Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling.

Tissue-specific somatic stem cells are characterized by their ability to reside in a state of prolonged reversible cell cycle arrest, referred to as quiescence. Maintenance of a balance between cell quiescence and division is critical for tissue homeostasis at the cellular level and is dynamically regulated by numerous extrinsic and intrinsic factors. Analysis of the activation of quiescent stem cells has been challenging because of a lack of methods for direct detection of de novo dividing cells. Here, we present and experimentally verify a novel method based on double labeling with thymidine analogues to detect de novo dividing stem cells in situ. In a proof of concept for the method, we show that memantine, a drug widely used for Alzheimer's disease therapy and a known strong inducer of adult hippocampal neurogenesis, increases the recruitment into the division cycle of quiescent radial glia-like stem cells-primary precursors of the adult-born neurons in the hippocampus. Our method could be applied to assess the effects of aging, pathology, or drug treatments on the quiescent stem cells in stem cell compartments in developing and adult tissues.

Synthetic Thymidine Analog Labeling without Misconceptions.

Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach.

TDP-43 interacts with mitochondrial proteins critical for mitophagy and mitochondrial dynamics.

Transactive response DNA-binding protein of 43 kDa (TDP-43) functions as a heterogeneous nuclear ribonucleoprotein and is the major pathological protein in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis/motor neuron disease (ALS/MND). TDP-43 pathology may also be present as a comorbidity in approximately 20-50% of sporadic Alzheimer's disease cases. In a mouse model of MND, full-length TDP-43 increases association with the mitochondria and blocking the TDP-43/mitochondria interaction ameliorates motor dysfunction. Utilizing a proteomics screen, several mitochondrial TDP-43-interacting partners were identified, including voltage-gated anion channel 1 (VDAC1) and prohibitin 2 (PHB2), a crucial mitophagy receptor. Overexpression of TDP-43 led to an increase in PHB2 whereas TDP-43 knockdown reduced PHB2 expression in cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inducer of mitophagy. These results suggest that TDP-43 expression contributes to metabolism and mitochondrial function however we show no change in bioenergetics when TDP-43 is overexpressed and knocked down in HEK293T cells. Furthermore, the fusion protein mitofusin 2 (MFN2) interacts in complex with TDP-43 and selective expression of human TDP-43 in the hippocampus and cortex induced an age-dependent change in Mfn2 expression. Mitochondria morphology is altered in 9-month-old mice selectively expressing TDP-43 in an APP/PS1 background compared with APP/PS1 littermates. We further confirmed TDP-43 localization to the mitochondria using immunogold labeled TDP-43 transmission electron microscopy (TEM) and mitochondrial isolation methods There was no increase in full-length TDP-43 localized to the mitochondria in APP/PS1 mice compared to wild-type (littermates); however, using C- and N-terminal-specific TDP-43 antibodies, the N-terminal (27 kDa, N27) and C-terminal (30 kDa, C30) fragments of TDP-43 are greatly enriched in mitochondrial fractions. In addition, when the mitochondrial peptidase (PMPCA) is overexpressed there is an increase in the N-terminal fragment (N27). These results suggest that TDP-43 processing may contribute to metabolism and mitochondrial function.