Intracellular accumulations of altered, misfolded proteins in neuronal and other cells are pathological hallmarks shared by many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in several genes give rise to familial forms of ALS. Mutations in Sigma receptor 1 have been found to cause a juvenile form of ALS and frontotemporal lobar degeneration (FTLD). We recently described altered localization, abnormal modification and loss of function of SigR1 in sporadic ALS. In order to further elucidate the molecular mechanisms underlying SigR1-mediated alterations in sporadic and familial ALS, we extended our previous studies using neuronal SigR1 knockdown cell lines. We found that loss of SigR1 leads to abnormal ER morphology, mitochondrial abnormalities and impaired autophagic degradation. Consistent with these results, we found that endosomal trafficking of EGFR is impaired upon SigR1 knockdown. Furthermore, in SigR1-deficient cells the transport of vesicular stomatitis virus glycoprotein is inhibited, leading to the accumulation of this cargo protein in the Golgi apparatus. Moreover, depletion of SigR1 destabilized lipid rafts and associated calcium mobilization, confirming the crucial role of SigR1 in lipid raft and intracellular calcium homeostasis. Taken together, our results support the notion that loss of SigR1 function contributes to ALS pathology by causing abnormal ER morphology, lipid raft destabilization and defective endolysosomal pathways.
Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons (MNs) and their target muscles. Misfolded proteins which often form intracellular aggregates are a pathological hallmark of ALS. Disruption of the functional interplay between protein degradation (ubiquitin proteasome system and autophagy) and RNA-binding protein homeostasis has recently been suggested as an integrated model that merges several ALS-associated proteins into a common pathophysiological pathway. The E102Q mutation in one such candidate gene, the endoplasmic reticulum (ER) chaperone Sigma receptor-1 (SigR1), has been reported to cause juvenile ALS. Although loss of SigR1 protein contributes to neurodegeneration in several ways, the molecular mechanisms underlying E102Q-SigR1-mediated neurodegeneration are still unclear. In the present study, we showed that the E102Q-SigR1 protein rapidly aggregates and accumulates in the ER and associated compartments in transfected cells, leading to structural alterations of the ER, nuclear envelope and mitochondria and to subsequent defects in proteasomal degradation and calcium homeostasis. ER defects and proteotoxic stress generated by E102Q-SigR1 aggregates further induce autophagy impairment, accumulation of stress granules and cytoplasmic aggregation of the ALS-linked RNA-binding proteins (RBPs) matrin-3, FUS, and TDP-43. Similar ultrastructural abnormalities as well as altered protein degradation and misregulated RBP homeostasis were observed in primary lymphoblastoid cells (PLCs) derived from E102Q-SigR1 fALS patients. Consistent with these findings, lumbar α-MNs of both sALS as well as fALS patients showed cytoplasmic matrin-3 aggregates which were not co-localized with pTDP-43 aggregates. Taken together, our results support the notion that E102Q-SigR1-mediated ALS pathogenesis comprises a synergistic mechanism of both toxic gain and loss of function involving a vicious circle of altered ER function, impaired protein homeostasis and defective RBPs.
Abstract. To study the reproductive potential of workers in the stingless bee Trigona (Tetragonisca) angustula Illiger (Apidae, Meliponinae), we examined the morphological quality of their eggs. Worker‐eggs were all placed on the inside of the upper wall of brood cells. Normally, such eggs are consumed by the queen and are therefore referred to as trophic. The provisioning and oviposition processes in queenright colonies are characterized by the occurrence of circular aggregations of workers, ‘rosettes’, around the broodcell opening immediately after the release of a worker‐egg. The ovaries of 35% of these rosette workers contained mature, chorionated eggs. In a single worker ovary, always only one mature egg was found. Some of the eggs, dissected from the ovarioles, showed a reticulate chorion pattern. Worker‐eggs which lacked this pattern were significantly bigger than patterned eggs. Scanning electron micrographs revealed that the patterned worker‐eggs are similar in appearance to queen‐eggs. After a worker‐egg had been removed experimentally from a broodcell, the same cell could be oviposited by a worker again. Light microscopic analysis revealed that all these worker‐eggs lacked the reticulate chorion pattern and were very similar in their morphology. In a colony without a laying queen workers laid eggs which had a reticulate pattern on the chorion. Since these eggs developed into males, we assume that the pattern on the chorion is characteristic for reproductive eggs. We also assume that the queen prevents the release of reproductive eggs by the workers. However, she does not inhibit the development of this type of egg.
Abstract. As in many other stingless bees, Melipona bicolor bicolor Lepeletier (Apidae: Meliponinae) workers lay two morphologically distinct types of eggs: slender ones that have a typical patterned chorion, and larger ones that lack this pattern. In this paper we report on the relation between egg morphology and the behaviour of the workers that lay such eggs. In most cases, the laying of each of these egg types is accompanied by a unique sequence of behaviours. After a worker has laid the unpatterned type of egg, she generally leaves the cell, giving the queen the possibility of eating this egg. In the case of the patterned egg type, the worker usually closes the cell immediately after her egg laying. When worker egg laying occurs right after a series of regurgitations, it stops the queen from ovipositing. When, instead, a worker lays an egg after queen oviposition, the cell contains two eggs. This study also revealed cases in which workers laid slender, patterned eggs without closing the cell, and other cases where workers laid large, spherical, unpatterned eggs and instantly closed the cell. Experiments in which worker eggs, destined to be eaten by the queen, were protected by covering the cell artificially with a piece of wax showed that some of these eggs developed into larvae, although they were occasionally relatively small. The occurrence of a range of combinations of egg‐laying behaviours and egg morphologies in M. b. bicolor workers is discussed from the perspective of worker egg laying in other stingless bees.
The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.
The eggs of the mollusc Lymnaea stagnalis generate weak extracellular ionic currents, which have been mapped from oviposition through first cleavage. Throughout this period the current is inward in the animal hemisphere, with highest density at the animal pole, and outward in the vegetal hemisphere, with highest density at the vegetal pole. Peak current densities are measured at the time of first and second polar body formation. During anaphase and telophase of the first mitotic cell cycle, the outward current at the vegetal pole reaches its minimum density and its direction is reversed in most eggs, whereas the inward current at the animal pole gradually increases. This coincides with the segregation of the so-called animal pole plasm to the animal pole (Raven, 1970). The organic calcium channel blockers diltiazem and D600 cause abnormal maturation division(s) and/or first cleavage. At the same time they reduce and eventually abolish the associated ionic currents. These results suggest the existence of a cell-cycle correlated, calcium-dependent component of ionic currents in Lymnaea eggs.
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