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P1‐166: Glutamate dyshomeostasis is associated with cognitive impairment, abnormalities in Insulin Degrading Enzyme levels, and peripheral fasting insulin levels
Author(s) -
Cook David G.,
Watson G. Stennis,
Green Pattie S.,
Zhu Ping,
Li Ning
Publication year - 2010
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2010.05.716
Subject(s) - insulin degrading enzyme , glutamate receptor , medicine , endocrinology , morris water navigation task , genetically modified mouse , insulin , transgene , forebrain , neurotoxicity , in vivo , chemistry , biology , biochemistry , toxicity , central nervous system , hippocampus , receptor , microbiology and biotechnology , gene
recovered in the sarkosyl-insoluble fraction of brain and spinal cord homogenates, it has been hypothesized that they serve to seed TDP-43 aggregation and play an important role in the pathogenesis of ALS and FTLD-U. We and others have found that TDP-43 is a substrate of caspases, and then identified caspase-cleaved TDP-43 fragment (amino acid residues 220-414) is component of inclusions in ALS and FTLD-U brain tissue. But it lacks a cellular model to study the aggregation, degradation and toxicity of TDP-43 Cterminal fragment. Methods: ByusingTet-off system, wehavegenerateda human neuroblastoma stable cell line (termed M17D3) that inducible expresses TDP220-414 fused to enhanced GFP under control of doxycycline. Results: Removal of Dox from the culture media leads to the time-dependent expression of GFP-TDP220-414 and the formation of cytoplasmic GFP-TDP220-414 inclusions. Of interest, GFP-TDP220-414 is phosphorylated at Ser409/Ser410, two sites that are abnormally phosphorylated in ALS and FTLD-U, and high-molecular weight (HMW) GFP-TDP220-414 species are observed by 6 days of induction, even under reducing conditions. Numerous small cytoplasmic inclusions are observed within cells as early as 2 days following the induction of GFPTDP220-414. That little diffuse GFP fluorescence is observed at this time-point highlights the high propensity for GFP-TDP220-414 to aggregate upon expression. At later time-points, larger, but fewer, inclusions are present per cell, suggesting that the larger inclusions are formed by the assembly of small inclusions. Moreover, the inclusions composed of GFP-TDP220-414 are colocalized with ubiqutin and S409/S410-phosphorylated TDP-43 staining. By using this cell line, we tried to answer impacts of ubiquitination and phosphorylation on aggregation of TDP-43 C-terminal fragment, and the degradation pathways of TDP-43 C-terminal fragment. Conclusions: Taken together, our cell model recapitulates a pathological hallmark of ALS and FTLD-U. By using this model, we can dissect the mechanisms of aggregation, degradation and toxicity of TDP-43 C-terminal fragment