Probes for INS

Some compulsive disorders have been considered to stem from the loss of control over coping strategies, such as displacement. However, the cellular mechanisms involved in the acquisition of coping behaviours and their subsequent compulsive manifestation in vulnerable individuals have not been elucidated. Considering the role of the locus coeruleus (LC) noradrenaline-dependent system in stress and related excessive behaviours, we hypothesised that neuroplastic changes in the LC may be associated with the acquisition of an adjunctive polydipsic water drinking, a prototypical displacement behaviour, and the ensuing development of compulsion in vulnerable individuals. Thus, male Sprague Dawley rats were characterised for their tendency, or not, to develop compulsive polydipsic drinking in a schedule-induced polydipsia (SIP) procedure before their fresh brains were harvested. A new quantification tool for RNAscope assays revealed that the development of compulsive adjunctive behaviour was associated with a low mRNA copy number of the plasticity marker Arc in the LC which appeared to be driven by specific adaptations in an ensemble of tyrosine hydroxylase (TH)+, zif268- neurons. This ensemble was specifically engaged by the expression of compulsive adjunctive behaviour, not by stress, because its functional recruitment was not observed in individuals that no longer had access to the water bottle before sacrifice, while it consistently correlated with the levels of polydipsic water drinking only when it had become compulsive. Together these findings suggest that downregulation of Arc mRNA levels in a population of a TH+/zif268- LC neurons represents a signature of the tendency to develop compulsive coping behaviours.

Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, microvascular dysfunction, and myocardial fibrosis with recent evidence implicating the immune system in orchestrating cardiac remodeling. Here, we show the mouse model of deoxycorticosterone acetate (DOCA)-salt hypertension induces key elements of HFpEF, including diastolic dysfunction, exercise intolerance, and pulmonary congestion in the setting of preserved ejection fraction. A modified single cell sequencing approach, CITE-seq, of cardiac immune cells reveals an altered abundance and transcriptional signature in multiple cell types, most notably cardiac macrophages. The DOCA-salt model results in differential expression of several known and novel genes in cardiac macrophages, including upregulation of Trem2, which has been recently implicated in obesity and atherosclerosis. The role of Trem2 in hypertensive heart failure, however, is unknown. We found that mice with genetic deletion of Trem2 exhibit increased cardiac hypertrophy, diastolic dysfunction, renal injury, and decreased cardiac capillary density after DOCA-salt treatment compared to wild-type controls. Moreover, Trem2-deficient macrophages have impaired expression of pro-angiogenic gene programs and increased expression of pro-inflammatory cytokines. Furthermore, we found that plasma levels of soluble TREM2 are elevated in DOCA-salt treated mice and humans with heart failure. Together, our data provide an atlas of immunological alterations that can lead to improved diagnostic and therapeutic strategies for HFpEF. We provide our dataset in an easy to explore and freely accessible web application making it a useful resource for the community. Finally, our results suggest a novel cardioprotective role for Trem2 in hypertensive heart failure.

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

The SUSD4 (Sushi domain-containing protein 4) gene encodes a complement inhibitor that is frequently deleted in 1q41q42 microdeletion syndrome, a multisystem congenital disorder that includes neurodevelopmental abnormalities. To understand SUSD4's role in the mammalian nervous system, we analyzed Susd4 knockout (KO) mice. Susd4 KO mice exhibited significant defects in motor performance and significantly higher levels of anxiety-like behaviors. Susd4 KO brain had abnormal "hairy" basket cells surrounding Purkinje neurons within the cerebellum and significantly reduced dendritic spine density in hippocampal pyramidal neurons. Neurons and oligodendrocyte lineage cells of wild-type mice were found to express Susd4 mRNA. Protein expression of the complement component C1q was increased in the brains of Susd4 KO mice. Our data indicate that SUSD4 plays an important role in neuronal functions, possibly via the complement pathway, and that SUSD4 deletion may contribute to the nervous system abnormalities in patients with 1q41q42 deletions

Mutations in Kv1.1 (Kcna1) voltage-gated potassium channels in humans and mice generate network hyperexcitability, enhancing aberrant postnatal neurogenesis in the dentate subgranular zone, resulting in epilepsy and hippocampal hypertrophy. While Kcna1 loss stimulates proliferation of progenitor cell subpopulations, the identity of extrinsic molecular triggers linking network hyperexcitability to aberrant postnatal neurogenesis remains incomplete. System x-c (Sxc) is an inducible glutamate/cysteine antiporter that regulates extracellular glutamate. Here, we find that the functional unit of Sxc, xCT (Slc7a11), is upregulated in regions of Kcna1 knockout (KO) hippocampus, suggesting a contribution to both hyperplasia and epilepsy. However, Slc7a11 KO suppressed and rescued hippocampal enlargement without altering seizure severity in Kcna1-Slc7a11-KO mice. Microglial activation, but not astrocytosis, was also reduced. Our study identifies Sxc-mediated glutamate homeostasis as an essential non-synaptic trigger coupling aberrant postnatal neurogenesis and neuroimmune crosstalk, revealing that neurogenesis and epileptogenesis in the dentate gyrus are not mutually contingent events.

The prostate gland produces prostatic fluid, high in zinc and citrate and essential for the maintenance of spermatozoa. Prostate cancer is a common condition with limited treatment efficacy in castration-resistant metastatic disease, including with immune checkpoint inhibitors. Using single-cell RNA-sequencing to perform an unbiased assessment of the cellular landscape of human prostate, we identify a subset of tumor-enriched androgen receptor-negative luminal epithelial cells with increased expression of cancer-associated genes. We also find a variety of innate and adaptive immune cells in normal prostate that were transcriptionally perturbed in prostate cancer. An exception is a prostate-specific, zinc transporter-expressing macrophage population (MAC-MT) that contributes to tissue zinc accumulation in homeostasis but shows enhanced inflammatory gene expression in tumors, including T cell-recruiting chemokines. Remarkably, enrichment of the MAC-MT signature in cancer biopsies is associated with improved disease-free survival, suggesting beneficial antitumor functions.

JNJ-64794964 (JNJ-4964/AL-034/TQ-A3334), an oral toll-like receptor 7 agonist, is being investigated for the treatment of chronic hepatitis B (CHB), a condition with a high unmet medical need. The anti-hepatitis B (HBV) activity of JNJ-4964 was assessed preclinically in an adeno-associated virus vector expressing HBV (AAV/HBV) mouse model. Mice were treated orally with 2, 6 or 20 mg/kg of JNJ-4964 once-per-week for 12 weeks and then followed up for 4 weeks. At 6 mg/kg, a partial decrease in plasma HBV-DNA and plasma hepatitis B surface antigen (HBsAg) were observed, and anti-HBs antibodies and HBsAg-specific T cells were observed in 1/8 animals. At 20 mg/kg, plasma HBV-DNA and HBsAg levels were undetectable for all animals 3 weeks after start of treatment, with no rebound observed 4 weeks after JNJ-4964 treatment was stopped. High anti-HBs antibody levels were observed until 4 weeks after JNJ-4964 treatment was stopped. In parallel, HBsAg-specific immunoglobulin G-producing B cells and interferon-γ-producing CD4+ T cells were detected in the spleen. In 2/4 animals, liver HBV-DNA and HBV-RNA levels, and liver hepatitis B core antigen expression dropped 4 weeks after JNJ-4964 treatment-stop. In these animals, HBsAg-specific CD8+ T cells were detectable. Throughout the study, normal levels of alanine aminotransferase were observed, with no hepatocyte cell death (end of treatment and 4 weeks later) and minimal infiltrations of B and T cells into the liver, suggesting induction of cytokine-mediated, non-cytolytic mechanisms.

Recent interest in astrocyte activation states has raised the fundamental question of how these cells, normally essential for synapse and neuronal maintenance, become pathogenic. Here, we show that activation of the unfolded protein response (UPR), specifically phosphorylated protein kinase R-like endoplasmic reticulum (ER) kinase (PERK-P) signaling-a pathway that is widely dysregulated in neurodegenerative diseases-generates a distinct reactivity state in astrocytes that alters the astrocytic secretome, leading to loss of synaptogenic function in vitro. Further, we establish that the same PERK-P-dependent astrocyte reactivity state is harmful to neurons in vivo in mice with prion neurodegeneration. Critically, targeting this signaling exclusively in astrocytes during prion disease is alone sufficient to prevent neuronal loss and significantly prolongs survival. Thus, the astrocyte reactivity state resulting from UPR over-activation is a distinct pathogenic mechanism that can by itself be effectively targeted for neuroprotection

Nonarteritic anterior ischemic optic neuropathy (NAION) is a common acute optic neuropathy and cause of irreversible vision loss in those older than 50 years of age. There is currently no effective treatment for NAION and yet the biological mechanisms leading to neuronal loss are not fully understood. Glial cells activation and intercommunication mediated by molecules such as gap junction protein Connexin 43 (Cx43) is thought to modulate neuronal fate in central nervous system disorders. In this study, we investigated retinal glial changes and neuronal loss following a novel NAION animal model using a 577 nm laser. We induced unilateral photochemical thrombosis using rose bengal at the optic nerve head vasculature in adult C57BL/6 mice using a 577 nm laser and performed morphometric analysis of the retinal structure using serial in vivo optical coherence tomography (OCT) and histology for glial and neuronal markers. OCT imaging revealed peripapillary thickening of the retinal ganglion cell complex (GCC, baseline: 79.5 ± 1.0 μm, n = 8; NAION: 93.0 ± 2.5 μm, n = 8, P < 0.01) and total retina (baseline: 202.9 ± 2.4 μm, n = 8; NAION: 228.1 ± 6.8 μm, n = 8, P < 0.01) at day 1 after NAION, and significant GCC thinning (baseline 78.3 ± 2.1 μm, n = 6; NAION: 72.2 ± 1.9 μm, n = 5, P < 0.05) at day 21. NAION induced a significant increase in retinal VEGF levels at day 1 (control: 2319 ± 195, n = 5; NAION: 4549 ± 683 gray mean value, n = 5, P < 0.05), which correlated with retinal thickness (r = 0.89, P < 0.05). NAION led to increased mRNA levels for Cx43 (Gj1a) at day 1 (control: 1.291 ± 0.38; NAION: 3.360 ± 0.58 puncta/mm2, n = 5, P < 0.05), which was not associated with changes in mRNA levels of glial fibrillary acidic protein (Gfap) at the same time (control: 2800 ± 0.59; NAION: 4690 ± 0.90 puncta/mm2 n = 5, P = 0.19). Retinal ganglion cell loss at day 21 was confirmed by a 30% decrease in Brn3a+ cells (control: 2844 ± 235; NAION: 2001 ± 264 cells/mm2, n = 4, P < 0.05). We described a novel protocol of NAION induction by photochemical thrombosis using a 577 nm laser, leading to retinal edema and VEGF increase at day 1 and RGCs loss at day 21 after injury, consistent with the pathophysiology of human NAION. Early changes in glial cells intercommunication revealed by increased Cx43+ gap junctions are consistent with a retinal glial role in mediating cell-to-cell signaling after an ischemic insult. Our study demonstrates an early glial response in a novel NAION animal model and reveals glial intercommunication molecules such as Cx43 as a promising therapeutic target in acute NAION.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodelling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in IPF patient lung and in CD14+ circulating monocytes obtained from IPF patient blood. After bleomycin administration, the myeloid-specific deletion of Atf6α altered pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. Further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.This article is protected by

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.