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  • 1.
    Beckman, Marie
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Temporal events in neuronal differentiation and cell death: expression and processing of the Alzheimer's amyloid precursor protein family and a protein at the nuclear pore2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The present study had two major objectives: 1) to elucidate the involvement of Alzheimer’s amyloid precursor protein (APP) family in neuronal differentiation, and the effect of the Alzheimer’s disease (AD)-linked mutation APPV642I on signal transduction; 2) to investigate the fate of the nuclear pore complex protein POM121 during apoptosis and to examine the possibility of using green fluorescent protein (GFP)-labelled POM121 as a non-invasive sensor of apoptosis in living (non-fixed) cells.

    APP is the parent protein of the b-amyloid peptide, which is the major peptide constituent in the “senile plaques” of AD. APP and the homologous amyloid precursor-like proteins, APLP1 and APLP2, are members of the APP family. We compared the temporal expression patterns of these proteins during retinoic acid (RA)-induced neuronal differentiation of human neuroblastoma cells. To this end, we established a quantitative non-radioactive Northern blot assay for APLP1, APLP2 and APP. We found that the transcripts of all three APP family members were increased in response to RA. This occurred simultaneously with progressive neurite outgrowth and increased expression of neuronal markers. In addition, we observed that the increase in APLP2 mRNA was similar to that of APP mRNA, whereas the increase in APLP1 mRNA was significantly higher. The elevated mRNA levels also resulted in an in-creased protein expression of APLP1, APLP2 and the neuronal APP695 isoform. Studies using curcumin (diferuloylmethane), an inhibitor of the transcription factors NFkB/AP-1, and the mitogen-activated protein kinase (MAPK) JNK (c-Jun N-terminal kinase), revealed a diffe-rential regulation of APLP1 and APLP2. Curcumin suppressed the RA-induced mRNA expression of the APP family, in particular that of APLP1. On the protein level, curcumin also reduced the expression of APLP1. In contrast, curcumin induced an accumulation of APLP2, which we propose is due to inhibition of its proteolytic processing. Furthermore, curcumin induced neurite retraction in RA-differentiated cells without affecting their viability. Our results suggest that NFkB/AP-1 signal transduction pathways mediate a co-ordinated regula-tion of the mRNA expression of the APP family and that APLP1 processing is not regulated by the same mechanisms as the processing of APLP2 and APP. Our results are in agreement with important functions for APLP1, APLP2 and APP within the period of neurite extension and synaptic maturation, and a proposed role for these proteins in neuronal differentiation and synaptic plasticity.

    Using a doxycycline-controlled gene expression system (Tet-On), we investigated the effect of wild-type APP695 and the pathogenic familial AD-linked APPV642I mutant on signal transduction. Overexpression was induced at different levels in rat pheochromocytoma (PC12) Tet-On cells. We observed a nerve growth factor-dependent increase in the levels of phosphorylated extracellular regulated kinases 1 and 2 in response to expression of mutant APP. Our results support that increased signalling via MAPKs may have a role in the development of AD. In addition, we found that the inducing agent doxycycline in itself affected cell signalling and protected against oxidative stress. This information is critical for evaluation of the effects of transgene expression using Tet systems.

    Finally, we showed that POM121 is cleaved by a caspase-3-dependent mechanism at aspartate 531 during apoptosis. Characterising the degradation of POM121-GFP in relation to other apoptotic events, revealed that it can be applied as an early non-invasive sensor of nuc-lear apoptosis in living cells using fluorescence microscopy or fluorimetric analysis.

  • 2.
    Beckman, Marie
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kihlmark, Madeleine
    Södertörn University, Stockholm, Sweden.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Nucleus and Nuclear Envelope: Methods for Preparation2010Book (Other academic)
    Abstract [en]

    The cell nucleus of eukaryotic organisms contains the genome surrounded by a nuclear envelope consisting of a double-lipid membrane with embedded nuclear pores and an underlying nuclear lamina. The uniformity in size and density makes it possible to isolate pure intact nuclei at high yields from tissue homogenates by centrifugation through a sucrose cushion. Nuclear envelopes can be prepared from isolated nuclei by enzymatic degradation of their nucleic acid content. The resulting nuclear envelope preparations contain structurally well-conserved inner and outer nuclear membranes with attached ribosomes, nuclear pore complexes and nuclear lamina. Reliable methods for preparation of nuclei and nuclear envelopes play an important role in the successful identification of components that are located in nuclei and in nuclear subcompartments.

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