Nancy J. Grant

How botulinum and tetanus neurotoxins block neurotransmitter release

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.

Cultured glial cells express the SNAP-25 analogue SNAP-23.

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP‐25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP‐23, an analogue of SNAP‐25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP‐23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t‐SNAREs SNAP‐23 and syntaxin 1 and the v‐SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis. GLIA 27:181–187, 1999.

Differential expression of SNAP-25 isoforms and SNAP-23 in the adrenal gland.

Abstract : In the rat adrenal gland, we previously observed that SNAP‐25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP‐25 isoform expression is different in the two phenotypes. Expression of SNAP‐25 isoforms and SNAP‐23 was examined by immunoblotting, immunofluorescence, and RT‐PCR. Amplifications of SNAP‐25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP‐25 isoform expression in rat and bovine adrenal glands. SNAP‐25 and SNAP‐23 mRNA and protein are expressed in the glands ; SNAP‐23 is enriched in the adrenal cortex, whereas SNAP‐25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP‐25 and low levels of SNAP‐23 are observed in the PC12 cells, whereas both SNAP‐25 and SNAP‐23 are expressed in adrenal medullary cultures. In all extracts, the SNAP‐23 mRNA corresponded to SNAP‐23a. SNAP‐25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP‐25a and SNAP‐25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP‐25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.

Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one‐half, whereas NGF increased L1 protein levels ∼2.3‐fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10−7M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down‐regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.

SNAP‐25 is differentially expressed by noradrenergic and adrenergic chromaffin cells

This study examines chromaffin cell expression of the synaptosomal‐associated protein SNAP‐25 in the adrenal medulla by immunoblotting, immunocytochemistry and PCR. Both mRNAs coding for the SNAP‐25 isoforms a and b were detected and SNAP‐25 was found to be present in all chromaffin cells in adult rat adrenal gland sections. It was essentially restricted to a zone close to the cytoplasmic face of the plasma membrane in the majority of cells, but located extensively throughout the cytoplasm in a chromaffin cell sub‐population, identified by double immunofluorescence labelling to have a noradrenergic phenotype. This differential SNAP‐25 expression may reflect different stages in the phenotypic development of the sympathoadrenal lineage and be related to an additional functional role in noradrenergic chromaffin cells not associated with secretion.

NGF enhances depolarization effects on SNAP-25 expression: induction of SNAP-25b isoform.

The 25 kDa synaptosomal associated protein (SNAP-25), which is implicated in neuronal plasticity and neurosecretion, exists as two isoforms generated by alternative splicing of exons 5a and 5b. The aim of the present study was to characterize factors influencing isoform expression. We report that chronic depolarization of PC12 cells alone or in the presence of NGF induces the expression of isoform-b, in addition to a 1.8- to 3-fold increase in SNAP-25 mRNA and protein as determined by immunoblotting and combined RT-PCR and Southern blot analysis. When cerebellar granule neurons were cultured in elevated K+, the predominant isoform switched from SNAP-25a to SNAP-25b. Taken together these results suggested that chronic depolarization regulates the transcription and processing of SNAP-25 mRNA.

Noradrenergic, but not adrenergic chromaffin cells in the adrenal gland express neuromodulin (GAP-43)

Neuroendocrine chromaffin cells of the adrenal gland express certain molecular markers either transiently during development or permanently. In the present study, the expression of neuromodulin (GAP‐43), a neuronal protein often associated with neurite outgrowth, was examined in adult adrenals. Neuromodulin was detected by Western blot analysis in extracts of both rat adrenals and cultured bovine chromaffin cells, and was localized in situ in a subpopulation of chromaffin cells, as well as in nerve fibres and Schwann cells. The use of anti‐tyrosine hydroxylase or anti‐phenylethanolamine N‐methyltransferase antibodies in combination with anti‐neuromodulin antibodies in double immunofluorescent labelling of cryostat sections of rat glands demonstrated that neuromodulin is expressed by noradrenergic, and not by adrenergic chromaffin cells. The results provide further evidence that neuromodulin is not limited to neurons; it is also expressed in a subpopulation of neuroendocrine chromaffin cells. Neuromodulin may play a role in the development of the adrenal medulla or in the specific regulation of noradrenalin secretion from chromaffin cells.

L1 Cell Adhesion Molecule is Expressed by Noradrenergic but not Adrenergic Chromaffin Cells: A Possible Major Role for L1 in Adrenal Medullary Design

The adrenal medulla of higher animals is constituted of homotypic groups of chromaffin cells secreting either adrenalin or noradrenalin. Since not all chromaffin cells are individually innervated by fibres of the splanchnic nerve, this tissue characteristic is crucial to the physiological function of the gland. In an attempt to analyse differences between these chromaffin cell types which might underlie the establishment of this tissue pattern, we examined the expression of the adhesion molecule L1 in this gland by immunocytochemistry at the optical and ultrastructural levels in rats. L1, an adhesion molecule abundant in the central nervous system, was found to be present in the adrenal medulla of adults; it was strongly expressed on innervating axons and their surrounding Schwann cells and also on a subpopulation of chromaffin cells. The nature of these chromaffin cells was examined by immunocytochemistry using antibodies against the catecholamine‐synthesizing enzyme phenylethanolamine N‐methyltransferase (PNMT), which are capable of distinguishing between adrenergic and noradrenergic cells. Immunofluorescence labelling of sequential frozen sections demonstrated that chromaffin cells which express L1 do not express PNMT; conversely, L1 was not detected in any chromaffin cells expressing PNMT. Ultrastructural immunocytochemistry confirmed the existence of two non‐overlapping populations of chromaffin cells. It is concluded that, in the adrenal medulla, noradrenergic but not adrenergic chromaffin cells express this adhesion molecule. These data, together with our previous observations that all chromaffin cells express the neural cell adhesion molecule, NCAM, suggest that L1, in cooperation with NCAM, could be responsible for the association of noradrenergic cells in the form of homotypic aggregates segregated from groups of adrenergic cells within the adrenal medulla.

Expression of cell adhesion molecules and catecholamine synthesizing enzymes in the developing rat adrenal gland.

Cell adhesion molecules play a major role in determining tissue architecture during histogenesis. This immunocytochemical study of the adrenal gland examines the embryonic and early postnatal cellular expression of two neural cell adhesion molecules, NCAM and L1, which are widely expressed in brain and have been found also to be expressed in the adult rat adrenal gland. In parallel, antibodies directed against two neuroendocrine cell markers, tyrosine hydroxylase and phenylethanolamine N-methyltransferase, were employed to verify the phenotypic nature of developing chromaffin cells in order to correlate cell adhesion molecule expression with the state of chromaffin cell differentiation. NCAM was found to be expressed by chromoblasts within extra-adrenal blastema (i.e. before their migration into the cortical primordium) at the 16th day of embryonic life. It continued to be expressed by all developing chromaffin cells after their infiltration into the developing adrenal gland at all ages. L1 was also expressed by chromoblasts in extra-adrenal sites, but was found only in a subpopulation of chromaffin cells within the cortical primordium from the 16th embryonic day onwards. Those chromoblasts which expressed L1 constituted relatively large compact cell clusters within the gland at this stage, while intra-adrenal chromaffin cells not expressing L1 were dispersed in small cell groups. L1 was also strongly expressed by nerve fibres (and their surrounding Schwann cells) which appeared to innervate cell groups as early as the 16th embryonic day. Both extra- and intra-adrenal chromoblasts expressed tyrosine hydroxylase, but the large L1-positive cell aggregates were less intensely immunoreactive for tyrosine hydroxylase than were cells in small groups. PNMT expression was restricted to L1-negative intra-adrenal chromoblasts present in small groups. Ultrastructural observations demonstrated that cells expressing L1 contained few secretory granules at the 18th embryonic day. It is concluded from these data that these chromoblasts are the precursors of the noradrenergic cells found in the mature gland. In addition, the arrangement of noradrenergic chromaffin cells in the form of homotypic cell groups throughout the course of histogenesis of the adrenal medulla is likely to be a direct consequence of the exclusive co-expression of both NCAM and L1 by this subpopulation of maturing chromaffin cells.

α-Melanocyte stimulating hormone promotes neurite outgrowth in chromaffin cells

Chromaffin cells from adult bovine adrenal medulla were found to develop neurites when cocultured with pituitary intermediate lobe (IL) cells. In coculture 51.7% of the chromaffin cells extended neurites compared with 12% in control cultures (chromaffin cells alone). A soluble factor released by IL cells was apparently involved as medium conditioned by contact with IL cells also promoted neurite outgrowth. Moreover, the addition of αMSH, one of the pro‐opiomelanocortin‐derived peptides secreted by IL cells, alone reproduced this effect in a dose‐dependent manner. The data provide evidence for a neurotrophic role of αMSH.