Transactivation Response Research Articles

Slow exchange model of nonrigid rotational motion in RNA for combined solid-state and solution NMR studies.

Functional RNA molecules are conformationally dynamic and sample a multitude of dynamic modes over a wide range of frequencies. Thus, a comprehensive description of RNA dynamics requires the inclusion of a broad range of motions across multiple dynamic rates which must be derived from multiple spectroscopies. Here we describe a slow conformational exchange theoretical approach to combining the description of local motions in RNA that occur in the nanosecond to microsecond window and are detected by solid-state NMR with nonrigid rotational motion of the HIV-1 transactivation response element (TAR) RNA in solution as observed by solution NMR. This theoretical model unifies the experimental results generated by solution and solid-state NMR and provides a comprehensive view of the dynamics of HIV-1 TAR RNA, a well-known paradigm of an RNA where function requires extensive conformational rearrangements. This methodology provides a quantitative atomic level view of the amplitudes and rates of the local and collective displacements of the TAR RNA molecule and provides directly motional parameters for the conformational capture hypothesis of this classical RNA-ligand interaction.

Sigma nonopioid intracellular receptor 1 mutations cause frontotemporal lobar degeneration–motor neuron disease

Frontotemporal lobar degeneration (FTLD) is the most common cause of early-onset dementia. Pathological ubiquitinated inclusion bodies observed in FTLD and motor neuron disease (MND) comprise trans-activating response element (TAR) DNA binding protein (TDP-43) and/or fused in sarcoma (FUS) protein. Our objective was to identify the causative gene in an FTLD-MND pedigree with no mutations in known dementia genes. A mutation screen of candidate genes, luciferase assays, and quantitative polymerase chain reaction (PCR) was performed to identify the biological role of the putative mutation. Neuropathological characterization of affected individuals and western blot studies of cell lines were performed to identify the pathological mechanism of the mutation. We identified a nonpolymorphic mutation (c.672*51G>T) in the 3'-untranslated region (UTR) of the Sigma nonopioid intracellular receptor 1 (SIGMAR1) gene in affected individuals from the FTLD-MND pedigree. The c.672*51G>T mutation increased gene expression by 1.4-fold, corresponding with a significant 1.5-fold to 2-fold change in the SIGMAR1 transcript or Sigma-1 protein in lymphocyte or brain tissue. Brains of SIGMAR1 mutation carriers displayed a unique pathology with cytoplasmic inclusions immunopositive for either TDP-43 or FUS but not Sigma-1. Overexpression of SIGMAR1 shunted TDP-43 and FUS from the nucleus to the cytoplasm by 2.3-fold and 5.2-fold, respectively. Treatment of cells with Sigma-1 ligands significantly altered translocation of TDP-43 by up to 2-fold. SIGMAR1 is a causative gene for familial FTLD-MND with a unique neuropathology that differs from other FTLD and MND cases. Our findings also suggest Sigma-1 drugs as potential treatments for the TDP-43/FUS proteinopathies.

Substrate-Specific Kinetics of Dicer-Catalyzed RNA Processing

The specialized ribonuclease Dicer plays a central role in eukaryotic gene expression by producing small regulatory RNAs—microRNAs (miRNAs) and short interfering RNAs (siRNAs)—from larger double-stranded RNA (dsRNA) substrates. Although Dicer will cleave both imperfectly base-paired hairpin structures (pre-miRNAs) and perfect duplexes (pre-siRNAs) in vitro, it has not been clear whether these are mechanistically equivalent substrates and how dsRNA binding proteins such as trans-activation response (TAR) RNA binding protein (TRBP) influence substrate selection and RNA processing efficiency. We show here that human Dicer is much faster at processing a pre-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates ( V max) calculated by Michaelis–Menten analysis differed by more than 100-fold under multiple turnover conditions. TRBP was found to enhance dicing of both substrates to similar extents, and this stimulation required the two N-terminal dsRNA binding domains of TRBP. These results demonstrate that multiple factors influence dicing kinetics. While TRBP stimulates dicing by enhancing the stability of Dicer–substrate complexes, Dicer itself generates product RNAs at rates determined at least in part by the structural properties of the substrate.

Pathological 43-kDa Transactivation Response DNA-Binding Protein in Older Adults With and Without Severe Mental Illness

Major psychiatric diseases such as schizophrenia and mood disorders have not been linked to a specific pathology, but their clinical features overlap with some aspects of the behavioral variant of frontotemporal lobar degeneration. Although the significance of pathological 43-kDa (transactivation response) DNA-binding protein (TDP-43) for frontotemporal lobar degeneration was appreciated only recently, the prevalence of TDP-43 pathology in patients with severe mental illness vs controls has not been systematically addressed. To examine patients with chronic psychiatric diseases, mainly schizophrenia, for evidence of neurodegenerative TDP-43 pathology in comparison with controls. Prospective longitudinal clinical evaluation and retrospective medical record review, immunohistochemical identification of pathological TDP-43 in the central nervous system, and genotyping for gene alterations known to cause TDP-43 proteinopathies including the TDP-43 (TARDBP) and progranulin (GRN) genes. University health system. One hundred fifty-one subjects including 91 patients with severe mental illness (mainly schizophrenia) and 60 controls. Clinical medical record review, neuronal and glial TDP-43 pathology, and TARDP and GRN genotyping status. Significant TDP-43 pathology in the amygdala/periamygdaloid region or the hippocampus/transentorhinal cortex was absent in both groups in subjects younger than 65 years but present in elderly subjects (29% [25 of 86] of the psychiatric patients and 29% [10 of 34] of control subjects). Twenty-three percent (8 of 35) of the positive cases showed significant TDP-43 pathology in extended brain scans. There were no evident differences between the 2 groups in the frequency, degree, or morphological pattern of TDP-43 pathology. The latter included (1) subpial and subependymal, (2) focal, or (3) diffuse lesions in deep brain parenchyma and (4) perivascular pathology. A new GRN variant of unknown significance (c.620T>C, p.Met207Thr) was found in 1 patient with schizophrenia with TDP-43 pathology. No known TARDBP mutations or other variants were found in any of the subjects studied herein. The similar findings of TDP-43 pathology in elderly patients with severe mental illness and controls suggest common age-dependent TDP-43 changes in limbic brain areas that may signify that these regions are affected early in the course of a cerebral TDP-43 multisystem proteinopathy. Finally, our data provide an age-related baseline for the development of whole-brain pathological TDP-43 evolution schemata.

A modified fluorescent intercalator displacement assay for RNA ligand discovery

Fluorescent intercalator displacement (FID) is a convenient and practical tool for identifying new nucleic acid-binding ligands. The success of FID is based on the fact that it can be fashioned into a versatile screening assay for assessing the relative binding affinities of compounds to nucleic acids. FID is a tagless approach; the target RNAs and the ligands or small molecules under investigation do not need to be modified in order to be examined. In this study, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. Electrospray ionization mass spectrometry (ESI-MS) results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs (A-site, TAR [transactivation response element], h31 [helix 31], and H69 [helix 69] were identified.

Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein

The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained β-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T1 occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved >6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.

Wild-Type Human TDP-43 Expression Causes TDP-43 Phosphorylation, Mitochondrial Aggregation, Motor Deficits, and Early Mortality in Transgenic Mice

Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the CNS and for studying TDP-43-associated neurotoxicity.

RNA Dynamics by Design: Biasing Ensembles Towards the Ligand‐Bound State

Window of opportunity: Using a single A-U to G-C mutation, the local and global dynamic characteristics of the transactivation response element (TAR) RNA was rationally altered over timescales extending up to milliseconds. This procedure allows it to mimic its bound state with the ligand argininamide (ARG). The mutant binds ARG with slightly enhanced affinity using a conformation indistinguishable from the wild-type sequence.

ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS

Dominant mutations in two functionally related DNA/RNA-binding proteins, trans-activating response region (TAR) DNA-binding protein with a molecular mass of 43 KDa (TDP-43) and fused in sarcoma/translocation in liposarcoma (FUS/TLS), cause an inherited form of ALS that is accompanied by nuclear and cytoplasmic aggregates containing TDP-43 or FUS/TLS. Using isogenic cell lines expressing wild-type or ALS-linked TDP-43 mutants and fibroblasts from a human patient, pulse-chase radiolabeling of newly synthesized proteins is used to determine, surprisingly, that ALS-linked TDP-43 mutant polypeptides are more stable than wild-type TDP-43. Tandem-affinity purification and quantitative mass spectrometry are used to identify TDP-43 complexes not only with heterogeneous nuclear ribonucleoproteins family proteins, as expected, but also with components of Drosha microprocessor complexes, consistent with roles for TDP-43 in both mRNA processing and microRNA biogenesis. A fraction of TDP-43 is shown to be complexed with FUS/TLS, an interaction substantially enhanced by TDP-43 mutants. Taken together, abnormal stability of mutant TDP-43 and its enhanced binding to normal FUS/TLS imply a convergence of pathogenic pathways from mutant TDP-43 and FUS/TLS in ALS.

TDP-43 Mediates Degeneration in a NovelDrosophilaModel of Disease Caused by Mutations in VCP/p97

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP7) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster, and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA(+) (ATPases associated with multiple cellular activities) ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. To elucidate the pathogenic mechanism, we developed and characterized a Drosophila model of IBMPFD (mutant-VCP-related degeneration). Based on genetic screening of this model, we identified three RNA-binding proteins that dominantly suppressed degeneration; one of these was TBPH, the Drosophila homolog of TAR (trans-activating response region) DNA-binding protein 43 (TDP-43). Here we demonstrate that VCP and TDP-43 interact genetically and that disease-causing mutations in VCP lead to redistribution of TDP-43 to the cytoplasm in vitro and in vivo, replicating the major pathology observed in IBMPFD and other TDP-43 proteinopathies. We also demonstrate that TDP-43 redistribution from the nucleus to the cytoplasm is sufficient to induce cytotoxicity. Furthermore, we determined that a pathogenic mutation in TDP-43 promotes redistribution to the cytoplasm and enhances the genetic interaction with VCP. Together, our results show that degeneration associated with VCP mutations is mediated in part by toxic gain of function of TDP-43 in the cytoplasm. We suggest that these findings are likely relevant to the pathogenic mechanism of a broad array of TDP-43 proteinopathies, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

The Mechanism of HIV-1 Tat-Directed Nucleic Acid Annealing Supports its Role in Reverse Transcription

The main function of the HIV-1 trans-activator of transcription (Tat protein) is to promote the transcription of the proviral DNA by the host RNA polymerase which leads to the synthesis of large quantities of the full length viral RNA. Tat is also thought to be involved in the reverse transcription (RTion) reaction by a still unknown mechanism. The recently reported nucleic acid annealing activity of Tat might explain, at least in part, its role in RTion. To further investigate this possibility, we carried out a fluorescence study on the mechanism by which the full length Tat protein (Tat(1-86)) and the basic peptide (44-61) direct the annealing of complementary viral DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, essential for the early steps of RTion. Though both Tat(1-86) and the Tat(44-61) peptide were unable to melt the lower half of the cTAR stem, they strongly promoted cTAR/dTAR annealing through non-specific attraction between the peptide-bound oligonucleotides. Using cTAR and dTAR mutants, this Tat promoted-annealing was found to be nucleated through the thermally frayed 3′/5′ termini, resulting in an intermediate with 12 intermolecular base pairs, which then converts into the final extended duplex. Moreover, we found that Tat(1-86) was as efficient as the nucleocapsid protein NCp7, a major nucleic acid chaperone of HIV-1, in promoting cTAR/dTAR annealing, and could act cooperatively with NCp7 during the annealing reaction. Taken together, our data are consistent with a role of Tat in the stimulation of the obligatory strand transfers during viral DNA synthesis by reverse transcriptase.

Nuclear import impairment causes cytoplasmic trans-activation response DNA-binding protein accumulation and is associated with frontotemporal lobar degeneration

Trans-activation response DNA-binding protein (TDP-43) accumulation is the major component of ubiquitinated protein inclusions found in patients with amyotrophic lateral sclerosis, and frontotemporal lobar degeneration with TDP-43 positive ubiquitinated inclusions, recently relabelled the 'TDP-43 proteinopathies'. TDP-43 is predominantly located in the nucleus, however, in disease it mislocalizes to the cytoplasm where it aggregates to form hallmark pathological inclusions. The identification of TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis cases confirms its pathogenic role; but it is wild-type TDP-43 that is deposited in the vast majority of TDP-43 proteinopathies, implicating other unknown factors for its mislocalization and aggregation. One such mechanism may be defective nuclear import of TDP-43 protein, as a disruption of its nuclear localization signal leads to mislocalization and aggregation of TDP-43 in the cytoplasm. In order to explore the factors that regulate the nuclear import of TDP-43, we used a small interfering RNA library to silence 82 proteins involved in nuclear transport and found that knockdowns of karyopherin-beta1 and cellular apoptosis susceptibility protein resulted in marked cytoplasmic accumulation of TDP-43. In glutathione S-transferase pull-down assays, TDP-43 bound to karyopherin-alphas, thereby confirming the classical nuclear import pathway for the import of TDP-43. Analysis of the expression of chosen nuclear import factors in post-mortem brain samples from patients with TDP-43 positive frontotemporal lobar degeneration, and spinal cord samples from patients with amyotrophic lateral sclerosis, revealed a considerable reduction in expression of cellular apoptosis susceptibility protein in frontotemporal lobar degeneration. We propose that cellular apoptosis susceptibility protein associated defective nuclear transport may play a mechanistic role in the pathogenesis of the TDP-43 positive frontotemporal lobar degeneration.

Caspase-Cleaved Transactivation Response DNA-Binding Protein 43 in Parkinson’s Disease and Dementia with Lewy Bodies

Background: Transactivation response DNA-binding protein 43 (TDP-43) proteinopathies are classified based upon the extent of modified TDP-43 and include a growing number of neurodegenerative diseases such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration with ubiquitin-immunoreactive, tau-negative inclusions and frontotemporal lobar degeneration with motor neuron disease. Objective: The purpose of the study was to examine whether proteolytic modifications of TDP-43 are a relevant finding in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Methods: A novel site-directed caspase cleavage antibody, termed TDP caspase cleavage product antibody (TDPccp), was utilized based upon a known caspase 3 cleavage consensus site within TDP-43 at position 219. Results: Application of this antibody to postmortem brain sections from PD and DLB patients revealed the presence of caspase-cleaved TDP-43 in Lewy bodies and Hirano bodies in all cases examined. Colocalization of TDPccp with an antibody to α-synuclein (α-Syn), which served as a general marker for Lewy bodies, was evident within the substantia nigra in both α-synucleinopathies. Interestingly, the TDPccp antibody detected a greater number of Lewy bodies in PD and DLB compared to the α-Syn antibody. In addition, a semiquantitative analysis in both diseases confirmed this finding by indicating that the percentage of caspase-cleaved TDP-43 single-labeled Lewy bodies was approximately twice that of α-Syn labeling (in DLB 13.4 vs. 5.5%, while in PD 34.6 vs. 17.6%). Conclusion: Collectively, these data have identified caspase-cleaved TDP-43 as a primary component of Lewy and Hirano bodies in PD and DLB, and suggest that the TDPccp antibody is an effective marker for the detection of Lewy bodies in these neurodegenerative diseases.

Identification of 2 Loci at Chromosomes 9 and 14 in a Multiplex Family With Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative brain disorder that can be accompanied by signs of amyotrophic lateral sclerosis (ALS). To identify a novel gene for FTLD-ALS. Genome-wide linkage study in a multiplex family with FTLD-ALS with subsequent fine mapping and mutation analyses. Memory Clinic of the Middelheim General Hospital. An extended Belgian family with autosomal dominant FTLD-ALS, DR14, with a mean age at onset of 58.1 years (range, 51-65 years [n = 9]) and mean disease duration of 6.4 years (range, 1-17 years [n = 9]). The proband with clinical FTLD showed typical FTLD pathology with neuronal ubiquitin-immunoreactive inclusions that were positive for the transactivation response DNA-binding protein 43 (TDP-43). Linkage to chromosome 9 and 14. We found significant linkage to chromosome 9p23-q21 (multipoint logarithm of odds [LOD] score = 3.38) overlapping with a known FTLD-ALS locus (ALSFTD2) and nearly significant linkage to a second locus at chromosome 14q31-q32 (multipoint LOD score = 2.79). Obligate meiotic recombinants defined candidate regions of 74.7 megabase pairs (Mb) at chromosome 9 and 14.6 Mb near the telomere of chromosome 14q. In both loci, the disease haplotype segregated in all patients in the family. Mutation analysis of selected genes and copy number variation analysis in both loci did not reveal segregating pathogenic mutations. Family DR14 provides additional significant evidence for the importance of the chromosome 9 gene to FTLD-ALS and reveals a possible novel locus for FTLD-ALS at chromosome 14. The identification of the underlying genetic defect(s) will significantly contribute to the understanding of neurodegenerative disease mechanisms in FTLD, ALS, and associated neurodegenerative disorders.

Formation of Tat–TAR containing ribonucleoprotein complexes for biochemical and structural analyses

Viruses manipulate multiple processes of the host cell machinery in order to replicate successfully in the infected cell. Among these, stimulation of transcription of the viral genes is crucial for lentiviruses such as HIV for increased protein expression levels and generation of escape mutants. The transactivation response (TAR) element at the 5′-end of HIV, SIV, BIV, EIAV or JDV retroviruses forms a unique RNA based promoter element that together with the transcription activator protein Tat stimulates viral gene expression at the level of transcription elongation. TAR is a double stranded non-coding RNA of typically 24–40 nucleotides length. Together with Tat it interacts with the Cyclin T subunit of the positive transcription elongation factor P-TEFb to recruit Cyclin T and its corresponding Cyclin-dependent kinase Cdk9 to the RNA polymerase II. In vitro formations of these Tat–TAR containing ribonucleoprotein complexes are a key requisite for biochemical characterizations and interaction studies that eventually will allow structural analyses. Here, we describe purification methods of the different factors employed and chromatography techniques that yield highly specific complex assemblies suitable for crystallization.

Kinetic analysis of the nucleic acid chaperone activity of the Hepatitis C virus core protein

The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters.

Repression of Glucocorticoid Receptor (GCR) Transactivation and Glucocorticoid Response Elements (GREs) Activities Within Human HDAC2 Gene Promoter by GCR-beta

RATIONALE: Reduced HDAC2 expression and activity is reported to correlate with corticosteroid resistance (CR). Recently we demonstrated a significant increase in HDAC2 mRNA expression following inhibition of dominant negative GCR isoform GCR-beta by siRNA in BAL cells from asthmatics. The molecular role of GCR-beta in HDAC2-mediated CR is unknown.

Phosphorylated and cleaved TDP-43 in ALS, FTLD and other neurodegenerative disorders and in cellular models of TDP-43 proteinopathy

Transactivation response (TAR) DNA-binding protein of Mr 43 kDa (TDP-43) is a major component of the tau-negative and ubiquitin-positive inclusions that characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration which is now referred to as FTLD-TDP. Concurrent TDP-43 pathology has been reported in a variety of other neurodegenerative disorders such as Alzheimer's disease, forming a group of TDP-43 proteinopathy. Accumulated TDP-43 is characterized by phosphorylation and fragmentation. There is a close relationship between the pathological subtypes of FTLD-TDP and the immunoblot pattern of the C-terminal fragments of phosphorylated TDP-43. These results suggest that proteolytic processing of accumulated TDP-43 may play an important role for the pathological process. In cultured cells, transfected C-terminal fragments of TDP-43 are more prone to form aggregates than full-length TDP-43. Transfecting the C-terminal fragment of TDP-43 harboring pathogenic mutations of TDP-43 gene identified in familial and sporadic ALS cases into cells enhanced the aggregate formation. Furthermore, we found that methylene blue and dimebon inhibit aggregation of TDP-43 in these cellular models. Understanding the mechanism of phosphorylation and truncation of TDP-43 and aggregate formation may be crucial for clarifying the pathogenesis of TDP-43 proteinopathy and for developing useful therapeutics.

Exaggerated inflammation, impaired host defense, and neuropathology in progranulin-deficient mice

Progranulin (PGRN) is a widely expressed protein involved in diverse biological processes. Haploinsufficiency of PGRN in the human causes tau-negative, ubiquitin-positive frontotemporal dementia (FTD). However, the mechanisms are unknown. To explore the role of PGRN in vivo, we generated PGRN-deficient mice. Macrophages from these mice released less interleukin-10 and more inflammatory cytokines than wild type (WT) when exposed to bacterial lipopolysaccharide. PGRN-deficient mice failed to clear Listeria monocytogenes infection as quickly as WT and allowed bacteria to proliferate in the brain, with correspondingly greater inflammation than in WT. PGRN-deficient macrophages and microglia were cytotoxic to hippocampal cells in vitro, and PGRN-deficient hippocampal slices were hypersusceptible to deprivation of oxygen and glucose. With age, brains of PGRN-deficient mice displayed greater activation of microglia and astrocytes than WT, and their hippocampal and thalamic neurons accumulated cytosolic phosphorylated transactivation response element DNA binding protein–43. Thus, PGRN is a key regulator of inflammation and plays critical roles in both host defense and neuronal integrity. FTD associated with PGRN insufficiency may result from many years of reduced neutrotrophic support together with cumulative damage in association with dysregulated inflammation.

Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5α Protein, a CCR5 shRNA, and a TAR Decoy

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.