Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure

Summary Mitochondria not only supply the energy for cell function, but also take part in cell signaling. This review describes the dysfunctions of mitochondria in aging and neurodegenerative diseases, and the signaling pathways leading to mitochondrial biogenesis (including PGC‐1 family proteins, SIRT1, AMPK) and mitophagy (parkin‐Pink1 pathway). Understanding the regulation of these mitochondrial pathways may be beneficial in finding pharmacological approaches or lifestyle changes (caloric restrict or exercise) to modulate mitochondrial biogenesis and/or to activate mitophagy for the removal of damaged mitochondria, thus reducing the onset and/or severity of neurodegenerative diseases.


| MITO CHONDRIA IN THE NORMAL CONDITION
The mitochondrion is a double-membrane-bound organelle in most eukaryotic cell. Generally, mitochondria are between 0.75 and 3 μm in diameter but differ in lengths and structure. Hundreds of mitochondria form cable-like structures and supply the energy demand of cells through the oxidative phosphorylation (OXPHOS) process.
Mitochondria constantly divide, fuse, and alter their size and shape, forming a dynamic network to maintain their integrity and quantity. Normal mammalian cells maintain a balance between fusion and fission. Mitofusin 1 and 2 (Mfn1/2) proteins are involved in fusion of the outer mitochondrial membrane, and optic atrophy 1 (OPA1) protein mediates fusion of the inner mitochondrial membranes. Mitochondrial fission 1 (Fis1) and dynamin-related protein 1 (Drp1) are associated with mitochondrial fission. Fis1 localizes primarily on the outer mitochondrial membrane. Drp1is a cytoplasmic protein that translocates to mitochondria and interacts with Fis1 to enhance fission. 1 Figure 1 illustrates these general features of mitochondrial fission and fusion.
A positive mitochondrial membrane potential (Δψm) of 120-200 mV is fundamental for the normal performance and survival of cells, especially those that have a high-energy requirement.
Thus, loss of Δψm is an indicator of reduced cell health. The collapse of Δψm due to the opening of a high-conductance pore in the inner mitochondrial membrane is part of the molecular mechanism in apoptosis. The mitochondrial uncoupler carbonyl cyanide-mchlorophenylhydrazone (CCCP) depolarizes the inner mitochondrial membrane, reducing Δψm and ATP production, thus increasing the level of AMP and the phosphorylated (active) AMP-activated protein kinase (pAMPK). This sequence of events elevates the level of reactive oxygen species (ROS), leading to oxidative damage.
While mitochondria are critical in regulating cellular energy balancem, they also have well-described roles in the maintenance of essential cellular functions. These include processes such as cellular differentiation, regulation of the cellular growth cycle, and cell death. 2

| MITOCHONDRIAL B IOG ENE S IS AND ITS REG UL ATOR S
Mitochondrial biogenesis is regulated to adapt the mitochondrial population to a cell's energy demands in response to the conditions of growth, cell division, and changes in oxidative stimuli and F I G U R E 1 General features of mitochondria fusion and fission. Left: Outer mitochondrial membrane proteins Mfn1 and Mfn2 mediate fusion of the outer mitochondrial membrane while the inner membrane protein OPA1 regulates the fusion of the inner mitochondrial membranes. Right: Fis1 and Drp1 contribute to mitochondrial fission process F I G U R E 2 The proteins involved in the process of mitochondrial biogenesis and mitophagy that help to maintain the mitochondria quality. Caloric restriction (CR), physical exercise, and energetic status elevate SIRT1 or NAD + levels that promote the nuclear translocation of PGC-1α, which would enhance the transcription of genes associated with the mitochondrial function and biogenesis. Supplementing with NAD + also enhance SIRT1 activity and trigger mitophagy hormones. Mitochondrial biogenesis depends on the coordinated expression of nuclear and mitochondrial DNA. Mitochondrial transcription factor A (TFAM) and nuclear respiratory factors 1 and 2 (NRF1 and NRF2) regulate the biogenesis of mitochondria. 3,4 The PPARγ coactivator-1 family of transcription coactivators, sirtuins and AMPK, also are involved in regulating gene expression during mitochondria biogenesis ( Figure 2).

| PGC-1 families
There are three members in the PPARγ coactivator-1 (PGC-1) family: PGC-1α, PGC-1β, and PGC-1-related coactivator (PRC). The PGC-1 coactivators respond to different stimuli to promote mitochondrial gene expression. 5,6 PGC-1α, the master regulator of mitochondrial biogenesis, directly interacts with NRF1 to increase the transcription of the genes regulated by NRF1. Overexpression of PGC-1α in cerebellar neurons increases mitochondrial density by 30% and protects the neurons against mutant synuclein A53T-α or mutant Huntingtin gene (Htt)-induced degeneration. 7 The brains of mice lacking PGC-1α presents microvacuolation and neuronal loss, which highlights the important role of PGC-1α in the nervous system. 8,9 In fact, the common character of several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, is the impaired function of PGC-1α.
PGC-1β plays a role in the maintenance of basal mitochondrial functions. 7 PRC, which is expressed ubiquitously, also enhances the gene transcription of NRF1. PRC mainly regulates gene expression in proliferating cells and in growth-regulated mitochondrial biogenesis. 10 PRC depletion results in the aggregation of atypical mitochondria and severe respiratory chain dysfunction. 11

| Sirtuins
Sirtuins are class III protein deacetylases that consume one molecule of NAD + during each deacetylation cycle. Evidence form the past research indicates that sirtuins regulate the aging process and extend life span. 12,13 Sirtuin levels are influenced by diet, exercise, and environmental stress. There are seven mammalian sirtuins, SIRT1-7.
In addition to deacetylating histones or other specific transcription factors to regulate gene expression, the mammalian sirtuins also regulate the activities of metabolic enzymes to response the calorie restriction or other stresses. SIRT1 is the most studied sirtuin. Research has demonstrated that overexpression of SIRT1 can alleviate diseases, including neurodegenerative diseases, diabetes, and liver steatosis. 14,15 SIRT1 deacetylates various transcription factors and coactivators, including PGC-1α, the tumor suppressor p53, and FOXO to enhance the transcription of genes regulated by these factors. 16 In the energydeficient condition caused by disease or injury, SIRT1 activation has neuroprotective effects by promoting mitochondrial biogenesis and triggering the turnover of damaged mitochondria. 17 Unlike SIRT1, which mostly localizes to the nucleus, SIRT3 localizes to mitochondria and is essential to mediate the response to oxidative stress by activating superoxide dismutase-2 (SOD2).In response to oxidative stress, SIRT3 deacetylates SOD2 to enhance its ability to scavenge ROS. 18 Normally, the endogenous SOD2 is inactivated by acetylation and it is important for antioxidant enzymes to counteract cellular ROS. SIRT3 deficiency dramatically exacerbates the degeneration of nigro-striatal dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson Disease mice. 19 Overexpression of SIRT3 decreases the excitotoxicity of N-methyl-d-aspartic acid (NMDA) to the cultured mouse cortical neurons. 20

| AMP-activated protein kinase
AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in eukaryotes. AMPK is activated by the increased AMP/ATP ratio which leads to enhanced glucose transport, fatty acid oxidation, and so on. 21

| MITOCHONDRIAL ANOMALIE S WITH AG IN G
Aging induces many potentially interconnected defects and is a common risk factor for adult human diseases. During the aging process, multiple mitochondrial anomalies may occur, including bioenergetic deficiency, the increased oxidative stress from respiratory chain, and the accumulation of the dysfunctional mitochondria. 23,24 Damaged mitochondrial DNA (mtDNA) and the accumulation of injured mitochondria are considered major contributors to aging.
Mutation in mtDNA reduces the lifespan of mice and humans. 27 According to the mathematical models, there is only a limited clonal expansion of somatic mtDNA mutations that can occur in short-lived organisms like fruit flies. The experimentally obtained very high mtDNA mutation levels, which are unlikely to be found in nature, also reduce the lifespan of fruit flies. Additionally, adulthood is less sensitive to mtDNA mutations than is embryonic development. 28

| Mitochondrial damage and mutation caused by ROS and replication errors
Impairment of mitochondrial dynamics can result in reduced oxidative phosphorylation and cell death. Mitochondria produce ATP for cellular energy requirements, and ROS are toxic by-products generated during OXPHOS in mitochondria. Mitochondria generate about 90% of the ROS present in cells. Excess ROS may cause oxidative damage to both nuclear DNA (nDNA) and mtDNA, which may contribute to many age-related disease states. 29 Compared to nDNA, mtDNA is prone to be damaged by oxidative stress because it is not protected by associated histones and other chromatin proteins and is near the ROS-generating respiratory chain.
Besides ROS, replication errors and failure of the repair mechanisms may be the more important reason for accumulation of mtDNA mutations. 30 mtDNA replication is independent of cellular division, so the replication rate is higher than that of nDNA. 31 In the meanwhile, there is a decrease in the DNA repair enzymes such as DNA polymerase 1 and endonuclease in aged tissue. 26 Ultra-deep sequencing was used to study genome-wide mtDNA mutation load; the sequence analysis showed that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life. 32

| Accumulation of dysfunctional mitochondria
Fusion and fission proteins control mitochondrial dynamics.
Dysfunctional mitochondria are marked and selectively removed by the specific autophagic process called mitophagy. Through the process of fission, the dysfunctional mitochondria are selected for mitochondrial fragmentation, then turnovered by mitophagy and degraded in lysosomes. Therefore, fission is important for keeping mitochondrial quality and integrity. The level of both autophagy and mitophagy decline with aging, 33,34 which results in an accumulation of dysfunctional mitochondria, advanced oxidative stress, and increased cell apoptosis. Dysfunctional mitochondrial accumulation occurs in all tissues during aging, including skeletal muscle, liver, and brain.

| Parkinson's disease
Parkinson's disease is a widespread neurodegenerative disease.

| Mitophagy maintains the quality of mitochondria in cells
Mitophagy is part of the mitochondrial quality control system and is regulated by mitochondria fission-and fusion-promoting proteins.
In these processes, impaired mitochondria are engulfed into an autophagosome, which then fuses with a lysosome for degradation by lysosomal enzymes (Figure 2). Mitochondrial dynamics is a crucial process in maintaining proper mitochondrial morphology and in regulating mitochondrial function, responses to apoptotic stimuli, and monitoring mitochondrial quality. 60,61 The PINK1/Parkin interaction is crucial in regulating mitophagy in mammalian cells. PTEN-induced putative kinase 1 (PINK1) is a serine/threonine protein kinase present in the cytosol but also targeted to the outer mitochondrial membrane. Mitochondria with positive mitochondrial membrane potential import and degrade PINK1, preventing its accumulation on the outer mitochondrial membrane.

PINK1 accumulates on the impaired mitochondria with a decreased
Δψm. Parkin, a component of a multi-protein E3 ubiquitin ligase complex, binds with PINK1 accumulated on the impaired mitochondria and tags the damaged mitochondria with ubiquitin for degradation through mitophagy. 62,63 In addition, increasing evidence supports, that mitophagy also can occur in a Parkin-independent way. Without the participation of Parkin, some proteins, such as NIX, FUNDC1, or BNIP3, and cardiolipin directly interact with LC3 protein and engulf the dysfunctional mitochondria into autophagosome. 64,65 Meanwhile, other E3 ubiquitin ligases such as SMURF1 and MUL1 also can ubiquitinate the damaged mitochondria and promote mitophagy. 67 Endosomes also play a role in mitochondrial elimination.
Dysfunctional mitochondria are marked with ubiquitin by parkin, sequestered into Rab5-positive early endosomes, and ultimately delivered into lysosomes for degradation. In certain cell types, disruption of the endosomal pathway through loss of Rab5 function increases the likelihood of cell death due to mitochondrial stress. 68

| Abnormal mitochondrial function affects lysosomal activity
Maintaining mitochondrial homeostasis requires the cooperation of mitochondrial biogenesis, mitochondrial fusion, fission, and mitophagy. The lysosome is the main degradation and recycling organelle. Mitophagy tags and delivers the dysfunctional mitochondria to the lysosome for degradation. Meanwhile, mitochondrial dysfunction also affects the structure and function of lysosomes. 69 Mfn2 promotes lysosomal autophagocytosis. Mfn2 depletion in cardiomyocytes retards the fusion of autophagosomes with lysosomes. 70 Phagocytosis also can be impaired by the depletion of proteins AIF, OPA1, or PINK1, and by chemical inhibition of the electron transport chain, causing the enlarged lysosomal vacuoles.
CCCP increases mitochondrial ROS levels via membrane depolarization and specifically activates the lysosomal TRPML1 channels, causing lysosomal Ca 2+ release. This activation triggers nuclear translocation of transcription factor EB (TFEB). This calcineurindependent transcription factor can activate the autophagy/lysosome pathway by regulating the biogenesis of autophagy/lysosome organelles. 71

| Pharmacological methods
There are several pharmacological strategies aimed at triggering mitochondria biogenesis to treat neurodegenerative diseases.
Rosiglitazone and bezafibrate can activate the PPAR-PGC-1α axis, and the SIRT1 agonists quercetin and resveratrol can activate sirtuins and AMPK. 72,73 Bezafibrate increase the mitochondrial proteins and mitochondrial ATP generating capacity; as the result, it had a neuroprotective effect in this mouse model of mitochondrial encephalopathy. 76 Rosiglitazone can increase mitochondrial mass and attenuate mitochondrial dysfunction in mutant Htt-expressing cells. 77 Several antioxidants such as creatine, coenzyme Q10, and mitochondrially-targeted antioxidants/peptides seems to improve the patient's ankle mobility in PD clinical trials. 78 However, most of the antioxidants, such as dimebon, seem to have beneficial effects in the AD preclinical research, but failed in the AD clinical trials. 79,80 Since mitochondrial dysfunction occurs at the early stage of diseases, the method of pharmacological interventions should be considered.
Besides enhancing mitochondria biogenesis and the scavenging of ROS, inducing mitophagy would be another strategy to maintain mitochondria homeostasis. Urolithin A, a metabolite of ellagitannins produced by the human gut microbiota, induces mitophagy, prevents the accumulation of dysfunctional mitochondria with age, and prolongs lifespan in Caenorhabditis elegans and increases muscle function in rodents. 81 Urolithin A can go through the blood-brain barrier, which may have neuroprotective effects against neurodegenerative diseases. Spermidine is produced from putrescine and could be a precursor of spermine generation. Spermidine acts as acetylase inhibitor and induces autophagy in a SIRT1-independent manner. 82 Therefore, spermidine promotes basal autophagic flux and stimulates mitophagy repairing mitochondrial activity in aged cardiomyocytes. 83 Spermidine feeding protects from age-induced memory impairment in an autophagy-dependent manner. 84 Spermidine induces the formation of mitophagosomes and decreases the aggregation of dysfunctional mitochondrial through the PINK1/Parkin pathway. 85

| Caloric restriction
Caloric restriction (CR) is to decrease the calories intake but maintain all the essential nutrients and without malnutrition. CR decreases the production of ROS and reduces oxidative DNA damage, slows down the transcriptional changes associated with aging. 86 Sirtuins are considered to have an important role in mediating the beneficial effects of CR on longevity. 87 90 CR delayed the onset of prion disease mice but failed to delay the onset in the SIRT1 knockout strain. 91 Knocking out the mitochondrial SIRT3 prevents the protective effect of CR against hearing loss. 92 Caloric restriction enhances the number of mitochondrial cristae as well as the number of mitochondria in per cell. 93 CR also prevents excitotoxic conditions through the indirect decrease in mitochondrial permeability and calcium retention. These are mediated through CR-activated SIRT3 deacetylation and inhibition of cyclophilin D, a peptidylprolyl isomerase. 94 Additionally, CR inhibits the PI3K/AKT pathway, induces autophagy, which may increase mitophagy and maintain mitochondria homeostasis.

| Physical exercise
Some research results show that exercise might be helpful in retarding the progress of neurodegenerative diseases, 95,96 which may be associated with the recovery of mitochondrial function by exercise.
Exercise stimulates brain mitochondrial activity. Exercise not only increases resistance against rotenone, an inhibitor of complex I activity, but also increases mRNA expression of TFAM and Ndufa6, subunits of mitochondrial complex I. 97 At the same time, exercise increases mtDNA repair capacity in the mouse hippocampus and activates mitochondrial uncoupling proteins (UCP) which can regulate mitochondrial proliferation 98 and control the production of mitochondrial-derived ROS. Exercise upregulates UCP2 levels in the hippocampus, lowers cellular oxidative stress 99 and can activate autophagy, which is helpful in maintaining muscle mass. 100, 101 We have found that exercise could ameliorate the detrimental effect of chloroquine on skeletal muscles through restoring autophagic flux 104 and activating the autophagy/lysosomal pathway through AMPK pathway in cerebral cortex and striatum. 105 The enhanced mitophagy lessens the level of dysfunctional mitochondria to maintain a high quality of mitochondria in cells.

| CON CLUDING REMARK S
Mitochondrial dysfunction, the downstream oxidative stress and impaired autophagy/lysosomal activity are the main factors involved in neurodegeneration. Thus, drugs that improve mitochondria function, scavenge the excessive ROS, or enhance the autophagic flux may have the potential to treat neurodegenerative diseases. However, pharmacological agents enhancing mitochondria integrity to treat neurodegenerative diseases remain to be developed. At this point, both CR and exercise, which can enhance mitochondria biogenesis and the autophagy/lysosome pathway (including mitophagy), maybe helpful in retarding the onset and progression of neurodegenerative diseases.