A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

The simultaneous occurrence of gestational diabetes and hypertensive disorders of pregnancy affects fetal growth and neonatal morbidity.

BACKGROUND: Gestational diabetes is associated with increased risk of hypertensive disorders of pregnancy, but there are limited data on fetal growth and neonatal outcomes when both conditions are present. OBJECTIVE: We evaluated the risk of abnormal fetal growth and neonatal morbidity in pregnancies with co-occurrence of gestational diabetes and hypertensive disorders of pregnancy. STUDY DESIGN: In a retrospective study of 47,093 singleton pregnancies, we compared the incidence of appropriate for gestational age birthweight in pregnancies affected by gestational diabetes alone, hypertensive disorders of pregnancy alone, or both gestational diabetes and hypertensive disorders of pregnancy with that in pregnancies affected by neither disorder using generalized estimating equations (covariates: maternal age, nulliparity, body mass index, insurance type, race, marital status, and prenatal care site). Secondary outcomes were large for gestational age birthweight, small for gestational age birthweight, and a neonatal morbidity composite outcome (stillbirth, hypoglycemia, hyperbilirubinemia, respiratory distress, encephalopathy, preterm delivery, neonatal death, and neonatal intensive care unit admission). RESULTS: The median (interquartile range) birthweight percentile in pregnancies with both gestational diabetes and hypertensive disorders of pregnancy (50 [24.0-78.0]; N=179) was similar to that of unaffected pregnancies (50 [27.0-73.0]; N=35,833). However, the absolute rate of appropriate for gestational age birthweight was lower for gestational diabetes/hypertensive disorders of pregnancy co-occurrence (78.2% vs 84.9% for unaffected pregnancies). Adjusted analyses showed decreased odds of appropriate for gestational age birthweight in pregnancies with both gestational diabetes and hypertensive disorders of pregnancy compared with unaffected pregnancies (adjusted odds ratio, 0.72 [95% confidence interval, 0.52-1.00]; P=.049), and in pregnancies complicated by gestational diabetes alone (adjusted odds ratio, 0.78 [0.68-0.89]; P<.001) or hypertensive disorders of pregnancy alone (adjusted odds ratio, 0.73 [0.66-0.81]; P<.001). The absolute risk of large for gestational age birthweight was greater in pregnancies with both gestational diabetes and hypertensive disorders of pregnancy (14.5%) than in unaffected pregnancies (8.2%), without apparent difference in the risk of small for gestational age birthweight (7.3% vs 6.9%). However, in adjusted models comparing pregnancies with gestational diabetes/hypertensive disorders of pregnancy co-occurrence with unaffected pregnancies, neither an association with large for gestational age birthweight (adjusted odds ratio, 1.33 [0.88-2.00]; P=.171) nor small for gestational age birthweight (adjusted odds ratio, 1.32 [0.80-2.19]; P=.293) reached statistical significance. Gestational diabetes/hypertensive disorders of pregnancy co-occurrence carried an increased risk of neonatal morbidity that was greater than that observed with either condition alone (gestational diabetes/hypertensive disorders of pregnancy: adjusted odds ratio, 3.13 [2.35-4.17]; P<.001; gestational diabetes alone: adjusted odds ratio, 2.01 [1.78-2.27]; P<.001; hypertensive disorders of pregnancy alone: adjusted odds ratio, 1.38 [1.26-1.50]; P<.001). CONCLUSION: Although pregnancies with both gestational diabetes and hypertensive disorders of pregnancy have a similar median birthweight percentile to those affected by neither condition, pregnancies concurrently affected by both conditions have a higher risk of abnormal fetal growth and neonatal morbidity.

Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing.

Adipocyte size and fragility and commercial kit costs impose significant limitations on single-cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.

A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed many neuronal subtypes' unique marker genes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard (http://harvard.heavy.ai:6273/) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

A single-cell atlas of human and mouse white adipose tissue.

White adipose tissue, once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic and heterogenous, and is involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control and host defence1. High-fat feeding and other metabolic stressors cause marked changes in adipose morphology, physiology and cellular composition1, and alterations in adiposity are associated with insulin resistance, dyslipidemia and type 2 diabetes2. Here we provide detailed cellular atlases of human and mouse subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells, vascular and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease and provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits and cell types in the function of white adipose tissue across species, depots and nutritional conditions.

PPARγ-induced upregulation of subcutaneous fat adiponectin secretion, glyceroneogenesis and BCAA oxidation requires mTORC1 activity.

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.

Astrocytes Do Not Forfeit Their Neuroprotective Roles After Surviving Intense Oxidative Stress.

In order to fulfill their evolutionary role as support cells, astrocytes have to tolerate intense oxidative stress under conditions of brain injury and disease. It is well known that astrocytes exposed to mild oxidative stress are preconditioned against subsequent stress exposure in dual hit models. However, it is unclear whether severe oxidative stress leads to stress tolerance, stress exacerbation, or no change in stress resistance in astrocytes. Furthermore, it is not known whether reactive astrocytes surviving intense oxidative stress can still support nearby neurons. The data in this Brief Report suggest that primary cortical astrocytes surviving high concentrations of the oxidative toxicant paraquat are completely resistant against subsequent oxidative challenges of the same intensity. Inhibitors of multiple endogenous defenses (e.g., glutathione, heme oxygenase 1, ERK1/2, Akt) failed to abolish or even reduce their stress resistance. Stress-reactive cortical astrocytes surviving intense oxidative stress still managed to protect primary cortical neurons against subsequent oxidative injuries in neuron/astrocyte co-cultures, even at concentrations of paraquat that otherwise led to more than 80% neuron loss. Although our previous work demonstrated a lack of stress tolerance in primary neurons exposed to dual paraquat hits, here we show that intensely stressed primary neurons can resist a second hit of hydrogen peroxide. These collective findings suggest that stress-reactive astroglia are not necessarily neurotoxic, and that severe oxidative stress does not invariably lead to stress exacerbation in either glia or neurons. Therefore, interference with the natural functions of stress-reactive astrocytes might have the unintended consequence of accelerating neurodegeneration.

Evidence for cross-hemispheric preconditioning in experimental Parkinson's disease.

Dopamine loss and motor deficits in Parkinson's disease typically commence unilaterally and remain asymmetric for many years, raising the possibility that endogenous defenses slow the cross-hemispheric transmission of pathology. It is well-established that the biological response to subtoxic stress prepares cells to survive subsequent toxic challenges, a phenomenon known as preconditioning, tolerance, or stress adaptation. Here we demonstrate that unilateral striatal infusions of the oxidative toxicant 6-hydroxydopamine (6-OHDA) precondition the contralateral nigrostriatal pathway against the toxicity of a second 6-OHDA infusion in the opposite hemisphere. 6-OHDA-induced loss of dopaminergic terminals in the contralateral striatum was ablated by cross-hemispheric preconditioning, as shown by two independent markers of the dopaminergic phenotype, each measured by two blinded observers. Similarly, loss of dopaminergic somata in the contralateral substantia nigra was also abolished, according to two blinded measurements. Motor asymmetries in floor landings, forelimb contacts with a wall, and spontaneous turning behavior were consistent with these histological observations. Unilateral 6-OHDA infusions increased phosphorylation of the kinase ERK2 and expression of the antioxidant enzyme CuZn superoxide dismutase in both striata, consistent with our previous mechanistic work showing that these two proteins mediate preconditioning in dopaminergic cells. These findings support the existence of cross-hemispheric preconditioning in Parkinson's disease and suggest that dopaminergic neurons mount impressive natural defenses, despite their reputation as being vulnerable to oxidative injury. If these results generalize to humans, Parkinson's pathology may progress slowly and asymmetrically because exposure to a disease-precipitating insult induces bilateral upregulation of endogenous defenses and elicits cross-hemispheric preconditioning.

Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe.

BACKGROUND: α-synucleinopathy emerges quite early in olfactory structures such as the olfactory bulb and anterior olfactory nucleus (OB/AON) in Parkinson's disease. This may contribute to smell impairments years before the commencement of motor symptoms. We tested whether α-synucleinopathy can spread from the OB/AON to regions of the limbic telencephalon that harbor connections with olfactory structures. FINDINGS: α-synuclein fibrils were infused into the OB/AON. Inclusions containing pathologically phosphorylated α-synuclein (pSer129) were observed three months later in the piriform and entorhinal cortices, amygdala, and hippocampal formation. The retrograde tract-tracer FluoroGold confirmed the existence of first-order afferents at these sites. Some sites harbored FluoroGold(+) neurons but no inclusions, suggestive of selective vulnerabilities. Multiple areas close to the injection site but not connected with the OB/AON remained free of inclusions, suggesting a lack of widespread uptake of fibrils from interstitial diffusion. Two independent pSer129 antibodies revealed the same labeling patterns and preadsorption control experiments confirmed a loss of pSer129 staining. Dense total α-synuclein (but not pSer129) staining was apparent in the OB/AON 1.5 h following fibril infusions, suggesting that pSer129(+) staining did not reflect exogenously infused material. Waterbath sonication of fibrils for 1 h improved α-synucleinopathy transmission relative to 1 min-long probe sonication. Electron microscopy revealed that longer sonication durations reduced fibril size. The Thioflavin stain labeled cells at the infusion site and some, but not all inclusions contained ubiquitin. Three-dimensional confocal analyses revealed that many inclusions ensconced NeuN(+) neuronal nuclei. Young and aged mice exhibited similar topographical spread of α-synucleinopathy. CONCLUSIONS: 1) α-synucleinopathy in this model is transmitted through some, but not all neuroanatomical connections, 2) pathology is largely confined to first-order afferent sites at three months and this is most parsimoniously explained by retrograde transport, and 3) transmission in aged animals is largely similar to that in young control animals at three months post-infusion.

Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury.

Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury.