mTORC2 Loss in Oligodendrocyte Progenitor Cells Results in Regional Hypomyelination in the Central Nervous System.

In the CNS, oligodendrocyte progenitor cells (OPCs) differentiate into mature oligodendrocytes to generate myelin, an essential component for normal nervous system function. OPC differentiation is driven by signaling pathways, such as mTOR, which functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), containing Raptor or Rictor, respectively. In the current studies, mTORC2 signaling was selectively deleted from OPCs in PDGFRα-Cre X Rictorfl/fl mice. This study examined developmental myelination in male and female mice, comparing the impact of mTORC2 deletion in the corpus callosum and spinal cord. In both regions, Rictor loss in OPCs resulted in early reduction in myelin RNAs and proteins. However, these deficits rapidly recovered in spinal cord, where normal myelin was noted at P21 and P45. By contrast, the losses in corpus callosum resulted in severe hypomyelination and increased unmyelinated axons. The hypomyelination may result from decreased oligodendrocytes in the corpus callosum, which persisted in animals as old as postnatal day 350. The current studies focus on uniquely altered signaling pathways following mTORC2 loss in developing oligodendrocytes. A major mTORC2 substrate is phospho-Akt-S473, which was significantly reduced throughout development in both corpus callosum and spinal cord at all ages measured, yet this had little impact in spinal cord. Loss of mTORC2 signaling resulted in decreased expression of actin regulators, such as gelsolin in corpus callosum, but only minimal loss in spinal cord. The current study establishes a regionally specific role for mTORC2 signaling in OPCs, particularly in the corpus callosum.SIGNIFICANCE STATEMENT mTORC1 and mTORC2 signaling has differential impact on myelination in the CNS. Numerous studies identify a role for mTORC1, but deletion of Rictor (mTORC2 signaling) in late-stage oligodendrocytes had little impact on myelination in the CNS. However, the current studies establish that deletion of mTORC2 signaling from oligodendrocyte progenitor cells results in reduced myelination of brain axons. These studies also establish a regional impact of mTORC2, with little change in spinal cord in these conditional Rictor deletion mice. Importantly, in both brain and spinal cord, mTORC2 downstream signaling targets were impacted by Rictor deletion. Yet, these signaling changes had little impact on myelination in spinal cord, while they resulted in long-term alterations in myelination in brain.

Nutritional regulation of oligodendrocyte differentiation regulates perineuronal net remodeling in the median eminence.

The mediobasal hypothalamus (MBH; arcuate nucleus of the hypothalamus [ARH] and median eminence [ME]) is a key nutrient sensing site for the production of the complex homeostatic feedback responses required for the maintenance of energy balance. Here, we show that refeeding after an overnight fast rapidly triggers proliferation and differentiation of oligodendrocyte progenitors, leading to the production of new oligodendrocytes in the ME specifically. During this nutritional paradigm, ME perineuronal nets (PNNs), emerging regulators of ARH metabolic functions, are rapidly remodeled, and this process requires myelin regulatory factor (Myrf) in oligodendrocyte progenitors. In genetically obese ob/ob mice, nutritional regulations of ME oligodendrocyte differentiation and PNN remodeling are blunted, and enzymatic digestion of local PNN increases food intake and weight gain. We conclude that MBH PNNs are required for the maintenance of energy balance in lean mice and are remodeled in the adult ME by the nutritional control of oligodendrocyte differentiation.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism.

The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ß-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.

A real-time method for measuring cAMP production modulated by Gαi/o-coupled metabotropic glutamate receptors.

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Triple threat treatment: Exploiting the dependence receptor properties of metabotropic glutamate receptor 1 against melanoma.

Melanoma cells that express metabotropic glutamate 1 (mGlu1) receptors depend on glutamate for their survival and proliferation. The dependence receptor properties of mGlu1 allow us to propose and justify three promising approaches for melanoma treatment: glutamate depletion, mGlu1 receptor antagonism, and targeting of mGlu1 receptor signaling.

Microglial activation and antioxidant responses induced by the Parkinson's disease protein α-synuclein.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder typified by tremor, rigidity, akinesia and postural instability due in part to the loss of dopamine within the nigrostriatal system. The pathologic features of this disorder include the loss of substantia nigra dopamine neurons and attendant striatal terminals, the presence of large protein-rich neuronal inclusions containing fibrillar α-synuclein and increased numbers of activated microglia. Evidence suggests that both misfolded α-synuclein and oxidative stress play an important role in the pathogenesis of sporadic PD. Here we review evidence that α-synuclein activates glia inducing inflammation and that Nrf2-directed phase-II antioxidant enzymes play an important role in PD. We also provide new evidence that the expression of antioxidant enzymes regulated in part by Nrf2 is increased in a mouse model of α-synuclein overexpression. We show that misfolded α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression. Importantly, we demonstrate that the precise structure of α-synuclein is important for induction of this proinflammatory pathway. This complex α-synuclein-directed glial response highlights the importance of protein misfolding, oxidative stress and inflammation in PD and represents a potential locus for the development of novel therapeutics focused on induction of the Nrf2-directed antioxidant pathway and inhibition of protein misfolding.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish ß-arrestin-mediated from G protein-mediated signaling.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

Anti-Xa stability of diluted dalteparin for pediatric use.

BACKGROUND: The low-molecular-weight heparin (LMWH) dalteparin is approved by the Food and Drug Administration for prophylaxis of venous thromboembolism (VTE) in adults and has recently received an indication for acute VTE therapy in adults with cancer. Published reports of experience with dalteparin use in European children suggest that this LMWH agent is safe and effective in the prophylaxis and treatment of VTE in the pediatric population. However, dalteparin is commonly available in the US in a concentrated form that requires dilution for accurate administration in infants and young children. OBJECTIVE: To investigate the in vitro stability of diluted dalteparin for pediatric use, as measured by serial anti-Xa activity assays over the course of 4 weeks. METHODS: At 2 clinical research pharmacies, dalteparin multidose vials (anti-Xa concentration 25,000 U/mL) of the 2 distinct lots presently available for clinical use were diluted 1:10 with preservative-free NaCl 0.9% and maintained in tuberculin syringes at 4 degrees C. Syringes were then sampled for anti-Xa activity by chromogenic assay at baseline and weekly over the course of 4 weeks. RESULTS: For each lot of dalteparin, there was strong agreement in anti-Xa activity between corresponding diluted syringes prepared at the 2 pharmacy sites. No statistically significant difference in anti-Xa activity was detected from baseline to any time point, nor was a trend of change detected in anti-Xa activity with time for either lot of dalteparin. CONCLUSIONS: These data indicate that the anti-Xa activity of diluted dalteparin for pediatric use is stable over the course of 4 weeks.