Crosstalk between mitochondria–ER contact sites and the apoptotic machinery as a novel health meter

1 Journal: Trends in Cell Biology 2 Date and volume: 2025 Jan;35(1):33-45. 3 doi: 10.1016/j.tcb.2024.08.007. 4 Title: Crosstalk between mitochondria-ER contact sites and the apoptotic 5 machinery as a novel health meter 6 Authors: Alvaro Larrañaga-SanMiguel, Nora Bengoa-Vergniory, Hector Flores7 Romero 8 PMID: 39379268 9 10 11 12 13 14 15 16 17 18 19 20 21 Crosstalk between Mitochondria-ER contact sites and apoptotic machinery as 22 a novel health-meter 23 Alvaro Larrañaga-SanMiguel1, Nora Bengoa-Vergniory1, 2, 3, 4 and Hector Flores24 Romero1-2,* 25 1. Achucarro Basque Center for Neuroscience, Leioa, Spain. 26 2. Ikerbasque, Basque Foundation for Science, 48013, Bilbao, Spain. 27 3. Oxford Parkinson’s Disease Centre and Department of Physiology, Anatomy and 28 Genetics, University of Oxford, South Park Road, Oxford OX1 3QU, UK. 29 4. University of the Basque Country (UPV/EHU), Department of Neuroscience, 30 Leioa, Spain. 31 * correspondence: [email protected] (Flores-Romero, H) 32 33 Keywords: MERCS, MAMs, Apoptosis, Inflammation, Neurodegeneration, cancer. 34 35 Abstract 36 Mitochondria-ER Contact Sites (MERCS) function as transient signalling platforms that 37 regulate essential cellular functions. MERCS are enriched in specific proteins and 38 lipids that connect mitochondria and the ER together, modulating their activities. 39 Dysregulation of MERCS is associated with several human pathologies, including 40 Alzheimer’s disease, Parkinson’s disease and cancer. BCL-2 family proteins can 41 locate at MERCS and control essential cellular functions such as calcium signalling 42 and autophagy in addition to their role in mitochondrial apoptosis. Moreover, the BCL43 2-mediated apoptotic machinery was recently found to trigger cGAS/STING pathway 44 activation and proinflammatory response, a recognized hallmark in these diseases that 45 requires mitochondria-ER interplay. This Opinion article underscores the pivotal role of 46 MERCS in regulating essential cellular functions, focusing on their crosstalk with BCL47 2 family proteins and discusses how their dysregulation is linked to disease. 48 49 MERCS, fundamental dynamic structures required for cellular homeostasis 50 The interface between cellular organelles is increasingly recognized as a pivotal 51 platform governing essential biological processes in eukaryotic cells. Specifically, the 52 contact sites between mitochondria outer membrane (MOM) and the endoplasmic 53 reticulum (ER), referred to as Mitochondria-Endoplasmic Reticulum Contact Sites 54 (MERCS) also known as mitochondria-associated ER membranes (MAMs), represent 55 mostly transient dynamic modules enriched in specific lipids and specialized proteins 56 that define their structure and functions (see Glossary) [1,2]. These ER-mitochondrial 57 junctions, where the interorganellar distance ranges between 10 and 80 nm, serve as 58 key locations for calcium (Ca2+) signalling, lipid transfer, and play crucial roles in 59 cellular bioenergetics, proteostasis, mitochondrial quality control, autophagy and 60 apoptotic cell death [3,4]. 61 Research employing techniques such as live-cell fluorescence and electron 62 microscopy has revealed several features of these tightly tethered, yet separate, 63 membrane structures. It is becoming clear that the number of contacts, the surface 64 area of contact and the interorganellar distance are three fundamental aspects of 65 MERCS mediated signalling affecting its triggering, amplification and kinetics [4,5]. 66 Since both the ER and mitochondria are dynamic organelles, it is unclear how these 67 contact areas are established. In yeast, MERCS are maintained through a complex of 68 known composition called ER-mitochondria encounter structure, ERMES [6]. However, 69 in mammals the molecular architecture of MERCS formation and stabilization is more 70 intricate and poorly understood [7]. A handful of proteins have been described to 71 maintain MERCS, acting like membrane-tethers, including inositol 1,4,5-triphosphate 72 receptors (IP3Rs) [8] together with VDACs (Voltage dependent anion channels) [9]; 73 the vesicle-associated membrane protein B (VAPB) associated to PTPIP51 (protein 74 tyrosine phosphatase-interacting protein-51) [10], mitofusin-2 (MFN2) and related 75 isoforms [11,12], B-cell receptor-associated protein-31 (BAP-31) [13] and PDZ 76 domain-containing protein 8 (PDZD8) [14] (Figure 1). 77 Each of these MERCS-tethers exhibit distinct characteristics and are intricately linked 78 to specific cellular functions and regulated by adaptors expressed in response to 79 diverse stimuli. Importantly, the proteins within these contact sites are neither exclusive 80 nor specific to MERCS, which presents technical challenges for understanding their 81 exact nature. Moreover, within a single MERCS, multiple specialized interaction 82 domains may coexist, further underscoring their dynamic nature being able to remodel 83 in response to changes in the environment and meet the metabolic needs of the cell 84 [15]. For example, IP3Rs are key mediators of ER-mitochondria Ca2+ homeostasis, 85 acting as channels for Ca2+ transport. IP3R forms a tetrameric complex with VDAC, 86 the molecular chaperone GRP75 (Glucose-regulated protein 75) and the deglycase 87 DJ-1, to modulate the Ca2+ transfer from the ER to the mitochondrial matrix, using the 88 mitochondrial calcium uniporter (MCU) [16–18], which can result in stimulation of Krebs 89 cycle enzymes and OxPhos [19,20]. The VAPB-PTPIP51 tether complex is related to 90 Ca2+ signalling as well, but also to alterations in mitochondrial morphology and 91 aggregation [10,21]. Depletion of either VAPB or PTPIP51 leads to the disruption of 92 MERCS, perturbation of Ca2+ transport and autophagy stimulation [10,22]. Some 93 authors also reported that MFN2 and its splicing variants can act as a physical tether 94 stabilizing the contact between both organelles, favouring lipid transfer and regulating 95 mitochondrial dynamics [11,12]. However, this remains a contentious topic within the 96 field [23–26]. Finally, BAP-31 has been shown to modulate mitochondrial oxygen 97 consumption, autophagy and apoptotic cell death [13]. These studies collectively 98 reveal that MERCS-tethers have distinct roles in regulating specific cellular processes. 99 While their investigation is technically demanding, it is crucial for uncovering their vital 100 contributions to maintaining cellular homeostasis. 101 These diverse MERCS-mediated functions are known to be dysregulated in multiple 102 human pathologies including cancer and neurodegenerative diseases such as 103 Alzheimer’s disease (AD) and Parkinson’s disease (PD) [3,27–29]. Interestingly, recent 104 evidence unravelled that MERCS are altered during cellular senescence, which could 105 mediate their impact on aging and age-related diseases [30]. However, the driving 106 cellular mechanisms behind these roles remain poorly defined. Proteins belonging to 107 the BCL-2 family, which are considered the main regulators of mitochondrial apoptosis 108 [31–34], are key regulators of MERCS-associated functions and strongly contribute to 109 the cell death imbalance observed in these age-related diseases. In this Opinion 110 article, we will discuss the role of MERCS in health and disease, with a novel 111 perspective emphasizing the crosstalk between MERCS and the BCL-2 mediated 112 apoptotic machinery, regulating cell death and inflammation, and highlighting non113 apoptotic BCL-2 roles in cellular homeostasis. 114 115 BCL-2 and MERCS, a novel cellular health-meter? 116 BCL-2 family proteins form a complex interaction network in which mitochondrial and 117 ER membranes play a key modulatory role. These proteins are typically classified into 118 three main groups: i) the anti-apoptotics (e.g. BCL-2, BCL-XL, or MCL1), also known 119 as guardians, that prevent MOM permeabilization (MOMP) and consequently 120 apoptosis; ii) the proapoptotic multidomain or effectors (e.g. BAX and BAK), which 121 directly induce MOMP and apoptosis by forming a pore at the mitochondria; and iii) the 122 BH3-only proteins (e.g. BIM, BID or BAD), which trigger apoptosis by either blocking 123 anti-apoptotics or directly activating effectors [35–38]. 124 During apoptosis, BAX and BAK converge at the mitochondria to form the apoptotic 125 pore, inducing MOMP and the subsequent release of pro-apoptogenic factors to the 126 cytosol [36]. The tunable protein:lipid nature of the formed pores, allows the passage 127 of small molecules such as cytochrome c or SMAC, but also larger molecules, such as 128 mitochondrial DNA (mtDNA) [39–42]. Once in the cytosol, released cytochrome c and 129 SMAC promote the assembly of the apoptosome, inducing caspase activation and 130 apoptotic cell death (Figure 2). Importantly, sublethal activation of this machinery, with 131 only partial release of the mitochondrial content, a phenomenon that is known as 132 minority MOMP, is associated with a sublethal caspase activation, damage in the 133 nuclear DNA and genomic instability [43]. Released mtDNA in the cytosol, is 134 recognised by nucleic acid sensors like cGAS (cytosolic cyclic GMP–AMP synthase). 135 cGAS activation leads to the synthesis of 2′3′ cyclic GMP–AMP (cGAMP), which 136 activates the ER-localized Stimulator of interferon genes (STING). Activated, STING 137 translocates to the Golgi, inducing TANK-binding kinase 1 (TBK1) and Interferon 138 regulatory factor 3 (IRF3) phosphorylation, inducing the expression of type I interferons 139 [44–46]. 140 Of note, this pro-inflammatory response is initiated within the mitochondria but relies 141 on the ER for its amplification, underscoring the role of MERCS (Figure 2). In this 142 regard, knock down of the MERCS tether VAPB substantially decreases interferon143 mediated inflammation [47] and STING depletion potentiates VDAC2/GRP75144 mediated MERCS formation impairing mitochondrial function [48]. Indeed, 145 mitochondrial VDAC2 was recently identified as a new STING binding partner able to 146 regulate BAX/BAK apoptotic activities [48–50]. This highlights the importance of the 147 crosstalk between MERCS-tethers, BCL-2 proteins and STING machinery. Moreover, 148 during infection BAX can interact with IRF3, impacting STING signalling and 149 inflammation [51–53]. In addition, MAVS (mitochondrial antiviral-signalling protein) and 150 the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), 151 responsible for inflammasome activation, interact at MERCS, suggesting that these 152 contact areas also play a role in innate immunity [54–56]. 153 On the other hand, the BCL-2 interaction network and downstream cascades can be 154 influenced by MERCS-mediated lipid transport. Of note, the majority of mitochondrial 155 lipids, including phospholipids, sphingolipids, and cholesterol, are synthesized in the 156 ER and then transferred to the mitochondria via MERCS [57,58]. Despite their minor 157 presence at the mitochondria, sphingolipids (e.g. ceramide and sphingosine-1P) have 158 been reported to play a crucial role in fine-tuning the apoptotic machinery: ceramide is 159 frequently associated with promoting BAX/BAK-dependent apoptosis, while 160 sphingosine-1P is linked to cell survival (reviewed in [59]). Additionally, elevated levels 161 of cholesterol at the mitochondria are correlated to impaired BAX activation and cell 162 survival [58,60]. In addition, cardiolipin (CL) plays a key role during apoptosis in the 163 mitochondrial targeting of BAX [61], its oligomerization [62], and the apoptotic pore 164 formation [63]. Importantly, CL (which is essential for mitochondrial cristae 165 maintenance) represents around 4-8% of the MOM lipids, but is highly enriched at 166 MERCS with 25% of the total lipid content [64], highlighting the importance of these 167 structures for BAX activation and the apoptotic pore formation. 168 In addition, BCL-2 family proteins are known to regulate essential MERCS-associated 169 activities such as Ca2+ signalling, mitochondrial dynamics, bioenergetics, the unfolded 170 protein response (UPR), mitochondrial quality control and autophagy (reviewed in 171 [34,65]). For example, the antiapoptotic protein BCL-2 and proapoptotic BOK, interact 172 with the MERCS-tether IP3R, displaying opposing roles in Ca2+ regulation, as both the 173 upregulation of BCL-2 and low levels of BOK are linked to limited Ca2+ fluxes to the 174 mitochondria [66,67]. Other studies support the idea that BOK interacts with IP3R at 175 MERCS modulates primarily mitochondrial dynamics and bioenergetics [68]. 176 Antiapoptotic members such as BCL-XL, MCL-1 and BCL-B are also reported to bind 177 to IP3R limiting Ca2+ transport to the mitochondria [69–71]. Regarding the UPR, 178 several BCL-2 family proteins function as stress rheostats [72,73]. The BH3-only 179 proteins BID, PUMA, NOXA, BAD and BIM, are transcriptionally and post180 translationally induced under acute ER stress [74–77] and the apoptotic effector BOK 181 translocates to the mitochondria inducing MOMP [67,78,79]. BCL-2 family members 182 are also implicated in regulating mitochondrial dynamics, interacting with MFN2 and 183 the dynamin-related protein 1 (DRP1), affecting mitochondrial fusion and fission events 184 [80–82]. Finally, BCL-2 is reported to interact with Beclin-1 impairing autophagy [83]. 185 Overall, independent studies report a continuous crosstalk between MERCS resident 186 proteins and BCL-2 family members which is responsible for fine-tuning their 187 respective activities and influencing cell fate. Alterations in the normal function of both 188 MERCS and BCL-2 family proteins are frequently associated with severe human 189 diseases. However, due to the dynamic nature of these structures and their 190 overlapping functions, it is complicated to discern whether alterations in MERCS are a 191 cause or a consequence of the disease. In the following sections, we will explore 192 variations in the composition of MERCS in relation to cancer, AD, and PD. These are 193 age-related diseases, in which alterations in MERCS, BCL-2 proteins, and 194 cGAS/STING-induced inflammation coexist, and so their interplay could play crucial 195 previously overlooked roles in disease. 196 MERCS as a crucial axis in human disease 197 MERCS and BCL-2 proteins in Cancer: Twisting homeostasis towards proliferation. 198 The communication between the ER and mitochondria plays a crucial role in cancer 199 development and progression. MERCS serve as signalling platforms that are involved 200 in rewiring normal cellular processes towards malignancy (Figure 3A-B). Aberrant 201 expression or mislocalization of MERCS-resident proteins is observed in several types 202 of cancer. For instance, the overexpression and increased MERCS localization of ER 203 stress sensors like ER oxidoreductin 1-alpha (ERO1-α) and RNA-dependent protein 204 kinase (PKR)-like ER kinase (PERK), are implicated in tumour initiation, progression, 205 and resistance to chemotherapy [84,85]. In breast cancer, the stress-activated 206 chaperone sigma-1 receptor (Sig1R) exhibits higher expression and increased 207 MERCS localization in metastatic cancer cells compared to normal tissues, modifying 208 IP3R-mediated Ca2+ dynamics and promoting cell invasiveness and migration [86–88]. 209 Conversely, reduced expression of the MERCS-tether MFN2 is correlated with worse 210 prognosis in colon and renal cancer subtypes [81,89], which taken together highlight 211 an overarching role for MERCS in cancer. 212 Regarding BCL-2 family proteins, the reduced apoptosis, excessive proliferation and 213 tumour chemotherapy resistance observed in cancer, are certainly linked to altered 214 signal crosstalk between the ER and the mitochondria [90,91]. For example, the 215 upregulation of antiapoptotic BCL-2 and BCL-XL is correlated with a reduction in IP3R216 mediated Ca2+ signalling and reduced cell death in several cancer types [66,92,93]. 217 Proapoptotic BOK also reported to interact with the IP3R, is deleted across several 218 cancer types and associated with impaired cell death [94]. On the other hand, 219 mitochondrial lipid composition, fine-tuned by mitochondria-ER communication, is also 220 altered in cancer conditions. For example, elevated cholesterol levels at the 221 mitochondria are linked to reduced activation of the proapoptotic BCL-2 member BAX 222 and cell survival in cancer [60,95]. Moreover, dysregulation of CL and ceramide 223 metabolism, key lipids in promoting BAX/BAK activation and mitochondrial apoptosis 224 has been observed in several types of cancer (reviewed in [96,97]). 225 Finally, BAX/BAK-mediated cGAS/STING-regulated immune responses can impact 226 most of the aspects associated with tumorigenesis, from malignant cell transformation 227 to metastasis (reviewed in [98]). In this regard, BAX/BAK induced minority MOMP with 228 partial activation of the apoptotic machinery has been linked to the generation of 229 genomic instability, causing a cell death resistance phenotype that includes 230 colonization and metastasis in vivo [99], and drives aggressive features in residual 231 cancer cells [100]. On the other hand, sublethal BAX/BAK-dependent cGAS/STING 232 signalling activation can induce a chronic senescence-associated secretory phenotype 233 (SASP) [101]. Although originally believed to halt cancer progression due to their 234 characteristic growth arrest, senescent cells remain metabolically active and secrete 235 different inflammatory agents, generating a microenvironment that can promote tumour 236 growth [102]. 237 An imbalance in MERCS-mediated functions and BCL-2 family proteins can therefore 238 lead to a plethora of cancer-driving processes, both within cells and in their 239 surroundings, including tumour initiation, progression, survival, and metastasis. This 240 underscores the importance of maintaining MERCS homeostasis to putatively predict 241 and prevent cancer-related features. 242 243 MERCS, BCL-2 proteins and Alzheimer’s Disease: Proximity fails to reveal the whole 244 picture. 245 Alzheimer’s Disease (AD) is the most prevalent neurodegenerative disorder worldwide, 246 leading to cognitive deficits and dementia [103]. Central to AD diagnosis is the 247 formation of intracellular neurofibrillary tangles composed of hyperphosphorylated tau 248 and extracellular plaques consisting of amyloid-β (Aβ) peptides [103]. These 249 aggregates and the subsequent inflammatory process are thought to lead to neuronal 250 dysfunction and loss. The γ-secretase complex, comprising presenilin 1 (PS1) or 251 presenilin 2 (PS2) as catalytic subunits, plays a crucial role in the cleavage of amyloid 252 precursor protein (APP), yielding Aβ fragments. Importantly, both APP and the γ253 secretase complex have been identified at MERCS in vitro, suggesting a potential 254 involvement of MERCS in AD pathogenesis [104–107] (Figure 3C). 255 Mutant forms of APP, PS1 and PS2 enhance ER–mitochondrial tethering, affecting 256 Ca2+ dynamics and lipid metabolism, contributing to mitochondrial dysfunction and 257 neuronal cell death which are observed during AD [108–110]. For example, mutants of 258 PS2 modulate Ca2+ transfer to the mitochondria through interaction with MFN2 [108]. 259 Studies in MEF cells lacking MFN2, showed diminished MERCS and reduced 260 interorganellar cholesterol trafficking [110]. However, in apparent contradiction to its 261 putative tethering function, knocking down MFN2 has also been associated with an 262 increase in mitochondria-ER contacts, altered mitochondrial Ca2+ transfer and reduced 263 APP processing [111]. Similar discrepancies exist regarding the putative role that Aβ 264 exerts over MERCS. In vivo studies in hippocampal neurons from AD rat models 265 indicate a reduction in MERCS upon Aβ exposure, associated with diminished lipid 266 metabolism and alterations in the mitochondrial lipidome [112]. In accordance with this 267 finding, in Drosophila models, forced expression of an artificial linker that enhances 268 MERCS in vivo rescues locomotion and prolongs the survival in AD models [113]. 269 However, other studies in primary hippocampal neurons show that Aβ treatment 270 increases MERCS and disrupts Ca2+ homeostasis [114]. This was also the case in 271 fibroblasts from both familial and sporadic AD patients that showed an increase in ER272 mitochondria tethering with MERCS dysfunction, including alterations in phospholipid 273 and cholesterol metabolism [110]. While some of these opposing results are difficult to 274 interpret, they are perhaps not entirely unexpected as MERCS are mostly dynamic and 275 plastic cell signalling structures and as such will highly depend on the studied model 276 and disease snapshot. Importantly, studies in AD models have shown changes in the 277 MERCS proteome preceding the onset of cognitive symptoms, indicating a potential 278 role of MERCS dysregulation in driving disease progression [115], and underscoring 279 their role in AD. 280 At MERCS, antiapoptotic BCL-2 plays a crucial role in Ca2+ homeostasis and cell 281 survival, and its downregulation is associated with AD [116]. Reduced expression 282 levels of other antiapoptotic proteins such as BCL-XL, and MCL1, alongside elevated 283 levels of proapoptotic proteins like BIM or BAX, tilting the BCL-2 interaction network 284 towards death, is also associated with AD and the neuronal death observed during 285 disease progression [117] (and reviewed in [116]). Finally, recent findings indicate that 286 activation of the innate immune cGAS-STING pathway contributes to AD in 5×FAD 287 mice [118]. In this study, cGAS KO mice were significantly protected from cognitive 288 impairment, amyloid-β pathology, neuroinflammation, and other aspects associated 289 with AD [118]. Whilst the specific impact of BCL-2 proteins on the cGAS-STING 290 pathway related to AD pathogenesis remains poorly described, recent evidence linked 291 AD risk alleles with BAX-dependent mtDNA release and elevated microglial cGAS 292 pathway activation [119], which again points towards an interplay between MERCS 293 and apoptotic machinery proteins in AD. Non-cell autonomous effects downstream of 294 MERCS dysregulation may be particularly important in AD given its strong glial and 295 peripheral components. 296 297 MERCS and BCL-2 in Parkinson’s Disease: Mitophagy to the rescue 298 Parkinson’s Disease (PD) is a progressive neurodegenerative disorder characterized 299 by the loss of neurons, particularly in the substantia nigra pars compacta (SNpc), 300 leading to symptoms such as tremors, dyskinesia and dementia [120]. The disruption 301 of MERCS is associated with PD pathogenesis, thereby affecting the communication 302 between mitochondria and the ER (Figure 3D). Indeed, α-Synuclein, a protein which 303 is able to aggregate and is found in Lewy bodies, which are a hallmark of PD, is 304 localized at MERCS and implicated in their malfunction [121]. Wildtype α-Synuclein as 305 well as its PD-associated mutants A30P and A53T are shown to target MERCS through 306 the interaction with the VAPB/PTPIP51 tether-complex, and yet the impact of the 307 different mutations remains under investigation [122,123]. Nonetheless, the 308 impairment of VAPB and PTPIP51 tethering activity is associated with neuronal 309 synaptic dysfunction [124]. Furthermore, genetic mutations associated with familial PD 310 such as those in DJ-1, PINK1, and Parkin, proteins that are located at MERCS 311 [18,82,125]. Mutations in DJ-1 impair its role in maintaining Ca2+ homeostasis and 312 facilitating ER-mitochondrial tethering, altering the IP3R-GRP75-DJ1-VDAC complex 313 [17,18]. Importantly, ablation of DJ-1 increases mitochondria-ER distance, thereby 314 reducing mitochondria-ER communication, which can be rescued by wild-type DJ-1 315 but not by PD-related mutants [17,18]. In addition, mutations in the mitophagy 316 mediators PINK1 and Parkin, as well as in their associated proteins MIRO1 and MFN2, 317 are also related to MERCS dysregulation in PD [82,126–128]. Parkin expression 318 increases MERCS in HeLa cells, thereby enhancing Ca2+ signalling and ATP 319 production [126] and conversely, loss of Parkin in human fibroblasts leads to a 320 decrease in MERCS [82]. In Parkin KO mice and patients with Parkin mutations, the 321 absence of Parkin, however, was linked to increased connectivity between 322 mitochondria and the ER along with increased Ca2+ transport [127]. Mutations in 323 MIRO1 are associated with decreased MERCS and impaired Ca2+ transport to the 324 mitochondria in PD patients [128]. Furthermore, the MERCS tether MFN2 gets 325 phospho-ubiquitinylated in Parkin/Pink1 dependent mitophagy. Mutations that inhibit 326 this posttranslational modification are reported to decrease MERCS, suggesting loss 327 of Parkin or ubiquitination-preventing mutations in MFN2, reduced MERCS and 328 mitophagy [82]. Importantly, recent studies have underscored the importance of 329 mitophagy in mitigating cytosolic mtDNA-dependent activation of cGAS/STING 330 inflammation during aging signalling [129], a pathway implicated in accelerating 331 neurodegeneration progression in a mouse model of PD [130]. As previously 332 mentioned, while the association between MERCS and PD is irrefutable, contradictory 333 findings could be explained by the dynamic and transient nature of MERCS associated 334 signalling, which highlights the need to carefully interpret these findings. This is 335 especially relevant for PD, which is tightly associated with mitochondrial malfunction, 336 through both genetics and toxic exposure to compounds such as rotenone. 337 BCL-2 proteins contribute to defects in mitophagy, neuronal cell death and the 338 pathogenesis of PD (reviewed in [131]). Although the precise molecular mechanisms 339 remains debated for PD, BCL-2 has been associated with MERCS localised 340 PINK1/Parkin and Beclin-1 to mediate autophagy [125,131]. Expression levels of BCL341 2 as well as other antiapoptotic proteins such as BCLXL are significantly decreased in 342 PD patients, with a negative correlation observed with disease duration and severity 343 [132,133]. Consistently, accumulation of the proapoptotic BAX has been observed in 344 neurons of the SNpc in post-mortem brains from PD patients [134], and in neurons 345 containing Lewy bodies [135]. Despite the specific impact of BCL-2 family proteins 346 directly activating cGAS-STING pathway and subsequent inflammation, this has not 347 yet been described in PD; their role in mitophagy could play an important role by 348 silencing this pathway [129,131]. Indeed, restoring autophagy through modulation of 349 BCL-2, has been already proposed for PD treatment [136]. 350 Concluding Remarks 351 There is a clear link between alterations in MERCS and important human pathologies. 352 Here, we discuss cancer, PD, and AD, which are becoming increasingly prevalent in 353 our society. Enriched with stimuli-specific proteins and lipids, MERCS play a vital role 354 in various cellular functions such as Ca2+ signalling, lipid transfer, ROS production and 355 mitophagy under healthy conditions. This complexity poses a challenge in discerning 356 whether their dysregulation is a potential cause or a consequence of the disease. 357 However, some studies already suggest MERCS dynamics and composition preceding 358 the onset of cognitive symptoms, indicating a potential role of MERCS dysregulation 359 in driving disease progression. Therefore, designing molecules that improve 360 mitochondria-ER communication could lead to new therapeutic opportunities. Despite 361 their fundamental importance, there is significant controversy surrounding the 362 composition of MERCS. This is likely due to technical challenges associated with 363 studying these dynamic structures in vivo, the use of different experimental models, 364 and the low temporal resolution during their characterization. Some disease 365 developments may take years or even decades to manifest, suggesting that analysing 366 MERCS at a single time point may be insufficient and that some of the observations 367 could be an epiphenomenon. Understanding the sequence of events would provide 368 not only mechanistic details but could also provide a broader window for treatment. 369 BCL-2 family proteins, the main regulators of mitochondrial apoptosis, are responsible 370 for several MERCS-related functions and they are also associated with the imbalance 371 in apoptotic cell death observed in cancer, PD, and AD. Recently, the mitochondrial 372 apoptotic machinery has been associated to the release of mtDNA and the activation 373 of the cGAS/STING pathway. As this signalling requires mitochondria-ER 374 communication, it suggests that MERCS could orchestrate or sense this pathway prior 375 to its effect at the cell or organism level. Connected to this, sublethal activation of the 376 26. Filadi, R. et al. 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Autophagy 14, 848 845–861 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 Figure 1. 870 871 872 Figure 2. 873 874 875 876 Figure 3. 877 878 879 Highlights 880 881 - MERCS serve as signaling hubs involved in vital cellular processes such as calcium 882 signaling, lipid metabolism, autophagy, and apoptosis. 883 - Alterations in MERCS dynamics and composition are closely associated with severe 884 human diseases such as neurodegenerative disorders and cancer. 885 - The BCL-2 protein family, primary regulators of mitochondrial apoptosis and key 886 players in maintaining cellular homeostasis, operate at MERCS to fulfill essential 887 cellular functions. 888 - Apoptotic machinery-induced cGAS-STING pathway activation and proinflammatory 889 signaling, requires mitochondria-ER communication and is affected in cancer, AD and 890 PD. 891 892 Outstanding questions 893 - Is the composition of MERCS tailored to specific tissues or cell-types, adapting to 894 specific needs? Do different MERCS tethers work simultaneously? Do they compete 895 for the same locations? Can they exhibit redundancy? What is their conformational 896 status when they are not bridging interorganellar connections? 897 - Are changes in the structure or function of MERCS a potential cause of disease, a 898 result of disease, or are they adaptive adjustments to compensate cellular imbalances 899 during disease development? Do those changes remain constant throughout the entire 900 process? Could restoring MERCS function effectively ameliorate disease progression? 901 - Can correlations be established between disruptions in MERCS-resident proteins and 902 diseases, potentially leading to their use as diagnostic biomarkers? Could the 903 interactions between BCL-2 family proteins and MERCS functions be leveraged to 904 assess overall cellular health or susceptibility to disease? 905 - Is the activation of sublethal apoptotic machinery causing chronic inflammation and 906 cellular stress, contributing to the excessive accumulation of Aβ or α-synuclein 907 observed in AD and PD or a consequence of these pathologies? Would inhibiting low908 level inflammation reduce the protein aggregation and disease progression? 909 - Considering the role of MERCS in transferring signals to mitochondria and modifying 910 mitochondrial membranes, are they responsible for the selective content release from 911 the apoptotic pores? Do MERCS directly mediate mtDNA release and pro912 inflammatory signalling upon apoptotic machinery activation? 913 - Could targeting the non-canonical roles of BCL-2 proteins in MERCS dynamics and 914 apoptotic signaling unlock groundbreaking therapies for halting or even reversing 915 neurodegenerative diseases like Parkinson's and Alzheimer's? 916 917 918