People used to be using APP over-expression mice or APP PS1 over-expression mice and actually they showed a number of artificial phenotypes because transgenes used make these transgenic mice destroy endogenous gene loci and showed artificial phenotypes such as endoplasmic reticulum stress or calpain activation. And also some of the transgenic mice showed behavioral abnormality before any pathology takes place...
People used to be using APP over-expression mice or APP PS1 over-expression mice and actually they showed a number of artificial phenotypes because transgenes used make these transgenic mice destroy endogenous gene loci and showed artificial phenotypes such as endoplasmic reticulum stress or calpain activation. And also some of the transgenic mice showed behavioral abnormality before any pathology takes place. I think this was caused by the destruction of endogenous gene and also overexpression of APP produced a number of non-physiological APP fragments and like a soluble APP interacts with GABA receptor under the normal conditions. And this is superinduced by the overexpression paradigm. And also APP interacts with kinesin and GIP1 affecting axonal transport. So if you overexpress APP, then these interactions will be physiologically affected. So we made APP knock-in mice, in which we humanized the A-beta sequence without, you know, using the overexpression paradigm. So it’s a kind of knock-in technique that used the homologous recombination. So after humanizing the A-beta sequence, we introduced pathogenic mutations in this mouse locus. So these mice express, show A-beta pathology without any overexpression artifacts. And the mice show, there are two types of mice and the first one starts showing A-beta pathology at nine months and shows massive A-beta deposition at 18 months. And the second type of the mice shows A-beta pathology three times faster than the first type. So these two have been used by more than 1,000 groups in the world because they can avoid overexpression artifacts. And then what we found was a connection between APP, I mean, A-beta pathology and tau pathology. And, again, people used to make mice that overexpressed mutant tau. And the most frequently used one has been PS-19, for which I was also one of the co-authors published about 20 years ago. And many people have been using these mice. And the mice don’t accumulate tau, you know, show tau pathology. But they live only up to 12 months and then die. So there’s something wrong about this model. So we made a mutant MAPT knock-in mice. It was necessary to humanize the entire MAPT gene because there are, I mean, messenger RNA splicing affects the biology and pathology of tau protein. And because pathogenic mutations have been identified in the exon and intron. So after humanization of the entire MAPT gene, we introduced pathogenic mutations, actually two mutations or three mutations. And then these mice start to show tau pathology. And then we crossed these mice with APP knock-in mice and have shown that A-beta deposition actually induces tau pathologies in some of these mouse lines. And then analyzing the messenger RNA using, you know, a single RNA sequencing technology, identifying some candidate genes. So now we are in the stage of identifying, you know, there are several candidate molecules that are involved in the A-beta tau transition. And it’s very important to identify these molecules because they can be a good target for stopping A-beta pathology, inducing tau pathology. So we have identified some candidates and then we are now in the process of using reverse genetics to identify the mechanisms or cause and effect relationships. And we have identified one molecule, but the sample size is still one, only one. So there is no statistical significance at this point. So we need, still need another six months to make sure that this is a, you know, response, I mean, molecule responsible for the A-beta tau transition. And also it takes about 24 months for the transition to take place. So we believe there are multiple cellular and molecular phases involved in this process. So I’m sure there will be more than one. And we now have made another model that shows A-beta-dependent tau pathology in six months. So use of these mice will make it faster to identify the, you know, transition mediators. So now we’re in the middle of this process. I think that’s about all that we have been doing.
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