Conditional overexpression of four-repeat human tau containing the P301L missense mutation in the rTg4510 mouse model of tauopathy leads to progressive accumulation of neurofibrillary tangles and hyperphosphorylated, sarkosyl-insoluble tau species, which are biochemically comparable to abnormal tau characteristic of hereditary tauopathies termed FTDP-17. To fully understand the impact of tau species at different stages of self-assembly on neurodegeneration, we fractionated rTg4510 brain representing several stages of tauopathy to obtain TBS-extractable (S1), high salt/sarkosyl-extractable (S3), and sarkosyl-insoluble (P3) fractions. Under reducing condition, the S1 fraction was demonstrated by Western blotting to contain both 50–60 kDa normally-sized and 64 kDa tau. Both are thermo-stable, but the 64 kDa tau showed a higher degree of phosphorylation. Under non-reducing condition, nearly all TBS-extractable 64 kDa tau were detected as ~130 kDa species consistent with the size of dimer. Quantitative analysis showed ~80 times more 64 kDa tau in S1 than P3 fraction. Immunoelectron microscopy revealed tau-positive granules/short filaments in S1 fraction. These structures displayed MC1 immunoreactivities indicative of conformational/pathological change of tau. MC1 immunoreactivity was detected by dot blotting in samples from 2.5 month-old mice, whereas Ab39 immunoreactivity indicative of late stages of tau assembly was detected only in P3 fraction. Quantitative analysis also demonstrated a significant inverse correlation between brain weight and 64 kDa tau, but the level of TBS-extractable 64 kDa tau reflects neurodegeneration better than that of sarkosyl-insoluble 64 kDa tau. Together, the findings suggest that TBS-extractable 64 kDa tau production is a potential target for therapeutic intervention of tauopathies.

Disruption of retinal pigment epithelial (RPE barrier integrity is a hallmark feature of various retinal blinding diseases, including diabetic macular edema and age-related macular degeneration, but the underlying causes and pathophysiology are not completely well-defined. One of the most conserved phenomena in biology is the progressive decline in mitochondrial function with aging leading to cytopathic hypoxia, where cells are unable to use oxygen for energy production. Therefore, this study aimed to thoroughly investigate the role of cytopathic hypoxia in compromising the barrier functionality of RPE cells. We used Electric Cell-Substrate Impedance Sensing (ECIS) system to monitor precisely in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with various concentrations of cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). We further investigated how the resistance across ARPE-19 cells changes across three separate parameters: Rb (the electrical resistance between ARPE-19 cells), α (the resistance between the ARPE-19 and its substrate), and Cm (the capacitance of the ARPE-19 cell membrane). The viability of the ARPE-19 cells and mitochondrial bioenergetics were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and seahorse technology, respectively. ECIS measurement showed that CoCl2 reduced the total impedance of ARPE-19 cells in a dose dependent manner across all tested frequencies. Specifically, the ECIS program’s modelling demonstrated that CoCl2 affected Rb as it begins to drastically decrease earlier than α or Cm, although ARPE-19 cells’ viability was not compromised. Using seahorse technology, all three concentrations of CoCl2 significantly impaired basal, maximal, and ATP-linked respirations of ARPE-19 cells but did not affect proton leak and non-mitochondrial bioenergetic. Concordantly, the expression of a major paracellular tight junction protein (ZO-1) was reduced significantly with CoCl2-treatment in a dose-dependent manner. Our data demonstrate that the ARPE-19 cells have distinct dielectric properties in response to cytopathic hypoxia in which disruption of barrier integrity between ARPE-19 cells precedes any changes in cells’ viability, cell-substrate contacts, and cell membrane permeability. Such differences can be used in screening of selective agents that improve the assembly of RPE tight junction without compromising other RPE barrier parameters.

Background: The BCL2-mediated anti-apoptotic phenotype is a contributor to the genesis and maintenance of a broad variety of tumors. BCL2 is also implicated in the regulation of the cell cycle by playing a role in the transition between quiescence and the cycling state. Further, chromosomal rearrangements, including t(14;18), up-regulate BCL2 transcription, preventing tumor cell death in B-cell lymphomas. PNT2258 is a DNA interference (DNAi) therapeutic targeted against BCL2 that is undergoing clinical evaluation in patients with hematological malignancies. PNT2258 contains PNT100, a single-stranded phosphodiester DNA oligodeoxynucleotide, encapsulated in protective liposomes. Material and Methods: Lymphomas cell lines with distinct genetic characteristics, namely, WSU-FSCCL, characterized by t(14;18) BCL2 and t(8;14) CMYC rearrangements, WSU-DLCL2, characterized by t(14;18) BCL2 rearrangement, and WSU-WM, characterized as lacking t(14;18) BCL2, but having t(8;14) CMYC and t(12;17) rearrangements were tested for cell viability at 24, 48, 72, and 96 hours post-exposure to PNT2258 at concentrations of 2.5, 5, and 10 μM. Normalized maximum cell kill at 96 hours was used in each cell line to adjust for differences in growth rates and to calculate sensitivity. Results: Dose-dependent effects were observed across all three cell lines. WSU-FSCCL was the most proliferative cell line, and correspondingly most sensitive to the effects of PNT2258, with a control doubling time of 29.6h and 1% of viable control cells remaining at 96h at 10 μM. The next most sensitive cell line was WSU-DLCL2, having a 60.6h control doubling time and 11% of the viable control cells remaining at 96h at 10 μM. WSU-WM exhibited a control doubling time of 35.5h and 21% of viable control cells remaining at 96h at 10 μM. These findings parallel the single agent activity of PNT2258 against xenograft tumor models containing BCL2 or CMYC chromosomal rearrangements (AACR Meeting Abstracts, Apr 2007; 2007: 4889). Conclusions: The effects of the clinical therapeutic PNT2258 against cell lines with well-characterized growth and genetic drivers were examined. Findings show lymphoma cell lines harboring the t(14;18) rearrangement (WSU-FSCCL and WSU-DLCL2) were most sensitive to PNT2258 and in this context a higher proliferative rate is linked to sensitivity. These data suggest that commonly used clinical assays of proliferation and tumor “aggressiveness,” such as Ki-67 and PET/CT standardized uptake values (SUVs), may be useful in conjunction with cytogenetic analysis (e.g. for t(14;18) and/or t(8,14) by fluorescence in situ hybridization (FISH), to select patients with tumors that may be responsive to the effects of PNT2258. Citation Format: Michael J. Woolliscroft, Abdul-Shukkur Ebrahim, Richard A. Messmann, Shari K. Gaylor, Mina P. Sooch, Ayad Al-Katib, Wendi V. Rodrigueza. The sensitivity of targeting genomic BCL2 by PNT2258 is linked to chromosomal rearrangements and proliferative rate of tumor types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2014-5473

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