Biology subjects

Serra, R.

Publications and source records attributed to Serra, R..

7 recordsLinked to original sources

bioRxiv2025

Single-nucleus RNA sequencing reveals the cellular diversity of cerebrospinal fluid in the context of intraventricular hemorrhage

BackgroundIntraventricular hemorrhage (IVH) is a common and severe complication of hemorrhagic brain injury. Current treatments offer limited improvement in long-term neurological outcomes. Inflammatory responses in the cerebrospinal fluid (CSF) after IVH are thought to drive secondary injury, but the cellular mechanisms underlying this inflammation remain poorly defined. MethodsWe performed single-nucleus RNA sequencing of leukocytes isolated from CSF collected through external ventricular drains in subjects with intracerebral (n = 6) or subarachnoid (n = 1) hemorrhage. We characterized transcriptionally distinct subpopulations of neutrophils, monocytes, and lymphocytes by comparison to reference datasets. Cell-cell signaling networks were analyzed to infer cytokine-mediated communication, and a flow cytometry panel was developed to validate transcriptomic findings in independent CSF samples. ResultsWe obtained 11,191 high-quality nuclei comprising neutrophils (53.8%), monocytes (26.1%), lymphocytes (17.8%), and non-immune cells (2.4%). Neutrophils segregated into Nascent, Quiescent, and Interferon-Activated states. Monocytes exhibited classical phenotypes that include interferon-activated states (characterized by expression of VCAN or PROK2) and CXC-chemokine expressing states (characterized by expression of CXCL5 or CXCL8). Lymphocytes were mainly naive and central memory CD4 T cells. Cell-cell signaling analysis predicted strong CXC chemokine signaling from monocytes to neutrophil subsets and IL-1 family-driven inflammatory responses across multiple populations. Type I and III interferon signaling defined a neutrophil population not previously described in the central nervous system. ConclusionThis study delineates the diverse cellular immune landscape of CSF after IVH. Transcriptomic profiles reveal interferon, IL-1, and CXC chemokine signaling networks as potential therapeutic targets to mitigate secondary injury.

genomics↗

bioRxiv2024

RiboTag RNA Sequencing Identifies Local Translation of HSP70 In Astrocyte Endfeet After Cerebral Ischemia

Brain ischemia causes disruption in cerebral blood flow and blood-brain barrier (BBB) integrity which are normally maintained by the astrocyte endfeet. Emerging evidence points to dysregulation of the astrocyte translatome during ischemia, but its effects on the endfoot translatome are unknown. In this study, we aimed to investigate the early effects of ischemia on the astrocyte endfoot translatome in a rodent model of cerebral ischemia-reperfusion. To do so, we immunoprecipitated astrocyte-specific tagged ribosomes (RiboTag IP) from mechanically isolated brain microvessels. In mice subjected to middle cerebral artery occlusion and reperfusion and contralateral controls, we sequenced ribosome-bound RNAs from perivascular astrocyte endfeet and identified 205 genes that were differentially expressed in the translatome after ischemia. Pathways associated with the differential expressions included proteostasis, inflammation, cell cycle, and metabolism. Transcription factors whose targets were enriched amongst upregulated translating genes included HSF1, the master regulator of the heat shock response. The most highly upregulated genes in the translatome were HSF1-dependent Hspa1a and Hspa1b, which encode the inducible HSP70. We found that HSP70 is upregulated in astrocyte endfeet after ischemia, coinciding with an increase in ubiquitination across the proteome. These findings suggest a robust proteostasis response to proteotoxic stress in the endfoot translatome after ischemia. Modulating proteostasis in endfeet may be a strategy to preserve endfeet function and BBB integrity after ischemic stroke.

neuroscience↗

bioRxiv2024

SRY-Box Transcription Factor 9 regulates 3'-Phosphoadenosine 5'-Phosphosulfate Synthase 2 mRNA expression through derepression of the transcriptional repressor, CCAAT/enhancer-binding protein beta

Previously, we showed that Papss2 expression is regulated through a Sox9-dependent pathway. Here we explore molecular mechanisms whereby Sox9 regulates mouse Papss2. A 509bp Sox9-responsive DNA element (Region C) was identified upstream of the Papss2 second start site using co-transfection and luciferase reporter assays. A Sox9 responsive element was narrowed down to 32bps within Region C (Sox9RE). Putative SoxE and C/EBP{beta} binding sites were identified within S9RE. C/EBP{beta} was identified as a repressor for Sox9-mediated activity. In cells transfected with expression vectors for C/EBP{beta} and Sox9, increasing amounts of C/EBP{beta} resulted in attenuation of Sox9-mediated activation of Region C while increasing amounts of Sox9 activated transcription in the presence of C/EBP{beta}. Using electromobility shift assays, three protein complexes were identified on S9RE after incubation with nuclear extracts from ATDC5 cells. Super shift assays indicated that under basal conditions C/EBP{beta} was present in the DNA-protein complexes observed. Unlabeled S9RE with point mutations in the predicted SoxE binding site competed with protein complex formation on the S9RE while excess oligo corresponding to the predicted SoxE binding site did not, suggesting that proteins do not bind to SoxE motiff under basal conditions. Under conditions of high Sox9 expression, the formation of protein-DNA complexes on S9RE was inhibited. We then showed by western blot that increasing Sox9 protein resulted in reduced C/EBP{beta} protein levels. Co-immunoprecipitation indicated interaction of Sox9 and C/EBP{beta} proteins. We propose that Sox9 acts to derepress C/EBP{beta}-inhibited transcription of Papss2 by first interacting with C/EBP{beta} to prevent it from binding DNA, then reducing C/EBP{beta} expression.

molecular biology↗

bioRxiv2021

TGFβ signaling is required for sclerotome resegmentation during development of the spinal column in Gallus gallus.

We previously showed the importance of TGF{beta} signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGF{beta} signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGF{beta}R1 inhibitor, SB431542, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in SB431542 injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGF{beta} signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro- caudal markers within sclerotome was disrupted at E3.5 after treatment with SB431542 with rostral domains expressing both rostral and caudal markers. We propose that TGF{beta} signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

developmental biology↗

bioRxiv2020

Canonical and noncanonical TGF-β signaling regulate fibrous tissue differentiation in the axial skeleton

Previously, we showed that embryonic deletion of TGF-{beta} type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-{beta} regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-{beta} stimulated expression of Scleraxis mRNA by two hours and Fibromodulin and Adamtsl2 mRNAs by eight hours of treatment. Regulation of Scleraxis by TGF-{beta} did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-{beta}-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-{beta}-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-{beta} treatment. Next, the role of several noncanonical TGF-{beta} pathways were tested. We found that ERK1/2 was activated by TGF-{beta} and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-{beta} regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGF{beta}-mediated fibrous differentiation in sclerotome.

developmental biology↗

bioRxiv2020

Tgfbr2 in dental pulp cells guides sensory innervation in developing teeth

Transforming growth factor {beta} (TGF{beta}) plays an important role in tooth morphogenesis and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in the dental pulp (DP) mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2cko). These mice survived postnatally, but had significant defects in bones and teeth, including reduced mineralization and short roots. H&E staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity was localized within the tooth and was active in the DP and derivatives, but not in neurons. Previous research has established that paracrine signals from the dental pulp attract trigeminal axons toward and into the tooth during postnatal development, yet very little is known about the signals that regulate tooth innervation. Immunofluorescent staining for a neuronal marker, {beta}3 tubulin, was performed on serial cryosections from control and mutant molars at two different postnatal stages. Confocal imaging and pixel quantification of {beta}3 tubulin demonstrated reduced innervation in P7 and P24 Tgfbr2cko first molars compared to controls, indicating that the signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. Quantitative real time PCR showed that the mRNA expression levels of several neuronal genes were reduced. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2f/f DP cells. Tgfbr2 in the DP was deleted with an adenovirus expressing Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured in the presence of Tgfbr2-positive DP cells compared to neurons cultured alone. Further, axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. These results indicate that Tgfbr2 in the DP mesenchyme regulates paracrine signals that guide sensory innervation of the tooth. Note: A list of abbreviations is included in the appendix.

neuroscience↗

bioRxiv2020

Cytosine methylation dynamics during post-testicular sperm maturation in mammals

Beyond the haploid genome, mammalian sperm contribute a payload of epigenetic information which can modulate offspring phenotypes. Recent studies have shown that the small RNA payload of sperm undergoes extensive remodeling during post-testicular maturation in the epididymis. Intriguingly, epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in remodeling the sperm epigenome. Here, we build on prior studies of the maturing sperm methylation landscape, further characterizing the genome-wide methylation landscape in seven germ cell populations collected from throughout the male reproductive tract. Overall, we find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is largely stable throughout post-testicular maturation. Intriguingly, although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, was present only in the caput epididymis of virgin males, where it was associated with citrullinated histone H3 and presumably resulted from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of the cytosine methylation landscape in mammalian sperm, and identify a surprising but transient period during which immature sperm are associated with extracellular DNA.

developmental biology↗