Christensen, Trace

Alzheimer’s disease (AD) has no cure. Earlier, we showed that partial inhibition of mitochondrial complex I (MCI) with the small molecule CP2 induces an adaptive stress response, activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, Aβ and pTau accumulation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD. Here, using serial block-face scanning electron microscopy (SBFSEM) and three-dimensional (3D) EM reconstructions combined with Western blot analysis and next-generation RNA sequencing, we demonstrate that CP2 treatment also restores mitochondrial morphology and mitochondria-endoplasmic reticulum (ER) communication, reducing ER and unfolded protein response (UPR) stress in the APP/PS1 mouse brain. Using 3D EM volume reconstructions, we show that in the hippocampus of APP/PS1 mice, dendritic mitochondria primarily exist as mitochondria-on-a-string (MOAS). Compared to other morphological phenotypes, MOAS have extensive interaction with the ER membranes, forming multiple mitochondria-ER contact sites (MERCS) known to facilitate abnormal lipid and calcium homeostasis, accumulation of Aβ and pTau, abnormal mitochondrial dynamics, and apoptosis. CP2 treatment reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reductions in MERCS, ER/UPR stress, and improved lipid homeostasis. These data provide novel information on the MOAS-ER interaction in AD and additional support for the further development of partial MCI inhibitors as a disease-modifying strategy for AD.

Abstract Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1.

Key Points In heme protein–induced AKI, mitochondrial functional integrity, as reflected by ATP and NAD + content and NAD + /NADH ratio, is impaired. Mitochondrial quality control is compromised as reflected by impaired biogenesis, exaggerated fission, and marked ultrastructural damage. Modern concepts regarding mitochondria and AKI apply to heme protein–induced AKI, with the possibility of novel therapeutic strategies. Background Mitochondrial injury occurs in and underlies acute kidney injury (AKI) caused by ischemia-reperfusion and other forms of renal injury. However, to date, a comprehensive analysis of this issue has not been undertaken in heme protein–induced AKI (HP-AKI). We examined key aspects of mitochondrial function, expression of proteins relevant to mitochondrial quality control, and mitochondrial ultrastructure in HP-AKI, along with responses to heme in renal proximal tubule epithelial cells. Methods The long-established murine glycerol model of HP-AKI was examined at 8 and 24 hours after HP-AKI. Indices of mitochondrial function (ATP and NAD + ), expression of proteins relevant to mitochondrial dynamics, mitochondrial ultrastructure, and relevant gene/protein expression in heme-exposed renal proximal tubule epithelial cells in vitro were examined. Results ATP and NAD + content and the NAD + /NADH ratio were all reduced in HP-AKI. Expression of relevant proteins indicate that mitochondrial biogenesis (PGC-1 α , NRF1, and TFAM) and fusion (MFN2) were impaired, as was expression of key proteins involved in the integrity of outer and inner mitochondrial membranes (VDAC, Tom20, and Tim23). Conversely, marked upregulation of proteins involved in mitochondrial fission (DRP1) occurred. Ultrastructural studies, including novel 3D imaging, indicate profound changes in mitochondrial structure, including mitochondrial fragmentation, mitochondrial swelling, and misshapen mitochondrial cristae; mitophagy was also observed. Exposure of renal proximal tubule epithelial cells to heme in vitro recapitulated suppression of PGC-1 α (mitochondrial biogenesis) and upregulation of p-DRP1 (mitochondrial fission). Conclusions Modern concepts pertaining to AKI apply to HP-AKI. This study validates the investigation of novel, clinically relevant therapies such as NAD + -boosting agents and mitoprotective agents in HP-AKI.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes.

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

Introduction: Characterization of acute ischemic stroke thrombi has typically focused on the two dimensional (2D) histological characterization of the thrombus. Little is known about the three dimensional (3D) structure of these occlusive thrombi. Electron Microscopy (EM) can be used to obtain 3D information about the organization of clot components. Individual structures can be segmented and qualitative and quantitative information obtained from the clots. Methods: Following IRB approval from Mayo Clinic, acute ischemic stroke clot samples were collected from 10 patients treated at Mayo Clinic. Samples were immediately fixed in fresh modified Trumps fixative solution. A representative fragment of clot was taken and split longitudinally into two pieces; one piece was sent for 3D-EM analysis and the other for histological processing and analysis. Hematoxylin and eosin stain (H&E) and Martius Scarlett Blue (MSB) histological stains were used for conventional histopathology evaluation and components analysis. Immunohistochemical (IHC) staining using anti-CD42b (Platelets) was applied to confirm the presence of platelets in each clot. Serial block-face scanning electron microscopy was used to generate high-resolution three-dimensional images. Results: The 2D morphological features of the clot were identified using H&E, MSB and anti-CD42b staining. The 3D organization of thrombi varies greatly, both within and between clots depending on the constituents. Red Blood Cell-rich areas are mainly composed of Red Blood Cells packaged tightly together, with a small number of Fibrin strands interwoven between the cells. Fibrin-rich areas are characterized by dense networks of fibrin strands along with platelets and White Blood Cells with some sparse Red Blood Cells present. Platelet-rich areas are composed of large volumes of de-granulated platelets, some fibrin stands and white blood cells and an absence of Red Blood Cells. Conclusions: 3D electron microscopy can provide important information on the organization of components within thrombi. This could provide key information on the age and etiology of the thrombus. In addition, it may help to inform thrombolytic treatment and medical device design.

Background Characterization of acute ischemic stroke (AIS) clots has typically focused on two-dimensional histological analysis of the thrombus. The three-dimensional (3D) architecture and distribution of components within emboli have not been fully investigated. The aim of this study was to examine the composition and microstructure of AIS clots using histology and serial block-face scanning electron microscopy (SBFSEM). Methods As part of the multi-institutional STRIP registry, 10 consecutive AIS emboli were collected from 10 patients treated by mechanical thrombectomy. Histological and immunohistochemical analysis was performed to determine clot composition. SBFSEM was used to assess the ultrastructural organization of the clots and specific features of individual components. Results Quantification of Martius Scarlett Blue stain identified fibrin (44.4%) and red blood cells (RBCs, 32.6%) as the main components. Immunohistochemistry showed a mean platelet and von Willebrand factor content of 23.9% and 11.8%, respectively. The 3D organization of emboli varied greatly depending on the region analyzed. RBC-rich areas were composed mainly of tightly packed RBCs deformed into polyhedrocytes with scant fibrin fibers interwoven between cells. The regions with mixed composition showed thick fibrin fibers along with platelets, white blood cells and RBC clusters. Fibrin-rich areas contained dense fibrin masses with sparse RBC. In three cases, the fibrin formed a grid-like or a sponge-like pattern, likely due to thrombolytic treatment. Segmentation showed that fibrin fibers were thinner and less densely packed in these cases. Conclusions 3D-SEM provides novel and potentially clinically relevant information on clot components and ultrastructure which may help to inform thrombolytic treatment and medical device design.

Abstract Viruses spread between hosts through particles, but within hosts, viral genomes can spread from cell to cell through fusion, evading antiviral defenses and obviating costly infectious virion production1-3. Billions of electromechanically coupled cardiomyocytes (CMs) make myocardium inherently vulnerable to pathological electromechanical short circuits caused by intercellular viral spread 4-6. Beyond respiratory illness, COVID-19 affects the heart7 and cardiac injury and arrhythmias are serious public health concerns8-12. By studying myocardium of a young woman who died suddenly, diagnosed postmortem with COVID-19, we discovered highly focal myocardial SARS-CoV-2 infection spreading from one CM to another through intercellular junctions identified by highly concentrated sarcolemmal t-tubule viral spike glycoprotein. SARS-CoV-2 permissively infected beating human induced pluripotent stem cell (hiPSC)-CMs building multinucleated cardiomyotubes (CMTs) through cell type-specific fusion driven by proteolytically-activated spike glycoprotein. Recombinant spike glycoprotein, co-localizing to sarcolemma and sarcoplasmic reticulum, produced multinucleated CMTs with pathological structure, electrophysiology and Ca2+ excitation-contraction coupling. Blocking cleavage, a peptide-based protease inhibitor neutralized SARS-CoV-2 spike glycoprotein pathogenicity. We conclude that SARS-CoV-2 spike glycoprotein, efficiently primed, activated and strategically poised during biosynthesis, can exploit the CM’s inherent membranous connectivities to drive heart damage directly, uncoupling clinically common myocardial injury from lymphocytic myocarditis, often suspected but rarely confirmed in COVID-19.