BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.

Exonic Sp1 sites are required for neural-specific expression of the glycine receptor beta subunit gene.

The gene encoding the beta subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5' non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5' region of the beta subunit gene and the corresponding regions of the homologous GlyR alpha subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5'-untranslated portion of the GlyR beta mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58 kb of upstream flanking region and 180 bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5' flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR beta subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites.

Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse.

Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABA(A) receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABA(A) receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABA(A) receptors. Because homozygous (geph -/-) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (-/-) mouse retina. In the case of GABA(A) receptors, there was a significant reduction of clusters incorporating the gamma2, alpha2, and alpha3 subunits; however, a substantial number of hot spots was still present in geph (-/-) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABA(A) receptors. In heterozygous geph (+/-) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit.

Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.

Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.