Abstract. The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus.The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAG-QAKKKKLD, and the sequence AVKRPAATKKAG-QAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism. IN addition to its established role in chromatin assembly and histone storage in the Xenopus oocyte and egg (4,16,18,21), nucleoplasmin has emerged as the protein of choice for studies of protein transport into the cell nucleus (reviewed in reference 5). Investigations using this acidic, thermostable, pentameric protein provided the decisive evidence that a mechanism of uptake involving selective entry into the nucleus operates in oocytes (8).Nucleoplasmin has also been used to demonstrate that entry into the Xenopus oocyte nucleus occurs through the nuclear pore complex (9), to evaluate the transport properties of nuclei reconstituted in vitro, and to show that ATP is required for the transport process (28). Primary amino acid sequences specifying entry into the nucleus have been identified in a number of proteins (5,23,25,30). The sequence that has been studied in greatest molecular detail is the SV-40 large T antigen nuclear location sequence (3,11,14,15,19,20,33,37). The earlier studies in this series demonstrated that the amino acid sequence PKK~28KRKV is able to translocate chicken muscle pyruvate kinase to the cell nucleus (15).The chicken muscle pyruvate kinase expression vector system has also been used to map the polyoma virus large T nu-C. Dingwall's present address is Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, England.clear location sequences (32) and more recently to investigate the effect of protein context upon the function of the SV-40 large T antigen nuclear location sequence (33). Therefore we chose this system to identify the nuclear location sequence or sequences in the nucleoplasmin "tail" region since a large body of data exists against which we can interpret our results.We have sequenced nucleoplasmin and shown that the tail regi...
Our understanding of the molecular processes underlying Alzheimer’s disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investigate how CLU mediates Aβ-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate Aβ-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aβ using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with Aβ25-35 peptides and Aβ1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment. However, in CLU knockout neurons Aβ exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to Aβ in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aβ and Dickkopf-1 (DKK1) proteins in an Aβ-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in Aβ treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aβ compared to the wild type neurons supports the role of clusterin in Aβ-mediated AD pathogenesis.
Human-induced pluripotent stem cells (iPSCs) offer a novel, timely approach for investigating the aetiology of neuropsychiatric disorders. Although we are starting to gain more insight into the specific mechanisms that cause Alzheimer's disease and other forms of dementia, this has not resulted in therapies to slow the pathological processes. Animal models have been paramount in studying the neurobiological processes underlying psychiatric disorders. Nonetheless, these human conditions cannot be entirely recapitulated in rodents. Human cell models derived from patients' cells now offer new hope for improving our understanding of the early molecular stages of these diseases, through to validating therapeutics. The impact of dementia is increasing, and a new model to investigate the early stages of this disease is heralded as an essential, new platform for translational research. In this paper, we review current literature using iPSCs to study Alzheimer's disease, describe drug discovery efforts using this platform, and discuss the future potential for this technology in psychiatry research.
We have investigated a pathogenic mutation in D-amino acid oxidase (DAO), DAOR199W, associated with familial Amyotrophic Lateral Sclerosis (ALS) that impairs D-serine metabolism and causes protein aggregation, autophagy and cell death in motor neuron cell lines. These features are consistent with the pathogenic processes occurring in ALS but most importantly, we have demonstrated that activation of the formation of ubiquitinated protein inclusions, increased autophagosome production and apoptotic cell death caused by the mutation in cell lines are attenuated by 5,7-dichlorokynurenic acid (DCKA), a selective inhibitor of the glycine/D-serine binding site of the NMDA receptor. D-serine is an essential co-agonist at this glutamate receptor. This data provides insight into potential upstream mechanisms that involve the action of D-serine at the NMDA receptor and might contribute to neurodegeneration. This is highly relevant to sporadic ALS (SALS), familial ALS, as well as ALS models, where elevated levels of D-serine have been reported and hence has broader clinical therapeutic implications. In order to investigate this further, we have generated a transgenic line expressing the pathogenic mutation, in order to determine whether mice expressing DAOR199W develop a motor phenotype and whether crossing the SOD1G93A model of ALS with mice expressing DAOR199W affects disease progression. We found that heterozygous expression of DAOR199W led to a significant loss of spinal cord motor neurons at 14 months, which is similar to that found in homozygous mice expressing DAOG181R. We hypothesize that DAO has potential for development as a therapeutic agent in ALS.
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