The Current Status Of Neuroprotection in Congenital Heart Disease Part 3

3.2. Semi-Quantitative Evaluation of Ectopic Mitochondrial DNA

Images of Hsp60 staining (the mitochondrial matrix) and histone-H2B staining (the nuclear marker) were subtracted from images of dsDNA staining. Then, using the "AdjustThreshold" function of the lmage] software (National Institutes of Health), images were binarized. 

Mitochondria are the energy factories in cells, responsible for synthesizing ATP to provide the energy needed by cells. The mitochondrial matrix is the internal liquid of mitochondria, which contains a variety of enzymes and ions and is a key site for various biochemical reactions of mitochondria. Memory is one of the most important cognitive abilities of human beings. Although the two may seem unrelated, they are closely related.

Research shows an interaction between the mitochondrial matrix and memory. Enzymes and ions in the mitochondrial matrix can directly affect the excitability of neurons by regulating the energy metabolism and ion channel status in neuronal cells, thus affecting memory. In addition, there are a variety of signaling molecules in the mitochondrial matrix, which can be transmitted to downstream neurons through the axons and synapses of neurons, affecting the signal transduction efficiency of neurons, thus affecting the formation and consolidation of memory.

At the same time, memory activity also directly affects the state of the mitochondrial matrix. Memory activities will increase the energy consumption of neurons and promote the redox reaction in the mitochondrial matrix, thereby affecting the metabolic level of mitochondria and intracellular ion balance, further affecting the excitability and signal transduction efficiency of neurons.

Therefore, maintaining good mitochondrial health is crucial to maintaining good memory function. By maintaining good living habits, such as regular diet, moderate exercise, adequate sleep, etc., you can help maintain the health of the mitochondrial matrix. In addition, memory function can also be enhanced through appropriate pharmaceutical intervention to promote biochemical reactions and signaling within the mitochondrial matrix.

In daily life, we can exercise memory in many ways, such as reading, studying, thinking, games, etc. At the same time, we should also pay attention to maintaining good health to better maintain the health of the mitochondrial matrix. I believe that through these efforts, we will surely have better memory and better cope with various challenges in life and work. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Cistanche deserticola can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.

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We selected dsDNA puncta ranging in size from 2 to 20 um' (circularity 0.1-1.0)using the "Analyze-Analyze Particles" function. if necessary, the original images were examined for the final count. lf the signal of histone H2B was small. then the subtracted images showed donut-like structures. 

These represent cytosolic dsDNA of nuclear origin and should be eliminated from the quantity of cytosolic dsDNA of mitochondrial origin. The range of size can be modified according to the cell type or the origin of histological specimens. Figure lE shows an example of the counting of ectopic mitochondrial DNA

dots using 1B D.

Alternatively, images of Hsp60 staining were subtracted from images of dsDNA staining. Then, the number of dsDNA puncta was counted using the "Adjust-Threshold "Analyze-Analyze Particles" functions. Subsequently, dsDNA puncta without the histone-H2B signal were counted by direct observation. The latter method is suitable when the histone-H2B images show a low signal-to-noise ratio.

Altogether, this method can clearly show ectopic mitochondrial DNA leaking into the cytoplasm and allows an objective and statistical evaluation of such ectopic mitochondrial DNA in cells.

4. Discussion

In our study, we developed a method that enables the counting of cytoplasmic mitochondrial DNA. Detection of cytoplasmic mitochondrial DNA has been reported by using immunofluorescence techniques, but the methods were not quantitative for the evaluation of cytoplasmic mitochondrial DNA in most cases [15,25]. In another study, droplet PCR was conducted to quantitate circulating mitochondrial DNA [9]. 

recently, Sato et al. [26,27] published an excellent protocol to evaluate cytoplasmic DNA by immunofluorescence microscopy using Lamin B1 (LMNB1) antibodies as a nuclear structure marker and cytochrome c oxidase subunit 4 (COX4) as a mitochondrial structure marker. This protocol seems very useful for quantitating cytoplasmic dsDNA of nuclear origin. 

However, when we tried COX4 or other mitochondrial membrane markers, including both inner and outer markers, it was very difficult to precisely discriminate the cytoplasm from the mitochondria. 

For example, if we observed circular signals using a mitochondrial membrane marker and observed dsDNA signals within, then this dsDNA could represent either mitochondrial DNA inside the circular mitochondria (normal) or cytoplasmic mitochondrial DNA outside the donut-like mitochondria (ectopic). We thus used Hsp60 as a marker labeling the mitochondrial matrix to evaluate the ectopic mitochondrial DNA in the cytoplasm. 

As described in a recent review [23], there are many different types of cytoplasmic dsDNA of nuclear origin, and most of them are positive for histone markers. Due to the limitation of the combination of antibodies used for triple staining, we used histone H2B as a histone marker. 

However, if this limitation is solved, γH2AX may be a better marker for cytoplasmic dsDNA of nuclear origin. Histone-H2B-negative cytoplasmic dsDNA was seldom observed in the control HeLa cells used in this study, but such dsDNA of nuclear origin may be present if other cell lines or conditions are used. 

The in situ hybridization of mitochondrial DNA used in our recent paper is suitable for qualitative evaluation, but it might be difficult to utilize in situ hybridization for quantitative evaluation [8]. 

In situ hybridization exposes the specimen to high temperatures, which can result in deformation or noise of some parts of the specimen. On the other hand, the immunocytochemical method presented in this paper can obtain signals with low background noise. In our recent paper, in the human cells and zebrafish used as Parkinson's disease models, we showed that mitochondrial DNA was leaked out into the cytoplasm, which is toxic to the cells. We also observed that the quantity of cytosolic mitochondrial DNA was increased in the post-mortem brain tissues of patients with Parkinson's disease [8]. 

These lines of evidence suggest that an increased quantity of ectopic mitochondrial DNA leaked out from the mitochondria can induce acute and chronic inflammation resulting in various diseases, including Parkinson's disease and heart failure; thus, upregulation of cytosolic-DNA degradation or inhibition of its sensors could be a potential therapeutic target for these diseases [8,28]. 

The method introduced in this paper for the quantitative evaluation of ectopic mitochondrial DNA serves as an important tool for further research in this field.

Author Contributions: H.M. and M.T.H. formulated the hypotheses, designed the majority of the experiments, performed the experiments, analyzed the data, and wrote the manuscript. N.M. performed the experiments related to cultured cells. All authors have read and agreed to the published version of the manuscript.

Funding: This work was supported by grants from AMED (grant numbers JP21gm6110028 and JP19dm0107154 (H.M.)), the Takeda Science Foundation (H.M.), JSPS KAKENHI (grant numbers JP 14516799 (H.M.), JP 16690735 (H.M.), and JP 17925674 (H.M.)), JST (Moonshot R&D; grant number JPMJMS2024 (H.M.)), and Grants-in-Aid from TBRF (grant number TBRF-RF 21-135 (H.M.)).

Data Availability Statement: The data and tools described in this manuscript are available upon request.

Acknowledgments: We acknowledge Shinano Kobayashi for participating in helpful discussions and providing continuous support. We acknowledge the Tokyo Biochemical Research Foundation (TBRF) for providing a TBRF postdoctoral fellowship for foreign researchers to M.H. (TBRF-RF 21-135).

Conflicts of Interest: The authors declare no conflict of interest.

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