2.1. Biogenesis
Mitochondria do not originate de novo; rather, proteins involved in the maintenance of mitochondrial population and mass regulate the biogenesis of mitochondria. These proteins are encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mitochondrial transcription factor A (TFAM), transcription factor B2, mitochondrial (TFB2M), nuclear respiratory factor 1 (NRF1) and NRF2, estrogen-related receptors (ERRs), and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) play important roles in activating the transcription of genes required for mitochondrial biogenesis.
TFAM is encoded by nDNA and transported from the cytosol to mitochondria. In mitochondria, TFAM plays dual roles: as a transcription factor (TF) for mitochondrial genes and as a regulator of mtDNA replication [
3,
4,
5]. TFB2M is also encoded by nDNA and transported from the cytosol to mitochondria to function as TF for mitochondrial genes. By contrast, transcription factor B1, mitochondrial (TFB1M), a paralog of TFB2M, is a dimethyltransferase that methylates adenine residues of mt12S rRNA [
6]. TFAM binds to mtDNA and changes its structure. TFB2M and mitochondrial RNA polymerase (POLRMT) interact with TFAM to induce target gene expression. Mitochondrial transcription machinery, composed of TFAM, TFB2M, and POLRMT, initiates the expression of mtDNA [
7,
8,
9]. A recent study has shown that POLRMT also functions as a switch between the transcription and replication of mtDNA [
10].
NRF1 and NRF2 are nuclear TFs. NRF1 binds to GC-rich palindromes [
11,
12], whereas NRF2 binds to direct tandem repeats with GGAA core motifs [
13,
14]. Human NRF2 consists of two subunits: α and β (β1 or β2) or γ (γ1 or γ2). Subunit α contains the DNA-binding domain, whereas subunit β or γ contains the transcription activation domain [
15]. Both NRF1 and NRF2 positively regulate the expression of genes encoding proteins related to the oxidative phosphorylation system (OXPHOS) complexes, heme biosynthesis, mitochondrial protein import and assembly, and mitochondrial translation [
16]. NRF1 and NRF2 also regulate the expression of TFAM [
17] and TFB2M [
18].
ERRs α, β, and γ are nuclear receptors (NRs) whose endogenous ligands are unknown [
19]. ERRs bind to the ERR response element with a consensus DNA sequence of TCAAGGTCA. ERRα and ERRγ bind to promoters of genes encoding mitochondrial proteins. However, the role of ERRβ in the expression of mitochondrial genes is unknown. The transcriptional function of ERRs is positively regulated by PGC-1α [
20,
21].
PGC-1α is a coactivator that lacks DNA-binding activity, but activates transcription of TFs or NRs by binding to these proteins. PGC-1α interacts with NRF1, NRF2, and ERRs to positively regulate these TFs and NRs for mitochondrial biogenesis [
22]. Although PGC-1α is not a coactivator of TFAM and TFB2M, it indirectly activates the expression of TFAM and TFB2M via the activation of NRFs [
17,
18]. Thus, PGC-1α is considered a master regulator of mitochondrial biogenesis. PGC-1α is a member of the PGC1 family, which also includes PGC-1β and PGC-related coactivator (PRC). PGC-1β associates with NRF1 and ERRs, and positively regulates the expression of mitochondrial biogenesis proteins [
22]. PRC also binds to NRF1 and ERRα. In addition, PRC forms a complex with NRF2 by binding to the host cell factor 1 (HCF-1) [
23]. Post-translational modifications of PGC-1α include phosphorylation, methylation, acetylation, and deacetylation, whereas those of PGC-1β and PRC remain unclear [
24].
2.3. Degradation (Mitophagy)
Damaged and dysfunctional mitochondria are deleterious to the cell. Degradation of such mitochondria is, therefore, crucial. Mitochondrial degradation is executed via the process of autophagy, which removes unwanted cytosolic components [
43,
44,
45]. Selective degradation of mitochondria via autophagy is called mitophagy. Several proteins mediate the process of mitophagy, including phosphatase and tensin homolog (PTEN), induced putative kinase 1 (PINK1), Parkin, BCL2 interacting protein 3 (BNIP3), NIX [also known as BNIP3 like (BNIP3L)], Bcl2-like protein 13 (Bcl2-L-13), and FUN14 domain containing 1 (FUNDC1).
PINK1 is a serine/threonine kinase localized to mitochondria [
46]. In healthy mitochondria, presenilin-associated rhomboid-like (PARL) processes PINK1, leading to the degradation of PINK1. In depolarized mitochondria, the processing of PINK1 by PARL is blocked. This results in the accumulation of PINK1 on the OMM [
47,
48,
49]. PINK1 undergoes autophosphorylation and phosphorylates ubiquitin moieties of originally ubiquitinated OMM proteins. In addition, PINK1 phosphorylates and activates Parkin, an E3 ubiquitin ligase that adds ubiquitin molecules to originally ubiquitinated OMM proteins [
50,
51,
52]. PINK1 also phosphorylates the ubiquitin molecules added by Parkin. The ubiquitin-binding autophagic adaptor proteins, nuclear dot protein 52 kDa (NDP52) and optineurin (OPTN), recruit microtubule-associated protein 1 light chain 3 (LC3) to the OMM proteins ubiquitinated and phosphorylated by PINK1 and Parkin, leading to mitophagy [
53,
54,
55].
BNIP3, NIX, Bcl2-L-13, and FUNDC1 are localized to the OMM and mediate mitophagy by associating with the LC3 subfamily proteins, including LC3 alpha (LC3A), LC3 beta (LC3B), and LC3C, and the γ-aminobutyric-acid-type-A receptor-associated protein (GABARAP) subfamily proteins, including GABARAP, GABARAP-like 1 (GABARAPL1), and GABARAP-like 2 (GABARAPL2) [
56]. BNIP3, NIX, and Bcl2-L-13 belong to the Bcl-2 family. Although Bcl-2 harbors four Bcl-2 homology motifs (BH1–4), BNIP3 harbors only the BH3 motif [
57]. Overexpression of BNIP3 leads to the induction of mitophagy [
58,
59], whereas BNIP3 knockdown suppresses mitophagy [
60]. Phosphorylation of Ser17 of BNIP3 is necessary for the binding of BNIP3 to LC3B, whereas phosphorylation of both Ser17 and Ser24 mediates the binding of BNIP3 to GABARAPL2 (also known as GATE-16) [
61]. Like BNIP3, NIX also harbors only the BH3 motif. Deletion of NIX results in defective mitophagy [
62,
63]. Although NIX associates with all members of the LC3 and GABARAP subfamilies [
56], phosphorylation of Ser34 and Ser35 residues of NIX enhances its ability to bind to LC3A and LC3B [
64]. Bcl2-L-13 is a Bcl-2 homolog protein that also plays key roles in mitophagy and mitochondrial fragmentation. Phosphorylation at Ser272 of Bcl2-L-13 is necessary for its interaction with LC3B [
65]. FUNDC1, which is also located in the OMM, functions to link mitochondria with LC3. Casein kinase 2 (CK2) and Src kinase phosphorylate Ser13 and Tyr18 residues of FUNDC1, respectively, blocking the induction of mitophagy. By contrast, dephosphorylation of Ser13 and Tyr18 residues of FUNDC1 by PGAM5, a mitochondrial phosphatase, or inhibition of CK2 and Src kinase results in the induction of mitophagy [
66,
67].
Mitochondrial-derived vesicles (MDVs) and mitochondrial spheroids are also important pathways for mitochondrial degradation and quality control, in addition to canonical autophagy [
68]. MDVs deliver oxidized components of the mitochondria to the lysosome in response to oxidative stress. MDV formation is dependent on PINK1 and Parkin. However, key proteins controlling canonical autophagy, such as ATG5 and LC3, are not necessary for MDV formation. In addition, MDV formation and turnover precede mitophagy [
69,
70]. A recent study has shown that Syntaxin-17 is required for fusing the MDV and the lysosome [
71]. Mitochondrial spheroids are the transformed structures of the mitochondria. They have a ring or cup-like shape and are generated in response to oxidative stress and mitochondrial damage [
72,
73,
74]. As mitochondrial spheroids are formed independently of ATG5 or ATG7, their formation is distinct from canonical autophagy. Although mitochondrial spheroids contain lysosomal proteins, the degradation of contents via mitochondrial spheroids remains to be elucidated. MFN1 and MFN2 are required for mitochondrial spheroid formation, and Parkin inhibits mitochondrial spheroid formation by degrading MFN1 and MFN2. Thus, Parkin induces mitophagy and prevents mitochondrial spheroid formation [
68,
72,
74].