Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia.

SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta.

There is growing recognition regarding the role of intracellular amyloid beta (Aß) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aß likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aß and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aß accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aß. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aß. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aß-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aß targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aß, its promotion of ROS, and Aß metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aß oligomer formation.

MeCP2 regulates activity-dependent transcriptional responses in olfactory sensory neurons.

During postnatal development, neuronal activity controls the remodeling of initially imprecise neuronal connections through the regulation of gene expression. MeCP2 binds to methylated DNA and modulates gene expression during neuronal development and MECP2 mutation causes the autistic disorder Rett syndrome. To investigate a role for MeCP2 in neuronal circuit refinement and to identify activity-dependent MeCP2 transcription regulations, we leveraged the precise organization and accessibility of olfactory sensory axons to manipulation of neuronal activity through odorant exposure in vivo. We demonstrate that olfactory sensory axons failed to develop complete convergence when Mecp2 is deficient in olfactory sensory neurons (OSNs) in an otherwise wild-type animal. Furthermore, we demonstrate that expression of selected adhesion genes was elevated in Mecp2-deficient glomeruli, while acute odor stimulation in control mice resulted in significantly reduced MeCP2 binding to these gene loci, correlating with increased expression. Thus, MeCP2 is required for both circuitry refinement and activity-dependent transcriptional responses in OSNs.

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

PURPOSE: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. METHODS: Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. RESULTS: Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results. CONCLUSIONS: Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.

Post-death Vesicles of Senescent Bone Marrow Mesenchymal Stromal Polyploids Promote Macrophage Aging and Breast Cancer.

Potential systemic factors contributing to aging-associated breast cancer (BC) remain elusive. Here, we reveal that the polyploid giant cells (PGCs) that contain more than two sets of genomes prevailing in aging and cancerous tissues constitute 5-10% of healthy female bone marrow mesenchymal stromal cells (fBMSCs). The PGCs can repair DNA damage and stimulate neighboring cells for clonal expansion. However, dying PGCs in advanced-senescent fBMSCs can form "spikings" which are then separated into membraned mtDNA-containing vesicles (Senescent PGC-Spiking Bodies; SPSBs). SPSB-phagocytosed macrophages accelerate aging with diminished clearance on BC cells and protumor M2 polarization. SPSB-carried mitochondrial OXPHOS components are enriched in BC of elder patients and associated with poor prognosis. SPSB-incorporated breast epithelial cells develop aggressive characteristics and PGCs resembling the polyploid giant cancer cells (PGCCs) in clonogenic BC cells and cancer tissues. These findings highlight an aging BMSC-induced BC risk mediated by SPSB-induced macrophage dysfunction and epithelial cell precancerous transition. SIGNIFICANCE: Mechanisms underlying aging-associated cancer risk remain unelucidated. This work demonstrates that polyploid giant cells (PGCs) in bone marrow mesenchymal stromal cells (BMSCs) from healthy female bone marrow donors can boost neighboring cell proliferation for clonal expansion. However, the dying-senescent PGCs in the advanced-senescent fBMSCs can form "spikings" which are separated into mitochondrial DNA (mtDNA)-containing spiking bodies (senescent PGC-spiking bodies; SPSBs). The SPSBs promote macrophage aging and breast epithelial cell protumorigenic transition and form polyploid giant cancer cells. These results demonstrate a new form of ghost message from dying-senescent BMSCs, that may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk.

Rap1gap2 regulates axon outgrowth in olfactory sensory neurons.

Olfactory sensory neurons (OSNs) extend their axons from the nasal epithelium to their odorant receptor-dependent locations in the olfactory bulb. Previous studies have identified several membrane proteins along the projection pathway, and on OSN axons themselves, which regulate this process; however, little is known about the signaling mechanisms through which these factors act. We have identified and characterized Rap1gap2, a novel small GTPase regulator, in OSNs during early postnatal mouse development. Rap1gap2 overexpression limits neurite outgrowth and branching in Neuro-2a cells, and counteracts Rap1-induced augmentation of neurite outgrowth. Rap1gap2 expression is developmentally regulated within OSNs, with high expression in early postnatal stages that ultimately drops to undetectable levels by adulthood. This temporal pattern coincides with an early postnatal plastic period of OSN innervation refinement at the OB glomerular layer. Rap1gap2 stunts OSN axon outgrowth when overexpressed in vitro, while knock-down of Rap1gap2 transcript results in a significant increase in axon length. These results indicate an important role of Rap1gap2 in OSN axon growth dynamics during early postnatal development.

Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium.

The olfactory neuroepithelium (OE) is one of the few neuronal tissues where environmental pathogens can gain direct access. Despite this vulnerable arrangement, little is known about the protective mechanisms in the OE to prevent viral infection and its antiviral responses. We systematically investigated acute responses in the olfactory mucosa upon exposure to vesicular stomatitis virus (VSV) via RNA-seq. VSVs were nasally inoculated into C57BL/6 mice. Olfactory mucosae were dissected for gene expression analysis at different time points after viral inoculation. Interferon functions were determined by comparing the viral load in interferon receptor knockout (Ifnar1-/- and Ifnlr1-/-) with wildtype OE. Antiviral responses were observed as early as 24 h after viral exposure in the olfactory mucosa. The rapidly upregulated transcripts observed included specific type I as well as type III interferons (Ifn) and interferon-stimulated genes. Genetic analyses demonstrated that both type I and type III IFN signaling are required for the suppression of viral replication in the olfactory mucosa. Exogenous IFN application effectively blocks viral replication in the OE. These findings reveal that the OE possesses an innate ability to suppress viral infection. Type I and type III IFNs have prominent roles in OE antiviral functions.

Novel Stilbene-Nitroxyl Hybrid Compounds Display Discrete Modulation of Amyloid Beta Toxicity and Structure.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These "toxic oligomers" have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AßO) and the oxidative stress elicited by AßO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer's disease in a powerful, synergistic manner. Examination of AßO structure by various biophysical tools shows that each stilbene candidate uniquely alters AßO conformation and toxicity, providing insight towards the future development of structural correctors for AßO. Correlations of AßO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer's, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AßO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.

Disruption of nuclear architecture as a cause of COVID-19 induced anosmia.

Olfaction relies on a coordinated partnership between odorant flow and neuronal communication. Disruption in our ability to detect odors, or anosmia, has emerged as a hallmark symptom of infection with SARS-CoV-2, yet the mechanism behind this abrupt sensory deficit remains elusive. Here, using molecular evaluation of human olfactory epithelium (OE) from subjects succumbing to COVID-19 and a hamster model of SARS-CoV-2 infection, we discovered widespread downregulation of olfactory receptors (ORs) as well as key components of their signaling pathway. OR downregulation likely represents a non-cell autonomous effect, since SARS-CoV-2 detection in OSNs is extremely rare both in human and hamster OEs. A likely explanation for the reduction of OR transcription is the striking reorganization of nuclear architecture observed in the OSN lineage, which disrupts multi-chromosomal compartments regulating OR expression in humans and hamsters. Our experiments uncover a novel molecular mechanism by which a virus with a very selective tropism can elicit persistent transcriptional changes in cells that evade it, contributing to the severity of COVID-19.

Immunohistochemistry and In Situ Hybridization in the Developing Chicken Brain.

One of the first steps in studies of gene function is the spatiotemporal analysis of patterns of gene expression. Indirect immunohistochemistry is a method that allows the detection of a protein of interest by incubating a histological section with an antibody or antiserum raised against the protein, and then localizing this primary antibody with a tagged secondary antibody. To determine the cellular source of a protein of interest, or if a specific antibody is not available, specific transcripts can be localized using in situ hybridization. A histological section is incubated with a labeled RNA probe that is complementary to the target transcript; after hybridization with the target transcript the labeled RNA probe can be identified with an antibody. Here we describe materials and methods used to perform basic indirect immunohistochemistry and in situ hybridization on frozen sections through the developing chicken brain, emphasizing controls and potential problems that may be encountered.

A mouse model and 19 F NMR approach to investigate the effects of sialic acid supplementation on cognitive development.

Researchers have observed that a sialic acid (Sia)-supplemented neonatal diet leads to improved cognition in weanling piglets. However, whether cognitive improvement appears with different physiological backgrounds and persists into adulthood is not known. Here, we have established a convenient mouse model and used an 19 F NMR approach to address these questions, test the conditionally essential nutrient hypothesis about Sia supplementation, and assess the prospect of measuring Sia metabolism directly in vivo. Indeed, the neonatal mouse brain uptakes more Sia than the adult brain, and Sia supplementation of neonatal mice improves the cognitive performance of adult mice. The non-invasive 19 F NMR approach and viable mouse model opens unique opportunities for clarifying the interplay of nutritional supplementation, metabolism, and cognitive development.

MeCP2 regulates gene expression through recognition of H3K27me3.

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation.

Secreted TARSH regulates olfactory mitral cell dendritic complexity.

Olfactory sensory neurons synapse with mitral cells to form stereotyped connections in the olfactory bulb (OB). Mitral cell apical dendrites receive input from olfactory sensory neurons expressing the same odorant receptor. During development, this restricted dendritic targeting of mitral cells is achieved through eliminating elaborated dendritic trees to a single apical dendrite. Through a genome-wide microarray screen, we identified TARSH (Target of NESH SH3) as a transiently expressed molecule in mitral cells during the dendritic refinement period. TARSH expression is restricted to pyramidal neurons along the main olfactory pathway, including the anterior olfactory nucleus and piriform cortex. The dynamic TARSH expression is not altered when odor-evoked activity is blocked by naris closure or in AC3 knockout mice. We also demonstrate that TARSH is a secreted protein. In dissociated OB cultures, secreted TARSH promotes the reduction of mitral cell dendritic complexity and restricts dendritic branching and outgrowth of interneurons. Dendritic morphological changes were also observed in mitral cells overexpressing TARSH themselves. We propose that TARSH is part of the genetic program that regulates mitral cell dendritic refinement.

A Metal-Free Method for Producing MRI Contrast at Amyloid-ß.

Alzheimer's disease (AD) is characterized by depositions of the amyloid-ß (Aß) peptide in the brain. The disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. It is therefore imperative to develop methods that permit early detection and monitoring of disease progression. In addition, the multifactorial pathogenesis of AD has identified several potential avenues for AD intervention. Thus, evaluation of therapeutic candidates over lengthy trial periods also demands a practical, noninvasive method for measuring Aß in the brain. Magnetic resonance imaging (MRI) is the obvious choice for such measurements, but contrast enhancement for Aß has only been achieved using Gd(III)-based agents. There is great interest in gadolinium-free methods to image the brain. In this study, we provide the first demonstration that a nitroxide-based small-molecule produces MRI contrast in brain specimens with elevated levels of Aß. The molecule is comprised of a  fluorene (a molecule with high affinity for Aß) and a nitroxide spin label (a paramagnetic MRI contrast species). Labeling of brain specimens with the spin-labeled fluorene produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of spin-labeled fluorene into live mice resulted in good brain penetration, with the compound able to generate contrast 24-h post injection. These results provide a proof of concept method that can be used for early, noninvasive, gadolinium-free detection of amyloid plaques by MRI.

Sequence features accurately predict genome-wide MeCP2 binding in vivo.

Methyl-CpG binding protein 2 (MeCP2) is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2-binding data, we show that DNA sequence features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported genome-wide association with methylation is in part due to MeCP2's affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localizes with nucleosomes. Finally, MeCP2 binding downstream of promoters correlates with increased expression in Mecp2-deficient neurons.

Immunohistochemistry and in situ hybridization in the developing chicken brain.

One of the first steps in studies of gene function is the spatiotemporal analysis of patterns of gene expression. Indirect immunohistochemistry is a method that allows the detection of a protein of interest by incubating a histological section with an antibody or antiserum raised against the protein and then localizing this primary antibody with a tagged secondary antibody. To determine the cellular source of a protein of interest, or if a specific antibody is not available, specific transcripts can be localized using in situ hybridization. A histological section is incubated with a labeled RNA probe that is complementary to the target transcript; after hybridization with the target transcript, the labeled RNA probe can be identified with an antibody. Here we describe materials and methods used to perform basic indirect immunohistochemistry and in situ hybridization on frozen sections through the developing chicken brain, emphasizing controls and potential problems that may be encountered.

Integrins of the starlet sea anemone Nematostella vectensis.

Integrins are extracellular matrix receptors composed of α and ß subunits. Here we describe two α subunits and four ß subunits from the starlet sea anemone Nematostella vectensis. Phylogenetic analysis suggests that the α subunits are most closely related to RGD- and LDV-dependent α subunits of chordates. The ß subunits cluster with the previously described ß integrins of the hard coral Acropora millepora. The expression of one of the α subunits and three of the ß subunits was confirmed by reverse transcription PCR and in situ hybridization. The α subunit is primarily expressed in cells near muscles, by a subset of gastrodermal cells, and in the gonad. The three ß subunits each have distinctive patterns of expression: one is concentrated in the gonad and mesenteric filament, another is found in a subset of cells in the epidermis of the oral region and in a subset of gastrodermal cells in the mesenteries, and a third is expressed widely. Changes in expression were also studied 48 h after horizontal transection by quantitative reverse transcription PCR and in situ hybridization. One of the ß subunits is expressed 8-fold higher during regeneration, and its expression is observed in cells within both the epidermis and the gastrodermis at the site of regeneration. Our observations confirm that complex patterns of integrin expression were already present in basal metazoans. The integrins expressed in the gonads may play roles in mediating sperm-egg interactions in N. vectensis, while others may play a role in regulating proliferation during regeneration.

A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration.

Thrombospondins are multimeric extracellular matrix glycoproteins that play important roles in development, synaptogenesis and wound healing in mammals. We previously identified four putative thrombospondins in the genome of the starlet sea anemone Nematostella vectensis. This study presents the first analysis of these thrombospondins, with the goals of understanding fundamental roles of thrombospondins in the Eumetazoa. Reverse transcriptase PCR showed that each of the N. vectensis thrombospondins (Nv85341, Nv22035, Nv168100 and Nv30790) is transcribed. Three of the four thrombospondins include an RGD or KGD motif in their thrombospondin type 3 repeats at sites equivalent to mammalian thrombospondins, suggesting ancient roles as RGD integrin ligands. Phylogenetic analysis based on the C-terminal regions demonstrated a high level of sequence diversity between N. vectensis thrombospondins. A full-length cDNA sequence was obtained for Nv168100 (NvTSP168100), which has an unusual domain organization. Immunohistochemistry with an antibody to NvTSP168100 revealed labeling of neuron-like cells in the mesoglea of the retractor muscles and the pharynx. In situ hybridization and quantitative PCR showed that NvTSP168100 is upregulated during regeneration. Immunohistochemistry of the area of regeneration identified strong immunostaining of the glycocalyx, the carbohydrate-rich matrix coating the epidermis, and electron microscopy identified changes in glycocalyx organization during regeneration. Thus, N. vectensis thrombospondins share structural features with thrombospondins from mammals and may have roles in the nervous system and in matrix reorganization during regeneration.

Culture of mouse olfactory sensory neurons.

Olfactory sensory neurons, located in the nasal epithelium, detect and transmit odorant information to the central nervous system. This requires that these neurons form specific neuronal connections within the olfactory bulb and express receptors and signaling molecules specific for these functions. This protocol describes a primary olfactory sensory neuron culture technique that allows in vitro investigation of olfactory sensory neuron differentiation, axon outgrowth, odorant receptor expression, and function. Olfactory epithelium is obtained from the nasal cavity and is enzymatically treated to reduce stroma tissue. Dissociated olfactory sensory neurons are cultured directly on a layer of cortical astrocytes to support their survival. Using this method, cultured olfactory sensory neurons maintain their bipolar morphology and express odorant signal transduction molecules, which are specific for olfactory sensory neurons.

Lentivirus-mediated genetic manipulation and visualization of olfactory sensory neurons in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function during olfactory development.