Biology subjects

Lambert, G.

Publications and source records attributed to Lambert, G..

7 recordsLinked to original sources

bioRxiv2024

Alteration of nociceptive Schwann cells in a mouse model of peripheral neuropathy in prediabetic condition

Diabetic peripheral neuropathy (DPN) is characterized by progressive and symmetrical sensory abnormalities and constitutes one of the earliest and main complications of diabetes. DPN is characterized by heterogeneous sensory symptoms such as chronic pain, tingling, burning or loss of sensation. Nociceptive Schwann cells (nSCs), a recently identified subtypes of dermal Schwann cells support terminal nerve fibers in mouse skin and contribute to mechanical sensation and neuropathic pain. While terminal nerve fibers density is evaluated in DPN models, there is currently no data about nSCs number and integrity during the early stages of the disease. In the present study, we determined the quantitative differences in terminal nerve fiber density as well as nSCs number and cellular extensions between control and high-fat diet (HFD) induced diabetic mice presenting with a neuropathic phenotype. We also characterized L1CAM as a reliable and specific marker of nSCs, a cell type previously assessed by the expression of S100{beta} and Sox10. Interestingly, we observed a decrease in intraepidermal nerve fiber density (IENFD) associated with a significant reduction of nSCs in the glabrous foot skin of neuropathic mice. Overall, this study identifies L1CAM as a new marker of nSCs and indicates that these cells are impacted in diabetic peripheral neuropathy.

neuroscience↗

bioRxiv2024

PCSK9 deficiency promotes the development of peripheral neuropathy

PCSK9 best-known and studied function is to induce the hepatic degradation of the low-density lipoprotein receptor (LDLR), thereby increasing the concentration of LDL-cholesterol (LDL-C) in the blood. Beyond its effects on LDL, recent studies have reported pleiotropic effects of PCSK9 notably in septic shock, vascular inflammation, viral infection, and cancer. While the functional and structural integrity of peripheral nerves are critically influenced by circulating lipids, the impact of PCSK9 in the peripheral nervous system is unknown. In this study, we investigated the consequences of PCSK9 deficiency on peripheral nerves. We found that PCSK9 deletion in mice leads to peripheral neuropathy characterized by a reduction of thermal and mechanical pain sensations. PCSK9 deficient mice also presented skin structural changes with a reduction of number of terminal nociceptive Schwann cells, Remak fiber axonal swelling, as well as hypomyelination of small nerve fibers. Interestingly, peripheral nerves of PCSK9 deficient mice presented an upregulation of the fatty acid transporter CD36 expression which correlated with an increase in nerve lipid contents and structural mitochondrial abnormalities. Our findings demonstrate that PCSK9 plays a critical role in the peripheral nerves by regulating lipid homeostasis, and its deficiency could lead to the development of symptoms related to peripheral neuropathy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/583154v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1469ef8org.highwire.dtl.DTLVardef@16ebcd9org.highwire.dtl.DTLVardef@1305cbborg.highwire.dtl.DTLVardef@181758_HPS_FORMAT_FIGEXP M_FIG C_FIG PCSK9 modulates nerve energy metabolism and health. Created with BioRender.com.

neuroscience↗

bioRxiv2022

Overcoming leak sensitivity in CRISPRi circuits using antisense RNA sequestration and regulatory feedback

The controlled binding of the catalytically-dead CRISPR nuclease (dCas) to DNA can be used to create complex, programmable transcriptional genetic circuits, a fundamental goal of synthetic biology. This approach, called CRISPR interference (CRISPRi), is advantageous over existing methods because the programmable nature of CRISPR proteins enables the simultaneous regulation of many different targets without crosstalk. However, such gene circuit elements are limited by 1) the sensitivity to leaky repression of CRISPRi logic gates and 2) retroactive effects owing to a shared pool of dCas proteins. By utilizing antisense RNAs (asRNAs) to sequester guide RNA transcripts, as well as CRISPRi feedback to self-regulate asRNA production, we demonstrate a mechanism that suppresses unwanted CRISPRi repression and improve logical gene circuit function in E. coli. This improvement is particularly pronounced during stationary expression when CRISPRi circuits do not achieve the expected regulatory dynamics. Further, the use of dual CRISPRi/asRNA inverters restores logical performance of layered circuits such as a double inverter. By studying circuit induction at the single cell level in microfluidic channels, we provide insight into the dynamics of antisense sequestration of gRNA and regulatory feedback on dCas-based repression and derepression. These results demonstrate how CRISPRi inverters can be improved for use in more complex genetic circuitry without sacrificing the programmability and orthogonality of dCas proteins.

synthetic biology↗

bioRxiv2021

A Plasmid System with Tunable Copy Number

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.

synthetic biology↗

bioRxiv2020

Genome-wide association of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) plasma levels in the ELSA-Brasil study

Pharmacological inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9) is an established therapeutic option to treat hypercholesterolemia and plasma PCSK9 levels have been implicated in cardiovascular disease incidence. A number of genetic variants within the PCSK9 gene locus have been shown to modulate PCSK9 levels, but these only explain a very small percentage of the overall PCSK9 interindividual variation. Here we present data on the genetic association structure between PCSK9 levels and genome-wide genetic variation in a healthy sample from the general population. We performed a genome-wide association study of plasma PCSK9 levels in a sample of Brazilian individuals enrolled in the ELSA-Brasil cohort (n=810). Enrolled individuals were free from cardiovascular disease, diabetes and were not under lipid-lowering medication. Genome-wide genotyping was conducted using the Axiom_PMRA.r3 array and imputation used the TOPMED multi-ancestry sample panel. Total PCSK9 plasma concentrations were determined using the Quantikine SPC900 ELISA kit. We observed two genome-wide significant loci and seven loci that reached the pre-defined p value threshold of 1 x 10-6. Significant variants were near KCNA5 and KCNA1, and LINC00353. Genetic variation at the PCSK9 locus was able to explain approximately 4% of the overall interindividual variation in PCSK9 levels. Colocalization analysis using eQTL data suggested RWDD3, ATXN7L1, KCNA1, and FAM177A1 to be potential mediators of some of the observed associations. Our results suggest that PCSK9 levels may be modulated by trans genetic variation outside of the PCSK9 gene and this may have clinical implications. Understanding both environmental and genetic predictors of PCSK9 levels may help identifying new targets for cardiovascular disease treatment and contribute to a better assessment of the benefits of long-term PCSK9 inhibition.

genetics↗

bioRxiv2020

βPix sequesters IDOL and prevents LDL receptor degradation through a β2AR-regulated signaling pathway in Alport Syndrome

Alport syndrome (AS) is a rare disease of the glomerular basement membrane type IV collagen causing progressive renal failure. We reported increased accumulation of low-density lipoprotein (LDL) receptor (LDLR) and subsequent LDL cholesterol (LDL-C) uptake in renal tubular epithelial cells (TEC) in Alport mice, but the mechanisms regulating LDLR stability and function remain unknown. Here, we show that a selective {beta}2-Adrenoceptor ({beta}2AR) agonist, salbutamol, decreased LDLR levels and LDL-C uptake in Alport kidneys accompanied with reduced albuminuria and improved cardiac systolic and diastolic function. Similarly, salbutamol decreased LDL-C uptake in HK2 and HEK293 human renal epithelial cell lines, in smooth muscle cells from an X-linked hereditary nephropathy dog model (a large animal model of AS), and in TECs differentiated from AS patient-derived iPSCs. We show that the Rac1/Cdc42 guanine nucleotide exchange factor {beta}1Pix blocked {beta}2AR-induced LDLR degradation and, hence, increased LDL-C uptake. {beta}1Pix also abrogated ubiquitination and degradation of LDLR induced by the inducible degrader of the LDLR (IDOL), an E3 ubiquitin ligase that promotes lysosomal LDLR ubiquitination and degradation. We identify a multimolecular complex comprised of {beta}Pix, IDOL, and LDLR and demonstrate that {beta}Pix counteracts {beta}2AR-mediated LDLR degradation by sequestering IDOL. Our findings show {beta}Pix acts as a significant post-transcriptional regulator of IDOL-mediated LDLR degradation and identify {beta}2AR activation as a potential treatment for Alport pathology.

molecular biology↗

bioRxiv2019

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has lead to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable non-specific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of Francisella novicida Cas12a (FnCas12a) as it searches for its DNA target. Our results reveal concealed thermodynamic factors affecting FnCas12a DNA binding which should guide the design and optimization of crRNA that limit off-target effects, including the crucial role of an extended PAM sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

biophysics↗