Signalling pathways involved in the inhibition and promotion of axonal regeneration

Axonal regeneration following nerve injury requires the complex orchestration of various molecular events. Neurite outgrowth can be initiated by a variety of cues from the extracellular environment, including neurotrophins (e.g. nerve growth factor; NGF) and the extracellular matrix (ECM). Biological responses to neurotrophins are mediated by two distinct receptors: Trks, which initiate distinct signalling for the promotion of growth and survival, and p75NTR. Signalling pathways initiated by p75NTR exhibit considerable complexity and can elicit a variety of paradoxical physiological responses depending upon cellular context. Biological responses to ECM components, including laminin (LN), are mediated by a group of receptors known as integrins, which facilitate signal transmission to regulate cellular behaviour. Signalling pathways initiated by NGF and LN, have been previously reported to synergize resulting in optimized axonal regeneration of sensory neurons. The present series of studies sought to explore the molecular mechanisms underlying the enhanced growth initiated by stimulation with neurotrophins and LN, with a particular focus on the roles of the NGF receptors; specifically, TrkA phosphorylation-induced signalling cascades, and events associated with p75NTR ligand-dependent and independent signalling. -- My initial studies utilized a series of PC 12 cell derivatives expressing TrkA phosphorylation mutants, to investigate the potential role of TrkA in the regulation of p75NTR expression. I determined that TrkA played a role in the regulation of constitutive p75NTR expression, and further, controlled the upregulation of p75NTR in response to neurotrophin stimulation. In a subsequent study, I demonstrated that this occurred via a Phospholipase C γ-Protein Kinase C δ-dependent mechanism, and confirmed the existence of this regulatory pathway in cerebellar granule neurons (CGN). I further investigated the contribution of the ECM to regenerative growth, in both its capacity to signal synergistically with the TrkA receptor for the enhancement of early signalling intermediates, and its ability to elicit growth in a neurotrophin-independent scenario. Strikingly, integrin activation in the absence of neurotrophins was responsible for the promotion of neurite outgrowth via a rapid and potent Egr-1-dependent increase in the expression of the phosphatase PTEN, which relocalized to the nucleus where it dephosphorylated transcription factor Spl, thereby decreasing its ability to bind to the p75NTR promoter, resulting in the subsequent downregulation of p75NTR and depression of Rho activity. This novel ECM-induced signalling paradigm was also determined to occur in CGNs, and following the development of a unique motility assay, I demonstrated that interference with this cascade impaired motility, suggesting that this signalling cascade may contribute to the developmental migration of CGNs. -- p75NTR is a unique and flexible pleiotropic receptor which may promote or inhibit cell growth depending upon the presence or absence of neurotrophins. Taken together, the results of these studies detail the mechanisms involved in the upregulation of p75NTR expression in the presence of neurotrophins, but additionally present a novel signalling paradigm initiated by the ECM for the downregulation of the p75NTR in the absence of neurotrophin stimulation. Interestingly, both scenarios result in the promotion of neurite outgrowth and cellular motility as a result of the flexible signalling interactions of p75NTR, particularly those involving Rho, which can directly influence cytoskeletal dynamics

Cooperativity within and among Pten, p53, and Rb Pathways Induces High-Grade Astrocytoma in Adult Brain

SummaryMutations in the PTEN, TP53, and RB1 pathways are obligate events in the pathogenesis of human glioblastomas. We induced various combinations of deletions in these tumor suppressors in astrocytes and neural precursors in mature mice, resulting in astrocytomas ranging from grade III to grade IV (glioblastoma). There was selection for mutation of multiple genes within a pathway, shown by somatic amplifications of genes in the PI3K or Rb pathway in tumors in which Pten or Rb deletion was an initiating event. Despite multiple mutations within PI3K and Rb pathways, elevated Mapk activation was not consistent. Gene expression profiling revealed striking similarities to subclasses of human diffuse astrocytoma. Astrocytomas were found within and outside of proliferative niches in the adult brain

Homeostatic control of metabolic and functional fitness of Treg cells by LKB1 signaling

Regulatory T cells (Treg cells) play a pivotal role in the establishment and maintenance of immunological self-tolerance and homeostasis1,2. Transcriptional programming of regulatory mechanisms facilitates Treg cell functional activation in the prevention of diverse types of inflammatory responses3,4. How Treg cells orchestrate their homeostasis and interplay with environmental signals remains poorly understood. Here we show that liver kinase B1 (LKB1) programs proper metabolic and functional fitness of Treg cells in the control of immune tolerance and homeostasis. Mice with Treg-specific deletion of LKB1 developed a fatal inflammatory disease characterized by excessive TH2-dominant responses. LKB1 deficiency disrupted Treg cell survival and mitochondrial fitness and metabolism, but also induced aberrant expression of immune regulatory molecules including the negative co-receptor PD-1, and TNF receptor (TNRF) superfamily proteins GITR and OX40. Unexpectedly, LKB1 function in Treg cells was independent of conventional AMPK signaling or the mTORC1-HIF-1α axis, but contributed to the activation of β-catenin signaling for the proper control of PD-1 and TNFR proteins. Blockade of PD-1 activity reinvigorated the suppressive capability of LKB1-deficient Treg cells in the repression of TH2 responses and the interplay with thymic stromal lymphopoietin (TSLP)-primed dendritic cells (DCs). Thus, Treg cells employ LKB1 signaling to coordinate their metabolic and immunological homeostasis and to prevent apoptotic and functional exhaustion, thereby orchestrating the balance between immunity and tolerance

Amino Acids License Kinase mTORC1 Activity and Treg Cell Function via Small G Proteins Rag and Rheb

Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and Rheb GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or Rheb1-Rheb2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while Rheb1-Rheb2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells

Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine

T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)–ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR–Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI—enzymes in the CDP–ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)—as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP–ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP–choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs