The effect of V‐ATPase function defects in pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) is a neurological degenerative disease characterized by a decrease in cognitive function and associated with a decline in life function and abnormal mental behavior.1 The aggregation of Aβ in brain parenchyma is the pathogenic factor and key link of AD.2 Aβ degradation has been gradually proven to be dependent on the degradation function of the small glial cell lysosomes in brain.3 The aggregation of Aβ in brain involves the rate balance between the generating and eliminating Aβ.4 As the final‐degradation organelles of Aβ, lysosomes are the most important organelles of the Aβ degradation.5 The activity of protease in intracellular lysosomes is mainly determined by the pH value in lysosomes.6 To maintain the pH value in a variety of important organelles is inseparable from the vacuolar‐type H⁺‐ATPase (V‐ATPase), which belongs to the family of the ATP dependent proton pump. And, we have already known that bafilomycin A1 (Baf A1) is a proton‐pump inhibitor which can inhibit the V‐ ATPase transporting H+ form cytoplasm to lysosomes. The pH value will increase when the H+ cannot be transported into lysosomes. The Aβ degradation would be negatively influenced by the acid environment changed in lysosomes. Accordingly, the Aβ aggradation aggravates.

Segregate and culture the primary microglia from TgAPPsw (Tg2576) mice and wild mice brain.7 Because the fluorescence intensity of pH‐sensitive fluorescein reduces with the pH value, the pH assessment of cytoplasm and lysosome is based on the fluorescence ratio of pH‐sensitive fluorescein to pH‐insensitive rose essence. According to this principle, we detect the differences in intracellular pH value and lysosome pH value between the TgAPPsw (Tg2576) mice and wild mice. We observe the fluorescence intensity of these fluoresceins by confocal microscope and calculate fluorescence intensity ratio of pH‐sensitive fluorescein to pH‐insensitive rose essence. According to the ratio, we get the pH value of cytoplasm and lysosom. Then, we find out the functional variation of the V‐ATPase. HT22 cells are divided into control group, no serum culture group, Aβ processing group, and Aβ+Bafilomycin A1 group. Stimulate the HT22 cells with 40 μmol/L and 10 μmol/L of Aβ25‐35, then measure the changes of cell viability, WBC spots, LC3, and V‐ATPase protein.

HT22 cell viability measurement results show that HT22 cell viability is dependent on Aβ25‐35 dose (P < 0.05). High concentration of Aβ25‐35 has a significant toxic effect on cells, and the cytotoxicity is weaker with concentration decreasing (Figure 1A). In addition, the toxicity of the Aβ25‐35 to HT22 cells also dependents on time. Cell vigor decreases in 24 hours after treating cells with 40 μmol/L Aβ25‐35(Figure 1B). The cell viability increases after add 100 nmol/L proton‐pump inhibitor BafA1 to cells (Figure 1C). In vitro experiments of TgAPPsw mice, we found there was an abnormality in the function of small glial cells in the rat brain isolated and cultured in mice. Aβ can induce significant autophagy in cells, and this autophagy has a certain correlation with the concentration and reaction time of Aβ.

Figure 1.

A, Effect on HT22 cell viability in different capacity of Aβ25‐35 for 24 hours; B, Effect of A beta 25‐35 (40 mol/L) on the viability of HT22 cells at different time; C, Effect of Bafilomycin A1 (100 nmol/L) on viability of HT22 cells; D, Expression of LC3‐protein in HT22 cells treated with A beta 25‐35 within 24 hours. 171 × 159 mm (300 × 300 DPI)

The LC3 (Figure 1D) and DAPI staining results observed by fluorescence microscope show that some blob‐like autophagosomes, characterized by brightly colored plaques, appear in HT22 cells by Aβ25‐35 effect. Higher Aβ25‐35 concentration (40 μmol/L) led to the increase in plaques in HT22 cells (Figure 2A and B). The number of visible spots increases significantly after adding 100 nmol/L BafA1 in higher and lower Aβ25‐35 concentration HT22 cells.

Figure 2.

A, Immunofluorescence spots of HT22 cells in different treatment groups (x1000); B, Statistical analysis of immunofluorescence spots of HT22 cells in different treatment groups; C, Expression of LC3 and V‐ATPase in A beta (40 mol/L) group and Aβ+ Baf A1 treatment group; D, Column graphs of the expression of LC3 and V‐ATPase; E, Determination of cytosolic pH in primary cultured microglia from 18‐days‐old fetal rats of APP transgenic mice and WT wild‐type mice; F, Determination of cytosolic pH in primary cultured microglia of TgAPPsw (Tg2576) mice and fetal rats at the gestational age of 18 days; G, Differential expression of V‐ATPase in lysosomal membrane of primary cultured microglia from TgAPPsw (Tg2576) mice and normal mice at 18 gestational age; H, Columnar graphs of V‐ATPase expression. 73 × 137 mm (300 × 300 DPI)

24 hours after adding Aβ25‐35 to HT22cells, the Western blotting results of Aβ25‐35, Aβ25‐35 + BafA1, and control group indicate that the LC3‐II/Iand V‐ATPase expression quantity in Aβ25‐35 and Aβ25‐35 + BafA1 group are higher than the control group(P < 0.05) especially in the Aβ25‐35 + BafA1 group (Figure 2C and D).

The difference in pH value in primary cultured microglia between TgAPPsw (Tg2576) and 18 days wild‐type mice is showed in the line chat. The pH values of TgAPPsw(Tg2576) are higher than wild type. With the addition of the ascorbic acid, the intracellular pH values of each group appear to drop significantly (Figure 2E and F).

After we know the difference in pH, the change in V‐ATPase is obvious too. The expression of V‐ATPase in the lysosomal membrane of primary cultured microglia of APP mice is significantly lower than that of wild type, and the expression of V‐ATPase is increased in TgAPPsw model and wild type after using MSCF in situ activated microglia, and the increase is more obvious in TgAPPsw mice model (Figure 2G and H).

In the TgAPPsw (Tg2576) mice study, it has been found that the degradation ability of Aβ in microglia cells is obviously weakened. The pH value of cytoplasm, lysosome, and the V‐ATPase protein expression is significantly different from wild‐type mice.8 The LC3 staining results show that more blob‐like autophagy structures emerge in higher concentration Aβ25‐35 processed group than the lower concentration group which indicates that a high concentration of Aβ25‐35 is more capable of stimulating cells to produce autophagy structure than a low concentration of Aβ25‐35 (Figure 2A).

The expression of V‐ATPase results also show that a distinct autophagy can be induced by Aβ in HT22 cells, and this kind of autophagy is related to Aβ dose and lasting time of reaction. The result also indicates that the quantity of autophagosomes increases with the rise of V‐ATPase expression level. Furthermore, Bafilomycin A1 can induce V‐ATPase expression to increase the accumulation of autophagy within the cell. It is also found that H₂O₂ and Aβ can cause the increase in autophagosomes in HT22 cells, suggesting oxidative stress can initiate early autophagy. It also indicates the free radical scavenger can reduce the damage caused by oxidative stress9 (Figure 2C and D).

From the determination results of the pH value of the cytoplasm, we envisage that the reduction in Aβ hydrolysis ability may be due to changes of intracellular and lysosomal acidic environment, which result in abnormal function of acid hydrolase and the cell autophagy (Figure 2E). Because of the abnormal expression of V‐ATPase on the membrane of TgAPPsw, the acidic environment of cytoplasm and lysosome turn into more alkaline than normal, which causes the abnormal function of lysosomes leading to the decrease in autophagy. Finally, when the Aβ cannot be hydrolyzed and eliminated by cells, the AD occurs.

The expression changes of V‐ATPase can affect the autophagy ability of cells.10 The brain microglia cytoplasm and lysosome pH value of AD animal model TgAPPsw (Tg2576) mouse and wild‐type mouse are significantly different, and the differences are determined by the different expression of V‐ATPase in TgAPPsw and wild‐type mouse.

Altogether, there are significant differences between TgAPPsw (Tg2576) mice and wild‐type mice on pH value and V‐ATPase expression level in the cytoplasm and lysosome of microglia in brain. The expression of V‐ATPase increased with the addition of inhibitor Baf A1 in microgila. This study may provide new insights into the pathogenesis and treatment of AD.

CONFLICT OF INTEREST

The authors declare no conflict of interest.