4 and dentate gyrus of the hippocampus, and in several amygdaloid nuclei. There was only weak labeling in the nucleus accumbens and caudate putamen. In the diencephalon, there was in general a strong labeling in the epithalamus, in several thalamic nuclei, including the anteriodorsal, anterioventral, anteriomedial, reticular, paravetricular, intermediodorsal, anteriomedial, interanteriomedial, rhomboid, reuniens, and parafascicular thalamic nuclei. Several hypothalamic regions, including the subfornical organ, the medial preoptic area, the suprachiasmatic, supraoptic, paraventricular, dorsomedial, ventromedial nuclei, and the zona incerta, were strongly labeled. In the mesencephalon, there was particularly strong labeling in the pars compacta and reticulata of the substantia nigra, central gray, and red nucleus, in the Darkschewitsch nucleus, and in the medial accessory oculomotor nucleus. In the rhombencephalon, there was strong hybridization in all raphe nuclei, pontine, tegmental, lateral parabrachial, olivary nuclei, and several cranial motor nuclei. All neurons of the locus ceruleus were heavily labeled. Very strong labeling was present in Purkinje and granular cells of the cerebellar cortex. Neurons of the medulla oblongata area postrema, nucleus tractus solitarius, reticular nucleus, nucleus cuneatus and several motor nuclei were strongly labeled. In the spinal cord, labeled cells were present in all laminae, and also neurons of the dorsal root ganglion were heavily labeled. Hexokinase I mRNA was also demonstrated in the epithelium lining the the choroid plexus. In the E15 fetus, very strong labeling was seen in the liver, heart, and trigeminal ganglion, with less intense labeling in in the brain and other tissues having more moderate labeling. Administration of 2% saline as drinking water resulted in a marked increase in hexokinase I mRNA in the magnocellular neurons of the supraoptics and paraventricular nuclei. In summary, the results show extensive neuronal distribution of hexokinase I mRNA with regional differences in the expression pattern.