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Verma, R.

Publications and source records attributed to Verma, R..

At least 19 recordsLinked to original sources

PIE: Generalizing perturbation effects across unseen perturbations, contexts and datasets

Predicting cellular response to perturbations is key to understanding biological mechanisms and selecting therapeutic targets. However, generalizing across cellular contexts, perturbations, and experimental datasets remains challenging due to incomplete representations of the system being perturbed and the difficulty of measuring true perturbation effects within that system. We introduce PIE, a model that addresses these limitations by reformulating the learning task around population level perturbation effects and incorporating auxiliary inputs that better characterize the biological system. PIE directly predicts differentially expressed genes and changes in gene expression for each (context, perturbation) pair, using external biological knowledge, baseline gene expression, and observed perturbation responses. Its architecture accommodates input sources with variable feature sets and predicts effects on genes seen and unseen during training. On the Replogle Nadig dataset, PIE achieves state of the art performance on most metrics across all tested generalization settings, including the most challenging setting where both the context and perturbation are unseen during training. For predicting differentially expressed genes, PIE achieves 1.2 to 3.2 times the AUPRC of the strongest baseline in each setting: across unseen contexts, perturbations, experimental datasets, and jointly unseen contexts and perturbations. Overall, PIE provides a framework for generalizing perturbation effects across diverse experimental conditions.

bioinformatics↗

Robean: A Standalone Freeware for Automated Rodent Neurobehavioural Analysis with Integrated Tracking, Visualization, and Reporting

Quantitative analysis of rodent behaviour is fundamental to neuroscience, preclinical drug discovery and neurotoxicology research. Although several commercial and open-source software packages are available for behavioural assessment, some are expensive, some require programming expertise, and some provide limited flexibility for user-defined experimental configurations. To address these limitations, we developed Robean, a freely available standalone software platform for automated rodent neurobehavioural analysis from both live camera feeds and pre-recorded videos. Robean provides an intuitive graphical user interface that enables users to design experimental arenas, define custom analysis zones, perform spatial calibration, and automatically track rodent movement without requiring programming knowledge. The software currently supports automated analysis of three widely used behavioural paradigms: the Morris Water Maze, Elevated Plus Maze, and Open Field Test. Robean extracts behavioural metrics including escape latency, path efficiency, platform crossings, target quadrant preference, thigmotaxis, locomotor activity, zone occupancy, arm entries, and centre exploration specific to behavioural tests. In addition, the software generates trajectory maps, occupancy heatmaps, comma-separated value (CSV) datasets, comprehensive PDF reports, and batch study summaries for multiple experimental sessions. Developed using open-source software technologies and distributed as a standalone freeware application, Robean provides an accessible and reproducible solution for behavioural neuroscience laboratories. Its modular architecture facilitates future integration of additional behavioural paradigms and analytical modules, making it a flexible platform for automated rodent behavioural assessment.

animal behavior and cognition↗

PIGMENT: A deep learning framework for Porcine Immunohistochemistry seGMENTation

Traumatic brain injury produces widespread axonal damage can be assessed histologically using amyloid precursor protein (APP) immunohistochemistry, which labels injured axonal profiles at cellular resolution [1, 2]. However, quantification of APP pathology remains a major bottleneck: annotation is manual, time-consuming, spatially localized, and variable across raters, limiting scalability and reproducibility. This limitation is particularly important in studies that use histology as a reference for neuroimaging or other tissue-level measurements, where cellular APP pathology must be quantified in a spatial form that can be aligned with imaging abnormalities. Here, we introduce PIGMENT, an annotation-efficient deep-learning framework for automated segmentation and quantification of APP-positive pathology in porcine white matter histology. PIGMENT uses a compact SegFormer-B0 architecture trained on 525 expert-annotated 512 x 512-pixel tiles from four APP-stained sections across three pigs. Because APP-positive profiles are sparse, fragmented, stain-variable, and morphologically diverse, PIGMENT combines limited expert labels with APP-specific augmentation designed to model variation in APP-positive intensity, size, continuity, fragmentation, and local tissue context. We evaluated PIGMENT using an instance-level detection rate that measures whether discrete APP-positive components are localized. Across held-out APP-stained data, PIGMENT achieved a mean instance-level detection rate of 0.86. Across the configurations tested, the highest mean detection rate was achieved by a training set that included sections from different animals, suggesting that annotation diversity may be an important factor under limited-label conditions. By extending limited high-confidence expert annotations into whole-section APP burden maps, PIGMENT provides a scalable framework for characterizing the extent and spatial distribution of traumatic axonal injury. These maps may support future studies that align histological injury burden with imaging-derived measures.

neuroscience↗

ANCHOR : Atlas of Neurochemical Characterization of the Human Brainstem with 3D Reconstruction

The human brainstem is a complex division of the brain comprised of more than 200 nuclei and fiber tracts. The brainstem is essential for the functioning of the entire body. We introduce here the most detailed human brainstem Atlas across the human lifespan: fetus, child, adult. ANCHOR, the Atlas of Neurochemical Characterization of the Human Brainstem, is an online platform that includes more than 800 serial histological sections, stained for Nissl and seven immunochemical (IHC) markers, from the human brainstem of three ages: 25 fetal gestational weeks (GW), 9 years old, and 54 years old. This makes ANCHOR the most comprehensive human brainstem Atlas to date. In these three brainstems, we identified and manually annotated over 200 structures. We further characterized these structures with the seven IHC markers. We specifically describe the catecholaminergic groups in the human brainstem across all three age groups. In addition, we identified the protoplasmic commissural dendrites of the hypoglossal nucleus and we describe the pretectal nuclei in the Nissl-stained fetal 25 GW brainstem. ANCHOR includes an online viewer that integrates multimodal data, from magnetic resonance imaging and block face imaging to Nissl- and IHC-stained serial sections and 3D reconstruction of the entire brainstem. For the 9-year-old specimen, the online viewer allows simultaneous navigation of annotated sections with corresponding IHC, for viewing the specific region-wise cellular features accessible at https://anchor.humanbrain.in/.

neuroscience↗

Embryonic origin of cancer in newborn twins

Studying monozygotic twins who present with identical cancers informs on the developmental origins of childhood tumours. Here, we performed whole genome sequencing on multiple tumour, normal, and placental samples to reconstruct the phylogeny of a soft tissue cancer that spread in utero between monozygotic twins. This generalisable and scalable approach allows us to dissect the earliest stages of twinning, revealing unexpected asymmetrical contributions of embryonic lineages to the placenta and each twin.

cancer biology↗

Morphotype-Resolved 3D Morphometry Reveals a Structure-Density-Location Coupling in Mitochondrial Networks

Mitochondrial architecture plays a critical role in cellular function, yet how organelle structure, metabolic density, and subcellular position are jointly remodeled across whole cells remains poorly understood. We applied quantitative 3D soft X-ray tomography to analyze intact INS-1E cells in a native, cryo-hydrated state. By integrating morphometric profiling with voxel-level linear absorption coefficients (LAC) and contour-based radial mapping, we tracked the structural, biochemical, and spatial remodeling of fragmented, intermediate, and interconnected mitochondrial morphotypes under high glucose and Exendin-4 stimulation. High glucose induces morphotype-specific hypertrophy, fission, and perinuclear redistribution of low-density fragments. Co-stimulation with Exendin-4 stabilizes interconnected networks and increases metabolic density at the cell periphery. Morphotype-resolved analysis uncovers a structure-density-location coupling in which mitochondrial shape, macromolecular packing, and radial position shift in concert. These results offer a quantitative framework and high-resolution spatial constraints for whole-cell modeling of organelle dynamics. HighlightsO_LIMitochondrial morphotypes occupy distinct regions during metabolic stimulation C_LIO_LIHigh glucose drives early mitochondrial swelling, fission, and spatial dispersion C_LIO_LIExendin-4 prevents structural collapse and preserves dense interconnected networks C_LIO_LIMorphotype-resolved analysis uncovers a structure-density-location coupling in mitochondrial networks. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/712811v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d495ecorg.highwire.dtl.DTLVardef@1bee80dorg.highwire.dtl.DTLVardef@74df9dorg.highwire.dtl.DTLVardef@104db6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Integrated biophysical and spatial remodeling during insulin secretory granule maturation at the mitochondrial network

Effective insulin secretion and blood glucose homeostasis depend on the multistep maturation of insulin secretory granules (ISGs), a process that includes lumen acidification, enzymatic insulin processing, and biophysical remodeling of the granule. An under studied aspect of ISG maturation is the role of inter-organelle contacts in organelle remodeling. While a correlation between ISG-mitochondria contacts and ISG maturation has been observed, many questions remain on how this interaction may impact maturation (1-5). We sought to address this gap in knowledge by using multi-scale imaging approaches (fluorescent microscopy, soft X-ray tomography, and cryo-electron tomography) to examine how the biophysical properties and spatial organization of ISGs change around the mitochondrial network. Our data suggests that ISGs in proximity to mitochondria exhibit lower pH, higher biomolecular density, and smaller vesicle diameter. Time-resolved imaging using a SNAP tag labelling system also shows that as ISGs age, their proximity to the mitochondria network is increased between 3-6 hours after biosynthesis, suggesting that ISG-mitochondria association is dynamically spatiotemporally regulated in pancreatic {beta}-cells. These data suggest that mitochondrial proximity contributes to the maturation and remodeling of ISGs in pancreatic beta cells.

cell biology↗

Cellular mechanisms of acquired drug resistance against regorafenib in colon cancer cells.

Colorectal cancer is the third most common cancer across the world. Acquired resistance to therapeutics is one of the major challenges in cancer cure. With the development of resistance to organometallic drugs there was switch for the usage of inhibitors of kinases which play a crucial role in cellular activities. Regorafenib is a multi-kinase inhibitor used as an oral anti-cancer drug for treating advanced colorectal cancer (CRC). With increasing reports of acquired resistance to regorafenib in long-term use it is inevitable to understand the mechanisms underlying in the development of resistance. To understand the molecular mechanism of acquired drug resistance towards regorafenib we have developed a regorafenib resistant HCT116 cell line (Reg-R-HCT116) and an integrated quantitative proteomic and phospho-proteomics approach is used to elucidate the molecular signaling mechanisms that help in drug tolerance. Proteome and Phosphoproteome analysis revealed an extensive remodelling of signaling pathways associated with metabolism, protein synthesis and stress adaptations. This also revealed a large set of phosphorylated proteins as well as proteins that might be associated with aberrant activation or differential alteration of PI3K-AKT-mTOR, EIF2, HIF-1, Apoptosis inhibition, Glucose metabolism, amino acid metabolism and DNA-repair-associated signaling. Differential phosphorylation of downstream molecules of the mentioned pathways NDRG1, ACINUS and RICTOR and enhanced cell survival further confirmed their role in drug tolerance. Targeted inhibition of both the mTOR complexes using Torin1 resensitized the cells to regorafenib. Combination treatment of regorafenib with torin1 showed a synergistic cytotoxicity and attenuated the expression of key survival proteins. These findings provide mechanistic insight into acquired resistance to regorafenib in colorectal cancer and identified mTOR/eIF2 signaling as one of the critical drivers of resistance phenotype. The results suggest combinatorial targeting of these pathways could be an effective strategy to overcome regorafenib resistance and improve clinical outcomes in CRC.

cancer biology↗

Immune signatures of protection and progression in the airways of recent human tuberculosis household contacts

Whilst the majority of individuals infected with M. tuberculosis control the infection and remain asymptomatic, only 5-10% progress to active tuberculosis (TB)1. Previous or current infection with M. tuberculosis is detected using antigen-specific interferon (IFN)-{gamma} release assays (IGRA), which cannot identify those who will remain healthy or progress to active TB1. However, 18F-Fluorodeoxyglucose positron emission-computed tomography (PET-CT) can detect increased immune cell metabolic activity in lung parenchyma and intrathoracic lymph nodes associated with infection2. The local early immune factors dictating protection or disease progression have not been defined. To address this, we interrogated the airway immune response at single cell resolution in bronchoalveolar lavage (BAL) from extensively clinically characterised recent household contacts of TB patients, who either controlled the infection or progressed to TB disease. Using unbiased analysis of bulk and scRNA-seq of BAL samples, we define type I IFN-dependent and -independent neutrophil signatures in active TB patients and contacts that progressed to TB. We additionally report an inverse relationship between airway neutrophils and T cells, with T cells showing signatures of exhaustion, cytotoxicity and cell death in progressors and TB patients with a neutrophil dominated airway profile. Conversely, T cell signatures of protection in contacts who remained healthy were dominated by genes related to regulation, quiescence and a stem-like profile. We show that both the inflammatory neutrophil signature of TB progression and the stem-like T cell signature of non-progressors from human airways were recapitulated in scRNA-seq data from non-human primate (NHP) granulomas, associated with disease or immune protection, respectively. Our findings from early human airway responses in TB contacts reveal genes, pathways and cell states that may dictate infection outcome and inform strategies for host-directed therapy and vaccine studies.

immunology↗

Contrastive modelling of transcription and transcript abundance in legumes using PlanTT

Predicting the impacts of sequence variation on gene expression remains a challenging task. Further, in plants, we have a limited understanding of the relative contributions of different gene expression regulatory mechanisms. To address these limitations we generated a comparative multiomic dataset comprising matched 3-RNA-seq and PRO-seq data from matched tissues of reference genotypes of four legumes of the invert repeat lacking clade (Pisum sativum, Vicia faba, Lathyrus sativa and Medicago truncatula). Focused on the challenging task of predicting expression differences between ortholog pairs from unseen orthogroups, we used this dataset and a novel prediction framework to build contrastive models that predict quantitative differences (effect size differences) in transcription and transcript abundance.

bioinformatics↗

Classifying Biophysical Subpopulations of Insulin Secretory Granules using Quantitative Whole Cell Structure Analysis

Pancreatic {beta}-cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft x-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole {beta}-cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts towards mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary {beta}-cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

biophysics↗

Redefined Strategies to generate Conditional miR-141/200c miRNA cluster Knockout mice to eliminate confounding neo cassettes

MicroRNAs (miRNAs) of the miR-200 family specifically miR-141 and miR-200c regulate neurogenesis, differentiation, and epithelial-mesenchymal transitions in development. Dysregulation of these miRNAs is associated with several diseases including cancer and stroke. The Mirc13tm1Mtm/Mmjax mouse line, which targets the miR-141/200c cluster, was originally generated and described by Park et al. 2012 as a knockout-first, reporter-tagged insertion with conditional potential (conditional-ready) mouse line. Harnessing its full potential requires a two-step breeding process: breeding with FLP mice to excise the lacZ/neo cassette, then breeding with Cre to delete the floxed miRNA cluster (Park, et al. 2012). However, many studies either bypassed removal of the lacZ/Neo cassettes and treated the mouse line as Mirc13 knockouts or bred directly with Cre mouse lines, which could lead to unpredictable recombination and genotypes. Here we show that retention of the lacZ/Neo cassette is associated with reduced expression of the neighboring genes Ptpn6, Phb2 and Atn1 in the olfactory bulb, and that these genes are expressed normally once the cassette is excised. We therefore recommend a validated two-step FLPo-then-Cre breeding plan for this line, together with case-by-case allele validation for other knockout-first, reporter-tagged mouse lines.

neuroscience↗

Electron transport chain complex I and mitochondrial fusion regulate ROS for differentiation in Drosophila neural stem cells

Mitochondrial activity and dynamics play crucial roles in regulating neuronal differentiation. Neural stem cells (NSCs) or neuroblasts in Drosophila require the regulation of mitochondrial fusion, along with the activity of the electron transport chain (ETC), to meet the metabolic demands of differentiation. However, the mechanisms by which mitochondrial fusion and activity together regulate NSC differentiation are not well understood. We investigated the relationship between mitochondrial fusion, and ETC complex I activity during Drosophila neuroblast differentiation. We found that depletion of complex I subunits did not affect the number of type II neuroblasts but reduced their proliferation, thereby decreasing the numbers of mature intermediate precursor cells (mINPs), ganglion mother cells (GMCs), and neurons in each lineage. Complex I depletion decreased the mitochondrial membrane potential and cristae numbers, and increased mitochondrial fragmentation and ROS. Increased ROS resulting from the depletion of antioxidant enzymes also led to a decrease in mINPs, GMCs, and neurons in each type II neuroblast lineage. Both complex I and antioxidant protein depletion led to delayed G1/S transition and decreased nuclear cyclin E levels. Interestingly, the defects in proliferation, differentiation, and ROS in complex I and anti-oxidant protein depleted neuroblasts could be restored by fused mitochondrial morphology obtained through additional depletion of the fission protein Drp1. Further, overexpression of anti-oxidant proteins could alleviate the ROS and rescue the differentiation defect in complex I depleted type II neuroblasts. Together, this study reveals a role for complex I and mitochondrial fusion in restricting ROS for differentiation in Drosophila neuroblasts. Significance statementThe electron transport chain complex I regulates differentiation in the Drosophila type II neuroblast lineage. Complex I and anti-oxidant scavenger protein depletion lead to mitochondrial fragmentation and an increase in ROS, thereby affecting the G1/S phase transition and decreasing the formation of lineage cells. Mitochondrial fusion induced by depletion of mitochondrial fission protein Drp1 in complex I and anti-oxidant protein depleted type II neuroblasts leads to suppression of the differentiation defects. This study confirms a crucial role of mitochondrial fusion in restricting ROS for appropriate division and differentiation in the type II neuroblast lineage in Drosophila.

developmental biology↗

Secretory stimuli distinctly regulate insulin secretory granule maturation through structural remodeling

Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic {beta}-cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G-protein coupled receptor 40 (GPR40) promote ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell altering overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically.

biophysics↗

A three-dimensional histological cell atlas of the developing human brain

The human brain is believed to contain a full complement of neurons by the time of birth together with a substantial amount of the connectivity architecture, even though a significant amount of growth occurs postnatally. The developmental process leading to this outcome is not well understood in humans in comparison with model organisms. Previous magnetic resonance imaging (MRI) studies give three-dimensional coverage but not cellular resolution. In contrast, sparsely sampled histological or spatial omics analyses have provided cellular resolution but not dense whole brain coverage. To address the unmet need to provide a quantitative spatiotemporal map of developing human brain at cellular resolution, we leveraged tape-transfer assisted serial section histology to obtain contiguous histological series and unbiased imaging with dense coverage. Interleaved 20 thick Nissl and H&E series and MRI volumes are co-registered into multimodal reference volumes with 60 isotropic resolution, together with atlas annotations and a stereotactic coordinate system based on skull landmarks. The histological atlas volumes have significantly more contrast and texture than the MRI volumes. We computationally detect cells brain-wide to obtain quantitative characterization of the cytoarchitecture of the developing brain at 13-14 and 20-21 gestational weeks, providing the first comprehensive regional cell counts and characterizing the differential growth of the different brain compartments. Morphological characteristics permit segmentation of cell types from histology. We detected and quantified brain-wide distribution of mitotic figures representing dividing cells, providing an unprecedented spatiotemporal atlas of proliferative dynamics in the developing human brain. Further, we characterized the abundance and distribution of Cajal-Retzius cells, a transient cell population that plays essential roles in organizing glutamatergic cortical neurons into layers. Together, our study provides an unprecedented quantitative window into the developing human brain and the reference volumes and coordinate space should be useful for integrating spatial omics data sets with dense histological context.

neuroscience↗

The heart rate dynamics and antioxidant capacity under cognitive load in Obsessive-Compulsive disorder

The research of the oxidants-antioxidants homeostasis and its connection to autonomic nervous system (ANS) dysfunction in patients with obsessive-compulsive disorder (OCD) is a relatively uncharted field of study. We recruited 31 patients diagnosed with OCD and 29 healthy volunteers to analyze their saliva antioxidant capacity before and after a three-block behavioral task with cognitive and emotional load, during which we assessed heart rate (HR) and heart rate variability (HRV) patterns. We found that the OCD group had a significantly lower antioxidant capacity (AOC) and showed no increase in AOC after the cognitive task as compared with healthy volunteers. The notable negative correlation between HR and AOC changes led us to select two subgroups inside the OCD and control group: one with high HR and another with low HR. The AOC changes for OCD and healthy participants with high HR were similar, while OCD sub-group with low HR exhibited significantly different changes in AOC compared with the control group. Although HRV did not show a correlation with AOC, the OCD group demonstrated substantially higher increase of very low frequencies (VLF) of HRV during the cognitive task performance and significantly elevated low frequency powers of HRV expressed in normal units (LFnu). OCD sub-group with lower HR exhibited higher LFnu compared to both the patient and the control group with high HR. Our findings imply that OCD patients with elevated sympathetic tone of ANS reflected by initially increased HR show more adaptive AOC to cognitive load

neuroscience↗

Human iPSC-derived motor neuron innervation enhances the differentiation of muscle bundles engineered with benchtop fabrication techniques

Engineered skeletal muscle tissues are critical tools for disease modeling, drug screening, and regenerative medicine, but are limited by insufficient maturation. Because innervation is a critical regulator of skeletal muscle development and regeneration in vivo, motor neurons are hypothesized to improve the maturity of engineered skeletal muscle tissues. Although motor neurons have been added to pre-engineered muscle constructs, the impact of motor neurons added prior to the onset of muscle differentiation has not been evaluated. In this study, benchtop fabrication equipment was used to facilely fabricate chambers for engineering 3-dimensional (3-D) skeletal muscles bundles and measuring their contractile performance. Primary chick myoblasts were embedded in an extracellular matrix hydrogel solution and differentiated into engineered muscle bundles, with or without the addition of human induced pluripotent stem cell (hiPSC)-derived motor neurons. Muscle bundles differentiated with motor neurons had neurites distributed throughout their volume and a higher myogenic index compared to muscle bundles without motor neurons. Innervated muscle bundles also generated significantly higher twitch and tetanus forces in response to electrical field stimulation after one and two weeks of differentiation compared to non-innervated muscle bundles cultured with or without neurotrophic factors. Non-innervated muscle bundles also experienced a decline in rise and fall times as the culture progressed, whereas innervated muscle bundles and non-innervated muscle bundles with neurotrophic factors maintained more consistent rise and fall times. Innervated muscle bundles also expressed the highest levels of the genes for slow myosin light chain 3 (MYL3) and myoglobin (MB), which are associated with slow twitch fibers. These data suggest that motor neuron innervation enhances the structural and functional development of engineered skeletal muscle constructs and maintains them in a more oxidative phenotype.

bioengineering↗

Axonal transcriptome reveals upregulation of PLK1 as a protective mechanism in response to increased DNA damage in FUSP525L spinal motor neurons

Mutations in the gene FUSED IN SARCOMA (FUS) are among the most frequently occurring genetic forms of amyotrophic lateral sclerosis (ALS). Early pathogenesis of FUS-ALS involves impaired DNA damage response and axonal degeneration. However, it is still poorly understood how these gene mutations lead to selective spinal motor neuron (MN) degeneration and how nuclear and axonal phenotypes are linked. To specifically address this, we applied a compartment specific RNA-sequencing approach using microfluidic chambers to generate axonal as well as somatodendritic compartment-specific profiles from isogenic induced pluripotent stem cells (iPSCs)-derived MNs. We demonstrate high purity of axonal and soma fractions and show that the axonal transcriptome is unique and distinct from that of somas including significantly fewer number of transcripts. Functional enrichment analysis revealed that differentially expressed genes (DEGs) in axons were mainly enriched in key pathways like RNA metabolism and DNA damage, complementing our knowledge of early phenotypes in ALS pathogenesis and known functions of FUS. In addition, we demonstrate a strong enrichment for cell cycle associated genes including significant upregulation of polo-like kinase 1 (PLK1) in FUSP525L mutant MNs. PLK1 was increased upon DNA damage induction and PLK1 inhibition further increased the number of DNA damage foci in etoposide-treated cells, an effect that was diminished in case of FUS mutant MNs. In contrast, inhibition of PLK1 increased late apoptotic or necrosis-induced neuronal cell death in mutant neurons. Taken together, our findings provide insights into compartment-specific transcriptomics in human FUS-ALS MNs and we propose that specific upregulation of PLK1 might represent an early event in the pathogenesis of ALS, possibly modulating DNA damage response and other associated pathways.

neuroscience↗