Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia.

Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers.

Members of the superfamily of nuclear receptors share the greatest homology in their DNA-binding domains. We have used reverse transcription-polymerase chain reaction and highly degenerate primers based on the amino acid sequence of the zinc finger motif of known nuclear receptors to identify novel members of the family. Starting with rat brain RNA, we have isolated an orphan receptor that we call RZR beta. The sequence of its nearly full-length complementary DNA shows great similarity to RZR alpha, a receptor we recently identified from human umbilical vein endothelial cells. These RZR subtypes represent members of a new family of orphan nuclear receptors that most likely regulate specific gene expression. Sequence comparison with other known nuclear receptors reveals great similarity for both RZR subtypes to retinoic acid and retinoid-X receptors. By Northern blot analyses, we found RZR beta messenger RNA only in brain, whereas RZR alpha is expressed in many tissues. We show here that the RZRs bind as monomers to natural retinoid response elements formed by (A/G)GGTCA half-sites. However, a T-residue in the -1 position of this motif greatly enhances the DNA binding affinity of RZRs, whereas the -2 position has no influence. We show that RZRs can bind as homodimers on response elements formed by palindromes, inverted palindromes, or direct repeats of two TAGGTCA half-sites. Interestingly, these response elements display dramatically reduced affinity for retinoic acid receptor-retinoid-X receptor heterodimers. Thus, the 5'-flanking sequence of hexameric half-sites appears to be crucial to direct the activity of several nuclear receptors. On monomeric as well as dimeric binding sites, RZRs show constitutive transactivational activity that can be enhanced by unidentified components of fetal calf serum.

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.

Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons.

To study how an increase in axon number influences the number of glial cells in the mammalian optic nerve, we have analyzed a previously described transgenic mouse that expresses the human bcl-2 gene from a neuron-specific enolase promoter. In these mice, the normal postnatal loss of retinal ganglion cell axons is greatly decreased and, as a consequence, the number of axons in the optic nerve is increased by approximately 80% compared with wild-type mice. Remarkably, the numbers of oligodendrocytes, astrocytes, and microglial cells are all increased proportionally in the transgenic optic nerve. The increase in oligodendrocytes apparently results from both a decrease in normal oligodendrocyte death and an increase in oligodendrocyte precursor cell proliferation, whereas the increase in astrocytes apparently results from an increase in the proliferation of astrocyte lineage cells. Unexpectedly, the transgene is expressed in oligodendrocytes and astrocytes, but this does not seem to be responsible for the increased numbers of these cells. These findings indicate that developing neurons and glial cells can interact to adjust glial cell numbers appropriately when neuronal numbers are increased. We also show that the expression of the bcl-2 transgene in retinal ganglion cells protects the cell body from programmed cell death when the axon is cut, but it does not protect the isolated axon from Wallerian degeneration, even though the transgene-encoded protein is present in the axon.

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.

Localization and targeting of SCG10 to the trans-Golgi apparatus and growth cone vesicles.

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles. In isolated growth cone particles, SCG10 was found to be particularly concentrated in the growth cone vesicle fraction. To evaluate the molecular determinants of the specific targeting of SCG10 to growth cones, we have transfected PC12 cells and primary neurons in culture with mutant and fusion cDNA constructs. Deletion of the amino-terminal domain or mutations within this domain that prevented palmitoylation at cysteines 22 and 24 abolished Golgi localization as well as growth cone targeting, suggesting that palmitoylation of the amino-terminal domain is a necessary signal for Golgi sorting and possibly transport of SCG10 to growth cones. Fusion proteins consisting of the amino-terminal domain of SCG10 and the cytosolic proteins stathmin or glutathione-S-transferase colocalized with a Golgi marker, alpha-mannosidase II, and accumulated in growth cones of both axons and dendrites. These results reveal a novel axonal/dendritic growth cone targeting sequence that involves palmitoylation.

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.

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.