Apolipoprotein E4 Reduction with Antisense Oligonucleotides Decreases Neurodegeneration in a Tauopathy Model.

OBJECTIVE: Apolipoprotein E (ApoE) genotype is the strongest genetic risk factor for late-onset Alzheimer's disease, with the ε4 allele increasing risk in a dose-dependent fashion. In addition to ApoE4 playing a crucial role in amyloid-ß deposition, recent evidence suggests that it also plays an important role in tau pathology and tau-mediated neurodegeneration. It is not known, however, whether therapeutic reduction of ApoE4 would exert protective effects on tau-mediated neurodegeneration. METHODS: Herein, we used antisense oligonucleotides (ASOs) against human APOE to reduce ApoE4 levels in the P301S/ApoE4 mouse model of tauopathy. We treated P301S/ApoE4 mice with ApoE or control ASOs via intracerebroventricular injection at 6 and 7.5 months of age and performed brain pathological assessments at 9 months of age. RESULTS: Our results indicate that treatment with ApoE ASOs reduced ApoE4 protein levels by ~50%, significantly protected against tau pathology and associated neurodegeneration, decreased neuroinflammation, and preserved synaptic density. These data were also corroborated by a significant reduction in levels of neurofilament light chain (NfL) protein in plasma of ASO-treated mice. INTERPRETATION: We conclude that reducing ApoE4 levels should be explored further as a therapeutic approach for APOE4 carriers with tauopathy including Alzheimer's disease. ANN NEUROL 2021;89:952-966.

Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease.

Increasing evidence supports a role of neuroinflammation in the pathogenesis of Alzheimer's disease (AD). Previously, we identified a neuron-glia signaling pathway whereby Aß acts as an upstream activator of astroglial nuclear factor kappa B (NF-κB), leading to the release of complement C3, which acts on the neuronal C3a receptor (C3aR) to influence dendritic morphology and cognitive function. Here we report that astrocytic complement activation also regulates Aß dynamics in vitro and amyloid pathology in AD mouse models through microglial C3aR. We show that in primary microglial cultures, acute C3 or C3a activation promotes, whereas chronic C3/C3a treatment attenuates, microglial phagocytosis and that the effect of chronic C3 exposure can be blocked by cotreatment with a C3aR antagonist and by genetic deletion of C3aR. We further demonstrate that Aß pathology and neuroinflammation in amyloid precursor protein (APP) transgenic mice are worsened by astroglial NF-κB hyperactivation and resulting C3 elevation, whereas treatment with the C3aR antagonist (C3aRA) ameliorates plaque load and microgliosis. Our studies define a complement-dependent intercellular cross talk in which neuronal overproduction of Aß activates astroglial NF-κB to elicit extracellular release of C3. This promotes a pathogenic cycle by which C3 in turn interacts with neuronal and microglial C3aR to alter cognitive function and impair Aß phagocytosis. This feedforward loop can be effectively blocked by C3aR inhibition, supporting the therapeutic potential of C3aR antagonists under chronic neuroinflammation conditions. SIGNIFICANCE STATEMENT: The complement pathway is activated in Alzheimer's disease. Here we show that the central complement factor C3 secreted from astrocytes interacts with microglial C3a receptor (C3aR) to mediate ß-amyloid pathology and neuroinflammation in AD mouse models. Our study provides support for targeting C3aR as a potential therapy for Alzheimer's disease.

Dose-dependent micronuclei formation in normal human fibroblasts exposed to proton radiation.

Micronuclei are small extranuclear bodies resulting from chromosome fragments or the whole chromosomes secluded from daughter nuclei during mitosis. The number of radiation-induced micronuclei reflects the level of chromosomal damage and relates to an absorbed dose and quality of incident ionizing radiation. The aim of the present study was to determine the micronucleus formation as a specific biological marker for acute radiation-induced DNA damage in normal human fibroblasts exposed to 30-MeV protons and Co-60 gamma radiation. We found a linear increase in binuclear cells containing micronuclei for absorbed doses from 1 to 5 Gy for both radiation modalities. However, the total number of micronuclei in binuclear cells follows a linear-quadratic dose dependence. In case of human exposure to mixed radiation fields or high LET radiation, the proportion of binuclear cells containing micronuclei from all binuclear cells can thus serve as a good biomarker of radiation-induced DNA damage.

Amelioration of Tau and ApoE4-linked glial lipid accumulation and neurodegeneration with an LXR agonist.

Apolipoprotein E (APOE) is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). APOE4 increases and APOE2 decreases risk relative to APOE3. In the P301S mouse model of tauopathy, ApoE4 increases tau pathology and neurodegeneration when compared with ApoE3 or the absence of ApoE. However, the role of ApoE isoforms and lipid metabolism in contributing to tau-mediated degeneration is unknown. We demonstrate that in P301S tau mice, ApoE4 strongly promotes glial lipid accumulation and perturbations in cholesterol metabolism and lysosomal function. Increasing lipid efflux in glia via an LXR agonist or Abca1 overexpression strongly attenuates tau pathology and neurodegeneration in P301S/ApoE4 mice. We also demonstrate reductions in reactive astrocytes and microglia, as well as changes in cholesterol biosynthesis and metabolism in glia of tauopathy mice in response to LXR activation. These data suggest that promoting efflux of glial lipids may serve as a therapeutic approach to ameliorate tau and ApoE4-linked neurodegeneration.

Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging.

Dysfunction of immune and vascular systems has been implicated in aging and Alzheimer disease; however, their interrelatedness remains poorly understood. The complement pathway is a well-established regulator of innate immunity in the brain. Here, we report robust age-dependent increases in vascular inflammation, peripheral lymphocyte infiltration, and blood-brain barrier (BBB) permeability. These phenotypes were subdued by global inactivation and by endothelial cell-specific ablation of C3ar1. Using an in vitro model of the BBB, we identified intracellular Ca2+ as a downstream effector of C3a/C3aR signaling and a functional mediator of vascular endothelial cadherin junction and barrier integrity. Endothelial C3ar1 inactivation also dampened microglia reactivity and improved hippocampal and cortical volumes in the aging brain, demonstrating a crosstalk between brain vasculature dysfunction and immune cell activation and neurodegeneration. Further, prominent C3aR-dependent vascular inflammation was also observed in a tau-transgenic mouse model. Our studies suggest that heightened C3a/C3aR signaling through endothelial cells promotes vascular inflammation and BBB dysfunction and contributes to overall neuroinflammation in aging and neurodegenerative disease.

Complement C3aR Inactivation Attenuates Tau Pathology and Reverses an Immune Network Deregulated in Tauopathy Models and Alzheimer's Disease.

Strong evidence implicates the complement pathway as an important contributor to amyloid pathology in Alzheimer's disease (AD); however, the role of complement in tau modulation remains unclear. Here we show that the expression of C3 and C3a receptor (C3aR1) are positively correlated with cognitive decline and Braak staging in human AD brains. Deletion of C3ar1 in PS19 mice results in the rescue of tau pathology and attenuation of neuroinflammation, synaptic deficits, and neurodegeneration. Through RNA sequencing and cell-type-specific transcriptomic analysis, we identify a C3aR-dependent transcription factor network that regulates a reactive glial switch whose inactivation ameliorates disease-associated microglia and neurotoxic astrocyte signatures. Strikingly, this C3aR network includes multiple genes linked to late-onset AD. Mechanistically, we identify STAT3 as a direct target of C3-C3aR signaling that functionally mediates tau pathogenesis. All together our findings demonstrate a crucial role for activation of the C3-C3aR network in mediating neuroinflammation and tau pathology.

Structural and functional characterizations of mung bean mitochondrial nucleoids.

Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a gamma-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.

A native interactor scaffolds and stabilizes toxic ATAXIN-1 oligomers in SCA1.

Recent studies indicate that soluble oligomers drive pathogenesis in several neurodegenerative proteinopathies, including Alzheimer and Parkinson disease. Curiously, the same conformational antibody recognizes different disease-related oligomers, despite the variations in clinical presentation and brain regions affected, suggesting that the oligomer structure might be responsible for toxicity. We investigated whether polyglutamine-expanded ATAXIN-1, the protein that underlies spinocerebellar ataxia type 1, forms toxic oligomers and, if so, what underlies their toxicity. We found that mutant ATXN1 does form oligomers and that oligomer levels correlate with disease progression in the Atxn1(154Q/+) mice. Moreover, oligomeric toxicity, stabilization and seeding require interaction with Capicua, which is expressed at greater ratios with respect to ATXN1 in the cerebellum than in less vulnerable brain regions. Thus, specific interactors, not merely oligomeric structure, drive pathogenesis and contribute to regional vulnerability. Identifying interactors that stabilize toxic oligomeric complexes could answer longstanding questions about the pathogenesis of other proteinopathies.

NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I.

Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).

Mitochondrial DNA and functional investigations into the radiosensitivity of four mouse strains.

We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive), C57BL/6 (radioresistant), and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survival in vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survival in vivo among the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP) measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.

Mitochondrially-encoded protein Var1 promotes loss of respiratory function in Saccharomyces cerevisiae under stressful conditions.

Stressed Saccharomyces cerevisiae cells easily lose respiratory function due to deletions in mitochondrial DNA, and this increases their general stress resistance. Is the loss active? We found that erythromycin (an inhibitor of mitochondrial translation) prevents the loss in control cells but not in the ones expressing mitochondrially-encoded protein Var1 in the nucleus. Var1 is a component of mitochondrial ribosomes; it is hydrophilic, positively charged, and prone to aggregation. Addition of DNase altered Var1 content in a preparation of mitochondrial nucleoids. Our data indicate that Var1 physically interacts with mitochondrial DNA and under stress negatively regulates its maintenance.

Characterization of the structure and DNA complexity of mung bean mitochondrial nucleoids.

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50-200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fast-moving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.

The homologous recombination protein RAD51D mediates the processing of 6-thioguanine lesions downstream of mismatch repair.

Thiopurines are extensively used as immunosuppressants and in the treatment of childhood cancers, even though there is concern about therapy-induced leukemias and myelodysplastic syndromes resulting from thiopurine use. Following metabolic activation, thiopurines are incorporated into DNA and invoke mismatch repair (MMR). Recognition of 6-thioguanine (6-thioG) in DNA by key MMR proteins results in cell death rather than repair. There are suggestions that homologous recombination (HR) is involved downstream of MMR following thiopurine treatment, but the precise role of HR is poorly understood. In this study, we demonstrate that cells deficient in RAD51D (a RAD51 paralogue) are extremely sensitive to 6-thioG. This sensitivity is almost completely rescued by the deletion of Mlh1, which suggests that HR is involved in the repair of the 6-thioG-induced recombinogenic lesions generated by MMR. Furthermore, 6-thioG induces chromosome aberrations in the Rad51d-deficient cells. Interestingly, Rad51d-deficient cells show a striking increase in the frequency of triradial and quadriradial chromosomes in response to 6-thioG therapy. The presence of these chromatid exchange-type aberrations indicates that the deficiency in RAD51D-dependent HR results in profound chromosomal damage precipitated by the processing of 6-thioG by MMR. The radials are notable as an important source of chromosomal translocations, which are the most common class of mutations found in hematologic malignancies. This study thus suggests that HR insufficiency could be a potential risk factor for the development of secondary cancers that result from long-term use of thiopurines in patients.

Differential immunoscreening identifies a glycosylation variant of the epidermal growth factor receptor in ME-180 cervical carcinoma cells.

In this report, we describe the development and characterization of an anti-ME-180 cervical cancer-specific epidermal growth factor (EGF) receptor monoclonal antibody (MAb). This MAb, 6C7, specifically binds to ME-180 cervical cancer cells and not to normal cervical epithelial cells. By immunoaffinity chromatography, we have shown that the 6C7 antibody binds to a 205-kDa protein. Subsequent mass spectrometry sequencing analysis identified this protein as an EGF receptor. In addition, treatment of the ME-180 EGF receptor with N- and O-linked glycosidases indicated that this antibody binds to the carbohydrate portion of the glycoprotein. Moreover, Western blotting analysis with an anti-EGF receptor antibody indicated that this protein is present in abundance in all cervical cancer cell lines, including ME-180, HeLa, Ca Ski, HT-3, SiHa, and Hs 588.T. However, the 6C7 antibody only binds to the EGF receptor from ME-180 cells, suggesting that this protein is differentially glycosylated in ME-180 cells, compared to other cervical cancer cell lines. Finally, we have shown that this antibody could selectively block EGF-mediated cell proliferation in ME-180 cells but not in HeLa cells. Overall, our study suggests that the differentially glycosylated EGF receptor could potentially serve as a unique target for the immunotherapeutic treatment of cervical cancer.

Generation and characterization of monoclonal antibodies directed against the surface antigens of cervical cancer cells.

In this report, we describe the development and characterization of monoclonal antibodies against the surface antigens of cervical cancer cells. Using HeLa cervical carcinoma cells as the immunogen, we have developed a number of antibodies that specifically label cervical cancer cells but not normal cervical epithelial cells. These antibodies displayed differential reactivity towards various cervical cancer cell lines as determined by immunofluorescence labeling and western blotting analyses. One of these antibodies, 13G4, which showed the strongest labeling to HeLa cells and has the widest range of reactivity to other cervical cancer cell lines, was extensively characterized. By immunoaffinity chromatography, we purified a 90-kDa protein that appears to be the principal target recognized by this antibody. This protein was subsequently identified as decay accelerating factor (DAF) or CD55 by the mass spec sequencing analysis of the tryptic peptides derived from this protein. Digestion of HeLa DAF with glycosidases that removed its N- and O-linked carbohydrates has revealed that the 13G4 antibody binds to the peptide portion of this glycoprotein. Overall, our approach of generating and characterizing monoclonal antibodies directed against the surface antigens of cervical cancer cells serves as a stepping stone towards the eventual development of a unique panel of monoclonal antibodies that could potentially be used for the detection and therapeutic treatment of cervical cancer.

Sequential structural changes of Escherichia coli thioesterase/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate.

Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC). NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC. NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. The largest chemical shift perturbation occurred around the catalytic Ser(10). In MC, the conformation of the mobile Ser(10) was stabilized, and its amide resonance became observable. From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser(10) and Gly(44) serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP. The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins.