PI 3-kinase independent role for AKT in F-actin regulation during outer segment phagocytosis by RPE cells.

Daily phagocytosis of photoreceptor outer segment fragments (POS) by the retinal pigment epithelium (RPE) is essential for vision. RPE cells use an uptake machinery that is highly similar to the one macrophages use to phagocytose apoptotic cells. In both forms of phagocytosis, particle binding induces phagocyte signaling that is required for F-actin assembly and re-arrangement beneath bound particles. Macrophage binding of apoptotic cells stimulates PI3 kinases (PI3K) and AKT kinases (AKT), which may be downstream of PI3K, and PI3K inhibition decreases engulfment. Here, we used specific inhibitory agents to investigate whether and how PI3K and AKT contribute to RPE phagocytosis. Either PI3K or AKT inhibition eliminated AKT activation by RPE cells in response to POS and increased the numbers of POS bound by RPE cells. Analyzing the quality of bound POS, we found a higher fraction of POS associated with F-actin phagocytic cups and myosin II in RPE receiving AKT inhibitor. In these cells, individual POS also recruited more F-actin and myosin II than POS in control cells. In contrast, PI3K inhibition did not alter frequency of phagocytic cups but individual cups contained less F-actin (but similar levels of myosin II) compared to control cups. Annexin AII, another phagocytic cup protein of RPE cells, associated with bound POS regardless of inhibitor treatment. POS engulfment proceeded normally if cells already carried surface-bound POS when receiving inhibitors. However, PI3K inhibition during POS binding blocked subsequent POS engulfment. In striking contrast, AKT inhibition had no effect on POS engulfment. Taken together, these results suggest distinct regulatory roles of PI3K and AKT during POS phagocytosis by RPE cells.

Insulin-degrading enzyme sorting in exosomes: a secretory pathway for a key brain amyloid-beta degrading protease.

The accumulation of amyloid-beta (Abeta) peptides in senile plaques is one of the hallmarks of Alzheimer's disease (AD) progression. The endocytic pathway has been proposed as a major subcellular site for Abeta generation while the compartments in which Abeta-degrading proteases interact with Abeta are still elusive. It was suggested that extracellular Abeta degradation may take place by plasma-membrane associated proteases or by extracellular proteases, among which insulin-degrading enzyme (IDE) is the most relevant. However, the mechanisms of IDE secretion are poorly understood. In the present study we used N2a cells to explore if IDE is indeed released through exosomes and the effect of exosomes release on extracellular levels of Abeta. We demonstrated that proteolytically-active plasma membrane associated-IDE is routed in living N2a cells to multivesicular bodies and subsequently, a major fraction is sorted to exosomes. We described that extracellular IDE levels decrease if the generation of multivesicular bodies is interfered and may be positively modulated by exosomes release under stress-induced conditions. Our results reinforce the relevance of functional IDE in the catabolism of extracellular Abeta.

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau.

BACKGROUND: Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. RESULTS: Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. CONCLUSION: Tumor suppressor PTEN can alleviate the phosphorylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity null PTEN increases the mutant tau phosphorylation at these sites. The changes of the tau phosphorylation status by ectopic expression of PTEN correlate to the alteration of the mutant tau's cellular distribution. In addition, the overexpression of the mutant PTEN can increase the level of the mutant tau aggregates and lead to the formation of visible aggregates in the cells.

Tumor-suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules.

Neurofibrillary tangles (NFTs), consisting of abnormally hyperphosphorylated tau, are implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). The molecular mechanisms underlying the regulation of tau phosphorylation are largely unknown. While the PI3K/Akt pathway has been shown to regulate multiple cellular events pertinent to AD pathogenesis, potential functions of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been explored. Here, we examine the effects of PTEN on tau phosphorylation, its microtubule association and formation of aggregates, and consequentially neuronal morphology. In cultured cells, overexpression of wild-type (WT) PTEN alters tau phosphorylation at several sites, increases tau-microtubule association and decreases formation of tau aggregates. In addition, the phosphatase-null PTEN increases tau aggregation and impairs tau binding to microtubule and neurite outgrowth of neurons expressing the mutant PTEN. We also found a significant loss of PTEN in AD patient brains correlated with a dramatically increased concentration of phospho-tau at Ser-214 in NFTs. Together, our results demonstrate that PTEN regulates tau phosphorylation, binding to microtubules and formation of aggregates and neurite outgrowth. These findings suggest a link between malfunction of PTEN and tauopathy, and imply PTEN as a therapeutic target for tauopathy.

Amyloid beta degradation: a challenging task for brain peptidases.

Amyloid beta (Abeta) accumulates in the neuropil and within the walls of cerebral vessels in association with normal aging, dementia or stroke. Abeta is released from its precursor protein as soluble monomeric species yet, under pathological conditions, it self-aggregates to form soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Abeta levels could be lowered by inhibiting its generation or by promoting its clearance by transport or degradation. Here we will summarize recent findings on brain proteases capable of degrading Abeta, with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence supporting a role in the proteolysis of Abeta in vivo.