1,721,015 research outputs found
Modulating immune system activation in neurodegenerative disease
No full text2024In Alzheimer’s disease and related dementias (ADRD), chronic neuroinflammation contributes to cellular dysfunction and neurodegeneration. During cellular stress, RNA-binding proteins (RBPs) interact with tau and other disease-related proteins to promote toxic protein aggregation. We hypothesize that modulating these stress pathways can alleviate the contribution of RBPs to disease pathology in models of ADRD. In this dissertation, two different approaches to modulating the immune system were explored. In the first study, the RBP T-cell intracellular 1 (TIA1) was selectively knocked out in microglia of P301S tauopathy mice. TIA1 decreases inflammation in the peripheral immune system; therefore, we hypothesized that TIA1 could regulate inflammation in the central immune system. To explore this, P301S tauopathy mice overexpressing human microtubule associated protein tau (MAPT) with P301S mutation were bred with TIA1 conditional knockout mice in microglia using a CX3CR1 cre recombinase (P301S/TIA1cKO). We assessed the effect of conditionally mutating TIA1 in microglia on immune system activation and tau pathology using immunoblot, transcriptional assays, and immunohistochemistry. TIA1 is required in microglia for activation of the microglial sensome and transcription of immune receptors. Receptors that respond to tau pathology (TREM2, CLEC7A, TYROBP) and inflammatory cytokines (TNFα, IL-1β) were significantly downregulated in P301S/TIA1cKO mice compared to P301S mice. This was corroborated by a distinct lack of microglial morphological changes and CD68+ microgliosis. P301S/TIA1cKO mice also had significantly less tau pathology than P301S mice. In response to lipopolysaccharide stimulation, TIA1 null microglia exhibited decreased microglial activation, in contrast with peripheral TIA1 null macrophages which have exacerbated immune activation. In conclusion, microglial TIA1 is critical for transcription of the microglial sensome and mutating microglial TIA1 reduces tau aggregation.
In the second project, the integrated stress response (ISR) was inhibited using the viral protein open reading frame 57 (ORF57) from Human herpesvirus-8. Activation of the ISR is one of the precipitating events for protein aggregation in ADRDs. The ISR is activated by many stressors, including protein aggregation, dsRNA, viruses, and oxidative stress. In the ISR, protein kinase R (PKR) phosphorylates eIF2α which halts global translation, facilitates stress granule (SG) assembly, and induces apoptosis. Chronic activation of the ISR leads to protein aggregation of TAR DNA-binding protein 43kDa (TDP-43) through this SG pathway. ORF57 binds to and inhibits protein kinase R (PKR), preventing PKR from phosphorylating eIF2α and affecting translation. We hypothesized that ORF57 would inhibit TDP-43 aggregation through this blockade of PKR activation. To study this, the viral protein, ORF57, was optimized for expression in human neuroblastoma cells overexpressing TDP-43. These cells were treated with sodium arsenite oxidative stress causing TDP-43 to form insoluble aggregates. The impact of ORF57 on immune activation and TDP-43 aggregation was explored using immunofluorescence, immunoblot, and proteomics. ORF57 reduced phosphorylation of eIF2α, improved protein synthesis rates, and decreased SG assembly. ORF57 also affected TDP-43 pathology by altering TDP-43 aggregate morphology and increasing solubility of those aggregates. TDP-43 aggregates in cells with ORF57 disassembled at a significantly higher rate and had a faster recovery after photobleaching than those without ORF57. Proteomics revealed that ORF57 interacts with TDP-43, and other RBPs, in the presence of stress. In conclusion, my work demonstrates how the anti-stress functions of viral proteins can be re-engineered to inhibit disease processes. ORF57 blocks the ISR and reduces insoluble TDP-43 aggregation, demonstrating a novel approach to decreasing immune activation in disease. Together these two projects support a benefit of modulating immune activation in ADRDs
Proteostasis Deregulation in Neurodegeneration and Its Link with Stress Granules: Focus on the Scaffold and Ribosomal Protein RACK1
Full text linkThe role of protein misfolding, deposition, and clearance has been the dominant topic in the last decades of investigation in the field of neurodegeneration. The impairment of protein synthesis, along with RNA metabolism and RNA granules, however, are significantly emerging as novel potential targets for the comprehension of the molecular events leading to neuronal deficits. Indeed, defects in ribosome activity, ribosome stalling, and PQC—all ribosome-related processes required for proteostasis regulation—can contribute to triggering stress conditions and promoting the formation of stress granules (SGs) that could evolve in the formation of pathological granules, usually occurring during neurodegenerating effects. In this review, the interplay between proteostasis, mRNA metabolism, and SGs has been explored in a neurodegenerative context with a focus on Alzheimer’s disease (AD), although some defects in these same mechanisms can also be found in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are discussed here. Finally, we highlight the role of the receptor for activated C kinase 1 (RACK1) in these pathologies and note that, besides its well characterized function as a scaffold protein, it has an important role in translation and can associate to stress granules (SGs) determining cell fate in response to diverse stress stimuli
Mitochondrial antiviral signaling (MAVS) is essential for elevated type I interferon signaling in the aging central nervous system (CNS)
Full text linkAging is amongst the strongest risk factors for neurodegenerative disease and elevated Type I interferon (IFN) signaling has been associated with both normal aging and central nervous system (CNS) diseases. Type I IFN is normally produced by nucleated cells in response to the detection of viral pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs). More recently it has been appreciated that Type I IFNs are also produced in response to endogenous stimuli, in the absence of viral pathogens. While Type I IFN signaling has been shown to be elevated in human and murine brains during normal aging, the underlying cause was unknown. Here we demonstrate by flow cytometry that aging results in increased size and numbers of mitochondria in the murine brain. Despite identifying increased mitochondrial number and mitochondrial DNA content, we found no change to mitochondrially-encoded transcripts, suggesting either deficits in mitophagy or augmented biogenesis due to insufficient oxidative phosphorylation. Interestingly, mitochondrial numbers correlated with elevated Type I IFN signaling in aging, linking mitochondria to the age-dependent innate immune response in the CNS. Using genetically engineered mice, we excluded roles for two critical innate immune pathways, STING and IRAK4, in the age-dependent increase in Type I IFN signaling in the brain. Notably, we subsequently identified a mitochondrially restricted innate immune protein, mitochondrial antiviral signaling (MAVS) as an essential molecular mediator of the age-dependent Type I IFN response; MAVS deficiency in aged mice restored Type I IFN signaling in the CNS to the levels observed in adult wildtype mice. Further, using intracerebroventricular (icv) administration of antisense oligonucleotides (ASO) as an orthogonal approach, we reduced MAVS transcript and protein expression within the CNS and thereby reduced Type I IFN signaling. Our data demonstrate a specific and selective role of MAVS expression in the CNS in Type I IFN signaling in aging. To investigate the relationship between mitochondrial aging and MAVS activation, we isolated cytoplasmic and mitochondrial RNA from young and aged animals as MAVS is most studied for its response to RNA ligands. Upon transfection into reporter cells, we found that mitochondrial RNA, but not cytoplasmic RNA, from both young and aged mice was sufficient to induce Type I IFN reporter activity in a MAVS-dependent manner. Furthermore, we attempted to mimic the increase of mitochondria observed in the aging CNS by transferring mitochondria from young and aged animals to recipient cells. Mitochondrial transfer also induced MAVS-dependent Type I IFN signaling in wildtype, but not MAVS null, mouse embryonic fibroblasts (MEFs). Collectively, our findings suggest that the accumulation of mitochondria in aging serves as a robust source of MAVS pathway ligands and implicate a novel link between mitochondrial aging and MAVS-mediated innate immune signaling in the CNS
KSHV-ORF57 inhibits stress granule assembly and may be a novel biotherapeutic for neurodegenerative diseases
Full text linkOpen reading frame 57 (ORF57), a protein found in the genome of Kaposi’s Sarcoma Virus (KSV), has been shown to interact with protein kinase R (PKR) and inhibit its activation by blocking PKRs interaction with PKR activating protein (PACT). PKR activation is a key step in stress granule (SG) assembly, a pathway implicated in the pathology of many neurodegenerative diseases, including Alzheimer’s Disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Expression of ORF57 in human neuroblastoma cells (SH-SY5Y) shows promise as an inhibitor of this pathway. ORF57 expression in SH-SY5Y cells showed decreases in SG-associated protein aggregates and phosphorylation of eukaryotic initiation factor 2 (eIF2), a necessary phosphorylation event upstream of SG assembly when treated with oxidative stress. ORF57 expression also shows increased translation and decreased apoptosis when cells are exposed to oxidative stress. These changes indicate a potential role of ORF57 as a stress granule pathway inhibitor. To further investigate ORF57’s potential as a biotherapeutic for neurodegenerative disease, and specifically ALS, ORF57 was expressed in a cell line overexpressing cytoplasmic TDP43 (SH-SY5Y TDP43 NLS). These cells expressing ORF57 under stress conditions showed decreases in SG-associated protein aggregates and phosphorylation of eIF2. SH-SY5Y TDP43 NLS cells expressing ORF57 under stress also showed an increase in the soluble fraction of TDP43. Taken together, ORF57 shows promise as a PKR inhibitor with potential for treating SG-associated neurodegenerative pathologies
Establishing RNA binding proteins as key components of Alzheimer's disease pathophysiology
Full text linkAggregation of the microtubule associated protein tau (MAPT) into filamentous neurofibrillary tangles (NFTs) is a defining molecular hallmark of neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Despite the discovery of NFTs decades ago, the molecular mechanisms underpinning their formation and neurotoxicity have remained elusive. Recently, our lab has shown that stress granule (SG) associated RNA binding proteins (RBPs) co-deposit with pathological tau in mouse models and human disease; reduction of the SG associated protein TIA1 also protects against tau-mediated behavioral deficits and degeneration. Here we demonstrate that RBPs facilitate tau pathogenesis and exhibit prominent signs of dysfunction early in disease. By immunoprecipitating pathological tau from the transgenic rTg4510 AD mouse model, we have found that tau associates with many RBPs and ribosomal subunits, and these associations change as tau pathology develops. These RBPs also become increasingly insoluble in tauopathy, consistent with the formation of fibrillar aggregates. We also show by immunohistochemistry that as tau forms mature neurofibrillary tangles, RBPs lose their interaction with tau and aggregate to the periphery of the tangle in mouse and human tissue, suggesting that RBPs contribute to earlier stages of tau aggregation. Seeing this, we sought to determine at what point of tau pathogenesis RBPs become relevant to the disease process. Using the PS19 mouse line, which develops tangle pathology more slowly than the rTg4510, we have found that RBP immunohistochemistry is highly sensitive to tissue fixation methods, that different brain regions have unique localization patterns of canonically nuclear RBPs, and that transgenic tau mice show striking changes in hippocampal RBP regulation very early in tau pathogenesis. This precedes an eventual destabilization and disruption of the nuclear lamina. We further show that overexpression of TIA1 accelerates the somatodendritic accumulation of phosphorylated tau in vivo, and that TIA1 granules co-localize with granules of misfolded tau. Together these findings support the idea that ribonucleoprotein granules contribute significantly to early pathological tau formation and that misregulation of these proteins progresses in tandem with tau pathology. RBPs thus offer promising new therapeutic targets for Alzheimer’s disease and related tauopathies
Dysregulation of the N6-methyladenosine epitranscriptome in Alzheimer’s disease and its implications in aging, synaptic function, and RNA metabolism
No full text2026Alzheimer’s disease (AD) is the most common cause of dementia and is characterized by cognitive decline and behavioral deficits. Despite recent therapeutic advances with amyloid-β–targeting antibodies and tau antisense oligonucleotides, there remains a critical need to investigate alternative mechanisms contributing to AD. Extensive work has been done in the field of AD analyzing disease-linked changes in transcription, splicing, DNA epigenetics and chromatic structure. Altered RNA metabolism is also well documented in AD, with increased aggregation of RNA-binding proteins. However, the epigenetics of RNA are only beginning to be studied in AD. N6-methyladenosine (m6A) is the most common epigenetic modification of mRNA and a key determinant of mRNA fate, including synaptic localization and activity-dependent translation. The site-specific regulation of m6A in the cognitively normal or AD brain has yet to be determined. Here, we mapped the m6A epitranscriptome in post-mortem human AD and Control cases using deamination adjacent to RNA modification targets and sequencing (DART-seq), an antibody-independent approach for nucleotide-resolution detection of m6A-modified sites and transcripts. DART-seq reveals a 3’ untranslated region (UTR)-enriched, age-associated increase in the number of m6A sites in Control cases, which is absent in AD cases. We hypothesize that this age-related increase in m6A labeling reflects a protective role of age-associated methylation. Instead in the AD brain, m6A-modified transcripts are globally hypomethylated. We observed a group of transcripts whose m6A methylation correlates with gene or protein expression levels; these correlations occur selectively in genes encoding astrocytic glutamate importers and GABAergic ionotropic receptors, implicating m6A in regulation of the tripartite synapse, and potentially contributing to excitotoxicity. These findings are consistent with a known imbalance between excitatory glutamatergic and inhibitory GABAergic signaling that is an important feature of AD leading to excitotoxicity, synapse loss, and disease progression. We also revealed differentially abundant m6A sites on translational stress response genes and a loss of m6A-governed transcript regulation for GABRA1, consistent with altered RNA metabolism in AD. These findings provide the first nucleotide-specific m6A landscape in the human brain, show clear changes associated with AD, and open novel therapeutic strategies, such as gene editing, to site-selectively modify m6A levels with the goal of restoring synaptic function and RNA metabolism in the AD brain
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Evaluation of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway in human myelomonocytic THP1 cells
Full text linkActivation of the NOD-like receptor protein 3 (NLRP3) inflammasome complex causes the processing and release of mature IL-1β, with mitochondria playing key roles in its assembly. An orally active NLRP3 inflammasome inhibitor would be a significant advance in therapy for IL-1β-driven diseases. To overcome both, the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. We established that undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages. CP-456,773 is a potent and selective inhibitor of the NLRP3 inflammasome, and it is an analogue of glyburide, a sulfonylurea receptor (SUR) inhibitor. Despite the extensive experimental use of CP-456,773, its molecular target remains unknown. Here we tested the hypothesis that mitochondrial ABCb7 or ABCb10 could be the pharmacologic targets of CP-456,773. We optimized a viral shRNA transduction method for genetic manipulations in THP1 cells and generated ABCb7 and ABCb10 knockdown (KD) THP1 cells. We demonstrate that NLRP3 inflammasome activation and CP-456,773 pharmacology are not altered in ABCb7- or ABCb10-deficient THP1 cells. For ABCb10, we confirmed these results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In studies of mitochondrial fitness, we found that a previously observed reduction in oxygen consumption rate (OCR) following nigericin treatment was completely blocked in NLRP3 KO cells. Our data demonstrating that CP-456,773 rescues the NLRP3-dependent nigericin-induced decline in OCR and protects undifferentiated THP1 cells from nigericin-induced pyroptosis are consistent with the possibility that the NLRP3 protein itself may be the molecular target of CP-456,773. Moreover, we showed that ABCb10 KO THP1 cells exhibit increased rates of basal ATP production and glycolysis, suggesting an important role for ABCb10 in mitochondrial metabolism. Finally, RNA-Seq analysis of ABCb7 and ABCb10 KD in undifferentiated THP1 cells indicate new functions for these proteins, including cell communication and migration, apoptosis and cell adhesion. Overall, our findings demonstrate that undifferentiated THP1 cells are an ideal system in which to study the NLRP3 inflammasome
Structural and mechanistic analyses of a nicotine- degrading enzyme from Pseudomonas putida: towards design of tools and biotherapeutics
Full text linkTobacco-soil bacteria have evolved not only to tolerate high concentrations of nicotine, but to degrade it as a primary growth source. The genomes of several of these species have been sequenced, allowing for the identification of unique bacterial degradation pathways. In the Gram-negative bacteria, Pseudomonas putida, the nicotine-degrading gene cluster has been described; the encoded enzymes catabolize nicotine via the pyrrolidine pathway, ultimately forming malate and fumarate. In previous studies, the flavoenzyme, nicotine oxidoreductase (NicA2), has been identified as the first committed step of nicotine catabolism in this organism. Preliminary kinetic analysis reported that NicA2 has high specificity for S-nicotine, but a slow catalytic rate. Taking advantage of its unique evolutionary adaptation, we aim to refine the inherent catalytic function and structural features of NicA2 towards the development of a biotherapeutic for nicotine addiction, nicotine poisoning and tools for nicotine biosensor development. Our goal is to identify the factors contributing to the mechanistic and substrate-binding properties of NicA2 to improve its biotherapeutic potential. This work presents the first crystal structure of NicA2, resolved to 2.2 Å resolution, establishing it as a member of the flavin-dependent amine oxidase family with a conserved amine oxidase fold. Structural analysis identified a unique composition of the canonical aromatic cage (W427 and N462), which flanks the flavin isoalloxazine ring. Additionally, the X-ray crystallographic structure of the NicA2/S-nicotine complex was refined to 2.6 Å resolution, revealing a hydrophobic active site in support of a hydride-transfer mechanism. Analysis of enzyme activity with a series of substrate analogs and kinetic analysis of active-site residues reveal the determinants of substrate binding affording the remarkable specificity of this enzyme. Using site-directed mutagenesis of aromatic cage residues, along with analysis of the kinetics of the reductive and oxidative steps, we demonstrate that the rate-limiting reaction step is in the oxidative half-reaction. Structural analysis of an active-site variant revealed a secondary binding site consistent with kinetic analysis demonstrating substrate inhibition. Together, our findings provide kinetic and structural evidence for the catalytic mechanism of NicA2, expanding the possibilities for the generation of catalytically-efficient variants and supporting its role as a promising therapeutic strategy.2021-01-30T00:00:00
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