phosphothreonine and Disease-Models--Animal

Hyperphosphorylated tau is the main component of neurofibrillary tangles and involved in the pathogenesis of Alzheimer's disease (AD). Increasing evidences suggest close associations between Porphyromonas gingivalis (P. gingivalis) and AD, but the relationship between P. gingivalis and tau hyperphosphorylation is still unclear. In this study, we investigated whether peripheral infection with P. gingivalis caused tau hyperphosphorylation by using wild Sprague-Dawley (SD) rats and HT-22 cells. The rats were injected with P. gingivalis suspension or phosphate-buffered saline 3 times per week. After 4 weeks or 12 weeks, the rats were sacrificed for analyzing systemic inflammation, neuroinflammation, and tau hyperphosphorylation. The results showed that the severity of phosphorylated tau at the AD-related sites Thr181 and Thr231 and the number of activated astrocytes were notably greater in the hippocampus of rats with P. gingivalis injection. And the levels of the inflammatory cytokines interleukin (IL)-1β and IL-6 and tumor necrosis factor-α in serum and hippocampus were also increased in the rats with P. gingivalis injection. In addition, the activity of protein phosphatase 2A (PP2A) was significantly inhibited in the hippocampus of rats with P. gingivalis injection. In vitro, IL-1β induced tau hyperphosphorylation by inhibiting the activity of PP2A in HT-22 cells and application of the PP2A promoter efficiently attenuated IL-1β-induced tau hyperphosphorylation in HT-22 cells. These results indicated that P. gingivalis could induce tau hyperphosphorylation via, in part, attenuating the activity of PP2A through triggering systemic inflammation and neuroinflammation in wild-type SD rats.

Topics: Alzheimer Disease; Animals; Astrocytes; Bacteremia; Bacteroidaceae Infections; Cell Line; Cytokines; Disease Models, Animal; Enzyme Activation; Hippocampus; Inflammation; Male; Nerve Tissue Proteins; Neurons; Phosphorylation; Phosphothreonine; Porphyromonas gingivalis; Protein Phosphatase 2; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Specific Pathogen-Free Organisms; tau Proteins; Tumor Necrosis Factor-alpha

Recent reports indicate that autophagy serves as a stress response and may participate in pathophysiology of cerebral ischemia. Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), the rate-limiting enzyme in mammalian NAD (+) biosynthesis, protects against ischemic stroke through inhibiting neuronal apoptosis and necrosis. This study was taken to determine the involvement of autophagy in neuroprotection of Nampt in cerebral ischemia. Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in cultured cortical neurons were performed. Nampt was overexpressed or knocked-down using lentivirus-mediated gene transfer in vivo and in vitro. Immunochemistry (LC3-II), electron microscope and immunoblotting assays (LC3-II, beclin-1, mammalian target of rapamycin [mTOR], S6K1 and tuberous sclerosis complex-2 [TSC2]) were performed to assess autophagy. We found that overexpression of Nampt increased autophagy (LC3 puncta immunochemistry staining, LC3-II/beclin-1 expression and autophagosomes number) both in vivo and in vitro at 2 hours after MCAO. At the early stage of OGD, autophagy inducer rapamycin protected against neuronal injury induced by Nampt knockdown, whereas autophagy inhibitor 3-methyladenine abolished the neuroprotective effect of Nampt partly. Overexpression or knockdown of Nampt regulated the phosphorylation of mTOR and S6K1 signaling pathway upon OGD stress through enhancing phosphorylation of TSC2 at Ser1387 but not Thr1462 site. Furthermore, in cultured SIRT1-knockout neurons, the regulation of Nampt on autophagic proteins LC3-II and beclin-1 was abolished. Our results demonstrate that Nampt promotes neuronal survival through inducing autophagy via regulating TSC2-mTOR-S6K1 signaling pathway in a SIRT1-dependent manner during cerebral ischemia.

Topics: Adenine; Animals; Autophagy; Brain Ischemia; Cells, Cultured; Disease Models, Animal; Glucose; Infarction, Middle Cerebral Artery; Mice; Neurons; Neuroprotective Agents; Nicotinamide Phosphoribosyltransferase; Oxygen; Phagosomes; Phosphorylation; Phosphoserine; Phosphothreonine; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirtuin 1; Stress, Physiological; Stroke; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Cerebral insulin receptors play an important role in regulation of energy homeostasis and development of neurodegeneration. Accordingly, type 2 diabetes characterized by insulin resistance is associated with an increased risk of developing Alzheimer's disease. Formation of neurofibrillary tangles, which contain hyperphosphorylated tau, represents a key step in the pathogenesis of neurodegenerative diseases. Here, we directly addressed whether peripheral hyperinsulinemia as one feature of type 2 diabetes can alter in vivo cerebral insulin signaling and tau phosphorylation. Peripheral insulin stimulation rapidly increased insulin receptor tyrosine phosphorylation, mitogen-activated protein kinase and phosphatidylinositol (PI) 3-kinase pathway activation, and dose-dependent tau phosphorylation at Ser202 in the central nervous system. Phospho-FoxO1 and PI-3,4,5-phosphate immunostainings of brains from insulin-stimulated mice showed neuronal staining throughout the brain, not restricted to brain areas without functional blood-brain barrier. Importantly, in insulin-stimulated neuronal/brain-specific insulin receptor knockout mice, cerebral insulin receptor signaling and tau phosphorylation were completely abolished. Thus, peripherally injected insulin directly targets the brain and causes rapid cerebral insulin receptor signal transduction and site-specific tau phosphorylation in vivo, revealing new insights into the linkage of type 2 diabetes and neurodegeneration.

Topics: Animals; Brain; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Hyperinsulinism; Insulin; Mice; Mice, Inbred C57BL; Phosphorylation; Phosphoserine; Phosphothreonine; Receptor, Insulin; Signal Transduction; tau Proteins

The amount of non-collagenous proteins is increased greatly during the pathological calcification of rat skin experimentally induced by dihydrotachysterol (DHT) and Ovalbumin (topical cutaneous calciphylaxis). This is accompanied by an increase in the total amount and concentrations of protein-bound serine phosphate [Ser(P)], threonine phosphate [Thr(P)] and gamma-carboxyglutamic acid (Gla), almost all of which can be extracted from the tissue and can be dissociated from collagen in 0.5M EDTA. The EDTA-soluble, non-collagenous proteins are rich in aspartic and glutamic acids, similar to the non-collagenous, EDTA-soluble proteins of bone, cementum and calcified cartilage, and quite distinct from those of dentin and enamel.

Topics: 1-Carboxyglutamic Acid; Animals; Calciphylaxis; Dihydrotachysterol; Disease Models, Animal; Female; Glutamates; Ovalbumin; Phosphoserine; Phosphothreonine; Rats; Serine; Skin; Skin Diseases; Threonine