Splicing of intron 3 of human BACE requires the flanking introns 2 and 4.

Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer's disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.

Clouded leopards, the secretive top-carnivore of South-East Asian rainforests: their distribution, status and conservation needs in Sabah, Malaysia.

BACKGROUND: The continued depletion of tropical rainforests and fragmentation of natural habitats has led to significant ecological changes which place most top carnivores under heavy pressure. Various methods have been used to determine the status of top carnivore populations in rainforest habitats, most of which are costly in terms of equipment and time. In this study we utilized, for the first time, a rigorous track classification method to estimate population size and density of clouded leopards (Neofelis nebulosa) in Tabin Wildlife Reserve in north-eastern Borneo (Sabah). Additionally, we extrapolated our local-scale results to the regional landscape level to estimate clouded leopard population size and density in all of Sabah's reserves, taking into account the reserves' conservation status (totally protected or commercial forest reserves), their size and presence or absence of clouded leopards. RESULTS: The population size in the 56 km2 research area was estimated to be five individuals, based on a capture-recapture analysis of four confirmed animals differentiated by their tracks. Extrapolation of these results led to density estimates of nine per 100 km2 in Tabin Wildlife Reserve. The true density most likely lies between our approximately 95 % confidence interval of eight to 17 individuals per 100 km2. CONCLUSION: We demonstrate that previous density estimates of 25 animals/100 km2 most likely overestimated the true density. Applying the 95% confidence interval we calculated in total a very rough number of 1500-3200 clouded leopards to be present in Sabah. However, only 275-585 of these animals inhabit the four totally protected reserves that are large enough to hold a long-term viable population of > 50 individuals.

Spontaneous synaptic activity in an organotypic culture of the mouse retina.

PURPOSE: Many strains of mutant mice die at birth, when the retina is still very immature. The retinas of such mice can be studied in organotypic cultures. After a preceding anatomic study of the synaptic development, the electrical activity of the synaptic circuits within such cultures was studied in wild-type and gephyrin-deficient mice. METHODS: Organotypic cultures of newborn mouse retinas were grown for 14 days in vitro. Spontaneous postsynaptic currents (sPSCs) of amacrine cells were measured by using the whole-cell configuration of the patch-clamp technique. GABAergic and glycinergic currents that were isolated with specific antagonists, and retinas from wild-type (geph(+/+)) and gephyrin-deficient (geph(-/-)) mice were compared. RESULTS: Rapidly decaying sPSCs that were blocked by kynurenic acid were mediated by ionotropic glutamate receptors, whereas sPSCs with significantly higher peak amplitudes and slow-decay kinetics were identified as spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) and glycine receptors (GlyRs). In gephyrin-deficient (geph(-/-)) cultures, we found no sIPSCs mediated by GlyRs. sIPSCs mediated by GABA(A)Rs expressed in amacrine cells of geph(-/-) retinas decayed significantly faster than GABAergic sIPSCs recorded in amacrine cells of geph(+/+) retinas. CONCLUSIONS: The different decay kinetics of GABA(A)Rs expressed in amacrine cells of geph(+/+) and of geph(-/-) retinas suggests that these cells express at least two types of GABA(A)R subtypes. In amacrine cells of geph(-/-) mice, a specific GABA(A)R subtype that may contain the alpha2 subunit, is impaired by the absence of gephyrin, whereas other GABA(A)Rs appear to function normally.

Localization and possible function of the glutamate transporter, EAAC1, in the rat retina.

We investigated the localization and possible function of EAAC1 in the rat retina. Immunocytochemical localization of EAAC1 at the light-microscopic level revealed a fine dust-like labelling pattern across the two synaptic layers. Horizontal cell and subpopulations of amacrine cell somata were labelled, as were some somata within the ganglion cell layer. Some immunoreactive puncta were observed within the cytoplasm of amacrine cells, in regions well away from synaptic sites. At the ultrastructural level, EAAC1 immunolabelled one postsynaptic element at synapses and also processes well away from the synaptic release site. Since EAAC1 was localized away from synaptic sites, we evaluated the role EAAC1 plays in GABA formation by measuring GABA concentrations via reversed-phase high-performance liquid chromatography following incubation of retinae in enzyme and glutamate uptake inhibitors. Incubation of retinae in D-threo-beta-hydroxyaspartate or D/ L-threo-beta-benzyloxyaspartate, which are known to inhibit the glutamate transporters GLAST1, GLT1, and EAAC1, caused a decrease in GABA synthesis by around 50%. Incubation in 6-diazo-5-oxo- L-norleucine, a phosphate-activated glutaminase inhibitor, decreased GABA formation by 40%. Taken together with the anatomical data, the results of this study suggest that EAAC1 plays very little role in GABA synthesis - indeed GABA formation occurs predominantly from glutamine. By virtue of its location both near and well away from synaptic release sites, EAAC1 may regulate glutamate uptake differentially.

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity.

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.

Cloning, transport properties, and differential localization of two splice variants of GLT-1 in the rat CNS: implications for CNS glutamate homeostasis.

At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid.

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartyl protease and is responsible for the beta-secretase cleavage of APP producing different endoproteolytic fragments referred to as the carboxy-terminal C99, C89 and the soluble ectodomain sAPPbeta. Here we describe two transgenic mouse lines expressing human BACE in the brain. Overexpression of BACE augments the amyloidogenic processing of APP as demonstrated by decreased levels of full-length APP and increased levels of C99 and C89 in vivo. In mice expressing huBACE in addition to human APP wild-type or carrying the Swedish mutation, the induction of APP processing characterized by elevated C99, C89 and sAPPbeta, results in increased brain levels of beta-amyloid peptides Abeta40 and Abeta42 at steady-state.