| Alzheimer disease(AD)is a neurodegenerative disease.It is well known that the pathological features of AD are associated with increased levels of Aβ42,an amphiphilic molecule,and thus as its levels increase,it is readily induced by itself or other molecules to assume a misfolded conformation in vivo or in vitro,leading to self-aggregation and the formation of soluble oligomers and large insoluble fibers that eventually accumulate into plaques deposited in the brain.The aggregation of Aβ42 in the brain and deposition are considered to be the main and initiating pathogenic factors driving the pathogenesis of AD,while Aβ42 aggregates,especially Aβ42 oligomers,have been recognized as extracellular toxic agents for neuronal cells,which progressively damage neuronal cells and ultimately the normal function of brain cells.Since different species of extracellular Aβ42,such as monomers,oligomers,fibrils,and different forms,such as soluble or suspended,attached or deposited,are present at different levels during different stages of AD development,the presence of Aβ42 oligomers and fibrils is an important factor in the development of AD.What are their effects on neurite motility,adhesion,and neurite outgrowth,as well as on the neurite scaffold proteins that are closely related to these cellular functions,and what are the correlations between these effects and the neurotoxicity of the corresponding Aβ42 aggregates? The correlation between these effects and the neurotoxicity of the corresponding Aβ42 aggregates is unclear,and the similarities and differences in their neurotoxic mechanisms have not yet been revealed.These elements have fundamental theoretical and experimental implications for determining the targeting and efficacy of AD therapeutics.Since it is not possible to accurately localize and assess the damage properties of different aggregates(types and forms)of Aβ42 on nerve cells and their differences in AD model animals,it is difficult to reveal their associated cytological effects,so it is necessary to conduct corresponding toxicological studies on these different forms and types of Aβ42 and their aggregates at the molecular and cellular levels to reveal their effects on nerve cell motility,survival,and neurogenesis.Therefore,it is necessary to conduct toxicological studies on these different forms and types of Aβ42 and their aggregates at the molecular and cellular levels to reveal their effects on the motility,survival,and neurogenesis of neuronal cells,and to reveal their toxicological mechanisms and internal linkages,to provide experimental and theoretical guidance for the development of AD therapeutic drugs.In this thesis,the effects of Aβ42 monomer and its two typical aggregates(oligomer and fiber)on neuronal cell migration,adhesion,and neurite outgrowth(or neuritis extension)were systematically investigated using differentiated and undifferentiated neuronal cell line SH-SY5 Y and primary hippocampal neuronal cell line HT22,respectively,and the effects of membrane integrin and neuronal scaffold protein,which are closely related to these cellular behaviors or processes,were also investigated.The aim is to determine the connection between the neurotoxic mechanisms of different extracellular Aβ42 aggregates and the corresponding cytological events,to condense the nature of the biological effects of extracellular Aβ42 and its aggregates at the molecular and cellular levels,and to promote research and development in the field of β-enter amyloid neurotoxicity as well as AD.The main findings of this thesis are as follows The main findings of this thesis are as follows.1.extracellular Aβ42 monomers do not negatively affect the motility of neuronal cells,but on the contrary,they promote to some extent the migratory escape(or avoidance)of neuronal cells from extracellular toxic Aβ42 aggregates.However,extracellular Aβ42aggregates,especially Aβ42 oligomers,significantly reduced the migratory ability of neuronal cells,while decreasing their survival rate.In contrast,extracellular soluble(or suspended)Aβ42 aggregates had a greater effect on neuronal cell survival,whereas extracellular sedimentary(or adherent)Aβ42 aggregates had a greater effect on neuronal cell migratory capacity.Furthermore,the above adverse effects of all forms of extracellular Aβ42 aggregates on neuronal cells were significantly attenuated(or inhibited)by the anti-oligomerized Aβ42 antibody HT7(a small single chain antibody developed by our group to inhibit/neutralize neurotoxic Aβ42 aggregates).2.Soluble(or suspended)and deposited(or attached)forms of Aβ42 aggregates showed different effects in causing nerve cells to become inert(decreased motility),suggesting some differences in their mechanisms of action on nerve cells.It was further found that extracellular sedimentary(or adherent)Aβ42 aggregates were higher than soluble(or suspended)Aβ42 aggregates for the proportion of integrins co-localized with nerve cell membranes,but at the same time,extracellular soluble(or suspended)Aβ42aggregates were higher than sedimentary(or adherent)Aβ42 aggregates for the proportion of integrins co-localized with nerve cell membranes.3.Molecular docking analysis revealed that Aβ42 disrupts the functional chain of neuronal integrins mainly through its RHDS motif,which eventually leads to inert neuronal cells,thus preliminarily revealing the mechanism by which extracellular soluble(or adherent)Aβ42 aggregates have a greater effect on the migration ability of neuronal cells than soluble(or suspended)Aβ42 aggregates.Therefore,this thesis proposes the idea of "anchoring" extracellular neurotoxic Aβ42 aggregates,i.e.,the appearance(or presence)of extracellular Aβ42 aggregates is like the appearance(or presence)of an unfavorable "anchor" in the brain,which binds the neuronal cells through anchoring and makes them inert.They anchor nerve cells and render them inert.If this anchoring effect of extracellular Aβ42 aggregates on nerve cells is severed or reduced,motor-damaged nerve cells can recover or be repaired even if the "anchors" themselves are not eliminated.4.Based on the close correlation between cell migration and adhesion and their close correlation with membrane integrins,the effect of extracellular Aβ42 aggregates on neuronal cell adhesion was further investigated in this thesis.At the same concentration,the effect of extracellular soluble(or suspended)Aβ42 aggregates on neuronal cell adhesion was greater than that of sedimentary(or adherent)Aβ42 aggregates.By further comparing the re-adhesion rates and mortality rates of target cells,it was found that the greater the neurotoxicity of extracellular Aβ42,the more pronounced the differences in these effects between its soluble(or suspended)and sedimentary(or adherent)forms,and also the presence of re-adherent cells(live cells that break away from adhesion)suggests that the damage to neuronal cells by extracellular toxic Aβ42 aggregates is progressive.It can be concluded that the higher the neurotoxicity of extracellular Aβ42 aggregates,the shorter the survival time of the de-adherent living cells and the faster they go to death,which inevitably leads to a high mortality rate of neuronal cells.In addition,the physical barrier effect of extracellular Aβ42 aggregates,especially of Aβ42 oligomers or fibers,can also adversely affect neuronal cell adhesion,and the physical barrier effect may remain even when binding by antibodies against oligomerized Aβ42.Thus,the physical barrier effect of extracellular Aβ42 aggregates has different effects on the migration and adhesion of neuronal cells.5.Extracellular Aβ42 aggregates have complex and diverse adverse effects on neurite outgrowth(or neuritis extension)through their biological and possibly physical effects,with the soluble(or suspended)form of Aβ42 aggregates mainly affecting neurite outgrowth(or neuritis extension)and the deposited(or adherent)form of Aβ42 aggregates mainly affecting neurite outgrowth(or neuritis extension).This implies that the different extracellular forms of Aβ42 aggregates adversely affect neurite outgrowth in similar but not identical patterns and mechanisms,and consequently damage neurite outgrowth with different focuses(from different aspects).However,the result is that a negative effect of either of these two aspects ultimately damages the synaptic network of neurite.6.The different extracellular species and forms of Aβ42 aggregates induce different changes in intracellular palladin levels and subcellular localization,which also suggests that the mechanisms by which they affect neuronal cells may differ and thus suggests that the extracellular soluble(or suspended)forms of Aβ42 aggregates exert their toxic effects through the larger surface area of neuronal cells,whereas the extracellular deposited(or attached)forms of Aβ42 aggregates exert their toxic effects only through the limited surface area of neuronal cells.Although scaffold protein palladin is important for cell migration,adhesion,and neurogenesis/growth,its upregulation is beneficial only under specific conditions,otherwise,its upregulation may eventually induce apoptosis,which may be a physiologically programmed process that is strictly regulated spatially and temporally,so scaffold protein palladin seems to be a double-edged sword for regulating neuronal cells.double-edged sword.Thus,the combination of the level and distribution of each palladin in response to a specific species and form of extracellular Aβ42 in neuronal cells produces a unique pattern of neurite outgrowth(length and number).7.Several palladin isoforms exist in mammalian cells,and the more important ones in neuronal cells are isoform 3(140 k Da)and isoform 4(90 k Da).To further analyze the specific changes in these two isoforms in the presence of different extracellular Aβ42aggregates,antibodies targeting this sequence were prepared based on a segment of amino acid sequence PalΔ11-15(amino acid residues 656-879)that is unique to these two isoforms.The antibody was tested to have good specificity for palladin isoform 3(140 k Da)and isoform 4(90 k Da).The antibodies prepared against PalΔ11-15 were applied to determine the changes in the levels of palladin isoform 3(140 k Da)and isoform 4(90 k Da)in the target cells of all the above groups,where the changes in isoform 3(140 k Da)were more pronounced and consistent with the changes in total palladin levels,while isoform 4(90 k Da)was relatively stable and almost unchanged.This suggests that palladin isoform3(140k Da)may be the palladin molecule in neuronal cells that primarily responds to extracellular Aβ42 aggregation and is associated with impairment of neuronal cell migration,adhesion,and neuronal synapse growth(both number and length).Extracellular Aβ42 aggregates may function as extracellular binding matrices,and the anchoring effect(or binding effect)they produce induces corresponding changes in the intracellular material of binding neuronal cells.The results of this thesis suggest that the extracellular aggregation and deposition of Aβ42 disrupts the normal interaction between neuronal cells and the extracellular matrix while tethering the neuronal cells,which may be part of the nature of the neurocytotoxicity of these extracellular Aβ42 aggregates,especially the Aβ42 oligomers.Of course,under physiological conditions,the aggregation and deposition of extracellular Aβ42 may be very slow,so the process of tethering to neuronal cells may also be slow and prolonged.This is where the difficulty of preventing and treating neurodegenerative diseases such as Alzheimer disease.Based on all the above findings and the proposed "anchoring" of neuronal cells by extracellular Aβ42 aggregates,we will further investigate how to effectively eliminate these unfavorable "anchors" from extracellular-intracellular and in vitro-in vivo levels in our future studies.In the future,we will study how to effectively eliminate these unfavorable "anchors" and how to protect the motor function,metabolic function,and neural network integrity of neuronal cells,etc.,and continue to make a small contribution to the early conquest of Alzheimer disease. |
PDF Full Text Request