Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.

Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.

Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions.

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Molecular and functional diversity at synapses of individual neurons in vitro.

We have quantified activity-dependent uptake of the fluorescent dye FM1-43 in combination with immunocytochemistry for synaptic vesicle-associated proteins (SVPs) at individual synapses in primary cultures of rat cortical neurons. We show that expression of synaptic proteins is highly variable and that the levels of synaptophysin (p38), synapsin I and sv2, but not synapsin II, correlate with the extent of FM1-43 labelling at synapses. The data indicate that SVP levels affect the uptake of FM1-43 with different efficacy (p38 > synapsin I > sv2 or synapsin II). We also found that the relative levels of SVPs vary at individual boutons of single neurons grown in isolation, which indicates that differential regulation of specific SVPs may contribute to the selective modulation of activity at synapses of the same neuron.

Differential distribution of stathmin and SCG10 in developing neurons in culture.

The neuron-specific protein SCG10 and the ubiquitous protein stathmin are two members of a family of microtubule-destabilizing factors that may regulate microtubule dynamics in response to extracellular signals. To gain insight into the function of these proteins in the nervous system, we have compared their intracellular distribution in cortical neurons developing in culture. We have used double-immunofluorescence microscopy with specific antibodies for stathmin and SCG10 in combination with antibodies for axonal, microtubule, and synaptic marker proteins. Stathmin and SCG10 were coexpressed in individual neurons. While both proteins were highly expressed in developing cultures during differentiation, their subcellular localization was strikingly different. Stathmin showed a cytosolic distribution, mainly in cell bodies, whereas SCG10 strongly labeled the growth cones of axons and dendrites. During neurite outgrowth, SCG10 appeared as a single concentrated spot in a region of the growth cone where the microtubules are known to be particularly dynamic. Disassembly of labile microtubules by nocodazole caused a dispersal of the SCG10 staining into punctate structures, indicating that its subcellular localization is microtubule-dependent. Upon maturation and synapse formation, the levels of both stathmin and SCG10 decreased to become undetectable. These observations demonstrate that the expression of both proteins is associated with neurite outgrowth and suggest that they perform their roles in this process in distinct subcellular compartments.

Common and distinct fusion proteins in axonal growth and transmitter release.

We have used the proteolytic properties of botulinum and tetanus neurotoxins (BoNT, TeNT) to cleave three proteins of the membrane fusion machinery, SNAP-25, VAMP/synaptobrevin, and syntaxin, in developing and differentiated rat central neurons in vitro. Then, we have studied the capacity of neurons to extend neurites, make synapses, and release neurotransmitters. All the toxins showed the expected specificity with the exception that BoNT/C cleaved SNAP-25 in addition to syntaxin and induced rapid neuronal death. In developing neurons, cleavage of SNAP-25 with BoNT/A inhibited axonal growth and prevented synapse formation. In contrast, cleavage of VAMP with TeNT or BoNT/B had no effects on neurite extension and synaptogenesis. All the toxins tested inhibited transmitter release in differentiated neurons, and cleavage of VAMP resulted in the strongest inhibition. These data indicate that SNAP-25 is involved in vesicle fusion for membrane expansion and transmitter release, whereas VAMP is selectively involved in transmitter release. In addition, our results support the hypothesis that synaptic activity is not essential for synapse formation in vitro.

Antisense Blockade of Expression : SNAP-25 In Vitro and In Vivo.

With the advent of modern molecular genetics and molecular biology, we will face more and more situations where novel gene products with unknown functions are identified. Genetic linkage analysis will allow the association of novel or known genes to Important diseases (1). Similarly, sensitlve differential cloning procedures will identify rare genes expressed in specific physiological or pathological situations (1, 3). In both cases, establishing the precise function of the identified gene is an essential step for the understanding of the cellular mechanisms that either lead to the disease or are pivotal in important physiological processes.

Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo.

Axonal elongation and the transformation of growth cones to synaptic terminals are major steps of brain development and the molecular mechanisms involved form the basis of the correct wiring of the nervous system. The same mechanisms may also contribute to the remodelling of nerve terminals that occurs in the adult brain, as a morphological substrate to memory and learning. We have investigated the function of the nerve terminal protein SNAP-25 (ref. 2) during development. We report here that SNAP-25 is expressed in axonal growth cones during late stages of elongation and that selective inhibition of SNAP-25 expression prevents neurite elongation by rat cortical neurons and PC-12 cells in vitro and by amacrine cells of the developing chick retina in vivo. These results demonstrate that SNAP-25 plays a key role in axonal growth. They also suggest that high levels of SNAP-25 expression in specific areas of the adult brain may contribute to nerve terminal plasticity.