Here is an example for studying of Axon initial segment. The Axon initial segment is the very beginning part of Axon labeled as red in this picture. So in this segment, in this area, the material here, the substance are very compact. And then the unique character of this region is this region is responsible for actual potential generation. So if we take a close look of this region here, inside of this region we have very complicated cytal skeleton network. And on this membrane, plasma membrane, we have concentrated ion channels, including potassium channel and sodium channel. And then between the cytoskeleton and ion channels there are some adaptor proteins. For example, AnkG And beta spectrum, to connect cytoskeleton and ion channels. To concentrate this ion channel within this small region to make the action potential generation possible. And in this cytoskeleton and the adapter proteins can together form a kind of selective filter at this region. Some protein can pass through this filter get into the distill area of axon. Some protein cannot pass through this filter, will be blocked by this filter and will be only localized in the soma and dendrites region. So this part here is like the gating machinery controlling the material transfer within the axon. And in my lab we first confirm the existence of this selective filter at the AIS region, Axon Initial Segment region. We inject two different sides of dextrane into the cell body. Into camphoneurons. One is the small molecule, 10 kilodelta dextrane and the other is the big molecule 70 kilodelta dextrane and in what type neuron we can see only the small molecule 10 kilodelta can pass through this selective filter, get into the distal area of axon. But the big molecule the 70 kilodelta dextrin cannot pass through this filter and. Will only localize in the soma and dendrite region. But in a. Disease model, animal APPPS1 this disease model animal harbors two familial Alzheimer's disease mutation. One is related to a gene called app, [INAUDIBLE] protein and the other is associated with 91. So in this Alzheimer's disease Mouse neuron. We can see both small molecule 10 kDa in one and 7 kDa in one and the big molecule can pass through this filter get into the axon. So suggest that in the neuron the selective filter at the AIS region is broken. Because the mouse line we used APPPS1 harbors two familiar Alzheimer's disease mutations. One is related to APP, the other one is related to purse 91. And we want to know which one, which gene is contributing to our phenotype, the broken filter phenotype. So we use a single, we use an alliance only harbors single mutations. The one was IPP only or the one's with PS1. Mutants only. And from data we can know only the one with PS1 mutation or only the lines with PS1 mutation, give us the similar broken filter phenotype. So we know the selective filter is impaired in the PS1 mutant lines. And in this PS1 mutant lines, the critical adaptive protein at as region AnkG. The level of AnkG is decreased in this PS1 mutant lines. We confirm the decrease of AnkG level in different age of the animal. And if we put back AnkG, AnkG can rescue the impaired filtering as written in AD neurons. We said it further we want to know the mechanism of PS one induces the AnkG, down regulation and in the broken filtering phenotype. So. We did a micro [INAUDIBLE] micro compare one type neuron and APPPS1 [INAUDIBLE] PS1 neuron and then there's one micro [INAUDIBLE] called [INAUDIBLE] 3425P, it's increased. 22 fold compared in 80 neuron compare to the multi neuron. And then we confirm the op regulation of this particular micron in different ages of animals. And how PS1 mutation induced up regulation of this particular micron. We know this miR is produced from its parent miR. MiR-342. And this pairing to miR is expressed within an in region of this EBL gene. And the EVL gene is regulated by a transcription factor called IRF9. And then PS1 mutation can mediate upregulation of beta pathway and PS1 mutants can upregulate the level of iR-F9. So iR-F9 then can upregulate, can increase the level of expression of miR-342. And then the pairing to miR-342 get spliced or get cleaved and then if the protection of miR-342-5p. And then the AnkG mRNA region is one of the target of this MiR-342-5p. And then here we confirm the miR-342-5p can target the three prime UTR region of mRNA of AnkG. By the luciferous reporter asking [INAUDIBLE] and but our is the [INAUDIBLE] if we decrease the level of this miR-342-5p and we can rescue the impaired filtering phenotype. At AIS region. So what's the functional consequence of this broken filtering at the air as region in aiding neurons? For example, one sodium channel, sodium b 1.6 in the neuron, this sodium channel only concentrates in the AS region. But in the 80 neuron, this sodium channel is not as concentrated as [INAUDIBLE] and it's [INAUDIBLE] to the and another example is an R2B. An R2B is a subunit of an empty eight receptor. Normally, in the wild half cell R2B only localizes in the soma and dendrites. But in the AD neurons and there are to be. Can be localized in the distal area of axon. And then these missed localization of these I channels or neurotransmitter receptors can lead to the dysfunction of action potential generation, action potential transmission, synaptic transmission and then the function of neurons. And then taken together our stories like this. In the world have neurons AnkG and other settle skeleton proteins can form a selective filter at the air as region and the some protein can pass through this filter get into axon. But other protein cannot pass through this filter will only localize at the soma and the dendrite region. But in the AD neuron in the Alzheimer diseased neuron, because of the PS1 mutation through this complicated pathway, cause the increased level of mIR-342-5p. And the target, one of the target of this it's the 3 point mpr nre of interim g. And then decrease a level of interim g in AD neuron. So the down regulation of interim g can cause the broken filter at the AIS region in AD neuron. So lots of the protein can pass through this broken filter now and get into a distal area of axon. And then this may lead to the alteration of actual potential generation and synaptic transmission. So before people always think Alzheimer disease is an age related disease. The cognitive dysfunction or impairment only appear happen during the late stage of life. But here our results, so, if you carry the familial mutation that your neurons are wrong from the very beginning, even during development and [INAUDIBLE] stage. And now we, the other interest issue is. The AIS region is not myelinated and why is that and how the AIS region is become vacate. It's quite obvious that AIS region because action potential is initiated here. So loss of ion channels are concentrated here. And then there are a lot of ions that flow through this region, so this region needs to be naked and not myelinated normally. And we studied the mechanism of why this region is not myelinated. And the critical protein here is also AnkG, AnkG is this domain. Interact with other proteins that has this domain. And recruit caspases like caspase 6 or caspase 3. And then these caspase can cut, can a protein called one type three. And one type three is a protein localized at the axon memory and recruit [INAUDIBLE] to come closer to the axon and then makes the myelin sheath and then makes the wrap around the axon. So through the high level of AnkG at the AIS region, the neuron can decrease the level of Neuregulin-1 type III. So this S region is normally not myelinated. Okay.
