HSP70 protects human neuroblastoma cells from apoptosis and oxidative stress induced by amyloid peptide isoAsp7-Aβ(1–42)

The initial stage of Alzheimer ’ s disease (AD) is char-acterized by aggregation of monomeric amyloid- β (A β ) and accumulation of A β as insoluble amyloid plaques, 1 which are in dynamic equilibrium with soluble oligomers of A β . 2 Formation of neurotoxic oligomers most likely proceeds via a nucleation-dependent mechanism and is initiated by structurally and/or chemically modified forms of A β . 3 The A β isoform with isomerized aspartic acid residue at position 7 (isoAsp7-A β (1 – 42)) is one of the most common in amyloid plaques. In contrast to intact A β , a synthetic peptide corresponding to isoAsp7-A β (1 – 42) causes cerebral amyloidosis in AD animal models 4 and induces neuronal cell death by apoptosis. 5 These findings point to isoAsp7-A β (1 – 42) as an agent triggering the initial step of AD – oligomerization of endogenous A β . 1 Oxidative stress has a significant role in the development of AD, 6 leading to an increase in the major stress protein HSP70. We have previously shown that recombinant human HSP70 reduces oxidative stress in innate immunity cells, 7 prevents death of neurons and reduces amyloid plaque burden in the brain of 5xFAD mice when administered intranasally. 8 This study examines the protective effect of recombinant human HSP70 on apoptosis and generation of reactive oxygen species (ROS) in human neuroblastoma cells, SK-N-SH, induced by isoAsp7-A β


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The initial stage of Alzheimer's disease (AD) is characterized by aggregation of monomeric amyloid-β (Aβ) and accumulation of Aβ as insoluble amyloid plaques, 1 which are in dynamic equilibrium with soluble oligomers of Aβ. 2 Formation of neurotoxic oligomers most likely proceeds via a nucleation-dependent mechanism and is initiated by structurally and/or chemically modified forms of Aβ. 3 The Aβ isoform with isomerized aspartic acid residue at position 7 (isoAsp7-Aβ(1-42)) is one of the most common in amyloid plaques. In contrast to intact Aβ, a synthetic peptide corresponding to isoAsp7-Aβ(1-42) causes cerebral amyloidosis in AD animal models 4 and induces neuronal cell death by apoptosis. 5 These findings point to isoAsp7-Aβ(1-42) as an agent triggering the initial step of ADoligomerization of endogenous Aβ. 1 Oxidative stress has a significant role in the development of AD, 6 leading to an increase in the major stress protein HSP70. We have previously shown that recombinant human HSP70 reduces oxidative stress in innate immunity cells, 7 prevents death of neurons and reduces amyloid plaque burden in the brain of 5xFAD mice when administered intranasally. 8 This study examines the protective effect of recombinant human HSP70 on apoptosis and generation of reactive oxygen species (ROS) in human neuroblastoma cells, SK-N-SH, induced by isoAsp7-Aβ(1-42).
In the concentration range of 1-10 μM, isoAsp7-Aβ(1-42) induced a dose-dependent increase in the proportion of apoptotic cells and production of ROS (Figures 1a and b). However, the proportion of necrotic cells in the population treated with isoAsp7-Aβ(1-42) increased by no more than 6% compared with the control. Predominant cell death by the apoptosis pathway indicates a specific effect of isoAsp7-Aβ(1-42), and the mechanism of this effect is associated with the induction of oxidative stress. Preincubation of the cells with HSP70 resulted in a significant decrease in the proportion of apoptotic cells and the level of ROS induced by isoAsp7-Aβ(1-42) ( Figure 1b). We have not detected any interaction between the isoAsp7-Aβ(1-42) and HSP70 by ITC and MST (data not shown). In addition, in experiments with cells the concentration of HSP70 (30 nM) was significantly lower than that of isoAsp7-Aβ(1-42). Thus, the protective effect of HSP70 could not be attributed to the interaction with isoAsp7-Aβ(1-42).
We have investigated the impact of various inhibitors of intracellular signaling pathways that regulate apoptosis and oxidative stress on the effect of isoAsp7-Aβ(1-42). We used wortmannin, U73122, PD98059, SB203580 and SP600125 for inhibition of PI3K, PLC, ERK, p38MAPK and JNK, correspondingly.
Although isoAsp7-Aβ(1-42) increased the level of ROS and the percentage of apoptotic cells, all inhibitors reduced these parameters in SK-N-SH cells (Figure 1c). This indicates that isoAsp7-Aβ(1-42) causes a response in the cells, linked to activation of the intracellular signaling pathways mediated by JNK, ERK, PI3K, p38MAPK and PLC. Although U73122 had no effect on the HSP70 protection against isoAsp7-Aβ(1-42), SB203580 reduced the protective effect of HSP70 against apoptosis, but did not affect the level of ROS. Shutting down protein kinases JNK, ERK and PI3K completely eliminated the HSP70 protective effect against oxidative stress and apoptosis induced by isoAsp7-Aβ(1-42). These data suggest that the observed protective effects of HSP70 are due to the activity of protein kinases JNK, ERK and PI3K.
In conclusion, we have demonstrated for the first time that the protective effect of HSP70 is realized via two main mechanismsreduction of the oxidative stress and apoptosis induced by the peptide isoAsp7-Aβ(1-42) in human neuroblastoma cells. Signaling pathways involving protein kinases JNK, ERK and PI3K have a key role in these mechanisms. It is reasonable to believe that the protective effect of HSP70 against isoAsp7-Aβ(1-42) can also be observed at the level of the whole organism, and used as an approach for the prevention of AD by utilizing recombinant HSP70.