MagnetoFectamine O2 Transfection Kit

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22-sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca2+ rise that was blocked by RS-102221, an antagonist of 5-HT2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

Symptoms and complications associated with severe SARS-CoV-2 infection such as acute respiratory distress syndrome (ARDS) and organ damage have been linked to SARS-CoV-2 spike glycoprotein S1-induced increased production of pro-inflammatory cytokines by immune cells. In this study, the effects of an extract of Garcinia kola seeds and garcinoic acid were investigated in SARS-CoV-2 spike glycoprotein S1-stimulated human PBMCs. Results of ELISA experiments revealed that Garcinia kola extract (6.25, 12.5 and 25 μg/mL) and garcinoic acid (1.25, 2.5 and 5 μM) significantly reduced SARS-CoV-2 spike glycoprotein S1-induced increased secretion of TNFα, IL-6, IL-1β and IL-8 in PBMCs. In-cell western assays showed that pre-treatment with Garcinia kola extract and garcinoic acid reduced elevated expressions of both phospho-p65 and phospho-κBα proteins, as well as NF-κB DNA binding capacity and NF-κB-driven luciferase expression following stimulation of PBMCs with spike glycoprotein S1. Furthermore, pre-treatment of PBMCs with Garcinia kola extract prior to stimulation with SARS-CoV-2 spike glycoprotein S1 resulted in reduced damage to adjacent A549 lung epithelial cells. Gas Chromatography-Mass Spectrometry (GCMS) and HPLC-PDA confirmed the presence of garcinoic acid in the Garcinia kola extract used in this study. These results suggest that the seed of Garcinia kola and garcinoic acid are natural products which may possess pharmacological/therapeutic benefits in reducing cytokine storm during the late stage of severe SARS-CoV-2 and other coronavirus infections.

We provide in vivo evidence that NMNAT2 in cortical glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies we demonstrate that NMNAT2 protects axons by ensuring the proper NAD-redox potential in distal axons of cortical neurons to support glycolysis on vesicular cargos, thus ensuring “onboard” ATP production fueling axonal transport. Exogenous NAD+ supplementation to NMNAT2 KO cortical neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.

Endocytic recycling controls the return of internalised cargoes to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargoes to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well-characterised cargoes and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock-sideways relocalisation approach, we were further able to confirm novel links between Rab11, Rab25 and the ESCPE-1 and retromer multiprotein sorting complexes, and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in the 3D matrix.

One major factor regulating the strength of GABA B receptor signaling and thereby neuronal excitability is the dynamic control of their cell surface expression. GABA B receptors are constitutively internalized and recycled back to the plasma membrane to maintain a stable number of receptors at cell surface for appropriate signaling. Protein phosphatase 2A (PP2A) dependent dephosphorylation of serine 783 (S783) in the GABA B2 subunit is a key event for downregulating GABA B receptor cell surface expression particularly under conditions associated with excitotoxicity. Here, we investigated the role of PP2A in regulating GABA B receptor cell surface expression under physiological and excitotoxic conditions. For this purpose, we developed an interfering peptide (PP2A-Pep) that inhibits the interaction of GABA B receptors with PP2A. Using cultured cortical neurons, we found that PP2A downregulates GABA B receptor cell surface expression by inhibiting recycling of the receptors and thereby promoting degradation of the receptors. Inhibition of the GABA B receptor/PP2A interaction by PP2A-Pep in cultured cortical neurons restored GABA B receptor cell surface expression after excitotoxic stress and inhibited progressing neuronal death even when added 48 h after the insult. To explore the therapeutic potential of PP2A-Pep, we further analyzed effect of PP2A-Pep in the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. Incubation of brain slices prepared from MCAO-treated mice with PP2A-Pep restored normal GABA B receptor expression and GABA B receptor-mediated inhibition, reduced ischemic-induced overexcitability of neurons, and prevented neuronal death in the ischemic penumbra. This data illustrates the crucial role of regulating GABA B receptor phosphorylation by PP2A for controlling neuronal inhibition and excitability. The results further suggest that interfering with the GABA B receptor/PP2A interaction is a promising strategy for the development of specific therapeutic interventions to treat neurological diseases associated with a disturbed excitation/inhibition balance and downregulation of GABA B receptors.