3.1 Ca²⁺ Homeostasis

Ca²⁺ is an important intracellular signaling molecule involved in multiple physiological processes, such as muscle contraction, cell secretion, and gene expression regulation. The ER and mitochondria are the main participants in cellular Ca²⁺ homeostasis. MAMs serve as platforms for Ca²⁺ transport between the ER and mitochondria, coordinating ER–mitochondrial interactions to avoid adverse effects caused by Ca²⁺ overload or depletion [125]. The ER is the primary Ca²⁺ storage site in the cell; upon stimulation, Ca²⁺ is released from the ER through ryanodine receptors (RyRs) or inositol-1,4,5-trisphosphate receptors (IP3Rs), or taken up by the sarcoplasmic/ER Ca²⁺-ATPase (SERCA) to regulate cellular Ca²⁺ homeostasis [37]. A portion of the released Ca²⁺ is taken up by the mitochondria through MAMs. After uptake, mitochondria regulate their metabolic activities and buffer changes in cytosolic Ca²⁺ concentrations, preventing Ca²⁺ overload and potential damage to the cell [126-128].

The mitochondrial calcium uniporter (MCU), which has low affinity for Ca²⁺, requires high Ca²⁺ concentrations for Ca²⁺ influx into mitochondria [129]. In MAMs, the interaction between IP3Rs on the ER and the voltage-dependent anion channels (VDAC1) on the mitochondria forms an efficient Ca²⁺ transport channel, allowing Ca²⁺ to be transported from the ER to the mitochondria along the electrochemical gradient [38]. Chaperone glucose-regulated protein 75 (GRP75) enhances the interaction between IP3R and VDAC1, thereby facilitating Ca²⁺ flow between the ER and mitochondria [39]. Several proteins enriched in MAMs are associated with the IP3R–GRP75–VDAC1 complex. For example, inositol-requiring enzyme 1α (IRE1α) and Fun14 domain-containing protein 1 (FUNDC1) act as scaffolding proteins at MAMs, physically interacting with IP3R to increase its abundance and stability at MAMs, thereby regulating Ca²⁺ flux between the ER and mitochondria [40, 41]. Given the importance of MAMs in the transfer of Ca²⁺ from the ER to the mitochondria, the length and thickness of tethering proteins may be critical factors affecting Ca²⁺ transport. Studies have shown that at an optimal distance (approximately 20 nm), ER and mitochondrial Ca²⁺ channels effectively interact and promote rapid Ca²⁺ transfer [40, 41]. Therefore, the binding of MAM-tethering proteins, such as VAPB–PTPIP51, is essential for efficient mitochondrial Ca²⁺ uptake and may influence the interaction between IP3R and VDAC1 [42]. Under certain conditions, MAMs may also facilitate the reverse transfer of Ca²⁺ from the mitochondria to the ER. This has been demonstrated in studies where the ER Ca²⁺ pump, SERCA, was inhibited by TG, and considerable Ca²⁺ accumulation was still observed in the ER, possibly due to dynamic changes in the cellular demand for Ca²⁺ [130]. In summary, MAMs coordinate Ca²⁺ transport between the ER and mitochondria, which is crucial for maintaining cellular homeostasis.

3.2 Lipid Synthesis and Transport

Lipids play a vital role in regulating various life processes; serve as major components of cell and organelle membranes; and participate in energy metabolism, signal transduction, cell recognition, and material transport [131]. The ER and mitochondria are the primary hubs for eukaryotic membrane biogenesis and rely on lipid exchange at membrane contact sites. In addition to providing energy to the ER [132], mitochondria coordinate the synthesis of key membrane phospholipids, such as PE and phosphatidylcholine (PC) [133]. The biogenesis and respiratory functions of mitochondrial membranes are entirely dependent on lipids or lipid precursors provided by the ER [134]. The ER is capable of synthesizing nearly all membrane lipids, including phospholipids and cholesterol [135], and transporting them to the mitochondria through MAMs via nonvesicular transport [136].

Studies have shown that the protein kinase RNA-like ER kinase (PERK) recruits ER–plasma membrane tethers and lipid transfer proteins such as E-Syt1 at ER–mitochondria contact sites for mitochondrial lipid transport and respiration [48]. PC, PE, and CL are the main lipids constituting the mitochondrial membrane and account for 75–95% of the total lipids [137, 138]. PE is synthesized via three of four pathways in the ER [139], but for unknown reasons, almost no PE in mitochondria is derived from the ER [140]; instead, it is produced mainly from PS through the action of PS decarboxylase (PSD) in the inner mitochondrial membrane (IMM) [139]. The PS necessary for this process is synthesized by PS synthase (PSS) within the ER, and is subsequently transported to the mitochondria through lipid transfer proteins located on MAMs [43].

Multiple studies have indicated that proteins located in MAMs, such as oxysterol-binding protein-related proteins 5 and 8 (ORP5/8), MFN2, and CDP-diacylglycerol synthase-2 (CDS2), mediate the nonvesicular transport of PS from the ER to the mitochondria [44-46]. A small portion of PE produced in the mitochondria is sent back to the ER, where it is converted to PC through the CDP-choline pathway or by the action of PE N-methyltransferase (mainly expressed in the liver) [141].

Like PS, PC is produced in the ER and transported to the mitochondria through MAMs rather than being synthesized in the mitochondria [138]. CL biosynthesis occurs exclusively in the IMM and requires PA as a precursor, which is an input from the ER [142]. Studies have shown that the VAPB–PTPIP51 complex and lipocalin 2 play key roles in regulating the transport of PA from the ER to mitochondria through MAMs [33, 143]. Although cholesterol is present in low amounts in the mitochondria, it can be rapidly transported from the ER to other organelles. The ER, which lacks cholesterol, synthesizes cholesterol, which is quickly delivered to other organelles [144]. MAMs are rich in cholesterol, sphingolipids, and related enzymes, and form a membrane domain known as the intracellular lipid raft, which may create a local gradient conducive to the transport of cholesterol from the ER to the mitochondrial membranes [145]. The acute regulatory protein of steroidogenesis (StAR) associated with MAMs interacts with the ER chaperone, GRP78, to form a cholesterol complex at the MAMs, which is then transferred to the OMM and interacts with VDAC1/2, facilitating the transport of substrate cholesterol into the mitochondria for steroidogenesis [146]. However, excessive accumulation of cholesterol in the mitochondria can affect membrane fluidity and indirectly affect energy production efficiency. Knocking out molecules such as Caveolin-1 and GRAMD1C may promote the transfer of cholesterol at mitochondrial–ER contact sites [49, 50], suggesting their role in regulating mitochondrial cholesterol metabolism.

In summary, MAMs are indispensable for lipid metabolism by promoting the transfer of lipids between the ER and mitochondria, regulating mitochondrial function, and affecting cellular metabolic pathways. The dynamic nature of MAMs and the participation of specific protein complexes highlight the importance of these structures in maintaining lipid homeostasis and overall cellular health.

3.3 Mitochondrial Homeostasis

MAMs regulate mitochondrial mass homeostasis. The regulation of mitochondrial mass homeostasis involves processes such as mitochondrial fusion, fission, and autophagy, which together maintain the number, structure, and function of mitochondria in a relatively stable, dynamic balance [147]. The core regulator of mitochondrial dynamics is the dynamin-related GTPase family, which includes the fission-promoting mitochondrial fission protein DRP1, the fusion-promoting MFN1, MFN2, and optic atrophy 1 [148]. Mitochondrial fission occurs when the ER tubules surrounding the mitochondria contract before mitochondrial fission, marking the fission site [149]. DRP1 is recruited to the OMM, where it associates with its adaptors, mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 kDa and 51 kDa (MiD49, MiD51), to form ring-like or spiral structures around the site of mitochondrial constriction [51].

Several MAM proteins are involved in recruiting DRP1 to regulate fission processes, including syntaxin 17 (STX17) [54], disulfide bond A oxidoreductase-like protein (DsbA-L) [55], and phosphofurin acidic cluster sorting protein 2 (PACS-2) [56]. Additionally, MAMs coordinate the licensing of mitochondrial DNA (mtDNA) replication with downstream mitochondrial fission events, distributing newly replicated mtDNA to daughter mitochondria [150]. DRP1 is not essential for mitochondrial fission; in the absence of DRP1, Fis1 can promote mitochondrial fission by inhibiting mitochondrial fusion [57]. Mitochondrial fusion results in mitochondrial elongation, a process controlled by MFN1 and MFN2 on OMM, and Opal on IMM [53]. MFN2 is sometimes located in the ER and physically interacts with MFN1 or MFN2 on OMM, where it participates in the connection between the ER and mitochondria [151]. MFN2 also interacts with other MAM components such as Sig-1R, and promotes MFN2 oligomerization to regulate mitochondrial dynamics [52]. The tubular ER-shaping protein, REEP5, interacts with MFN1/2 to regulate the “hitchhiking” behavior of mitochondria on the tubular ER, promoting the even distribution of mitochondria within the cell [58]. MFN2 is also involved in mitochondrial transfer mediated by mitochondrial–ER contact [152]. Intracellularly redundant or malfunctioning mitochondria are selectively eliminated through mitophagy, a highly conserved cellular process that maintains the equilibrium between mitochondrial quantity and the stability of cellular energy metabolism [153]. MAMs plays a key role in this process by regulating mitophagy via both ubiquitin-dependent and nonubiquitin-dependent pathways. PTEN-induced putative kinase 1 (Pink1)/Parkin is the main protein in the ubiquitin-dependent pathway. When mitochondria are damaged, Pink1 accumulates and phosphorylates Parkin, which ubiquitinates many OMM proteins, initiating mitophagy [154]. In the late stage of autophagy, BECN1 interacts with Pink1 in MAMs, promoting the formation of MAMs and autophagosomes [155]. FUNDC1 is a key protein in the nonubiquitin-dependent pathway; under hypoxic conditions, Lon accumulates in MAMs and interacts with the FUNDC1–unc-51-like autophagy-activating kinase 1 (ULK1) complex, inducing mitochondrial autophagy through chaperone activity [59]. In summary, MAMs regulate mitochondrial fission, fusion, and autophagy and participate in maintaining mitochondrial mass homeostasis.

3.4 ER Stress

ERS is an important defense mechanism by which cells respond to the accumulation of misfolded and unfolded proteins in the ER lumen and disturbances in Ca²⁺ balance to aid in the restoration of cellular homeostasis. To cope with ERS, the unfolded protein response (UPR) dynamically adjusts the folding and degradation capacity of the ER, restoring protein folding functions and reducing the concentration of unfolded proteins [156]. Moderate ERS can help restore normal ER function through UPR [157], whereas prolonged ERS can lead to cell death [158]. Under normal physiological conditions, PERK, IRE1α, and activating transcription factor 6 (ATF6) in the ER lumen are in an inactive monomeric state and are bound to BiP and GRP78. However, the accumulation of many unfolded proteins in the ER and their binding to BiP/GRP78 lead to the release of PERK, IRE1α, and ATF6α, initiating the UPR [159].

IRE1α accumulates in MAMs during ERS and splices XBP1 mRNA, upregulating the expression of folding enzymes and degradation enzymes and promoting the degradation of misfolded proteins [61]. The ERS sensor, ER Sig-1R, associates with IRE1α in MAMs at the onset of ERS, allowing IRE1α to dimerize into a persistent, active nucleic acid endonuclease that enhances cell survival [60]. The mitochondrial ubiquitin ligase (MITOL) is enriched at the MAMs during ERS and interacts with IRE1α, which promotes the ubiquitination of IRE1α, thereby effectively suppressing the excessive activation of IRE1α-induced ERS [62] Another ERS sensor, PERK, interacts with other proteins in the MAMs region, undergoes phosphorylation, and activates eukaryotic translation initiation factor 2α (eIF2α), inhibiting global protein synthesis, reducing the influx of nascent proteins into the ER, and selectively increasing the translation of some stress-related proteins to alleviate ERS [63, 64]. Key proteins in MAMs, such as MFN2, act as upstream inhibitors of PERK activity and maintain ER homeostasis by directly interacting with and inhibiting PERK activation [65]. These findings indicate that proteins in MAMs play crucial roles in UPR signal transduction and the maintenance of cellular homeostasis.

3.5 Apoptosis

Apoptosis is a highly regulated programmed cell death process that is essential for maintaining homeostasis, promoting development, and defending against diseases in organisms [160]. Both mitochondria and ER are key organelles that mediate apoptosis [161], and the interaction between them is particularly important in the complex and finely regulated process of programmed cell death. The transfer of Ca²⁺ from the ER to mitochondria is crucial for maintaining mitochondrial function, but excessive accumulation of Ca²⁺ ions can induce mitochondrial Ca²⁺ overload, leading to the opening of the mitochondrial permeability transition pore (mPTP), mitochondrial swelling, membrane depolarization, rupture of the OMM, and release of cytochrome c, ultimately triggering apoptosis [162]. Cytochrome c released from the mitochondria into the cytosol is transferred to the ER, enhancing the function of IP3Rs and amplifying apoptotic signals [66].

IP3Rs play a central role in the regulation of mitochondrial–ER Ca²⁺ transfer and are regulated by multiple factors. For example, the serine–threonine kinase, Akt, preferentially phosphorylates IP3R3 in MAMs, inhibiting IP3R-mediated ER–mitochondrial Ca²⁺ release and blocking apoptosis [67]. The BCL-2 family of proteins also regulates IP3Rs. Specifically, Bcl-xL can interact with IP3R, inhibiting Ca²⁺ release, thus affecting the initiation and progression of Ca²⁺-dependent apoptosis [68, 69]. Another proapoptotic member of the Bcl-2 family, Bax, also depends on MAMs for translocation to the mitochondrial membrane. Bax induces cytochrome c release and mediates apoptosis via mitochondrial Ca²⁺ accumulation and mPTP opening [70]. MAM-associated proteins also play key roles in apoptosis. During apoptosis, the mitochondrial fission factor, Fis1, binds to Bap31, promotes ER–mitochondrial tethering, recruits and activates procaspase-8, and initiates early apoptotic events [71]. Caspase-8 further cleaves Bap31 into its proapoptotic form, stimulating ER Ca²⁺ release, leading to mitochondrial Ca²⁺ overload, and ultimately triggering apoptosis [72]. MAMs also appear to mediate apoptosis via the mitochondrial fission pathway. Studies have shown that BAX and DRP1 colocalize in the mitochondria during apoptosis, where they mediate mitochondrial permeabilization and fragmentation [73].

In summary, mitochondria–ER Ca²⁺ signaling, molecular platform regulation, and MAMs stability collectively form the core of the apoptotic signaling network, finely regulating the life and death decisions of cells.

3.6 Autophagy

Autophagy is a highly regulated, intracellular, self-digestion process in which double-membrane autophagosomes form and fuse with lysosomes to degrade cellular proteins, organelles, and other components, achieving material recycling and maintaining cellular homeostasis [67]. MAMs are key platforms for the formation of autophagosomes, bringing together various autophagy-related proteins, such as autophagy-related gene 14 (Atg14), Atg5, and BECN1, to regulate autophagosome formation and maturation [78]. In the early stages of autophagosome formation, starvation-induced ULK1 translocates to MAMs, driving the assembly of the phosphatidylinositol 3-kinase (PI3K) complex with Atg14 and BECN1, and initiating the generation of PI3P, thereby initiating autophagosome formation [74, 75]. Atg5, a marker protein for autophagosome formation, also translocates to MAMs where it elongates the autophagosome membrane and promotes transmembrane translocation of LC3 (microtubule-associated protein 1 light chain 3) [76].

The elongation phase of autophagosomes involves the coordinated action of multiple key factors and processes. During the elongation phase of autophagosomes, Atg 2A (a subtype of Atg2) translocates to MAMs with the assistance of the TOM40 and TOM70 components of the mitochondrial translocase, where it interacts with Atg 9 to promote the growth of the phagophore and expand the volume of the autophagosome, thereby increasing autophagic flux [77]. ER-resident STX17 interacts with Atg14 in MAMs, facilitating the final fusion of autophagosomes with lysosomes, whereas disruption of the mitochondria–ER contact site hinders the formation of Atg14 puncta [78].

Ca²⁺ transport between the ER and mitochondria is an important factor affecting autophagy. During autophagy, protein 2.4 induced by etoposide, translocates to MAMs and interacts with the core complex of IP3R–Grp75–VDAC, which helps maintain the integrity of MAMs structure and promotes autophagy [163]. When Ca²⁺ transport from the ER to mitochondria is inhibited, AMP-activated protein kinase (AMPK), localized in MAMs, is activated by BECN1, promoting local autophagy [79]. Promyelocytic leukemia protein (PML) localizes to MAMs in a Ca²⁺-dependent manner in P53-driven cells and inhibits autophagy via the AMPK/mTOR/ULK1 pathway [80].

MFN2 is also involved in autophagy. Under energy stress, when mitochondrial fission occurs, a large quantity of AMPK translocates from the cytosolic solution to MAMs and interacts with MFN2, phosphorylating MFN2 and activating autophagy [81]. Autophagy also occurs in the absence of MFN2. For example, RTN3L, an effector of Rab32, promotes selective autophagy of proteins in MAMs [82].

In summary, autophagy is a complex process involving multiple proteins and organelles that work together and are precisely regulated. MAMs plays a pivotal role in this process by linking various autophagy-related events.

3.7 Ferroptosis

Ferroptosis is a novel form of programmed cell death that is closely associated with abnormalities in iron metabolism and lipid peroxidation [164]. Ferroptosis is induced by lipid peroxidation in various subcellular membranes, including the ER, mitochondria, and lysosomes, with the ER being the key initial site of peroxidation [165]. In cells undergoing ferroptosis, the mitochondria often exhibit shrinkage, decreased size, increased membrane density, reduced cristae, degeneration or disappearance, and outer membrane rupture [166]. Therefore, communication between the ER and mitochondria may mediate ferroptosis. MAMs are involved in mediating intracellular iron homeostasis and that deletion of MAM components can activate an Aft1-dependent iron deficiency response, leading to excessive iron accumulation within the cell [167]. Li et al. [83] reported that acute arsenic exposure induced the production of mitochondrial ROS (mtROS) in mouse lung epithelial cells, activated the PERK pathway, inhibited MFN2 expression, and ultimately led to MAMs dysfunction and ferroptosis, suggesting that mtROS-induced MAMs dysfunction is at least partially related to arsenic-induced ferroptosis and acute processes.

Similarly, increased MAMs formation was observed in ethanol-exposed mouse liver cells with upregulated PERK expression and interaction with ACSL4, promoting lipid peroxidation and exacerbating ferroptosis [86]. In a recent study, the use of a fluorescent probe increased the number of MAMs during ferroptosis [168], further confirming the correlation between MAMs and ferroptosis. MAMs are also associated with cancer cell resistance to ferroptosis. The mechanisms by which MAMs regulate ferroptosis have been investigated. For example, tetrabromobisphenol A exposure-induced ferroptosis in liver cells was found to upregulate the expression of GRP75, VDAC1, and MFN2, suggesting that Ca²⁺ between the ER and mitochondria may be involved in regulating ferroptosis [85]. This was confirmed in a study by Zhang et al. [169], who reported that defects in MAMs inhibited Ca²⁺ transfer between the ER and mitochondria, promoting the accumulation of DAG-containing polyunsaturated fatty acids (PUFA) and increasing the resistance of cells to ferroptosis. These findings indicate that the resistance of cancer cells to ferroptosis is partly related to the Ca²⁺ transport function of MAMs. Under conditions of ferroptosis, the peripheral mitochondrial membrane protein A-kinase anchoring protein 1 (AKAP1) anchors protein kinase A (PKA) in the mitochondrial MAMs, phosphorylating the S148 site of GRP75, causing GRP75 to be sequestered outside the mitochondria and interact with nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1, leading to the transcriptional activation of a series of antiferroptosis genes [84]. Therefore, targeting MAMs may be a potential approach for promoting ferroptosis in cancer cells.

Zhuang et al. [170] discovered that the prodrug, LSA (LA-SS-ART, linoleic acid–disulfide–artemisinin), could promote MAMs formation and increase Ca²⁺ influx from the ER to the mitochondria by enhancing the interaction between IP3R and VDAC1, inhibiting mitochondrial β-oxidation, triggering lipid peroxidation, simultaneously increasing the expression of Lpcat3 and MFN2, promoting phospholipid synthesis and increasing the production of PUFA–phospholipids, and synergistically enhancing ferroptosis [170]. In summary, MAMs play a key role in ferroptosis via calcium ion transport and lipid remodeling.

3.8 Inflammation

Inflammation is an essential mechanism by which the body combats injury and infection; however, chronic inflammatory responses can damage cells and tissues, leading to various diseases [171]. MAMs are necessary molecular platforms for formation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome [172]. In the resting state, NLRP3 is located in the mitochondria and ER. When cells are subjected to stress, NLRP3 and its adaptor protein, apoptosis-associated speck-like protein, are relocalized to MAM components [87]. Possible reasons for the relocation of NLRP3 to MAMs include the release of DAMPs [173] or ROS [174] from mitochondria. ERS is also involved in mediating inflammation, and ERS-induced activation of the NLRP3 inflammasome occurs through a Ca²⁺-dependent and ROS-independent mechanism associated with the upregulation of MAM-resident chaperone proteins, increased mitochondria–ER contact, mitochondrial depolarization, and impaired dynamics [175].

The activation of the innate immune interferon gene stimulator (STING) also depends on MAM-mediated Ca²⁺ signaling [176, 177]. Under high-glucose conditions, the IP3R1‒GRP75‒VDAC1 axis mediates increased Ca²⁺ flow from the ER to the mitochondria, leading to mitochondrial Ca²⁺ overload, opening of the mPTP, release of mtDNA into the cytoplasm, binding to cyclic GMP‒AMP synthase (cGAS), and activation of STING [88]. The upregulation of STING also promotes its interaction with VDAC2, inhibiting MAMs formation mediated by VDAC2/GRP75, reducing mitochondrial Ca²⁺ uptake, and protecting mitochondrial function [89]. MAMs also enrich various inflammation-responsive miRNAs including miR-146a, miR-142-3p, and miR-142-5p [178]. In summary, MAMs play a central role in the cellular responses to inflammation and stress, which is crucial for understanding and treating diseases.

4.1 Role of MAMs in Nervous System Diseases

Neurological diseases are a group of disorders affecting the brain, spinal cord, and peripheral nerves, leading to dysfunction in the corresponding controlled areas and potentially other systemic conditions. MAMs influence not only the metabolism and secretion of lipids and proteins in neurons, calcium homeostasis, and energy production but also axonal transport and neurotransmission through interactions between the ER and mitochondria [179, 180]. Therefore, defects in MAMs levels may be the initial triggers of neurological diseases. Research on MAMs in neurological diseases is evolving rapidly, and MAMs dysfunction is associated with various neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Wolfram syndrome. AD, one of the most common and earliest discovered neurodegenerative diseases associated with MAMs, is characterized by the accumulation of abnormally hyperphosphorylated Tau protein aggregates and extracellular amyloid-beta (Aβ) plaques [181]. MAMs are enriched with amyloid precursor protein, presenilin-1/2, and γ-secretase-activating protein, enzymes, or precursors involved in Aβ generation, and may influence the clearance rate of Aβ and Tau aggregates through autophagy regulation [182, 183]. Transcriptional modifications induced by inhibiting or knocking down class I histone deacetylases have potential therapeutic effects on improving mitochondrial–ER crosstalk in AD models [184].

An increase in Aβ aggregates can lead to a decrease in the mRNA levels of MAM proteins and a reduction in MAM length, causing intracellular calcium imbalance [184]. The binding of hyperforin to Aβ and its interaction with RyRs can regulate ER–mitochondria calcium levels and related signaling cascades, reducing Aβ-induced neuronal apoptosis [185]. MAMs also play an important role in the pathogenesis of PD, with studies finding loose, shortened, and reduced MAM tethers in PD mouse models [186]. A proteomic analysis of MAMs in the midbrain identified 158 differentially expressed proteins [186]. Additionally, α-synuclein, a marker of PD, can localize to MAMs and interact with VAPB, reducing its binding to PTPIP51 and ATP production [187]. MAMs disruption is a common pathological mechanism in ALS, potentially leading to the inactivation of TANK-binding kinase 1 and exacerbating protein homeostasis stress [188]. Sig-1R deficiency causes the dissociation of MAM components and mislocalization of IP3R, leading to calcium homeostasis imbalance in MAMs, activation of calpain, mitochondrial dysfunction, possibly causing ALS [189].

The primary defect in Wolfram syndrome involves poor ER calcium handling, which may be related to reduced Ca²⁺ transfer from the ER to the mitochondria through IP3R–GRP75–VDAC1 and decreased ATP production due to WFS1 or CISD2 deficiency [128]. These findings indicate that MAMs play crucial roles in various neurological diseases by regulating calcium signaling, energy metabolism, and other mechanisms that influence neuronal function and survival. A deeper understanding of these mechanisms will aid in the development of new therapeutic strategies to improve the prognosis of neurological diseases.

4.2 Roles of MAMs in Digestive System Diseases

The liver is the most studied organ of the digestive system in the field of MAMs. The liver plays a pivotal role in the digestive system, serves as a crucial metabolic organ, and performs functions, such as detoxification, bile secretion, and nutrient storage [190]. Additionally, the liver acts as a central regulator of lipid metabolism, primarily by coordinating the uptake of circulating lipids, de novo lipogenesis, fatty acid oxidation, and lipid export in very low density lipoprotein particles, to maintain a balance between lipid acquisition and disposal [191]. Consequently, lipid metabolism disorders caused by impaired MAM function are an important cause of liver disease. For example, a deficiency in liver MFN2 results in reduced PS transfer and phospholipid synthesis, leading to ERS and the development of alcoholic steatohepatitis (NASH)-like phenotypes and hepatocellular carcinoma [45]. Similarly, the depletion of Mic19 in mice leads to decreased mitochondria–ER contact, disrupted mitochondrial lipid metabolism, and crista disintegration, damaging mitochondrial fatty acid β-oxidation and lipid metabolism and potentially triggering NASH and liver fibrosis in mice [192].

Dysregulation of MAM-mediated calcium regulation can also result in digestive system diseases. For example, CyD knockout disrupts MAMs integrity and interrupts IP3R-mediated mitochondrial–ER calcium transport, ultimately leading to hepatic insulin resistance [193]. A similar situation occurs in intestinal diseases, in which enhanced IP3R‒MCU calcium axis-mediated excessive calcium transfer in MAMs induces intestinal barrier damage, mitochondrial calcium overload, and dysfunction, which is one of the mechanisms by which deoxynivalenol damages the jejunum of piglets [194].

MAMs also mediate digestive system inflammation. Methane saline has been shown to inhibit the activation of MAMs and the NLRP3 inflammasome through the regulation of the PERK signaling pathway, thereby improving intestinal ischemia–reperfusion injury [195]. Furthermore, MAMs play an important role in viral replication. For example, hepatitis C viral proteins are specifically associated with MAMs in hepatitis C, although this association does not affect calcium signaling or glucose homeostasis between the ER and mitochondria [188]. These studies highlight the critical role of MAMs in digestive system diseases.

4.3 Roles of MAMs in Urinary System Diseases

MAMs have become a focal point of research in urinary system diseases, particularly kidney diseases. Wang et al. reported that vanadium exposure in duck kidneys resulted in alterations in 412 MAM proteins, indicating a high level of MAMs involvement in renal injury processes [21]. In a mouse model of diabetic kidney disease (DKD), high glucose levels increased the expression of mitogen-activated protein kinase 1, which, in turn, reduced the levels of PACS-2. This induced MAMs disruption and mitochondrial fragmentation. However, increasing PACS-2 levels in HK-2 cells ameliorated this effect [196]. Overexpression of PACS-2 in HK-2 cells prevents mitochondrial recruitment of DRP1, thus alleviating excessive mitochondrial division caused by high glucose concentrations; this subsequently restores MAMs integrity and enhances mitochondrial autophagy [56].

Excessive Ca²⁺ uptake by podocytes owing to mitochondrial dysfunction is a key feature of DKD. TRPV1-mediated transient Ca²⁺ influx is reported to AMPK, thereby inhibiting the transcription of Fundc1. This reduces MAMs formation and Ca²⁺ transport from the ER to the mitochondria, a mechanism by which dietary capsaicin improves DKD [197]. Reticulon-1A (RTN1A) expression is increased in human patients with DKD and RTN1A interacts with VDAC1, leading to the detachment of hexokinase-1 from VDAC1. This triggers apoptosis and activation of the inflammatory pathway, contributing to tubular epithelial cell injury and loss [198]. Moreover, RTN1A-mediated ERS may be a crucial determinant of the severity and maladaptive repair of acute kidney injury (AKI) [199]. These findings suggest that MAMs have promising research prospects in the field of urinary system diseases.

4.4 Roles of MAMs in Reproductive System Diseases

The reproductive system is an organ system tasked with the production of gametes (spermatozoa and ova) and their transport, fertilization, and the sustenance of nascent life [200]. Proteomic analysis of testicular MAMs in humans and mice indicated the highly conserved expression of 1,347 proteins specific to MAMs in the testes [201]. These testis-specific MAM proteins have been implicated in spermatogenesis, male gamete production, and sexual reproduction. Latino et al. [202] demonstrated that d-aspartate enhanced MAMs function by increasing lipid transfer from the ER to the mitochondria and calcium signaling, while reducing ERS, which is essential for efficient steroidogenesis in the testes. Disruption of MAMs function can lead to reproductive system diseases. For example, exposure of female mice to di-(2-ethylhexyl) phthalate increased the risk of ERS in atretic follicles, disturbed intracellular calcium homeostasis in oocytes, altered the structure and function of MAMs, and ultimately induced apoptosis [203]. These findings suggest that targeting MAMs could be a potential therapeutic strategy for the treatment of reproductive system diseases. For example, the chlorpromazine derivative, JX57, has shown promising effects in the treatment of endometrial cancer, possibly by targeting GRP75. Mechanistically, JX57 directly binds to GRP75, inhibiting the formation of the IP3R‒GRP75 complex, damaging the structure of MAMs and disrupting calcium homeostasis between the ER and mitochondria, leading to a mitochondrial energy crisis and AMPK activation [204].

Overall, MAMs are crucial for normal reproductive system function and is closely associated with the development of various reproductive system diseases. Targeting MAMs are considered a potential therapeutic strategy, although research is still in its early stages and further exploration is needed to advance related treatments.

4.5 Roles of MAMs in Circulatory System Diseases

Cardiovascular diseases, including heart diseases, are among the most prevalent circulatory system disorders. Cardiac myocytes contain chloride intracellular channel protein (CLIC4), which plays a role in regulating calcium homeostasis between the ER and mitochondria. The absence of CLIC4 is associated with an increased risk of myocardial infarction [205]. In obese cardiomyopathy, elevated STX17 levels promote MAMs formation, leading to mitochondrial Ca²⁺ overload, accumulation of mitochondrial O²⁻, lipid peroxidation, and subsequent cardiac damage [206]. Research on diabetic cardiomyopathy has indicated that high glucose levels inhibit AMPK, promoting MAMs formation mediated by Fundc1 and excessive mitochondrial fission, resulting in myocardial injury [11].

MAMs are also implicated in angiogenesis; for example, TMEM215 was found to protect endothelial cells from BIK-induced mitochondrial apoptosis through the downregulation of EZH2 in response to shear stress derived from blood flow [207]. Additionally, the cytoplasmic F-actin-polymerizing protein, DIAPH1, facilitates ischemia–reperfusion-induced cardiac injury by increasing the proximity of mitochondria to the ER through interaction with MFN2 [208]. FUNDC1-mediated MAMs contribute to cardiac protection against DIC by restoring the biosynthesis of obstructed autophagosomes [209].

In summary, studies on circulatory system diseases have indicated the mechanisms of action of MAMs and other factors, thereby providing potential directions for disease treatment.

4.6 Roles of MAMs in Endocrine System Diseases

The endocrine system is a complex network composed of glands and tissues in the human body that secretes hormones into the blood to regulate various physiological functions. Diabetes mellitus (DM), one of the most common endocrine disorders [210] and is characterized by hyperglycemia, with manifestations of insulin secretion defects and insulin resistance [211]. Pancreatic β-cells rely on Ca²⁺ signals to trigger the release of insulin granules. Overexpression of IP3Rs may lead to the abnormal uptake of mitochondrial Ca²⁺, resulting in abnormal morphology and function of the mitochondria, thus causing a reduction in insulin secretion or a disordered secretion pattern [212].

MAMs are also involved in mediating pancreatic β-cell death. Tiwary et al. [213] reported that in palmitate-induced pancreatic β-cell apoptosis, the overexpression of GRP75 increased the physical coupling between the ER and mitochondria, promoting the transfer of Ca²⁺ to mitochondria, which is related to increases in the mitochondrial membrane potential and ROS. Similarly, PDZD8 is highly expressed in DM and regulates mitochondria–ER contact and calcium dynamics through enhanced interactions with cyclophilin D and IP3R‒GRP75‒VDAC, inducing pancreatic β-cell death [96]. Therefore, targeting the calcium channels in MAMs may offer therapeutic potential for the treatment of endocrine disorders.

Urolithin A has been shown to protect against severe acute pancreatitis by modulating ER–mitochondrial calcium channels and reducing acinar cell necroptosis [214]. MAMs also participate in gluconeogenesis. The lncRNA H19 can suppress the impairment of mitochondria–ER junctions in hepatocytes and potentiate gluconeogenesis by regulating the expression level of VDAC1 [215]. Collectively, these studies suggest that MAMs hold great promise as therapeutic targets for endocrine system-related diseases.

4.7 Roles of MAMs in Locomotor System Diseases

The human locomotor system, namely, the musculoskeletal system, consists of bones, skeletal muscles, ligaments, tendons, joints, cartilage, and other connective tissues and plays crucial roles in locomotion, support, and protection [216]. Abnormalities in MAMs are closely linked to skeletal muscle dysfunction. For example, SEPN1-related myopathy caused by SEPN1 mutations is characterized by muscle weakness and fatigue, leading to scoliosis and potentially life-threatening respiratory failure [217]. SEPN1 is highly concentrated in MAMs, and its deficiency results in reduced mitochondria–ER connections, decreased intracellular calcium concentrations in organelles, and impaired oxidative phosphorylation [218]. Furthermore, BCL2L13, a member of the BCL-2 family, is highly expressed in endurance-trained individuals and localized to MAMs. BCL2L13 interacts with calcium ion signaling channels, such as VDAC1, potentially playing an important role in maintaining calcium homeostasis and skeletal muscle function [219].

MAMs are vital for maintaining normal spinal function. Sphingosine-1-phosphate depletion can lead to intervertebral disc degeneration (IDD) through vesicular transport, mitochondria–ER calcium flux, and oxidative stress pathways [220]. SYNJ2BP, located in nucleus pulposus (NP) cells, maintains MAM connections and the formation of the NLRX1–SLC39A7 complex, aiding in the maintenance of zinc ion (Zn²⁺) homeostasis within mitochondria. The loss of SYNJ2BP exacerbates MAMs damage and inhibits the formation of the NLRX1–SLC39A7 complex, potentially reducing the localization of SLC39A7 in the mitochondria, increasing mitochondrial zinc ion accumulation, and ultimately leading to the exacerbation of mitochondrial dysfunction, accelerated aging of NP cells, and progression of IDD [221].

Overall, the structure of MAMs is essential for proper muscle and bone function. Research on molecules such as SEPN1 and BCL2L13 has indicated new mechanisms underlying related diseases, thus offering new avenues for therapeutic strategies.

4.8 Role of MAMs in respiratory System Diseases

The respiratory system consists of the airways and lungs, the latter being the primary organ responsible for the inhalation of oxygen and exhaled carbon dioxide, thereby maintaining normal physiological functions in the body [222]. The role of MAMs in respiratory diseases is being increasingly recognized. MAMs dysfunction is a key factor in lung injury. For example, Xiao et al. showed that paraquat was toxic to rat lung tissue cells. Paraquat can induce ER Ca²⁺ overload, and the activated immunoglobulin binding protein (BIP) and transcription factor C/EBP-homologous protein (CHOP) pathways can directly or indirectly affect the expression of apoptosis-related caspase family factors [223]. Moreover, paraquat promoted ER Ca²⁺ release and induced mitochondrial Ca²⁺ uptake. This process triggers a response of Bax/Bcl-2 channel proteins to the IP3R/VDAC/MCU calcium axis, resulting in the disruption of this axis and cytochrome c release, ultimately leading to ERS and cell apoptosis [223]. In addition, arsenic-induced pulmonary ferroptosis and acute lung injury have been proven to be, at least partially, related to MAMs dysfunction triggered by mtROS [83]. MAMs dysfunction is also associated with bronchial disease. Titanium dioxide nanoparticles can induce ERS in human bronchial epithelial cells and disrupt the MAMs and Ca²⁺ balance, thereby increasing autophagy [224]. These findings illustrate the complex relationship between MAMs dysfunction and various respiratory diseases.

4.9 Role of MAMs in Cancer

Research on the involvement of MAMs in different types of cancer and their roles in tumor growth and metastasis is a field of exponential growth. Owing to their rapid proliferation and invasive nature, cancer cells require various methods to meet their energy and material demands, such as increasing glucose uptake and glycolytic activity (known as the Warburg effect), lipid synthesis and degradation, and modulation of Ca²⁺ signaling [225]. Therefore, communication between ER and mitochondria plays a crucial role in cancer initiation and progression. Aberrant expression of MAM proteins is commonly observed in tumors, where changes in the cellular microenvironment are often associated with the activation of transmembrane receptors and Ca²⁺-permeable channels involved in cancer progression, epithelial–mesenchymal transition, invasion, and resistance to apoptosis [226].

The IP3R–GRP75–VDAC–MCU complex, which plays a central role in MAMs Ca²⁺ transport, is regulated by oncogenes, such as PTEN, PML, and BCL2. In MAMs, PTEN binds to IP3R and prevents its degradation, thus promoting Ca²⁺ transport into mitochondria, which is crucial for apoptosis [227]. Loss of PTEN function and subsequent inappropriate Ca²⁺ transport can lead to resistance to apoptosis [228, 229]. Similarly, when PML is directed to MAMs by p53, it affects Ca²⁺ transfer from the ER to the mitochondria, thereby regulating apoptosis and autophagy. Therefore, its absence can promote tumor development by providing a growth advantage to tumor cells that use autophagy as a survival strategy under stress conditions [80]. Conversely, BCL2, another oncogene enriched in MAMs, directly interacts with IP3R, preventing Ca²⁺ transport from the ER to the mitochondria and interfering with the release of cytochrome c from the mitochondria, thus playing a role in resisting apoptosis in cancer [230]. High IP3R expression is associated with increased invasiveness and metastasis of cancer cells [231].

MAMs also influence tumor angiogenesis by modulating Ca²⁺ transfer [207]. For example, increased MAMs formation in cancer cells leads to elevated cytosolic Ca²⁺ levels, promoting the phosphorylation of serum response factor (SRF) and enhancing the binding of SRF to the VEGFR2 promoter, resulting in increased VEGFR2 production and subsequent angiogenesis. Silencing of FUNDC1 reduces MAMs formation and inhibits tumor angiogenesis [232]. In breast cancer, the extracellular matrix enhances ER-mediated Ca²⁺ transport to the mitochondria, promoting DRP1 recruitment to the mitochondria and phosphorylation at serine 616, and inducing mitochondrial fragmentation and Parkin/PINK1-mediated mitophagy, which is beneficial for tumor cell survival [233]. Furthermore, cancer cells often exhibit alterations in inner membrane CL and cholesterol levels, which may confer resistance to apoptosis or metabolic reprogramming [234]. The tumor suppressor, p53, regulates PA transport to the mitochondria through MAMs [235]. In drug-resistant cells, the transfer of Ca²⁺ from MAMs to the mitochondria is not essential for reducing apoptosis; however, disrupting sphingolipid biosynthesis in MAMs and lipid transfer to the OMM can decrease apoptotic signaling and enhance multidrug resistance in recurrent cells [236]. These studies highlight the diverse roles of MAMs in cancer.