The Current Status Of Neuroprotection in Congenital Heart Disease Part 2

The microscopic images were then subjected to image calculation using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The signal of Hsp60 was multiplied by "x". 

Hsp60 is an important protein molecule that plays an important biological role in the human body. Research in recent years has shown that the presence of Hsp60 is closely related to human memory.

Hsp60 is a molecular chaperone protein that is mainly involved in protein folding, unfolding, stabilization, and degradation. These processes are very important for normal metabolism and cellular function of the human body. At the same time, Hsp60 also has antioxidant, anti-inflammatory, and immunomodulatory effects, and plays a great role in maintaining human health and the balance of the immune system.

Research has found that Hsp60 plays a crucial role in memory formation and maintenance. During the process of learning and memory, brain cells produce a large number of Hsp60 molecules. These molecules can protect cells from interference and damage from the external environment to ensure the smooth progress of learning and memory. In addition, Hsp60 can also affect human thinking and behavior by changing the signal transduction of brain cells and the development of neurons and has a positive impact on cognition and stress resistance.

Although the role of Hsp60 has shown positive effects on the human body, people also need to realize that both excess and deficiency of Hsp60 may have negative effects on human health. Therefore, we should actively explore and study the mechanism of Hsp60 in various physiological and pathological conditions of the human body to better understand the position and role of Hsp60 in human health. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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The "x" value was determined as follows: subtracting the "x"-folded Hsp60 signal from the mitochondrial DNA signal in the control group made the mitochondrial DNA value less than or equal to zero. The signal of histone H2B was multiplied by "y". 

The "y" value was determined as follows: subtracting the "y"-folded histone-H2B signal from the nuclear-DNA signal in the control group made the nuclear-DNA value less than or equal to zero. If the background signal becomes high from the multiplication, the contrast of the multiplied image can be adjusted for the same conditions in all the samples. 

The image of dsDNA staining, minus the adjusted image of multiplied Hsp60 and histone H2B, was used for subsequent analysis. Using the "Adjust-Threshold" function, these images were binarized. We selected dsDNA puncta ranging in size from 2 to 20 µm2 (circularity 0.1–1.0), and they were counted using the "Analyze-Analyze Particles" function.

2.3. Electron Microscopy

One day before transfection, HeLa cells were plated at a density of 600 cells/µL. The expression of genes of interest was silenced in HeLa cells using Stealth RNAi siRNA (Thermo Fisher Scientific) in Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions (with a final RNA concentration of 10 nM). 

Four days after siRNA knockdown, cells were subjected to fixation. Cell-culture samples were fixed with 2% PFA and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 37 ◦C, then placed in a 4 ◦C refrigerator for 30 min. The samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer overnight at 4 ◦C. The samples were washed three times with 0.1 M phosphate buffer for 30 min and post-fixed with 2% osmium tetroxide in 0.1 M phosphate buffer at 4 ◦C for 1 h. 

The samples were dehydrated in graded ethanol solutions, transferred to resin (Quetol-812, Nisshin EM Co., Tokyo, Japan), and polymerized at 60 ◦C for 48 h. The polymerized resins were cut into ultrathin 70-nm sections with a diamond knife using an ultramicrotome (Ultracut UCT, Leica Microsystems, Wetzlar, Germany) and were then mounted on copper grids. 

The sections were stained with 2% uranyl acetate at RT for 15 min and washed with distilled water, followed by secondary staining with a lead staining solution (Sigma-Aldrich, Tokyo, Japan) at RT for 3 min.

The grids were observed under a transmission electron microscope (JEM-1400Plus, JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 80 kV (zebrafish brain) or 100 kV (cell), and images were recorded using a charge-coupled-device (CCD) camera (EM-14830RUBY2, JEOL Ltd.).

2.4. Immunoelectron Microscopy

The samples on the gold disks were frozen in liquid propane at −175 ◦C. After the samples were frozen, they were freeze-substituted with 1% tannic acid in ethanol and 2% distilled water at −80 ◦C for 24 hr. 

The samples were then kept at −20 ◦C for 4 hr, followed by incubation at 4 ◦C for 1 hr. Next, the samples were dehydrated in anhydrous ethanol 3 times for 30 min. each time, followed by infiltration with a 50:50 mixture of ethanol and resin (LR white: London Resin Co. Ltd., Berkshire, UK) at 4 ◦C for 1 hr. The samples were transferred to fresh 100% resin and incubated at 4 ◦C for 30 min, and this process was repeated three times. 

The samples were transferred to fresh 100% resin and were polymerized at 50 ◦C O/N. The polymerized resins were ultra-thin sectioned at 90 nm with a diamond knife using an ultramicrotome (Ultracut UCT; Leica), and the sections were placed on nickel grids. The grids were incubated with anti-dsDNA antibody (35I9 DNA) (1/800, Abcam, Cat# ab27156, RRID: AB_470907) in 1% BSA/PBS at 4 ◦C O/N, followed by three rinses with 1% BSA/PBS for 1 min each. 

They were subsequently incubated with the secondary antibody conjugated to 15-nm gold particles (Goat anti-Mouse IgG polyclonal antibody) for 2 hr at RT. After rinsing with PBS, the grids were placed in 2% glutaraldehyde in 0.1 M cacodylate buffer. 

Afterward, the grids were dried and then stained with 2% uranyl acetate for 15 min and with a lead stain solution (Sigma-Aldrich) at RT for 3 min. The grids were observed through a transmission electron microscope (JEM-1400plus; JEOL Ltd.) at an acceleration voltage of 100 kV. Digital images were obtained with a CCD camera (EM-14830RUBY2, JEOL Ltd.).

3. Results

3.1. Decreased TFAM or GBA Induces an Increase in Ectopic Mitochondrial DNA

First, we showed an example of the detection of mitochondria-derived cytoplasmic DNA in HeLa cells in which the TFAM protein was knocked down by siRNA (Figure 1A). TFAM is a key mitochondrial transcription factor and also functions in mitochondrial DNA replication and repair [20]. 

Sequence polymorphisms in this gene are reported to be associated with Parkinson's disease [21,22]. After 3 days of TFAM protein knockdown, a lot of double-stranded DNA dots were detected in the cytoplasm of the HeLa cells. 

Histones are positive markers for nuclear-origin DNA in the cytoplasm, and negative markers for mitochondrial DNA and mitochondrial-origin DNA in the cytoplasm [23]. These dots did not co-localize with histones, suggesting that they were not nuclear-derived DNA. Furthermore, some of these cytoplasmic, ectopic DNA dots did not co-localize with Hsp60, a marker for the mitochondrial matrix, indicating that they resided outside the mitochondria (Figure 1B, C). 

Electron microscopy also showed that the ectopic DNA leaked from the mitochondria (Figure 2A, B). In summary, the increase in ectopic DNA dots in the cytoplasm by TFAM knockdown was due to mitochondrial DNA leaking from the mitochondria. Second, we showed another example of the detection of mitochondria-derived cytoplasmic DNA in HeLa cells in which GBA protein was knocked down by siRNA (Figure 1A). 

GBA is the causative gene for Gaucher disease, but a heterozygous mutation of GBA is also a risk factor for Parkinson's disease [24]. A lot of double-stranded DNA dots were detected in the cytoplasm of the HeLa cells after 3 days of GBA protein knockdown. Some DNA dots were co-localized with histones, suggesting that they were nuclear-derived DNA. 

Other DNA dots were not co-localized with histones, suggesting that they were not nuclear-derived DNA but mitochondria-derived DNA (Figure 1D). In summary, the increase in ectopic DNA dots in the cytoplasm by GBA knockdown was at least partly due to mitochondrial DNA leaking from the mitochondria.

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