Introduction Information processing in cortical cell microcircuits involves regulation of excitatory pyramidal (PYR) cells by inhibitory Somatostatin- (SST), Parvalbumin- (PV), and Vasoactive intestinal peptide- (VIP) expressing interneurons. Human post-mortem and rodent studies show impaired PYR-cell dendritic morphology and decreased SST-cell markers in MDD or after chronic stress. However, knowledge of coordinated changes across microcircuit cell-types is virtually absent. Methods We investigated the transcriptomic effects of unpredictable chronic mild stress (UCMS) on distinct microcircuit cell-types in the medial prefrontal cortex (Cingulate regions 24a/b and 32) in mice. C57Bl/6 mice, exposed to UCMS or control housing for five weeks, were assessed for anxiety- and depressive-like behaviors. Microcircuit cell-types were laser-microdissected and processed for RNA-sequencing. Results UCMS induced predicted elevations in behavioral emotionality in mice. DESeq2 analysis revealed unique differentially-expressed genes in each cell-type after UCMS. Pre-synaptic functions, oxidative stress response, metabolism, and translational regulation were differentially dysregulated across cell-types, whereas nearly all cell-types showed downregulated post-synaptic gene signatures. Across the cortical microcircuit, we observed a shift from a distributed transcriptomic coordination across cell-types in controls towards UCMS-induced increased coordination between PYR-, SST- and PV-cells, and hub-like role for PYR-cells. Lastly, we identified a microcircuit-wide coexpression network enriched in synaptic, bioenergetic, and oxidative stress response genes that correlated with UCMS-induced behaviors. Conclusions These findings suggest cell-specific deficits, microcircuit-wide synaptic reorganization, and a shift in cells regulating the cortical excitation-inhibition balance, suggesting increased coordinated regulation of PYR-cells by SST- and PV-cells.

Precise control of lineage segregation is critical for the development of multicellular organisms, but our quantitative understanding of how variable signaling inputs are integrated to activate lineage-specific gene programs remains limited. Here, we show how precisely two out of eight ectoderm cells adopt neural fates in response to ephrin and FGF signals during ascidian neural induction. In each ectoderm cell, FGF signals activate ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a bimodal transcriptional response of the immediate early gene, Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS family transcriptional repressor, ERF2. Ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and "switch on" Otx transcription.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

1q21.3 amplification, which is frequently observed in metastatic melanoma, is associated with cancer progression. Interleukin enhancer-binding factor 2 (ILF2) is located in the 1q21.3 amplified region, but its functional role or contribution to tumour aggressiveness in cutaneous melanoma is unknown.In silico analyses were performed using the TCGA SKCM dataset with clinical annotations and three melanoma microarray cohorts from the GEO datasets. RNA in situ hybridisation and immunohistochemistry were utilised to validate the gene expression in melanoma tissues. Four stable melanoma cell lines were established for in vitro ILF2 functional characterisation.Our results showed that the ILF2 copy number variation (CNV) is positively correlated with ILF2 mRNA expression (r = 0.68, p < .0001). Additionally, ILF2 expression is significantly increased with melanoma progression (p < .0001), and significantly associated with poor overall survival for metastatic melanoma patients (p = .026). The overexpression of ILF2 (ILF2-OV) promotes proliferation in metastatic melanoma cells, whereas ILF2 knockdown decreases proliferation by blocking the cell cycle. Mechanistically, we demonstrated the interaction between ILF2 and the splicing factor U2AF2, whose knockdown reverses the proliferation effects mediated by ILF2-OV. Stage IIIB-C melanoma patients with high ILF2-U2AF2 expression showed significantly shorter overall survival (p = .024). Enhanced ILF2/U2AF2 expression promotes a more efficient DNA-damage repair by increasing RAD50 and ATM mRNA expression. Paradoxically, metastatic melanoma cells with ILF2-OV were more sensitive to ATM inhibitors.Our study uncovered that ILF2 amplification of the 1q21.3 chromosome is associated with melanoma progression and triggers a functional downstream pathway in metastatic melanoma promoting drug resistance.

In Parkinson's disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics.

Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.

Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). MACE-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset "Heart Cell Atlas" and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis.MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The GO term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response.This study reveals, that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte-destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.Cardiac injury can be documented in COVID-19, regardless the direct cardiac virus infection and is known to be associated with outcome. However, the direct virus infection of the myocardium leads to transcriptomic alterations and might therefore additionally contribute to pathophysiological processes in COVID-19. Therefore, consequences of cardiac virus infection need to be investigated in future studies, since they might also contribute to long-term effects in case of survival.

Background: Kidneys with chronic inflammation develop tertiary lymphoid structures (TLSs). Infectious pyelonephritis is characterized by renal pelvis (RP) inflammation. However, the pathological features of TLSs, including their formation and association with non-infectious nephritis, are unclear. Methods: RPs from humans and mice that were healthy or had non-infectious chronic nephritis, were analyzed for TLS development, and the mechanism of TLS formation investigated using urothelium or lymphoid structure cultures. Results: Regardless of infection, TLSs in the RP, termed urinary tract-associated lymphoid structures (UTALSs), formed in humans and mice with chronic nephritis. Moreover, urine played a unique role in UTALS formation. Specifically, we identified urinary IFN-γ as a candidate factor affecting urothelial barrier integrity because it alters occludin expression. In a nephritis mouse model, urine leaked from the lumen of the RP into the parenchyma. In addition, urine immunologically stimulated UTALS-forming cells via cytokine (IFN-γ, TNF-α) and chemokine (CXCL9, CXCL13) production. CXCL9 and CXCL13 were expressed in UTALS stromal cells and urine stimulation specifically induced CXCL13 in cultured fibroblasts. Characteristically, type XVII collagen (BP180), a candidate autoantigen of bullous pemphigoid, was ectopically localized in the urothelium covering UTALSs and associated with UTALS development by stimulating CXCL9 or IL-22 induction via the TNF-α/FOS/JUN pathway. Notably, UTALS development indices were positively correlated with chronic nephritis development. Conclusion: TLS formation in the RP is possible and altered urine-urothelium barrier-basedUTALS formation may represent a novel mechanism underlying the pathogenesis of chronic nephritis, regardless of urinary tract infection.

The long-term outcome of COVID-19-associated collapsing glomerulopathy is unknown.We retrospectively identified 76 native kidney biopsies from patients with history of COVID-19 between March 2020 and April 2021. Presenting and outcome data were obtained for all 23 patients with collapsing glomerulopathy and for seven patients with noncollapsing podocytopathies. We performed APOL1 genotyping by Sanger sequencing, immunostaining for spike and nucleocapsid proteins, and in situ hybridization for SARS-CoV-2.The 23 patients with COVID-19-associated collapsing glomerulopathy were median age 57 years (range, 35-72), included 16 men, and were predominantly (91%) Black. Severity of COVID-19 was mild or moderate in most (77%) patients. All but one patient presented with AKI, 17 had nephrotic-range proteinuria, and six had nephrotic syndrome. Fourteen (61%) patients required dialysis at presentation. Among 17 patients genotyped, 16 (94%) were high-risk APOL1. Among 22 (96%) patients with median follow-up at 155 days (range, 30-412), 11 (50%) received treatment for COVID-19, and eight (36%) received glucocorticoid therapy for podocytopathy. At follow-up, 19 (86%) patients were alive, and 15 (68%) were dialysis free, including seven of 14 who initially required dialysis. The dialysis-free patients included 64% (seven of 11) of those treated for COVID-19 and 75% (six of eight) of those treated with glucocorticoids for podocytopathy. Overall, 36% achieved partial remission of proteinuria, 32% had no remission, and 32% reached combined end points of ESKD or death. Viral infection of the kidney was not detected.Half of 14 patients with COVID-19-associated collapsing glomerulopathy requiring dialysis achieved dialysis independence, but the long-term prognosis of residual proteinuric CKD remains guarded, indicating a need for more effective therapy.

Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame (uORF) isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains β-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP and its amyloid formation was inhibited in vitro by Cα-peptide more efficiently than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus (T2DM) autopsy donors. Intriguingly, 100 years after the discovery of insulin we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

A variety of mechanosensory neurons are involved in touch, proprioception, and pain. Many molecular components of the mechanotransduction machinery subserving these sensory modalities remain to be discovered. Here, we combine recordings of mechanosensitive (MS) currents in mechanosensory neurons with single-cell RNA sequencing. Transcriptional profiles are mapped onto previously identified sensory neuron types to identify cell-type correlates between datasets. Correlation of current signatures with single-cell transcriptomes provides a one-to-one correspondence between mechanoelectric properties and transcriptomically defined neuronal populations. Moreover, a gene-expression differential comparison provides a set of candidate genes for mechanotransduction complexes. Piezo2 is expectedly found to be enriched in rapidly adapting MS current-expressing neurons, whereas Tmem120a and Tmem150c, thought to mediate slow-type MS currents, are uniformly expressed in all mechanosensory neuron subtypes. Further knockdown experiments disqualify them as mediating MS currents in sensory neurons. This dataset constitutes an open resource to explore further the cell-type-specific determinants of mechanosensory properties.