Biology subjects

Scudese, E.

Publications and source records attributed to Scudese, E..

7 recordsLinked to original sources

bioRxiv2024

Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments

Mitochondria are hubs of metabolism and signaling, playing crucial roles in tumorigenesis, therapeutic resistance, and metastasis in many types of cancer. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the full extent of the complexity of the mitochondrial network, nor its regulation upon exposure to therapeutic stressors. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Herein we present the first three-dimensional analysis of mitochondrial networks in human tumor tissues. Use of two experimentally tractable orthotopic patient-derived xenograft (PDX) models TNBC enabled us to conduct longitudinal analyses to construct mitochondrial networks in treatment-naive and residual tumors persisting after exposure to a variety of conventional chemotherapies. Further, we modeled lipid droplet (LD) structures and their physical contacts with mitochondria. In total, we reconstructed 3,750 mitochondria and 800 LDs in three dimensions using serial block-face scanning electron microscopy (SBF-SEM), providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Both carboplatin (CRB) and docetaxel (DTX) chemotherapies produced residual tumors that harbored mitochondria with significantly increased areas, volumes, and perimeters in both PDX models. Additionally, treatment with the conventional combinations DTX plus CRB or Adriamycin plus cyclophosphamide (AC), led to reduced mitochondrial branching and elongation. In contrast, DTX or CRB alone elicited model-specific changes in mitochondrial complexity. Further, the extensive intra-tumoral heterogeneity of mitochondrial structure in untreated PDX tumors significantly decreased in residual tumors. Analyses of LDs revealed significant and consistent elevation of the number and physical proximity of MLCs in residual tumors, congruent with our previous studies providing evidence for transcriptomic and proteomic rewiring of lipid metabolism in residual TNBC. These results highlight the potential for structure-based monitoring of chemotherapeutic metabolic reprogramming and suggest unique molecular mechanisms that may underlie chemoresistance in TNBC. Furthermore, our findings provide novel insights into a new type of intratumoral heterogeneity, that of mitochondrial intratumoral heterogeneity, which complements our understanding of the genomic, epigenomic, transcriptomic, and proteomic complexity of TNBC.

cancer biology↗

bioRxiv2024

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

Background & AimsAging is associated with a significant decline in mitochondrial function in the liver, leading to an increased risk of liver disease. This study examines age-related changes in the mitochondrial structure of human and murine livers using a combination of Serial Block-Face Scanning Electron Microscopy (SBF-SEM) and mass spectrometry approaches. MethodsThis study integrates mitochondrial structure analysis in a murine model with an analysis of liver architecture, lipogenesis, and genetically regulated gene expression in human cohorts. We explored the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex using SBF-SEM, three-dimensional reconstruction with Amira software, and mass spectrometry techniques. ResultsAging leads to a reduction in mitochondrial size and complexity, resulting in changes in the metabolomic and lipidomic profiles of murine liver cells that are comparable to those observed in aged human samples. We find that genetically modeled expression of MICOS complex genes OPA1 and CHCHD3 is associated with chronic liver disease phenotypes within a large biobank population. Furthermore, we observed dysregulated mitochondrial calcium handling and increased oxidative stress due to the disruption of the MICOS complex. ConclusionOur study highlights the age-associated decline in mitochondrial complexity and metabolic regulation within the aging murine liver and the human population. We have identified that these changes are partially attributable to the age-related loss of the MICOS complex. Impact and implicationsThis study offers new insights into the changes to mitochondrial ultrastructure that occur during aging. Using SBF-SEM, the quantification of young and aged murine mitochondrial structure was performed, which had previously been an underexplored avenue for measuring mitochondrial changes. The discovery of mitochondrial ultrastructural changes, in conjunction with measurements of age-associated metabolic alterations and gene association data, provides a model for how changes in MICOS expression may modulate age-related impairment of hepatic mitochondria. These results provide a new model by which changes in MICOS protein expression may both cause and be a potential therapeutic target for age-related impairment in hepatic function. HighlightsDecreased modeled expression of CHCHD3 in individuals of European genetic ancestry is linked to liver transplant and cirrhosis, while decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic liver disease and cirrhosis. Aging alters liver lipid accumulation, MICOS mRNA levels, and disease markers. Aging reduces the volume and complexity of murine liver ultrastructure. Aging and diet significantly alter the MICOS complex in mice. Knockdown of Mic60 and Chchd6 lowers Ca2+ uptake, retention, and induces oxidative stress in HepG2 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/599846v3_ufig1.gif" ALT="Figure 1000"> View larger version (47K): org.highwire.dtl.DTLVardef@1cdd61corg.highwire.dtl.DTLVardef@a3fb74org.highwire.dtl.DTLVardef@1d1ad36org.highwire.dtl.DTLVardef@c2e55f_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver aging causes metabolic, lipidomic, and mitochondrial structural alterations, reflecting age-dependent losses in the MICOS complex. Key components of the MICOS complex (MIC60, CHCHD3 and CHCHD6) are illustrated.

physiology↗

bioRxiv2024

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

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed 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 with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a868f0org.highwire.dtl.DTLVardef@1817bfborg.highwire.dtl.DTLVardef@1f2a1a3org.highwire.dtl.DTLVardef@520692_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/566502v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1d2be0eorg.highwire.dtl.DTLVardef@bba6caorg.highwire.dtl.DTLVardef@1c335e5org.highwire.dtl.DTLVardef@10789c9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Age-related skeletal muscle atrophy shows morphological alterations in mitochondrial structure associated with declining function. Our findings propose that exercise intervention may counteract these structural declines by reinstating levels of mitofusin 2, thus highlighting a potential mechanism by which exercise attenuates age-induced mitochondrial dysfunction. C_FIG

biophysics↗

bioRxiv2023

Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/559135v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e5b7org.highwire.dtl.DTLVardef@e965e5org.highwire.dtl.DTLVardef@120c7e9org.highwire.dtl.DTLVardef@124bfb5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Workflow highlighting the process of murine interscapular BAT extraction, processing and imaging serial block facing-scanning electron microscopy and using Amira software for 3D reconstruction of LDs and mitochondrial lipid contact sites to elucidate their structure across aging. C_FIG

biophysics↗

bioRxiv2023

Alterations in Cardiovascular and Cerebral Pulse Wave Velocity in 5XFAD Murine Model of Alzheimer's Disease

Alzheimers Disease (AD) is a global health issue, affecting over 6 million in the United States, with that number expected to increase as the aging population grows. As a neurodegenerative disorder that affects memory and cognitive functions, it is well established that AD is associated with cardiovascular risk factors beyond only cerebral decline. However, the study of cerebrovascular techniques for AD is still evolving. Here, we provide reproducible methods to measure impedance-based pulse wave velocity (PWV), a marker of arterial stiffness, in the systemic vascular (aortic PWV) and in the cerebral vascular (cerebral PWV) systems. Using aortic impedance and this relatively novel technique of cerebral impedance to comprehensively describe the systemic vascular and the cerebral vascular systems, we examined the sex-dependent differences in 5x transgenic mice (5XFAD) with AD under normal and high-fat diet, and in wild-type mice under a normal diet. Additionally, we validated our method for measuring cerebrovascular impedance in a model of induced stress in 5XFAD. Together, our results show that sex and diet differences in wildtype and 5XFAD mice account for very minimal differences in cerebral impedance. Interestingly, 5XFAD, and not wildtype, male mice on a chow diet show higher cerebral impedance, suggesting pathological differences. Opposingly, when we subjected 5XFAD mice to stress, we found that females showed elevated cerebral impedance. Using this validated method of measuring impedance-based aortic and cerebral PWV, future research may explore the effects of modifying factors including age, chronic diet, and acute stress, which may mediate cardiovascular risk in AD. New and NoteworthyHere, we presented a new technique which is an application of the concept of aortic impedance to determining cerebral impedance. While aortic PWV is typically utilized to study aortic stiffness, we also developed a technique of cerebral PWV to study cerebral vascular stiffness. This method may be useful in improving the rigor of studies that seek to have a dual focus on cardiovascular and cerebral function.

physiology↗

bioRxiv2023

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/540609v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@96099borg.highwire.dtl.DTLVardef@50b4f4org.highwire.dtl.DTLVardef@983092org.highwire.dtl.DTLVardef@19ceb9b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Overview of serial block facing-scanning electron microscopy (SBF-SEM) workflow, data segmentation, and 3D analysis of mitochondria using Amira software for murine interscapular BAT. C_FIG

biophysics↗