Abstract
TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition in vitro and in vivo. However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRed-tagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro.
Highlights
Protein aggregation is one of the pathological hallmarks of neurodegenerative diseases such as prion disease, Alzheimer’s disease, frontotemporal lobar degeneration (FTLD), Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS) [1,2,3,4]
We have previously demonstrated that proteasome inhibition enhanced the formation of adenovirus-transduced artificial TAR DNA-binding protein 43 (TDP-43) cytoplasmic aggregates both in vitro and in vivo, suggesting that impairment of protein degradation pathways accelerates the formation of TDP-43-positive aggregates [26]
The 1464R cell line used in the present study proliferates indefinitely without morphological alterations and, in the presence of retinoic acid, ceases dividing and differentiates into neuronal and glial cells that are very similar to primary cultured brain cells [38]
Summary
Protein aggregation is one of the pathological hallmarks of neurodegenerative diseases such as prion disease, Alzheimer’s disease, frontotemporal lobar degeneration (FTLD), Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS) [1,2,3,4]. Formation and spreading of TDP-43 aggregates in cultured cells demonstrated by time-lapse imaging no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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