A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology.

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors-reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon-gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology

Novel Functions for Reelin Signaling in Mammary Gland Development and Breast Cancer Progression

The reelin signaling pathway is a critical regulator of cell migration and positioning throughout the central nervous system. Recent studies identified non-neuronal functions for reelin, and reported alterations in reelin expression in cancers. It is not known whether the reelin signaling pathway plays a role in the development of the mammary gland or in progression of breast cancer. Using mice with mutations in reelin and Disabled-1 (Dab1), the intracellular adaptor protein activated in response to the reelin signal, we showed that the reelin signaling pathway is required for correct ductal patterning during mammary gland morphogenesis. Reelin and Dab1 are expressed in the developing and the mature mammary duct in a complementary pattern. Mutations in reelin or Dab1 resulted in delayed ductal elongation and disorganized mammary epithelium in mice. Mammary epithelial cells exhibited decreased migration in response to exogenous reelin in a Dab1-dependent manner in vitro. Using a syngeneic mouse mammary tumor transplantation model, we examined the effect of host-derived reelin on breast cancer progression. We found that transplanted tumors grew and metastasized more slowly in reelin-deficient mice. Tumors grown in reelin-deficient animals had fewer blood vessels and increased macrophage infiltration. Loss of host-derived reelin altered the balance of M1- and M2-associated macrophage markers in primary tumors, suggesting that reelin may influence the polarization of tumor-associated macrophages. These results indicated an important function for the reelin protein in progression of breast cancer. Furthermore, we found that several alternatively spliced variants of Dab1 exist in the developing and the mature mammary gland. In addition, we showed that alternative splicing of Dab1 is spatially and temporally regulated during puberty, pregnancy, and lactation. Taken together, our findings suggest that the reelin signaling pathway is an essential regulator of mammary gland development and has critical functions in progression of breast cancer

Novel Functions for Reelin Signaling in Mammary Gland Development and Breast Cancer Progression

The reelin signaling pathway is a critical regulator of cell migration and positioning throughout the central nervous system. Recent studies identified non-neuronal functions for reelin, and reported alterations in reelin expression in cancers. It is not known whether the reelin signaling pathway plays a role in the development of the mammary gland or in progression of breast cancer. Using mice with mutations in reelin and Disabled-1 (Dab1), the intracellular adaptor protein activated in response to the reelin signal, we showed that the reelin signaling pathway is required for correct ductal patterning during mammary gland morphogenesis. Reelin and Dab1 are expressed in the developing and the mature mammary duct in a complementary pattern. Mutations in reelin or Dab1 resulted in delayed ductal elongation and disorganized mammary epithelium in mice. Mammary epithelial cells exhibited decreased migration in response to exogenous reelin in a Dab1-dependent manner in vitro. Using a syngeneic mouse mammary tumor transplantation model, we examined the effect of host-derived reelin on breast cancer progression. We found that transplanted tumors grew and metastasized more slowly in reelin-deficient mice. Tumors grown in reelin-deficient animals had fewer blood vessels and increased macrophage infiltration. Loss of host-derived reelin altered the balance of M1- and M2-associated macrophage markers in primary tumors, suggesting that reelin may influence the polarization of tumor-associated macrophages. These results indicated an important function for the reelin protein in progression of breast cancer. Furthermore, we found that several alternatively spliced variants of Dab1 exist in the developing and the mature mammary gland. In addition, we showed that alternative splicing of Dab1 is spatially and temporally regulated during puberty, pregnancy, and lactation. Taken together, our findings suggest that the reelin signaling pathway is an essential regulator of mammary gland development and has critical functions in progression of breast cancer

Decreased Reelin Expression and Organophosphate Pesticide Exposure Alters Mouse Behaviour and Brain Morphology

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders, including ASDs (autism spectrum disorders). In this study, we examined the combinatorial effect of two factors thought to be involved in autism – reduction in the expression of the extracellular matrix protein reelin and prenatal exposure to an organophosphate pesticide, CPO (chlorpyrifos oxon). Mice with reduced reelin expression or prenatal exposure to CPO exhibited subtle changes in ultrasound vocalization, open field behaviour, social interaction and repetitive behaviour. Paradoxically, mice exposed to both variables often exhibited a mitigation of abnormal behaviours, rather than increased behavioural abnormalities as expected. We identified specific differences in males and females in response to both of these variables. In addition to behavioural abnormalities, we identified anatomical alterations in the olfactory bulb, piriform cortex, hippocampus and cerebellum. As with our behavioural studies, anatomical alterations appeared to be ameliorated in the presence of both variables. While these observations support an interaction between loss of reelin expression and CPO exposure, our results suggest a complexity to this interaction beyond an additive effect of individual phenotypes

Reelin Deficiency Delays Mammary Tumor Growth and Metastatic Progression

Reelin is a regulator of cell migration in the nervous system, and has other functions in the development of a number of non-neuronal tissues. In addition, alterations in reelin expression levels have been reported in breast, pancreatic, liver, gastric, and other cancers. Reelin is normally expressed in mammary gland stromal cells, but whether stromal reelin contributes to breast cancer progression is unknown. Herein, we used a syngeneic mouse mammary tumor transplantation model to examine the impact of host-derived reelin on breast cancer progression. We found that transplanted syngeneic tumors grew more slowly in reelin-deficient (rl Orl -/- ) mice and had delayed metastatic colonization of the lungs. Immunohistochemistry of primary tumors revealed that tumors grown in rl Orl -/- animals had fewer blood vessels and increased macrophage infiltration. Gene expression studies from tumor tissues indicate that loss of host-derived reelin alters the balance of M1- and M2-associated macrophage markers, suggesting that reelin may influence the polarization of these cells. Consistent with this, rl Orl -/- M1-polarized bone marrow-derived macrophages have heightened levels of the M1-associated cytokines iNOS and IL-6. Based on these observations, we propose a novel function for the reelin protein in breast cancer progression

Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling.

It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals

Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

Summary: It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals. : Argus et al. developed Fatty Acid Source Analysis (FASA), a model that quantifies cellular fatty acid synthesis, elongation, and import. FASA is used to demonstrate that elongation can be a major contributor to cellular fatty acid content and that different stimuli reprogram macrophage fatty acid elongation pathways in distinct ways. Keywords: fatty acid homeostasis, fatty acid modeling, stable isotope labelin

Toll-Like Receptors Induce Signal-Specific Reprogramming of the Macrophage Lipidome

Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems-level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary "shotgun" and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. "Shotgun" lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity