Pathway In Mouse Model Research Articles

Anti-nociceptive effects of Paecilomyces hepiali via multiple pathways in mouse models.

Paecilomyces hepiali (PH), a well-known medicinal fungus, has various pharmacological efficacies. In our study, the antinociceptive effects of PH and underlying mechanisms were evaluated using various mouse models. An acetic acid-induced writhing test, hot plate test, and formalin test were employed to evaluate the antinociceptive activities of PH. The levels of neuronal nitric oxide synthase (nNOS) in the hypothalamus and monoamine neurotransmitters in the serum and hypothalamus of experimental mice were examined. Additionally, hot plate tests using mice pretreated with various antagonists were used to determine the mechanisms of PH-mediated antinociception. The PH-enhanced latency period of mice in the hot plate test was significantly blocked by pretreatment with atropine and glibenclamide. PH shortened the phase I and phase II reaction times of formalin-treated mice. Strongly reduced writhing and stretching induced by acetic acid were observed in PH-treated mice, indicating that PH mainly exerts antinociceptive activity on neurogenic pain. After thermal pain stimulation for 30 s, compared to control mice, 7-day PH-treated mice had lower nNOS and dopamine levels, and increased levels of serotonin in both the serum and hypothalamus. Collectively, our data showed that PH mediated antinociceptive activities via multiple pathways, including monoamines, nNOS/ATP-sensitive K+ channels, and M-type acetylcholine receptors.

YAPing Hippo Forecasts a New Target for Lung Cancer Prevention and Treatment.

YAPing Hippo Forecasts a New Target for Lung Cancer Prevention and Treatment.

Noninvasive imaging of tumor burden and molecular pathways in mouse models of cancer.

Imaging plays a central role in the diagnosis of cancer and the evaluation of therapeutic efficacy in patients with cancer. Because macroscopic imaging is noninvasive and quantitative, the development of specialized instruments for small animals has spurred increasing utilization in preclinical cancer studies. Some small-animal imaging devices are miniaturized derivatives of clinical imaging modalities, including computed tomography, magnetic resonance imaging, positron-emission tomography, single-photon emission computed tomography, and ultrasonography. Optical imaging, including bioluminescence imaging and fluorescence imaging, has evolved from microscopic cellular imaging technologies. Here, we review how current imaging modalities are enabling high-resolution structural imaging with micrometer-scale spatial resolution, thus allowing for the quantification of tumor burden in genetically engineered and orthotopic models of cancer, where tumors develop within organs not typically accessible to measurements with calipers. Beyond measuring tumor size, imaging is increasingly being used to assess the activity of molecular pathways within tumors and to reveal the pharmacodynamic efficacy of targeted therapies. Each imaging technology has particular strengths and limitations, and we discuss how studies should be carefully designed to match the imaging approach to the primary experimental question.

γ-Secretase Inhibitor Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation.

T helper 17 (Th17) cells play an important role in the pathogenesis of allergic asthma. Th17 cell differentiation requires Notch signaling. γ-Secretase inhibitor (GSI) blocks Notch signaling; thus, it may be considered as a potential treatment for allergic asthma. The aim of this study was to evaluate the effect of GSI on Th17 cell differentiation in a mouse model of allergic asthma. OVA was used to induce mouse asthma model in the presence and absence of GSI. GSI ameliorated the development of OVA-induced asthma, including suppressing airway inflammation responses and reducing the severity of clinical signs. GSI also significantly suppressed Th17-cell responses in spleen and reduced IL-17 levels in serum. These findings suggest that GSI directly regulates Th17 responses through a Notch signaling-dependent pathway in mouse model of allergic asthma, supporting the notion that GSI is a potential therapeutic agent for the treatment of allergic asthma.

Abstract 5023: Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy

Abstract Combination immunotherapy regimens incorporating agents that target more than a single regulatory pathway or immune inhibitory checkpoint can confer marked enhancement of anti-tumor immune responses when combined with active immunotherapy. We have tested the antitumor effect of the IDO pathway inhibitor NLG-919 (currently in Phase I clinical trial) in combination with indoximod (a different IDO pathway inhibitor currently in Phase II clinical trials), chemotherapy, vaccination and/or PD-1/PD-L1/PD-L2 blockade. Here we show that the highly potent, orally-bioavailable IDO pathway inhibitor NLG-919 is synergistic with blockade of the PD-1/PD-L1/PD-L2 pathway in mouse models of large established tumors. The combination of NLG-919 plus PD-1/PD-L1/PD-L2 blockade showed significantly enhanced anti-tumor effect compared to either agent alone. This synergy was particularly evident in the setting of large established tumors treated with normally ineffective immunotherapy regimens (e.g., a single dose of chemotherapy plus a vaccine against a poorly immunogenic shared-self antigen). In vivo, administration of NLG-919 enhanced anti-tumor vaccine responses against B16F10 melanoma treated with hgp100 vaccine plus resting, non-activated pmel-1 T cells NLG-919 also reduced Treg-mediated suppression in tumor-bearing hosts, and enhanced dendritic cell activation in tumors and TDLNs. In combination with chemotherapy, treatment with NLG-919 allowed effector T cell responses against endogenous tumor antigens released by chemotherapy. By each of the preceding readouts, the in vivo biologic effect of NLG-919 was qualitatively identical to that of indoximod, but the same immunologic effects could be achieved at lower plasma concentrations in vivo. In multiple models, a combination of oral NLG-919 with oral indoximod produced synergistic anti-tumor effects, which was further enhanced by blockade of the PD 1/PD L1/PD-L2 pathway. In a more stringent B16F10 melanoma tumor model that did not involve adoptive transfer of pmel-1 cells, a combination of immune checkpoint inhibition involving NLG-919, indoximod and anti-PD1/PD-L1/PD-L2 antibodies with chemotherapy and hgp100 peptide vaccine was able to achieve a significant antitumor effect. The current preclinical studies suggest a mechanistic rationale for a combining NLG919 with indoximod and with agents targeting the PD 1/PD L1/PD-L2 pathway. Citation Format: Mario R. Mautino, Charles J. Link, Nicholas N. Vahanian, James T. Adams, Clarissa Van Allen, Madhav D. Sharma, Theodore S. Johnson, David Munn. Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2014-5023

The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker.

Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.

Heterogeneity and convergence: the synaptic pathophysiology of autism

Autism is a developmental disorder characterised by a high heterogeneity of clinical diagnoses and genetic associations. This heterogeneity is a challenge for the identification of the pathophysiology of the disease and for the development of new therapeutic strategies. New conceptual approaches are being used to try to challenge this complexity and gene cluster analysis studies suggest that the pathophysiology of autism is associated with a dysregulation of specific cellular mechanisms. This review will present the experimental evidence for a convergence of synaptic pathophysiology between syndromic and non-syndromic forms of autism, grouped under the generic term of autism spectrum disorders. In particular I will highlight the results from genetic mouse models identifying a convergence of dysregulation of the synaptic type I metabotropic glutamate receptor pathway in mouse models for autism spectrum disorders. These results help to build a new conceptual framework for the study of the synaptic phenotype of autism, which is important for the identification of new therapeutic strategies.

Identification of Disease Specific Pathways Using in Vivo SILAC Proteomics in Dystrophin Deficient mdx Mouse

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with (13)C6-lysine or stable isotope labeling in mammals (SILAM) with (15)N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of human skeletal muscle disease.

A novel strategy for integrating gene expression profiles derived from multiple publicly available datasets highlights novel hub genes and pathways in mouse models of allergic asthma

Event Abstract Back to Event A novel strategy for integrating gene expression profiles derived from multiple publicly available datasets highlights novel hub genes and pathways in mouse models of allergic asthma Michela Riba1*, Jose M. Garcia Manteiga1, Berislav Bosniak2, Michelle Epstein2 and Elia Stupka1 1 San Raffaele Scientific Institute, Center for Translational Genomics and Bioinformatics, Italy 2 University of Vienna, Department of Dermatology, Austria There are 30 studies of gene expression profiling of lungs from mice with allergic asthma, which shed new light on the molecular mechanisms of disease, but the experimental designs between them differ. Nevertheless, relevant genes and pathways underlying disease pathogenesis may emerge from the integration of these datasets. From 12 publically available datasets, we selected 6 for meta-­analysis on the basis of the microarray platform and in vivo experimental protocol. Our strategy was to combine a top down pathway­centered and a bottom up gene­centered analyses. The first approach consisted of selecting asthma­specific pathways from a combination of ‘enriched biological terms’ in each individual study. Whereas, secondly, we combined differentially expressed genes from individual datasets to create a gene list. From 22,690 genes and 131 samples, we obtained a core-­network of more than 300 interconnected genes unraveling known (STAT1, JAK2) and unknown hubs (PU.1, CDK1). Additionally, we successfully selected known and unknown disease-­related pathways, such as T­cell activation during an immune response, B­cell mediated immunity, phagocytosis and complement activation. The power of this strategy is the possibility of uncovering key molecules underlying disease pathogenesis utilizing datasets derived from disparate experiments Keywords: Asthma, Gene Expression, Network analysis, Meta-analysis, pathway analysis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Riba M, Garcia Manteiga JM, Bosniak B, Epstein M and Stupka E (2013). A novel strategy for integrating gene expression profiles derived from multiple publicly available datasets highlights novel hub genes and pathways in mouse models of allergic asthma. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00881 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Michela Riba, San Raffaele Scientific Institute, Center for Translational Genomics and Bioinformatics, Milano, Italy, riba.michela@hsr.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Michela Riba Jose M Garcia Manteiga Berislav Bosniak Michelle Epstein Elia Stupka Google Michela Riba Jose M Garcia Manteiga Berislav Bosniak Michelle Epstein Elia Stupka Google Scholar Michela Riba Jose M Garcia Manteiga Berislav Bosniak Michelle Epstein Elia Stupka PubMed Michela Riba Jose M Garcia Manteiga Berislav Bosniak Michelle Epstein Elia Stupka Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd (mdx), SJL/J, Large (myd) and Lama2 (dy2J) /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-β1 and Pro-collagen 1α2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-β1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease

Studies of human diseased aortic valves have demonstrated increased expression of genetic markers of valve progenitors and osteogenic differentiation associated with pathogenesis. Three potential mouse models of valve disease were examined for cellular pathology, morphology, and induction of valvulogenic, chondrogenic, and osteogenic markers. Osteogenesis imperfecta murine (Oim) mice, with a mutation in Col1a2, have distal leaflet thickening and increased proteoglycan composition characteristic of myxomatous valve disease. Periostin null mice also exhibit dysregulation of the ECM with thickening in the aortic midvalve region, but do not have an overall increase in valve leaflet surface area. Klotho null mice are a model for premature aging and exhibit calcific nodules in the aortic valve hinge-region, but do not exhibit leaflet thickening, ECM disorganization, or inflammation. Oim/oim mice have increased expression of valve progenitor markers Twist1, Col2a1, Mmp13, Sox9 and Hapln1, in addition to increased Col10a1 and Asporin expression, consistent with increased proteoglycan composition. Periostin null aortic valves exhibit relatively normal gene expression with slightly increased expression of Mmp13 and Hapln1. In contrast, Klotho null aortic valves have increased expression of Runx2, consistent with the calcified phenotype, in addition to increased expression of Sox9, Col10a1, and osteopontin. Together these studies demonstrate that oim/oim mice exhibit histological and molecular characteristics of myxomatous valve disease and Klotho null mice are a new model for calcific aortic valve disease.

IL‐13 regulates cancer invasion and metastasis through IL‐13Rα2 via ERK/AP‐1 pathway in mouse model of human ovarian cancer

Previously, we have demonstrated that a variety of human cancers including the ovarian cancer express IL-13Rα2, a high affinity receptor for IL-13. Herein, we have examined if IL-13 regulates invasion and metastasis of ovarian cancer through IL-13Rα2 in vitro and in vivo in animal models of human ovarian cancer. We tested cell invasion and protease activity in IL-13Rα2-overexpressing and IL-13Rα2-negative ovarian tumor cell lines. IL-13 treatment significantly augmented both cell invasion and enzyme activities in only IL-13Rα2-positive cells but not in IL-13Rα2-negative cells in vitro. Mechanistically, IL-13 enhanced ERK1/2, AP-1 and MMP activities only in IL-13Rα2-positive cells but not in IL-13Rα2-negative cells. In contrast, other signaling pathways such as IRS1/2, PI3K and AKT do not seem to be involved in IL-13 induced signaling in ovarian cancer cell lines. Highly specific inhibitors for MMP and AP-1 efficiently inhibited both invasion and protease activities without impacting the basal level invasion and protease activities in vitro. In orthotopic animal model of human ovarian cancer, IL-13Rα2-positive tumors metastasized to lymph nodes and peritoneum earlier than IL-13Rα2-negative tumors. Interestingly, the IL-13Rα2-positive tumor bearing mice died earlier than mice with IL-13Rα2-negative tumor. Intraperitoneal injection of IL-13 further shortened survival of IL-13Rα2-positive tumor bearing mice compared to IL-13Rα2-negative tumor mice. IL-13Rα2-positive tumors and lymph node metastasis expressed higher levels of MMPs and higher ERK1/2 activation compared to IL-13Rα2-negative tumors. Taken together, IL-13Rα2 is involved in cancer metastasis through activation of ERK/AP-1 and that targeting IL-13Rα2 might not only directly kill primary tumors but also prevent cancer metastasis.

Two-Photon Imaging of the Mouse Eye

To image the conventional aqueous outflow pathway and adjacent structures within the intact enucleated mouse eye using a noninvasive microscopy technique. Two-photon microscopy (2PM) techniques, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to obtain images of the trabecular meshwork (TM) region within an intact mouse eye. Cardiac perfusion of fluorescein-conjugated dextran was used to label blood vessels within the eye to serve as an anatomic reference. Eyes were subsequently fixed, paraffin embedded, sectioned, and stained for comparison to the 2PM images. Three-dimensional analyses of multiple 2PM images revealed a well-defined region adjacent to the iris and cornea that is free of SHG signal and consistent with the location of Schlemm's canal. This open region is continuous with smaller tube structures consistent with collector channels. These structures do not label in mice perfused with the vascular probe dextran, supporting the hypothesis that the enclosed spaces are filled with aqueous humor rather than circulating blood. The TM region in the mouse eye was also visible, with a clear SHG signal representing collagen fibers. These results support the hypothesis that 2PM may be useful for noninvasively imaging the conventional aqueous outflow pathway in mouse models of glaucoma. Studies are ongoing to validate our methodology in live animals.

Deletion of the p85α Regulatory Subunit of Phosphoinositide 3-Kinase in Cone Photoreceptor Cells Results in Cone Photoreceptor Degeneration

Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit. Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy. An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice. The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Abstract 715: TGF-beta blockade improves the distribution and efficacy of chemotherapeutic agents in breast carcinoma

Abstract Although the role of TGF-β has been studied extensively, its impact upon drug delivery within tumors remains poorly understood. In this study, we blocked the TGF-β pathway in mouse models of breast cancer and studied the effect on delivery and efficacy of chemotherapeutics. We used both genetic (overexpression of sTβRII, a soluble TGF-β type II receptor) and pharmaceutical (1D11, a TGF-β antibody) approaches to block the TGF-β signaling in two orthotopic breast cancer models (human MDA-MB-231 cells and murine 4T1 cells). TGF-β blockade significantly decreased the growth and metastasis of orthotopic human breast carcinoma xenografts. In addition, TGF-β blockade increased the recruitment and incorporation of myofibroblasts into tumor blood vessels and restored vascular perfusion. Moreover, TGF-β blockade normalized the tumor extracellular matrix (ECM) by decreasing collagen I content. As a result of this vessel and ECM normalization, TGF-β blockade improved the transvascular and interstitial transport of both small and large molecular weight chemotherapeutic agents, and consequently enhanced their efficacy. In conclusion, our study shows that TGF-β blockade might represent a new approach to improve delivery of both low molecular weight conventional drugs as well as nanomedicines in order to maximize therapeutic outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 715. doi:10.1158/1538-7445.AM2011-715

Altered Balance of Activity in the Striatal Direct and Indirect Pathways in Mouse Models of Huntington's Disease

Imbalance in the activity of striatal direct and indirect pathway neurons contributes to motor disturbances in several neurodegenerative diseases. In Huntington's disease (HD), indirect pathway [dopamine (DA) D2 receptor-expressing] medium-sized spiny neurons (MSNs) are believed to show earlier vulnerability than direct pathway MSNs. We examined synaptic activity and DA modulation in MSNs forming the direct and indirect pathways in YAC128 and BACHD mouse models of HD. To visualize the two types of MSNs, we used mice expressing enhanced green fluorescent protein under the control of the promoter for the DA D1 or D2 receptor. Experiments were performed in early symptomatic (1.5 months) and symptomatic (12 months) mice. Behaviorally, early symptomatic mice showed increased stereotypies while symptomatic mice showed decreased motor activity. Electrophysiologically, at the early stage, excitatory and inhibitory transmission onto D1-YAC128 and D1-BACHD MSNs were increased, while there was no change in D2 MSNs. DA modulation of spontaneous excitatory postsynaptic currents (sEPSCs) in slices was absent in YAC128 cells at the early stage, but was restored by treating the slices with the DA depleter tetrabenazine (TBZ). In BACHD mice TBZ restored paired-pulse ratios and a D1 receptor antagonist induced a larger decrease of sEPSCs than in D1-WT cells, suggesting increased DA tone. Finally, TBZ decreased stereotypies in BACHD mice. These results indicate that by reducing DA or antagonizing D1 receptors, increases in inhibitory and excitatory transmission in early phenotypic direct pathway neurons can be normalized. In symptomatic YAC128 mice, excitatory synaptic transmission onto D1 MSNs was decreased, while inhibitory transmission was increased in D2 MSNs. These studies provide evidence for differential and complex imbalances in glutamate and GABA transmission, as well as in DA modulation, in direct and indirect pathway MSNs during HD progression.

Treating cancer cachexia to treat cancer

Skeletal muscle wasting is a major component of cachectic states found in a variety of disease settings, including cancer. As increasing caloric intake often provides little benefit in combating muscle loss in cachectic patients, a major research focus has been to develop strategies stimulating muscle anabolic pathways - in an attempt to fight the catabolic pathways induced during cachexia. Two recent papers have reported the beneficial effects of blocking the myostatin/activin signalling pathway in mouse models of cancer cachexia. We discuss the implications of their findings both with respect to the role that this signalling pathway may play in the aetiology of cachexia and with respect to the prospects for targeting this pathway as a therapeutic strategy in patients with cachexia.

Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders

Glomerular diseases account for 90% of end-stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowman's capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S-phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G2/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch-regulated balance between podocyte death and regeneration provided by renal progenitors. Stem Cells 2010; 28:1674–1685.

Canonical and Non Canonical Activation of Hedgehog Pathway in Multiple Myeloma

To identify genes or Tumor-Associated Antigens (TAA) potentially involved in the transition from Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) and in the MM pathogenesis, we used SEREX and screened serum of MGUS and MM patients with a MM cDNA library. We identified 10 novel antigens and focused our attention on Oral-facial-digital syndrome 1 gene (Ofd1) due to a specific antibody response in MGUS (20.6%) as well as post-transplant MM patients (27.2%). Ofd1 is an embryonal antigen with an important role in the organogenesis and fetal survival; however, in the adult life its expression decreases significantly and is restricted to few tissues. Additionally it is a basal body/centrosome-related protein and a component of Tip60-HAT complex which is involved in cilia formation and functional regulation of Hedgehog (Hh)- and Wnt- pathways in mouse models. We first studied the expression of Ofd1 by western blotting analysis and observed that 11 MM cell lines and 4 primary MM patient cells express high levels compared with PBMCs. To define its role in MM pathogenesis, we specifically knocked-down Ofd1 using 100nM and 200nM of specific siRNA in MM1s cell line. Time-dependent but dose-independent inhibition of Ofd1 does not affect viability and proliferation of MM1s at 48h, as assayed by MTT and thymidine-uptake, respectively. Since mutations in proteins of cilia may block the Hh-signal transduction downstream to Smoothened (Smo) but upstream to Gli transcription factors, we next evaluated the effects of Ofd1 inhibition on the expression of Gli1 and Patched1 (Ptch1), hallmarks of Hh-activity. Western blotting analysis showed that specific knock-down of Ofd1 leads to down-regulation of Gli1 or Ptch1, and β-catenin. Therefore Ofd1 has a role in the functional regulation of Hh- and Wnt-pathway, and its overexpression might lead to constitutive activation of these tumorigenic pathways. These data strongly indicate the existence of an alternative and not canonical activation of Hh-pathway in MM. We also investigated if the canonical way is active in MM. To demonstrate that Hh-pathway is important in CD138+ MM patient cells, we used double staining with antibody anti-CD138 and anti-Sonic (Shh) to show that MM cells co-express Shh, the most common Hh-ligand, suggesting that MM cells might have autocrine and/or paracrine Hh-activity. We confirmed the expression of Gli1 and Ptch1, by western blotting, in 10 on 11 MM cell lines tested and using antibody anti-Gli1 we demonstrated, by immunofluorescence, predominant nuclear localization of Gli1 suggesting constitutive Hh-pathway activation. Treatment with 5μM of a specific Smo-inhibitor for 12–24 and 36 hours, induces down-regulation of Gli1 and Ptch1 in 50% MM cell lines. While down-regulation of Ptch1, a Gli-target gene, is an early event starting at 12 hours, down-regulation of Gli1 starts later at 36 hours. Therefore Gli1 is not the most important transducer of Hh-signaling in MM. In 50% MM cell lines, however, we didn't observe any down-regulation of Gli1 and Ptch1 after treatment with Smo-inhibitor; in these MM cell lines, the predominant nuclear localization of Gli1 is completely abrogated by treatment for 24 hours with 10μM of forskolin, a compound inhibiting the nuclear translocation of Gli1 via PKA. Taken together our data show canonical as well as alternative not canonical activation of Hh-pathway associated with overexpression of ciliary protein Ofd1. Further delineation of the role of primary cilia in regulating cellular response to Hh-signaling in MM will both define their role in disease pathogenesis and support novel therapies targeting Hh-signaling to improve patient outcome in MM.

Respiratory virus and asthma: The role of immunoglobulin E

Background: Atopic diseases, including asthma, have increased to epidemic proportions in the westernized world. The reasons for this increase are not known, nor are the mechanisms behind the development of these diseases. An interesting aspect of atopic disease is the role of respiratory viruses in the development of asthma and atopy. In fact, severe respiratory viral infection in infancy has been associated with a greatly increased risk of asthma. Objective: This paper explores potential mechanisms through which viruses impart an increased risk of asthma, focusing on new pathways in mouse models of atopy. Methods: A search of MEDLINE (1950-March 2008) was conducted using terms that included viral-induced wheeze, respiratory virus, asthma, IgE, and dendritic cells. Results: A total of 1643 publications were identified that contained ≥ 1 of the search terms; however, only 7 of these focused on immunoglobulin E (IgE) and the viral risk of asthma, and only 1 of the 7 explored the role of dendritic cells in this process. The latter study suggested a mechanistic link between lung dendritic cells and the development of postviral atopic disease. Important in this pathway is the generation of IgE, its high-affinity receptor, and the T-cell chemoattractant CCL28. Conclusions: Data from recent mouse models suggest that the development of asthma after severe respiratory viral infection may be the result of a response generated by production of antiviral IgE, which is capable of engaging dendritic cells to form a chemoattractant for interleukin-13-producing T cells. This new paradigm points to a focus for development of future therapies to prevent or at least ameliorate post- viral atopic disease.