Biology subjects

Dhingra, A.

Publications and source records attributed to Dhingra, A..

At least 19 recordsLinked to original sources

bioRxiv2026

The p53-p21-Cyclin D2 regulatory axis drives metabolic reprogramming and a distinct senescent macrophage senotype during aging and MASLD.

Aging drives chronic disease in part through senescent cells, including macrophages, which fuel inflammation. Senescent macrophages are functionally heterogeneous: canonical p16-high macrophages promote tumorigenesis or, in other contexts, disease tolerance, whereas we previously identified a distinct p21-high, p16-low senotype that drives metabolic dysfunction-associated steatotic liver disease (MASLD). The molecular basis of this senotype has remained undefined. Using genetic and multi-omic approaches, we show that a p53-p21-dependent program actively represses p16 and is required for senescent macrophage viability. We identify Cyclin D2 as a non-canonical downstream effector that redistributes from the nucleus to mitochondria and lipid droplets, where it partners with MIC60 to drive metabolic reprogramming and modulates AKT1-mTORC1 signaling that sustains the SASP. Cyclin D2-p21 double-positive macrophages accumulate with aging and MASLD in mice and in human liver cirrhosis, and can be selectively depleted by senolytic treatment. Together, these findings define a druggable p53-p21-Cyclin D2 axis that specifies macrophage senotype.

immunology↗

bioRxiv2026

Bioengineering of Pea (Pisum sativum) for the Expression of Myoglobin, a Heme-containing Animal Protein

Myoglobin, an oxygen-binding animal protein, was engineered in Pisum sativum (pea) to explore its potential as a food ingredient and balance the amino acid profile. In this study, minimal expression cassettes and binary vectors were used to express bovine myoglobin using particle gun and Agrobacterium-mediated transformation, respectively. Successful integration and expression of the myoglobin gene was achieved in P. sativum, with both methods yielding similar transformation efficiencies (~1%). Expression analysis of T2 seeds revealed that Agrobacterium-mediated transformation-derived transgenic lines that expressed myoglobin under the regulation of a Soybean 7S seed-specific promoter and Tobacco Etch Virus (TEV) translation enhancer and a chimeric Rb7MAR Terminator (Ps-BpRG13 events) consistently yielded the highest level of expression (0.32-1.57% of TSP), while transgenic lines with myoglobin expression under the regulation of a Soybean Phaseolin promoter and Rb7MAR Terminator (Ps-BpRG14 events) resulted in moderate levels of heterologous protein expression (0.13-0.83% TSP). Transgenic events with constitutive 2xCaMV35S promoter, TEV translation enhancer and Rb7MAR terminator (Ps-BpRG15 events) exhibited the lowest level of myoglobin expression (0.09-0.14% TSP). Co-bombardment of two minimal expression cassettes - one with myoglobin under the regulation of the Phaseolin promoter and Rb7MAR Terminator and the other with the nptII selectable marker under the regulation of a 2X constitutive CaMV35S promoter, TEV translational enhancer and TNOS Terminator, yielded lines that exhibited variable expression (0.03-0.77% TSP), with some events comparable in expression to Agrobacterium-derived Ps-pRG14 events. To the best of our knowledge, this is the first report of producing a heme-containing animal protein, myoglobin, in peas, with potential implications for sustainable production of food ingredients and nutritionally fortified and value-added plant products using molecular farming.

plant biology↗

bioRxiv2026

A Cesium Chloride Gradient Ultracentrifugation-Based Method for the Isolation of DNA from Diverse Recalcitrant Plant Species for Nanopore Sequencing

Developments in Nanopore sequencing have enabled telomere to telomere genomic assembly as a routine technique in genomic research. Nanopore DNA sequencing for genomic assembly is typically performed on native DNA molecules, making it particularly sensitive to the quality of input DNA, with contaminating molecules limiting data yields and reducing read quality. As pangenome analysis gains interest, particularly in non-model plant species which are often rich in inhibitory secondary metabolites, the development of methods which can improve the quality and throughput of nanopore sequencing is essential. Here we describe a method for isolation of total DNA from the leaf tissues of diverse Viridiplantae species. The initial lysis buffer consists of a modified CTAB buffer, incorporating dimethyl sulfoxide for the reduction of viscosity, which can be problematic in many plant DNA preparations. An organic extraction with 2-butoxyethanol is utilized to further extract phenolic compounds which may be sufficiently hydrophilic to evade chloroform extraction, while reducing aqueous phase volume. Further cleanup via cesium chloride (CsCl) ultracentrifugation is performed to minimize the carryover of residual contaminating macromolecules. Samples prepared using this method are of consistent high quality, even when extracted from challenging late season leaf tissue or secondary metabolite rich species. Sequencing results from samples prepared by this method outperform those obtained from typical modified CTAB DNA isolation techniques in both quantity and quality. We tested sequencing performance from Vitis DNA isolated using a modified CTAB method and Vitis DNA isolated using the CsCl ultracentrifugation-based method described here. DNA isolated via the method described here produced 83% more >Q10 sequence data (52.61 Gb vs. 28.8 Gb), resulted in a 60% greater read N50 despite more handling steps (32.78kb vs. 20.45kb), and resulted in a higher modal read quality (Q27 vs. Q24). The consistency of this method across diverse plant taxa suggests its use as a general method for DNA isolation prior to Nanopore sequencing and genomic assembly for diverse plant taxa.

molecular biology↗

bioRxiv2026

Comprehensive investigation of AAV tropism across human iPSC-derived neuronal subtypes

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

neuroscience↗

bioRxiv2026

Extensive Novel Genomic Variations in Mutant European Pear Individuals Revealed by Mapping to a Pangenome Reference

European pear (Pyrus communis) is the most widely cultivated Pyrus species outside of Asia; however, production has been declining in Europe, Oceania, and North America. Most European pear cultivars are over 100 years old and face pressure from disease, a changing climate, and challenging postharvest characteristics, while demand for increased production efficiency rises. To generate germplasm with desirable characteristics to address these concerns, a mutation breeding approach was chosen, with crosses made between four economically significant cultivars ( Bartlett, dAnjou, Abbe Fetel, and Comice) using gamma-irradiated pollen. This resulted in 49 viable offspring, of which 37 have survived at least 10 years. Nanopore whole-genome sequencing was used to test the success of this approach and screen for variants of interest. Sequence reads were mapped to both a lightweight, purpose-built pangenome derived from assemblies of parental haplotypes and a linear reference genome, enabling the high-quality discovery of variants of all sizes, ranging from single-base substitutions to megabase-scale deletions, with the overwhelming majority being small variants. The overall rate of mutation was 153 novel small variants and 0.228 novel structural variants per Gray of absorbed gamma radiation. Alternate ploidy levels were detected in four lines, which included three triploids and one tetraploid. While the resulting individuals appear incapable of floral development, they may be of utility as rootstock cultivars and a valuable genetic resource for understanding the underlying basis of structural traits.

genomics↗

bioRxiv2026

Pangenome Analysis of Pyrus Reveals Potential Role of Transcription Factors in Recent Adaptation to Arid Environments

Pears (genus Pyrus) are among the most extensively cultivated tree fruits with a wide-reaching economic impact. Despite this, the genetic basis of most pear traits of interest, including abiotic stress tolerance, tree architecture, precocity, parthenocarpy, disease resistance, and fruit ripening, remains poorly understood. Although extensive efforts have been made to identify quantitative trait loci (QTLs) that explain the genetic basis of pear traits, many are poorly transferable, limiting their utility for informing genetic improvement or management of pears across most genetic backgrounds. To provide a whole-genome context and enable the exploration of functional variation in Pyrus, we developed a pangenome graph using 31 accessions representing 23 Pyrus species from the National Clonal Germplasm Repository. Whole-genome sequencing was performed solely with Oxford Nanopore, generating highly contiguous assemblies for pangenome construction, demonstrating the viability of a single-platform approach to pangenomic analysis in Viridiplantae. Exploration of the pangenome graph reveals genes present in some lineages, with potential functional implications. A group of arid-adapted Pyrus species exhibits signs of selective sweeps in regions associated with transcription factors, likely impacting abiotic stress tolerance. With the development of this pangenomic resource, resequencing analysis in Pyrus is now possible without the limitations imposed by single-reference genome assemblies.

genomics↗

bioRxiv2025

Both conventionally and organically fertilized tomatoes maintain fruit quality through uncontrolled green peach aphid infestation, with a transcriptional shift towards catabolism

Aphids are a major pest of greenhouse-grown temperate crops, responsible for billions in crop damage yearly. As organic agriculture rapidly grows in popularity, understanding how plants grown under organic systems respond to insect pest pressure may give insights into better management practices and information about the genes of interest for crop improvement. We measured the response of tomato (Solanum lycopersicum) leaf and fruit transcriptome, as well as a few selected metabolites in the mature fruit, to an infestation of the generalist green peach aphid (Myzus persicae). The aphids were introduced approximately halfway through the lifecycle of the plants that were grown under conventional and organic fertilizer regimes. While plants provided with conventional fertilizer experienced greater aphid infestation, neither group suffered a significant loss in total yield or fruit quality. This result is likely a consequence of ample nutrient and water availability. Co-expression network analysis using WGCNA revealed that in leaf tissue, both treatment groups showed a general shift from diverse anabolic processes to catabolism, while fruit tissue experienced relatively minor changes. At the stage of infestation investigated, abscisic acid appeared to be the main phytohormone response. One coexpression network module showed a correlation with both organic fertilizer treatment and aphid infestation; its hub gene (Solyc02g078940.3) may be of interest in exploring unique responses to phloem feeding insect infestation under an organic production regime.

plant biology↗

bioRxiv2025

The Insertion of an ATTTC Repeat in an Alu Element Hyperactivates a Primate-Specific Neurodevelopmental Enhancer in Spinocerebellar Ataxia Type 37

Alu are evolutionarily very old primate-specific interspersed repeat elements that constitute [~]11% of the human genome. They are a source of short tandem repeats (STRs), which often expand in size and originate inherited neuromuscular and neurodegenerative disorders. How expanded STR insertion mutations within Alu STRs culminate in disease remains unknown. Here we report an Alu STR located in an intron of DAB1 that functions as a neurodevelopmental enhancer. We demonstrated that an ATTTC repeat insertion in this DAB1 Alu STR, known to cause spinocerebellar ataxia type 37 (SCA37), hyperactivates a neurodevelopmental DAB1 enhancer. Importantly, we showed that neurons derived from SCA37 subjects have higher levels of DAB1 expression and DAB1 overexpression causes abnormal axonal pathfinding in vivo. Overall, these results establish that neuronal dysregulation of a developmental DAB1 Alu STR enhancer contributes to SCA37 pathogenesis, an unexplored mechanism likely acting in many Alu STR diseases, potentially reshaping the therapeutic landscape.

genetics↗

bioRxiv2024

C9orf72 repeat expansion-carrying iPSC-microglia from FTD patients show increased phagocytic activity concomitantly with decreased number of autophagosomal-lysosomal vesicles

C9orf72 hexanucleotide repeat expansion (HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The role of microglia in these C9orf72 HRE-associated diseases is understudied. To elucidate effects of C9orf72 HRE on microglia, we have characterized human induced pluripotent stem cell-derived microglia (iMG) from behavioral variant frontotemporal dementia (bvFTD) patients carrying the C9orf72 HRE. C9orf72 HRE iMG were compared to iMG from healthy controls and sporadic bvFTD patients. The phenotypes of iMG were analyzed using bulk RNA sequencing, biochemical and immunofluorescence analyses, and live cell imaging. C9orf72 HRE-carrying iMG showed nuclear RNA foci and poly-GP dipeptide repeat proteins but no decreased C9orf72 mRNA or protein expression. TDP-43 pathology was absent from all bvFTD iMG. As compared to healthy control iMG, quantitative immunofluorescence analyses indicated that all bvFTD iMG had reduced number, size, and intensity of LAMP2-A-positive vesicles. C9orf72 HRE-carrying iMG additionally showed decreased number, size, and intensity of p62/SQSTM1-positive vesicles. These changes were accompanied by increased phagocytic activity of the C9orf72 HRE-carrying iMG. Serum starvation increased phagocytic activity also in the iMG of sporadic bvFTD patients. RNA sequencing revealed that iMG of C9orf72 HRE-carrying bvFTD patients as compared to the iMG of sporadic bvFTD patients showed differential gene expression in pathways related to RNA and protein regulation and mitochondrial metabolism. Our data suggest potential alterations in the autophagosomal/lysosomal pathways in bvFTD patient iMG, which are further reinforced by the C9orf72 HRE and functionally manifest as increased phagocytic activity.

neuroscience↗

bioRxiv2024

Single-cell transcriptomic changes in oligodendrocytes and precursors derived from Parkinson's disease patient-iPSCs with LRRK2-G2019S mutation

Despite extensive research, the contribution of the LRRK2 p.G2019S mutation to Parkinsons disease (PD) remains unclear. Recent findings indicate oligodendrocytes (ODCs) and their progenitors are vulnerable in PD pathogenesis. Notably, oligodendrocyte precursor cells (OPCs) exhibit high endogenous expression of LRRK2. We induced PD patient-iPSCs with the LRRK2 p.G2019S mutation into oligodendroglial lineages and performed single-cell RNA sequencing. Cell type composition analysis revealed an increase in OPCs, proliferating OPCs and ciliated ependymal cells in LRRK2 lines, all of which are characterized by LRRK2 expression. Differential expression analysis revealed transcriptomic changes in several pathways, including down-regulation of genes related to myelin assembly in ODCs, semaphorin-plexin pathway in OPCs, and cilium movement in proliferating OPCs. Cell-cell communication analysis identified significant alterations in several signaling pathways including a deactivation of PSAP signaling and an activation of MIF signaling in LRRK2 lines. Additionally, we observed an overall increase in SEMA6 signaling communication in LRRK2 cell lines; however, OPCs derived from these LRRK2 lines specifically lost SEMA6 signaling due to a down-regulation of SEMA6A and PLXNA2. Pseudotemporal trajectory analysis revealed that SHH had significantly altered expression along the pseudotime, accompanied by higher expression levels in LRRK2 lines. We propose that dysfunctional semaphorin-plexin signaling, along with cilia movement and SHH signaling, might represent early events in PD pathology.

neuroscience↗

bioRxiv2024

Direct and indirect regulation of β- glucocerebrosidase by the transcription factors USF2 and ONECUT2

Mutations in the GBA gene, which encodes the lysosomal enzyme {beta}-glucocerebrosidase (GCase), are the most prevalent genetic susceptibility factor for Parkinsons disease (PD). However, only approximately 20% of carriers develop the disease, suggesting the presence of genetic modifiers influencing the risk of developing PD in the presence of GBA mutations. Here we screened 1,634 human transcription factors (TFs) for their effect on GCase activity in cell lysates of the human glioblastoma line LN-229, into which we introduced the pathogenic GBA L444P variant via adenine base editing. Using a novel arrayed CRISPR activation library, we uncovered 11 TFs as regulators of GCase activity. Among these, activation of MITF and TFEC increased lysosomal GCase activity in live cells, while activation of ONECUT2 and USF2 decreased it. Conversely, ablating USF2 increased GBA mRNA and led to enhanced levels of GCase protein and activity. While MITF, TFEC, and USF2 affected GBA transcription, ONECUT2 was found to control GCase trafficking by modulating the guanine exchange factors PLEKHG4 and PLEKHG4B. Hence, our study provides a systematic approach to identifying modulators of GCase activity, expands the transcriptional landscape of GBA regulation, and deepens our understanding of the mechanisms involved in influencing GCase activity.

molecular biology↗

bioRxiv2024

Frontotemporal dementia patient-derived iPSC neurons show cell pathological hallmarks and evidence for synaptic dysfunction and DNA damage

Frontotemporal dementia (FTD) is the second most common cause of dementia in patients under 65 years, characterized by diverse clinical symptoms, neuropathologies, and genetic background. Synaptic dysfunction is suggested to play a major role in FTD pathogenesis. Disturbances in the synaptic function can also be associated with the C9orf72 repeat expansion (C9-HRE), the most common genetic mutation causing FTD. C9-HRE leads to distinct pathological hallmarks, such as C9orf72 haploinsufficiency and development of toxic RNA foci and dipeptide repeat proteins (DPRs). FTD patient brains, including those carrying the C9-HRE, are also characterized by neuropathologies involving accumulation of TDP-43 and p62/SQSTM1 proteins. This study utilized induced pluripotent stem cell (iPSC)-derived cortical neurons from C9-HRE-carrying or sporadic FTD patients and healthy control individuals. We report that the iPSC neurons derived from C9-HRE carriers developed typical C9-HRE-associated hallmarks, including RNA foci and DPR accumulation. All FTD neurons demonstrated increased TDP-43 nucleus-to-cytosolic shuttling and p62/SQSTM1 accumulation, and changes in nuclear size and morphology. In addition, the FTD neurons displayed reduced number and altered morphologies of dendritic spines and significantly altered synaptic function indicated by a decreased response to stimulation with GABA. These structural and functional synaptic disturbances were accompanied by upregulated gene expression in the FTD neurons related to synaptic function, including synaptic signaling, glutamatergic transmission, and pre- and postsynaptic membrane, as compared to control neurons. Pathways involved in DNA repair were significantly downregulated in FTD neurons. Only one gene, NUPR2, potentially involved in DNA damage response, was differentially expressed between the sporadic and C9-HRE-carrying FTD neurons. Our results show that the iPSC neurons from FTD patients recapitulate pathological changes of the FTD brain and strongly support the hypothesis of synaptic dysfunction as a crucial contributor to disease pathogenesis in FTD.

neuroscience↗

bioRxiv2023

Chloroplast Genome Data of Tecticornia indica, a Hygrohalophytic C4 Plant Species

Tecticornia indica is a hygrohalophytic, NAD-malic enzyme-type C4 plant species with near-aphyllous photosynthetic shoots. Total cellular DNA was extracted from the aphyllous shoots and branches of Tecticornia indica for subsequent High Throughput Sequencing (HTS). Illumina HiSeq 2000 technology produced a total of 91,825,666 reads. Reads were trimmed and filtered to retain base calls corresponding to a 99.9% base call accuracy. A de novo assembly produced 413,729 contigs and 45,238 scaffolds with a maximum length of 120,548 nucleotides, an N50 of 881, and an average depth of coverage of 16.29 nucleotides. A subset of 2,196,299 reads were used for assembling the chloroplast genome at an average depth of coverage of 1,094 nucleotides. Sanger sequencing was performed to validate the LSC-IR-SSC junctions and to complete the missing regions of the chloroplast genome. The assembled chloroplast genome map and T. indica genomic sequence is expected to facilitate genetics and genomic research focused on understanding function of genes involved in halophytic and drought tolerant traits. SPECIFICATIONS TABLE O_TBL View this table: org.highwire.dtl.DTLVardef@c070bborg.highwire.dtl.DTLVardef@293fd8org.highwire.dtl.DTLVardef@1e9ab6org.highwire.dtl.DTLVardef@13f3126org.highwire.dtl.DTLVardef@1b3c4ca_HPS_FORMAT_FIGEXP M_TBL C_TBL VALUE OF THE DATAO_LIGenomic sequence information of Tecticornia indica and its chloroplast map will be useful for genomics research. C_LIO_LIGenomics and genetics scientists, bioinformatics and biotechnologists in the fields of photosynthesis, taxonomy and drought tolerance will benefit from these data. C_LIO_LIThese data can be used in developing molecular markers and quantitative trait loci in identification and selection of halophyte and drought tolerance, taxonomic resolution, and variables in photosynthetic genes. C_LI

genomics↗

bioRxiv2023

Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or to age-related disease triggers lipid accumulation within the RPE, on Bruchs membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (apoB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPE{Delta}Mttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor -associated cholesterol deposits and photoreceptor cell death, and loss of rod but not cone function. RPE-specific ablation of Mttp had no significant effect on plasma lipids and lipoproteins. While, apoB was decreased in the RPE, ocular retinoid concentrations remained unchanged. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but not directly involved in ocular retinoid and plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

cell biology↗

bioRxiv2023

Transcriptomic changes predict metabolic alterations in LC3 associated phagocytosis in aged mice

LC3b (Map1lc3b) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b, to pro-mote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis and neuroprotection. In a mouse model of retinal lipid steatosis - mice lacking LC3b (LC3b-/-), we observed increased lipid deposition, metabolic dysregulation and enhanced inflammation. Herein we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b-/- mice revealed 1533 DEGs, with ~73% upregulated and 27% down-regulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.

biochemistry↗

bioRxiv2022

Rescue of the increased susceptibility to Mild Chronic Oxidative Stress of iNeurons carrying the MAPT Chromosome 17q21.3 H1/H1 risk allele by FDA-approved compounds

The microtubule associated protein tau (MAPT) chromosome 17q21.31 locus lies within a region of high linkage disequilibrium (LD) conferring two extended haplotypes commonly referred to as H1 and H2. The major haplotype, H1 has been genetically associated with an increased risk for multiple neurodegenerative disorders, including Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), APOE {varepsilon}4-negative Alzheimers disease (AD) and Parkinsons disease (PD). The mechanism causing this increased risk is largely unknown. Here, we investigated the role of Mild Chronic Oxidative Stress (MCOS) in neurogenin 2 (NGN2) induced neurons (iNeurons) derived from iPS (induced pluripotent stem cells) from carriers of both haplotypes. We identified that iNeurons of the H1 homozygous haplotype showed an increased susceptibility to MCOS compared to homozygous H2 carriers, leading to cell death through ferroptosis. We performed a cellular screen in H1 iNeurons using a FDA-approved Drug Library and identified candidate molecules that rescued the increased susceptibility to MCOS and prevented ferroptosis in H1 iNeurons. HighlightsO_LIMild Chronic Oxidative Stress induces neurotoxicity via ferroptosis on iNGN2 neurons C_LIO_LIAxonal degeneration, disordered microtubules, blebs precede neurotoxicity C_LIO_LIMAPT-17q21.3 locus H1/H1, risk allele for NDD is more vulnerable to MCOS C_LIO_LIFDA-approved drugs reverse MCOS induced ferroptosis on H1/H1 risk allele C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/515284v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@eddb2eorg.highwire.dtl.DTLVardef@1d0c00dorg.highwire.dtl.DTLVardef@1f0b00eorg.highwire.dtl.DTLVardef@b68f1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrPC. Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.

genomics↗

bioRxiv2021

The landscape of PrPC biosynthesis revealed by an arrayed genome-wide interference screen

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

neuroscience↗