TDP-43 knockdown impairs neurite outgrowth dependent on its target histone deacetylase 6

<p>Abstract</p> <p>Background</p> <p>Trans-activation response element (TAR) DNA binding protein of 43kDa (TDP-43) is causally related to the neurodegenerative diseases frontotemporal dementia and amyotrophic lateral sclerosis being the hallmark protein in the disease-characteristic neuropathological lesions and via genetic linkage. Histone deacetylase 6 (HDAC6) is an established target of the RNA-binding protein TDP-43. HDAC6 is an unusual cytosolic deacetylase enzyme, central for a variety of pivotal cellular functions including aggregating protein turnover, microtubular dynamics and filopodia formation. All these functions are important in the context of neurodegenerative proteinopathies involving TDP-43. We have previously shown in a human embryonic kidney cell line that TDP-43 knockdown significantly impairs the removal of a toxic, aggregating polyQ ataxin-3 fusion protein in an HDAC6-dependent manner. Here we investigated the influence of TDP-43 and its target HDAC6 on neurite outgrowth.</p> <p>Results</p> <p>Human neuroblastoma SH-SY5Y cells with stably silenced TDP-43 showed a significant reduction of neurite outgrowth induced by retinoic acid and brain-derived neurotrophic factor. Re-transfection with TDP-43 as well as HDAC6 rescued retinoic acid-induced neurite outgrowth. In addition, we show that silencing of HDAC6 alone is sufficient to reduce neurite outgrowth of <it>in vitro </it>differentiated SH-SY5Y cells.</p> <p>Conclusions</p> <p>TDP-43 deficiency leads to impairment of neurite growth in an HDAC6-dependent manner, thereby contributing to neurodegenerative events in TDP-43 diseases.</p

TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5′-GA-3’ and 5′-UG-3′ repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis

miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1)

Computational prediction of miRNA candidates for human PINK1. a Computation prediction of miRNAs expressed in human midbrain with putative binding sites in the 3′UTR of human PINK1 mRNA. We first searched miRNAs that have putative binding sites in the 3′UTR of human PINK1 mRNA by utilizing several miRNA-target prediction algorithms, such as miRanda [67], miRWalk [68], RNAhybrid [37], and Targetscan [69]. Among 49 miRNAs commonly predicted by different algorithms, 7 miRNAs were known to be expressed in human midbrain [34]. miR-27a/b are predicted to have 2 putative binding sites in the 3′UTR of human PINK1 mRNA, while all other miRNAs are predicted to have 1 putative binding site. b Computational binding prediction of miR-27a/b and their binding sites in the 3′UTR of human PINK1 mRNA. The binding free energies were determined by the RNAhybrid algorithm. (PDF 68 kb

Activation of the E3 ubiquitin ligase Parkin

Abstract The PINK1 (phosphatase and tensin homologue-induced putative kinase 1)/Parkin-dependent mitochondrial quality control pathway mediates the clearance of damaged organelles, but appears to be disrupted in Indeed, the obtained structures showed a sequential release of Parkin&apos;s intertwined domains and allowed docking of an Ub-charged E2 coenzyme, which could enable its enzymatic activity. In addition, using cellbased screening, select E2 enzymes that redundantly, cooperatively or antagonistically regulate Parkin&apos;s activation and/or enzymatic functions at different stages of the mitochondrial autophagy (mitophagy) process were identified [Fiesel et al. (2014) J. Cell Sci. 127, 3488-3504]. Other work that aims to pin-point the particular pathogenic dysfunctions of Parkin mis-sense mutations have been recently disseminated (Fabienne C. Fiesel, Thomas R. Caulfield, Elisabeth L. Moussaud-Lamodiere, Daniel F.A.R. Dourado, Kotaro Ogaki, Owen A. Ross, Samuel C. Flores, and Wolfdieter Springer, submitted). Such a structure-function approach provides the basis for the dissection of Parkin&apos;s regulation and a targeted drug design to identify small-molecule activators of this neuroprotective E3 Ub ligase

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.</p

TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity

Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear—a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function—but not ALS/FTLD-linked mutation, mislocalization, or truncation—for TDP-43-mediated neurotoxicity in vivo

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear