Viral-Mediated Knockdown of Nucleus Accumbens Shell PAC1 Receptor Promotes Excessive Alcohol Drinking in Alcohol-Preferring Rats

Alcohol use disorder (AUD) is a chronic, relapsing disorder whose genetic and environmental susceptibility components are not fully understood. Neuropeptidergic signaling has been repeatedly implicated in modulating excessive alcohol drinking, especially within sub-regions of the striatum. Here, we investigated the potential involvement of the selective receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), PAC1R, in the nucleus accumbens shell (NAcc Shell) in excessive alcohol drinking in alcohol-preferring rats, an established animal model of the genetic propensity for alcoholism. Scr:sP alcohol-preferring rats were trained to operantly self-administer alcohol and then either an AAV virus short-hairpin RNA (shRNA) targeted to knockdown PAC1R, or an AAV control virus were microinfused into the NAcc Shell. NAcc Shell PAC1R shRNA knockdown virus was confirmed to significantly decrease PAC1R levels in the NAcc Shell. The effects of NAcc Shell PAC1R shRNA knockdown on ethanol self-administration were investigated using a Fixed Ratio (FR) 1 and a Progressive Ratio (PR) schedule of reinforcement. The effect of PAC1R knockdown on self-administration of an alternative reinforcer, saccharin, was also assessed. The results showed that the reduction in PAC1R in the NAcc Shell led to excessive ethanol drinking, increased preference for ethanol, and higher motivation to drink. NAcc Shell PAC1R shRNA knockdown did not comparably increase saccharin self-administration, suggesting selectivity of action. These data suggest that NAcc Shell PAC1R may serves as a “brake” on alcohol drinking, and thereby the loss of function of PAC1R leads to excessive alcohol consumption. Therefore, the PACAP/PAC1R system may represent a novel target for the treatment of AUD.

Ionizing irradiation-induced Fgr in senescent cells mediates fibrosis

The role of cellular senescence in radiation-induced pulmonary fibrosis (RIPF) and the underlying mechanisms are unknown. We isolated radiation-induced senescent tdTOMp16 positive mesenchymal stem cells, established their absence of cell division, then measured levels of irradiation-induced expression of biomarkers of senescence by RNA-seq analysis. We identified a Log2 6.17-fold upregulation of tyrosine kinase Fgr, which was a potent inducer of biomarkers of fibrosis in target cells in non-contact co-cultures. Inhibition of Fgr by shRNA knockdown did not block radiation-induced senescence in vitro; however, both shRNA knockdown, or addition of a specific small-molecule inhibitor of Fgr, TL02-59, abrogated senescent cell induction of profibrotic genes in transwell-separated target cells. Single-cell RNA-seq (scRNAseq) analysis of mouse lungs at day 150 after 20 Gy thoracic irradiation revealed upregulation of Fgr in senescent neutrophils, and macrophages before detection of lung fibrosis. Thus, upregulated Fgr in radiation-induced senescent cells mediates RIPF and is a potential therapeutic target for the prevention of this radiation late effect.

Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells

Intestinal NaCl, HCO3- and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical NHE isoforms, NHE2, NHE3 and NHE8. We addressed the role of NHE8 (SLC9A8) and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis, and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated vs. undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state pHi, an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi-control.

Important role of long non-coding RNA UBE2R2-AS1 in diabetes-induced renal injury

IntroductionThe present study examined the effects and mechanisms underlying long non-coding RNA (lncRNA) UBE2R2-AS1 in diabetes-induced renal injury.Material and methodsHigh glucose was used to imitate diabetes-induced renal injury in a cell model. Cell apoptosis rate was measured using flow cytometry, tumour necrosis factor-a (TNF-a) and interleukin-6 (IL-6) concentrations were evaluated using ELISA, and relative protein expression and amount were measured using western blot (WB) analysis and immunofluorescence, respectively. Correlations between lncRNA UBE2R2-AS1, miRNA-877-3p and TLR4 were analysed using the luciferase reporter assay.ResultsCell apoptosis rate and TNF-a and IL-6 concentrations were significantly higher (p < 0.001) in the high glucose (model) group compared with those of the Control group (CG) group. Following shRNA knockdown of lncRNA UBE2R2-AS1, the cell apoptosis rate and TNF-a and IL-6 concentrations were significantly lower (p < 0.001, respectively) compared with those of the model group. However, under high-glucose conditions, shRNA knockdown of UBE2R2-AS1 and miRNA-877-3p significantly increased (p < 0.001) the cell apoptosis rate as well as TNF-a and IL-6 concentrations compared with the shRNA UBE2R2-AS1 knockdown group. WB and immunofluorescence analysis showed that toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kB (p65) (NF-kB(p65)) pathway proteins were significantly stimulated in the model group compared with those in the CG, whereas shRNA transfection with miRNA-877-3p stimulation suppressed the TLR4/MyD88/NF-kB(p65) pathway.ConclusionsKnockdown of lncRNA UBE2R2-AS1 improves diabetes-induced renal injury via regulation of the miRNA-877-3p/TLR4 axis in vitro.

Spatiotemporal Mislocalization of Nuclear Membrane-Associated Proteins in γ-Irradiation-Induced Senescent Cells

Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence nor changes in the LINC protein distribution before irradiation. However, the cytological changes following irradiation were more pronounced in shRNA knockdown cells compared to original cell lines. We conclude that mislocalization of LINC complex proteins is a significant characteristic of cellular senescence phenotypes and may influence complex events at the nuclear membrane, including trafficking and heterochromatin attachment.