Glutamate treatment and p25 transfection increase Cdk5 mediated tau phosphorylation in SH-SY5Y cells

Neurofibrillary tangles (NFT) of hyperphosphorylated tau protein are a major pathological hallmark of Alzheimer's disease (AD). One of the tau phosphorylating kinases with pathological relevance in AD has been suggested to be the cyclin-dependent kinase 5 (Cdk5). The proposed mechanism leading to pathological Cdk5 activity is through induced cleavage of p35 to a proteolytic product, p25. To further study activation of Cdk5 and its role in tau phosphorylation in vitro, we used differentiated SH-SY5Y cells treated with neurotoxic stimuli or transfected with p25. We show that glutamate increased tau phosphorylation, concomitant with an increased Cdk5 activity achieved by upregulation of Cdk5 and p35 protein levels. Treatment with the calcium ionophore A23187 generated the calpain cleaved p25 fragment but only in toxic conditions that caused dephosphorylation and loss of tau. When p25 was transfected to the cells, increased tau phosphorylation was achieved. However, application of the Cdk5 inhibitor Roscovitine did not result in inhibition of tau phosphorylation possibly due to activation of extracellular regulated kinase 1/2 (Erk1/2), which also is capable of phosphorylating tau. Cdk5 and Erk1/2 kinases share some common substrates but impact of their cross talk on tau phosphorylation has not previously been demonstrated. We also show that p25 is degraded via the proteasome in Roscovitine treated cells.     

Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats

Background

There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells.

Methodology/Principal Findings

Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally) once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin), plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium.

Conclusions/Significance

Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.     

Presenilin dependence of phospholipase C and protein kinase C signaling

Presenilins (PSs) are involved in processing several proteins such as the amyloid precursor protein (APP), as well as in pathways for cell death and survival. We previously showed that some familial Alzheimer's disease PS mutations cause increased basal and acetylcholine muscarinic receptor-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha and PKCgamma activations were significantly lower in PS1 and PS2 double knockout MEFs after PLC stimulation. Protein levels of PKCalpha and PKCgamma were lower in PS1 and PS2 double knockout MEFs. In contrast, PKCdelta levels were significantly elevated in PS1 and PS2 double knockout as well as in PS1 knockout MEFs. Also, PKCdelta levels were lowered after transfection of PS1 into PS1 knockout or PS double knockout MEFs. Using APP knockout MEFs we showed that the expression of PKCalpha, but not the other PKC isoforms is partially dependent on APP and can be regulated by APP intracellular domain (AICD). These results show that PLC and PKC activations are modulated by PS and also that PSs differentially regulate the expression of PKC isoforms by both APP/AICD-dependent and independent mechanisms.     

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.     

Amyloid precursor protein accumulates in aggresomes in response to proteasome inhibitor

Aggresomes are cytoplasmic inclusions which are localized at the microtubule organizing center (MTOC) as a result of induced proteasome inhibition, stress or over-expression of certain proteins. Aggresomes are linked to the pathogenesis of many neurodegenerative diseases. Here we studied whether amyloid precursor protein (APP), a type-I transmembrane glycoprotein, is localized in aggresomes after exposure to stress condition. Using confocal microscopy we found that APP is located in aggresomes and co-localized with vimentin, γ-tubulin, 20S and ubiquitin at the MTOC in response to proteasome dysfunction. An interaction between vimentin and APP was found after proteasome inhibition suggesting that APP is an additional protein constituent of aggresomes. Suppression of the proteasome system in APP-HEK293 cells overexpressing APP or transfected with APP Swedish mutation caused an accumulation of stable, detergent-insoluble forms of APP containing poly-ubiquitinated proteins. In addition, brain homogenates from transgenic mice expressing human APP with the Arctic mutation demonstrated an interaction between APP and the aggresomal-marker vimentin. These data suggest that malfunctioning of the proteasome system caused by mutation or overexpression of pathological or non-pathological proteins may lead to the accumulation of stable aggresomes, perhaps contributing to the neurodegeneration.     

Oxidative stress induces macroautophagy of amyloid β-protein and ensuing apoptosis

There is increasing evidence for the toxicity of intracellular amyloid β-protein (Aβ) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Aβ in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Aβ that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Aβ-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Aβ content (Vector < APPwt < APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.     

β-Adrenergic inhibition of contractility in L6 skeletal muscle cells

The β-adrenoceptors (β-ARs) control many cellular processes. Here, we show that β-ARs inhibit calcium depletion-induced cell contractility and subsequent cell detachment of L6 skeletal muscle cells. The mechanism underlying the cell detachment inhibition was studied by using a quantitative cell detachment assay. We demonstrate that cell detachment induced by depletion of extracellular calcium is due to myosin- and ROCK-dependent contractility. The β-AR inhibition of L6 skeletal muscle cell detachment was shown to be mediated by the β(2)-AR and increased cAMP but was surprisingly not dependent on the classical downstream effectors PKA or Epac, nor was it dependent on PKG, PI3K or PKC. However, inhibition of potassium channels blocks the β(2)-AR mediated effects. Furthermore, activation of potassium channels fully mimicked the results of β(2)-AR activation. In conclusion, we present a novel finding that β(2)-AR signaling inhibits contractility and thus cell detachment in L6 skeletal muscle cells by a cAMP and potassium channel dependent mechanism.