NELL-1 is a secreted, osteogenic protein first discovered to control ossification of the cranial skeleton. Recently, NELL-1 has been implicated in bone maintenance. However, the cellular determinants of NELL-1’s bone-forming effects are still unknown. Here, recombinant human NELL-1 (rhNELL-1) implantation was examined in a clinically relevant nonhuman primate lumbar spinal fusion model. Prolonged rhNELL-1 protein release was achieved using an apatite-coated β-tricalcium phosphate carrier, resulting in a local influx of stem cell antigen-1–positive (Sca-1+) mesenchymal progenitor cells (MPCs), and complete osseous fusion across all samples (100% spinal fusion rate). Murine studies revealed that
Aaron W. James, Jia Shen, Rebecca Tsuei, Alan Nguyen, Kevork Khadarian, Carolyn A. Meyers, Hsin Chuan Pan, Weiming Li, Jin H. Kwak, Greg Asatrian, Cymbeline T. Culiat, Min Lee, Kang Ting, Xinli Zhang, Chia Soo
Microglia play a critical role in the development and homeostasis of the CNS. While mobilization of microglia is critical for a number of pathologies, understanding of the mechanisms of their migration in vivo is limited and often based on similarities to macrophages. Kindlin3 deficiency as well as Kindlin3 mutations of integrin-binding sites abolish both integrin inside-out and outside-in signaling in microglia, thereby resulting in severe deficiencies in cell adhesion, polarization, and migration in vitro, which are similar to the defects observed in macrophages. In contrast, while Kindlin3 mutations impaired macrophage mobilization in vivo, they had no effect either on the population of microglia in the CNS during development or on mobilization of microglia and subsequent microgliosis in a model of multiple sclerosis. At the same time, acute microglial response to laser-induced injury was impaired by the lack of Kindlin3-integrin interactions. Based on 2-photon imaging of microglia in the brain, Kindlin3 is required for elongation of microglial processes toward the injury site and formation of phagosomes in response to brain injury. Thus, while Kindlin3 deficiency in human subjects is not expected to diminish the presence of microglia within CNS, it might delay the recovery process after injury, thereby exacerbating its complications.
Julia Meller, Zhihong Chen, Tejasvi Dudiki, Rebecca M. Cull, Rakhilya Murtazina, Saswat K. Bal, Elzbieta Pluskota, Samantha Stefl, Edward F. Plow, Bruce D. Trapp, Tatiana V. Byzova
Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide–binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell–specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116–/– mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.
Kari Brown, Alyssa Filuta, Marie-Gabrielle Ludwig, Klaus Seuwen, Julian Jaros, Solange Vidal, Kavisha Arora, Anjaparavanda P. Naren, Kathirvel Kandasamy, Kaushik Parthasarathi, Stefan Offermanns, Robert J. Mason, William E. Miller, Jeffrey A. Whitsett, James P. Bridges
Psychiatric patients treated with lithium (Li+) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li+-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li+ inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6–/–) mice and potentially novel connecting tubule/principal cell–specific AC6 knockout (AC6loxloxCre) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li+ administration increased water intake and reduced urine osmolality in control, AC6–/–, and AC6loxloxCre mice. Consistent with AC6–/– mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6loxloxCre mice compared with controls under standard conditions, and levels were further reduced after Li+ administration. AC6loxloxCre and control mice had a similar increase in the numbers of proliferating cell nuclear antigen–positive cells in response to Li+. However, AC6loxloxCre mice had a higher number of H+-ATPase B1 subunit–positive cells under standard conditions and after Li+ administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell–to–principal cell ratio.
Søren Brandt Poulsen, Tina Bøgelund Kristensen, Heddwen L. Brooks, Donald E. Kohan, Timo Rieg, Robert A. Fenton
Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release–activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated
Miriam Eckstein, Martin Vaeth, Cinzia Fornai, Manikandan Vinu, Timothy G. Bromage, Meerim K. Nurbaeva, Jessica L. Sorge, Paulo G. Coelho, Youssef Idaghdour, Stefan Feske, Rodrigo S. Lacruz
Loss of LKB1 activity is prevalent in
Melissa Gilbert-Ross, Jessica Konen, Junghui Koo, John Shupe, Brian S. Robinson, Walter Guy Wiles IV, Chunzi Huang, W. David Martin, Madhusmita Behera, Geoffrey H. Smith, Charles E. Hill, Michael R. Rossi, Gabriel L. Sica, Manali Rupji, Zhengjia Chen, Jeanne Kowalski, Andrea L. Kasinski, Suresh S. Ramalingam, Haian Fu, Fadlo R. Khuri, Wei Zhou, Adam I. Marcus
The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo–conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox–mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate–induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.
Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud
ice and humans lacking the caveolae component polymerase I transcription release factor (PTRF, also known as cavin-1) exhibit lipo- and muscular dystrophy. Here we describe the molecular features underlying the muscle phenotype for PTRF/cavin-1 null mice. These animals had a decreased ability to exercise, and exhibited muscle hypertrophy with increased muscle fiber size and muscle mass due, in part, to constitutive activation of the Akt pathway. Their muscles were fibrotic and exhibited impaired membrane integrity accompanied by an apparent compensatory activation of the dystrophin-glycoprotein complex along with elevated expression of proteins involved in muscle repair function.
Shi-Ying Ding, Libin Liu, Paul F. Pilch
Mitophagy occurs during ischemia/reperfusion (I/R) and limits oxidative stress and injury. Mitochondrial turnover was assessed in patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). Paired biopsies of right atrial appendage before initiation and after weaning from CPB were processed for protein analysis, mitochondrial DNA/nuclear DNA ratio (mtDNA:nucDNA ratio), mtDNA damage, mRNA, and polysome profiling. Mitophagy in the post-CPB samples was evidenced by decreased levels of mitophagy adapters NDP52 and optineurin in whole tissue lysate, decreased Opa1 long form, and translocation of Parkin to the mitochondrial fraction. PCR analysis of mtDNA comparing amplification of short vs. long segments of mtDNA revealed increased damage following cardiac surgery. Surprisingly, a marked increase in several mitochondria-specific protein markers and mtDNA:nucDNA ratio was observed, consistent with increased mitochondrial biogenesis. mRNA analysis suggested that mitochondrial biogenesis was traniscription independent and likely driven by increased translation of existing mRNAs. These findings demonstrate in humans that both mitophagy and mitochondrial biogenesis occur during cardiac surgery involving CPB. We suggest that mitophagy is balanced by mitochondrial biogenesis during I/R stress experienced during surgery. Mitigating mtDNA damage and elucidating mechanisms regulating mitochondrial turnover will lead to interventions to improve outcome after I/R in the setting of heart disease.
Allen M. Andres, Kyle C. Tucker, Amandine Thomas, David J.R. Taylor, David Sengstock, Salik M. Jahania, Reza Dabir, Somayeh Pourpirali, Jamelle A. Brown, David G. Westbrook, Scott W. Ballinger, Robert M. Mentzer Jr., Roberta A. Gottlieb
Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes, but it is not known whether molecular differences exist between polyfunctional and monofunctional cytokine-producing T cells. Here, we report that polyfunctional CD4+ T cells induced during
Julie G. Burel, Simon H. Apte, Penny L. Groves, James S. McCarthy, Denise L. Doolan
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