Targeting tauopathy with engineered tau-degrading intrabodies

BACKGROUND: The accumulation of pathological tau is the main component of neurofibrillary tangles and other tau aggregates in several neurodegenerative diseases, referred to as tauopathies. Recently, immunotherapeutic approaches targeting tau have been demonstrated to be beneficial in decreasing tauopathy in animal models. We previously found that passive immunotherapy with anti-tau antibody to human tau or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system of a mouse model of tauopathy decreased but did not remove all tau-associated pathology. Although these and other studies demonstrate that conventional immunotherapeutic approaches targeting tau can influence tau pathogenesis, the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody. Therefore, we reasoned targeting intracellular tau might be more efficacious in preventing or decreasing tauopathy. METHODS: By utilizing our anti-tau scFv, we generated anti-tau intrabodies for the expression in the cytosol of neurons. To enhance the degradation capacity of conventional intrabodies, we engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations that shuttle intracellular tau for either the proteasome or lysosomal mediated degradation. To evaluate the efficacy in delaying or eliminating tauopathy, we expressed our tau degrading intrabodies or controls in human tau transgenic mice by adeno-associated virus prior to overt tau pathology and after tau deposition. RESULTS: Our results demonstrate, the expression of chimeric anti-tau intrabodies significantly reduce tau protein levels in primary neuronal cultures expression human tau relative to a non-modified anti-tau intrabody. We found the expression of engineered tau-degrading intrabodies destined for proteasomal-mediated degradation are more effective in delaying or eliminating tauopathy than a conventional intrabody in aged human tau transgenic mice. CONCLUSION: This study, harnesses the strength of intrabodies that are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation providing a new immunotherapeutic approach with potentially improved efficacy

An Insight into Alpha-Synulcien's Biological Function and Its Pathogenesis in Neurodegenerative Disease

The discovery of two missense mutations (A53T and A30P) in alpha -synuclein that are genetically linked to Parkinson's disease, together with alpha -synuclein being the major component in Lewy bodies, has generated extensive interest in alpha -synuclein as a key component in neurodegenerative diseases. In recent years modeling this disease in transgenic mice and flies has lead to new understandings of alpha -synuclein function and pathogenesis in neurodegeneration. In the current study we analyzed transgenic mice overexpressing human alpha -synuclein and human alpha -synuclein mutations (A53T&A30P) to; First establish these transgenic mice as a model for degenerative diseases; second to identify potential contributing factors in neurodegeneration; third to decipher a potential function of alpha -synuclein. We first established that transgenic mice expressing human variants of alpha -synuclein developed an age dependant motor dysfunction with symptom logy characteristic of Parkinson's disease. Immunohistological studies revealed the presence of alpha -synuclein inclusions and a loss of motor neurons. Biochemical analysis identified a 4-5 fold increase in ubiquitin with altered expression of proteasomal subunits, characteristic of proteasomal impairment. In addition, we identified a significant increase in amyloid beta -peptides. Protein quantification of apolipoprotein E (ApoE) a protein that has been associated with the development of Alzheimer's disease, demonstrated a 5-15 fold increase in symptomatic transgenic mice. Ablation of ApoE in alpha -synuclein transgenic mice by genetic crosses revealed a delayed onset for motor dysfunction and an overall increase in survival. ApoE deficient transgenic mice displayed a decrease in ubiquitin and amyloid beta -peptides. This study illustrates ApoE, ubiquitin and Abeta - peptides contribute to the onset and progression of the neurodegeneration in transgenic alpha -synuclein mice. Genetic crosses of transgenic alpha -synuclein with a csystine string protein-alpha (CSPalpha ) knockout mouse revealed a potential function for alpha -synuclein. CSPalpha deficient mice develop an early age neurodegenerative disease that is lethal at 3-4 months. Transgenic expression of human alpha -synuclein prevented the deleterious effects of CSPalpha deficiency. Immunofluorescence studies illustrated alpha -synuclein function in a cell autonomous manner. Biochemical analysis demonstrated CSPalpha deficient mice have impaired SNARE complexes that are partially reverted by transgenic alpha -synuclein. This study illustrates a protective function of alpha -synuclein in preventing neurodegeneration

Purification of Nuclear Poly(A)-binding Protein Nab2 Reveals Association with the Yeast Transcriptome and a Messenger Ribonucleoprotein Core Structure

Nascent mRNAs produced by transcription in the nucleus are subsequently processed and packaged into mRNA ribonucleoprotein particles (messenger ribonucleoproteins (mRNPs)) before export to the cytoplasm. Here, we have used the poly(A)-binding protein Nab2 to isolate mRNPs from yeast under conditions that preserve mRNA integrity. Upon Nab2-tandem affinity purification, several mRNA export factors were co-enriched (Yra1, Mex67, THO-TREX) that were present in mRNPs of different size and mRNA length. High-throughput sequencing of the co-precipitated RNAs indicated that Nab2 is associated with the bulk of yeast transcripts with no specificity for different mRNA classes. Electron microscopy revealed that many of the mRNPs have a characteristic elongated structure. Our data suggest that mRNPs, although associated with different mRNAs, have a unifying core structure

AAV-mediated expression of anti-tau scFvs decreases tau accumulation in a mouse model of tauopathy

Tauopathies are characterized by the progressive accumulation of hyperphosphorylated, aggregated forms of tau. Our laboratory has previously demonstrated that passive immunization with an anti-tau antibody, HJ8.5, decreased accumulation of pathological tau in a human P301S tau-expressing transgenic (P301S-tg) mouse model of frontotemporal dementia/tauopathy. To investigate whether the

Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation

The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid β (Aβ) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aβ pathology in mice producing both human Aβ and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aβ deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aβ pathology and may represent a selective approach to treat AD

An OBSL1-Cul7Fbxw8 Ubiquitin Ligase Signaling Mechanism Regulates Golgi Morphology and Dendrite Patterning

The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7Fbxw8 localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7Fbxw8 by independent approaches including Fbxw8 knockdown reveals that Cul7Fbxw8 is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7Fbxw8 also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7Fbxw8 in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7Fbxw8 in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7Fbxw8 ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development

Measurement of the polarisation of W bosons produced in top-quark decays using dilepton events at root s=13 TeV with the ATLAS experiment

A measurement of the polarisation of $W$ bosons produced in top-quark decays is presented, using proton-proton collision data at a centre-of-mass energy of $\sqrt{s} = 13$ TeV. The data were collected by the ATLAS detector at the Large Hadron Collider and correspond to an integrated luminosity of 139 fb$^{-1}$. The measurement is performed selecting $t\bar{t}$ events decaying into final states with two charged leptons (electrons or muons) and at least two $b$-tagged jets. The polarisation is extracted from the differential cross-section distribution of the $\cos{\theta^{*}}$ variable, where $\theta^{*}$ is the angle between the momentum direction of the charged lepton from the $W$ boson decay and the reversed momentum direction of the $b$-quark from the top-quark decay, both calculated in the $W$ boson rest frame. Parton-level results, corrected for the detector acceptance and resolution, are presented for the $\cos{\theta^{*}}$ angle. The measured fractions of longitudinal, left- and right-handed polarisation states are found to be $f_{0} = 0.684 \pm 0.005\,\mathrm{(stat.)} \pm 0.014\,\mathrm{(syst.)}$, $f_{\mathrm{L}} = 0.318 \pm 0.003\,\mathrm{(stat.)} \pm 0.008\,\mathrm{(syst.)}$ and $f_{\mathrm{R}} = -0.002 \pm 0.002\,\mathrm{(stat.)} \pm 0.014\,\mathrm{(syst.)}$, in agreement with the Standard Model prediction

Author Correction: A detailed map of Higgs boson interactions by the ATLAS experiment ten years after the discovery

In the version of this article initially published, the ATLAS Collaboration author names, affiliations and acknowledgements were omitted and have now been included in the HTML and PDF versions of the article

Measurements of differential cross-sections in top-quark pair events with a high transverse momentum top quark and limits on beyond the Standard Model contributions to top-quark pair production with the ATLAS detector at √s = 13 TeV

Cross-section measurements of top-quark pair production where the hadronically decaying top quark has transverse momentum greater than 355 GeV and the other top quark decays into ℓνb are presented using 139 fb−1 of data collected by the ATLAS experiment during proton-proton collisions at the LHC. The fiducial cross-section at s = 13 TeV is measured to be σ = 1.267 ± 0.005 ± 0.053 pb, where the uncertainties reflect the limited number of data events and the systematic uncertainties, giving a total uncertainty of 4.2%. The cross-section is measured differentially as a function of variables characterising the tt¯ system and additional radiation in the events. The results are compared with various Monte Carlo generators, including comparisons where the generators are reweighted to match a parton-level calculation at next-to-next-to-leading order. The reweighting improves the agreement between data and theory. The measured distribution of the top-quark transverse momentum is used to search for new physics in the context of the effective field theory framework. No significant deviation from the Standard Model is observed and limits are set on the Wilson coefficients of the dimension-six operators OtG and Otq(8), where the limits on the latter are the most stringent to date. [Figure not available: see fulltext.]

Medium-Induced Modification of Z-Tagged Charged Particle Yields in Pb+Pb Collisions at 5.02 TeV with the ATLAS Detector

The yield of charged particles opposite to a Z boson with large transverse momentum ( p T ) is measured in 260     pb − 1 of p p and 1.7     nb − 1 of Pb + Pb collision data at 5.02 TeV per nucleon pair recorded with the ATLAS detector at the Large Hadron Collider. The Z boson tag is used to select hard-scattered partons with specific kinematics, and to observe how their showers are modified as they propagate through the quark-gluon plasma created in Pb + Pb collisions. Compared with p p collisions, charged-particle yields in Pb + Pb collisions show significant modifications as a function of charged-particle p T in a way that depends on event centrality and Z boson p T . The data are compared with a variety of theoretical calculations and provide new information about the medium-induced energy loss of partons in a p T regime difficult to measure through other channels