Metabolic partitioning in the brain and its hijacking by glioblastoma.

The different cell types in the brain have highly specialized roles with unique metabolic requirements. Normal brain function requires the coordinated partitioning of metabolic pathways between these cells, such as in the neuron-astrocyte glutamate-glutamine cycle. An emerging theme in glioblastoma (GBM) biology is that malignant cells integrate into or "hijack" brain metabolism, co-opting neurons and glia for the supply of nutrients and recycling of waste products. Moreover, GBM cells communicate via signaling metabolites in the tumor microenvironment to promote tumor growth and induce immune suppression. Recent findings in this field point toward new therapeutic strategies to target the metabolic exchange processes that fuel tumorigenesis and suppress the anticancer immune response in GBM. Here, we provide an overview of the intercellular division of metabolic labor that occurs in both the normal brain and the GBM tumor microenvironment and then discuss the implications of these interactions for GBM therapy.

Microglia regulate chandelier cell axo-axonic synaptogenesis.

SignificanceChandelier cells (ChCs) are a unique type of GABAergic interneuron that form axo-axonic synapses exclusively on the axon initial segment (AIS) of neocortical pyramidal neurons (PyNs), allowing them to exert powerful yet precise control over PyN firing and population output. The importance of proper ChC function is further underscored by the association of ChC connectivity defects with various neurological conditions. Despite this, the cellular mechanisms governing ChC axo-axonic synapse formation remain poorly understood. Here, we identify microglia as key regulators of ChC axonal morphogenesis and AIS synaptogenesis, and show that disease-induced aberrant microglial activation perturbs proper ChC synaptic development/connectivity in the neocortex. In doing so, such findings highlight the therapeutic potential of manipulating microglia to ensure proper brain wiring.

Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

TGF-ß/Smad signaling through DOCK4 facilitates lung adenocarcinoma metastasis.

The mechanisms by which TGF-ß promotes lung adenocarcinoma (ADC) metastasis are largely unknown. Here, we report that in lung ADC cells, TGF-ß potently induces expression of DOCK4, but not other DOCK family members, via the Smad pathway and that DOCK4 induction mediates TGF-ß's prometastatic effects by enhancing tumor cell extravasation. TGF-ß-induced DOCK4 stimulates lung ADC cell protrusion, motility, and invasion without affecting epithelial-to-mesenchymal transition. These processes, which are fundamental to tumor cell extravasation, are driven by DOCK4-mediated Rac1 activation, unveiling a novel link between TGF-ß and Rac1. Thus, our findings uncover the atypical Rac1 activator DOCK4 as a key component of the TGF-ß/Smad pathway that promotes lung ADC cell extravasation and metastasis.

The paraventricular thalamus controls a central amygdala fear circuit.

Appropriate responses to an imminent threat brace us for adversities. The ability to sense and predict threatening or stressful events is essential for such adaptive behaviour. In the mammalian brain, one putative stress sensor is the paraventricular nucleus of the thalamus (PVT), an area that is readily activated by both physical and psychological stressors. However, the role of the PVT in the establishment of adaptive behavioural responses remains unclear. Here we show in mice that the PVT regulates fear processing in the lateral division of the central amygdala (CeL), a structure that orchestrates fear learning and expression. Selective inactivation of CeL-projecting PVT neurons prevented fear conditioning, an effect that can be accounted for by an impairment in fear-conditioning-induced synaptic potentiation onto somatostatin-expressing (SOM(+)) CeL neurons, which has previously been shown to store fear memory. Consistently, we found that PVT neurons preferentially innervate SOM(+) neurons in the CeL, and stimulation of PVT afferents facilitated SOM(+) neuron activity and promoted intra-CeL inhibition, two processes that are critical for fear learning and expression. Notably, PVT modulation of SOM(+) CeL neurons was mediated by activation of the brain-derived neurotrophic factor (BDNF) receptor tropomysin-related kinase B (TrkB). As a result, selective deletion of either Bdnf in the PVT or Trkb in SOM(+) CeL neurons impaired fear conditioning, while infusion of BDNF into the CeL enhanced fear learning and elicited unconditioned fear responses. Our results demonstrate that the PVT-CeL pathway constitutes a novel circuit essential for both the establishment of fear memory and the expression of fear responses, and uncover mechanisms linking stress detection in PVT with the emergence of adaptive behaviour.

The Rho-linked mental retardation protein oligophrenin-1 controls synapse maturation and plasticity by stabilizing AMPA receptors.

Oligophrenin-1 (OPHN1) encodes a Rho-GTPase-activating protein (Rho-GAP) whose loss of function has been associated with X-linked mental retardation (MR). The pathophysiological role of OPHN1, however, remains poorly understood. Here we show that OPHN1 through its Rho-GAP activity plays a critical role in the activity-dependent maturation and plasticity of excitatory synapses by controlling their structural and functional stability. Synaptic activity through NMDA receptor activation drives OPHN1 into dendritic spines, where it forms a complex with AMPA receptors, and selectively enhances AMPA-receptor-mediated synaptic transmission and spine size by stabilizing synaptic AMPA receptors. Consequently, decreased or defective OPHN1 signaling prevents glutamatergic synapse maturation and causes loss of synaptic structure, function, and plasticity. These results imply that normal activity-driven glutamatergic synapse development is impaired by perturbation of OPHN1 function. Thus, our findings link genetic deficits in OPHN1 to glutamatergic dysfunction and suggest that defects in early circuitry development are an important contributory factor to this form of MR.

The X-linked mental retardation protein OPHN1 interacts with Homer1b/c to control spine endocytic zone positioning and expression of synaptic potentiation.

At glutamatergic synapses, local endocytic recycling of AMPA receptors (AMPARs) is important for the supply of a mobile pool of AMPARs required for synaptic potentiation. This local recycling of AMPARs critically relies on the presence of an endocytic zone (EZ) near the postsynaptic density (PSD). The precise mechanisms that couple the EZ to the PSD still remain largely elusive, with the large GTPase Dynamin-3 and the multimeric PSD adaptor protein Homer1 as the two main players identified. Here, we demonstrate that a physical interaction between the X-linked mental retardation protein oligophrenin-1 (OPHN1) and Homer1b/c is crucial for the positioning of the EZ adjacent to the PSD, and present evidence that this interaction is important for OPHN1's role in controlling activity-dependent strengthening of excitatory synapses in the rat hippocampus. Disruption of the OPHN1-Homer1b/c interaction causes a displacement of EZs from the PSD, along with impaired AMPAR recycling and reduced AMPAR accumulation at synapses, in both basal conditions and conditions that can induce synaptic potentiation. Together, our findings unveil a novel role for OPHN1 as an interaction partner of Homer1b/c in spine EZ positioning, and provide new mechanistic insight into how genetic deficits in OPHN1 can lead to impaired synapse maturation and plasticity.

Chronic stress increases metastasis via neutrophil-mediated changes to the microenvironment.

Chronic stress is associated with increased risk of metastasis and poor survival in cancer patients, yet the reasons are unclear. We show that chronic stress increases lung metastasis from disseminated cancer cells 2- to 4-fold in mice. Chronic stress significantly alters the lung microenvironment, with fibronectin accumulation, reduced T cell infiltration, and increased neutrophil infiltration. Depleting neutrophils abolishes stress-induced metastasis. Chronic stress shifts normal circadian rhythm of neutrophils and causes increased neutrophil extracellular trap (NET) formation via glucocorticoid release. In mice with neutrophil-specific glucocorticoid receptor deletion, chronic stress fails to increase NETs and metastasis. Furthermore, digesting NETs with DNase I prevents chronic stress-induced metastasis. Together, our data show that glucocorticoids released during chronic stress cause NET formation and establish a metastasis-promoting microenvironment. Therefore, NETs could be targets for preventing metastatic recurrence in cancer patients, many of whom will experience chronic stress due to their disease.

Next Directions in the Neuroscience of Cancers Arising outside the CNS.

SUMMARY: The field of cancer neuroscience has begun to define the contributions of nerves to cancer initiation and progression; here, we highlight the future directions of basic and translational cancer neuroscience for malignancies arising outside of the central nervous system.

Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins.

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.

Dialogue between RhoA/ROCK and members of the Par complex in cell polarity.

Cell polarity is essential for many biological processes and is regulated by conserved protein complexes, including the Par complex, Rho GTPases, and their regulators. In this issue of Developmental Cell, studies by Nakayama et al. and Zhang and Macara examine how interplay between Rho GTPases and the Par complex control polarized cell migration and dendritic spine morphogenesis in alternate ways.

Functional analysis of the protein phosphatase activity of PTEN.

In vitro, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) displays intrinsic phosphatase activity towards both protein and lipid substrates. In vivo, the lipid phosphatase activity of PTEN, through which it dephosphorylates the 3 position in the inositol sugar of phosphatidylinositol derivatives, is important for its tumour suppressor function; however, the significance of its protein phosphatase activity remains unclear. Using two-photon laser-scanning microscopy and biolistic gene delivery of GFP (green fluorescent protein)-tagged constructs into organotypic hippocampal slice cultures, we have developed an assay of PTEN function in living tissue. Using this bioassay, we have demonstrated that overexpression of wild-type PTEN led to a decrease in spine density in neurons. Furthermore, it was the protein phosphatase activity, but not the lipid phosphatase activity, of PTEN that was essential for this effect. The ability of PTEN to decrease neuronal spine density depended upon the phosphorylation status of serine and threonine residues in its C-terminal segment and the integrity of the C-terminal PDZ-binding motif. The present study reveals a new aspect of the function of this important tumour suppressor and suggest that, in addition to dephosphorylating the 3 position in phosphatidylinositol phospholipids, the critical protein substrate of PTEN may be PTEN itself.

Antibody-directed extracellular proximity biotinylation reveals Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Connecting copper and cancer: from transition metal signalling to metalloplasia.

Copper is an essential nutrient whose redox properties make it both beneficial and toxic to the cell. Recent progress in studying transition metal signalling has forged new links between researchers of different disciplines that can help translate basic research in the chemistry and biology of copper into clinical therapies and diagnostics to exploit copper-dependent disease vulnerabilities. This concept is particularly relevant in cancer, as tumour growth and metastasis have a heightened requirement for this metal nutrient. Indeed, the traditional view of copper as solely an active site metabolic cofactor has been challenged by emerging evidence that copper is also a dynamic signalling metal and metalloallosteric regulator, such as for copper-dependent phosphodiesterase 3B (PDE3B) in lipolysis, mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 in cell growth and proliferation and the kinases ULK1 and ULK2 in autophagy. In this Perspective, we summarize our current understanding of the connection between copper and cancer and explore how challenges in the field could be addressed by using the framework of cuproplasia, which is defined as regulated copper-dependent cell proliferation and is a representative example of a broad range of metalloplasias. Cuproplasia is linked to a diverse array of cellular processes, including mitochondrial respiration, antioxidant defence, redox signalling, kinase signalling, autophagy and protein quality control. Identifying and characterizing new modes of copper-dependent signalling offers translational opportunities that leverage disease vulnerabilities to this metal nutrient.

Regulation of the postsynaptic cytoskeleton: roles in development, plasticity, and disorders.

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during experience-dependent plasticity. This mini-symposium review will feature ongoing research into how spines are regulated by actin-signaling pathways during development and plasticity. It will also highlight evolving studies into how disruptions to these pathways might be functionally coupled to congenital disorders such as mental retardation.

Molecular mechanisms of axo-axonic innervation.

One of the most intriguing features of inhibitory synapses is the precision by which they innervate their target, not only at the cellular level but also at the subcellular level (i.e. axo-dendritic, axo-somatic, or axo-axonic innervation). In particular, in the cerebellum, cortex, and spinal cord, distinct and highly specialized GABAergic interneurons, such as basket cells, chandelier cells, and GABApre interneurons, form precise axo-axonic synapses, allowing them to directly regulate neuronal output and circuit function. In this article, we summarize our latest knowledge of the cellular and molecular mechanisms that regulate the establishment and maintenance of axo-axonic synapses in these regions of the CNS. We also detail the key roles of the L1CAM family of cell adhesion molecules in such GABAergic subcellular target recognition.

Oligophrenin-1 moderates behavioral responses to stress by regulating parvalbumin interneuron activity in the medial prefrontal cortex.

Ample evidence indicates that individuals with intellectual disability (ID) are at increased risk of developing stress-related behavioral problems and mood disorders, yet a mechanistic explanation for such a link remains largely elusive. Here, we focused on characterizing the syndromic ID gene oligophrenin-1 (OPHN1). We find that Ophn1 deficiency in mice markedly enhances helpless/depressive-like behavior in the face of repeated/uncontrollable stress. Strikingly, Ophn1 deletion exclusively in parvalbumin (PV) interneurons in the prelimbic medial prefrontal cortex (PL-mPFC) is sufficient to induce helplessness. This behavioral phenotype is mediated by a diminished excitatory drive onto Ophn1-deficient PL-mPFC PV interneurons, leading to hyperactivity in this region. Importantly, suppressing neuronal activity or RhoA/Rho-kinase signaling in the PL-mPFC reverses helpless behavior. Our results identify OPHN1 as a critical regulator of adaptive behavioral responses to stress and shed light onto the mechanistic links among OPHN1 genetic deficits, mPFC circuit dysfunction, and abnormalities in stress-related behaviors.

The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18.

The polarization of a neuron generally results in the formation of one axon and multiple dendrites, allowing for the establishment of neuronal circuitry. The molecular mechanisms involved in priming one neurite to become the axon, particularly those regulating the microtubule network, remain elusive. Here we report the identification of DOCK7, a member of the DOCK180-related protein superfamily, as a Rac GTPase activator that is asymmetrically distributed in unpolarized hippocampal neurons and selectively expressed in the axon. Knockdown of DOCK7 expression prevents axon formation, whereas overexpression induces formation of multiple axons. We further demonstrate that DOCK7 and Rac activation lead to phosphorylation and inactivation of the microtubule destabilizing protein stathmin/Op18 in the nascent axon and that this event is important for axon development. Our findings unveil a pathway linking the Rac activator DOCK7 to a microtubule regulatory protein and highlight the contribution of microtubule network regulation to axon development.

Role of Dok-1 and Dok-2 in leukemia suppression.

Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr/abl oncoprotein that shows elevated and constitutive protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Although several p210bcr/abl substrates have been identified, their relevance in the pathogenesis of the disease is unclear. We have identified a family of proteins, Dok (downstream of tyrosine kinase), coexpressed in hematopoietic progenitor cells. Members of this family such as p62dok (Dok-1) and p56dok-2 (Dok-2) associate with the p120 rasGTPase-activating protein (rasGAP) upon phosphorylation by p210bcr/abl as well as receptor and nonreceptor tyrosine kinases. Here, we report the generation and characterization of single and double Dok-1 or Dok-2 knockout (KO) mutants. Single KO mice displayed normal steady-state hematopoiesis. By contrast, concomitant Dok-1 and Dok-2 inactivation resulted in aberrant hemopoiesis and Ras/MAP kinase activation. Strikingly, all Dok-1/Dok-2 double KO mutants spontaneously developed transplantable CML-like myeloproliferative disease due to increased cellular proliferation and reduced apoptosis. Furthermore, Dok-1 or Dok-2 inactivation markedly accelerated leukemia and blastic crisis onset in Tec-p210bcr/abl transgenic mice known to develop, after long latency, a myeloproliferative disorder resembling human CML. These findings unravel the critical and unexpected role of Dok-1 and Dok-2 in tumor suppression and control of the hematopoietic compartment homeostasis.