Biology subjects

Namboori, S. C.

Publications and source records attributed to Namboori, S. C..

6 recordsLinked to original sources

bioRxiv2026

Single-cell transcriptomics uncovers chromatin dysfunction in a human TDP-43 proteinopathy model of Amyotrophic Lateral Sclerosis.

TDP-43 proteinopathy, characterised by nuclear depletion and cytoplasmic aggregation of TDP-43, is the defining pathological hallmark of amyotrophic lateral sclerosis (ALS) and a shared pathology across frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), limbic-predominant age-related TDP-43 encephalopathy (LATE), and a substantial subset of Alzheimers disease. We recently developed a human model of TDP-43 proteinopathy that enables inducible mislocalisation of endogenous TDP-43 in iPSC-derived neurons without chemical stress or mutant protein overexpression. Using single-cell RNA sequencing of this model, we dissected the transcriptomic consequences of TDP-43 nuclear depletion across motor neurons as well as V1 and V2 interneurons at single-cell resolution. This approach uncovered disruption of ATP-dependent chromatin remodelling as a convergent downstream pathway across all three spinal neuron subtypes. Master regulator analysis identified ACTL6B, the neuron-specific subunit of the nBAF (neuronal BRG1/BRM-associated factor) chromatin remodelling complex, as the most consistently inhibited transcription factor following TDP-43 mislocalisation. ACTL6B downregulation emerges early in the mislocalisation cascade and is confirmed in post-mortem ALS spinal cord. ACTL6B knockdown in post-mitotic motor neurons phenocopies both the morphological and transcriptional consequences of TDP-43 pathology. Together, these findings establish nBAF complex dysfunction as a principal, spinal cord-enriched driver of TDP-43-associated neurodegeneration and reveal chromatin remodelling defects as a key mechanism in ALS.

neuroscience↗

bioRxiv2023

Rapid and Inducible Mislocalization of Endogenous TDP43 in a Novel Human Model of Amyotrophic Lateral Sclerosis

Transactive response DNA binding protein 43 kDa (TDP43) proteinopathy, characterized by the mislocalization and aggregation of TDP43, is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). In this study, we describe the development of a new model of TDP43 proteinopathy using human induced pluripotent stem cell (iPSC)-derived neurons. Utilizing a genome engineering approach, we induced the mislocalization of endogenous TDP43 from the nucleus to the cytoplasm without mutating the TDP43 gene or using chemical stressors. Our model successfully recapitulates key early and late pathological features of TDP43 proteinopathy, including neuronal loss, reduced neurite complexity, and cytoplasmic accumulation and aggregation of TDP43. Concurrently, the loss of nuclear TDP43 leads to splicing defects, while its cytoplasmic gain adversely affects microRNA expression. Strikingly, our observations suggest that TDP43 is capable of sustaining its own mislocalization, thereby perpetuating and further aggravating the proteinopathy. This innovative model provides a valuable tool for the in-depth investigation of the consequences of TDP43 proteinopathy. It offers a clinically relevant platform that will accelerate identification of potential therapeutic targets for the treatment of TDP43-associated neurodegenerative diseases including sporadic ALS.

neuroscience↗

bioRxiv2023

Exonuclease assisted mapping of protein-RNA interactions (ePRINT)

RNA processing is a fundamental mode of gene regulation that is perturbed in a variety of diseases including cancer and neurodegenerative disorders. RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding RNA molecules. Current methods to map these interactions, such as CLIP, rely on purifying single proteins at a time. We have developed a new method (ePRINT) to map RBP-RNA interaction networks on a global scale without purifying individual RBPs. ePRINT allows precise mapping of the 5 end of the RBP binding site, and can uncover direct and indirect targets of an RBP of interest. Importantly, ePRINT can also uncover RBPs that are differentially activated between cell fate transitions, for instance, as neural progenitors differentiate into neurons. Given its versatility, ePRINT has vast application potential as an investigative tool for RNA regulation in development, health and disease.

genomics↗

bioRxiv2023

Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons.

SummaryNeuronal circuits evolve as a precisely patterned network. In this context, a growing neuron must locate the appropriate target area on a neurite of a neighbouring cell with which to connect. Controlled target selection involves dendritic filopodial contacts and requires the exact apposition of synaptic components. Calcium signalling has been postulated to trigger the transformation from dendritic filopodia into functional synapses. However, calcium is a rather unspecific signalling system, and it needs to be clarified how the exact development of synaptic connections is controlled. Similarly, Wnt/{beta}-catenin signalling promotes synapse formation; however, how secreted Wnts induce and maintain synapses on neuronal dendrites is not well understood. Here, we show that Wnt-7a is tethered to the tips of dynamic dendritic filopodia during spine formation in human cortical neurons. These filopodia can activate Wnt signalling precisely at the contact sites on the dendrites of an adjacent neuron. Subsequently, local calcium transients can be observed at these Wnt-positive contact sites. Depleting either the filopodial-loaded Wnt or the extracellular calcium pool blocks the clustering of pre- and post-synaptic markers, hence the establishment of stable connections. Therefore, we postulate that local Wnt-7a signalling from the tip of the dendritic filopodia, verified by simultaneous calcium signalling, provides an elegant mechanism for orchestrating focal synapse maturation.

neuroscience↗

bioRxiv2020

Novel epigenetic clock for fetal brain development predicts fetal epigenetic age for iPSCs and iPSC-derived neurons.

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and iPSC-derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.

neuroscience↗

bioRxiv2019

Single cell transcriptomics identifies master regulators of dysfunctional pathways in SOD1 ALS motor neurons

BackgroundBulk RNA-Seq has been extensively utilized to investigate the molecular changes accompanying motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). However, due to the heterogeneity and degenerating phenotype of the neurons, it has proved difficult to assign specific changes to neuronal subtypes and identify which factors drive these changes. Consequently, we have utilized single cell transcriptomics of degenerating motor neurons derived from ALS patients to uncover key transcriptional drivers of dysfunctional pathways. ResultsSingle cell analysis of spinal neuronal cultures derived from SOD1 E100G ALS and isogenic iPSCs allowed us to classify cells into neural subtypes including motor neurons and interneurons. Differential expression analysis between disease and control motor neurons revealed downregulation of genes involved in synaptic structure, neuronal cytoskeleton, mitochondrial function and autophagy. Interestingly, interneurons did not show similar suppression of these homeostatic functions. Single cell expression data enabled us to derive a context-specific transcriptional network relevant to ALS neurons. Master regulator analysis based on this network identified core transcriptional factors driving the ALS MN gene dysregulation. Specifically, we identified activation of SMAD2, a downstream mediator of the TGF-{beta} signalling pathway as a potential driving factor of ALS motor neuron degeneration. Our phenotypic analysis further confirmed that an activated TGF-{beta} signalling is major driver of motor neuron loss in SOD1 ALS. Importantly, expression analysis of TGF{beta} target genes and computational analysis of publicly available datasets indicates that activation of TGF{beta} signalling may be a common mechanism shared between SOD1, FUS and sporadic ALS. ConclusionsOur results demonstrate the utility of single cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We find that ALS-associated mutant SOD1 targets transcriptional networks that perturb motor neuron homeostasis.

genomics↗