Mutation-induced loss of APP function causes GABAergic depletion in recessive familial Alzheimer's disease: analysis of Osaka mutation-knockin mice.

The E693Δ (Osaka) mutation in APP is linked to familial Alzheimer's disease. While this mutation accelerates amyloid ß (Aß) oligomerization, only patient homozygotes suffer from dementia, implying that this mutation is recessive and causes loss-of-function of amyloid precursor protein (APP). To investigate the recessive trait, we generated a new mouse model by knocking-in the Osaka mutation into endogenous mouse APP. The produced homozygous, heterozygous, and non-knockin littermates were compared for memory, neuropathology, and synaptic plasticity. Homozygotes showed memory impairment at 4 months, whereas heterozygotes did not, even at 8 months. Immunohistochemical and biochemical analyses revealed that only homozygotes displayed intraneuronal accumulation of Aß oligomers at 8 months, followed by abnormal tau phosphorylation, synapse loss, glial activation, and neuron loss. These pathologies were not observed at younger ages, suggesting that a certain mechanism other than Aß accumulation underlies the memory disturbance at 4 months. For the electrophysiology studies at 4 months, high-frequency stimulation evoked long-term potentiation in all mice in the presence of picrotoxin, but in the absence of picrotoxin, such potentiation was observed only in homozygotes, suggesting their GABAergic deficit. In support of this, the levels of GABA-related proteins and the number of dentate GABAergic interneurons were decreased in 4-month-old homozygotes. Since APP has been shown to play a role in dentate GABAergic synapse formation, the observed GABAergic depletion is likely associated with an impairment of the APP function presumably caused by the Osaka mutation. Oral administration of diazepam to homozygotes from 6 months improved memory at 8 months, and furthermore, prevented Aß oligomer accumulation, indicating that GABAergic deficiency is a cause of memory impairment and also a driving force of Aß accumulation. Our findings suggest that the Osaka mutation causes loss of APP function, leading to GABAergic depletion and memory disorder when wild-type APP is absent, providing a mechanism of the recessive heredity.

Neurodegenerative disorder FTDP-17-related tau intron 10 +16C → T mutation increases tau exon 10 splicing and causes tauopathy in transgenic mice.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative disorder caused by mutations in the tau gene. Many mutations identified in FTDP-17 have been shown to affect tau exon 10 splicing in vitro, which presumably causes pathologic imbalances in exon 10(-) [3-repeat (3R)] and exon 10(+) [4-repeat (4R)] tau expression and leads to intracellular inclusions of hyperphosphorylated tau in patient brains. However, no reports have investigated this theory using model mice with a tau intronic mutation. Herein, we generated new transgenic mice harboring the tau intron 10 +16C → T mutation. We prepared a transgene construct containing intronic sequences required for exon 10 splicing in the longest tau isoform cDNA. Although mice bearing the construct without the intronic mutation showed normal developmental changes of the tau isoform from 3R tau to equal amounts of 3R and 4R tau, mice with the mutation showed much higher levels of 4R tau at the adult stage. 4R tau was selectively recovered in insoluble brain fractions in their old age. Furthermore, these mice displayed abnormal tau phosphorylation, synapse loss and dysfunction, memory impairment, glial activation, tangle formation, and neuronal loss in an age-dependent manner. These findings provide the first evidence in a mouse model that a tau intronic mutation-induced imbalance of 3R and 4R tau could be a cause of tauopathy.

RodZ regulates the post-transcriptional processing of the Shigella sonnei type III secretion system.

The expression of the type III secretion system-a main determinant of virulence in Shigella-is controlled by regulator cascades VirF-InvE (VirB) and CpxAR two-component system. A screen for mutants that restore virulence in the cpxA background led to the isolation of a mutant of rodZ, a cytoskeletal protein that maintains the rod-shaped morphology of bacilli. InvE is normally repressed at 30 °C because of decreased messenger RNA (mRNA) stability, but rodZ mutants markedly increase invE-mRNA stability. Importantly, the inhibition of InvE production by RodZ can be genetically separated from its role in cell-shape maintenance, indicating that these functions are distinguishable. Thus, we propose that RodZ is a new membrane-bound RNA-binding protein that provides a scaffold for post-transcriptional regulation.

Regulation of TCR Vγ2 gene rearrangement by the helix-loop-helix protein, E2A.

V(D)J recombination of Ig and TCR genes is strictly regulated by the accessibility of target gene chromatin in a lineage- and stage-specific manner. In the mouse TCRγ locus, rearrangement of the Vγ2 gene predominates over Vγ3 rearrangement in the adult thymus. This preferential rearrangement is likely due to the differential accessibility of the individual Vγ genes, because the levels of germ line transcription and histone acetylation of the Vγ genes are well correlated with the rearrangement frequency in adult thymocytes. However, factors responsible for the differential regulation of the Vγ gene rearrangement have been largely unknown. In this study, we demonstrated that Vγ2 rearrangement in the adult thymus was substantially reduced in mice deficient for the basic helix-loop-helix protein, E2A. The decreased rearrangement is likely caused by the reduced accessibility of Vγ2 chromatin, since germ line transcription and histone acetylation of the Vγ2 gene were reduced in an E2A dosage-dependent manner. We further showed that E2A bound around the Vγ2 gene in vivo and we identified two canonical E-box sites downstream of Vγ2, to which E2A can bind in vitro. Furthermore, these two E-box sites had the ability to activate transcription upon E2A over-expression. These data suggest that E2A directly binds to and increases accessibility of Vγ2 chromatin, thereby facilitating Vγ2 rearrangement in the adult thymus.

Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus.

The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.

A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo.

Although amyloid beta (Abeta) oligomers are presumed to cause synaptic and cognitive dysfunction in Alzheimer's disease (AD), their contribution to other pathological features of AD remains unclear. To address the latter, we generated APP transgenic mice expressing the E693Delta mutation, which causes AD by enhanced Abeta oligomerization without fibrillization. The mice displayed age-dependent accumulation of intraneuronal Abeta oligomers from 8 months but no extracellular amyloid deposits even at 24 months. Hippocampal synaptic plasticity and memory were impaired at 8 months, at which time the presynaptic marker synaptophysin began to decrease. Furthermore, we detected abnormal tau phosphorylation from 8 months, microglial activation from 12 months, astrocyte activation from 18 months, and neuronal loss at 24 months. These findings suggest that Abeta oligomers cause not only synaptic alteration but also other features of AD pathology and that these mice are a useful model of Abeta oligomer-induced pathology in the absence of amyloid plaques.

Structure and functional characterization of Vibrio parahaemolyticus thermostable direct hemolysin.

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 A resolution. The TDH tetramer forms a central pore with dimensions of 23 A in diameter and approximately 50 A in depth. Pi-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.