Morphological Representation of C1q in the Aging Central Nervous System.

INTRODUCTION: The complement protein C1q is essential for the innate immune system and neurophysiological and neuropathological processes. To gain more insight into these functions in the CNS, a comprehensive understanding of the morphological representation, especially of its cellular and subcellular target structures, is of great importance. METHODS: For a free-floating preparation, the brains of wild-type and ArcAß mice were cut into 100 µm slices. Living slices were incubated in Ringer's solution and then fixed in 4% paraformaldehyde (PFA) and stained with different primary and secondary antibodies or methoxy-X04. RESULTS: C1q was abundant in the entire brain. Interestingly, C1q accumulated around cell nuclei, with a perineuronal localization around neuronal somata and a paraneuronal accumulation around non-neuronal cells, e. g., microglia. Moreover, dendritic-like, linear, branched C1q signals were observed in the area between the dentate gyrus and the CA1 region of the hippocampus. Complementary staining revealed an overlap with ß-amyloid accumulation reflected by the deposition of C1q within plaques and modified basal C1q levels in the brains of transgenic ArcAß animals. DISCUSSION: The applied free-floating approach is suitable for C1q immunofluorescence imaging. The consistent colocalization of the complement protein C1q with ß-amyloid plaques may reflect an activated immune response, whereas the accumulation of C1q around neuronal structures such as somata and dendrites is still a matter of debate. Intriguingly, C1q surrounds those structures in older brains of both wild-type and ArcAß mice. Our results also indicate an involvement of C1q in neurophysiological and neurodegenerative processes.

CARM1 regulates senescence during airway epithelial cell injury in COPD pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a life-threatening lung disease. Although cigarette smoke was considered the main cause of development, the heterogeneous nature of the disease leaves it unclear whether other factors contribute to the predisposition or impaired regeneration response observed. Recently, epigenetic modification has emerged to be a key player in the pathogenesis of COPD. The addition of methyl groups to arginine residues in both histone and nonhistone proteins by protein arginine methyltransferases (PRMTs) is an important posttranslational epigenetic modification event regulating cellular proliferation, differentiation, apoptosis, and senescence. Here, we hypothesize that coactivator-associated arginine methyltransferase-1 (CARM1) regulates airway epithelial cell injury in COPD pathogenesis by controlling cellular senescence. Using the naphthalene (NA)-induced mouse model of airway epithelial damage, we demonstrate that loss of CC10-positive club cells is accompanied by a reduction in CARM1-expressing cells of the airway epithelium. Furthermore, Carm1 haploinsuffficent mice showed perturbed club cell regeneration following NA treatment. In addition, CARM1 reduction led to decreased numbers of antisenescent sirtuin 1-expressing cells accompanied by higher p21, p16, and ß-galactosidase-positive senescent cells in the mouse airway following NA treatment. Importantly, CARM1-silenced human bronchial epithelial cells showed impaired wound healing and higher ß-galactosidase activity. These results demonstrate that CARM1 contributes to airway repair and regeneration by regulating airway epithelial cell senescence.

Coactivator-Associated Arginine Methyltransferase-1 Function in Alveolar Epithelial Senescence and Elastase-Induced Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases worldwide. A major feature of COPD is emphysema, which is the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone methylation and the transcription of genes involved in senescence, proliferation, and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type II (ATII) cells. We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and small interfering (si)RNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5 ± 9.6 µm versus 38.8 ± 5.5 µm; P < 0.01) and lung compliance (2.8 ± 0.32 µl/cm H2O versus 2.4 ± 0.4 µl/cm H2O; P < 0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased sirtuin 1 expression (0.034 ± 0.003 versus 0.022 ± 0.001; P < 0.05) but increased expression of p16 (0.27 ± 0.013 versus 0.31 ± 0.010; P < 0.5) and p21 (0.81 ± 0.088 versus 1.28 ± 0.063; P < 0.01) and higher ß-galactosidase-positive senescent cells (50.57 ± 7.36% versus 2.21 ± 0.34%; P < 0.001) compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs transdifferentiation and wound healing (32.18 ± 0.9512% versus 8.769 ± 1.967%; P < 0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence as well as impaired regeneration, repair, and differentiation of ATII cells.

Multiplexed imaging and automated signal quantification in formalin-fixed paraffin-embedded tissues by ChipCytometry.

Deciphering the spatial composition of cells in tissues is essential for detailed understanding of biological processes in health and disease. Recent technological advances enabled the assessment of the enormous complexity of tissue-derived parameters by highly multiplexed tissue imaging (HMTI), but elaborate machinery and data analyses are required. This severely limits broad applicability of HMTI. Here we demonstrate for the first time the application of ChipCytometry technology, which has unique features for widespread use, on formalin-fixed paraffin-embedded samples, the most commonly used storage technique of clinically relevant patient specimens worldwide. The excellent staining quality permits workflows for automated quantification of signal intensities, which we further optimized to compensate signal spillover from neighboring cells. In combination with the high number of validated markers, the reported platform can be used from unbiased analyses of tissue composition to detection of phenotypically complex rare cells, and can be easily implemented in both routine research and clinical pathology.

Integrin αvß3-dependent thyroid hormone effects on tumour proliferation and vascularisation.

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvß3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvß3, in two murine xenograft tumour models with and without integrin αvß3 expression. Integrin αvß3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvß3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvß3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvß3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.