TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA-binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signaling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies.
TDP-43 is the major disease protein in ubiquitin-positive inclusions of amyotrophic lateral sclerosis and frontotemporal lobar degeneration (FTLD) characterized by TDP-43 pathology (FTLD-TDP). Accumulation of insoluble TDP-43 aggregates could impair normal TDP-43 functions and initiate disease progression. Thus, it is critical to define the signalling mechanisms regulating TDP-43 since this could open up new avenues for therapeutic interventions. Here, we have identified a redox-mediated signalling mechanism directly regulating TDP-43. Using in vitro and cell-based studies, we demonstrate that oxidative stress promotes TDP-43 cross-linking via cysteine oxidation and disulphide bond formation leading to decreased TDP-43 solubility. Biochemical analysis identified several cysteine residues located within and adjacent to the second RNA-recognition motif that contribute to both intra-and inter-molecular interactions, supporting TDP-43 as a target of redox signalling. Moreover, increased levels of cross-linked TDP-43 species are found in FTLD-TDP brains, indicating that aberrant TDP-43 cross-linking is a prominent pathological feature of this disease. Thus, TDP-43 is dynamically regulated by a redox regulatory switch that links oxidative stress to the modulation of TDP-43 and its downstream targets.
Tau proteins are the building blocks of neurofibrillary tangles (NFTs) found in a range of neurodegenerative tauopathies, including Alzheimer's disease. Recently, we demonstrated that tau is extensively post-translationally modified by lysine acetylation, which impairs normal tau function and promotes pathological aggregation. Identifying the enzymes that mediate tau acetylation could provide targets for future therapies aimed at reducing the burden of acetylated tau. Here, we report that mammalian tau proteins possess intrinsic enzymatic activity capable of catalyzing self-acetylation. Functional mapping of tau acetyltransferase activity followed by biochemical analysis revealed that tau uses catalytic cysteine residues in the microtubule-binding domain to facilitate tau lysine acetylation, thus suggesting a mechanism similar to that employed by MYST-family acetyltransferases. The identification of tau as an acetyltransferase provides a framework to further understand tau pathogenesis and highlights tau enzymatic activity as a potential therapeutic target.
Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source, potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 or 1 x 10 6 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly, in this study, a minimum of 1 x 10 5 ES cells in the myocardium and 1 x 10 4 cells in the skeletal muscle was observed to be requisite for teratoma development, suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic.
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