Biology subjects

Nair, R. R.

Publications and source records attributed to Nair, R. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional Mammary Gland Organoids From Human Induced Pluripotent Stem Cells

The human mammary gland arises through sequential lineage restrictions from surface ectoderm to hormone-responsive bilayered epithelium, yet a tractable in vitro model that captures these events has remained elusive. Here, we established a stepwise differentiation protocol that directs human induced pluripotent stem cells (hiPSCs) into mammary organoids (MGOs) by sequential induction of non-neural ectoderm, mammary placodes, and lineage maturation in a defined medium. The resulting MGOs reproducibly formed polarized basal (KRT14+, TP63+) and luminal (KRT8+, GATA3+) layers, with ~25% of luminal cells expressing oestrogen receptor (ER). MGOs accumulated lipids, expressed {beta}-casein upon prolactin stimulation, and retained lineage fidelity after serial passaging. Upon transplantation into cleared mouse mammary fat pads, MGOs generated branched mammary ductal networks. Single-cell transcriptomics confirmed the emergence of basal, luminal hormone-sensing, luminal secretory, and stromal populations, transcriptionally aligned with adult human breast epithelium and responsive to endocrine disruption. These findings demonstrate that hiPSCs can be reprogrammed into functional mammary epithelium, providing a platform to dissect development, endocrine signalling, and breast cancer pathogenesis.

cell biology↗

bioRxiv2026

Rpl12 paralog dependent TOR-signaling controls the expression of ribosome preservation factor Stm1

Ribosomes are increasingly recognized as heterogeneous regulators of gene expression, yet how ribosomal protein paralogs interface with nutrient signaling remains poorly understood. In Saccharomyces cerevisiae, ribosomal protein gene expression is governed by duplicated gene pairs, many of which exhibit functional divergence despite high sequence identity. A central regulator of ribosome biogenesis and translational control is the Target of Rapamycin (TOR) pathway, which integrates nutrient signals to modulate growth, stress adaptation, and lifespan. Target of Rapamycin Complex 1 (TORC1) influences ribosome activity by phosphorylating ribosomal protein S6 (Rps6), a modification that links nutrient availability to translational output. Here, we investigated the functional divergence of the Rpl12 ribosomal stalk protein paralogs RPL12a and RPL12b and found that rpl12b{Delta} produces phenotypes consistent with reduced TOR activity, including decreased Rps6 phosphorylation, G2/M cell-cycle accumulation, and significant extension of chronological lifespan as compared to rpl12a{Delta} and wildtype strain. Multi-omics analyses further indicate translational and metabolic reprogramming consistent with activation of a stress-adaptive program associated with Gcn4. Importantly, loss of RPL12b also reduces levels of the ribosome preservation factor Stm1, a TORC1-regulated protein required for stabilization of 80S ribosomes under stress. This finding links ribosomal stalk composition to ribosome stability and nutrient-responsive signaling. Together, our results demonstrate that ribosomal paralog specialization provides an additional regulatory layer connecting translation, TOR signaling, and cellular longevity.

cell biology↗

bioRxiv2025

CRISPR/Cas9-mediated generation of a de novo C9ORF72 knock-in isogenic iPSC cell bank to model ALS and FTD

BackgroundThe pathogenic G4C2 repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies focused on delineating the underlying perturbed mechanisms resulting from this genetic mutation are often confounded by the heterogeneity present in current disease models, such as patient-derived iPSC lines, with estimations of up to 50% of the variation in iPSC cell phenotypes resulting from inter-individual differences. Isogenic models, in which the pathogenic mutation is introduced into a defined genetic background, offer a powerful approach to isolating mutation-specific effects and enable high-resolution comparison across distinct ALS/FTD-associated mutations. Such models are essential for uncovering convergent disease mechanisms and improving reproducibility in ALS/FTD research. MethodsA two-step scarless CRISPR/Cas9 genome editing strategy was used to generate isogenic human iPSC lines carrying a de novo knock-in of a disease-length G4C2 repeat expansion in the C9ORF72 locus. The resulting lines underwent thorough quality control and were differentiated into lower motor neurons and assessed for the presence of key ALS/FTD pathologies, including changes to C9ORF72 mRNA and protein expression, RNA foci and dipeptide repeat proteins. ResultsTwo C9ORF72 knock-in iPSC lines were generated with 631 and 600 G4C2 repeats, alongside an isogenic genome editing control line. The C9ORF72 G4C2 repeat expansion knock-in iPSC lines exhibit both loss-of-function and gain-of-function pathological features characteristic of ALS/FTD. Compared to the parental wild-type KOLF2.1J line and isogenic (wild-type) CRISPR control line, these exhibit a significant reduction in C9ORF72 mRNA and protein levels, the presence of RNA foci accumulation, and a marked increase in poly(GA) and poly(GP) dipeptide repeat protein levels in iPSCs and motor neurons. ConclusionsThis is one of the first reports of a successful knock-in of the pathogenic C9ORF72 G4C2 repeat expansion into a human iPSC line, establishing a genetically defined and physiologically relevant model of ALS/FTD. These isogenic lines recapitulate both key loss- and gain-of-function disease pathologies, providing a crucial complement to existing patient-derived iPSC banks. By eliminating confounding genetic background variability, these cell lines will enable more precise interrogation of C9ORF72-linked pathomechanisms and offer a robust platform for comparative studies across the ALS and FTD spectrum, mechanistic investigations, and future therapeutic targeting with enhanced translational relevance.

neuroscience↗

bioRxiv2025

Accurately modelling RNase H-mediated antisense oligonucleotide efficacy

Antisense oligonucleotides (ASOs) are a powerful class of drugs with the potential to treat a wide range of human diseases. However, the prediction of ASO efficacy remains challenging, as large-scale and costly experimental screens are typically required to identify optimal candidates for a specific therapeutic target. To address this challenge, we compiled ASO Atlas, a database comprising 188,521 RNase H-mediated ASO sequences targeting 334 unique genes with corresponding knockdown efficacy measurements extracted from published patents. Using ASO Atlas, we trained OligoAI, a deep learning model capable of jointly modelling RNA target context, ASO sequence, sugar and backbone chemistries, and dosage to predict in vitro efficacy. We experimentally validated OligoAI by targeting KCNT2, achieving a 5.72-fold reduction in screening effort compared to random selection. ASO Atlas provides the first systematic resource to rigorously evaluate hypotheses regarding key parameters in ASO design, including sequence composition, chemical modifications, and target region selection. Both ASO Atlas and OligoAI have been made freely accessible through an online web-tool with the aim of facilitating the accelerated optimisation of ASO design.

bioinformatics↗

bioRxiv2025

The Mycobacterium tuberculosis ESX-5 secretion system enables carbon source utilization and growth in mice

Mycobacterium tuberculosis uses several ESX type VII protein secretion systems for pathogenesis. M. tuberculosis ESX-5 is poorly characterized because it is essential for growth in standard lab culture conditions. To circumvent ESX-5 essentiality, we made an M. tuberculosis strain in which the central ESX-5 membrane component EccD5 can be conditionally depleted. Here, we use this strain to demonstrate that M. tuberculosis requires the ESX-5 secretion system to grow using specific carbon sources in vitro, to grow in cultured macrophages, and to replicate and disseminate in aerosol-infected mice. M. tuberculosis requires ESX-5 to use glycerol or glucose as the sole carbon source. Use of glycerol and glucose also depends on the outer membrane protein PPE51. We show that M. tuberculosis requires ESX-5 activity for outer membrane export and surface exposure of PPE51. Expression of the outer membrane porin MspA enabled growth of ESX-5 deficient M. tuberculosis on glycerol, suggesting that the main function of ESX-5 in vitro is to export nutrient transporters to the outer membrane. Importantly, depletion of EccD5 in acutely infected mice resulted in clearance of M. tuberculosis from lung tissues, demonstrating the critical importance of ESX-5 activity during infection. Our findings suggest that ESX-5 promotes M. tuberculosis pathogenesis by mediating export of outer membrane proteins that enable nutrient acquisition. ImportanceMycobacterium tuberculosis ESX type VII secretion systems play important roles in pathogenesis, but the functions of ESX-5 are not well characterized because it is essential for growth in standard lab culture conditions. We used a strain that conditionally expresses a central membrane component of the ESX-5 secretion apparatus to determine how ESX-5 impacts growth in lab cultures and in a mouse infection model. We found that M. tuberculosis requires ESX-5 to grow using several carbon sources and to grow in the lungs of infected mice. Inhibiting production of the ESX-5 secretion system in mice also led to clearance of M. tuberculosis from lung tissues. Our results demonstrate that the M. tuberculosis ESX-5 system is a critical virulence factor and suggest that ESX-5 is a strong candidate for anti-tubercular drug development.

microbiology↗

bioRxiv2025

Generation of C9orf72h370 mice, an intron 1 humanised C9orf72 repeat-expansion knock-in model

An autosomal dominant GGGGCC repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we set out to engineer a gene targeted mouse model harbouring a pathogenic length humanised C9orf72 repeat expansion allele, in order to model pathological mechanisms in a physiological context. In human disease, pathogenic repeats typically range from the hundreds to thousands of units in length, representing a considerable challenge for cellular and in vivo model generation given the instability of GC rich and repetitive DNA sequences during molecular cloning. To overcome this challenge, we developed new methodology to synthetically and iteratively build pure GGGGCC repeats within a linear vector system, which we then seamlessly and scarlessly embedded within the native human genomic sequence. This created a gene targeting DNA vector for homologous recombination of the human sequence in mouse embryonic stem cells. We used this novel targeting vector to generate a new gene targeted mouse allele, C9orf72h370, that for the first time has mouse C9orf72 intron 1 scarlessly replaced with human intron 1 including a pure (GGGGCC)370 hexanucleotide repeat expansion. We confirm that the mouse model expresses human intron 1-derived RNA and produces dipeptide repeat proteins derived from the GGGGCC repeat expansion. We now provide this model as a new freely available resource for the field. In addition, we demonstrate the utility of our cloning method for engineering diverse repeat expansion sequences for modelling other disorders, such as Fragile X Syndrome.

molecular biology↗

bioRxiv2024

Glioblastoma Treatment by Systemic Actinium-225 alpha-particle Dendrimer-radioconjugates is Improved by Chemotherapy

RATIONALEThe poor prognosis of glioblastoma is largely due to drug resistance and tumor location that, together, make it difficult to treat aggressively without affecting the rest of the brain. METHODOLOGYHigh-energy, short-range (40-80{micro}m) dendrimer-delivered -particles could address both challenges, because (1) they cause complex, highly cytotoxic double-strand DNA breaks, and (2) irradiation of the neighboring brain is minimal, since dendrimers selectively delivers them to tumors. Since cancer cells that are not directly hit by -particles will likely not be killed, the patterns of tumor irradiation affect efficacy. Systemically injected dendrimers extensively accumulate in glioblastomas, where they are taken up by tumor associated macrophages (TAMs), which tend to infiltrate tumors. We hypothesized that dendrimers labeled with -particle emitters, when being carried by TAMs, could more evenly irradiate glioblastomas, improving survival. In this study, the efficacy of dendrimers radiolabeled with the -particle emitter actinium-225 (dendrimer-radioconjugates) was evaluated when administered alone and/or after temozolomide, in a syngeneic immune-competent orthotopic GL261-C57BL/6 mouse model. RESULTSSystemically-administered dendrimer-radioconjugates, at activities that did not result in long-term toxicities, prolonged survival of mice with orthotopic GL261 tumors, compared to standard-of-care temozolomide (39 vs 31 days mean survival, p=0.0061) and non-treated animals (30 days, p=0.0009). Importantly, injection of temozolomide 24 hours before administration of dendrimer-radioconjugates further improved survival remarkably (44 days). This improvement in efficacy was attributed to: (1) the significant increase (by 33%) in tumor absorbed doses delivered by dendrimer-radioconjugates when injected after chemotherapy, without altering normal organ dosimetry, while sparing the tumor-surrounding healthy brain; (2) the potentially deeper tumor penetration of dendrimer-radioconjugates, suggested by the enhancement of dendrimer penetration within GL261-spheroids, employed as model tumor-avascular regions and/or TAM-free regions; and/or (3) the formation of a more lethal cocktail when both modalities acted on same cancer cells, that was correlated with increased levels of dendrimer-radioconjugates associating with GL261 cells in vitro and with greater incidences of karyomegaly in vivo. CONCLUSIONSThis study demonstrates the potential of a brain tumor targeted systemic actinium-225 radiopharmaceutical therapy that inhibits growth of glioblastoma cells and prolongs survival of mice with orthotopic brain tumors, further improved by standard-of-care temozolomide, without notable toxicities.

bioengineering↗

bioRxiv2024

BLM Aggregation-Prone Ability and Similarity to Intrinsically Disordered Proteins Indicate its Potential Role in Neurodegenerative Diseases

BLM helicase is a member of the RecQ family of DNA helicases, enzymes that play a critical role in maintaining genome stability. The BLM protein is named after Bloom syndrome (BS), a rare genetic disorder caused by mutations in the helicase domain of BLM gene. BLM role has mostly been studied in DNA repair, replication, and recombination; however, a recent study has highlighted the RNA binding capability of the BLM protein. Several proteins, including TDP43, Tau, and alpha-synuclein, are known to play key roles in neurodegenerative diseases. These proteins possess residues prone to parallel aggregation and fibril formation, the key contributor to neurodegenerative disease development. We utilized various in-silico tools like PASTA 2.0, SIFT, PolyPhen 2, and PhD SNP, SNP&GO, Meta-SNP, and SNAP, etc., to identify the molecular signature of the BLMs protein. Our in-silico analysis suggests that the BLMs HRDC domain has ability of parallel aggregation and fibril formation. Moreover, structural similarity with proteins like -synuclein, TDP-43, and Tau and its interactions with PARP1 suggests it may have a role in neurodegenerative diseases. Furthermore, we identified deleterious mutations in the HRDC domain of BLM protein that may compromise its stability and alter its function. Hence, these findings suggest that in addition to BLMs well-known functions, the protein may have ability to form parallel aggregation and fibril formation and its role in neurodegenerative disease need to further be explored. Author SummaryThis paper higlights the importance of considering the structural and physical characteristics of the BLM protein, which may have been previously overlooked in the context of disease pathology. Through in-silico analyses, we identified that the BLM protein possesses residues within the HRDC domain that are prone to parallel aggregation and fibril formation. Moreover, the structural similarity of BLM to proteins such as -synuclein, TDP-43, and Tau indicates a potential role for BLM in neurodegenerative processes. Additionally, we identified deleterious mutations within the HRDC domain that could compromise the stability of the protein. Furthermore, our findings suggest that the association between BLM and PARP1 may involve a regulatory mechanism that could significantly influence BLMs function.

bioinformatics↗

bioRxiv2024

Comprehensive Analysis of Exportins Expression and its Prognostic Significance in Colon Adenocarcinoma: Insights from Public Databases

Colon cancer remains a significant health burden globally, necessitating deeper investigation. Identification and targeting of prognostic markers can significantly improve the current therapeutic approaches for colon cancer. The differential nuclear transport (import and export) of cellular proteins, plays an important role in tumor progression. Exportins, critical mediators of nuclear export, have emerged as potential players in cancer pathogenesis. However, their precise roles and prognostic significance in colon adenocarcinoma remain elusive. This study was designed to comprehensively analyse the expression and prognostic significance of all seven exportins in Colon Adenocarcinoma (COAD) using the online public database. We used public databases UALCAN, C-Bio portal, Human Protein Atlas (HPA), and DAVID, to investigate exportins in COAD patients. Kaplan-Meier plotter, Gene ontology (GO), TIMER, STRING, and KEGG were used to analyse data and draw conclusions. Our observations showed a significant correlation of exportins expression with clinical parameters, used to predict a patients prognosis in general, such as advancing tumor stage, overall/relapse-free survival, and immune cell infiltrations. Mutation analysis showed the presence of amplifications, missense mutations in XPO2 and XPO4, and deep deletions in XPO7 genes contributing to disease progression and patients survival. This study highlights the potential use of exportins as novel prognostic biomarkers and therapeutic targets for colon adenocarcinoma progression and management.

cancer biology↗

bioRxiv2024

Oxysterol accumulation in aging cells alters GPCR signalling

Organismal aging is accompanied by the accumulation of senescent cells in the body, which drives tissue dysfunction. Senescent cells have a distinctive profile, including proliferation arrest, resistance to apoptosis, altered gene expression, and high inflammation. Despite global signalling and metabolic dysregulation during senescence, the underlying reasons for changes in signalling remain unclear. GPCRs are pivotal in cellular signalling, dynamically mediating the complex interplay between cells and their surrounding environment to maintain cellular homeostasis. The chemokine receptor CXCR4 plays a crucial role in modulating immune responses and inflammation. It has been shown that expression of CXCR4 increases in cells undergoing senescence, which enhances inflammation post-activation. Here we examine CXCR4 signalling in deeply senescent cells, where cholesterol and its oxidized derivatives, oxysterols, affect receptor function. We report elevated oxysterol levels in senescent cells, which altered classical CXCL12-mediated CXCR4 signalling. Tail-oxidized sterols disrupted signalling more than ring-oxidized counterparts. Molecular dynamics simulations revealed that 27-hydroxycholesterol displaces cholesterol and binds strongly to alter the conformation of critical signalling residues to modify the sterol-CXCR4 interaction landscape. Our study provides a molecular view of the observed mitigated GPCR signalling in the presence of oxysterols, which switched G-protein signalling from Gi/o to Gs class. Overall, we present an altered paradigm of GPCR signalling where cholesterol oxidation alters the signalling outcome in aged cells. Significance StatementOur study brings to light a novel and significant discovery in aged cells: the accumulation of oxysterols, oxidized forms of cholesterol, critically impairs CXCR4-dependent signalling and alters G-protein coupling specificity. This effect of oxysterols is demonstrated for the first time in aged cellular models, providing a molecular basis for a multitude of observed alterations in senescence, such as compromised immune functions and a decline in cellular responsiveness with age. Our research not only fills a crucial gap in understanding the aging process at the molecular level but also identifies potential targets for therapeutic interventions aimed at mitigating age-related cellular dysfunctions and diseases.

cell biology↗

bioRxiv2023

TDP-43-M323K causes abnormal brain development and progressive cognitive and motor deficits associated with mislocalised and increased levels of TDP-43.

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as "TDP-43 proteinopathies". Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of [~]50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.

neuroscience↗

bioRxiv2023

FUSDelta14 mutation impairs normal brain development and causes systemic metabolic alterations

FUS (Fused in sarcoma) is a ubiquitously expressed DNA/RNA binding protein. Mutations in FUS cause aggressive juvenile forms of amyotrophic lateral sclerosis (ALS), as in the case with the FUSDelta14 mutation. While most studies have focused on the role of FUS in motor neuron degeneration, little is known about the effect of FUS mutations in the whole body, and the impact of FUS mutations in the correct development of the nervous system. We studied pleiotropic phenotypes in a physiological knock-in mouse model carrying the FUSDelta14 mutation in homozygosity. RNA sequencing was conducting in six different tissues (frontal cortex, spinal cord, tibialis anterior muscle, white and brown adipose tissue and liver) to identify the genes and pathways altered by the FUSDelta14 mutant protein in the systemic transcriptome. Additionally, brain structural magnetic resonance imaging (MRI) and histological characterisation was conducted in young mice to study the role of FUS mutation in the brain development. FUS mutant protein was upregulated and mislocalised in the cytoplasm in most cells of the tissues analysed. We identified few genes commonly altered in all tissues by this mutation, although most genes and pathways affected were generally tissue-specific. Phenotypic assessment of mice revealed systemic metabolic alterations related to the pathway changes identified. MRI brain scans revealed that homozygous FUSDelta14 brains were smaller and displayed significant morphological alterations including a thinner cortex, reduced neuronal number and increased gliosis, which correlated with early cognitive impairment and fatal seizures. We demonstrated that the disease aetiology of FUS mutations can include neurodevelopmental and systemic alterations, which should be taken into consideration in the clinic.

neuroscience↗

bioRxiv2022

Revisiting the significance of keratin expression in complex epithelia

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells, and biological context (e.g., normal vs. diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Further, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic vs. a post-mitotic, differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia.

bioinformatics↗

bioRxiv2022

A multifaceted architectural framework of the mouse claustrum complex

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CC) this has yet to be fully accomplished. The CC is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are highly debated. To address this, we conducted a multifaceted analysis of fiber- and cyto-architecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to the CC. We identified four distinct subregions within the CC, subdividing both the CL and the DEn into two. Additionally, we conducted brain-wide tracing of inputs to the entire CC using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of the CC and its subregions. Myelinated fibers were abundant in dorsal parts of the CL but absent in ventral parts, while parvalbumin labelled fibers occupied the entire CL. Calbindin staining revealed a central gap within the CL, which was also visible at levels anterior to the striatum. Furthermore, cells in the CL projecting to the retrosplenial-cortex were located within the myelin sparse area. By combining our own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework. Significance statementMice are a highly tractable model for studying the claustrum complex (CC). However, without a consensus on how to delineate the CC in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CC, to match the level of detail needed to study its functional properties. Using multiple strategies for identifying claustral borders, we created a comprehensive guide to delineate the CC and its subregions. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CC.

neuroscience↗

bioRxiv2022

Complete identity of the cis-asRNA StfZ and its influence on FtsZ protein level and cell division in Escherichia coli

Bacteria regulate FtsZ protein levels through transcriptional and translational mechanisms for proper cell division. A cis-antisense RNA, StfZ, produced from the ftsA-ftsZ intergenic region was proposed to regulate the FtsZ level in Escherichia coli. However, its structural identity remained unknown. In the present study, we determined the complete sequence, identified the promoters, and effects of StfZ on the FtsZ level. We show that StfZ is expressed at 1:6 ratio of StfZ:ftsZ mRNA at all growth phases from three promoters as three isoforms of 366 nt, 474 nt, and 552 nt. Overexpression of StfZ reduces FtsZ level, increases cell size, and blocks cell division. Thus, the cis-encoded StfZ emerges as a novel factor for maintaining the FtsZ level at different growth phases in E. coli.

microbiology↗

bioRxiv2021

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

Neuronal firing patterns are the result of inputs converging onto single cells. Identifying these inputs, anatomically and functionally, is essential to understand how neurons integrate information. Single-cell electroporation of helper genes and subsequent local injection of recombinant rabies viruses enable precise mapping of inputs to individual cells in superficial layers of the intact cortex. However, access to neurons in deeper structures requires more invasive procedures, including removal of overlying tissue. We have developed a method that through a combination of virus injections allows us to target [≤]4 hippocampal cells 48% of the time and a single cell 16% of the time in wildtype mice without the use of electroporation or tissue aspiration. We identify local and distant monosynaptic inputs that can be functionally characterised in vivo. By expanding the toolbox for monosynaptic circuit tracing, this method will help further our understanding of neuronal integration at the level of single cells.

neuroscience↗

bioRxiv2021

Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS

Amyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS. We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan. These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.

neuroscience↗

bioRxiv2020

Local circuits of layer Vb-to-Va neurons mediate hippocampal-cortical outputs in lateral but not in medial entorhinal cortex

The entorhinal cortex, in particular neurons in layer V, allegedly mediate transfer of information from the hippocampus to the neocortex, underlying long-term memory. Recently, this circuit has been shown to comprise a hippocampal output recipient layer Vb and a cortical projecting layer Va. With the use of in vitro electrophysiology in transgenic mice specific for layer Vb, we assessed the presence of the thus necessary connection from layer Vb-to-Va in the functionally distinct medial (MEC) and lateral (LEC) subdivisions; MEC, particularly its dorsal part, processes allocentric spatial information, whereas the corresponding part of LEC processes information representing elements of episodes. Using identical experimental approaches, we show that connections from layer Vb-to-Va neurons are more effective in LEC compared with dorsal MEC. This indicates that the hippocampal-cortex output circuit is more effective in LEC, suggesting that episodic systems-consolidation may use LEC-derived information more than allocentric spatial information from MEC.

neuroscience↗