Predicting Mitochondrial Dynamic Behavior in Genetically Defined Neurodegenerative Diseases Part 3

Mitochondria are not abnormal in all genetic neurodegenerative diseases, and mitochondrial dysdynamism is not necessarily a component of all neurodegenerative conditions that exhibit mitochondrial abnormalities. 

Mitochondria are an organ in cells that are mainly responsible for energy production and cell metabolism in cells. In recent years, studies have found that mitochondrial abnormalities may be related to memory loss. However, we should not worry too much about the negative effects of mitochondrial abnormalities, because modern science and technology are becoming more and more advanced, and we can prevent the occurrence of mitochondrial abnormalities through various methods and health products in the life cycle.

A study found that mitochondrial abnormalities may be related to memory loss in the elderly. This is because mitochondrial damage can cause neuronal damage and cell death, thereby affecting the normal function of brain neurons and leading to memory and cognitive impairment. However, this does not mean that mitochondrial abnormalities are the root cause of memory loss, because memory loss may also be caused by other factors, such as malnutrition, brain damage, aging, etc.

To prevent mitochondrial abnormalities, we can improve our health by changing our lifestyle and diet. For example, daily exercise and proper exercise can promote the generation of mitochondria, improve the body's metabolic level, maintain physical health, and improve memory. In addition, diet is also an important factor. We should have a regular diet, eat more nutritious foods such as fruits, vegetables, and grains, and pay attention to avoiding high-sugar and high-fat foods.

In addition, targeted drugs can be used to protect mitochondria in moderation. There are already some drugs targeting mitochondria on the market, such as diuretics, hypoglycemic drugs, nutritional supplements, etc.

In short, mitochondrial abnormalities are associated with memory loss, but we should not worry too much because we can protect mitochondria, improve physical health, and improve memory by changing our lifestyle, and diet, and using health supplements. Let us actively prevent and improve mitochondrial abnormalities to maintain a healthy body and a sharp brain. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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For example, metabolic abnormalities are described in Alzheimer's dementia [73], a disease of uncertain and complex etiology linked to the formation of extracellular deposits of β-amyloid peptide and neurofibrillary tangles of microtubular tau protein [74]. 

Reported mitochondrial defects, which include organelle fragmentation, are varied and inconsistent [73,75]. 

The sheer scope of different mitochondrial abnormalities described in Alzheimer's disease confounds conclusions about whether they contribute in a meaningful way to, or are largely a consequence of, the primary disease. 

By contrast, heritable Parkinson's disease is a disease of mitochondria, since the most common genetic forms of Parkinsonism are linked to mutations of genes encoding mitochondrial PINK1 kinase (PARK6) that initiates mitophagy, as well as the principal mitophagy effector protein, Parkin (PARK2) [76]. 

Again, mitochondrial fragmentation is reported in some, but not all, descriptions of Parkinson's disease [77], likely because of heterogeneity and multifactorial etiology. 

Finally, Friedreich's ataxia/spinocerebellar degeneration is unambiguously a neurodegenerative disease with a mitochondrial etiology; the autosomal recessive genetic defect is in the FXN gene that encodes the mitochondrial iron-binding protein, frataxin. 

Mitochondrial iron overload in Friedreich's ataxia compromises ATP formation by the electron transport chain and provokes excessive ROS elaboration [78,79]. It is not clear if mitochondrial dynamic dysfunction contributes to, or even exists, in this disease [80]. 

Variable mitochondrial phenotypes in neurodegenerative diseases and uncertainty regarding the pathophysiological impact of abnormal mitochondrial dynamics emphasize the need for analytical platforms directly relevant to individual human patients.

4. Evaluating Mitochondrial Dynamics

The most readily obtained and obvious evidence for mitochondrial dysdynamism is abnormal mitochondrial morphology in static images of live or fixed cells, typically imaged with a mitochondrial-specific fluorophore. 

The most commonly reported mitochondrial structural abnormality is "fragmentation" or structural shortening of the organelles, conventionally and conveniently measured as reduced aspect ratio (length/width) [81,82]. 

Although the "normal" mitochondrial aspect ratio depends both upon cell type and physiological status [83], measuring it is conceptually and technically straightforward. 

Thus, a decrease in mitochondrial aspect ratio observed in high-resolution images (reduced length/width or shortening) typically reflects a relative increase in mitochondrial fission to fusion ("fragmentation"), whereas an increase in aspect ratio ("elongation") reflects an increase in fusion relative to fission. However, the altered aspect ratio does not reveal the underlying cause of dysdynamism, i.e., whether the dynamic imbalance is the consequence of altered fission, or fusion, or both. 

Moreover, fragmentation of mitochondria in cells having interconnected mitochondrial networks, such as fibroblasts, can be a normal component of mitosis and apoptosis [6]. 

Finally, short, spherical, or "fragmented" mitochondria are the norm in striated muscle cells and neuronal axons [7,8,83]. The nature of mitochondrial fusion/fission disequilibrium is best revealed by live cell imaging, using either a single mitochondrial fluorophore (e.g., Mitotracker Red, Green, or Orange), fused cells expressing different mitochondrial-targeted fluorescent proteins (e.g., Mito-GFP and Mito-RFP), or a photo-switchable mitochondrial fluorophore (e.g., MitoDendra). 

When a single fluorophore is used, video confocal microscopy can document mitochondrial fusion and fission events [82]. 

When different groups of cells expressing mitochondrial-targeted GFP or RFP are fused with polyethylene glycol, it becomes possible to quantify mitochondria content mixing from fusion as a function of time [84]. 

This same approach is easier using photo-switchable mito-Dendra [85], enabling a subset of mitochondria within cells of interest to be photo-switched from green to red fluorescing, and the fate of individual mitochondria (i.e., whether they are fusing, undergoing fission, or being transported within the cell) to be assessed over time. 

However, the photo-switching approach requires special equipment and is limited by sample throughput. 

Although it is possible to infer from serial static images of differentially labeled mitochondria that subpopulations are being transported within cells, detailed quantitative assessment of the proportion as the velocity of motile mitochondria requires time-lapse videomicroscopy in living cells or tissue with the generation of kymographs [86]. 

Our laboratory found this to be feasible when applied to both in vitro and ex vivo preparations. Moreover, as described below, these techniques are readily applied to either genetically manipulated mouse neurons or patient-derived cells [25,57].

5. Patient-derived primary Fibroblasts Exhibit Disease-Related Imbalances in Mitochondrial Fission/Fusion

It was challenging to devise an optimal research platform to evaluate abnormal mitochondrial fusion, fission, and motility vis a vis their relationship to human neurodegenerative diseases. 

Intuitively, it seems that neurons would be the best system, but primary human-diseased neurons are difficult to obtain, and iPSC-derived neurons often lose seminal characteristics of the clinical disease [87,88]. 

The relationships between underlying genetic abnormality and mitochondrial phenotype were most extensively defined using mouse gene knockout approaches. 

Germline ablation of either Mfn1 or Mfn2 is embryonically lethal [89], but fibroblasts derived from mouse embryos revealed that impaired mitochondrial fusion caused by Mfn1 or Mfn2 deletion produces mitochondrial fragmentation through unopposed mitochondrial fission; functional impairment measured as a loss of inner membrane polarization is another consequence of defective mitochondrial content exchange [90]. 

When relating mitochondrial dysmorphology to underlying genetic causes, one must consider the differences between gene ablation that abrogates expression of the suspect protein and is a standard experimental approach to studying genetic loss of function, versus naturally occurring loss-of-function mutations wherein a dysfunctional protein is expressed and may exert dominant suppressive effects. 

Thus, heterozygous ablation of the Mfn2 gene is well tolerated in mice [89], whereas the majority of patients with CMT2A have a single mutant MFN2 allele that provokes early progressive neurodegeneration [53,55]. This difference was attributed to the dominant suppression of normal MFN1 and MFN2 functioning by the single mutant allele, amplifying its functional consequences [55–57]. 

Accordinglytransgenic expression of human CMT2A mutants in mice recapitulates some CMT2A neuronal phenotypes [57,91]. However, dominant inhibition of normal mitofusins is not the only possible explanation for differences between gene knockout and expressed loss of-function mutant effects: homozygous Mfn2 (or Mfn1) gene ablation in mice does not induce neurodegeneration; it provokes early embryonic lethality [89]. 

Thus, changing MFN2 gene dosage by experimental "knockout" is not the same as expressing an MFN2 missense mutation. 

Indeed, this may be a general biological principle as gene knockdown in Drosophila models is a straightforward manipulation that does not induce the same quantity or magnitude of compensatory responses as expression of genetic mutants [92]. 

The above observations support the use of patient-derived cells carrying authentic disease-producing mutations, rather than mouse gene knockout cells, for the investigative evaluation of disease-related mitochondrial phenotypes. 

However, recapitulation of prototypical mitochondrial abnormalities in patient-derived fibroblasts was inconsistent. 

In a companion article [93], we demonstrate how mitochondrial phenotypes can be evoked by metabolic stressing in patient dermal fibroblasts from some, but not all, genetic neurodegenerative diseases. 

Moreover, we present data supporting the use of patient-derived cells as platforms for individualized examination of mitochondrial-directed therapeutics.

6. Summary and Conclusions

Mitochondrial dynamism is ubiquitous, but the relative rates and importance of mitochondrial fusion, fission, and motility differ according to cell morphology and metabolic requirements. 

Thus, neurons with high metabolic requirements and long axons have unique dynamic profiles and are especially susceptible to dynamic dysfunction. Mitochondrial phenotypes are difficult to evaluate in human neurodegenerative diseases because human neurons are not readily available. 

Previous and accompanying data demonstrate that cultured human dermal fibroblasts can be induced to exhibit neurodegenerative disease-related mitochondrial fusion-fission phenotypes by forcing mitochondrial metabolism through the simple maneuver of substituting galactose for glucose in the culture medium. 

Moreover, the same fibroblasts can be directly reprogrammed into neurons that retain hallmark mitochondrial abnormalities, including dysmotility, which is best measured in neuronal axons. 

We suggest that integration of these platforms with genetic mouse models, as depicted in Figure 4, might be an effective means of evaluating candidate therapeutics in the multitude of genetic neurodegenerative conditions exhibiting characteristic mitochondrial abnormalities, or of different patients for a personalized medicine approach.

Author Contributions: Conceptualization, G.W.D.II; writing-original draft preparation, G.W.D.II; writing-review and editing, G.W.D.II and X.D.; supervision, G.W.D.II; funding acquisition, G.W.D.II. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by NIH R35135736, R42NS115184, and a research grant from the Muscular Dystrophy Association. The APC was funded by NIH R35135736.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Conflicts of Interest: GWD is the Philip- and Sima K. Needleman-endowed Professor at Washington University in St. Louis and a past Scholar-Innovator awardee of the Harrington Discovery Institute. 

GWD is an inventor on patents issued and pending owned by Washington University in St. Louis and Mitochondria Emotion, Inc. covering small molecule mitofusin activators, and is the founder of Mitochondria in Motion, Inc., a Saint Louis-based biotech research and development company focused on enhancing mitochondrial trafficking and fitness in neurodegenerative diseases. 

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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