Alzheimer disease : a super-resolved picture of the amyloid β-peptide producing machinery

Abstract

Alzheimer disease (AD) is the most common neurodegenerative disease, influencing 40 million patients in the world. Pathologically, AD is hallmarked by intracellular tau tangles and extracellular Amyloid-β peptide (Aβ) plaques. Aβ is generated from the amyloid precursor protein (APP) and its C-terminal fragment (APP-CTF). Aβ is involved in the pathology of AD and has been studied for over 30 years. However, detailed subcellular information of Aβ and the APP processing pathway is still lacking. In this thesis, we use super-resolution microscopy, which allows approximately ten times higher resolution than traditional confocal microscopy and therefore can be used to study the subcellular localization of full-length APP (FL-APP), APP-CTF, and Aβ in mature primary cultured mouse hippocampal neurons from both wild type and a disease model (AppNL-F knock in mice).

In Paper I, we showed super-resolution microscopy is a powerful technique to study the subcellular localization of Aβ42 in neurons, especially in synapses. With super-resolution microscopy, we can observe lots of details that cannot get by confocal microscopy, it is possible to distinguish between the pre- and the postsynapse and resolve the synaptic cleft. We found that Aβ42 was enriched in the presynaptic side, but not in the postsynaptic side. Aβ42 was only partly colocalized with synaptophysin, which means that some Aβ42 is localized to other vesicles besides the synaptic vesicles. In Paper II, we continued to illustrate the subcellular localization of Aβ42, FL-APP, APP-CTF and N-terminal fragment (APP-NTF) in hippocampal neurons and showed that Aβ42 was present in early endosomes in immature presynapse. In mature synapses, partly Aβ42 was localized in clathrin coated vesicles. Interestingly, in the soma region, Aβ42 was not present in early endosomes, but in late endosome/lysosome and autophagosome. We also found that some FL-APP, and a high percentage of APP-CTF/-NTF were present in early endosomes in the soma region. In the synapse, only APP-CTF, but not FL-APP, was located in presynapse, in line with the presynaptic localization of Aβ42. In Paper III, we investigated the subcellular localization of APP-CTF and Aβ42 in AppNL-F knock in neurons. We found that mature and immature APP levels were altered in AppNL-F neurons. In Paper IV, we used iPALM microscopy to further characterize the Aβ42 containing vesicles in the presynaptic regions of hippocampal neurons. With this technique, the resolution along the z-axis was improved, and therefore, we can get more details in three dimensions.

Overall, our studies use different types of super-resolution microscopy techniques to reveal the subcellular localization of FL-APP, APP-CTF, and Aβ. Subcellular details of APP amyloidogenic pathway in hippocampal neurons were illustrated, such as APP-CTF and Aβ42 are only enriched in presynapse, but not postsynapse; mostly Aβ42 in presynapse has different pool rather synaptic vesicles; No Aβ42, but FL-APP located in early endosome. These finds will be helpful to find a high targeting to specific site, disease modifying drug for AD.