Converging evidence points to a neural network that supports a range of abilities including remembering the past, thinking about the future, and introspecting about oneself and others. Neuroimaging studies find hippocampal activation during event construction tasks, and patients with hippocampal amnesia are impaired in their ability to (re)construct events of the past and the future. Neuroimaging studies of constructed experiences similarly implicate the medial prefrontal cortex (mPFC), but it remains unknown whether the mPFC is critical for such processes. The current study compares performance of five patients with bilateral mPFC damage, six patients with bilateral hippocampal damage, and demographically matched comparison participants on an event construction task. Participants were given a neutral cue word and asked to (re)construct events across four time conditions: real past, imagined past, imagined present, and future. These event narratives were analyzed for the number of internal and external details to quantify the extent of episodic (re)experiencing. Given the literature on the involvement of the mPFC in self-referential processing, we also analyzed the event narratives for self-references. The patients with mPFC damage did not differ from healthy comparison participants in their ability to construct highly detailed episodic events across time periods but displayed disruptions in their incorporation of the self. Patients with hippocampal damage showed the opposite pattern; they were impaired in their ability to construct highly detailed episodic events across time periods but not in their incorporation of the self. The results suggest differential contributions of hippocampus and medial prefrontal cortex to the distributed neural network for various forms of self-projection.
BackgroundMicroglia safeguard the CNS against injuries and pathogens, and in the presence of certain harmful stimuli are capable of inducing a disease-dependent inflammatory response. When exposed to anti-inflammatory cytokines, however, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth.ObjectiveThe tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies.MethodsSerum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGFβ, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways.ResultsThe proteomic analysis of HMC3 cells enabled the identification of ~10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGFβ, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, was indicative of ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that link ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, vesicular/viral transport, and secretory processes. Immunosuppressive and proangiogenic chemokines, as well as anti-angiogenic factors, were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors, signaling and ECM proteins indicated the presence of such capabilities.ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a spectrum of microglia phenotypes supportive of cancer development in the brain via microenvironment-dependent biological mechanisms.
The response of cells to physical or chemical stimuli is complex, unfolding on time-scales from seconds to days, with or without de novo protein synthesis, and involving signaling processes that are transient or sustained. By combining the technology of microfluidics that supports fast and precise execution of a variety of cell handling operations, with that of mass spectrometry detection that facilitates an accurate and complex characterization of the protein complement of cells, in this work, we developed a platform that supports (near) real-time sampling and proteome-level capturing of cellular responses to a perturbation such as treatment with mitogens. The geometric design of the chip supports three critical features: (a) capture of a sufficient number of cells to meet the detection limit requirements of mass spectrometry instrumentation, (b) fluid delivery for uniform stimulation of the resident cells, and (c) fast cell recovery, lysis and processing for accurate sampling of time-sensitive cellular responses to a stimulus. COMSOL simulations and microscopy were used to predict and evaluate the flow behavior inside the microfluidic device. Proteomic analysis of the cellular extracts generated by the chip experiments revealed that the identified proteins were representative of all cellular locations, exosomes, and major biological processes related to proliferation and signaling, demonstrating that the device holds promising potential for integration into complex lab-on-chip work-flows that address systems biology questions. The applicability of the chips to study time-sensitive cellular responses is discussed in terms of technological challenges and biological relevance.
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