*4.2. MCS Formation by MITOL*

MITOL can control signals contributing to various features outside mitochondria through the MCS between the ER and mitochondria. A primary finding that MITOL is enriched in the MCS between the ER and mitochondria led us to investigate MITOL with a focus on MCS.

In basal and physiological conditions, MITOL interacts with and ubiquitinates the mitochondrial GTPase MFN2 [71]. MFN2 acts as a tethering factor between the ER and mitochondria, as well as a factor for inter-mitochondrial fusion via its GTPase activity [72]. The MFN2 ubiquitination by MITOL contributes to its function related to ER–mitochondrion contacts through the GTPase activation of MFN2 (Figure 4A). Thus, MFN2 mutated at K192, the lysine specific for MITOL-mediated ubiquitination, leads to the inability to connect the membrane between the ER and mitochondria, resulting in a failure of calcium ion transfer from the ER to mitochondria in the cell. The disruption of MCS between the ER and mitochondria is not fatal for the cell but a ffects setting the threshold for the complementation of mitochondrial or ER defects triggered by pathological conditions. Therefore, abnormal formation of the MCS might initiate or aggravate progressive, not early-developmental, diseases in humans. Actually, mutations in the MFN2 gene lead to its catalytic inactivation and subsequently trigger Charcot-Marie-Tooth disease type 2A (CMT2A) in the peripheral nervous system [73]. Similarly, a primary mutation in Sig1R or SOD1, a cause of inherited juvenile ALS, was shown to result in the disruption of MCS between the ER and mitochondria [74].

What is the in vivo and physiological contribution of MCS between the ER and mitochondria? What is triggered by the perturbation of MCS between the ER and mitochondria in vivo remains poorly characterized. There are also serious concerns regarding the accuracy of the methods for analyzing the MCS structure in vivo. To obtain an understanding of the morphology of MCS between the ER and mitochondria in vivo, electron microscopy is mostly adopted (sometimes potentially being the only method available in vivo). However, both organelles, mitochondria and the ER, exhibit complex and diverse morphology. A single image obtained from an electron microscope was limited and restricts us to evaluating a whole picture of mitochondrial states. It is also di fficult to judge whether the membrane structure in a single image is part of the continuous ER or an independent part. Therefore, there is an urgen<sup>t</sup> need to accurately investigate the structure of MCS between continuous ER and mitochondria in the brain.

To obtain a precise understanding of the morphology of MCS between mitochondria and the ER in neurons, we recently performed three-dimensional (3D) reconstructions from serial electron microscopy

images of mitochondria using serial block—face scanning electron microscopy (SBF-SEM) [75]. Interestingly, over 95% of mitochondria had at least one contact site with the continuous ER in the brain regardless of the individual morphology of mitochondria, suggesting that MCS with the ER is pivotal for almost all mitochondria, at least in neurons (in this analysis, only neurons were selected morphologically from serial images). Larger mitochondria required more MCS with continuous ER. However, each MCS with continuous ER displayed morphological di fferences, such as large and small types. Contacts between the ER and mitochondria with distinct sizes might involve distinct roles. We also examined the physiological contribution of the MITOL-MFN2 axis to MCS using mice with nerve-specific ablation of MITOL. The MITOL-deleted brain showed the formation of fewer and smaller MCS between continuous ER and mitochondria. However, the phenotype of MITOL-deleted neurons regarding MCS appeared to be restricted to only larger mitochondria. When taking the findings as a whole, mitochondrial defects in the brain with MITOL deletion were mild, leading to slight developmental abnormalities in the brain. Currently, we are examining the pathological contribution of disrupted mitochondrion-ER connections during disease development by performing crossing with murine models for aging-related diseases.

**Figure 4.** (**A**) Membrane contact site (MCS) formation by MITOL-MFN2 axis. MITOL ubiquitinates mitochondrial MFN2. The ubiquitinated MFN2 enhances its GTPase activity, triggering trans-oligomerization between mitochondrial MFN2 and ER-localized MFN2 for tethering both organelles; mitochondria and the ER; (**B**) UPR regulation by MITOL-IRE1α axis. MITOL ubiquitinates IRE1α at ER-mitochondria contact site in unstressed and low-stressed conditions regarding the ER. The ubiquitination of IRE1α does not perturbate the oligomerization itself in response to ER stress, however, leading to short-term stabilization and smaller oligomerization. In contract, the ubiquitination by MITOL is reduced under severe or chronic ER stress, resulting in continuous oligomerization of IRE1α and apoptotic switching of IRE1α signaling.
