BDNF Activates mTOR to Upregulate NR2B Expression in the Rostral Anterior Cingulate Cortex Required for Inflammatory Pain-Related Aversion in Rats

The mechanistic target of rapamycin (mTOR) has been demonstrated to mediate pain-related aversion induced by formalin in the rostral anterior cingulate cortex (rACC). However, it remains unclear the signaling pathways and regulatory proteins involved. In the rACC, brain-derived neurotrophic factor (BDNF), an activity-dependent neuromodulator, has been shown to play a role in the development and persistence of chronic pain. In this study, we used a rat formalin-induced inflammatory pain model to demonstrate BDNF up-regulation in the rACC. Stimulation with exogenous BDNF up-regulated mTOR, whilst cyclotraxin B (CTX-B), a tropomyosin receptor kinase B (TrkB) antagonist, down-regulated mTOR. Our results suggest BDNF could activate an mTOR signaling pathway. Subsequently, we used formalin-induced conditioned place avoidance (F-CPA) training in rat models to investigate if mTOR activation was required for pain-related aversion. We demonstrated that BDNF/mTOR signaling could activate the NMDA receptor subunit episilon-2 (NR2B), which is required for F-CPA. Our results reveal that BDNF activates mTOR to up-regulate NR2B expression, which is required for inflammatory pain-related aversion in the rACC of rats.     

Acute BDNF Treatment Upregulates GluR1-SAP97 and GluR2-GRIP1 Interactions: Implications for Sustained AMPA Receptor Expression

Brain-derived neurotrophic factor (BDNF) plays several prominent roles in synaptic plasticity and in learning and memory formation. Reduced BDNF levels and altered BDNF signaling have been reported in several brain diseases and behavioral disorders, which also exhibit reduced levels of AMPAr subunits. BDNF treatment acutely regulates AMPA receptor expression and function, including synaptic AMPAr subunit trafficking, and implicates several well defined signaling molecules that are required to elicit long term potentiation and depression (LTP and LTD, respectively). Long term encoding of synaptic events, as in long term memory formation, requires AMPAr stabilization and maintenance. However, factors regulating AMPAr stabilization in neuronal cell membranes and synaptic sites are not well characterized. In this study, we examine the effects of acute BDNF treatment on levels of AMPAr-associated scaffolding proteins and on AMPAr subunit-scaffolding protein interactions. We also examine the effects of BDNF-dependent enhanced interactions between AMPAr subunits with their specific scaffolding proteins on the accumulation of both types of proteins. Our results show that acute BDNF treatment upregulates the interactions between AMPAr subunits (GluR1 and GluR2) with their scaffold proteins SAP97 and GRIP1, respectively, leading to prolonged increased accumulation of both categories of proteins, albeit with distinct mechanisms for GluR1 and GluR2. Our findings reveal a new role for BDNF in the long term maintenance of AMPA receptor subunits and associated scaffolding proteins at synapses and further support the role of BDNF as a key regulator of synaptic consolidation. These results have potential implications for recent findings implicating BDNF and AMPAr subunits in various brain diseases and behavioral disorders.     

Magnesium Activates Microsecond Dynamics to Regulate Integrin-Collagen Recognition

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Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.     

SIRT3 Deacetylates and Activates OPA1 To Regulate Mitochondrial Dynamics during Stress

Mitochondrial morphology is regulated by the balance between two counteracting mitochondrial processes of fusion and fission. There is significant evidence suggesting a stringent association between morphology and bioenergetics of mitochondria. Morphological alterations in mitochondria are linked to several pathological disorders, including cardiovascular diseases. The consequences of stress-induced acetylation of mitochondrial proteins on the organelle morphology remain largely unexplored. Here we report that OPA1, a mitochondrial fusion protein, was hyperacetylated in hearts under pathological stress and this posttranslational modification reduced the GTPase activity of the protein. The mitochondrial deacetylase SIRT3 was capable of deacetylating OPA1 and elevating its GTPase activity. Mass spectrometry and mutagenesis analyses indicated that in SIRT3-deficient cells OPA1 was acetylated at lysine 926 and 931 residues. Overexpression of a deacetylation-mimetic version of OPA1 recovered the mitochondrial functions of OPA1-null cells, thus demonstrating the functional significance of K926/931 acetylation in regulating OPA1 activity. Moreover, SIRT3-dependent activation of OPA1 contributed to the preservation of mitochondrial networking and protection of cardiomyocytes from doxorubicin-mediated cell death. In summary, these data indicated that SIRT3 promotes mitochondrial function not only by regulating activity of metabolic enzymes, as previously reported, but also by regulating mitochondrial dynamics by targeting OPA1.     

The Gastric CB1 Receptor Modulates Ghrelin Production through the mTOR Pathway to Regulate Food Intake

Over the years, the knowledge regarding the relevance of the cannabinoid system to the regulation of metabolism has grown steadily. A central interaction between the cannabinoid system and ghrelin has been suggested to regulate food intake. Although the stomach is the main source of ghrelin and CB1 receptor expression in the stomach has been described, little information is available regarding the possible interaction between the gastric cannabinoid and ghrelin systems in the integrated control of energy homeostasis. The main objective of the present work was to assess the functional interaction between these two systems in terms of food intake using a combination of in vivo and in vitro approaches. The present work demonstrates that the peripheral blockade of the CB1 receptor by rimonabant treatment decreased food intake but only in food-deprived animals. This anorexigenic effect is likely a consequence of decreases in gastric ghrelin secretion induced by the activation of the mTOR/S6K1 intracellular pathway in the stomach following treatment with rimonabant. In support of this supposition, animals in which the mTOR/S6K1 intracellular pathway was blocked by chronic rapamycin treatment, rimonabant had no effect on ghrelin secretion. Vagal communication may also be involved because rimonabant treatment was no longer effective when administered to animals that had undergone surgical vagotomy. In conclusion, to the best of our knowledge, the present work is the first to describe a CB1 receptor-mediated mechanism that influences gastric ghrelin secretion and food intake through the mTOR pathway.     

HAP1 Is Required for Endocytosis and Signalling of BDNF and Its Receptors in Neurons

When BDNF binds to its receptors, TrkB and p75^NTR, the BDNF-receptor complex is endocytosed and trafficked to the cell body for downstream signal transduction, which plays a critical role in neuronal functions. Huntingtin-associated protein 1 (HAP1) is involved in trafficking of vesicles intracellularly and also interacts with several membrane proteins including TrkB. Although it has been known that HAP1 has functions in vesicular trafficking and receptor stabilisation, it is not yet established whether HAP1 has a role in BDNF and its receptor endocytosis. In the present study, we found that HAP1 is in an interacting complex with p75^NTR, TrkB and BDNF, especially newly endocytosed BDNF. BDNF and TrkB internalisation is abolished in HAP1 knock-out (KO) cortical neurons. TrkB downstream signalling pathways such as ERK, Akt and PLCγ-1 are also impaired in HAP1 KO cortical neurons upon BDNF stimulation. Proliferation of cerebellar granule cells is also impaired in cell culture and cerebellum of HAP1 KO mice. Our findings suggest that HAP1 may play a key role in BDNF and its receptor endocytosis and may promote neuronal survival and proliferation.     

Duodenal Lipid Sensing Activates Vagal Afferents to Regulate Non-Shivering Brown Fat Thermogenesis in Rats

Previous evidence indicates that duodenal lipid sensing engages gut-brain neurocircuits to determine food intake and hepatic glucose production, but a potential role for gut-brain communication in the control of energy expenditure remains to be determined. Here, we tested the hypothesis that duodenal lipid sensing activates a gut–brain–brown adipose tissue neuraxis to regulate thermogenesis. We demonstrate that direct administration of lipids into the duodenum increases brown fat temperature. Co-infusion of the local anesthetic tetracaine with duodenal lipids abolished the lipid-induced increase in brown fat temperature. Systemic administration of the CCKA receptor antagonist devazepide blocked the ability of duodenal lipids to increase brown fat thermogenesis. Parenchymal administration of the N-methyl-d-aspartate receptor blocker MK-801 directly into the caudomedial nucleus of the solitary tract also abolished duodenal lipid-induced activation of brown fat thermogenesis. These findings establish that duodenal lipid sensing activates a gut–brain–brown fat axis to determine brown fat temperature, and thereby reveal a previously unappreciated pathway that regulates thermogenesis.     

Soluble Expression and Complex Formation of Proteins Required for HCMV DNA Replication Using the SFV Expression System

Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms—a precursor and a product—indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.     

Neurogenesis Inhibition Prevents Enriched Environment to Prolong and Strengthen Social Recognition Memory,But Not to Increase BDNF Expression

Hippocampus-dependent memories, such as social recognition (SRM), are modulated by neurogenesis. However, the precise role of newborn neurons in social memory processing is still unknown. We showed previously that 1 week of enriched environment (EE) is sufficient to increase neurogenesis in the hippocampus (HIP) and the olfactory bulb (OB) of mice. Here, we tested the hypothesis that 1 week of EE would enhance SRM persistence and strength. In addition, as brain-derived neurotrophic factor (BDNF) may mediate some of the neurogenesis effects on memory, we also tested if 1 week of EE would increase BDNF expression in the HIP and OB. We also predicted that neurogenesis inhibition would block the gain of function caused by EE on both SRM and BDNF expression. We found that EE increased BDNF expression in the HIP and OB of mice; at the same time, it allowed SRM to last longer. In addition, mice on EE had their SRM unaffected by memory consolidation interferences. As we predicted, treatment with the anti-mitotic drug AraC blocked EE effects on SRM. Surprisingly, neurogenesis inhibition did not affect the BDNF expression, increased by EE. Together, our results suggest that newborn neurons improve SRM persistence through a BDNF-independent mechanism. Interestingly, this study on social memory uncovered an unexpected dissociation between the effect of adult neurogenesis and BDNF expression on memory persistence, reassuring the idea that not all neurogenesis effects on memory are BDNF-dependent.