Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in health and disease. Most LD studies in vivo rely on staining methods, providing only a snapshot. We therefore developed a LD-reporter mouse by labelling the endogenous LD coat protein perilipin 2 (PLIN2) with tdTomato, enabling staining-free fluorescent LD visualisation in living and fixed tissues and cells. Here we validate this model under standard and high-fat diet conditions and demonstrate that LDs are highly abundant in various cell types in the healthy brain, including neurons, astrocytes, ependymal cells, neural stem/progenitor cells and microglia. Furthermore, we also show that LDs are abundant during brain development and can be visualized using live imaging of embryonic slices. Taken together, our tdTom-Plin2 mouse serves as a novel tool to study LDs and their dynamics under both physiological and diseased conditions in all tissues expressing Plin2.

The respiratory system is the main target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 19 (COVID-19) where acute respiratory distress syndrome is considered the leading cause of death. Changes in pulmonary blood vessels, among which an endothelialitis/endotheliitis has been particularly emphasized, have been suggested to play a central role in the development of acute lung injury. Similar vascular changes are also observed in animal models of COVID-19. The present study aimed to determine whether the latter are specific for SARS-CoV-2 infection, investigating the vascular response in the lungs of mice infected with SARS-CoV-2 and other respiratory viruses (influenza A and murine gammaherpesvirus) by in situ approaches (histology, immunohistology, morphometry) combined with RNA sequencing and bioinformatic analysis. Non-selective recruitment of monocytes and T and B cells from larger muscular veins and arteries was observed with all viruses, matched by a comparable transcriptional response. There was no evidence of endothelial cell infection in any of the models. Both the morphological investigation and the transcriptomics approach support the interpretation that the lung vasculature in mice mounts a stereotypic response to alveolar and respiratory epithelial damage. This may have implications for the treatment and management of respiratory disease in humans.

BACKGROUND: Smooth muscle cells (SMCs) regulate blood flow distribution via vasoconstriction mediated by α-ARs (α-adrenergic receptors). Plasma membrane cholesterol accumulation affects α ₁ -AR signaling and promotes loss of SMC contractile markers in vitro. ABCA1 and ABCG1 (ATP-binding cassette transporter A1 and G1) mediate cholesterol efflux to HDL (high-density lipoprotein). ABCA1/ABCG1 show high expression in medial and low expression in intimal SMCs of atherosclerotic plaques. The role of ABCA1 and ABCG1 in SMC-mediated vasoconstriction and atherogenesis remains poorly understood. METHODS: We generated mice with SMC-specific Abca1/Abcg1 deficiency on the low-density lipoprotein receptor–deficient ( Ldlr ^{− / −} ) background by crossbreeding Abca1 ^fl/fl Abcg1 ^fl/fl Ldlr ^−/− mice with Myh11Cre ^ERT2 transgenic mice. To induce SMC cholesterol accumulation and atherogenesis, we fed Myh11Cre ^ERT2 Abca1 ^fl/fl Abcg1 ^fl/fl Ldlr ^−/− , Myh11Cre ^ERT2 Abca1 ^fl/fl Ldlr ^−/− , Myh11Cre ^ERT2 Abcg1 ^fl/fl Ldlr ^−/− , and Myh11Cre ^ERT2 Ldlr ^−/− mice Western-type diet for 16 weeks. RESULTS: Combined SMC-Abca1/Abcg1 deficiency increased vasoconstriction in aortic rings induced by the α ₁ -AR agonist phenylephrine. Unexpectedly, SMC-Abca1/Abcg1 deficiency induced urinary bladder distension by >20-fold. This was reversed by the α ₁ -AR antagonist tamsulosin, indicating its dependence on bladder neck SMC constriction. Moreover, SMC-Abca1/Abcg1 deficiency decreased contractile markers and increased macrophage and fibroblast markers in bladder SMCs, indicating SMC transdifferentiation. This was accompanied by free cholesterol accumulation and increased endoplasmic reticulum stress. SMC-Abca1/Abcg1 deficiency did not induce thoracic aorta SMC transdifferentiation, presumably due to increased cholesteryl ester accumulation and no endoplasmic reticulum stress in thoracic aorta SMCs. Surprisingly, SMC-Abca1/Abcg1 deficiency did not affect atherosclerotic lesion size or composition in the aortic root or brachiocephalic artery. CONCLUSIONS: We uncover a new role of SMC cholesterol efflux pathways in suppressing α ₁ -AR–mediated vasoconstriction and bladder SMC transdifferentiation, decreasing urinary bladder distension. Our data may provide a mechanistic link for the association between urinary bladder distension and diabetes in humans, particularly because diabetes is associated with decreased cholesterol efflux. SMC-Abca1/Abcg1 deficiency did not affect atherosclerotic lesion size or plaque composition, presumably due to low expression of Abca1/Abcg1 in intimal SMCs.

Reptarenaviruses cause Boid inclusion body disease (BIBD), a lethal disease primarily affecting captive boa constrictors. The presence of cytoplasmic inclusion bodies (IBs), mainly composed of viral nucleoprotein (NP), in various cell types is characteristic to and a diagnostic criterion of BIBD. We have previously reported that reptarenavirus replication and IB formation are efficient in cell cultures that are maintained at 27–30 °C but not in cells that are kept at 37 °C, the temperature commonly used for mammalian cell cultures. Here, considering the poikilothermic nature of snakes, we studied the ideal temperature(s) for reptarenavirus propagation and the expression of reptarenavirus NP. We incubated Boa constrictor kidney-derived I/1Ki cells at different temperatures (24–36 °C), inoculated them with University of Giessen virus 1 (UGV-1) and monitored both cell growth and virus proliferation. Cell growth was optimal at 30–34 °C and was not significantly affected by UGV-1 infection. Viral RNA release per cell was highest at ambient temperatures between 28 and 32 °C, as determined by qRT-PCR. However, the cells passaged at day 15 post-inoculation released viral RNA at comparable levels even when kept at slightly lower temperatures (24–26 °C). Morphometric analyses undertaken on sections of cell pellets immunostained for reptarenavirus NP found the expression to be most intense at 32 and 34 °C in freshly inoculated cells, and at 28–32 °C in passaged cells. The NP expression positively correlated with the amount of viral RNA released per cell. Our results indicate that the optimal temperature ranges for boid cell growth and reptarenavirus replication (as judged based on antigen expression and RNA release) overlap at about 32 °C. They also suggest that environmental temperature modulation could represent a strategy to impair reptarenavirus replication and, potentially, the spread of reptarenaviruses within and between snake collections.

Hypertrophic cardiomyopathy (HCM) is the most prevalent acquired heart disease in cats and shares many clinical, phenotypical and pathological features with human HCM. Despite its relevance, knowledge on the pathomechanisms underlying the disease is limited. The present study aimed to characterize the molecular phenotypic changes in cardiomyocytes in feline HCM (fHCM) to better understand their contribution to the pathogenesis. To achieve this, the myocardium of the left ventricular free wall of 15 cats with confirmed fHCM and 30 control cats (two age groups: 16 cats 18-month-old, and 14 older adult cats without cardiac disease) were subjected to RT-qPCRs for markers representative of cardiomyocyte function. Overall, all markers were expressed at the highest level in young control cats, and increasing age correlated with decreased expression, regardless of sex. The comparison between the older adult control cats and those with HCM showed increased transcription levels for most markers associated with the disease, and higher expression of all markers in affected male cats compared to females. The constitutive transcription of all markers provides evidence of continuous myocardial adaptation throughout cats’ life. The high transcription values in the myocardium of young healthy cats and male cats affected by HCM suggest a particularly high myocardial responsiveness early in life and with HCM and reveal sex as relevant factor in the disease process. These results support the relevance of age and sex in the cardiac response to HCM in feline hearts.

Ronapreve, a combination of two neutralising monoclonal antibodies, casirivimab and imdevimab, was amongst the authorised treatments against SARS-CoV-2 early in the COVID-19 pandemic. Ronapreve has lost some of its efficiency with the rise of new virus variants, however, it remains a valuable tool for experimental studies to gain insights into the mechanisms and effects of anti-viral drugs. In this study we combined morphological, pharmacokinetic and molecular approaches (including multiomics) to investigate the biodistribution of Ronapreve in the K18-hACE2 murine model of SARS-CoV-2 neuroinvasion, as well as possible consequences for the brain. We also investigated the effect of the treatment on the infection status. Our results showed that after intraperitoneal injection, Ronapreve accumulates in the serum and is unable to cross the blood-brain barrier, thus not reaching the brain parenchyma; treatment has only a minimal effect on the brain transcriptome, with no significant changes in the brain lipidome or metabolome. Nonetheless, post-exposure Ronapreve treatment resulted in reduced viral loads in the lung and, in particular, the brain, with markedly reduced tissue response in the brain, as shown by the transcriptomic analysis. The results suggest a peripheral mode of action of Ronapreve to block brain infection, possibly by lowering viral replication in the nasal epithelium, reducing a subsequent spread to the brain.