Probes for INS

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.

Maternal obesity affects nearly one-third of pregnancies and is a major risk factor for nonalcoholic fatty liver disease (NAFLD) in adolescent offspring, yet the mechanisms behind NAFLD remain poorly understood. Here, we demonstrate that nonhuman primate fetuses exposed to maternal Western-style diet (WSD) displayed increased fibrillar collagen deposition in the liver periportal region, with increased ACTA2 and TIMP1 staining, indicating localized hepatic stellate cell (HSC) and myofibroblast activation. This collagen deposition pattern persisted in 1-year-old offspring, despite weaning to a control diet (CD). Maternal WSD exposure increased the frequency of DCs and reduced memory CD4+ T cells in fetal liver without affecting systemic or hepatic inflammatory cytokines. Switching obese dams from WSD to CD before conception or supplementation of the WSD with resveratrol decreased fetal hepatic collagen deposition and reduced markers of portal triad fibrosis, oxidative stress, and fetal hypoxemia. These results demonstrate that HSCs and myofibroblasts are sensitive to maternal WSD-associated oxidative stress in the fetal liver, which is accompanied by increased periportal collagen deposition, indicative of early fibrogenesis beginning in utero. Alleviating maternal WSD-driven oxidative stress in the fetal liver holds promise for halting steatosis and fibrosis and preventing developmental programming of NAFLD.

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.

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

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli - eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

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.

To understand the subcellular localization of RUNX2 and two lncRNAs, LINC02035 and LOC100130207, immunocytochemistry (for RUNX2 protein) and RNA _in situ_ hybridization assays (for both lncRNAs) were performed using human primary chondrocytes isolated from knee cartilage of OA patients. We confirmed that the RUNX2 protein was strongly detected in the nucleus of chondrocytes isolated from damaged cartilage (Figure 4A). The fractionated western blot results also showed that the RUNX2 protein was detected only in the nucleus of chondrocytes isolated from damaged cartilage (Figure 4B). To further understand the molecular mechanisms of the lncRNAs LINC02035 and LOC100130207, we performed an _in situ_ assay using primary chondrocytes derived from patients, because primary chondrocytes are a valuable model for studying OA pathogenesis. The results showed that both LINC02035 and LOC100130207 were highly expressed in chondrocytes isolated from the knee cartilage of patients with OA (Figure 4C). We then evaluated the mRNA levels and subcellular localization of both lncRNAs to elucidate their site of action using a commercially available kits in primary chondrocytes isolated from intact or damaged cartilage tissues. The results showed that both lncRNAs were more upregulated in primary chondrocytes isolated from damaged cartilage tissue than in intact cartilage tissue (Figure 4D). In primary chondrocytes, LINC02035 and LOC100130207 were merely detected in the cytoplasm of human primary chondrocytes and both lncRNAs were localized to nucleus (Figure 4E). Likewise, we also studied the subcellular localization of both lncRNAs in TC28a2 cells. The results showed that LINC02035 and LOC100130207 were evenly distributed in the nucleus and cytoplasm of normal chondrocytes (Figure 4F, left). However, both lncRNAs were preferentially localized to the nucleus and to a lesser extent to the cytoplasm after TC28a2 cells were treated with hypertrophic medium or TNF-α (Figure 4F, middle and right). To investigate whether RUNX2 is regulated at the post-translational level during hypertrophic changes in chondrocytes, human primary chondrocytes or TC28a2 cells were treated with the proteasome inhibitor MG132. The results showed that the protein level of RUNX2 was dose-dependently increased by MG132 treatment (Figure 4G-H), indicating that the upregulation of RUNX2 in osteoarthritic or hypertrophic chondrocytes occurs at the post-translational level. To examine whether both lncRNAs are involved in the stabilization of RUNX2 protein during hypertrophic differentiation and the inflammatory response in chondrocytes, IP was conducted to confirm the ubiquitination of RUNX2 protein. First, we investigated how the ubiquitination of RUNX2 protein is regulated during hypertrophic differentiation or the inflammatory response of chondrocytes, and as a result, it was confirmed that ubiquitination of RUNX2 was reduced by hypertrophic medium or TNF-α treatment (Figure 4I). However, ubiquitination of RUNX2 protein was clearly increased in TC28a2 cells transfected with siRNAs targeting LINC02035 or LOC100130207, even though the cells were treated with hypertrophic medium or TNF-α (Figure 4J-K). These results suggest that both lncRNAs upregulated during hypertrophic differentiation and the inflammatory response in chondrocytes contribute to the stabilization of the RUNX2 protein.

Activation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood.Bulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called IN flammatory M K L1 I nteracting L ong N oncoding RNA ( INKILN ). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation and human atherosclerosis and abdominal aortic aneurysm (AAA) samples. The transcriptional regulation of INKILN was determined through luciferase reporter system and chromatin immunoprecipitation assay. Both loss- and gain-of-function approaches and multiple RNA-protein and protein-protein interaction assays were utilized to uncover the role of INKILN in VSMC proinflammatory gene program and underlying mechanisms. Bacterial Artificial Chromosome (BAC) transgenic (Tg) mice were utilized to study INKLIN expression and function in ligation injury-induced neointimal formation.INKILN expression is downregulated in contractile VSMCs and induced by human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB site within its proximal promoter. INKILN activates the proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. Mechanistically, INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks ILIβ-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1, and the luciferase activity of an NF-κB reporter. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme, USP10. INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in BAC Tg mice.These findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8+ CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker.

Paralleling the activation of dorsal horn microglia after peripheral nerve injury is a significant expansion and proliferation of macrophages around injured sensory neurons in dorsal root ganglia (DRG). Here we demonstrate a critical contribution of DRG macrophages, but not those at the nerve injury site, to both the initiation and maintenance of the mechanical hypersensitivity that characterizes the neuropathic pain phenotype. In contrast to the reported sexual dimorphism in the microglial contribution to neuropathic pain, depletion of DRG macrophages reduces nerve injury-induced mechanical hypersensitivity and expansion of DRG macrophages in both male and female mice. However, fewer macrophages are induced in the female mice and deletion of colony-stimulating factor 1 from sensory neurons, which prevents nerve injury-induced microglial activation and proliferation, only reduces macrophage expansion in male mice. Finally, we demonstrate molecular cross-talk between axotomized sensory neurons and macrophages, revealing potential peripheral DRG targets for neuropathic pain management