Cancer-associated epithelial cell adhesion molecule (EpCAM; CD326) enables epidermal Langerhans cell motility and migration in vivo

After activation, Langerhans cells (LC), a distinct subpopulation of epidermis-resident dendritic cells, migrate from skin to lymph nodes where they regulate the magnitude and quality of immune responses initiated by epicutaneously applied antigens. Modulation of LC-keratinocyte adhesion is likely to be central to regulation of LC migration. LC express high levels of epithelial cell adhesion molecule (EpCAM; CD326), a cell-surface protein that is characteristic of some epithelia and many carcinomas and that has been implicated in intercellular adhesion and metastasis. To gain insight into EpCAM function in a physiologic context in vivo, we generated conditional knockout mice with EpCAM-deficient LC and characterized them. Epidermis from these mice contained increased numbers of LC with normal levels of MHC and costimulatory molecules and T-cell-stimulatory activity in vitro. Migration of EpCAM-deficient LC from skin explants was inhibited, but chemotaxis of dissociated LC was not. Correspondingly, the ability of contact allergen-stimulated, EpCAM-deficient LC to exit epidermis in vivo was delayed, and strikingly fewer hapten-bearing LC subsequently accumulated in lymph nodes. Attenuated migration of EpCAM-deficient LC resulted in enhanced contact hypersensitivity responses as previously described in LC-deficient mice. Intravital microscopy revealed reduced translocation and dendrite motility in EpCAM-deficient LC in vivo in contact allergen-treated mice. These results conclusively link EpCAM expression to LC motility/migration and LC migration to immune regulation. EpCAM appears to promote LC migration from epidermis by decreasing LC-keratinocyte adhesion and may modulate intercellular adhesion and cell movement within in epithelia during development and carcinogenesis in an analogous fashion

Testis Developmental Phenotypes in Neurotropin Receptor trkA and trkC Null Mutations: Role in Formation of Seminiferous Cords and Germ Cell Survival

The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation

A coordinate deregulation of microRNAs expressed in mucosa adjacent to tumor predicts relapse after resection in localized colon cancer

Up to 20% of colorectal cancer (CRC) node-negative patients develop loco-regional or distant recurrences within 5 years from surgery. No predictive biomarker able to identify the node-negative subjects at high risk of relapse after curative treatment is presently available.Forty-eight localized (i.e. stage I-II) colon cancer patients who underwent radical tumor resection were considered. The expression of five miRNAs, involved in CRC progression, was investigated by qRT-PCR in both tumor tissue and matched normal colon mucosa.Interestingly, we found that the coordinate deregulation of four miRNAs (i.e. miR-18a, miR-21, miR-182 and miR-183), evaluated in the normal mucosa adjacent to tumor, is predictive of relapse within 55 months from curative surgery.Our results, if confirmed in independent studies, may help to identify high-risk patients who could benefit most from adjuvant therapy. Moreover, this work highlights the importance of extending the search for tissue biomarkers also to the tumor-adjacent mucosa

Nedd4-2 haploinsufficiency causes hyperactivity and increased sensitivity to inflammatory stimuli

Nedd4-2 (NEDD4L in humans) is a ubiquitin protein ligase best known for its role in regulating ion channel internalization and turnover. Nedd4-2 deletion in mice causes perinatal lethality associated with increased epithelial sodium channel (ENaC) expression in lung and kidney. Abundant data suggest that Nedd4-2 plays a role in neuronal functions and may be linked to epilepsy and dyslexia in humans. We used a mouse model of Nedd4-2 haploinsufficiency to investigate whether an alteration in Nedd4-2 levels of expression affects general nervous system functions. We found that Nedd4-2 heterozygous mice are hyperactive, have increased basal synaptic transmission and have enhanced sensitivity to inflammatory pain. Thus, Nedd4-2 heterozygous mice provide a new genetic model to study inflammatory pain. These data also suggest that in human, SNPs affecting NEDD4L levels may be involved in the development of neuropsychological deficits and peripheral neuropathies and may help unveil the genetic basis of comorbidities

The Cytoskeletal Adaptor Protein Band 4.1B Is Required for the Maintenance of Paranodal Axoglial Septate Junctions in Myelinated Axons

Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here we report the generation and characterization of 4.1B null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axo-glial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after one year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at about one year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons

Overexpression of Parkinson's Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and alpha-Synuclein Pathology

Citation: Xiong, Y. L., Neifert, S., Karuppagounder, S. S., Stankowski, J. N., Lee, B. D., Grima, J. C., . . . Dawson, V. L. (2017). Overexpression of Parkinson's Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and alpha-Synuclein Pathology. Eneuro, 4(2), 10. https://doi.org/10.1523/eneuro.0004-17.2017Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an unambiguous cause of late-onset, autosomal-dominant familial Parkinson's disease (PD) and LRRK2 mutations are the strongest genetic risk factor for sporadic PD known to date. A number of transgenic mice expressing wild-type or mutant LRRK2 have been described with varying degrees of LRRK2-related abnormalities and modest pathologies. None of these studies directly addressed the role of the kinase domain in the changes observed and none of the mice present with robust features of the human disease. In an attempt to address these issues, we created a conditional LRRK2 G2019S (LRRK2 GS) mutant and a functionally negative control, LRRK2 G2019S/D1994A (LRRK2 GS/DA). Expression of LRRK2 GS or LRRK2 GS/DA was conditionally controlled using the tet-off system in which the presence of tetracycline-transactivator protein (tTA) with a CAMKII alpha promoter (CAMKII alpha-tTA) induced expression of TetP-LRRK2 GS or TetP-LRRK2 GS/DA in the mouse forebrain. Overexpression of LRRK2 GS in mouse forebrain induced behavioral deficits and alpha-synuclein pathology in a kinase-dependent manner. Similar to other genetically engineered LRRK2 GS mice, there was no significant loss of dopaminergic neurons. These mice provide an important new tool to study neurobiological changes associated with the increased kinase activity from the LRRK2 G2019S mutation, which may ultimately lead to a better understanding of not only the physiologic actions of LRRK2, but also potential pathologic actions that underlie LRRK2 GS-associated PD

Inhibition of CXCR2 Plays a Pivotal Role in Re-Sensitizing Ovarian Cancer to Cisplatin Treatment

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatinresistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients’ high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.acknowledge financial support from CAPES, FAPES and CNPq, as well as the Biotechnology Program/ RENORBIO from the Federal University of Espirito Santo, Espirito Santo, Brazil; Institute of Pathology and Molecular Immunology (IPATIMUP) and the Institute of Innovation and Health Research (I3s), Porto, Portugal

Deletion of the BDNF Truncated Receptor TrkB.T1 Delays Disease Onset in a Mouse Model of Amyotrophic Lateral Sclerosis

Brain Derived Neurotrophic Factor (BDNF) exerts strong pro-survival effects on developing and injured motoneurons. However, in clinical trials, BDNF has failed to benefit patients with amyotrophic lateral sclerosis (ALS). To date, the cause of this failure remains unclear. Motoneurons express the TrkB kinase receptor but also high levels of the truncated TrkB.T1 receptor isoform. Thus, we investigated whether the presence of this receptor may affect the response of diseased motoneurons to endogenous BDNF. We deleted TrkB.T1 in the hSOD1G93A ALS mouse model and evaluated the impact of this mutation on motoneuron death, muscle weakness and disease progression. We found that TrkB.T1 deletion significantly slowed the onset of motor neuron degeneration. Moreover, it delayed the development of muscle weakness by 33 days. Although the life span of the animals was not affected we observed an overall improvement in the neurological score at the late stage of the disease. To investigate the effectiveness of strategies aimed at bypassing the TrkB.T1 limit to BDNF signaling we treated SOD1 mutant mice with the adenosine A2A receptor agonist CGS21680, which can activate motoneuron TrkB receptor signaling independent of neurotrophins. We found that CGS21680 treatment slowed the onset of motor neuron degeneration and muscle weakness similarly to TrkB.T1 removal. Together, our data provide evidence that endogenous TrkB.T1 limits motoneuron responsiveness to BDNF in vivo and suggest that new strategies such as Trk receptor transactivation may be used for therapeutic intervention in ALS or other neurodegenerative disorders

Altering a Histone H3K4 Methylation Pathway in Glomerular Podocytes Promotes a Chronic Disease Phenotype

Methylation of specific lysine residues in core histone proteins is essential for embryonic development and can impart active and inactive epigenetic marks on chromatin domains. The ubiquitous nuclear protein PTIP is encoded by the Paxip1 gene and is an essential component of a histone H3 lysine 4 (H3K4) methyltransferase complex conserved in metazoans. In order to determine if PTIP and its associated complexes are necessary for maintaining stable gene expression patterns in a terminally differentiated, non-dividing cell, we conditionally deleted PTIP in glomerular podocytes in mice. Renal development and function were not impaired in young mice. However, older animals progressively exhibited proteinuria and podocyte ultra structural defects similar to chronic glomerular disease. Loss of PTIP resulted in subtle changes in gene expression patterns prior to the onset of a renal disease phenotype. Chromatin immunoprecipitation showed a loss of PTIP binding and lower H3K4 methylation at the Ntrk3 (neurotrophic tyrosine kinase receptor, type 3) locus, whose expression was significantly reduced and whose function may be essential for podocyte foot process patterning. These data demonstrate that alterations or mutations in an epigenetic regulatory pathway can alter the phenotypes of differentiated cells and lead to a chronic disease state