Biology subjects

Hegde, M.

Publications and source records attributed to Hegde, M..

14 recordsLinked to original sources

bioRxiv2026

A touch that lingers: synchronized patting strengthens neural tracking of auditory rhythm in newborns

Rhythmic auditory stimulation in early life rarely occurs in isolation. Caregivers naturally combine singing and speech with rhythmic touch, rocking, and visual cues. Newborns are sensitive to auditory rhythm from birth, yet little is known about how temporally aligned information across sensory modalities shapes the neural encoding of auditory rhythmic structure. Here, we tested whether rhythmic touch can alter how the newborn brain tracks an ongoing auditory signal beyond the period of tactile stimulation. We recorded high-density electroencephalography from full-term newborns (N = 40), 1-3 days after birth, while they were exposed to recorded French lullabies presented either alone or accompanied by gentle patting synchronized with the metrically strong beat. Patting was delivered intermittently, allowing us to compare neural responses to lullabies across conditions, notably during periods without concurrent tactile stimulation. We quantified neural tracking of the lullabies using brain-stimulus coherence and temporal response function modeling. Newborns' neural activity synchronized with the rhythmic structure of the lullabies at frequencies corresponding to both the beat and the metrically accented strong beat. Critically, neural tracking was stronger when the lullabies had been accompanied by synchronized touch than when they were presented alone, and this enhancement persisted during subsequent periods in which no touch was delivered. Although rhythmic touch enhanced tracking in newborns with both weaker and stronger auditory-only tracking, there was a larger gain in those who initially showed weaker auditory tracking. Together, our findings reveal that the newborn brain exploits rhythmic touch to strengthen its encoding of auditory rhythm, and that this benefit outlasts the touch itself. This provides neural evidence that patting an infant in time with a song, a caregiving practice shared across cultures, does more than simply provide a tactile stimulation. It shapes how the newborn brain encodes the song's rhythm.

neuroscience↗

bioRxiv2024

Fructose-2,6-bisphosphate restores TDP-43 pathology-driven genome repair deficiency in motor neuron diseases

TAR DNA-binding protein 43 (TDP-43) proteinopathy plays a critical role in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We recently reported that TDP-43 plays an essential role in DNA double-strand break (DSB) repair via the non-homologous end-joining (NHEJ) pathway. Here, we provide evidence that the brain of patients with ALS exhibit persistent DNA damage in the transcribed regions of the genome. While investigating the mechanistic basis, we found that the activity of polynucleotide kinase 3-phosphatase (PNKP) was severely impaired in the nuclear extracts of patient brains and TDP-43-depleted cells. PNKP is a key player in DSB repair within the transcribed genome, where its 3-phosphate termini processing activity is crucial for preventing persistent DNA strand breaks and neuronal death. The inactivation of PNKP was due to the reduced level of its interacting partner, phosphofructo-2-kinase fructose 2,6 bisphosphatase (PFKFB3), and its biosynthetic product, fructose-2,6-bisphosphate (F2,6BP), an allosteric modulator of glycolysis. Recently, we have demonstrated that F2,6BP acts as a positive modulator of PNKP activity in vivo. Furthermore, F2,6BP supplementation in cultured ALS patient-derived neural progenitor stem cells (NPSCs) reduced the toxic aggregation of polyubiquitinated TDP-43 and cytosolic pTDP-43 (S409/410). Notably, F2,6BP supplementation restored the PNKP activity in the nuclear extracts from autopsied ALS/FTD brain tissues and patient iPSC-derived NPSCs harboring TDP-43 mutations. Importantly, F2,6BP administration significantly restored the genome integrity and motor phenotypes in a Drosophila model of ALS-TDP-43. Collectively, these findings underscore the therapeutic potential of F2,6BP in TDP-43 pathology-associated motor neuron diseases.

neuroscience↗

bioRxiv2024

F2,6BP restores mitochondrial genome integrity in Huntingtons Disease

Several reports have indicated that impaired mitochondrial function contributes to the development and progression of Huntingtons disease (HD). Mitochondrial genome damage, particularly DNA strand breaks, is a potential cause for its compromised functionality. Here we show that the activity of polynucleotide kinase 3-phosphatase (PNKP), a critical DNA end-processing enzyme, is significantly decreased in the mitochondrial extract of HD patients brains due to a lower level of fructose-2,6 bisphosphate (F2,6BP), a biosynthetic product of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Such decrease in PNKP activity leads to persistent DNA strand breaks that are refractory to subsequent steps for repair completion. Both PFKFB3 and F2,6BP, an allosteric modulator of glycolysis, are also present in the mitochondria and PFKFB3 is part of a mitochondrial DNA repair complex containing HTT, PNKP, DNA Pol {gamma} (POLG) and Lig III. Notably, PNKP binds F2,6BP (Kd= 525{+/-}25 nM) and utilizes it as a cofactor. The levels of both F2,6BP and PFKFB3 are significantly decreased in the mitochondrial extract of HD mouse striatal neuronal cells and patients brain. Activity of PNKP is thus severely decreased in the mitochondrial extract; however, addition of F2,6BP restored its activity. Moreover, supplementation of F2,6BP in HD cells restored PFKFB3 level, mitochondrial genome integrity and partially restored mitochondrial membrane potential, mitochondrial respiration and prevented pathogenic aggregate formation. We also observed that supplementation with F2,6BP restored mitochondrial genome integrity in an HD Drosophila model. Our findings, therefore, suggest that F2,6BP-mediated restoration of PNKP activity could have a profound impact in ameliorating neurodegenerative symptoms in HD. SignificanceWe reported earlier the loss of PNKP activity in the nuclear extracts from HD patients brain. However, a glycolytic metabolite, F2,6BP, can restore PNKP activity and rescue organismal phenotypes in HD fly models. As PNKP is present in mitochondria and several reports indicate that mitochondrial dysfunction contributes to HD, we therefore analyzed PNKP activity in the mitochondrial extract. Surprisingly, we found that PFKFB3 and its product, F2,6BP are present in mitochondria, but significantly low in patients brains. Exogenous addition of F2,6BP restored PNKP activity in patients brain mitochondrial extract. Moreover, supplementing F2,6BP in HD cells and fruit flies restored mitochondrial genome integrity suggesting maintaining adequate intracellular F2,6BP levels is critical for proper functionality of PNKP and thereby of brain health.

neuroscience↗

bioRxiv2024

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

biochemistry↗

bioRxiv2023

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

genomics↗

bioRxiv2022

Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening

CRISPR technology is a powerful tool for studying genome function. To aid in picking sgRNAs that have maximal efficacy against a target of interest from many possible options, several groups have developed models that predict sgRNA on-target activity. Although multiple tracrRNA variants are commonly used for screening, no existing models account for this feature when nominating sgRNAs. Here we develop an on-target model, Rule Set 3, that makes optimal predictions for multiple tracrRNA variants. We validate Rule Set 3 on a new dataset of sgRNAs tiling essential and non-essential genes, demonstrating substantial improvement over prior prediction models. By analyzing the differences in sgRNA activity between tracrRNA variants, we show that Pol III transcription termination is a strong determinant of sgRNA activity. We expect these results to improve the performance of CRISPR screening and inform future research on tracrRNA engineering and sgRNA modeling.

bioengineering↗

bioRxiv2022

Phenotypic Characterization of Liver Sinusoidal Endothelial Cells on the Human Liver-Chip for Potential in vitro Therapeutic Antibody Pharmacology Applications

Liver plays a vital role in the human immune system, in the internalization and catabolic clearance of therapeutic antibodies and antibody-bound immune complexes via Fc-receptor (FcR) binding on the hepatic reticuloendothelial system cells. This Fc portion of the antibody binding to FcR in the liver initiates the clearance of these antibodies or immune complexes, which is vital in the context of half-life, dosing interval, efficacy, and safety of therapeutic antibodies. The liver sinusoidal endothelial cells (LSECs) express scavenging receptors that recognize, bind, and internalize an enormous diversity of extracellular ligands. The Fc gamma receptor Fc{gamma}RIIB or CD32B on LSECs is responsible for the clearance of a large majority of IgG-bound immune complexes in the liver. Investigating the pharmacological effects of antibody clearance via human liver in vitro has been challenging due to the lack of reliable long-term LSEC culture protocols. Human LSECs downregulate the expression of CD32B rapidly in vitro in traditional 2D LSEC mono- and co-cultures. We describe a Liver-Chip model with a co-culture of primary human LSECs and hepatocytes to recreate the liver microenvironment and extend the viability and function of LSECs, including CD32B expression levels, for a duration that is relevant for assessing the pharmacokinetics (PK) of therapeutic antibodies. Our results show that the expression of CD32B can differ based on experimental variables such as the source of primary cells (donor), passage number or source of detection antibodies used to visualize CD32B and shear stress. The CD32B expression was maintained for 14 days on the Liver-Chip in a donor-dependent but passage number independent manner. The Scanning Electron Microscopy (SEM) imaging showed the presence of fenestrae structures - one of the hallmarks of LSEC function. Key LSEC markers, including CD32B expression, were validated through flow cytometry.

bioengineering↗

bioRxiv2021

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we benchmark PAM preferences, on-target activity, and off-target susceptibility of 11 variants of SpCas9 in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A>G and C>T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new insights into these clinically-relevant genes. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

genomics↗

bioRxiv2021

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

microbiology↗

bioRxiv2020

Massively parallel assessment of human variants with base editor screens

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens and identify known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we conduct screens with BH3 mimetics and PARP inhibitors and identify point mutations that confer drug sensitivity or resistance. Finally, we create a library of 52,034 clinically-observed variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.

genetics↗

bioRxiv2019

Optimization of AsCas12a for combinatorial genetic screens in human cells

Cas12a enzymes have attractive properties for scalable delivery of multiplexed perturbations, yet widespread usage has lagged behind Cas9-based strategies. Here we describe the optimization of Cas12a from Acidaminococcus (AsCas12a) for use in pooled genetic screens in human cells. By assaying the activity of thousands of guides, we confirm on-target design rules and extend them to an enhanced activity variant, enAsCas12a. We also develop the first comprehensive set of off-target rules for Cas12a, and demonstrate that we can predict and exclude promiscuous guides. Finally, to enable efficient higher-order multiplexing via lentiviral delivery, we screen thousands of direct repeat variants and identify 38 that outperform the wildtype sequence. We validate this optimized AsCas12a toolkit by targeting 12 synthetic lethal gene pairs with up to 400 guide pairs each, and demonstrate effective triple knockout via flow cytometry. These results establish AsCas12a as a robust system for combinatorial applications of CRISPR technology.

genetics↗

bioRxiv2019

Genetic screens in isogenic mammalian cell lines without single cell cloning

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.

genetics↗

bioRxiv2018

Up, down, and out: next generation libraries for genome-wide CRISPRa, CRISPRi, and CRISPR-Cas9 knockout genetic screens

Advances in CRISPR-Cas9 technology have enabled the flexible modulation of gene expression at large scale. In particular, the creation of genome-wide libraries for CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) has allowed gene function to be systematically interrogated. Here, we evaluate numerous CRISPRko libraries and show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we developed genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese). Negative selection screens showed that Dolcetto substantially outperforms existing CRISPRi libraries with fewer sgRNAs per gene and achieves comparable performance to CRISPRko in the detection of gold-standard essential genes. We also conducted positive selection CRISPRa screens and show that Calabrese outperforms the SAM library approach at detecting vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.tracr

genomics↗

bioRxiv2018

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.

molecular biology↗