Isolation and analysis of fractions enriched in WrappER-associated mitochondria from mouse liver

Ilacqua, Nicolò; Anastasia, Irene

doi:10.1016/j.xpro.2021.100752

Cited by 4 publications

(6 citation statements)

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“…To gain insights into the effects of Synj2bp knockdown in hepatic metabolism, we performed quantitative proteomics analysis of the liver fractions enriched in wrap-pER-associated mitochondria (Fig. 2C and Additional file 2: Table S1) [42]. Compared to control fractions, WAM isolated from Synj2bp-silenced livers contained nearly half the amount of ER proteins (Fig.…”

Section: Upregulated Mitochondria Fatty Acid Respiration In Synj2bp-s...mentioning

confidence: 99%

“…WAM-enriched fractions were isolated as described [42]. Briefly, the mouse liver was quickly excided, washed three times with Buffer A (BA; 10 mM Tris-HCl, 1 mM MgCl 2 , 0.1 mM EGTA, pH 7.4) supplemented with 250 mM Sucrose (B10), minced in small pieces, and resuspended in 5 ml B10 supplemented with 1 × Protease Inhibitor Cocktail (PIC; Thermo Fisher Scientific, #78429).…”

Section: Preparation Of Fractions Enriched In Wrapper-associated Mito...mentioning

confidence: 99%

“…Livers of mice at 3 h postprandial were excised and WAM-enriched fractions were freshly prepared as described in [42]. The WAM-containing pellet was resuspended in 600 μl of TBS (150 mM NaCl, 10 mM Tris-HCl pH 7.6) supplemented with 1 × PIC (Thermo Fisher Scientific).…”

Section: Lipoprotein Imaging By Negative Staining and Immunogold Em A...mentioning

confidence: 99%

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Expression of Synj2bp in mouse liver regulates the extent of wrappER-mitochondria contact to maintain hepatic lipid homeostasis

et al. 2022

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Background In mouse liver hepatocytes, nearly half of the surface area of every mitochondrion is covered by wrappER, a wrapping-type of ER that is rich in fatty acids and synthesizes lipoproteins (VLDL) (Anastasia et al. in Cell Rep 34:108873, 2021; Hurtley in Science (80- ) 372:142–143, 2021; Ilacqua et al. in J Cell Sci 135:1–11, 2021). A disruption of the ultrastructure of the wrappER-mitochondria contact results in altered fatty acid flux, leading to hepatic dyslipidemia (Anastasia et al. 2021). The molecular mechanism that regulates the extent of wrappER-mitochondria contacts is unknown. Methods We evaluated the expression level of the mitochondrial protein Synj2bp in the liver of normal and obese (ob/ob) mice. In addition, we silenced its expression in the liver using an AAV8 vector. We coupled quantitative EM morphometric analysis to proteomics and lipid analyses on these livers. Results The expression level of Synj2bp in the liver positively correlates with the extent of wrappER-mitochondria contacts. A 50% reduction in wrappER-mitochondria contacts causes hepatic dyslipidemia, characterized by a gross accumulation of lipid droplets in the liver, an increased hepatic secretion of VLDL and triglycerides, a curtailed ApoE expression, and an increased capacity of mitochondrial fatty acid respiration. Conclusion Synj2bp regulates the extent of wrappER-mitochondria contacts in the liver, thus contributing to the control of hepatic lipid flux.

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Section: Upregulated Mitochondria Fatty Acid Respiration In Synj2bp-s...mentioning

confidence: 99%

Section: Preparation Of Fractions Enriched In Wrapper-associated Mito...mentioning

confidence: 99%

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Expression of Synj2bp in mouse liver regulates the extent of wrappER-mitochondria contact to maintain hepatic lipid homeostasis

et al. 2022

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“…These changes in sphericity and morphology may impact the ability of mitochondria to form contact sites. Particularly, liver mitochondria are known to exhibit phenotypes in which ER wraps around them, known as wrappER, to modulate lipid flux (63)(64)(65). To investigate this paradigm, we qualitatively identified wrappER within liver tissue (Figures 3G-H).…”

Section: Ultrastructural Changes In Murine Liver Reveals Aging Is Ass...mentioning

confidence: 99%

“…Qualitatively, we found that wrappER exists in both 3-month and 2-year murine liver tissue (Figures 3G'-H'), but in 3-month tissue wrappER contact is much more extensive (Figures 3G"-H"). This suggests that liver lipid homeostasis may be negatively impacted (63)(64)(65). However, to confirm these changes and further explore the implications of these mitochondrial morphological alterations, we analyzed metabolism and performed lipidomics in mice.…”

Section: Ultrastructural Changes In Murine Liver Reveals Aging Is Ass...mentioning

confidence: 99%

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes

Vue,

Murphy,

et al. 2024

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The liver, the largest internal organ and a metabolic hub, undergoes significant declines due to aging, affecting mitochondrial function and increasing the risk of systemic liver diseases. How the mitochondrial three-dimensional (3D) structure changes in the liver across aging, and the biological mechanisms regulating such changes confers remain unclear. In this study, we employed Serial Block Face-Scanning Electron Microscopy (SBF-SEM) to achieve high-resolution 3D reconstructions of murine liver mitochondria to observe diverse phenotypes and structural alterations that occur with age, marked by a reduction in size and complexity. We also show concomitant metabolomic and lipidomic changes in aged samples. Aged human samples reflected altered disease risk. To find potential regulators of this change, we examined the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex, which plays a crucial role in maintaining mitochondrial architecture. We observe that the MICOS complex is lost during aging, but not Sam50. Sam50 is a component of the sorting and assembly machinery (SAM) complex that acts in tandem with the MICOS complex to modulate cristae morphology. In murine models subjected to a high-fat diet, there is a marked depletion of the mitochondrial protein SAM50. This reduction in Sam50 expression may heighten the susceptibility to liver disease, as our human biobank studies corroborate that Sam50 plays a genetically regulated role in the predisposition to multiple liver diseases. We further show that changes in mitochondrial calcium dysregulation and oxidative stress accompany the disruption of the MICOS complex. Together, we establish that a decrease in mitochondrial complexity and dysregulated metabolism occur with murine liver aging. While these changes are partially be regulated by age-related loss of the MICOS complex, the confluence of a murine high-fat diet can also cause loss of Sam50, which contributes to liver diseases. In summary, our study reveals potential regulators that affect age-related changes in mitochondrial structure and metabolism, which can be targeted in future therapeutic techniques.Graphical AbstractLiver aging causes metabolic, lipidomic, and mitochondrial structural alterations, reflecting age-dependent losses in the MICOS complex. Diet-dependent losses of the SAM complex underlie genetic disease associations and mitochondrial structure.

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The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

Vue,

Prasad,

et al. 2024

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The kidney filters nutrient waste and bodily fluids from the bloodstream, in addition to secondary functions of metabolism and hormone secretion, requiring an astonishing amount of energy to maintain its functions. In kidney cells, mitochondria produce adenosine triphosphate (ATP) and help maintain kidney function. Due to aging, the efficiency of kidney functions begins to decrease. Dysfunction in mitochondria and cristae, the inner folds of mitochondria, is a hallmark of aging. Therefore, age-related kidney function decline could be due to changes in mitochondrial ultrastructure, increased reactive oxygen species (ROS), and subsequent alterations in metabolism and lipid composition. We sought to understand if there is altered mitochondrial ultrastructure, as marked by 3D morphological changes, across time in tubular kidney cells. Serial block facing-scanning electron microscope (SBF-SEM) and manual segmentation using the Amira software were used to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented, compared to the 3-month, with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we show that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney due to aging. Disruption of the MICOS complex shows altered mitochondrial calcium uptake and calcium retention capacity, as well as generation of oxidative stress. We found significant, detrimental structural changes to aged kidney tubule mitochondria suggesting a potential mechanism underlying why kidney diseases occur more readily with age. We hypothesize that disruption in the MICOS complex further exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, thus impacting kidney health.Translational StatementDue to aging, the efficiency of kidney functions begins to decrease and the risk of kidney diseases may increase, but specific regulators of mitochondrial age-related changes are poorly explained. This study demonstrates the MICOS complex may be a target for mitigating age-related changes in mitochondria. The MICOS complex can be associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies.Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations.

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Isolation and analysis of fractions enriched in WrappER-associated mitochondria from mouse liver

Cited by 4 publications

References 2 publications

Expression of Synj2bp in mouse liver regulates the extent of wrappER-mitochondria contact to maintain hepatic lipid homeostasis

Expression of Synj2bp in mouse liver regulates the extent of wrappER-mitochondria contact to maintain hepatic lipid homeostasis

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

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