Mitochondrial quality control as a key determinant of cell survival

Accepted Manuscript Mitochondrial quality control as a key determinant of cell survival

Lucia Sedlackova, Viktor I. Korolchuk PII: DOI: Reference:

S0167-4889(18)30541-X https://doi.org/10.1016/j.bbamcr.2018.12.012 BBAMCR 18418

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

16 October 2018 3 December 2018 21 December 2018

Please cite this article as: Lucia Sedlackova, Viktor I. Korolchuk , Mitochondrial quality control as a key determinant of cell survival. Bbamcr (2018), https://doi.org/10.1016/ j.bbamcr.2018.12.012

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Mitochondrial quality control as a key determinant of cell survival Lucia Sedlackova and Viktor I. Korolchuk* 1

Affiliations: Institute for Cell and Molecular Biosciences; Newcastle University; Newcastle upon Tyne NE4 5PL, UK *Correspondence to: [email protected]

PT

Highlights:

Mitochondria are the hubs of cellular metabolic, signalling and death pathways.



Mitophagy and mitochondrial dynamics maintain a healthy pool of mitochondria.



Age-related accumulation of damaged mitochondria can result in an aberrant response to

SC

RI



cellular cues and lead to the development of pathologies. 

Therapeutic targeting of mitochondria can be a promising strategy to ameliorate age-related

NU

diseases. Abstract:

MA

Mitochondria are the energy producing dynamic double-membraned organelles essential for cellular and organismal survival. A multitude of intra- and extra-cellular signals involved in the regulation of energy metabolism and cell fate determination converge on mitochondria to promote or prevent cell

D

survival by modulating mitochondrial function and structure. Mitochondrial fitness is maintained by

PT E

mitophagy, a pathway of selective degradation of dysfunctional organelles. Mitophagy impairment and altered clearance results in increased levels of dysfunctional and structurally aberrant mitochondria, changes in energy production, loss of responsiveness to intra- and extra-cellular signals and ultimately cell death. The decline of mitochondrial function and homeostasis with age is reported to be

CE

central to age-related pathologies. Here we discuss the molecular mechanisms controlling mitochondrial dynamics, mitophagy and cell death signalling and how their perturbation may

AC

contribute to ageing and age-related illness.

Keywords (6): mitochondria, mitophagy, cell death, ageing, BCL-2 family, ubiquitin.

1

ACCEPTED MANUSCRIPT Introduction All living organisms interact with their environment to gather the nutrients necessary for survival. Cells catabolise absorbed nutrients to generate building blocks and energy required for cellular anabolic processes thus driving cell growth and proliferation. However, cellular anabolism also results in damage accumulation which, with age, contributes to cell and tissue dysfunction and ultimately death. [1]. To combat this issue cells have evolved a number of quality control mechanisms which are designed to decrease the damage load and thus promote fitness. Viewed in the context of a single cell, the pathways of energy metabolism, quality control and cell demise all converge on cellular

PT

power stations, mitochondria.

Mitochondria, a dynamic network of double-membrane organelles, meet the energy and metabolic

RI

demands of the cell within which they reside. Energy generation in mitochondria occurs at the inner mitochondrial membrane (IMM). Cristae, micro-compartments generated by invagination of the IMM,

SC

change in response to metabolic cues or external insults and cristae width and length in turn determines the efficiency of the electron transport chain (ETC) [2]. ETC comprises a group of four -

-

NU

multimeric protein complexes embedded in the IMM and two membrane-permeable electron (e ) carriers [3]. Movement of e through the ETC to O2, its terminal acceptor, is accompanied by proton pumping across the IMM by complexes I, III and IV. Proton pumping across the IMM leads to an +

MA

electrochemical gradient of hydrogen ions (H ), also referred to as mitochondrial membrane potential (MMP) (ΔΨm). The resulting transmembrane potential of -160mV and concentration of protons in the intermembrane space drives proton flow through the F0 subunit of the ATP synthase to provide the rotational energy for ATP synthesis at the F1 subunit [4]. Aerobic respiration via the coupling of ETC

D

together with ATP-synthase is collectively referred to as oxidative phosphorylation (OXPHOS). In

PT E

addition to energy generation, mitochondria act as hubs for a number of metabolic and signalling pathways. It has long been recognized that mitochondria are sites of amino acid, fatty acid and carbon metabolism and thus supply the cytosol with precursors of essential building blocks for de novo biosynthesis of proteins, lipids, nucleotides and carbohydrates [5]. Intermediates of lipid β-

CE

oxidation and the tricarboxylic acid (TCA) cycle metabolites are also involved in retrograde signalling from mitochondria to the nucleus [6].

AC

The health of a mitochondrial population is maintained by the processes of biogenesis, targeted degradation and mitochondrial dynamics of fusion and fission [7]. Balance between fusion and fission events shapes mitochondrial networks to meet metabolic demands and allow for degradation of dysfunctional organelles. In addition, alteration of mitochondrial shape and content is important for the maintenance of redox status, mtDNA content and organelle integrity (reviewed in [8]). With regards to mitochondrial dynamics, only fusion is dependent on a healthy OXPHOS and MMP. Increased fusion and an increase of cristae content results in more efficient ETC function due to a tighter spatial organisation of the OXPHOS and solute carriers. Mitochondrial fusion protects the organelles from degradation, allows for inter-organelle content exchange and is protective in cells undergoing nutrient or oxidative stress, in which hyperfused networks have been observed [7, 9]. Conversely, mitochondrial fragmentation facilitated by an increase in fission events was observed in cells cultured

2

ACCEPTED MANUSCRIPT in full nutrient media [10] or in dying cells [11]. Fragmentation of the mitochondrial network in full nutrient conditions presumably occurs due to a lesser need for ETC efficiency and increased selection of depolarised mitochondria for degradation. In cell death, loss of MMP leads to unopposed fission and network fragmentation [11]. Mitochondrial dynamics The balance between fusion and fission events dictates the shape of mitochondrial networks. Fusion and fission are dependent on the action of a few key IMM and OMM guanosine triphosphatases

PT

(GTPases) and their interactors, many conserved from yeast to mammals [11]. Mitochondrial fusion is a three-step process of organelle approximation, OMM tethering and fusion followed by IMM fusion. Dynamin-related GTPases, mitofusin 1 and its paralog mitofusin 2 (Mfn1 and Mfn2) are anchored in

RI

the OMM by their C-terminal domains. At first, neighbouring mitofusins anchored in adjacent organelles oligomerise via their coiled coil domains in a reversible hydrogen bond-dependent manner

SC

[12]. Subsequently, mitofusins mediate OMM fusion in a GTPase dependent manner thus creating an organelle with a single intact OMM and two separate IMM compartments, a configuration which is

NU

immediately followed by IMM fusion [13].

The most widely studied protein involved in mitochondrial IMM shaping and fusion was first discovered by genetic mapping in dominant optic atrophy, a neurodegenerative disease of the optic

MA

nerve [14, 15]. Optic atrophy 1 (OPA1) is imported into mitochondrial intermembrane space where it adopts two peptide structures, a long peptide tethered to the IMM (L-OPA1) and a soluble short form localised in the intermembrane space (S-OPA1) [16]. The complexity of OPA1 function in fusion is

D

further increased by its transcriptional modifications, resulting in the formation of eight isoforms.

PT E

Imbalance of OPA1 isoforms affects mitochondrial IMM fusion, fission and cristae structure [17]. Under normal circumstances L-OPA1 isoforms are necessary and sufficient for IMM fusion, achieved by interactions with cardiolipin (CL), a unique, abundant lipid of the IMM predominantly present in the inner leaflet [18]. Increased levels of S-OPA1 peptides arising from excessive L-OPA1 cleavage in

CE

response to metabolic cues have been associated with mitochondrial fragmentation. It is however unclear whether S-OPA1 directly participates in mitochondrial fission or whether increased fission occurs as an indirect consequence of L-OPA1 loss. Intriguingly, transfection of cells with an artificial

AC

S-OPA1 construct leads to its localisation to OMM fission sites and accelerates mitochondrial fragmentation [19, 20].

Mitochondrial fission is dependent on dynamin related protein 1 (Drp1) [11]. Drp1, a cytosolic protein, is recruited to mitochondria, interacts with mitochondrial fission 1 protein (Fis1), tethered to the OMM via its C-terminal domain, and together these two proteins form foci for scission events. Drp1 oligomerises and changes its conformational state in a GTP-binding dependent manner [21, 22]. Drp1 oligomer constriction pinches the IMM and OMM upon GTP hydrolysis and thus separates the parent and the daughter organelle. The events preceding Drp1 recruitment to mitochondria are not entirely understood, though ER-mitochondria contacts and actin are thought to play a role [23].

3

ACCEPTED MANUSCRIPT Mitochondrial quality control Mitochondria are susceptible to oxidative damage and require efficient clearance mechanisms to remove dysfunctional mitochondria from the pool of organelles. These mechanisms are particularly important for post-mitotic cells of high energetic demand and low regenerative capacity such as neurons where mitochondrial damage can cause cell death [24, 25]. One of the key cellular mechanisms mediating removal of dysfunctional mitochondria is autophagy, a vesicle-mediated catabolic pathway of damaged cargo recognition, sequestration, and delivery to the lysosome for degradation and nutrient release [26]. Selective degradation of damaged mitochondria by autophagy

PT

is commonly referred to as mitophagy. This process can be triggered by mitochondrial depolarisation, proteotoxic stress, dysregulation of calcium signalling, absence of molecular oxygen (hypoxia) or

RI

occurs as a programmed event [27]. The little explored field of mitophagy gained traction due to association of mitochondrial dysfunction and neurodegeneration, following the discovery of the

SC

involvement of two proteins implicated in the pathology of Parkinson’s disease, PINK1 (PTEN-induced putative kinase 1) and Parkin, in mitochondrial degradation. Together these proteins were found to facilitate ubiquitin-dependent mitophagy of depolarised organelles. The initial discovery of

NU

ubiquitination-dependent mitophagy mediated by these proteins was followed by the exploration of specific ubiquitin-independent mitophagy events. Most recently, new reports describing partial organelle quality control mediated by mitochondria-derived vesicles have been published. Below we

MA

give a brief overview of different types of mitophagy discovered to date. Ubiquitin-dependent mitophagy

The canonical model of mitochondrial clearance, PINK1/Parkin-dependent mitophagy, relies on the

D

loss of membrane potential, for example due to inefficient proton pumping across the IMM. In

PT E

conditions of mitochondrial membrane depolarisation mitochondrial dynamics is shifted towards mitochondrial fission as a result of increased L-OPA1 cleavage and an accumulation of S-OPA1 [16]. Loss of MMP also results in a block of mitochondrial protein translocator pores and stabilisation of PINK1 at the OMM, which evades cleavage by presenilin-associated rhomboid-like protein (PARL), a

CE

mitochondrial matrix protease [28, 29]. Protein kinase activity of PINK1 results in

its

autophosphorylation, in ubiquitin phosphorylation, in Parkin recruitment and in ubiquitination of

AC

mitochondria-associated proteins in a positive feed-forward loop [30, 31]. In this process Parkin acts as an E3 ubiquitin ligase promoting ubiquitination of OMM proteins and antagonising the action of mitochondrial deubiquitinases USP30 and USP15 [32-34]. OMM protein ubiquitination results in several outcomes. First of all, ubiquitination and proteasomal degradation of mitochondrial Rho GTPase 1 (MIRO-1), a protein involved in the mitochondrial association with actin filaments, results in the stalling of mitochondrial movement [35]. Next, Parkin-dependent ubiquitination and subsequent degradation of Mfn1/Mfn2 inhibits OMM fusion [36]. Finally, ubiquitination of these and other Parkin substrates such as voltage-dependent anion channel 1 (VDAC1), B-cell lymphoma 2 (BCL-2) and Drp1 results in the recruitment of mitophagy receptors via their ubiquitin-binding domains (Figure 1A) [37, 38]. Amongst these are Optineurin (OPTN), NDP52 and TAX1BP1 [39] whilst AMBRA1 is recruited to OMM via its interaction with Parkin [40]. All autophagy receptors function to recruit autophagy machinery via their LC3 interacting region (LIR) which drives the formation of an 4

ACCEPTED MANUSCRIPT autophagic vesicle around the dysfunctional mitochondria. The formation of the autophagosome is also facilitated by the proteasomal degradation of a number of ubiquitinated OMM proteins. This process leads to the rupture of OMM followed by the exposure of prohibitin 2 (PHB2), an IMM protein which also contains a LIR domain [41]. IMM protein recognition and binding of the autophagy machinery hints at an additional layer of specificity in autophagic removal of dysfunctional organelles upon OMM rupture [41]. Ultimately, all these events ensure rapid sequestration of dysfunctional, and potentially damaging, mitochondria by autophagic vesicles and their delivery to lysosomes for

PT

degradation. To date the methods used to study PINK1/Parkin-mediated mitophagy rely largely on overexpression of either or both proteins, as well as acute artificial mitochondrial depolarisation. It has been

RI

suggested however that PINK1-Parkin mitophagy may also be triggered by unfolded protein stress in the matrix in the absence of mitochondrial depolarisation [42, 43]. The physiological significance and

SC

trigger mechanisms of PINK1-Parkin-dependent mitophagy in vivo remain to be discovered, though recent findings suggest the role of PINK1/Parkin and basal mitophagy in inflammation [44, 45].

NU

Conversely, recent generation of a mitophagy reporter mouse model expressing mito-QC led to the discovery of widespread and heterogeneous mitophagy events in vivo independent of PINK1 [46, 47]. Further studies of the mechanisms of basal mitophagy and the involvement of PINK1 and Parkin in

MA

this process will be required and are likely to be focused on the elucidation of the selective vulnerability of dopaminergic neurons to dysfunctional mitophagy. Ubiquitin-independent mitophagy

D

Stress-induced mitophagy pathways can proceed independently of ubiquitin-mediated cargo delivery

PT E

and require an alternative set of autophagy receptors. Oxygen depletion, or hypoxia, leads to increased expression of three autophagy receptors which localise to mitochondria (Figure 1B). Fun14 domain containing 1 (FUNDC1), a hypoxia responsive autophagy receptor is anchored to the OMM by its three transmembrane domains [48], while its LIR containing N-terminus is exposed to the

CE

cytoplasm. Regulation of FUNDC1-mediated mitophagy in hypoxia is two-fold. Firstly, hypoxia leads to increased expression of FUNDC1 via downregulation of microRNA-137 (miR-137) involved in the repression of FUNDC1 translation [49]. Secondly, hypoxia triggers dephosphorylation of a tyrosine

AC

residue within its LIR motif followed by increased FUNDC1-mediated LC3 recruitment [48]. In addition to FUNDC1, two members of the BCL-2 protein family regulate mitophagy initiation in response to hypoxia. BCL-2 and adenovirus E1B-19kDa (BNIP3) and BNIP3L (NIX) proteins are non-canonical BH3-only pro-apoptotic members (Figure 2) involved in cell death regulation at the mitochondrial OMM and endo/sarcoplasmic reticulum [50]. Both BNIP3 and NIX are capable of homodimerisation and insertion into the OMM via their transmembrane glycine zipper domain [50], of interaction with LC3 [51], and of indirect autophagy initiation by antagonising the binding of BCL-2 to Beclin1, an autophagy inducer. [50]. The role of NIX in mitophagy was established in NIX-deficient mice presenting with a defect in erythroid maturation [52]. The erythrocytes of NIX-deficient mice retain their mitochondria due to a defect in mitochondrial targeting to an autophagy-exocytosis pathway. BNIP3 dependent mitophagy was first described in chemotherapy-mediated tumour cell death

5

ACCEPTED MANUSCRIPT induced by ceramide [53] and arsenic trioxide [54], and followed up by studies in hypoxia-induced mitophagy [50]. Both BNIP3 and NIX expression is also upregulated in hypoxia [50]. Thus BNIP3 and NIX can promote development-specific and stress-induced ubiquitin-independent mitophagy. However, the role of BNIP3 and NIX in basal mitophagy is unknown, as is the exact mechanistic nature of mitophagy initiation and execution under stress conditions. Increased oxidative stress induces mitophagy by acting upon CL, an integral phospholipid of the mitochondrial IMM. CL consists of a glycerol molecule coupled to two phosphatidate moieties and four

PT

acyl chains [18]. In healthy mitochondria, CL is confined to the inner leaflet of the IMM where it is synthesized and, due to its unique structure, shapes the IMM by increasing its fluidity [55]. A recent study has shown that upon oxidation, CL localises to IMM/OMM contact sites and gets translocated

RI

from the IMM to the OMM by phospholipid scramblase 3 [56]. CL externalisation can act as a signal for mitophagy, for LC3 contains CL-binding residues and was shown to bind CL at the OMM leading

Mitophagy-independent Mitochondrial Quality Control

SC

to the recruitment of the autophagic machinery (Figure 1C) [57].

NU

Recent advances in the mitophagy field led to the identification of a population of mitochondriaderived vesicles (MDVs) which are trafficked in a PINK1-Parkin dependent manner [58]. MDVs contain cargo destined for the delivery to peroxisomes and lysosomes and thus fulfil the role of

MA

mitochondrial signalling and quality control (Figure 1D) [59]. They arise by a Drp1-independent membrane budding in response to oxidative protein damage, but not IMM depolarisation [58]. MDV budding occurs at the sites of tight IMM and OMM association and in the proximity to protein import

D

machinery [59]. The mechanism of negative membrane curvature which results in IMM and OMM

PT E

protrusion into the cytoplasm is unknown. MDVs tend to be of a uniform size and contain cargo from some or all mitochondrial compartments depending on the type of stress or defect occurring in mitochondria [60].

Coordinated mitochondrial stress response

CE

The majority of mitochondrial proteins are encoded in the nuclear DNA and require efficient expression and transport into their designated mitochondrial compartment to sustain mitochondrial

AC

function [61]. The mitochondrial import machinery is intimately linked to the energetic state of the organelle for it requires MMP and ATP to function efficiently [61]. Mitochondrial protein import thus acts as a sensor for mitochondrial dysfunction and its loss triggers two quality control events. Firstly, loss of MMP and inefficient degradation of PINK1 by PARL leads to its accumulation at the OMM and activation of mitophagy. Secondly, intramitochondrial stress leads to upregulation of a transcriptional mt

program called the mitochondrial unfolded protein response (UPR ). UPR

mt

was first identified in

Caenorhabditis elegans [62] and further explored in mammalian cells (reviewed in [63]). UPR

mt

in C.

elegans is regulated by a stress activated transcription factor (ATFS-1), which contains both, a mitochondrial and nuclear localisation signals. In a healthy system, ATFS-1 translocates to the mitochondrial matrix and is degraded by the Lon protease [62]. Upon inefficient protein import into mitochondria ATFS-1, and its mammalian homolog activating transcription factor 5 (ATF5), localise to the nucleus instead and relay the inefficiency of mitochondrial protein import, and by proxy 6

ACCEPTED MANUSCRIPT mitochondrial function, to the nuclear transcriptional programs of mitochondrial homeostasis, metabolism maintenance and ROS response [64]. Thus, mitochondrial dysfunction leading to inefficiency of protein import is a common initiation event for both, ATF5-regulated transcriptional adaptation to mitochondrial dysfunction and PINK1/Parkinmediated mitophagy, and results in a coordinated response to mitochondrial dysfunction aimed to protect the cell from mitochondrial stress, to improve mitochondrial health and to remove depolarised organelles.

PT

Mitochondria as hubs of cell death signalling

Cell death is an irreversible loss of cell function followed by cell remodelling which allows for the

RI

removal of superfluous or damaged cells triggered by intra- or extracellular signals [65]. Cell death was historically viewed as a programmed event, apoptosis, or accidental cell death, necrosis. More

SC

recently, programmed cell death pathways initiated upon persistent and unresolvable insults to cellular homeostasis, have been coined with an umbrella term regulated cell death (RCD). Conversely, accidental cell death is an immediate and irreversible loss of cell viability as a result of a physical or a

NU

chemical insult. RCD execution via a caspase protein family-dependent mechanism has been redefined as apoptosis, whereas all caspase independent cell death is referred to as necroptosis [65]. The nomenclature committee on cell death (NCCD), which meets annually since 2005, has published

MA

a new recommendation for cell death classification based on changes to molecular and signalling effectors [65]. Of the 12 recommended cell death classes, mitochondria directly participate in intrinsic apoptosis, MPT-driven necrosis and parthanatos by either outer mitochondrial outer membrane

D

permeabilisation (MOMP) or mitochondrial permeability transition (MPT) [65]. An indirect role of

PT E

mitochondria was described in extrinsic apoptosis, ferroptosis, and lysosome dependent cell death. The role of mitochondria was initially described in necroptosis, though a rising body of evidence disputes this finding [65]. No mitochondrial involvement has so far been described in cell and context specific RCD programes of pyroptosis, entosis, NETosis, immunogenic cell death and autophagy

Intrinsic apoptosis

CE

dependent cell death.

AC

By far the best explained mode of cell death which involves mitochondria is the pathway of intrinsic apoptosis. Intrinsic apoptosis is a programmed, energy-dependent cell death event which results in cell elimination without a release of harmful components into extracellular milieu. Morphologically, apoptosis involves cells shrinkage, chromatin condensation, nuclear fragmentation and cell breakage into small vesicle bound fragments [66]. Intrinsic apoptosis is now recognised as a process critical for normal tissue development and homeostasis. The key event in apoptotic cell death is MOMP, a protein-complex-mediated permeabilisation of the OMM which results in the release of pro-apoptotic factors from the intermembrane space to initiate a cascade of signalling events leading to cellular demise [65]. The apoptotic pathway is governed by a BCL-2 family of pro- and anti-apoptotic proteins, which permanently or transiently localise to the OMM and regulate events preceding or preventing MOMP

7

ACCEPTED MANUSCRIPT [65]. Three subfamilies of the BCL-2 proteins are described as the multidomain anti-apoptotic, the multidomain BH3 pro-apoptotic and the BH3-only pro-apoptotic families (Figure 2). Currently, over 25 members of the BCL-2 family are identified based on sequence homology to the four BCL-2 homology (BH) domains [67, 68]. The widely expressed anti-apoptotic members include BCL-2, BCL-XL and a less studied MCL-1 protein, all of which are substrates of cleavage by activated caspase 3 [69, 70]. Caspase 3-mediated cleavage upon apoptosis induction gives rise to shorter pro-apoptotic fragments of the anti-apoptotic family members. In addition, less studied and in some cases tissue-specific proteins which share BCL-2 homology have been identified, namely BFL-1 and BCL-W. Anti-apoptotic

PT

family members antagonise MOMP by binding the mitochondrial pool of multidomain pro-apoptotic members and BH3-only cell death activators [68].

RI

The multidomain pro-apoptotic family members include the widely studied BAX, BAK and BOK, three BCL-2 family members capable of pore formation by oligomerisation and insertion into the OMM thus

SC

triggering MOMP [65]. BAX transiently associates with the OMM in its non-active form either as a monomer or a dimer, BOK resides in the cytosol, whereas BAK resides at the OMM, anchored by a

NU

transmembrane domain [65, 68]. BAK is likely held in its inactive form by interaction with antiapoptotic MCL-1 and BCL-XL. Translocation and stabilisation of BAX and BOK at OMM is thought to be triggered by conformational changes upon binding of the BH3-only family members to a surface

MA

groove formed by the special organisation of their BH1, BH2 and BH3 domains [71]. Binding of proapoptotic BH3-only family members to the hydrophobic surface groove leads to their conformational change, displaces a transmembrane domain and allows their anchorage to the OMM [71].

D

The pro-apoptotic BH3-only protein family is a diverse group of proteins which share one common

PT E

feature, the BH3 domain [65]. Amongst these are BAD, BIK, BMF, HRK and PUMA, which bind and neutralise anti-apoptotic BCL-2 proteins to lower the threshold for apoptosis and thus sensitise cells to apoptotic signalling. Additionally, the interaction of activator pro-apoptotic BH3-only family members BID, BIM and NOXA with BAX leads to its oligomerisation which induces OMM pore formation [65].

CE

Finally, BID is a unique example of a BH3 only protein linking extrinsic apoptotic cues to the intrinsic apoptosis pathway. This function of BID is mediated by caspase 8-mediated BID cleavage, its

AC

localisation to mitochondria and interaction with CL to promote BAX and BAK pore formation [72, 73]. MOMP, coupled with morphological changes of the IMM, orchestrates the release of apoptogenic proteins which normally reside within the mitochondrion (Figure 3C) [65]. Cytochrome c (cyt c), an electron shuttle of the ETC; a second mitochondria-derived activator of caspase (SMAC/DIABLO) and a high temperature requirement protein A2 (HTRA2), binding partners of cytoplasmic inhibitor of apoptosis (XIAP); and AIF, an apoptosis inducing factor involved in mitochondrial protein import, are all released from the intermembrane space upon pore formation [65]. Released apoptogenic factors then participate in events preceding and triggering apoptosis. SMAC/DIABLO represses XIAPmediated caspase sequestration to modulate apoptosis [74]. Cyt c interacts with its cytosolic binding partner Apaf-1 leading to association with caspase 9 and the formation of an apoptosome [75]. Apoptosome formation results in caspase 9 activation and cleavage of executioner pro-caspases 3

8

ACCEPTED MANUSCRIPT and 7, responsible for DNA fragmentation, apoptotic body formation and the apoptotic cell death program completion [65]. MPT-driven necrosis MPT involves a sudden IMM permeability to small solutes and MMP dissipation in response to a burst 2+

of oxidative stress or calcium (Ca ) overload resulting in the loss of cristae structure, water intake and mitochondrial swelling [76]. Pore formation at the IMM was attributed to the assembly and conformational changes of ATP synthase, adenine nucleotide translocase (ANT), and VDAC in the

PT

close proximity of MAMs and mitochondrial import machinery, coined the permeability transition pore complex (PTPC) [77]. Initially confirmed to participate in MPT in cell models, knockdown of any of the proteins in mouse models failed to prevent MPT [77]. Up to date only cyclophilin D, a matrix protein,

RI

has been robustly shown to participate in MPT [77], a mode of cell death presenting with ATP

SC

depletion and necrotic morphology. Parthanatos

Parthanatos is an RCD dependent on the action of poly(ADP-ribose) polymerase 1 (PARP1) in

NU

response to DNA damage [65]. PARP1 hyperactivation as a result of extensive DNA damage leads to +

NAD depletion from the cytosol, loss of antioxidant defences, poly(ADP-ribosylation) of an apoptosis inducing factor (AIF) at the IMM, its release and relocation to the nucleus and DNA fragmentation. In

MA

addition to the release of AIF from the IMM, mitochondrial involvement was confirmed by the loss of MMP, MOMP and apoptotic cell death initiation [65]. Mitochondrial quality control at the OMM

D

The OMM encloses the whole organelle and forms a barrier between the mitochondrion and the

PT E

cytoplasm. A large number of porins in the OMM ensure its permeability to small solutes. The OMM also forms an interface for mitochondria associated membrane (MAM) formation and microdomain 2+

Ca

signalling. An intact OMM acts as a hindrance to release of apoptogenic factors from within the

IMS and the mitochondrial matrix (Figure 3A). In fact, mobilisation of cyt c from the IMM is only

CE

sufficient in apoptosis initiation if it coincides with OMM permeabilisation [78], which is controlled by the BCL-2 protein family. Anti-apoptotic family members sequester and prevent oligomerisation of

AC

pro-apoptotic BAX and BAK members (Figure 3B). Conversely, increased levels of pro-apoptotic BH3-only family members promote BAX and BAK oligomerisation and insertion into the OMM. The OMM then becomes permeable to apoptogenic factors released from within the IMS and mitochondrial matrix (Figure 3C). Mitophagy A growing body of evidence suggests a role of the BCL-2 protein family in autophagy regulation. BCL2 was shown to interact with Beclin1, an autophagy initiator which contains a BH3-like domain [79]. In healthy cells, a free pool of BCL-2 associates with Beclin1 and inhibits autophagy. Increased cellular stress, and subsequent BH3-only protein transcription or activation leads to BCL-2 displacement from Beclin1 and autophagy initiation to reconstitute cellular homeostasis [79]. In addition, BNIP3 and NIX, two BCL-2 family proteins atypical in their function and structure, stand at the crossroads of

9

ACCEPTED MANUSCRIPT mitophagy and cell death. Initially identified by their involvement in stress-induced mitophagy, both BNIP3 and NIX contain a transmembrane domain which tethers them to the OMM, a BH3 domain which allows them to interact with the BCL-2 family members and an LIR by which they recruit LC3 to initiate mitophagy (Figure 1) [50]. Both proteins are able to initiate cell death by localising and inserting into the OMM, though the exact mechanism is unknown. BNIP3-dependent cell death initiation is accompanied by an increase in reactive oxygen species (ROS) and IMM permeabilisation [80]. In contrast, NIX can localise to mitochondria or the ER to trigger apoptosis or necrosis, 2+

respectively [81]. ER-driven necrosis is initiated by NIX-dependent manipulation of ER Ca

stores,

PT

which are aberrantly released, buffered by mitochondria and trigger MPT [81]. Mitophagy in cell survival vs cell death

RI

Mitophagy is generally viewed as a survival-promoting cellular event which leads to reconstitution of overall health of the mitochondrial network by the removal of dysfunctional organelles. However, a

SC

new notion of lethal mitophagy has been coined in the treatment of acute myeloid leukaemia [82]. Lethal mitophagy is a concept whereby a bioactive sphingolipid, ceramide, induces excessive

NU

mitophagy by the recruitment of autophagic machinery and interaction with LC3B-II at the OMM to induce non-apoptotic death of cancer cells [82]. However, ceramide also induces expression of the BNIP3 protein and has previously been shown to induce IMM depolarisation and autophagic cell

MA

death of malignant melanoma cells [53]. Combined findings of the two studies suggest that it is the initial increase in BNIP3 expression and MMP dissipation followed by DRP1 mediated fission, which leads to ceramide accumulation at the OMM and excessive mitophagy events. So far, the concept of lethal mitophagy has been explored in cancer cells and treatments which target ceramide metabolism

D

are successful cancer treatment [83], but further work will be required to understand the relevance of

Mitochondrial dynamics

PT E

the concept in healthy cells.

Mitophagy and MOMP coincide with an increase in mitochondrial fragmentation [84] as a result of

CE

both, loss of IMM and OMM fusion and an increase in fission. In cell death, mitochondrial fission precedes caspase activation and chromosome condensation [65]. In the early stages of apoptosis initiation, Drp1 co-localises with BAX and Mfn2 at the OMM, forming a complex, which was

AC

hypothesized to prevent mitochondrial OMM fusion due to Mfn2 inhibition [85]. Drp1 localisation to OMM is also responsible for mitochondrial fission for Drp1 depletion or inhibition by expression of a dominant negative mutant (DrpK38A) prevents mitochondrial fragmentation, cyt c release, caspase activation and apoptosis [84]. In addition, overexpression of BAX, but not a BAX mutant lacking the BH3 domain leads to an accelerated mitochondrial fragmentation upon an apoptotic stimulus [84]. Calcium signalling Maintenance of a healthy Ca

2+

flow from the ER and cytosol into mitochondria is essential for cell 2+

survival. As mentioned above, aberrant release of Ca from the ER, its uptake by mitochondria and spread through the mitochondrial network can initiate large-scale ROS production and MPT-driven necrosis. Ca

10

2+

handling and signal propagation within the ER and mitochondrial networks are

ACCEPTED MANUSCRIPT regulated by the BCL-2 protein family at both organelles; at the ER via the inositol 1,4,5-trisphosphate receptors (IP3Rs) and at the mitochondrial OMM via the interaction with VDACs [86]. 2+

BCL-2 is capable of binding the IP3Rs via its BH4 domain to modulate Ca release from the ER [87]. 2+

The exact mechanism of BCL-2 mediated resistance to Ca toxicity is disputed and involves either a direct inhibition of the IP3Rs [87] or increased leak of Ca

2+

from the ER resulting in the lower steady-

2+

state levels of Ca [88]. In support of the latter hypothesis, authors of a BAX and BAK depletion study report decreased ER resting Ca

2+

levels due to increased Ca

2+

leakage from the ER, presumably as a

At the level of mitochondria the regulation of Ca

2+

PT

direct result of increased levels of non-bound BCL-2 [88]. levels involves interaction of BCL-2 with VDAC

which decreases its channel conductivity [89]. In contrast, VDAC interaction with BCL-XL [90], MCL-1

RI

2+

[91], BAX [92] and BNIP3 [93] leads to increased Ca conductance and elevated Ca

2+

uptake into the

SC

mitochondrial matrix which can lead to a boost of OXPHOS and/or cell death. NIX, depending on its localisation to mitochondria or to the ER, can trigger apoptotic or necrotic cell death linked to its involvement in Ca

2+

signalling, though the exact mechanisms remain elusive [81, 94]. BIK, a BH3-

lead to the leak of Ca

2+

NU

only member of the BCL-2 family (Figure 2), localises to the ER. Overexpression of this protein can from the ER stores, mitochondrial fragmentation and Drp1 recruitment to

MA

OMM followed by apoptotic cristae remodelling [95].

Taken together, these observations strongly implicate the BCL-2 family proteins in the regulation of the integrity of mitochondrial membranes, mitochondrial quality control, Ca

2+

signalling and cell

survival. This function is controlled by a complex net of factors such as their protein levels, localisation

PT E

D

and interaction with one another, altogether affecting their availability to other binding partners. Control of mitochondrial function at the IMM The IMM is a non-permeable barrier to ions and molecules. The IMM curves and invaginates into the mitochondrial matrix thus forming a third compartment in addition the IMS and the mitochondrial

CE

matrix, the so-called cristae lumen. The cristae lumen accounts for the largest proportion of the IMM and is separated from the IMS by mitochondrial contact site and cristae organising system (MICOS, previously known as MitOS and MINOS), a protein complex positioned at the interface of cristae

AC

junctions forming a link between the IMM and OMM [96]. MICOS subunits interact with import machinery, complexes III and IV of the ETC and with CL to shape mitochondrial cristae [97]. In addition, studies in yeast uncovered that cristae curvature is controlled by the arrangement of ATP synthase dimer rows (Figure 3A) and membrane CL content [98]. Proteins and lipids located at the IMM or enclosed within the cristae lumen participate in the maintenance of healthy mitochondrial energetics, membrane potential and cristae structure. Cristae structure link to changes in metabolism OPA1 is a GTPase well known for its role in mitochondrial inner membrane fusion via its interaction with CL [99]. Additionally, OPA1 regulates cristae structure via oligomerisation and interaction with MICOS complex subunit, Mic60, to regulate cristae width and length in response to nutrient availability [100]. As described above, OPA1 adopts eight isoforms and the proportion of long, 11

ACCEPTED MANUSCRIPT membrane-anchored, and short, soluble, isoforms is thought to influence cristae structure via formation and stabilisation of IMM invaginations (Figure 3A) [16, 101]. Starvation-induced elevation in energy demand signals converge on OPA1 trigger cristae remodelling via increased L-OPA1 oligomerisation and result in a tighter cristae structure [102]. To investigate the mechanism upstream of OPA1 oligomerisation status, immunoprecipitation and mass spectrometry-based proteomics studies were performed to identify proteins which interact with OPA1 in response to a change in energy demand [102]. This led to the identification of mitochondrial SLC5A3 (phosphate carrier protein), SLC5A5 and SLC5A6 (ADP/ATP translocases 2 and 3, respectively) as OPA1 binding

PT

partners. In addition, the screen identified ATP5A1, ATP5B and ATP5C1 (subunits α, β and γ of ATP synthase) as OPA1 binding partners, thus pointing towards its role in the assessment of nutrient

RI

availability for ATP generation [102]. Furthermore, modelling studies suggested that OPA1-mediated cristae remodelling can play a role in metabolite diffusion within mitochondria, and metabolite

SC

exchange between the mitochondria and cytoplasm [103]. In turn, loss of MMP leads to L-OPA1 cleavage and further processing to short isoforms which participate in mitochondrial fission and cell

NU

death. Cristae structure remodelling in apoptosis

-

Sequestered in the intermembrane space by OPA1 bridging is an ETC mobile e carrier cyt c. A -

MA

minority of the mitochondrial cyt c pool remains soluble and participates in e shuttling within the ETC and superoxide scavenging [104-106]. Most cyt c molecules are tethered to IMM by interactions with a non-oxidised form of CL [105]. The tightly bound form of cyt c undergoes a conformational change upon association with CL, which results in exposure of its heme and plays a role in cyt c-mediated

D

H2O2 scavenging [106]. For apoptosis to proceed, the large pool of tightly-bound cyt c must first be

PT E

released from CL, its IMM binding partner and form a large soluble cyt c fraction. Mobilisation of the tightly bound cyt c pool has been linked to high ROS production by mitochondria, upon which membrane-bound cyt c oxidises CL. CL oxidation lowers cyt c binding affinity and leads to cyt c dissociation from the IMM [106]. Release of soluble cyt c from within the cristae is facilitated by OPA1

CE

oligomer disassembly (Figure 3B) [107]. Excessive cleavage of L-OPA1 results in apoptotic remodelling of the IMM, opening of cristae junctions and cyt c release into the IMS and ultimately into

AC

the cytoplasm to initiate cell death pathways (Figure 3C). In contrast, OPA1 overexpression contributes to apoptosis resistance thanks to tight cristae structure and sequestration of apoptogenic factors within the cristae lumen [108]. Altogether, the IMM protein interactions form a network required for the maintenance of mitochondrial function as well as protection from apoptosis in normal conditions, and yet is able to prime mitochondria for apoptosis initiation in conditions of increased ROS generation by the ETC. OMM-IMM protein interactions Despite their distinct proteomes, the IMM and OMM do not exist entirely separated from each other and signals from the cytoplasm are often transmitted to the IMM via its interaction with the proteome of the OMM. Under normal circumstances, the extent of OMM and IMM protein interactions does not often exceed that of mitochondrial protein import machinery and metabolite import. In contrast, 12

ACCEPTED MANUSCRIPT harmful external stimuli and mitochondrial dysfunction can lead to a more extensive interaction of the IMM and OMM proteins. Recent evidence from studies of the role of the BCL-2 family members upon MOMP reveals a convergence of their localisation to the OMM on the disruption of CL, OPA1 and cyt c interactions to initiate apoptotic cristae remodelling and release of apoptosis inducing proteins, cyt c, SMAC/DIABLO and AIF, from within the cristae lumen. Firstly, BAX and BAK oligomerisation was shown to lead to LOPA1 proteolytic cleavage and result in the loss of cristae structure [109]. Secondly, BNIP3 was

PT

found to interact with OPA1 and disrupt its oligomers in a manner dependent on its BH3-only domain and the presence of BAX and BAK [110]. Alternatively, if the apoptotic pathway is initiated by Bid cleavage, tBid, the truncated product of Bid, contains a CL binding domain and is capable of

RI

displacing cyt c from CL and thus initiate apoptotic signalling [111]. Thus interaction between OMM and IMM proteins lead to coordination of OMM permeabilisation and IMM cristae loss which are both

SC

required for apoptosis initiation.

Mitochondria and mitochondrial quality control in ageing

increased ROS production and impaired Ca

2+

NU

Dysfunctional mitochondria affect cellular health not only by inefficient ATP synthesis, but also by buffering [112]. Aberrant ROS release at mitochondria

initially upregulates cellular catabolic pathways of protein degradation and selective autophagy to

MA

remove the dysfunctional organelles from the mitochondrial pool. Loss of proteins involved in the regulation of mitochondrial dynamics results in altered metabolism, further ROS production and cell death, all potentially contributing to ageing. Impaired removal of defective organelles is also linked to

D

the onset of complex age-related diseases, at least in part due to energy depletion which underpins

nervous system [112]. Skeletal muscle wasting

PT E

increased frailty of the most energy demanding tissues - the skeletal muscle, the heart and the central

Ageing of the skeletal muscle, or sarcopenia, is associated with the loss of muscle innervation, 2+

CE

muscle mass and muscle function [113]. On a cellular level, muscle contraction results in a release of Ca from sarcoplasmic reticulum (SR), its uptake to mitochondria and upregulation of TCA cycle and

AC

OXPHOS, which are central to the maintenance of muscle energy metabolism [114, 115]. Recent evidence identifies the roles of two IMM proteins, OPA1 and a mitochondrial calcium uniporter 2+

complex (MCU), an IMM Ca channel [116], in muscle ageing and its prevention. A study of healthy human skeletal muscle biopsies revealed that mitochondrial DNA (mtDNA) content, mRNA levels and ATP production decline with age [117]. In addition, authors observed increased levels of DNA oxidation and a significant reduction of the majority of mitochondrial proteins probed within the tissue from aged volunteers, most notably the NADH and pyruvate dehydrogenases. Furthermore, studies of aged human [118] and chronically disused mouse tissue [119] both reveal higher levels of mitochondrial fragmentation and altered expression levels of proteins involved in the regulation of mitochondrial dynamics, mainly OPA1, Mfn2 and DRP1. A recently published study identifies OPA1 as a factor contributing to muscle maintenance, or conversely, to muscle loss when

13

ACCEPTED MANUSCRIPT its levels decline [120]. Also within this study, age-related decline in OPA1 levels was observed in sedentary volunteers when compared to their active counterparts. Supporting the role of OPA1 deficiency in muscle ageing is the observation that patients carrying a mutation in OPA1 develop a late-onset myopathy [121]. Physical exercise is known to promote healthy muscle ageing and to be beneficial for the recovery of lost muscle mass and strength. In a recent study, muscle strengthening by electric stimulation or leg press in elderly volunteers led to increased levels of MCU [122]. Additionally, electric stimulation led

PT

to increased levels of COX IV and OPA1 and a concomitant increased fusion of the mitochondrial network. Interestingly, in contrast to the electric stimulation, leg press exercise resulted in decreased levels of OPA1 though its transcript levels remained unchanged. Despite the discrepancy of the OPA1

RI

level findings, both approaches resulted in increased muscle strength and MCU levels, not related to protein translation, but rather to increased stability. Further studies of the mechanisms of increased

SC

stability of OPA1 and MCU might lead to a better understanding of the pathways that underlie muscle ageing and aid in the development of therapeutic treatments for sarcopenia [118, 120, 122].

NU

Cardiac ischaemia/reperfusion injury

Ischaemia, a restriction of blood supply to a tissue, is associated with a shortage of oxygen and energy substrate delivery to cells and results in impaired energy metabolism and, if not corrected by

MA

reperfusion, in cell death. Ischaemia triggers release of Ca

2+

from the sarco/endoplasmic reticulum

into the cytoplasm [123]. Cells protect themselves by MCU-mediated Ca

2+

import into mitochondria

and rely on reperfusion to support cell survival. However, prolonged ischaemia, despite reperfusion

D

and re-establishment of MMP, can lead to ischaemia/reperfusion (I/R) injury and trigger MPT-driven

PT E

necrotic cell death [124].

Of the BCL-2 protein family, roles of BNIP3, Bid and PUMA have been explored in ischaemia. Bid and PUMA have been implicated in ischaemia-induced apoptosis and their levels were shown to correlate with cardiac infarct size [125, 126]. Increased BNIP3 expression was observed in the hearts of adult

CE

rats subjected to hypoxia and those with chronic heart failure [127]. In an in vitro model of simulated cardiac I/R, BNIP3 was found to induce both mitochondrial dysfunction and autophagy [128]. A

AC

boosted autophagy response by co-expression of BNIP3 and Atg5 in this model partially protected cells from apoptosis, corroborating findings of an earlier study of chronic myocardial ischaemia in swine in which regions of the myocardium with enhanced autophagy displayed lower levels of apoptosis [129].

Cardiac hypertrophy The ageing process in the human heart is characterised by the progressive loss of myocytes and pathological hypertrophy, fibrosis and altered Ca2+ signalling in the surviving tissue [130]. Aberrant 2+

release of Ca from the endo/sarcoplasmic reticulum (ER/SR), its uptake by mitochondria and spread through the mitochondrial network can initiate large-scale ROS production and MPT-driven necrosis [76]. Ca

2+

handling and signal propagation within the ER and mitochondrial networks are regulated by

the BCL-2 protein family at both organelles; at the ER via the inositol 1,4,5-trisphosphate receptors

14

ACCEPTED MANUSCRIPT (IP3Rs) and at the mitochondrial OMM via interaction with VDACs [86]. A dual localisation of NIX to mitochondria and to the ER/SR, and the role it plays in ER/SR/mitochondrial Ca

2+

flux regulation

made it a target for a study in cardiac hypertrophy. Overexpression of this protein can lead to the leak of Ca

2+

from the ER stores, mitochondrial fragmentation and Drp1 recruitment to OMM followed by

apoptotic cristae remodelling [95]. Increased NIX expression was found to coincide with elevated 2+

diastolic Ca

levels in a transgenic mouse model and in human hypertensive left ventricular

hypertrophy samples [131].

PT

In addition, decreased levels of CL and enzymes involved in its synthesis and remodelling were reported in rat models of spontaneous hypertensive heart failure and in failing human hearts upon explanation [132]. Deficient CL content in the IMM has been linked to alterations of cristae structure,

RI

decreases in supercomplex assembly and aberrations in oxidative phosphorylation [133]. Together these findings point towards the role of mitochondrial machinery in the age-related decline of the heart

SC

although a causal link between NIX, CL and cardiac hypertrophy is yet to be established. Supporting the role of BNIP3 and NIX proteins in healthy cardiac ageing is a study from Dorn’s group,

NU

in which aged BNip3 and Nix double cardiac-specific knockout mice are reported to accumulate mitochondria with aberrant cristae structure [134]. Altogether these findings suggest that BNIP3 and NIX expression and a functional autophagic response are required for the maintenance of a healthy

acute mitochondrial stress and cell death. Cerebral ischaemia/reperfusion injury

MA

mitochondrial pool in development and during healthy ageing and may together be protective against

D

Cerebral ischaemia is a shortage of blood flow to the brain observed in many pathologies, most

PT E

commonly an ischaemic stroke as a result of a blood vessel occlusion or blood flow interruption in cardiac arrest. I/R insult to the cell results in increased levels of mitophagy upon reperfusion which, when inhibited, leads to exacerbated cellular injury [135]. The molecular nature of mitophagy induction upon I/R injury is three-fold and involves the combined action of PINK1-/Parkin-, NIX- and

CE

CL-mediated mitophagy. Firstly, Parkin was first shown to induce mitophagy upon reperfusion in a cellular model of oxygen-glucose deprivation (OGD) [135] and further confirmed in Park2

AC

heterozygous knockout mice subjected to the transient middle cerebral artery occlusion (tMCAO) and ER stress [136].

Secondly, ischaemia leads to a hypoxic environment in regions where blood flow is occluded. Increased expression of NIX was reported from a variety of cell lines grown in hypoxia and was only -/-

recently linked to a pathological cerebral mitophagy [137]. Nix mice subjected to tMCAO presented with significantly lower levels of mitophagy and suffered from increased infarct volumes and neurological pathologies compared to controls. Further in vitro experiments confirmed that Nixinduced mitophagy is independent of Parkin, and vice versa [137]. Additionally, a double knockout of Park2 and Nix leads to a synergistic mitophagy impairment and a further decreased tissue viability [137].

15

ACCEPTED MANUSCRIPT Lastly, it has long been recognised that large-scale oxidation is associated with neuronal injury. More recently, CL externalisation and mitophagy induction was observed in injured neurons [57]. Fundamental changes to the diverse population of neuronal CL species upon experimental traumatic brain injury were detected in a lipidomics study [138]. Neuronal injury in both rat and human brains lead to extensive oxidation of CL species, but not other phospholipids of the brain. In vitro, introduction of oxidised CL species into neurons results in cell death, while prevention of CL oxidation by XJB-5-131, a blood-brain barrier permeable antioxidant, was shown to be neuroprotective in the

PT

context of experimental traumatic brain injury [138]. Neurodegeneration

Low regenerative ability, high energetic demand and large size of neuronal cells make neurons

RI

susceptible to damage accumulation [139-141]. Efficient, accurate and timely degradation of damaged cellular components, amongst them mitochondria, is essential for homeostasis maintenance

SC

and neuronal cell survival [139, 140]. Mitochondrial dysfunction in neurodegenerative diseases stems from the accumulation of mutations in mitochondrial DNA, decrease in mitochondrial function and Neurons of healthy aged humans and humans presenting with

NU

impairment of turnover [139].

neurodegenerative diseases suffer from deficiencies of OXPHOS complexes and aberrant mitochondrial dynamics [140]. The PINK1/Parkin-mediated mitophagy has long been considered to be

MA

the main pathway of mitochondrial turnover in neurons. However, conflicting evidence from nonneuronal and neuronal models and a lack of rapid depolarisation response of neurons in cell culture to parkinsonian toxins (rotenone, 6-hydroxydopamine) paints a complicated story which is likely to involve all, PINK1/Parkin mediated, CL initiated, LIR-adaptor-dependent and MDV-mediated modes of

D

mitophagy in response to different levels of damage severity [42, 141]. For a detailed review of

PT E

mitophagy impairment in neurodegenerative diseases see [141]. Studies of the ageing brain have revealed increased levels and stages of processing of members of the BCL-2 protein family. Firstly, elevated levels of BAK were detected in elderly cerebrum and

CE

cerebellum regions of the brain [142]. Secondly, increased BCL-2 levels were found in nigrostriatal dopaminergic regions of parkinsonian patients, while no significant difference was detected in the cerebral cortex [143]. Considering that the tissue was examined post-mortem, high levels of BCL-2 in

AC

surviving dopaminergic regions could indicate a protective effect of increased BCL-2 expression against cell death. Thirdly, tBid levels were elevated in temporal cortex of parkinsonian brain [144]. Together, elevated levels of anti-apoptotic and pro-apoptotic proteins could sensitise neurons to, and mediate, cell death. Therapeutic targeting of mitochondrial dysfunction Pharmacological or genetic manipulation of mitochondrial processes is indispensable for basic research and for clinical medicine. Small molecule delivery into the mitochondrial IMS or matrix is complex due to the double membraned nature of mitochondria, the negative charge of the organelle and, in the case of brain injury or neurodegeneration, compound permeability through the blood-brain barrier. Delivery of an active compound into mitochondria can be achieved by various methods. Firstly, a potent antioxidant targeting approach was developed by Murphy and co-workers [145]. 16

ACCEPTED MANUSCRIPT Triphenylphosphonium (TPP), a lipophilic cation, is driven into the cell based on plasma membrane potential and accumulates in mitochondria due to MMP. Conjugation of TPP to antioxidants leads to efficient mitochondrial delivery which led to the design of a plethora of antioxidants targeting various ROS moieties, including superoxide (MitoSOD, MitoCP), hydrogen peroxide (MitoPeroxidase), ferrous iron (MitoTEMPOL) and lipid peroxidation (MitoQ10, MitoE2, SkQ1) [145, 146]. Extensive effort has been dedicated to characterisation of the TTP conjugated compounds. While pre-treatment of mouse and rat models leads to protection from I/R injury [147, 148], their efficacy in age-related diseases is yet to be established. Secondly, liposome-like vesicles are explored as a method of mitochondrial

PT

compound and DNA delivery [149]. The method is based on the self-assembly of dequalinium, a mitochondriotropic, into vesicles (‘DQAsomes’), their entry into the cell and their disassembly upon

RI

interaction with mitochondrial membranes. Coupled with a mitochondrial localisation signal, DQAsomes were successfully used to deliver plasmid DNA into mitochondria in cell culture [149, 150].

SC

However, further research and evaluation will be needed to prevent the cytotoxic effect of dequalinium in vivo and establishment of the liposome-like vesicle delivery as a possible therapeutic intervention.

NU

Development of new therapeutics which do not scavenge, but rather prevent the production of oxidative damage provide an exciting avenue for a mitochondria-targeted approach. Prevention of ROS release would be beneficial for the treatment of a large spectrum of pathologies which are

MA

underpinned by mitochondrial dysfunction and oxidative stress. An increased body of evidence links CL content, localisation and oxidation status to mitochondrial function, dynamics and turnover [72, 73, 99, 111, 133, 151-153]. CL has become an exciting target for battling age-related decline in OXPHOS function. Initially, a proof of concept study demonstrated that age-related changes in mitochondrial

D

lipid content affect OXPHOS function [154]. Targeted addition of phospholipids to the IMM rescues an

PT E

age-related decline in CIV function in healthy aged hearts which was hypothesized to occur due to changes in CIV lipid environment and an underlying loss of membrane fluidity [154]. In contrast, phospholipid addition provided little benefit to ischaemia-induced CIV dysfunction in the aged heart

complex [154].

CE

and thus suggest that an ischaemic episode introduces oxidative damage to the peptides of the

Szeto-Schiller peptide 31 (SS-31) is a novel cell permeable compound which specifically binds the

AC

phospholipid head of CL. By its close proximity, SS-31 prevents cyt c unfolding and peroxidase activity upon interaction with CL, and leads to a boost of OXPHOS activity and ATP production in ischaemia [155, 156]. SS-31 has now been tested in a variety of disease models of heart failure, skeletal muscle ageing and neurodegeneration, (reviewed in [157]) in which treatment with SS-31 was found to mitigate mitochondrial dysfunction and promote recovery of cellular bioenergetics status. SS31 (Elamipretide, Bendavia, MTP-131) was recently a subject of a Phase 2 intervention clinical trial with patients with confirmed mitochondrial disease [158]. Recruitment is currently undergoing for a 24week Phase 3 trial [159]. In addition, SBT-20, another Szeto-Schiller peptide, with increased stability in the cerebral fluid, is currently investigated in Phase 1/2 trial of patients with early stage Huntington’s disease [160].

17

ACCEPTED MANUSCRIPT Canonical and atypical BCL-2 family members stand at the crossroad of multiple cellular fitness signalling pathways. Development and synthesis of inhibitory and activatory compounds acting on 2+

members involved in Ca

signalling and apoptotic cell death are currently explored for the benefit of

combined chemotherapy treatment and of senolytic treatment to clear cancer and senescent cells by apoptosis induction [161-163]. In an effort to prevent the initiation of apoptosis upon I/R injury, Chen laboratory succeeded in an in vivo delivery of BCL-XL and prevention of neuronal loss when administered post ischemia in a mouse model [164]. The delivery system is based on a fusion of the desired protein to a protein transduction domain (PTD) of the human trans- activator of transcription 2+

Ca

PT

(TAT) and its permeability through the blood-brain barrier. In contrast to BCL-2 which plays a role in signalling and localises to both ER and mitochondria, BCL-XL which is tethered to the OMM

addition, due to the role of aberrant Ca

2+

RI

provides a good target for apoptosis modulation by TAT-mediated BCL-XL delivery [164, 165]. In release from ER/SR and accumulation in mitochondria upon

SC

I/R injury, MCU has been identified as a target for therapy. Two studies from the 1982 [166] and 1996 [167] identified ruthenium red (RR), an MCU inhibitor as a compound capable of preventing reperfusion-induced accumulation of Ca

2+

in mitochondria and ameliorating the infarct size and

NU

recovery. The beneficial effects of MCU inhibition in myocardial I/R translate to cerebral I/R. MCU channel inhibition prior to MCAO in a rat model significantly reduced the infarct volume, neuronal damage and levels of apoptosis, while activation of MCU by spermine supplementation increased the

MA

injury load [168]. Concluding Remarks

Mitochondria play a vital role in cellular and tissue homeostasis by facilitating substrate metabolism,

D

energy production and cell death regulation. A multitude of recent research advances paint a more

PT E

nuanced picture of mitochondrial ageing and its impact on cellular health. Altered energy production, increased ROS release, impaired mitochondrial dynamics, dysfunctional quality control and increased levels of cell death have all been linked to the development of age-related pathologies. It is also well documented that mitochondrial dysfunction precedes or occurs concurrently with an onset of

CE

pathologies. However, it remains unclear how age-related damage accumulation within the organelles affects their responsiveness to cytoplasmic and extracellular signalling.

AC

In this review, we aimed to highlight the complexity of temporal and spatial interconnectedness of key mitochondrial processes which maintain cellular health and survival. An important factor in the study of mitochondrial dysfunction in ageing is the pleiotropic nature of nearly all mediators involved in mitochondrial function, quality control and cell death execution. Interventions targeted at a single protein could therefore lead to unexpected large-scale effects in multiple pathways, or elicit compensatory effects due to system redundancies. Altered levels of proteins regulating mitochondrial dynamics, elevated levels of pro-apoptotic BCL-2 family members and large-scale oxidation are present in aged mouse and human tissue. The underlying processes are currently explored as targets for small therapeutic compounds aimed to attenuate the effect of ageing on the human population. Current strategies tend to focus on alleviating the effects downstream of mitochondrial dysfunction. It will be interesting to see if future studies 18

ACCEPTED MANUSCRIPT highlight the age-related impairment in quality control pathways and focus on elucidating how impairment of autophagy/mitophagy acts upstream of mitochondrial dysfunction and contributes to disease pathology and the burden of human ageing.

Acknowledgements: This work was funded by BBSRC and the MRC Centre for Ageing and Vitality.

7. 8. 9.

10. 11. 12. 13.

14.

19

RI

SC

NU

MA

6.

D

5.

PT E

4.

CE

3.

López-Otín, C., et al., The Hallmarks of Aging. Cell, 2013. 153(6): p. 1194-1217. Cogliati, S., J.A. Enriquez, and L. Scorrano, Mitochondrial Cristae: Where Beauty Meets Functionality. Trends in Biochemical Sciences, 2016. 41(3): p. 261-273. Sazanov, L.A., A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat Rev Mol Cell Biol, 2015. 16(6): p. 375-88. Watt, I.N., et al., Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc Natl Acad Sci U S A, 2010. 107(39): p. 16823-7. Weinberg, S.E. and N.S. Chandel, Targeting mitochondria metabolism for cancer therapy. Nature chemical biology, 2015. 11(1): p. 9-15. Frezza, C., Mitochondrial metabolites: undercover signalling molecules. Interface Focus, 2017. 7(2). Youle, R.J. and A.M. van der Bliek, Mitochondrial Fission, Fusion, and Stress. Science, 2012. 337(6098): p. 1062-1065. McBride, H.M., M. Neuspiel, and S. Wasiak, Mitochondria: More Than Just a Powerhouse. Current Biology, 2006. 16(14): p. R551-R560. Gomes, L.C., G.D. Benedetto, and L. Scorrano, During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nature Cell Biology, 2011. 13: p. 589. Molina, A.J.A., et al., Mitochondrial Networking Protects β-Cells From NutrientInduced Apoptosis. Diabetes, 2009. 58(10): p. 2303. Westermann, B., Mitochondrial fusion and fission in cell life and death. Nature reviews Molecular cell biology, 2010. 11(12): p. 872. Koshiba, T., et al., Structural Basis of Mitochondrial Tethering by Mitofusin Complexes. Science, 2004. 305(5685): p. 858-862. Bertholet, A.M., et al., Mitochondrial fusion/fission dynamics in neurodegeneration and neuronal plasticity. Neurobiology of Disease, 2016. 90: p. 3-19. Alexander, C., et al., OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Genet, 2000. 26(2): p. 211-5.

AC

1. 2.

PT

References:

ACCEPTED MANUSCRIPT

22.

23. 24. 25.

26. 27.

28.

29. 30.

31. 32.

20

PT

RI

SC

21.

NU

20.

MA

19.

D

18.

PT E

17.

CE

16.

Delettre, C., et al., Nuclear gene OPA1, encoding a mitochondrial dynaminrelated protein, is mutated in dominant optic atrophy. Nat Genet, 2000. 26(2): p. 207-10. Song, Z., et al., OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L. The Journal of Cell Biology, 2007. 178(5): p. 749-755. Delettre, C., et al., Mutation spectrum and splicing variants in the OPA1 gene. Hum Genet, 2001. 109(6): p. 584-91. Ban, T., et al., Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat Cell Biol, 2017. 19(7): p. 856-863. Ishihara, N., et al., Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. The EMBO journal, 2006. 25(13): p. 2966-2977. Anand, R., et al., The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission. J Cell Biol, 2014. 204(6): p. 919-929. Basu, K., et al., Molecular mechanism of DRP1 assembly studied in vitro by cryoelectron microscopy. PLoS ONE, 2017. 12(6): p. e0179397. Francy, C.A., et al., The mechanoenzymatic core of dynamin-related protein 1 comprises the minimal machinery required for membrane constriction. Journal of Biological Chemistry, 2015. 290(18): p. 11692-11703. Friedman, J.R., et al., ER tubules mark sites of mitochondrial division. Science, 2011. 334(6054): p. 358-62. Lin, M.T. and M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature, 2006. 443: p. 787. Wallace, D.C., A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu. Rev. Genet., 2005. 39: p. 359-407. Ravikumar, B., et al., Regulation of Mammalian Autophagy in Physiology and Pathophysiology. Physiological Reviews, 2010. 90(4): p. 1383-1435. Palikaras, K. and N. Tavernarakis, Mitochondrial homeostasis: The interplay between mitophagy and mitochondrial biogenesis. Experimental Gerontology, 2014. 56: p. 182-188. Jin, S.M., et al., Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. The Journal of Cell Biology, 2010. 191(5): p. 933-942. Yamano, K. and R.J. Youle, PINK1 is degraded through the N-end rule pathway. Autophagy, 2013. 9(11): p. 1758-1769. Okatsu, K., et al., PINK1 autophosphorylation upon membrane potential dissipation is essential for Parkin recruitment to damaged mitochondria. Nature Communications, 2012. 3: p. 1016. Koyano, F., et al., Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature, 2014. 510(7503): p. 162-6. Bingol, B., et al., The mitochondrial deubiquitinase USP30 opposes parkinmediated mitophagy. Nature, 2014. 510(7505): p. 370-5.

AC

15.

ACCEPTED MANUSCRIPT

40. 41. 42.

43.

44. 45. 46. 47.

48.

49.

50.

21

PT

RI

SC

39.

NU

38.

MA

37.

D

36.

PT E

35.

CE

34.

Cornelissen, T., et al., The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy. Human Molecular Genetics, 2014. 23(19): p. 5227-5242. Marcassa, E., et al., Dual role of USP30 in controlling basal pexophagy and mitophagy. EMBO Rep, 2018. 19(7). Wang, X., et al., PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility. Cell, 2011. 147(4): p. 893-906. Tanaka, A., et al., Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin. The Journal of Cell Biology, 2010. Sarraf, S.A., et al., Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature, 2013. 496(7445): p. 372-6. Hamacher-Brady, A. and N.R. Brady, Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy. Cellular and Molecular Life Sciences, 2016. 73(4): p. 775-795. Lazarou, M., et al., The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature, 2015. 524(7565): p. 309-314. Van Humbeeck, C., et al., Parkin interacts with Ambra1 to induce mitophagy. J Neurosci, 2011. 31(28): p. 10249-61. Wei, Y., et al., Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor. Cell, 2017. 168(1): p. 224-238.e10. Whitworth, A.J. and L.J. Pallanck, PINK1/Parkin mitophagy and neurodegeneration—what do we really know in vivo? Current Opinion in Genetics & Development, 2017. 44: p. 47-53. Jin, S.M. and R.J. Youle, The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria. Autophagy, 2013. 9(11): p. 1750-1757. Sliter, D.A., et al., Parkin and PINK1 mitigate STING-induced inflammation. Nature, 2018. 561(7722): p. 258-262. Stolz, A. and I. Dikic, Elusive mitochondrial connection to inflammation uncovered. 2018, Nature Publishing Group. McWilliams, T.G., et al., mito-QC illuminates mitophagy and mitochondrial architecture in vivo. The Journal of Cell Biology, 2016. McWilliams, T.G., et al., Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand. Cell Metabolism, 2018. 27(2): p. 439-449.e5. Liu, L., et al., Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nature cell biology, 2012. 14(2): p. 177. Li, W., et al., MicroRNA-137 is a novel hypoxia-responsive microRNA that inhibits mitophagy via regulation of two mitophagy receptors FUNDC1 and NIX. Journal of Biological Chemistry, 2014. 289(15): p. 10691-10701. Zhang, J. and P.A. Ney, Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death And Differentiation, 2009. 16: p. 939.

AC

33.

ACCEPTED MANUSCRIPT

57.

58.

59. 60.

61.

62.

63.

64. 65.

22

PT

RI

SC

NU

56.

MA

55.

D

54.

PT E

53.

CE

52.

Novak, I., et al., Nix is a selective autophagy receptor for mitochondrial clearance. EMBO reports, 2010. 11(1): p. 45-51. Schweers, R.L., et al., NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Proceedings of the National Academy of Sciences, 2007. 104(49): p. 19500-19505. Daido, S., et al., Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells. Cancer Res, 2004. 64(12): p. 4286-93. Kanzawa, T., et al., Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene, 2005. 24(6): p. 980-91. Dowhan, W., M. Bogdanov, and E. Mileykovskaya, CHAPTER 1 - Functional roles of lipids in membranes, in Biochemistry of Lipids, Lipoproteins and Membranes (Fifth Edition), D.E. Vance and J.E. Vance, Editors. 2008, Elsevier: San Diego. p. 1-37. Liu, J., et al., Phospholipid scramblase 3 controls mitochondrial structure, function, and apoptotic response. Mol Cancer Res, 2003. 1(12): p. 892-902. Chu, C.T., et al., Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol, 2013. 15(10): p. 1197-1205. McLelland, G.L., et al., Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. The EMBO Journal, 2014. 33(4): p. 282-295. Sugiura, A., et al., A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. The EMBO Journal, 2014. 33(19): p. 2142-2156. Soubannier, V., et al., Reconstitution of Mitochondria Derived Vesicle Formation Demonstrates Selective Enrichment of Oxidized Cargo. PLOS ONE, 2012. 7(12): p. e52830. Harbauer, Angelika B., et al., The Protein Import Machinery of Mitochondria—A Regulatory Hub in Metabolism, Stress, and Disease. Cell Metabolism, 2014. 19(3): p. 357-372. Martinus, R.D., et al., Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome. European Journal of Biochemistry, 1996. 240(1): p. 98-103. Shpilka, T. and C.M. Haynes, The mitochondrial UPR: mechanisms, physiological functions and implications in ageing. Nature Reviews Molecular Cell Biology, 2017. 19: p. 109. Nargund, A.M., et al., Mitochondrial Import Efficiency of ATFS-1 Regulates Mitochondrial UPR Activation. Science, 2012. 337(6094): p. 587-590. Galluzzi, L., et al., Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ, 2018. 25(3): p. 486-541.

AC

51.

ACCEPTED MANUSCRIPT

73.

74.

75. 76. 77. 78.

79. 80.

81.

82. 83.

23

PT

RI

SC

72.

NU

71.

MA

70.

D

69.

PT E

68.

CE

67.

Elmore, S., Apoptosis: a review of programmed cell death. Toxicologic pathology, 2007. 35(4): p. 495-516. Hata, A.N., J.A. Engelman, and A.C. Faber, The BCL-2 family: key mediators of the apoptotic response to targeted anti-cancer therapeutics. Cancer discovery, 2015. 5(5): p. 475-487. Youle, R.J. and A. Strasser, The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology, 2008. 9: p. 47. Cheng, E.H.Y., et al., Conversion of Bcl-2 to a Bax-like Death Effector by Caspases. Science, 1997. 278(5345): p. 1966. Michels, J., P.W.M. Johnson, and G. Packham, Mcl-1. The International Journal of Biochemistry & Cell Biology, 2005. 37(2): p. 267-271. Czabotar, Peter E., et al., Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis. Cell, 2013. 152(3): p. 519-531. Lutter, M., et al., Cardiolipin provides specificity for targeting of tBid to mitochondria. Nature Cell Biology, 2000. 2: p. 754. Gonzalvez, F., et al., tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak. Cell Death Differ, 2005. 12(6): p. 614-26. Srinivasula, S.M., et al., A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature, 2001. 410: p. 112. Riedl, S.J. and G.S. Salvesen, The apoptosome: signalling platform of cell death. Nat Rev Mol Cell Biol, 2007. 8(5): p. 405-13. Vanden Berghe, T., et al., Regulated necrosis: the expanding network of nonapoptotic cell death pathways. Nat Rev Mol Cell Biol, 2014. 15(2): p. 135-47. Izzo, V., et al., Mitochondrial Permeability Transition: New Findings and Persisting Uncertainties. Trends in Cell Biology. 26(9): p. 655-667. Ott, M., et al., Cytochrome c release from mitochondria proceeds by a two-step process. Proceedings of the National Academy of Sciences, 2002. 99(3): p. 1259-1263. Pattingre, S., et al., Bcl-2 Antiapoptotic Proteins Inhibit Beclin 1-Dependent Autophagy. Cell, 2005. 122(6): p. 927-939. Vande Velde, C., et al., BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol, 2000. 20(15): p. 5454-68. Chen, Y., et al., Dual autonomous mitochondrial cell death pathways are activated by Nix/BNip3L and induce cardiomyopathy. Proceedings of the National Academy of Sciences, 2010. 107(20): p. 9035. Sentelle, R.D., et al., Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy. Nature chemical biology, 2012. 8(10): p. 831-838. Henry, B., et al., Targeting the ceramide system in cancer. Cancer Letters, 2013. 332(2): p. 286-294.

AC

66.

ACCEPTED MANUSCRIPT

85.

86. 87.

93.

94.

95.

96.

97. 98.

24

NU

MA

D

92.

PT E

91.

CE

90.

AC

89.

SC

RI

88.

Frank, S., et al., The Role of Dynamin-Related Protein 1, a Mediator of Mitochondrial Fission, in Apoptosis. Developmental Cell. 1(4): p. 515-525. Karbowski, M., et al., Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. The Journal of Cell Biology, 2002. 159(6): p. 931-938. Rasola, A. and P. Bernardi, Mitochondrial permeability transition in Ca2+dependent apoptosis and necrosis. Cell Calcium, 2011. 50(3): p. 222-233. Chen, R., et al., Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5trisphosphate. The Journal of Cell Biology, 2004. 166(2): p. 193-203. Oakes, S.A., et al., Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(1): p. 105-110. Arbel, N. and V. Shoshan-Barmatz, Voltage-dependent anion channel 1-based peptides interact with Bcl-2 to prevent antiapoptotic activity. J Biol Chem, 2010. 285(9): p. 6053-62. Huang, H., et al., An Interaction between Bcl-x(L) and the Voltage-dependent Anion Channel (VDAC) Promotes Mitochondrial Ca(2+) Uptake. The Journal of Biological Chemistry, 2013. 288(27): p. 19870-19881. Huang, H., et al., Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation. Cell Death Dis, 2014. 5: p. e1482. Shimizu, S., Y. Shinohara, and Y. Tsujimoto, Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator. Oncogene, 2000. 19(38): p. 4309-18. Chaanine, A.H., et al., Potential role of BNIP3 in cardiac remodeling, myocardial stiffness, and endoplasmic reticulum: mitochondrial calcium homeostasis in diastolic and systolic heart failure. Circ Heart Fail, 2013. 6(3): p. 572-83. Diwan, A., et al., Endoplasmic reticulum–mitochondria crosstalk in NIXmediated murine cell death. The Journal of Clinical Investigation, 2009. 119(1): p. 203-212. Germain, M., et al., Endoplasmic reticulum BIK initiates DRP1 ‐ regulated remodelling of mitochondrial cristae during apoptosis. The EMBO Journal, 2005. 24(8): p. 1546-1556. Pfanner, N., et al., Uniform nomenclature for the mitochondrial contact site and cristae organizing system. The Journal of Cell Biology, 2014. 204(7): p. 10831086. Friedman, J.R., et al., MICOS coordinates with respiratory complexes and lipids to establish mitochondrial inner membrane architecture. Elife, 2015. 4. Strauss, M., et al., Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. The EMBO Journal, 2008. 27(7): p. 1154-1160.

PT

84.

ACCEPTED MANUSCRIPT

106.

107.

108. 109.

110.

111.

112. 113. 114.

25

PT

RI

SC

105.

NU

104.

MA

103.

D

102.

PT E

101.

CE

100.

Ban, T., et al., Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nature Cell Biology, 2017. 19: p. 856. Glytsou, C., et al., Optic Atrophy 1 Is Epistatic to the Core MICOS Component MIC60 in Mitochondrial Cristae Shape Control. Cell Reports, 2016. 17(11): p. 3024-3034. Guillery, O., et al., Metalloprotease‐mediated OPA1 processing is modulated by the mitochondrial membrane potential. Biology of the Cell, 2008. 100(5): p. 315-325. Patten, D.A., et al., OPA1 ‐ dependent cristae modulation is essential for cellular adaptation to metabolic demand. The EMBO Journal, 2014. 33(22): p. 2676-2691. Germain, M., OPA1 and mitochondrial solute carriers in bioenergetic metabolism. Molecular & cellular oncology, 2015. 2(2): p. e982378. Hüttemann, M., et al., The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis. Mitochondrion, 2011. 11(3): p. 369-381. Berezhna, S., H. Wohlrab, and P.M. Champion, Resonance Raman investigations of cytochrome c conformational change upon interaction with the membranes of intact and Ca2+-exposed mitochondria. Biochemistry, 2003. 42(20): p. 614958. Kagan, V.E., et al., Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine. Free Radical Biology and Medicine, 2004. 37(12): p. 1963-1985. Yamaguchi, R., et al., Opa1-Mediated Cristae Opening Is Bax/Bak and BH3 Dependent, Required for Apoptosis, and Independent of Bak Oligomerization. Molecular Cell, 2008. 31(4): p. 557-569. Frezza, C., et al., OPA1 Controls Apoptotic Cristae Remodeling Independently from Mitochondrial Fusion. Cell, 2006. 126(1): p. 177-189. Jiang, X., et al., Activation of mitochondrial protease OMA1 by Bax and Bak promotes cytochrome c release during apoptosis. Proceedings of the National Academy of Sciences, 2014. 111(41): p. 14782-14787. Landes, T., et al., The BH3‐only Bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms. EMBO reports, 2010. 11(6): p. 459-465. Liu, J., et al., The cardiolipin-binding domain of Bid affects mitochondrial respiration and enhances cytochrome c release. Apoptosis, 2004. 9(5): p. 533541. Gonzalez-Freire, M., et al., Reconsidering the Role of Mitochondria in Aging. The Journals of Gerontology: Series A, 2015. 70(11): p. 1334-1342. Hepple, R.T. and C.L. Rice, Innervation and neuromuscular control in ageing skeletal muscle. The Journal of physiology, 2016. 594(8): p. 1965-1978. Berridge, M.J., P. Lipp, and M.D. Bootman, The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol, 2000. 1(1): p. 11-21.

AC

99.

ACCEPTED MANUSCRIPT

121. 122.

123.

124. 125.

126.

127.

128.

129.

26

PT

RI

SC

NU

120.

MA

119.

D

118.

PT E

117.

CE

116.

Denton, R.M., Regulation of mitochondrial dehydrogenases by calcium ions. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009. 1787(11): p. 13091316. Baughman, J.M., et al., Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature, 2011. 476(7360): p. 341-345. Short, K.R., et al., Decline in skeletal muscle mitochondrial function with aging in humans. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(15): p. 5618-5623. Crane, J.D., et al., The Effect of Aging on Human Skeletal Muscle Mitochondrial and Intramyocellular Lipid Ultrastructure. The Journals of Gerontology: Series A, 2010. 65A(2): p. 119-128. Iqbal, S., et al., Expression of mitochondrial fission and fusion regulatory proteins in skeletal muscle during chronic use and disuse. Muscle & Nerve, 2013. 48(6): p. 963-970. Tezze, C., et al., Age-Associated Loss of OPA1 in Muscle Impacts Muscle Mass, Metabolic Homeostasis, Systemic Inflammation, and Epithelial Senescence. Cell Metabolism, 2017. 25(6): p. 1374-1389.e6. Yu-Wai-Man, P., et al., Multi-system neurological disease is common in patients with OPA1 mutations. Brain, 2010. 133(3): p. 771-786. Zampieri, S., et al., Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics. Physiological Reports, 2016. 4(24): p. e13005. Murphy, E. and C. Steenbergen, Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiological reviews, 2008. 88(2): p. 581609. Halestrap, A.P., Calcium, mitochondria and reperfusion injury: a pore way to die. 2006, Portland Press Limited. Chen, M., et al., Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. Journal of Biological Chemistry, 2001. 276(33): p. 30724-30728. Toth, A., et al., Targeted deletion of Puma attenuates cardiomyocyte death and improves cardiac function during ischemia-reperfusion. American Journal of Physiology-Heart and Circulatory Physiology, 2006. 291(1): p. H52-H60. Regula, K.M., K. Ens, and L.A. Kirshenbaum, Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res, 2002. 91(3): p. 226-31. Hamacher-Brady, A., et al., Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell death and differentiation, 2007. 14(1): p. 146. Yan, L., et al., Autophagy in chronically ischemic myocardium. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(39): p. 13807-13812.

AC

115.

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

130. Shimizu, I. and T. Minamino, Physiological and pathological cardiac hypertrophy. Journal of Molecular and Cellular Cardiology, 2016. 97: p. 245-262. 131. Yussman, M.G., et al., Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy. Nat Med, 2002. 8(7): p. 725-30. 132. Saini-Chohan, H.K., et al., Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure. J Lipid Res, 2009. 50(8): p. 1600-8. 133. Zhang, M., E. Mileykovskaya, and W. Dowhan, Gluing the respiratory chain together. Cardiolipin is required for supercomplex formation in the inner mitochondrial membrane. J Biol Chem, 2002. 277(46): p. 43553-6. 134. Dorn, G.W., Mitochondrial Pruning by Nix and BNip3: An Essential Function for Cardiac-Expressed Death Factors. Journal of cardiovascular translational research, 2010. 3(4): p. 374-383. 135. Zhang, X., et al., Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance. Autophagy, 2013. 9(9): p. 1321-33. 136. Zhang, X., et al., Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury: Involvement of PARK2-dependent mitophagy. Autophagy, 2014. 10(10): p. 1801-13. 137. Yuan, Y., et al., BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2. Autophagy, 2017. 13(10): p. 1754-1766. 138. Ji, J., et al., Lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of brain injury. Nat Neurosci, 2012. 15(10): p. 1407-13. 139. Otten, E.G., D. Manni, and V.I. Korolchuk, Mitochondrial Degradation, Autophagy and Neurodegenerative Disease, in Mitochondrial Dysfunction in Neurodegenerative Disorders. 2016, Springer. p. 255-278. 140. Golpich, M., et al., Mitochondrial dysfunction and biogenesis in neurodegenerative diseases: pathogenesis and treatment. CNS neuroscience & therapeutics, 2017. 23(1): p. 5-22. 141. Martinez-Vicente, M., Neuronal mitophagy in neurodegenerative diseases. Frontiers in molecular neuroscience, 2017. 10: p. 64. 142. Obonai, T., M. Mizuguchi, and S. Takashima, Developmental and aging changes of Bak expression in the human brain. Brain Research, 1998. 783(1): p. 167-170. 143. Mogi, M., et al., bcl-2 Protein is increased in the brain from parkinsonian patients. Neuroscience Letters, 1996. 215(2): p. 137-139. 144. Jiang, H., et al., Bid signal pathway components are identified in the temporal cortex with Parkinson disease. Neurology, 2012. 79(17): p. 1767-1773. 145. Murphy, M.P. and R.A.J. Smith, Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu. Rev. Pharmacol. Toxicol., 2007. 47: p. 629-656. 146. Battogtokh, G., et al., Mitochondria-targeting drug conjugates for cytotoxic, anti-oxidizing and sensing purposes: Current strategies and future perspectives. Acta Pharmaceutica Sinica B, 2018. 27

ACCEPTED MANUSCRIPT

154.

155.

156.

157.

158.

159. 160.

161.

28

PT

RI

SC

153.

NU

152.

MA

151.

D

150.

PT E

149.

CE

148.

Adlam, V.J., et al., Targeting an antioxidant to mitochondria decreases cardiac ischemia-reperfusion injury. The FASEB Journal, 2005. 19(9): p. 1088-1095. Dare, A.J., et al., Protection against renal ischemia–reperfusion injury in vivo by the mitochondria targeted antioxidant MitoQ. Redox Biology, 2015. 5: p. 163168. D'Souza, G.G., S.V. Boddapati, and V. Weissig, Mitochondrial leader sequence-plasmid DNA conjugates delivered into mammalian cells by DQAsomes colocalize with mitochondria. Mitochondrion, 2005. 5(5): p. 352-8. Weissig, V., et al., Liposomes and liposome-like vesicles for drug and DNA delivery to mitochondria. J Liposome Res, 2006. 16(3): p. 249-64. Chu, C.T., H. Bayir, and V.E. Kagan, LC3 binds externalized cardiolipin on injured mitochondria to signal mitophagy in neurons: implications for Parkinson disease. Autophagy, 2014. 10(2): p. 376-8. Chu, C.T., et al., Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nature cell biology, 2013. 15(10): p. 1197. Unsay, J.D., et al., Cardiolipin Effects on Membrane Structure and Dynamics. Langmuir, 2013. 29(51): p. 15878-15887. Lesnefsky, E.J. and C.L. Hoppel, Ischemia–reperfusion injury in the aged heart: role of mitochondria. Archives of Biochemistry and Biophysics, 2003. 420(2): p. 287-297. Birk, A.V., et al., Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis. Br J Pharmacol, 2014. 171(8): p. 2017-28. Birk, A.V., et al., The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin. J Am Soc Nephrol, 2013. 24(8): p. 1250-61. Szeto, H.H., First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics. Br J Pharmacol, 2014. 171(8): p. 2029-50. A Study to Evaluate the Safety, Tolerability, and Efficacy of Subcutaneous Injections of Elamipretide (MTP-131) in Subjects With Genetically Confirmed Mitochondrial Disease Previously Treated in the Stealth BioTherapeutics SPIMM-201 Study. A Trial to Evaluate the Safety and Efficacy of Elamipretide in Subjects With Primary Mitochondrial Myopathy Followed by an Open-Label Extension. Register, E.C.T. A Two Part Study to Assess the Safety, Pharmacokinetics and Pharmacodynamics of SBT-020 in Patients with Early Stage Huntington’s Disease. 2018 [cited 2018 8 Oct]; Available from: https://www.clinicaltrialsregister.eu/ctr-search/search?query=2016-003730-25. Ashkenazi, A., et al., From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nature Reviews Drug Discovery, 2017. 16: p. 273.

AC

147.

ACCEPTED MANUSCRIPT

166.

167.

AC

CE

PT E

D

MA

168.

PT

165.

RI

164.

SC

163.

Zhu, Y., et al., The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging cell, 2015. 14(4): p. 644-658. Zhu, Y., et al., Identification of a novel senolytic agent, navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors. Aging Cell, 2016. 15(3): p. 428-435. Cao, G., et al., In vivo delivery of a Bcl-xL fusion protein containing the TAT protein transduction domain protects against ischemic brain injury and neuronal apoptosis. Journal of Neuroscience, 2002. 22(13): p. 5423-5431. Yin, W., et al., TAT-mediated delivery of Bcl-xL protein is neuroprotective against neonatal hypoxic–ischemic brain injury via inhibition of caspases and AIF. Neurobiology of Disease, 2006. 21(2): p. 358-371. Ferrari, R., et al., The effects of ruthenium red on mitochondrial function during post-ischaemic reperfusion. Journal of Molecular and Cellular Cardiology, 1982. 14(12): p. 737-740. Miyamae, M., et al., Attenuation of postischemic reperfusion injury is related to prevention of [Ca2+]m overload in rat hearts. Am J Physiol, 1996. 271(5 Pt 2): p. H2145-53. Zhao, Q., et al., The role of the mitochondrial calcium uniporter in cerebral ischemia/reperfusion injury in rats involves regulation of mitochondrial energy metabolism. Molecular medicine reports, 2013. 7(4): p. 1073-1080.

NU

162.

29

ACCEPTED MANUSCRIPT Figure Legends:

Figure 1. Mitochondrial Quality Control Degradation of whole mitochondrial organelles is preceded by a physical interaction of autophagy receptors with LC3-II and recruitment of nascent membranes for the formation of an autophagosome. (A) Loss of MMP leads to stabilisation of PINK1 at the OMM and recruitment of Parkin followed by ubiquitination of OMM proteins, followed by recognition and binding by autophagy receptors. (B)

PT

Oxygen depletion results in increased translation and activity of autophagy receptors resident at the OMM. (C) Oxidative stress promotes CL externalisation at the OMM and direct LC3-II binding. (D) Partial organelle quality control is achieved by budding and PINK1/Parkin-mediated delivery of the

RI

vesicle to the lysosome.

SC

Figure 2 - BCL-2 Protein Family

BCL-2 family members are regulators of mitophagy and cell death. Members all contain one or more of BCL-2 homology (BH) domains and an optional transmembrane (TM) domain. According to their

NU

levels of homology to BCL-2 and their function in cell death, members are divided into three subfamilies. The multidomain anti-apoptotic family members interact with pro-apoptotic members via a BH3 binding hydrophobic pocket formed by BH1-BH3 domains. Multidomain pro-apoptotic members

MA

homo-oligomerise to form pores in OMM. BH3-only pro-apoptotic proteins can activate cell death by direct binding to BAX, or sensitise cells to cell death via sequestration of anti-apoptotic family members. Non-canonical BH3-domain members BNIP3 and NIX are capable of inducing cell death in

D

the absence of multidomain pro-apoptotic BCL-2 members.

PT E

Figure 3. Changes in Mitochondrial IMM Integrity upon Apoptotic Cell Death IMM folding and cristae structure are controlled by the combined action of OPA1 oligomeric structure, ATP synthase dimers rows and CL localisation, all of which influence and anchor membrane curvature based on signals from the cytoplasm. (A) Mitochondrial IMM invaginates into tight cristae

CE

which allow for efficient OXPHOS in healthy cells. Anti-apoptotic members of the BCL-2 protein family outnumber pro-apoptotic members and prevent pore formation. (B) Cellular insults resulting in

AC

increased oxidative stress or elevated expression of pro-apoptotic BCL-2 family members promote MOMP initiation, dissociation of OPA1 oligomers, CL externalisation and increased solubility of cyt c. (C) Persistent damage signalling promotes BAX/BAK oligomerisation, MOMP and escape of apoptogens into the cytoplasm.

30

ACCEPTED MANUSCRIPT Conflict of Interest Statement:

AC

CE

PT E

D

MA

NU

SC

RI

PT

The authors declare no conflict of interest.

31

Figure 1

Figure 2

Figure 3