Heat shock-induced phosphorylation of TAR DNA-binding protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function

TAR DNA-binding protein (TDP-43) is a highly conserved and essential DNA- and RNA-binding protein that controls gene expression through RNA processing, in particular, regulation of splicing. Intracellular aggregation of TDP-43 is a hallmark of amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. This TDP-43 pathology is also present in other types of neurodegeneration including Alzheimer's disease. We report here that TDP-43 is a substrate of MEK, a central kinase in the MAPK/ERK signaling pathway. TDP-43 dual phosphorylation by MEK, at threonine 153 and tyrosine 155 (p-T153/Y155), was dramatically increased by the heat shock response (HSR) in human cells. HSR promotes cell survival under proteotoxic conditions by maintaining protein homeostasis and preventing protein misfolding. MEK is activated by HSR and contributes to the regulation of proteome stability. Phosphorylated TDP-43 was not associated with TDP-43 aggregation, and p-T153/Y155 remained soluble under conditions that promote protein misfolding. We found that active MEK significantly alters TDP-43-regulated splicing and that phosphomimetic substitutions at these two residues reduce binding to GU-rich RNA. Cellular imaging using a phospho-specific p-T153/Y155 antibody showed that phosphorylated TDP-43 was specifically recruited to the nucleoli, suggesting that p-T153/Y155 regulates a previously unappreciated function of TDP-43 in the processing of nucleolar-associated RNA. These findings highlight a new mechanism that regulates TDP-43 function and homeostasis through phosphorylation and, therefore, may contribute to the development of strategies to prevent TDP-43 aggregation and to uncover previously unexplored roles of TDP-43 in cell metabolism

Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from patients with sporadic ALS and familial ALS with GGGGCC repeat expansion in the C9orf72 gene, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS-FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability

Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation.

The normally soluble TAR DNA-binding protein 43 (TDP-43) is found aggregated both in reversible stress granules and in irreversible pathogenic amyloid. In TDP-43, the low-complexity domain (LCD) is believed to be involved in both types of aggregation. To uncover the structural origins of these two modes of β-sheet-rich aggregation, we have determined ten structures of segments of the LCD of human TDP-43. Six of these segments form steric zippers characteristic of the spines of pathogenic amyloid fibrils; four others form LARKS, the labile amyloid-like interactions characteristic of protein hydrogels and proteins found in membraneless organelles, including stress granules. Supporting a hypothetical pathway from reversible to irreversible amyloid aggregation, we found that familial ALS variants of TDP-43 convert LARKS to irreversible aggregates. Our structures suggest how TDP-43 adopts both reversible and irreversible β-sheet aggregates and the role of mutation in the possible transition of reversible to irreversible pathogenic aggregation

One-pot Enzymatic Synthesis of Deoxy-thymidine-diphosphate (TDP)-2-deoxy-∝-d-glucose Using Phosphomannomutase

Production of deoxy-thymidine-diphosphate (TDP)-sugars as substrates of glycosyltransferases, has been one of main hurdles for combinatorial antibiotic biosynthesis, which combines sugar moiety with aglycon of various antibiotics. Here, we report the one-pot enzymatic synthesis of TDP-2-deoxy-glucose employing high efficient TMP kinase (TMK; E.C. 2.7.2.12), acetate kinase (ACK; E.C. 2.7.1.21), and TDP-glucose synthase (TGS; E.C. 2.7.7.24) with phosphomannomutase (PMM; E.C. 5.4.2.8). In this study, replacing phosphoglucomutase (PGM; E.C. 5.4.2) by PMM from Escherichia coli gave four times higher specific activity on 2-deoxy-6-phosphate glucose, suggesting that the activity on 2-deoxy-glucose-6-phosphate was mainly affected by PMM activity, not PGM activity. Using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, TDP-2-deoxy-glucose (63% yield) was successfully synthesized. Considering low productivity of NDP-sugars from cheap starting materials, this paper showed how production of NDP-sugars could be enhanced by controlling mutase activity

Time Driven Priority Router Implementation and First Experiments

This paper reports on the implementation of Time-Driven Priority (TDP) scheduling on a FreeBSD platform. This work is part of a TDP prototyping and demonstration project aimed at showing the implications of TDP deployment in packet-switched networks, especially benefits for real-time applications. This paper focuses on practical aspects related to the implementation of the technology on a Personal Computer (PC)-based router and presents the experimental results obtained on a testbed network. The basic building blocks of a TDP router are described and implementation choices are discussed. The relevant results achieved and here presented can be categorized into two types: qualitative results, including the successful integration of all needed blocks and the insight obtained on the complexity related to the implementation of a TDP router, and quantitative ones, including measures of achievable network utilization and of jitter experienced on a fully-loaded TDP network. The outcome demonstrates the effectiveness of the presented implementation while confirming TDP points of strengt