TDP-43 Knockdown Research Articles

TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons.

Nuclear clearance of TDP‐43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP‐43 knockdown specifically reduces the number and motility of RAB11‐positive recycling endosomes in dendrites, while TDP‐43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP‐43‐knockdown neurons and decreased β2‐transferrin levels in patient CSF. Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP‐43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP‐43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP‐43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP‐43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP‐43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP‐43‐induced neurodegeneration by blocking trophic signaling.

CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS.

The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.

TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways.

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile.

TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA.

Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.

Staurosporine induced apoptosis rapidly downregulates TDP- 43 in glioma cells.

TDP-43 is a ubiquitously expressed DNA/RNA binding protein that has recently attracted attention for its involvement in neurodegenerative diseases. While TDP-43 has been found to participate in various important cellular activities including stress and apoptosis, little is known about its role in cancer cells. Here we report that staurosporine (STS) induced apoptosis in U87 glioma cells is associated with rapid downregulation of TDP-43 at both mRNA and protein levels. The latter is dependent on activation of caspase 3. More importantly, we have shown that knockdown of TDP-43 by specific siRNA dramatically enhanced cytotoxicity of STS. These results suggest that normal level of TDP-43 may be protective for cancer cells under apoptotic insult.

TDP-43 regulates the mammalian spinogenesis through translational repression of Rac1

Impairment of learning and memory is a significant pathological feature of many neurodegenerative diseases including FTLD-TDP. Appropriate regulation and fine tuning of spinogenesis of the dendrites, which is an integral part of the learning/memory program of the mammalian brain, are essential for the normal function of the hippocampal neurons. TDP-43 is a nucleic acid-binding protein implicated in multi-cellular functions and in the pathogenesis of a range of neurodegenerative diseases including FTLD-TDP and ALS. We have combined the use of single-cell dye injection, shRNA knockdown, plasmid rescue, immunofluorescence staining, Western blot analysis and patch clamp electrophysiological measurement of primary mouse hippocampal neurons in culture to study the functional role of TDP-43 in mammalian spinogenesis. We found that depletion of TDP-43 leads to an increase in the number of protrusions/spines as well as the percentage of matured spines among the protrusions. Significantly, the knockdown of TDP-43 also increases the level of Rac1 and its activated form GTP-Rac1, a known positive regulator of spinogenesis. Clustering of the AMPA receptors on the dendritic surface and neuronal firing are also induced by depletion of TDP-43. Furthermore, use of an inhibitor of Rac1 activation negatively regulated spinogenesis of control hippocampal neurons as well as TDP-43-depleted hippocampal neurons. Mechanistically, RT-PCR assay and cycloheximide chase experiments have indicated that increases in Rac1 protein upon TDP-43 depletion is regulated at the translational level. These data together establish that TDP-43 is an upstream regulator of spinogenesis in part through its action on the Rac1→GTP-Rac1→AMPAR pathway. This study provides the first evidence connecting TDP-43 with the GTP-Rac1→AMPAR regulatory pathway of spinogenesis. It establishes that mis-metabolism of TDP-43, as occurs in neurodegenerative diseases with TDP-43 proteinopathies, e.g., FTLD-TDP, would alter its homeostatic cellular concentration, thus leading to impairment of hippocampal plasticity.

Abberrant Posttranscriptional Regulation and Protein Degradtion of TDP-43 and FUS to Stress Respone in ALS (IN9-1.008)

Objective: To further analyze molecular interactions and posttranscriptional regulation of TDP-43, FUS with HuR in the presence of mutant SOD1 and its impact on protein degradation. Background The genetic link of TDP-43 and FUS with familial and sporadic ALS underscores the importance of RNA binding proteins (RBP) and post-transcriptional regulation in the pathogenesis of ALS. HuR, another RBP, shares structural and functional similarities to TDP-43 and FUS. These RBPs provide an important contribution to stress response. We have observed aberrant cross-regulation and protein degradation of a network of RBPs in associated with mutant SOD1. Recent identification of UBQLN 2 mutation accentuated abnormal protein degradation in ALS pathogenesis. Design/Methods: HuR was upregulated ectopically or knocked down using siRNA in primary astrocytes or U251 cells expressing wild-type or mutant SOD1. Cells were subjected to oxidative, hypoxic and hormonal stress. TDP-43 39UTR regulation was measured by a luciferase reporter. Protein stability was analyzed by Western blot after treating cells with cycloheximide for different time intervals. Ubiquitination was quantified with Western blot and an ELISA kit. Results: Following oxidative stress, we observed cytoplasmic association and stress granules of TDP43 and FUS with HuR in G93A astrocytes. In both U251 and astrocytes, TDP-43 and FUS are downregulated by HuR silencing. In the presence of mutant SOD1, their protein half life significantly increased compared to wt cells. With stress induction, we observed accumulation of these RBPs and significantly increased ubiliquitination. Knockdown of TDP-43 and FUS had no effect on HuR protein and RNA level. There was increasing neuronal toxicity when co-cultured with those conditional medium. Conclusions: 1. The posttranscriptional regulation and protein stability of a network of RBPs may play a role in the pathogenesis of ALS. 2. ALS-associated mutant SOD1 and HuR dysfunction may block the ubiquitin proteasome pathway of these RBPs that leads to their aberrant degradation. Supported by: NIH/NINDS Grant NS057664, NS064133 and a merit review from the Department of Veterans Affairs. Disclosure: Dr. Lu has nothing to disclose. Dr. Zheng has nothing to disclose. Dr. Si has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Luo has received personal compensation for activities with XenoPort as an employee. Dr. Oh has nothing to disclose. Dr. King has nothing to disclose.

The abnormal processing of TDP-43 is not an upstream event of reduced ADAR2 activity in ALS motor neurons

TDP-43 pathology in motor neurons is a hallmark of ALS. In addition, the reduced expression of an RNA editing enzyme, adenosine deaminase acting on RNA 2 (ADAR2), increases the expression of GluA2 with an unedited Q/R site in the motor neurons of patients with sporadic ALS. As the occurrence of these two disease-specific abnormalities in the same motor neurons suggests a molecular link between them, we examined the effects of altered TDP-43 processing on ADAR2 activity in TetHeLaG2m and Neuro2a cells. We found that ADAR2 activity did not consistently change due to the overexpression or knockdown of TDP-43 or the expression of abnormal TDP-43, including caspase-3-cleaved fragments, truncated TDP-43 lacking either nuclear localization or export signals and ALS-linked TDP-43 mutants. These results suggest that the abnormal processing of TDP-43 is not an upstream event of inefficient GluA2 Q/R site editing in the motor neurons of sporadic ALS patients.

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.

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

BackgroundTrans-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.ResultsHuman 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 in vitro differentiated SH-SY5Y cells.ConclusionsTDP-43 deficiency leads to impairment of neurite growth in an HDAC6-dependent manner, thereby contributing to neurodegenerative events in TDP-43 diseases.

Nuclear factor TDP‐43 can affect selected microRNA levels

TDP-43 has recently been described as the major component of the inclusions found in the brain of patients with a variety of neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 is a ubiquitous protein whose specific functions are probably crucial to establishing its pathogenic role. Apart from its involvement in transcription, splicing and mRNA stability, TDP-43 has also been described as a Drosha-associated protein. However, our knowledge of the role of TDP-43 in the microRNA (miRNA) synthesis pathway is limited to the association mentioned above. Here we report for the first time which changes occur in the total miRNA population following TDP-43 knockdown in culture cells. In particular, we have observed that let-7b and miR-663 expression levels are down- and upregulated, respectively. Interestingly, both miRNAs are capable of binding directly to TDP-43 in different positions: within the miRNA sequence itself (let-7b) or in the hairpin precursor (miR-663). Using microarray data and real-time PCR we have also identified several candidate transcripts whose expression levels are selectively affected by these TDP-43-miRNA interactions.

TDP-43 Depletion Induces Neuronal Cell Damage through Dysregulation of Rho Family GTPases

The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases.

A Novel CpG-free Vertebrate Insulator Silences the Testis-specific SP-10 Gene in Somatic Tissues: ROLE FOR TDP-43 IN INSULATOR FUNCTION

Regulation of cell type-specific gene transcription is central to cellular differentiation and development. During spermatogenesis, a number of testis-specific genes are expressed in a precise spatiotemporal order. How these genes remain silent in the somatic tissues is not well understood. Our previous studies using the round spermatid-specific mouse SP-10 gene, which codes for an acrosomal protein, revealed that its proximal promoter acts as an insulator and prevents expression in the somatic tissues. Here we report that the insulator tethers the SP-10 gene to the nuclear matrix in somatic tissues, sequestering the core promoter in the process, thus preventing transcription. In round spermatids where the SP-10 gene is expressed, this tethering is released. TAR DNA-binding protein of 43 kDa (TDP-43), previously shown to interact with the SP-10 insulator, was found to be in the 2 m NaCl-insoluble nuclear matrix fraction. TDP-43 prevented enhancer-promoter interactions when artificially recruited between the two by Gal4 strategy. Knockdown of TDP-43 using small interfering RNA released the enhancer-blocking effect of the SP-10 insulator in a stable cell culture model. Mutation of TDP-43 binding sites abolished this effect. Finally, a 50-bp subfragment of the SP-10 insulator, which includes TDP-43 binding sites, functioned as a minimal insulator in transgenic mice and silenced an otherwise ectopically expressed transgene in somatic tissues. The SP-10 insulator lacks CpG dinucleotides or CTCF binding sites. Thus, the present study characterized a novel vertebrate insulator in a physiological context and showed for the first time how a testis-specific gene is silenced in the somatic tissues by an insulator.