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The presenilin-1 gene is mutated in early-onset familial Alzheimer's disease. The mutation Pro117Leu is implicated in a very severe form of the disease, with an onset of less than 30 years. The consequences of this mutation on neurogenesis in the hippocampus of adult transgenic mice have already been studied in situ. The survival of neural progenitor cells was impaired resulting in decreased neurogenesis in the dentate gyrus. Our intention was to verify if similar alterations could occur in vitro in progenitor cells from the murine ganglionic eminences isolated from embryos of this same transgenic mouse model. These cells were grown in culture as neurospheres and after differentiation the percentage of neurons generated as well as their morphology were analysed. The mutation results in a significant decrease in neurogenesis compared to the wild type mice and the neurons grow longer and more ramified neurites. A shift of differentiation towards gliogenesis was observed that could explain decreased neurogenesis despite increased proliferation of neural precursors in transgenic neurospheres. A diminished survival of the newly generated mutant neurons is also proposed. Our data raise the possibility that these alterations in embryonic development might contribute to increase the severity of the Alzheimer's disease phenotype later in adulthood.

In NSE-Hu-Bcl-2 transgenic mice, line 71, retina undergoes early postnatal degeneration linked to the prior death of Müller cells. The purpose of this study was to complete the characterization of this retinal dysfunction by using electroretinographic (ERG) recordings in both scotopic and photopic conditions. Here, we showed that both rod and cone systems were profoundly affected in NSE-Hu-Bcl-2 transgenic mice as soon as 15 postnatal days in accordance with histological study performed previously.

We investigated apoptosis during early stages of in vitro differentiation of neuronal precursors generated by embryonic day 14 (E14) mouse striata stem cells. Differentiation was in conditions of suboptimal growth factor supply. Apoptosis reached 10-15% of cells and affected proliferating as well as postmitotic cells, including TUJ1-positive cells. Inhibition of apoptosis led to an increased proportion of TUJ1-positive cells generated by stem cells. K(+) current was reported to be related to apoptosis. Outward K(+) currents were present in differentiating neuronal precursors that were consistent with delayed rectifier and transient A-type currents. The amplitude of the delayed rectifier current varied during the first 4 days of stem cell differentiation. Current amplitude was greatly increased in the presence of staurosporine but reduced at elevated extracellular K(+) concentration. In addition, the amplitude of the current was significantly diminished by inhibiting several caspases, but not caspase 8. In Bax knock-out transgenic neuronal precursors, K(+) current was not decreased after the first day but at later stages of cell differentiation. At this early stage, apoptosis of proliferating cells and of TUJ1-positive cells was not reduced by the absence of Bax, but was by caspase 9 inhibition. Thus, activation of a delayed rectifier K(+) current in differentiating stem cells is related to apoptosis. Recordings of this current revealed that apoptosis at early stages of neuronal differentiation occurred in two phases that did not exhibit similar dependence on the proapoptotic protein Bax and that probably used different pathways.

We have generated a mouse strain carrying a transgene driven by a strong and ubiquitous promoter (human cytomegalovirus hCMV/beta-actin) and containing an enhanced green fluorescent protein (eGFP) coding sequence upstream of the 3' untranslated region (3'UTR) of tissue-type plasminogen activator (t-PA) mRNA. The 3'UTR of t-PA mRNA is known to be involved in the reversible deadenylation and translational repression of transcripts in mouse oocytes. hCMV/beta-actin-eGFP-3'UTR t-PA transgenic mice express eGFP mRNA in all brain structures analyzed but lack eGFP fluorescence, with the exception of blood vessels, choroid plexus, and Purkinje cells. Taking advantage of these features, we tested whether certain pathological conditions, in particular injuries of the nervous system, might trigger eGFP fluorescence in traumatized cells or neurons. From this perspective, we analyzed eGFP mRNA expression and eGFP fluorescence in experimental models of nervous system lesions, such as motoneuron axotomy and cerebral stroke induced by middle cerebral artery occlusion. We found an increase in eGFP fluorescence in specific brain areas in cells suffering or reacting to these injuries. This increased fluorescence is correlated with an increased transcription of eGFP in lesioned cells, presumably enhanced by a release of the translational silencing mediated by the 3'UTR region of the t-PA mRNA. This transgenic mouse model may prove useful to study the development of neurodegenerative lesions., (Copyright 2004 Wiley-Liss, Inc.)

Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.

Choline acetyltransferase (ChAT), the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine, exists in a soluble and membrane-bound form in cholinergic nerve terminals of different animal species. This study was performed on the enzyme present in Drosophila central nervous system. We show that the two forms of the enzyme have the same apparent molecular weight (75 kDa) when analysed by immunoblotting using an antibody we raised against the recombinant enzyme. According to different authors, membrane-bound enzyme might be associated with synaptic vesicles or plasma membrane. Subfractionation of Drosophila head homogenates in linear glycerol gradients showed that ChAT does not associate with synaptic vesicles. Analysis of ChAT activity and immunoreactivity showed that two peaks of ChAT were produced. One peak was present in fractions containing soluble components and the other was associated with rapidly sedimenting membranes containing plasma membranes. ChAT in the first peak was mainly hydrophilic. A large proportion of ChAT associated with rapidly sedimenting membranes was amphiphilic. Further fractionation of these membranes by flotation in sucrose gradients showed that membrane-associated ChAT sedimented in fractions containing plasma membrane marker. Membrane-bound ChAT was neither solubilized nor converted to hydrophilic enzyme after membrane treatment with 1 M hydroxylamine, suggesting that the enzyme is not palmitoylated and therefore not anchored to membrane through thioester-linked long chain fatty acid. Partial solubilization of ChAT present on membranes with urea and carbonate suggests that this form of ChAT is a peripheral membrane protein. Carbonate solubilization of membrane-bound ChAT converted the enzyme from hydrophobic to hydrophilic protein.

We have previously shown that the enzyme choline-O-acetyltransferase (ChAT) exists in a hydrophilic and an amphiphilic form in Drosophila head. A complementary DNA clone of 4.2 kb containing the entire coding region of ChAT was isolated from a cDNA library of Drosophila heads. The cDNA was subcloned in an expression vector and injected into the nucleus of Xenopus oocytes. Injected oocytes expressed high levels of ChAT activity. This activity was inhibited by bromoacetylcholine, a specific inhibitor of the enzyme. In the present study the non-ionic detergent Triton X-114 was used to analyse whether the expression of hydrophilic and amphiphilic ChAT was or was not directed by a single cDNA. The two forms of ChAT were found to be synthesized in injected oocytes. Approximately 9% of the recombinant enzyme partitioned as amphiphilic activity. This value was similar to that found for native amphiphilic ChAT in Drosophila heads. Sedimentation in sucrose gradients of amphiphilic enzyme was found to be influenced by the type of detergent present in the gradient whereas this was not the case for hydrophilic ChAT. Hydrophilic and amphiphilic enzyme activities differed in some of their biochemical properties. Amphiphilic ChAT was less sensitive to inhibition by the product acetylcholine than was hydrophilic ChAT. Moreover, amphiphilic ChAT was found to be more resistant than hydrophilic ChAT to heat inactivation at 45 degrees C. These properties were observed for the native as well as for recombinant ChAT. These results demonstrate that the hydrophilic and amphiphilic forms of ChAT are derived from one mRNA.

We show that in the central nervous system of the fly, Drosophila melanogaster, choline acetyltransferase (ChAT) activity exists under two molecular forms, a soluble, hydrophilic form and a membrane-bound, amphiphilic form. This is based on the following demonstrations of differential solubilization and interaction with non-denaturing detergents: sequential extraction of Drosophila heads produced low-salt-soluble (83-87%) and detergent-soluble (6-7%) ChAT activity. Sedimentation in sucrose gradients of detergent-soluble ChAT was found to be influenced by the type of detergent present in the gradient (Triton X-100 and Brij 96). This was not the case for low-salt-soluble ChAT. To further confirm these findings, we subjected Drosophila heads to Triton X-114 fractionation. This method, which yielded 12% of amphiphilic ChAT activity, separates hydrophilic from amphiphilic proteins. Compared to central nervous tissue of rat and Torpedo electric lobes, Drosophila head contained the highest proportion of amphiphilic ChAT activity. Synaptosomes isolated from Torpedo electric organ exhibited higher levels of amphiphilic ChAT than did electric lobes. Of the three animal species analyzed here, the Torpedo amphiphilic enzyme was the most hydrophobic and the rat enzyme the least hydrophobic. The proportion of amphiphilic ChAT was analyzed during Drosophila development. The percentage of this activity increased about 7 times from embryo to larva and then remained constant until the adult fly age.

1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2.6 +/- 0.4 ms and 6.0 +/- 1.1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 6.2 +/- 1.1 ms. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65.2 +/- 16.1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0.7 +/- 0.1 pA for the mean amplitude of the elementary event, and 4.7 +/- 0.2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro. 7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Due to Triton X-114 fractionation of synaptosomes isolated from the electric organ of the fish Torpedo, the existence of a hydrophilic and an amphiphilic form of the enzyme choline-O-acetyltransferase (ChAT) was revealed. Amphiphilic ChAT which represents about 10% of total enzyme activity in synaptosomes, reached 40% of ChAT activity measured in preparations of synaptosomal plasma membranes (SPM) which were washed with solutions of increasing ionic strength. ChAT activity bound to washed SPM could be partially solubilized using proteinase K but not phospholipase C. No ChAT solubilization occurred by treating intact synaptosomes with proteinase K. Water/Triton X-114 partition coefficients of hydrophilic and amphiphilic ChAT were found to be 6.5 and 0.17, respectively. Sedimentation coefficients determined by centrifugation in linear density gradients of sucrose containing Triton X-100, were 4.2S and 4.4S for amphiphilic and hydrophilic ChAT, respectively. On the other hand, removal of Triton X-114 from the detergent phase containing amphiphilic ChAT activity led to enzyme aggregation. Finally, amphiphilic ChAT was slightly more acidic (pH 6.6) than was hydrophilic enzyme (6.8-7.0). We conclude that in Torpedo synaptosomes two forms of ChAT activity, a soluble and a membrane-bound form, are indeed present which differ in their hydrophobicity. The soluble form is hydrophilic. The membrane-bound form is amphiphilic and it aggregates upon removal of detergent. These are two characteristics of integral membrane proteins. Membrane-bound ChAT is most probably intracellularly oriented and not bound to membrane through a 'receptor' protein.

Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of approximately 120,000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetylcholine and were able to synthesize [14C]acetylcholine from 1-[14C]acetate. With KCl depolarization and upon treatment with a Ca2+ ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca(2+)-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentration, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. Immunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh-releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances.

Choline acetyltransferase was purified approximately 18,000-fold from 300 g of bovine caudate nuclei to a specific activity of 21 ?mol min mg protein. The overall procedure used was: extraction of the enzyme by high salt concentration, chromatography on carboxy-methyl-Sephadex, precipitation by ammonium sulphate, affinity chromatography on Blue-Sepharose and, finally absorption on hydroxylapatite. When the enzyme absorbed on hydroxylapatite was injected into mice, it provoked reproducibly a transient production of 'inhibitory' antibodies, followed by higher antibody titres mainly of 'non-inhibitory' type. These responses were elicited by injecting less than a total of 20 ?g of immunogen. The highest antibody titre was obtained less than 2 months following the initial immunisation. Species cross reactivity was investigated. This procedure should prove to be of value in the production of monoclonal antibodies to choline acetyltransferase.

A monoclonal antibody raised against cholinergic synaptosomal plasma membranes isolated from Torpedo electric organ, inhibited completely amphiphilic and hydrophilic choline acetyltransferase (ChAT) activities extracted and separated by using Triton X-114 phase partition of synaptosomes. We tested whether ChAT inhibition was direct or not. We found that the antibody was inhibiting ChAT in preparations of very low purity as well as ChAT that was immunoprecipitated by using a non-inhibitory anti-ChAT polyclonal antibody. Also, inclusion of acetylcoenzyme A at 20 times its Km during incubation of ChAT and antibody, completely prevented ChAT inhibition. This same concentration of the ChAT substrate could significantly but not completely dissociate the complex enzyme-antibody. These results spoke in favour of a direct inhibition of ChAT; the antibody most probably binds to an epitope that may be located at or near the acetylcoenzyme A binding site. The inhibitory effect on hydrophilic and amphiphilic ChAT was dependent on the antibody concentration, but amphiphilic activity required higher concentrations to be affected to the same extent as hydrophilic activity. This was found not only with Torpedo, but also with rat and human ChAT activities. Thus, the antibody appears to be able to distinguish the two forms of ChAT activity.