Inflammation Induces TDP-43 Mislocalization and Aggregation

TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43^A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.     

ALS-Associated TDP-43 Induces Endoplasmic Reticulum Stress,Which Drives Cytoplasmic TDP-43 Accumulation and Stress Granule Formation

In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.     

Cell migration: Rho GTPases lead the way

Rho GTPases control signal transduction pathways that link cell surface receptors to a variety of intracellular responses. They are best known as regulators of the actin cytoskeleton, but in addition they control cell polarity, gene expression, microtubule dynamics and vesicular trafficking. Through these diverse functions, Rho GTPases influence many aspects of cell behavior. This review will focus specifically on their role in cell migration.     

The Function of Two Rho Family GTPases Is Determined by Distinct Patterns of Cell Surface Localization

Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.Cell polarity is a highly conserved feature of eukaryotic cells and is important for a number of events in animal cell biology, including embryonic development, cell migration, and epithelial function (21). The budding yeast Saccharomyces cerevisiae provides a simple model system in which to understand cell polarity, as much of the machinery that is responsible for polarity between yeast and animal cells is highly conserved. Rho/Cdc42 family GTPases are examples of this conservation and have been shown to be critical determinants of polarity in both yeast and animal cells. Rho GTPases are thought to exert their effects on cell polarization through regulation of a number of cellular processes, including the cytoskeleton and polarized delivery of new membrane to sites of active growth.Previous studies have demonstrated that Rho3 and Cdc42 have direct roles in regulating exocytosis which are independent of their role in regulating the polarity of the actin cytoskeleton (1, 2). Studies from a number of laboratories have shown that a multisubunit vesicle tethering complex known as the exocyst is likely to be a critical effector for Rho/Cdc42 signaling during polarized exocytosis (1, 2, 11, 22). A number of models have been suggested to describe the action of Rho GTPases in regulating exocytic function (28, 30). Analysis of specific loss-of-function alleles of RHO3 and CDC42 demonstrated that defects in secretion could be distinguished not only from actin polarity but from the polarization of the exocytic machinery as well. This led to the suggestion that Rho GTPases act by local activation rather than recruitment of the exocytic machinery (25).Genetic analysis suggests that the pathway by which Cdc42 regulates secretion is closely linked to that of Rho3. Secretion-defective alleles in each of these GTPases are suppressed by a common set of genes, and the mutants exhibit synthetic lethality when combined in the same cell (1). Recent work has provided direct evidence that the Exo70 subunit of the exocyst both genetically and physically interacts with both Rho3 and Cdc42 (29).Although Rho3 and Cdc42 share a effector and have overlapping functions, there are different characteristics in how these two proteins regulate exocytosis in yeast. Analysis of the Rho3 and Cdc42 secretory mutants by electron microscopy and secretory assays revealed that cdc42-6 mutants showed defects only in cells with small or emerging buds; in contrast, rho3-V51 mutants exhibited secretory defects throughout bud growth (1, 2). These phenotypes suggested that the exocytic function of Rho3 and Cdc42 is required at overlapping but distinct stages of bud growth.Most small GTPases require multiple elements to promote their association with the membrane on which they engage their downstream targets (26). Modification of the C-terminal CAAX motif by prenylation is common to both Rho3 and Cdc42, with Rho3 predicted to be farnesylated and Cdc42 shown to be geranylgeranylated (14, 17, 19). However, as with other small GTPases, C-terminal prenylation by itself is not sufficient for stable membrane association (10, 18). As with many other small GTPases, a second site of interaction is thought to be required for both Rho3 and Cdc42 GTPases. Sequence alignment of Rho3 and Cdc42 revealed that Rho3 has a long N-terminal extension, which contains a site (a cysteine at position 5) for palmitoylation (24). In contrast, Cdc42 is not palmitoylated but instead contains a polybasic domain adjacent to the CAAX motif at its C terminus, which is thought to act as a membrane targeting signal via the electrostatic interactions with phospholipids at the plasma membrane (6, 12).In this study we examine how the function and localization of two Rho GTPases are specified at distinct stages of polarized growth in yeast. Using a novel monoclonal antibody, we find that the pattern of cell surface localization observed for the Rho3 GTPase is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we find that the N terminus plays a particularly important role in this specification. The functional effect imparted by the N terminus appears to have two key elements. One element involves palmitoylation of a cysteine in the N terminus of Rho3 that is critical in generating the dispersed pattern of localization observed for Rho3. A second element regulates the affinity of the GTPases for a common effector, the exocyst complex. Taken together, this work provides a model for how these GTPases have evolved distinct functions by adopting sequence elements that affect both the pattern of localization and the ability to engage the downstream effector in a way that allows each GTPase to function at different stages of polarized growth.     

Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior

Pathological modifications in the highly conserved and ubiquitously expressed heterogeneous ribonucleoprotein TDP-43 were recently associated to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a late-onset disorder that affects predominantly motoneurons [Neumann, M. et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133, Sreedharan, J. et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668-1672, Kabashi, E. et al. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat. Genet. 40, 572-574]. However, the function of TDP-43 in vivo is unknown and a possible direct role in neurodegeneration remains speculative. Here, we report that flies lacking Drosophila TDP-43 appeared externally normal but presented deficient locomotive behaviors, reduced life span and anatomical defects at the neuromuscular junctions. These phenotypes were rescued by expression of the human protein in a restricted group of neurons including motoneurons. Our results demonstrate the role of this protein in vivo and suggest an alternative explanation to ALS pathogenesis that may be more due to the lack of TDP 43 function than to the toxicity of the aggregates.     

TSC1 Controls Distribution of Actin Fibers through Its Effect on Function of Rho Family of Small GTPases and Regulates Cell Migration and Polarity

The tumor-suppressor genes TSC1 and TSC2 are mutated in tuberous sclerosis, an autosomal dominant multisystem disorder. The gene products of TSC1 and TSC2 form a protein complex that inhibits the signaling of the mammalian target of rapamycin complex1 (mTORC1) pathway. mTORC1 is a crucial molecule in the regulation of cell growth, proliferation and survival. When the TSC1/TSC2 complex is not functional, uncontrolled mTORC1 activity accelerates the cell cycle and triggers tumorigenesis. Recent studies have suggested that TSC1 and TSC2 also regulate the activities of Rac1 and Rho, members of the Rho family of small GTPases, and thereby influence the ensuing actin cytoskeletal organization at focal adhesions. However, how TSC1 contributes to the establishment of cell polarity is not well understood. Here, the relationship between TSC1 and the formation of the actin cytoskeleton was analyzed in stable TSC1-expressing cell lines originally established from a Tsc1-deficient mouse renal tumor cell line. Our analyses showed that cell proliferation and migration were suppressed when TSC1 was expressed. Rac1 activity in these cells was also decreased as was formation of lamellipodia and filopodia. Furthermore, the number of basal actin stress fibers was reduced; by contrast, apical actin fibers, originating at the level of the tight junction formed a network in TSC1-expressing cells. Treatment with Rho-kinase (ROCK) inhibitor diminished the number of apical actin fibers, but rapamycin had no effect. Thus, the actin fibers were regulated by the Rho-ROCK pathway independently of mTOR. In addition, apical actin fibers appeared in TSC1-deficient cells after inhibition of Rac1 activity. These results suggest that TSC1 regulates cell polarity-associated formation of actin fibers through the spatial regulation of Rho family of small GTPases.     

Cell type-specific functions of Rho GTPases revealed by gene targeting in mice

Mammalian Rho family GTPases are intracellular signal transducers known to regulate multiple signaling pathways involved in actin organization and cell proliferation. However, previous knowledge of their cellular functions came mostly from studies using a dominant-negative or constitutively active mutant expression approach in various clonal cell lines. Such an approach has increasingly been recognized to impose experimental limitations related to specificity, dosage and/or clonal variation. Recent progress in mammalian Rho GTPase cell biology by gene targeting individual Rho GTPases in mice has provided more convincing evidence of their physiological roles and signaling pathways in diverse primary cells. Although adaptive compensation by related Rho GTPase members remains a potential concern in the gene targeting approach, in many cases these studies enable an elucidation of the unique functions of individual Rho GTPases in different cell types in vivo.     

Rho GTPases regulate axon growth through convergent and divergent signaling pathways

Rho GTPases are essential regulators of cytoskeletal reorganization, but how they do so during neuronal morphogenesis in vivo is poorly understood. Here we show that the actin depolymerization factor cofilin is essential for axon growth in Drosophila neurons. Cofilin function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase. Dephosphorylating cofilin appears to be the major function of Slingshot in regulating axon growth in vivo. Genetic data provide evidence that Rho or Rac/Cdc42, via effector kinases Rok or Pak, respectively, activate LIM kinase to inhibit axon growth. Importantly, Rac also activates a Pak-independent pathway that promotes axon growth, and different RacGEFs regulate these distinct pathways. These genetic analyses reveal convergent and divergent pathways from Rho GTPases to the cytoskeleton during axon growth in vivo and suggest that different developmental outcomes could be achieved by biases in pathway selection.     

TDP-43 regulates its mRNA levels through a negative feedback loop

TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.     

TDP-43: gumming up neurons through protein-protein and protein-RNA interactions

Since the discovery that 43 kDa TAR DNA binding protein (TDP-43) is involved in neurodegeneration, studies of this protein have focused on the global effects of TDP-43 expression modulation on cell metabolism and survival. The major difficulty with these global searches, which can yield hundreds to thousands of variations in gene expression level and/or mRNA isoforms, is our limited ability to separate specific TDP-43 effects from secondary dysregulations occurring at the gene expression and various mRNA processing steps. In this review, we focus on two biochemical properties of TDP-43: its ability to bind RNA and its protein-protein interactions. In particular, we overview how these two properties may affect potentially very important processes for the pathology, from the autoregulation of TDP-43 to aggregation in the cytoplasmic/nuclear compartments.     

Mutant TDP-43 Expression Triggers TDP-43 Pathology and Cell Autonomous Effects on Primary Astrocytes: Implications for Non-cell Autonomous Pathology in ALS

Neurochemical Research - Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular...     

Small molecules that regulate zymosan phagocytosis of macrophage through deactivation of Rho GTPases

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immune response to microbial infections. To find small molecule regulators for the investigation of complicated phagocytic process, we screened our in-house chemical library and found chemicals which inhibit phagocytosis of zymosan by macrophages. A representative compound 5a reduced phagocytosis of zymosan in both peritoneal macrophages and RAW264.7 cells in a dose-dependent manner. Treatment of 5a led to downregulate the key regulators of phagocytosis, Rac1, Rac2 and Cdc42, and slightly reduced phosphorylation of Akt by zymosan.     

TDP-43 Loss-of-Function Causes Neuronal Loss Due to Defective Steroid Receptor-Mediated Gene Program Switching in Drosophila

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Highlights? Gain or loss of dTDP-43 causes degeneration of neurons responsible for adult eclosion ? Gain or loss of dTDP-43 directly deregulates Map205 expression ? Map205-overexpression-defective EcR-dependent gene network switching ? dTDP-43-mediated neurodegeneration is caused by a loss of its normal function     

A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.     

Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.