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@PHDTHESIS{Kaniyappan:270420,
      author       = {Kaniyappan, Senthilvelrajan},
      title        = {{S}tructural and {F}unctional {C}haracterization of
                      {N}eurotoxic {O}ligomers of {P}ro-aggregant {T}au {R}epeat
                      {D}omain},
      school       = {Rheinische Friedrich-Wilhelms-Universität Bonn},
      type         = {Dissertation},
      reportid     = {DZNE-2024-00798},
      pages        = {127 pages, 41 figures},
      year         = {2016},
      note         = {Dissertation, Rheinische Friedrich-Wilhelms-Universität
                      Bonn, 2015},
      abstract     = {Tau protein is a microtubule associated protein present
                      abundantly in the neurons of the central nervous system
                      where it stabilizes the axonal microtubules thereby
                      providing structural architecture for the axons of neurons.
                      Aggregation of Tau occurs in many neurodegenerative diseases
                      collectively termed tauopathies including Alzheimer disease
                      (AD) and frontotemporal dementia (FTD). The mutation ΔK280
                      of Tau was originally discovered in cases of FTD (Rizzu et
                      al., 1999). In vitro it leads to a pronounced propensity of
                      the protein to aggregate (Barghorn et al., 2000). The repeat
                      domain of Tau protein with this pro-aggregant mutation
                      (TauRDΔ) induces toxicity in transgenic mice and
                      organotypic hippocampal slice culture models (Sydow et al.,
                      2011, Messing et al., 2013). One current concept of
                      Tau-mediated toxicity in Alzheimer disease and related tau
                      dependent pathologies is that it is based on low-n
                      oligomeric species, rather than higher aggregated forms
                      (fibers and neurofibrillary tangles). To test this we
                      characterized oligomers from TauRDΔ protein assembled and
                      purified in vitro. Since Tau oligomers are in dynamic
                      equilibrium during aggregation, we tried to capture and
                      stabilize only the oligomeric forms of Tau using EGCG
                      (Epigallocatechin gallate). EGCG reduces the formation of
                      fibrils and increases the SDS stable oligomers. However, the
                      oligomers are not separable by gel filtration
                      chromatography. Therefore we stabilized the tau oligomers
                      using a low concentration of glutaraldehyde as a
                      cross-linking reagent. This yielded SDS stable low-n
                      oligomers predominantly in the form of dimers, trimers,
                      tetramers with very low amounts of higher order species. The
                      cross-linked TauRDΔ oligomers can be purified by
                      hydrophobic interaction chromatography with $~95\%$ purity.
                      They exhibit enhanced fluorescence with the dye ANS, arguing
                      for an altered conformation (compared with monomers) and
                      possibly exposed hydrophobic surface patches. However, they
                      do not contain substantial ß-sheet structure, as analyzed
                      by thioflavin S fluorescence and circular dichroism. Atomic
                      force microscopy (AFM) of TauRDΔ oligomers reveals that the
                      particles are roughly globular in shape, with diameters in
                      the range 1.6-5.4 nm (AFM height values). The hydrodynamic
                      radius of TauRDΔ oligomers (~5.2 nm) is dominated by that
                      of tetramers, as measured by dynamic light scattering. The
                      size of TauRDΔ oligomers reveals that they contain up to
                      4-5 molecules of Tau, consistent with the SDS gel analysis.
                      The TauRDΔ oligomers do not exhibit global toxicity towards
                      rat primary neurons when applied to the extracellular
                      medium, as judged by MTT and LDH assays. However, functional
                      impairment can be deduced from a pronounced (up to $50\%)$
                      decrease of dendritic spines and a shift from
                      mushroom-shaped to stubby spines. Consistent with this, the
                      expression of cytoskeletal proteins which are necessary to
                      maintain the mushroom spines is reduced. The neurons also
                      show an increase in reactive oxygen species and influx of
                      calcium. In summary, low-n oligomers of TauRDΔ do not cause
                      gross changes in viability, but induce subtle functional
                      defects, leading to an increase in Ca++ and ROS, and
                      consequently to loss of spines and associated shape
                      changes.Since Tau is an intracellular protein and the
                      formation of oligomers occurs inside the cells, we
                      introduced low-n Tau oligomers by protein transfection into
                      SH-SY5Y cells and primary rat hippocampal neurons and
                      analyzed them by flow cytometry and western blot analysis.
                      This showed that only the cells transfected with TauRDΔ
                      oligomers (but not monomers) induce the intracellular
                      aggregation of Tau and recruitment of endogenous Tau into
                      the aggregates. This is accompanied by the
                      hyperphosphorylation of aggregated Tau. Although TauRDΔ
                      oligomer transfected cells do not undergo cell death within
                      15 h of transfection, we found the presence of annexin V
                      positive cells. When compared to monomers and fibrils, the
                      oligomer transfected cells show a 5 fold increase in annexin
                      V positive cells suggesting enhanced apoptosis. We conclude
                      that TauRDΔ oligomers applied extracellularly cause
                      degeneration of spines without affecting cell viability,
                      whereas introducing oligomers intracellularly leads to Tau
                      aggregation and apoptosis.},
      cin          = {AG (Eckhard) Mandelkow / AG (Eva) Mandelkow},
      cid          = {I:(DE-2719)1013014 / I:(DE-2719)1013015},
      pnm          = {899 - ohne Topic (POF4-899)},
      pid          = {G:(DE-HGF)POF4-899},
      typ          = {PUB:(DE-HGF)11},
      urn          = {https://nbn-resolving.org/urn:nbn:de:hbz:5n-43264},
      url          = {https://pub.dzne.de/record/270420},
}