Browsing by Author "Tsutsui, Shigeki"

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    Copper ions, prion protein and Aβ modulate Ca levels in central nervous system myelin in an NMDA receptor-dependent manner
    (2022-07-26) Tsutsui, Shigeki; Morgan, Megan; Tedford, Hugo; You, Haitao; Zamponi, Gerald W.; Stys, Peter K.
    Abstract As in neurons, CNS myelin expresses N-Methyl-D-Aspartate Receptors (NMDARs) that subserve physiological roles, but have the potential to induce injury to this vital element. Using 2-photon imaging of myelinic Ca in live ex vivo mouse optic nerves, we show that Cu ions potently modulate Ca levels in an NMDAR-dependent manner. Chelating Cu in the perfusate induced a substantial increase in Ca levels, and also caused significant axo-myelinic injury. Myelinic NMDARs are shown to be regulated by cellular prion protein; only in prion protein KO optic nerves does application of NMDA + D-serine induce a large Ca increase, consistent with strong desensitization of these receptors in the presence of prion protein limiting Ca overload. Aβ1-42 peptide induced a large Ca increase that was also Cu-dependent, and was blocked by NMDAR antagonism. Our results indicate that like in neurons, myelinic NMDARs permeate potentially injurious amounts of Ca, and are also potently regulated by micromolar Cu and activated by Aβ1-42 peptides. These findings shed mechanistic light on the important primary white matter injury frequently observed in Alzheimer's brain.
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    Investigations on the role of rab7 in prion infection
    (2022-06) Cherry, Pearl; Gilch, Sabine; Kar, Satyabrata; Braun, Janice; Haigh, Cathryn; Tsutsui, Shigeki
    Prion diseases are fatal and infectious neurodegenerative diseases caused by the misfolding of the cellular prion protein PrPc into its infectious isoform PrPSc, which comprises the main if not the only constituent of prions. In response to prion infection certain cellular impairments such as reduced membrane association of Rab7, compromised lysosomal acidification and elevated cholesterol levels are observed. Here, we found that prion infected primary neurons are marked by an increased expression of active Rab7 (Rab7.GTP) levels during the initial stages of the infection, followed by a loss in its levels as the infection progresses. However, astrocytes in prion infected terminal mice upregulate the active Rab7 expression. The reduced Rab7 activation is linked to its reduced ubiquitination status. In neurons, the loss in active Rab7 leads to a delay in the Rab7 mediated trafficking of LDL to the lysosomes and Golgi, resulting in a defective feedback regulation of cholesterol metabolism. This in turn triggers de novo cholesterol synthesis. Over-expression of a constitutively active mutant of Rab7 in prion infected neuronal cell-lines rescues the delay in LDL trafficking, restores the normal cholesterol levels and reduces prion propagation. Inspired by the differential kinetics of Rab7 activation during prion infection and the cell-type specific differences of Rab7 activation in the prion infected terminal mouse brain, we studied the Rab7 interactome using quantitative proteomics. We identified a loss in the Rab7 interactome associated with ubiquitination in prion infected cells and synaptic signalling in prion infected brains, whereas proteins in the mitochondrial respiratory chain were observed to be lost in both cases. The enriched pathways identified in Rab7 interactome were linked to mRNA transport and degradation to name some. In the prion-infected brain, we identified the cholesterol esterification pathway to be enriched in the Rab7 interactome in the brain. These studies indicate a possible involvement of Rab7 in the above-mentioned pathways and suggest candidate proteins to be evaluated for their potential to ubiquitinate Rab7, as a functional impairment of the Rab7 ubiquitin ligase can cause reduced activation observed in prion infections.
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    The Role of Quaternary Structure Organization in Prion Strain Selection and Adaptation
    (2023-08) Chang, Sheng Chun; Gilch, Sabine; Braun, Janice; Gilleard, John; Tsutsui, Shigeki; Booth, Stephanie
    Prion diseases are fatal and infectious neurodegenerative diseases caused by the misfolded, protease-resistant, and aggregated prion protein isoform, PrPSc. Unlike conventional pathogens, the propagation of prions does not depend on the replication of genetic material, but different cases of infection can lead to PrPSc adopting specific conformations resulting in different and stably transmissible phenotypic properties that are, by definition, the determinants of different strains. The distribution of PrPSc quaternary structure has previously been determined to vary across prion strains, and different PrPSc aggregate assemblies in turn harbour varying levels of infectivity. We were interested in determining how PrPSc aggregate size modulate strain properties and how these properties are affected by different inoculation routes, specifically in comparing peripheral versus intracerebral infection. Using in vivo murine models, we explored prion disease pathogenesis and strain selection upon inoculation of different PrPSc protein quaternary structures or prion strains. Transgenic mice overexpressing cervid prion protein inoculated with different elk chronic wasting disease PrPSc fractions exhibited strikingly different survival times and disease phenotypes, including variations in the prion biochemical, neuropathological, and biophysical profiles; interestingly, these properties converged upon passaging, though not the differing clinical signs. Gene-targeted mice expressing the cervid prion protein at physiological levels infected with CWD prions either directly into the brain or from the periphery resulted in different biochemical and neuropathological properties, notably with a converging conformational profile among the peripherally inoculated group. Similar observations were recorded where animals peripherally inoculated with CWD prion aggregates exhibited markedly reduced variations in survival times compared to infection directly into the brain. These results indicate that the direct selection of PrPSc conformers, or PrPSc conformational mutation, is likely to occur in the presence of a barrier interfering with the ideal replication setting as adaptation under a more restrictive set of circumstances takes place. This research demonstrates the intricacies of prion strain selection and adaptation that would provide novel insight into future investigations of the evolution and emergence of prion strains.