Jerry R. Mendell, Louise Rodino-Klapac, Zarife Sahenk, Vinod Malik, Brian K. Kaspar, Christopher M. Walker, K. Reed Clark

Monte M. Winslow, Talya L. Dayton, Roel G. W. Verhaak, Caroline Kim-Kiselak, Eric L. Snyder, David M. Feldser, Diana D. Hubbard, Michel J. DuPage, Charles A. Whittaker, Sebastian Hoersch, Stephanie Yoon, Denise Crowley, Roderick T. Bronson, Derek Y. Chiang, Matthew Meyerson, and Tyler Jacks

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS1 and inactivation of the p53 pathway2, using conditional alleles in mice3, 4, 5. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of KrasLSL-G12D/+;p53flox/flox mice initiates lung adenocarcinoma development4. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function6, 7, 8, 9, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Kathleen M. Schoch, Heather N. Evans, Jennifer M. Brelsfoard, Sindhu K. Madathil, Jiro Takano, Takaomi C. Saido, Kathryn E. Saatman

Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium–calmodulin dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury.

Sandrine Da Cruz, Philippe A. Parone, Vanda S. Lopes, Concepción Lillo, Melissa McAlonis-Downes, Sandra K. Lee, Anne P. Vetto, Susanna Petrosyan, Martin Marsala, Anne N. Murphy, David S. Williams, Bruce M. Spiegelman, Don W. Cleveland

The transcriptional coactivator PGC-1α induces multiple effects on muscle, including increased mitochondrial mass and activity. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, adult-onset neurodegenerative disorder characterized by selective loss of motor neurons and skeletal muscle degeneration. An early event is thought to be denervation-induced muscle atrophy accompanied by alterations in mitochondrial activity and morphology within muscle. We now report that elevation of PGC-1α levels in muscles of mice that develop fatal paralysis from an ALS-causing SOD1 mutant elevates PGC-1α-dependent pathways throughout disease course. Mitochondrial biogenesis and activity are maintained through end-stage disease, accompanied by retention of muscle function, delayed muscle atrophy, and significantly improved muscle endurance even at late disease stages. However, survival was not extended. Therefore, muscle is not a primary target of mutant SOD1-mediated toxicity, but drugs increasing PGC-1α activity in muscle represent an attractive therapy for maintaining muscle function during progression of ALS.

Yosefa Avraham, Johnny Amer, Sarit Doron, Lina Abu-Tair, Mahmud Mahamid, Areej A.S Khatib, Elliot M. Berry, and Rifaat Safadi

Cannabinoid CB2-receptors expressed on immune cells are considered to be anti-fibrogenic. Hepatic stellate cells (HSCs) directly interact with, and phagocytose lymphocytes, but the nature of this interaction is obscure. Aims: To study the effects of CB2-receptors on hepatic-fibrosis via their role in mediating immunity. Methods: (I) Hepatic-fibrosis was induced by carbon-tetrachloride (CCl4) administration in C57BL/6 wild-type (WT) and CB2-knockout (CB2-/-) mice. (II) Irradiated-animals were reconstituted with WT or CB2-/- lymphocytes. (III) Lymphocytes from naïve/ fibrotic WT animals; and healthy/cirrhotic HCV were pre-incubated in-vitro with/without CB2-antagonist, evaluated for proliferation and apoptosis, and then co-cultured with primary mouse HSCs or a human HSCs line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Results: (I) Following CCl4-administration; CB2-/- mice develop significant hepatic-fibrosis but less necro-inflammation. WT mice harbored decreased liver CD4+ and NK+ cells but increased CD8+ subsets. Naïve CB2-/- mice had significantly decreased T-cell subsets. (II) Adoptive transfer of CB2-/- lymphocytes led to decreased fibrosis in the irradiated WT recipient as compared to animals receiving WT lymphocytes. Moreover, necro-inflammation also tended to decrease. (III) In-vitro, a CB2-antagonist directly increased human HSCs activation and increased apoptosis, decreased proliferation of mice/ human T-cells (healthy/ fibrotic) and their phagocytosis. Conclusions: CB2-/- lymphocytes exert an anti-fibrotic activity while lack of CB2-receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.

Juan Pablo de Rivero Vaccari, Dominic Bastien, Geoffrey Yurcisin, Isabelle Pineau, W. Dalton Dietrich, Yves De Koninck, Robert W. Keane, and Steve Lacroix

P2X4 and P2X7 are the predominant purinergic P2X receptor subtypes expressed on immune and neural cells. These receptor subtypes traffic between intracellular compartments and the plasma membrane and form protein interactions with each other to regulate ATP-dependent signaling. Our recent studies have shown that P2X7 receptors in neurons and astrocytes activate NLRP1 inflammasomes, but whether P2X4 receptors regulate inflammasome signaling is essentially unknown. Here, we demonstrate that P2X4 receptors are expressed in neurons of the spinal cord. We provide direct evidence that spinal cord injury (SCI) induces an innate inflammatory response that leads to increased caspase-l cleavage and production of IL-1β but not IL-18. Consistent with these findings, P2X4 knock-out mice showed impaired inflammasome signaling in the cord, resulting in decreased levels of IL-1β and reduced infiltration of neutrophils and monocyte-derived M1 macrophages, resulting in significant tissue sparing and improvement in functional outcomes. These results indicate that P2X4 receptors influence inflammasome signaling involving caspase-1 activation and IL-1β processing in neurons after SCI. P2X4 might thus represent a potential therapeutic target to limit inflammatory responses associated with SCI and neurodegenerative disorders.

Ling Yu, Yan Su, Paisit Paueksakon, Huifang Cheng, Xiwu Chen, Hongtao Wang, Raymond C Harris, Roy Zent and Ambra Pozzi

Animal models that mimic human diabetic nephropathy are useful to identify key factors in pathogenesis of this disease, as well as the development of new therapies. Several mouse models of diabetes have features of human diabetic nephropathy, yet none of these completely fulfill the Animal Models of Diabetes Complications Consortium criteria and completely reproduce pathological and functional features of the human disease. The Akita mouse carries a mutation in the insulin-2 gene and, to date, only survives as heterozygotes that develop spontaneous type 1 diabetes. Here we show that Akita mice with mutation of both insulin-2 alleles (Akita knockout (KO)) survive if crossed onto the Balb/c background. These mice develop hyperglycemia, more severe albuminuria, and mesangial sclerosis compared with heterozygous mice on the same genetic background. Interestingly, crossing these AkitaKO mice with integrin α1KO mice, a model of exacerbated glomerulosclerosis after injury and also on the Balb/c background, resulted in a 16-fold increase in albuminuria, significant mesangial matrix expansion, nodular and diffuse glomerulosclerosis, and a 2-fold increase in glomerular basement membrane thickening when compared with nondiabetic mice. Moreover, a significant decline in glomerular filtration was evident in the α1KOAkitaKO mice at 6 months of age. Thus, the integrin α1KOAkitaKO Balb/c mouse represents a promising model presenting with most features of human diabetic nephropathy.

S. M. Rida, A. M. Youssef, M. H. Badr, A. Malki, Z. A. Sherif, A. S. Sultan

Novel benzimidazoles, benzothiazoles and benzofurans incorporating pyrazole moiety have been synthesized and screened for their antiangogenic activities, by testing their ability to inhibit human umbilical vein endothelial cell (HUVEC) proliferation, cord formation and migration in response to chemoattractant. 3 compounds 19, 23 and 26 showed antiangiogenic activities at non-cytotoxic concentrations. Compound 19 was the most active with chemotaxis activity data nearly comparable to that of the positive control, TNP-470. Compound 42 showed a significant cytotoxic effect on the tested cancer cell lines and less antiangiogenesis activity compared to compounds 19, 23 and 26. All the tested compounds, in contrary to TNP-470, interfered with the migratory function of HUVECs in response to vascular endothelial growth factor rather than the endothelial cells proliferation or cord formation. Moreover, a docked pose of compounds 19 and 26 was obtained bound to kinase insert domain receptor using Molecular Operating Environment module.

Jian Zou, Ryan P. Vetreno, Fulton T. Crews

Activation and proliferation of glial cells and their progenitors is a key process of neuroinflammation associated with many neurodegenerative disorders. Under neuropathological conditions where glial cell activation and proliferation is evident, controlling the population of glia might be of therapeutic importance. The proliferative action of the cytokine tumor necrosis factor alpha (TNFα) on microglia has been reported, but the molecular mechanism of TNFα regulation of glial cell proliferation is largely unknown. Using a model of organotypic hippocampal–entorhinal cortex (HEC) slice culture, we investigated the role of ATP-P2X7 receptor signaling in glial proliferation by TNFα. Populations of proliferating cells in HEC culture were labeled with 5-bromo-2′-deoxyuridine (BrdU). Treatment with TNFα induced strong expression of P2X7 receptor mRNA and immunoreactivity in BrdU+ cells while markedly increasing proliferation of BrdU+ cells. In addition, TNFα increased aquaporin 4 (AQP4) expression, an ion channel involved in glial proliferation. The proliferative action of TNFα was attenuated by blocking the P2X7 receptors with the specific antagonists oxATP, BBG, and KN62, or by lowering extracellular ATP with ATP hydrolysis apyrase. Basal proliferation of BrdU+ cells was also sensitive to blockade of ATP-P2X7 signaling. Furthermore, TNFα activation of P2X7 receptors appear to regulate AQP4 expression through protein kinase C cascade and down regulation of AQP4 expression can reduce TNFα-stimulated BrdU+ cell proliferation. Taken together, these novel findings demonstrate the importance of ATP-P2X7 signaling in controlling proliferation of glial progenitors under the pathological conditions associated with increased TNFα.

Nicholas C. Bambakidis, Mary Petrullis, Xu Kui, Brian Rothstein, Ioannis Karampelas, Youzhi Kuang, Warren R. Selman, Joseph C. LaManna, and Robert H. Miller