Biology subjects

Nielsen, S.

Publications and source records attributed to Nielsen, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Ocean depth modifies host-microbiome ecology and evolution

A major unsolved question in ecology and evolution is how similar free-living and symbiotic microbial communities are. In marine systems, depth homogenises free-living communities due to stronger dispersal and weaker environmental filtering compared to surface waters. We show that symbiotic communities of marine sponges follow a different pattern, with a subset of sponge species mirroring the increased similarity with depth seen in their free-living counterparts, while others remain highly host-specific both in shallow and deep waters. Moreover, in deep waters, more phylogenetically similar hosts harbor more similar microbiomes, yet host-symbiont cophylogeny nearly vanishes. This reveals two contrasting eco-evolutionary assembly rules: in more constant environments like the deep sea, community assembly is driven by host and environmental filtering with minimal signature of coevolution, whereas in variable environments, like shallow waters, host-symbiont coevolution is strong and key to maintain host-specific microbiomes.

microbiology↗

bioRxiv2026

Single-locus chromatin memory enables flexible spatial fate specification in the Drosophila visual system

Spatial patterning generates neuronal diversity by compartmentalizing progenitors into domains with distinct molecular identities. However, these patterning cues are often transient in neurogenic domains, raising the question of how spatial information can be preserved and how rigidly it constrains neuronal fates. Here we show that a single-locus chromatin memory in the Drosophila medulla enables spatial identity to be both faithfully executed and flexibly bypassed. Medulla progenitors are partitioned into three spatial domains marked by Vsx1, Optix and Bifid. Domain-resolved single-cell multiome profiling reveals that progenies from different neuroepithelial domains are nearly indistinguishable for both transcriptome and chromatin accessibility, although persistent, domain-specific accessibility is retained only at a single spatial-factor locus, either Vsx1/2 or Bifid. These same factors are absent when neuroepithelial cells are converted to neural stem cells but are re-expressed in postmitotic neurons to execute domain-specific fates. Because this bookmarking is so restricted, specific classes of neurons can skip the domain-specific re-expression program and default to a common ground state, adopting equivalent fates regardless of spatial origin. Other neurons reach the same domain-ignoring state by expressing Vsx1/2 through a program independent of their domain of origin. PRC2-mediated silencing restricts Vsx1 re-expression to its home domain, while temporal identity and Notch signaling in newborn neurons determine which neurons engage or bypass the spatial program. Thus, single-locus chromatin memory preserves spatial information without making it an obligatory determinant of every neuronal fate.

developmental biology↗

bioRxiv2026

A Common Pathogenic Founder Variant in Rwandan Breast Cancer Cases

Germline data from African populations remain sparse, limiting characterization of population-specific BRCA1/2 pathogenic variants. In a study of 175 Rwandan women with breast cancer, 7 unrelated carriers (4% of cases; 22% of pathogenic variant carriers) harbored the same BRCA1 frameshift variant, c.4065_4068del (p.Asn1355Lysfs*10), which is extremely rare in gnomAD yet recurrent in European, Asian, and Middle Eastern cohorts. Whole-exome sequencing and haplotype analysis of all 7 carriers revealed a shared ancestral block of approximately 581 kb surrounding the variant, and extended haplotype homozygosity and network analyses confirmed a common founder origin. Coalescent-based age estimation placed the founder event approximately 4,000--10,000 years ago. Comparison with 1000 Genomes Project data showed the founder haplotype is absent or exceedingly rare outside African and South Asian populations. These findings strongly suggest the c.4065_4068del variant as a pre-historical BRCA1 founder variant in Rwanda, with implications for targeted genetic testing, cascade screening, and cancer prevention in the region.

genetics↗

bioRxiv2025

An ancestral transmembrane transcription factor couples cell envelope regulation and the SOS response in Caulobacter crescentus

The DNA damage (SOS) response in bacteria involves derepression of a set of genes in order to activate mechanisms to tolerate stress, repair DNA and slow down cell division. While some of these genes are well characterized, many genes exist which are clearly induced by DNA damage but for which the function is unclear. In Caulobacter crescentus, the toxin-antitoxin (TA) system higBA and a closely associated downstream transcription factor (higX) are strongly induced as part of the SOS response, but the role of higX is unknown. We show that, unexpectedly, HigX functions independently of HigBA as a cell membrane-associated regulator and is toxic when overexpressed in filamentous cells. ChIP-Seq indicated that it binds to several promoters associated with cell envelope regulation. In cells with the SOS response constitutively activated, HigX was overproduced, but at the same time was unable to bind the majority of its target promoters. higX was highly conserved in genomic context among many alpha-proteobacteria, while higBA was only found upstream of it in a small number of Caulobacter genomes, including the universal laboratory strain C. crescentus NA1000. Compositional analysis suggested that higBA originated from a foreign source, while higX is likely ancestral to the alpha-proteobacteria. Our data support a model where dysregulation of HigX production and activity in filamentous {Delta}lexA cells contributes to cell envelope instability and antibiotic sensitivity. Thus, the protective effect of inhibiting cell division during the SOS response in order to repair the DNA, carries the hidden cost of interference with HigX-mediated cell envelope maintenance. Author summaryThe DNA damage (SOS) response in bacteria is key for their survival in harsh conditions, but the genes which are induced by this response and the molecular mechanisms involved are variable between different bacteria. We investigated the function of an uncharacterized SOS-induced gene in Caulobacter crescentus (higX) and found that it is a membrane-localized DNA binding protein, but that it did not help survival in DNA damage. Instead, it negatively affected cell viability when induced, unlike other SOS response genes. Genomic analysis showed that higX is highly conserved among alphaproteobacteria, but it is only under SOS control in a small number of genomes (including Caulobacter crescentus) where a toxin-antitoxin system and its SOS-regulatory promoter have been inserted upstream. This work uncovers HigX as a novel factor in cell envelope regulation, and shows that loss of cell shape regulation, eg. in filamentous {Delta}lexA cells, can influence the ability of transmembrane transcription factors to bind to their target genes.

microbiology↗

bioRxiv2025

Comparative analysis of convergent jellyfish eyes reveals extensive differences in expression of vision-related genes

Quantifying gene expression across convergent origins of traits clarifies the degree to which those traits arise from shared versus distinct genetic programs, revealing how gene re-use relates to the repeatability of evolution. Eyes are important traits that evolved in many distantly related lineages, including at least nine times within cnidarians. Here, we investigate gene expression in eye-bearing and non-visual tissues from three cnidarian species representing long-diverged lineages where eyes evolved convergently (Cubozoa, Scyphozoa, and Hydrozoa). We find gene expression in eye-bearing tissues to be mostly lineage-specific, with only a small proportion of genes having convergent expression across species. Nevertheless, all species express homologs of deeply conserved vision-related genes known from Bilateria, which likely reflects deep homology (parallel evolution across vast phylogenetic distances) of a metazoan phototransduction toolkit. A gene tree analysis of opsins--the prototypical animal photosensors--shows that convergent eyes recruited different opsin paralogs, with the potential exception of an opsin ortholog shared between scyphozoan and cubozoan eyes. Our results suggest that eyes have mostly lineage-specific patterns of gene expression, yet some key phototransduction components are repeatedly recruited across multiple independent eye origins in Medusozoa.

evolutionary biology↗

bioRxiv2025

Sex Hormone Binding Globulin Controls Gender Specific Lipolytic Activity in Human Abdominal Subcutaneous Adipocytes

Regulation of lipid metabolism is fundamental for metabolic health, and adipose tissue is a central component in this process. Adipose tissue differs dramatically between women and men with a higher subcutaneous capacity for storage and healthy metabolism in women. Sex hormone-binding globulin (SHBG) contributes to the regulation of circulating sex hormone bioavailability and has been shown to predict risk of metabolic dysfunction. We here investigate the sex-specific relationship of SHBG with metabolic status and adipocyte-dependent lipolysis. We measured serum concentrations of sex hormones, SHBG, fasting glucose and insulin in a cohort of 63 women and 27 men from which adipose biopsies were collected and mature adipocytes were isolated. We found that, in women, high serum SHBG concentrations were strongly associated with low HOMA-IR in vivo, and lower baseline lipolysis but higher responsiveness to isopropanol-induced lipolysis ex vivo. In contrast, no effect of SHBG on the above-mentioned parameters were observed in men. In vitro, cultured adipocytes also increased lipolytic capacity in response to SHBG, but only in the absence of testosterone, suggesting that testosterone inhibits the catecholmine-induced lipolysis of SHBG in adipose tissue. In conclusion, we here define a novel role for SHBG in adipocyte lipolysis. At the same time, our data emphasize sex-dependent differences in adipocyte lipid metabolism, and we propose testosterone binding to SHBG as a driving factor mediating these differences.

physiology↗

bioRxiv2024

Brown Bears activates a Satiety Hormone Cholecystokinin (CCK) pathway in adipose tissue during hibernation

The brown bear (Ursus arctos) hibernates to survive cold winters without access to food. It builds enormous subcutaneous fat stores during summer and relies on it for energy during winter. Remarkably, the weight loss during winter occurs without muscle loss despite inactivity. Studying brown bear biology can therefore provide insights for improving human health in obesity and weight loss treatments. We here investigate subcutaneous adipose tissue biopsies obtained during summer and winter from free-living brown bears. During winter, a signature of genes involved in food intake and digestion is upregulated. Among these are several regulators of satiety, substrate transport and lipid metabolism. Interestingly, in humans these genes are enriched in distinct metabolic organs including, brain, intestine, stomach, liver and even salivary glands. We focused on the satiety brain/intestinal hormone cholecystokinin (CCK), which we demonstrate is produced in adipocytes, accompanied by an upregulation of the CCK receptor CCKBR. Importantly, CCK was undetectable in the circulation during winter and presence of sensory neurons suggest a neuronal feedback mechanism within the adipose tissue. Using RNA sequencing, we predict additionally 537 secreted proteins to be seasonally regulated, 37 of which could be confirmed with plasma proteomics. In conclusion, we propose that brown bears have developed a strategy of healthy fat burning and satiety regulation through an adipose tissue-contained mechanism which includes digestion factors and satiety mediators to provide safe energy turnover during hibernation-dependent weight loss.

molecular biology↗

bioRxiv2024

Conserved protein Seb1 that interacts with RNA polymerase II and RNA is a bona fide transcription elongation factor

Maturation of protein-coding precursor messenger RNA (pre-mRNA) is closely linked to RNA polymerase II (Pol II) transcription. However, the mechanistic understanding of how RNA processing is coordinated with transcription is incomplete. Conserved proteins interacting with the C-terminal domain of the largest catalytic subunits of Pol II and nascent RNA (CID-RRM factors) were demonstrated to play a role in mRNA 3-end processing and termination of Pol II transcription. Here, we employ a fully reconstituted system to demonstrate that fission yeast CID-RRM factor Seb1 acts as a bona fide elongation factor in vitro. Our analyses show that Seb1 exhibits context-dependent regulation of Pol II pausing, capable of either promoting or inhibiting pause site entry. We propose that CID-RRM factors coordinate Pol II transcription and RNA 3-end processing by modulating the rate of Pol II transcription.

molecular biology↗

bioRxiv2024

Identification of novel plasma proteomic biomarkers of Dupuytren Disease

Dupuytren Disease (DD) is a chronic progressive disease that can cause disabling hand deformities. The most common treatments have either high complication rates or high early recurrence rates. Dupuytren lacks a staging biomarker profile to inform the development of preventive therapeutics to improve long-term outcomes. This multi-omic study aimed to create a DD blood proteomic biomarker profile by comparing DD plasma with that of a healthy control group. We measured circulating collagen metabolism peptides and found normal Collagen I synthesis but impaired Collagen I degradation in DD. We measured 6995 serum protein aptamers and identified 68 proteins that showed statistically significant differences compared with the control group. We developed two Diagnostic Proteomic Risk Scores (DPRS) based on hypothesis-free and hypothesis-based analyses. In independent data, our hypothesis-free and hypothesis-based DPRS distinguished Dupuytren from control subjects with accuracies of 76.5% and 70.6%, respectively. Our hypothesis-based DPRS also distinguished DD subjects with different disease progression rates by age at their first corrective procedure (p=0.0018). This pilot study is the first to provide evidence to suggest that Collagen I accumulation in DD results from impaired degradation rather than increased collagen synthesis. It also describes novel DPRS that have potential use as diagnostic and staging biomarker panels for Dupuytren disease.

molecular biology↗

bioRxiv2024

Mitochondria isolated from male skeletal muscle contain a distinct 1 population of miRNA that are differentially expressed following acute exercise

Initially thought to localise at the cytosol and nucleus only, emerging evidence indicates that miRNAs also localise within the mitochondria where they could regulate diverse pathological and physiological processes. Therefore, the aim of the current study was to use small RNA sequencing to profile and compare the entire population of miRNAs in human skeletal muscle of healthy males in whole-tissue and in isolated mitochondria at rest and in response to acute endurance exercise. Twelve healthy males (age 26 {+/-} 4 years, mean {+/-} SD) cycled for 60 min at 70% VO2peak and muscle biopsies were collected at rest, immediately after and 3 h after exercise. The mitochondria were isolated by immunoprecipitation, further purified, then the resident RNA was sequenced to assess the mitochondrial transcriptome. Small RNA sequencing revealed that mitochondria isolated from male skeletal muscle tissue contain a small and distinct population of miRNAs. Of the approximately 110 mature miRNAs detected in skeletal muscle mitochondria at each time-point, the canonical myo-miRs miR-1, miR-133 and miR-206 families constituted on average 45% of total mitochondria miRNA reads. However, none of these canonical myo-miRs were differentially expressed in mitochondria following endurance exercise. One miRNA, hsa-miR-146b-3p, was differentially expressed in both whole muscle tissue and mitochondria when adjusted for multiple testing (FDR <0.05). Future research is now required to investigate miRNA-mRNA interactions in the mitochondria of skeletal muscle tissue. KEY POINTS SUMMARYO_LIEmerging evidence suggests microRNA are localised in the mitochondria of skeletal muscle cells and may play a role in regulating mitochondrial function. C_LIO_LIWe recently optimised an approach to isolate RNA from mitochondria of human skeletal muscle that is free from contaminating cytosolic RNA and suitable for RNA sequencing. C_LIO_LIIn this study we examined the microRNA population from male skeletal muscle mitochondria before and in the hours following 60 minutes of moderate intensity cycling exercise. C_LIO_LIWe detected around 110 microRNAs in skeletal muscle mitochondria, with the muscle enriched myo-miR such as miR-1, miR-133 and miR-206 families constituting almost half of the reads. However, only one microRNA, hsa-miR-146b-3p, was differentially expressed whereby it increased [~] 10-fold following exercise. C_LIO_LIThe results provide new knowledge into how mitochondria might be regulated at the subcellular level and in response to physiological stressors such as exercise. C_LI

cell biology↗

bioRxiv2024

An ultrasound-guided biopsy technique for obtaining supraclavicular brown fat biopsies and preadipocytes

Studying activated human brown adipose tissue (BAT) in vivo poses challenges due to its intricate anatomical positioning. Through the implementation of an ultrasound-guided biopsy technique, we successfully collected BAT samples from the supraclavicular region of 27 healthy individuals. As a comparative control, subcutaneous white adipose tissue (WAT) was similarly extracted from the same participants. Furthermore, we isolated progenitor cells from four tissue biopsies in both regions, subsequently subjecting them to a 12-day in vitro differentiation protocol following stimulation with 10 {micro}M norepinephrine. To assess the mRNA expression of thermogenic genes within these small tissue samples, we employed a targeted cDNA amplification procedure, followed by conventional quantitative PCR (qPCR). Our study demonstrated that, with further refinement, this biopsy methodology can be used to obtain thermogenic adipose tissue. However, the expression data exhibited considerable diversity, and no statistically significant overall trends emerged for any of the five BAT marker genes (UCP1, PPARGC1A, PRDM16, CIDEA, CITED1), nor for the WAT marker HOXC8. The differentiation capacity of the progenitor cells revealed irregularities, with only three adipocyte cultures (two WAT and one BAT) displaying satisfactory differentiation potential. Remarkably, the differentiated BAT culture displayed a significantly elevated basal UCP mRNA expression level, further induced by 1.7-fold upon stimulation with norepinephrine. In summary, based on the in vitro data, brown adipose samples can be obtained using our ultrasound-guided biopsy technique approach. However, significant refinements are necessary before robust in vivo data can be generated in future intervention studies.

physiology↗

bioRxiv2024

DNA targeting by compact Cas9d and its resurrected ancestor

The type II-A CRISPR effector SpCas9 has gained widespread popularity as an efficient and programmable genome editing tool. However, much remains to be known about novel compact variants that may overcome some limitations of current systems1,2. Recently, alternative CRISPR-Cas systems with highly compact nucleases capable of genome editing in mammalian cells have been discovered through metagenomic analysis of uncultivated microbes, including Cas9d (a type II-D CRISPR-Cas effector)3. Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains and facilitate DNA target recognition. We find that the sgRNA can be truncated by up to [~]25% yet still retain activity in vivo. We also show that despite preferentially targeting an NGG PAM, Cas9d exhibits a unique mechanism for PAM recognition. Finally, we identify the first Cas9d smaller than 800 amino acids exhibiting robust nuclease activity in mammalian cells. Using ancestral sequence reconstruction, we demonstrate that it is possible to generate compact nucleases capable of efficient genome editing by expanding the diversity of Cas9d families. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a molecular blueprint for future rational re-engineering of minimal RNA-guided DNA nucleases.

biochemistry↗

bioRxiv2023

rCRUX: A Rapid and Versatile Tool for Generating Metabarcoding Reference libraries in R

Key to making accurate taxonomic assignments are curated, comprehensive reference barcode databases. However, the generation and curation of such databases has remained challenging given the large and continuously growing volumes of DNA sequence data and novel reference barcode targets. Monitoring and research applications require a greater diversity of specialized gene regions and targeted taxa to meet taxonomic classification goals then are currently curated by professional staff. Thus, there is a growing need for an easy to implement tool that can generate comprehensive metabarcoding reference libraries for any bespoke locus. We address this need by reimagining CRUX from the Anacapa Toolkit and present the rCRUX package in R. The typical workflow involves searching for plausible seed amplicons (get_seeds_local() or get_seeds_remote()) by simulating in silico PCR to acquire seed sequences containing a user-defined primer set. Next these seeds are used to iteratively blast search seed sequences against a local NCBI formatted database using a taxonomic rank based stratified random sampling approach (blast_seeds()) that results in a comprehensive set of sequence matches. This database is dereplicated and cleaned (derep_and_clean_db()) by identifying identical reference sequences and collapsing the taxonomic path to the lowest taxonomic agreement across all matching reads. This results in a curated, comprehensive database of primer specific reference barcode sequences from NCBI. We demonstrate that rCRUX provides more comprehensive reference databases for the MiFish Universal Teleost 12S, Taberlet trnl, and fungal ITS locus than CRABS, METACURATOR, RESCRIPt, and ECOPCR reference databases. We then further demonstrate the utility of rCRUX by generating 16 reference databases for metabarcoding loci that lack dedicated reference database curation efforts. The rCRUX package provides a simple to use tool for the generation of curated, comprehensive reference databases for user-defined loci, facilitating accurate and effective taxonomic classification of metabarcoding and DNA sequence efforts broadly.

bioinformatics↗

bioRxiv2022

Exercise Training Remodels Adipocyte-Specific Extracellular Matrix Proteins and Alters Innervation and Vascularization in Inguinal White Adipose Tissue (iWAT)

Inguinal white adipose tissue (iWAT) is essential for the beneficial effects of exercise training on metabolic health. Extracellular matrix (ECM) composition, innervation, and vascularization are all important regulators of iWAT function, yet whether exercise training improves these structural components of iWAT is unknown. Using biochemical, imaging, and multi-omics analyses we find that 11-days of wheel running in male mice causes profound iWAT remodeling including decreased ECM deposition and increased vascularization and innervation. We identify adipose stem cells as the main contributors to training-induced ECM remodeling, determine that training causes a shift from hypertrophic to insulin-sensitive adipocyte subpopulations, show that the PRDM16 transcriptional complex is necessary for iWAT remodeling and beiging, and discover neuronal growth regulator 1 (NEGR1) as a link between PRDM16 and neuritogenesis. Exercise training leads to remarkable adaptations to iWAT structure and cell-type composition that can confer beneficial changes in tissue metabolism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/503375v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@fd590eorg.highwire.dtl.DTLVardef@e1dd02org.highwire.dtl.DTLVardef@74be24org.highwire.dtl.DTLVardef@6f6e58_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Cold-induced expression of a truncated Adenylyl Cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity has been recognized as innovative therapeutic approach to improve obesity and metabolic disease. Whilst the molecular circuitry underlying thermogenic activation of BAT is well understood, the processes underlying rheostatic regulation of BAT to maintain homeostasis and avoid excessive energy dissipation remain ill-defined. Increasing cyclic AMP (cAMP) biosynthesis is key for BAT activation. Here, we demonstrate that ADCY3, an adenylyl cyclase whose expression is induced during cold exposure and regulates cAMP homeostasis in thermogenic fat, is dispensable for BAT function in lean mice, but becomes critical during obesity. Furthermore, by combining RNA-seq with epigenomic H3K4me3 profiling, we detected a novel, cold-inducible promoter that generates a 5 truncated Adcy3-at mRNA isoform, Adcy3-at. Mice lacking only Adcy3-at, but not full-length Adcy3, displayed increased energy expenditure already under lean conditions and were protected against obesity and ensuing metabolic imbalances. Subcellularly, translated ADCY3-AT proteins are retained in the endoplasmic reticulum (ER), did not translocate to the cell membrane, and lacked enzymatic activity. By interacting with ADCY3, ADCY3-AT retained ADCY3 in the ER and, thereby, reduced the plasma membrane pool of ADCYs available for G-protein mediated cAMP synthesis. Thereby, ADCY3-AT acts as a signaling rheostat in BAT, limiting adverse consequences of uncurbed cAMP activity after long-term BAT activation. Adcy3-at induction was driven by a cold-induced, truncated isoform of the transcriptional cofactor PPARGC1A (PPARG Coactivator 1 Alpha, PPARGC1A-AT). Expression of Ppargc1a-at and Adcy3-at are evolutionary conserved, indicating that transcriptional rewiring by commissioning of alternative promoters is key for thermogenic fat function.

cell biology↗

bioRxiv2022

Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localisation of nuclear-encoded non-coding RNAs

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small-RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small-RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

cell biology↗

bioRxiv2021

Polarized α-synuclein trafficking and transcytosis across Brain Endothelial Cells via Rab7-decorated carriers

Parkinsons disease is mainly caused by aggregation of -synuclein (-syn) in the brain. Exchange of -syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of -syn across the blood brain barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of -syn monomers and oligomers across an in vitro BBB model system. Both -syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric -syn in brain endothelial cells, we investigated co-localization of -syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric -syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric -syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric -syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric -syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric -syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinsons disease and related synucleinopathies. Significance StatementIn the submitted manuscript, we describe the use of a state-of-the-art porcine blood-brain barrier model based on primary cells to get information about this important issue. We identify several hitherto undescribed cellular pathways to mediate polarized transport of alpha-synuclein. One of these paths we find regulated by Rab7 and can be inhibited by targeting several intracellular proteins such as VPS35, Caveolin1 and Rab7. New knowledge describing brain endothelial intracellular transport systems are highly warranted for identifying new target to alleviate Parkinsons disease. We believe that our findings could be the seed to establish new therapeutic strategies against Parkinsons disease and related synucleinopathies.

cell biology↗

bioRxiv2021

The obesity-linked human lncRNA AATBC regulates adipocyte plasticity by stimulating mitochondrial dynamics and respiration

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, the activation of thermogenic brown and beige adipocytes is linked to improved cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this adipocyte plasticity could help improving energy balance and weight loss. Here, we show that the long non-coding RNA (lncRNA) Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Searching for new human lncRNAs implicated in adipocyte biology we compared transcriptional profiles of human adipose tissues and cultured adipocytes and discovered that AATBC was enriched in thermogenic conditions. Using primary human adipocytes and immortalized human adipocytes we found that gain-of-function of AATBC enhanced the thermogenic phenotype whereas loss-of-function diminished this effect. The AATBC-mediated increase in mitochondrial respiration was linked to a more fragmented mitochondrial network and vice versa. While we found that AATBC is predominantly located in the nucleus, its effect on global transcription was only marginal. As AATBC is specific to humans, we expressed AATBC in adipose tissue of mice to study its systemic impact, which led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, body mass index and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

molecular biology↗