Probes for INS

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope ) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-β (Aβ). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aβ. Furthermore, in mhCOs, we observed reduced expression of Aβ-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer's disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aβ using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.

Involvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum ACTH levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated.To evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes.SARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time RT-PCR, in situ hybridization, immunohistochemistry and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay.The SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2 positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus-positivity.Our study supports the tropism of SARS-CoV-2 for human pituitary and encourage to explore pituitary dysfunction post-COVID-19.

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive PACAP knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in wild-type mice that do not occur in knock-out mice (aPRGs). Comparing constitutive PACAP knock-out mice to a variety of temporally and regionally specific PACAP knock-outs, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knock-out mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Infant avoidance and aggression are promoted by activation of the Urocortin-3 expressing neurons of the perifornical area of hypothalamus (PeFAUcn3) in male and female mice. PeFAUcn3 neurons have been implicated in stress, and stress is known to reduce maternal behavior. We asked how chronic restraint stress (CRS) affects infant-directed behavior in virgin and lactating females and what role PeFAUcn3 neurons play in this process. Here we show that infant-directed behavior increases activity in the PeFAUcn3 neurons in virgin and lactating females. Chemogenetic inhibition of PeFAUcn3 neurons facilitates pup retrieval in virgin females. CRS reduces pup retrieval in virgin females and increases activity of PeFAUcn3 neurons, while CRS does not affect maternal behavior in lactating females. Inhibition of PeFAUcn3 neurons blocks stress-induced deficits in pup-directed behavior in virgin females. Together, these data illustrate the critical role for PeFAUcn3 neuronal activity in mediating the impact of chronic stress on female infant-directed behavior.

SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.

The development of mouse models for COVID-19 has enabled testing of vaccines and therapeutics and defining aspects of SARS-CoV-2 pathogenesis. SARS-CoV-2 disease is severe in K18 transgenic mice (K18-hACE2-Tg) expressing human ACE2 (hACE2), the SARS-CoV-2 receptor, under an ectopic cytokeratin promoter, with high levels of infection measured in the lung and brain. Here, we evaluated SARS-CoV-2 infection in hACE2 KI mice that express hACE2 under an endogenous promoter in place of murine ACE2 (mACE2). Intranasal inoculation of hACE2 KI mice with SARS-CoV-2 WA1/2020 resulted in substantial viral replication within the upper and lower respiratory tracts with limited spread to extra-pulmonary organs. However, SARS-CoV-2-infected hACE2 KI mice did not lose weight and developed limited pathology. Moreover, no significant differences in viral burden were observed in hACE2 KI mice infected with B.1.1.7 or B.1.351 variants compared to WA1/2020 strain. Because the entry mechanisms of SARS-CoV-2 in mice remains uncertain, we evaluated the impact of the naturally-occurring, mouse-adapting N501Y mutation by comparing infection of hACE2 KI, K18-hACE2-Tg, ACE2-deficient, and wild-type C57BL/6 mice. The N501Y mutation minimally affected SARS-CoV-2 infection in hACE2 KI mice but was required for viral replication in wild-type C57BL/6 mice in a mACE2-dependent manner and augmented pathogenesis in the K18-hACE2 Tg mice. Thus, the N501Y mutation likely enhances interactions with mACE2 or hACE2 in vivo. Overall, our study highlights the hACE2 KI mice as a model of mild SARS-CoV-2 infection and disease and clarifies the requirement of the N501Y mutation in mice. IMPORTANCE Mouse models of SARS-CoV-2 pathogenesis have facilitated the rapid evaluation of countermeasures. While the first generation of models developed pneumonia and severe disease after SARS-CoV-2 infection, they relied on ectopic expression of supraphysiological levels of human ACE2 (hACE2). This has raised issues with their relevance to humans as the hACE2 receptor shows a more restricted expression pattern in the respiratory tract. Here we evaluated SARS-CoV-2 infection and disease with viruses containing or lacking a key mouse-adapting mutation in the spike gene in hACE2 KI mice, which express hACE2 under an endogenous promoter in place of murine ACE2. While infection of hACE2 KI mice with multiple strains of SARS-CoV-2 including variants of concern resulted in viral replication within the upper and lower respiratory tracts, the animals did not sustain severe lung injury. Thus, hACE2 KI mice serve as a model of mild infection with both ancestral and emerging SARS-CoV-2 variant strains.

Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.

Social interactions play an important role in our daily lives and can profoundly impact our health for better and worse. To better understand the neural circuitry underlying social behavior, we focused on neural circuits involving vasopressin neurons of the bed nucleus of the stria terminalis (BNST) and serotonin neurons of the dorsal raphe (DR). Previous research shows that BNST vasopressin neurons are activated in male mice by interaction with a female and that vasopressin indirectly excites serotonin neurons. In our studies, we tested the hypothesis that specific social interactions would also activate neurons in the DR, specifically vasopressin 1A receptor (Avpr1a)-expressing neurons, which may be direct targets of the BNST vasopressin neurons. Using in separate experiments immunohistochemistry and in situ hybridization, we found that male and female subjects exposed to a female conspecific show activation in the DR, and the activated neurons include populations of Avpr1a-expressing and other non-serotonergic, non-Avpr1a neurons in roughly equal numbers. Avpr1a neurons in the DR constitute a largely undocumented neuron population. Electrophysiological data suggest that most DR Avpr1a neurons behave like fast spiking interneurons found in other brain regions. Examination of RNAseq and in situ hybridization data suggests that there are glutamatergic, GABAergic, and serotonergic subtypes of Avpr1a neurons in the DR. Together our data support a model in which a subset of vasopressin-responsive interneurons in the DR may relay stimulus specific social signals from the forebrain BNST to the serotonergic DR system, which could help direct prosocial stimulus specific behavioral responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease in mink similar to human COVID-19. We characterized the pathological findings in 72 mink from US farms with SARS-CoV-2 outbreaks, localized SARS-CoV-2 and its host cellular receptor angiotensin-converting enzyme 2 (ACE2) in mink respiratory tissues, and evaluated the utility of various test methods and specimens for SARS-CoV-2 detection in necropsy tissues. Of SARS-CoV-2-positive animals found dead, 74% had bronchiolitis and diffuse alveolar damage (DAD). Of euthanized SARS-CoV-2-positive animals, 72% had only mild interstitial pneumonia or minimal nonspecific lung changes (congestion, edema, macrophages); similar findings were seen in SARS-CoV-2-negative animals. Suppurative rhinitis, lymphocytic perivascular inflammation in the lungs, and lymphocytic infiltrates in other tissues were common in both SARS-CoV-2-positive and SARS-CoV-2-negative animals. In formalin-fixed paraffin-embedded (FFPE) upper respiratory tract (URT) specimens, conventional reverse transcription-polymerase chain reaction (cRT-PCR) was more sensitive than in situ hybridization (ISH) or immunohistochemistry (IHC) for detection of SARS-CoV-2. FFPE lung specimens yielded less detection of virus than FFPE URT specimens by all test methods. By IHC and ISH, virus localized extensively to epithelial cells in the nasal turbinates, and prominently within intact epithelium; olfactory mucosa was mostly spared. The SARS-CoV-2 receptor ACE2 was extensively detected by IHC within turbinate epithelium, with decreased detection in lower respiratory tract epithelium and alveolar macrophages. This study expands on the knowledge of the pathology and pathogenesis of natural SARS-CoV-2 infection in mink and supports their further investigation as a potential animal model of SARS-CoV-2 infection in humans.

Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of general anaesthesia. In order to identify potential phosphorylation events in the brain mediating general anaesthesia effects, we have explored the phosphoproteome responses in the rat SON, and compared these to cingulate cortex (CC) which displays no FOS activation is response to general anaesthetics.Adult Sprague-Dawley rats were treated with isoflurane for 15 minutes. Proteins from the CC and SON were extracted and processed for Nano-LC Mass Spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS.We found many changes in the phosphoproteomes of both the CC and SON in response to 15 minutes of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region-specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to general anaesthesia between the CC and SON.In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating general anaesthesia.S. Karger AG, Basel.