Okay, so the real life situation is how this knowledge can help us to solve a real problem in our research. So the real life situation, how does amyloid-beta peptide get into the cytosol area? This amyloid-beta peptide is very important in Alzheimer's disease. Alzheimer's disease is dementia, happened in the brain region. It's related to memory decline and it affect ability to learn, reasoning, judgment, communication, and the personality. So this is a comparison of healthy age-matched control brain and the Alzheimer brain. So from this picture we can see, in the Alzheimer disease, we have a shrinking volume of the brain. We have a bigger vascular structure in the brain. So indicating there are lots of cell deaths and neuronal loss in the Alzheimer disease. If we take a close look of this, what happened inside of Alzheimer brain, we can see this down here is the control picture of neurons. And then here the big picture is what happened in the Alzheimer disease brain. Inside of the cell we have lots of protein aggregations here, and in this aggregations are made of hyperphosphorylated tau protein and we call it neurofibrillary tangles. And outside the cell, in the extracellular region, we have these so-called senile plaques in the extracellular region. And these senile plaques are made with amyloid-beta peptide. So this picture shows us the same thing. The intracellular region, we have neurofibrillary tangle made with hyperphosphorylated tau protein. And in the extracellular region we have senile plaque, or amyloid plaque, made by amyloid-beta peptide. This is the staining for amyloid-beta peptide around the cellular region. So this peptide, amyloid-beta peptide, is produced, is cleaved from its precursor protein, amyloid precursor protein. This protein is single transmembrane protein. The green region is the cell membrane and then the N-terminal of this APP, this amyloid precursor protein, localized in extracellular region, and then the C terminal is in the intracellular region. So this protein are cleaved by beta secretase here and by gamma secretase here so make this 40 to 42 amino acid peptide, amyloid-beta. And then this amyloid-beta peptide is the major component for senile plaque. Because the senile plaque are extracellular, but the interesting thing is the A-beta is produced intracellularly. So we want to know if the intracellular A-beta is also toxic to the neuron. And there are some evidence that showing the intracellular A-beta is actually an early event in AD development. First is intracellular A-beta is observed in AD and a mild cognitive impairment patient brain. And second is in the Down's syndrome where the patient has three copies of chromosome 21. APP, the precursor protein of A-beta, is also located in chromosome 21. So in Down's syndrome we also see intercellular A-beta in the brain and muscle tissue. And also the only AD animal model showing neuronal loss has significant intracellular A-beta deposition. So we want to test if the intracellular A-beta is toxic to neurons. We simply put in the A-beta, put the A-beta into the cytosol region of human neurons. With the Tunel staining we see how many cells died with this injection. And then from the data we can see with amyloid-beta 1 to 42, after two days of injection we got great cytotoxicity. And now we know the intracellular A-beta can induce p53 phosphorylation and then up-regulate Bax level and then activate caspase-6 and then eventually induce cell deaths. And both androgen and estrogen can block this pathway. So the question now is how this normally membrane associated peptide or protein can get into the cytosol region. There are several hypotheses. One is normally the A-beta is associated with membrane, but A-beta can somehow increase the permeability of membrane structure. And it can break this vesicle structure, and get in to get released to the cytosol region. And then the other hypothesis is very much similar to this one. A-beta is associated with membrane and get released to the extracellular region, and then this released A-beta can be uptake, or endocytosed, back to the cell. And in the endosome, the membrane of endosome can be broken by the A-beta. And then A-beta get released from endosome to the cytosol region. And then the third hypothesis is the A-beta get released, gets secreted to extracellular region, and A-beta can bind to some binding proteins, or so-called receptors. For example, acetylcholine receptor, alpha-7 nicotine-like acetylcholine receptors, and then these receptors can uptake extracellular A-beta and internalize the A-beta into the cell. And then A-beta get into the cytosol area of the neuron.
