Stefan Catsicas

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.

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.

Stiffness Tomography by Atomic Force Microscopy

The atomic force microscope is a convenient tool to probe living samples at the nanometric scale. Among its numerous capabilities, the instrument can be operated as a nano-indenter to gather information about the mechanical properties of the sample. In this operating mode, the deformation of the cantilever is displayed as a function of the indentation depth of the tip into the sample. Fitting this curve with different theoretical models permits us to estimate the Youngs modulus of the sample at the indentation spot. We describe what to our knowledge is a new technique to process these curves to distinguish structures of different stiffness buried into the bulk of the sample. The working principle of this new imaging technique has been verified by finite element models and successfully applied to living cells.

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2.

Trafficking of AMPA‐type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1‐binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR‐mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21–GRIP1 binding is crucial for GluR2‐AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.

Developmental and plasticity-related differential expression of two SNAP-25 isoforms in the rat brain

In this article we study the relationship between the expression pattern of two recently identified isoforms of the 25‐kD synaptosomal‐associated protein (SNAP‐25a and SNAP‐25b) and the morphological changes inherent to neuronal plasticity during development and kainic acid treatment. SNAP‐25 has been involved in vescicle fusion in the nerve terminal, and most likely participates in different membrane fusion‐related processes, such as those involved in neurotransmitter release and axonal growth. In the adult brain, SNAP‐25b expression exceeded SNAP‐25a in distribution and intensity, being present in most brain structures. Moderate or high levels of SNAP‐25a hybridization signal were found in neurons of the olfactory bulb, the layer Va of the frontal and parietal cortices, the piriform cortex, the subiculum and the hippocampal CA4 field, the substantia nigra/pars compacta, and the pineal gland, partially overlapping SNAP‐25b mRNA distribution. In restricted regions of cerebral cortex, thalamus, mammillary bodies, substantia nigra, and pineal glands the two isoforms were distributed in reciprocal fashion. During development SNAP‐25a mRNA was the predominant isoform, whereas SNAP‐25b expression increased postnatally. The early expression of SNAP‐25a in the embryo and the decrease after P21 is suggestive of a potential involvement of this isoform in axonal growth and/or synaptogenesis. This conclusion is indirectly supported by the observation that SNAP‐25a mRNA, but not SNAP‐25b mRNA, was upregulated in the granule cells of the adult dentate gyrus 48 hours after kainate‐induced neurotoxic damage of the hippocampal CA3‐CA4 regions. Increase of SNAP‐25 immunoreactivity was observed as early as 4 days after kainate injection within the mossy fiber terminals of the CA3 region, and in the newly formed mossy fiber aberrant terminals of the supragranular layer. These data suggest an isoform‐specific role of SNAP‐25 in neural plasticity.

Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein–protein interactions.

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, α‐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.

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. J. Neurosci. Res. 50:1000–1009, 1997. © 1997 Wiley‐Liss, Inc.

Activity-Dependent Phosphorylation of SNAP-25 in Hippocampal Organotypic Cultures

Abstract: Synaptosomal‐associated protein of 25 kDa (SNAP‐25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP‐25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP‐25 phosphorylation. In addition, immunochemical analysis of two‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels shows that SNAP‐25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12, 13‐dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP‐25 among these spots. Phorbol 12, 13‐dibutryate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long‐term potentiation, a property involved in learning mechanisms, and production of seizures with a GABAA receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP‐25 can be phosphorylated in situ by PKC in an activity‐dependent manner.

Oscillation modes of microtubules

Abstract Microtubules are long, filamentous protein complexes which play a central role in several cellular physiological processes, such as cell division transport and locomotion. Their mechanical properties are extremely important since they determine the biological function. In a recently published experiment [Phys. Rev. Lett. 89 (2002) 248101], microtubules Youngs and shear moduli were simultaneously measured, proving that they are highly anisotropic. Together with the known structure, this finding opens the way to better understand and predict their mechanical behavior under a particular set of conditions. In the present study, we modeled microtubules by using the finite elements method and analyzed their oscillation modes. The analysis revealed that oscillation modes involving a change in the diameter of the microtubules strongly depend on the shear modulus. In these modes, the correlation times of the movements are just slightly shorter than diffusion times of free molecules surrounding the microtubule. It could be therefore speculated that the matching of the two timescales could play a role in facilitating the interactions between microtubules and MT associated proteins, and between microtubules and tubulins themselves.