The thymus originates from the third pharyngeal pouch endoderm, which also gives rise to respiratory tract elements. Here, we examined intrathymic cystic structures, long considered remnants of organogenesis. Through sequential histology and ultrastructural imaging, we uncovered that these 'cysts' are in fact continuous and structured epithelial networks embedded within the thymic parenchyma. These networks follow a conserved 'head-neck-funnel-tentacle' architecture spanning the trabeculae, cortex, corticomedullary junction (CMJ) and medulla. The head, typically glandular and ciliated, connects to a funnel enriched in diverse epithelial cell types-goblet, tuft, club, ionocyte-like, microfold and ciliated cells-at the CMJ. Tentacle-like projections sometimes extend into the medulla, often surrounding perivascular spaces. Luminal contents vary, with thymocytes and macrophages most abundant caudally. We also identified solitary medullary thymic epithelial cells with large ciliated cytoplasmic lumens, distinct from these epithelial networks. Electron microscopy suggested a respiratory identity and thymic-specific adaptations for the lining cells. These findings challenge the notion of thymic cysts as inert debris, and instead reveal a coherent, mimetic system with possible roles in thymocyte selection, maturation and egress.

Abstract The goal of this study was to develop an imaging approach to assess the integrity of the thymic epithelium in preclinical mouse models. The thymus is the most sensitive organ to cytoreductive treatments, and it rapidly involutes after chemotherapy insult in pediatric cancer patients. By using automated fluorescent whole slide scanning and complementary 3D SEM array tomography, we were able to visualize the biological structure of the thymic epithelial cell (TEC) network, analyze branching of this network, and obtain a more complete three-dimensional morphological structure of the cortical thymic epithelial cell (cTEC) compartment. The establishment of this imaging method will allow researchers to move forward in comparing the integrity of the cTEC in different conditions, such as different treatment conditions, for example, chemotherapy and irradiation, as well as different ages.

ABSTRACTThe thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1 Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, Rictor KO , NDRG1 Ser336Ala mutants and Cdc42 -deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

Endogenous thymic repair is quite complex, demanding coordinated thymocyte and thymic stromal responses for T cell development and establishment of central tolerance. While the importance of thymic stroma, notably cortical (cTEC) and medullary (mTEC) thymic epithelial cells, has been shown in gain- and loss-of-function studies, endogenous thymic epithelial repair has never been resolved ultrastructurally. Using Transmission Electron Microscopy (TEM), we analyzed morphometrically thymi in mice receiving Cyclophosphamide (CTX). In vehicle-treated mice, the cortex was populous with thymocytes neighboring cTEC, which extended long cytoplasmic processes into a dendritic meshwork, called “cytoreticulum”. In CTX-treated mice though, thymocytes were scarcely met in the cortex and TEC appeared more circular/ellipsoid. The cytoreticulum was evidently collapsed, increasing total contact surface area among cTEC/mTEC subsets. Despite that thymic macrophages primarily mediate clearance of thymocytes failing positive/negative selection, those in CTX-treated thymi were rich in secondary lysosomes and many were found phagocytosing TEC. The surviving TEC in CTX-treated mice had increased autophagolysosomes per surface area unit of TEC cytoplasm compared to vehicle-treated ones. Analysis via intensity thresholding revealed higher electron density of enclosed particles, consistent with the presence of partly-digested amorphous material, indicating membranous organelle self-digestion (i.e., stress macroautophagy). These data propose an emerging hypothesis that survival of thymic epithelium following cytotoxic insult is mediated via a collective stress response involving macroautophagy activation in TEC. Funded by: 1)AAI careers in immunology Fellowship - trainee 2)new investigator's start up funds (PI)

Summary Fasting triggers diverse cellular and metabolic adaptations to facilitate organismal survival 1,2 . During nutrient deprivation, increases in circulating fatty acids support mitochondrial respiration 2 . The mechanisms driving mitochondrial adaptations and respiratory sufficiency during nutrient deprivation remain incompletely understood. Here we show that extended periods of fasting, or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its target NDRG1 3 at S336 sustains mitochondrial fission and respiratory sufficiency. Timelapse imaging reveals that wildtype NDRG1, but not phosphorylation-deficient NDRG1 S336A mutant, engages with mitochondria to facilitate its scission. Using proteomics, and an siRNA screen, we show that mTORC2-phosphorylated NDRG1 cooperates with the small GTPase Cdc42 4 and Cdc42-specific effectors and regulators to orchestrate fission. Accordingly, Rictor KO , NDRG1 S336A mutants, and Cdc42 -deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions 5 ; however, paradoxical reactivation of mTORC2 during fasting plays an unexpected role in driving mitochondrial fission and respiration.

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer’s disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-β as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes (“MAM” domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.

Perturbations in mitochondrial dynamics have been observed in most neurodegenerative diseases. Here, we focus on manganese (Mn)-induced Parkinsonism-like neurodegeneration, a disorder associated with the preferential of Mn in the basal ganglia where the mitochondria are considered an early target. Despite the extensive characterization of the clinical presentation of manganism, the mechanism by which Mn mediated mitochondrial toxicity is unclear. In this study we hypothesized whether Mn exposure alters mitochondrial activity, including axonal transport of mitochondria and mitochondrial dynamics, morphology, and network. Using primary neuron cultures exposed to 100 μM Mn (which is considered the threshold of Mn toxicity in vitro) and intraperitoneal injections of MnCl2 (25mg/kg) in rat, we observed that Mn increased mitochondrial fission mediated by phosphorylation of dynamin-related protein-1 at serine 616 (p-s616-DRP1) and decreased mitochondrial fusion proteins (MFN1 and MFN2) leading to mitochondrial fragmentation, defects in mitochondrial respiratory capacity, and mitochondrial ultrastructural damage in vivo and in vitro. Furthermore, Mn exposure impaired mitochondrial trafficking by decreasing dynactin (DCTN1) and kinesin-1 (KIF5B) motor proteins and increasing destabilization of the cytoskeleton at protein and gene levels. In addition, mitochondrial communication may also be altered by Mn exposure, increasing the length of nanotunnels to reach out distal mitochondria. These findings revealed an unrecognized role of Mn in dysregulation of mitochondrial dynamics providing a potential explanation of early hallmarks of the disorder, as well as a possible common pathway with neurological disorders arising upon chronic Mn exposure.

Abstract: There is often a need to locate the same cellular structure of interest in light and electron microscopy, which can be a difficult task. Here we present a method that uses only commercially available reagents and standard epi-fluorescence and transmission electron microscopy (TEM) technology to make correlative light and electron microscopy (CLEM) available to a large group of researchers without specialized CLEM hardware. This was achieved by seeding cells on photo-etched gridded cover slips and staining the protein to be localized with a secondary antibody coupled to both a fluorophore and 10 nm gold. The presence of the grid allowed for the alignment of light microscopy images with TEM images and the double-labeled antibody revealed co-localization of the fluorophore with gold particles.

A significant number of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND) despite highly effective antiretroviral therapy (ART). Dysregulated macroautophagy (autophagy) is implicated in HAND pathogenesis. The viral protein Nef, expressed even with suppressive ART, and certain antiretrovirals affect autophagy in non-CNS cells. Astrocytes, vital for CNS microenvironment homeostasis and neuronal health, require autophagy for their own homeostasis. We hypothesized that extracellular Nef and/or ART impact astrocyte autophagy, thus contributing to HAND. We studied in-bulk and selective autophagic flux in primary human astrocytes treated with extracellular Nef and/or a combination of tenofovir+emtricitabine+raltegravir (ART) using Western blotting, a tandem fluorescent LC3 reporter, and transmission electron microscopy/morphometry. We show that after 24 h treatment, Nef and ART decrease autophagosomes through different mechanisms. While Nef accelerates autophagosome degradation without inducing autophagosome formation, ART inhibits autophagosome formation. Combination Nef+ART further depletes autophagosomes by inducing both abnormalities. Additionally, extracellular Nef and/or ART inhibit lysosomal degradation of p62, indicating Nef and/or ART affect in-bulk and selective autophagy differently. Dysregulation of both autophagic processes is maintained after 7 days of Nef and/or ART treatment. Persistent autophagy dysregulation due to chronic Nef and/or ART exposure may ultimately result in astrocyte and neuronal dysfunction, contributing to HAND.

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed-sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5-6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

Lipin-1 ( Lpin1)–deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the β-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1 -/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid–Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1 -/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1 -/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.

Journal Article Career Advancement: Microscopy Technician to Core Facility Director Get access Frank P Macaluso Frank P Macaluso Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, NY, USADepartment of Anatomy and Structural Biology, A. Einstein College of Medicine, Bronx, NY, USAGruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 2664–2665, https://doi.org/10.1017/S1431927619014053 Published: 01 August 2019

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In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β‐secretase to generate a 99‐aa C‐terminal fragment (C99) that is then cleaved by γ‐secretase to generate the β‐amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ‐secretase activity is enriched in mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM) and that ER–mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ‐secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.