Patrick Foran

Glial heterogeneity in expression of the inwardly rectifying K(+) channel, Kir4.1, in adult rat CNS.

Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K+ channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Müller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K+ through this channel, whereas nonexpressors might use other mechanisms. GLIA 30:362–372, 2000.

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N -ethyl-maleimide-sensitive factorattachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.

Rescue of Exocytosis in Botulinum Toxin A-poisoned Chromaffin Cells by Expression of Cleavage-resistant SNAP-25 IDENTIFICATION OF THE MINIMAL ESSENTIAL C-TERMINAL RESIDUES

Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1–197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1–206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxins protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1–201, 1–202, 1–203 afforded a significant return of exocytosis, unlike shorter forms 1–197, −198, −199, or −200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism.

Botulinum A Like Type B and Tetanus Toxins Fulfils Criteria for Being a Zinc‐Dependent Protease

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc‐binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc‐dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxins’ neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins’ degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc‐dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins’ effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.

The janus faces of botulinum neurotoxin: Sensational medicine and deadly biological weapon

The botulinum neurotoxins are the most dangerous toxins known (BoNTs serotypes A–G) and induce profound flaccid neuromuscular paralysis by blocking nerve–muscle communication. Poisoned motoneurons react by emitting a sprouting network known to establish novel functional synapses with the abutting muscle fiber. Understanding how our motoneurons are capable of bypassing such transmission blockade, thereby overcoming paralysis, by an astonishing display of plasticity is one of the research goals that have numerous therapeutic ramifications. This Mini‐Review aims at giving a brief update on the recent discoveries regarding the molecular mechanism of botulinum toxins intoxication. Curing botulism still is a challenge once the toxin has found his way inside motoneurons. In view of the potential use of botulinum toxins as biological weapon, more research is needed to find efficient ways of curing this disease.

Insights into a basis for incomplete inhibition by botulinum toxin A of Ca2+-evoked exocytosis from permeabilised chromaffin cells

Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cells. Both virtually abolished catecholamine release triggered from intact cells by depolarising stimuli, whereas the blockade by type A, but not B, was only partial after cell permeabilisation and direct stimulation of exocytosis by Ca(2+). Botulinum toxin A did not alter the [Ca(2+)]-dependency of exocytosis in permeabilised cells but, rather, proportionally reduced the amount of release at each concentration tested. Likewise, this toxin decreased the extents of Ca(2+)-induced structural changes in SNAP-25, synaptobrevin and syntaxin (known collectively as SNAREs), whilst leaving their [Ca(2+)]-sensitivity unaltered. Thus, botulinum toxin A does not reduce the Ca(2+)-sensitivity of the exocytosis sensor, but hinders transmission of the signal to the SNAREs which mediate fusion.