Biology subjects

Silva, G. M.

Publications and source records attributed to Silva, G. M..

10 recordsLinked to original sources

bioRxiv2025

Mitochondrial control of fuel switching via carnitine biosynthesis

Environmental adaptation often involves a shift in energy utilization toward mitochondrial fatty acid oxidation, which requires carnitine. Besides dietary sources of animal origin, carnitine biosynthesis from trimethyllysine (TML) is essential, particularly for those who consume plant-based diets; however, its molecular regulation and physiological role remain elusive. Here, we identify SLC25A45 as a mitochondrial TML carrier that controls carnitine biosynthesis and fuel switching. SLC25A45 deficiency decreased the carnitine pool and impaired mitochondrial fatty acid oxidation, shifting reliance to carbohydrate metabolism. Slc25a45-deficient mice were cold-intolerant and resistant to lipid mobilization by GLP1 receptor agonist (GLP-1RA), rendering them resistant to adipose tissue loss. Our study suggests that mitochondria serve as a regulatory checkpoint in fuel switching, with implications for metabolic adaptation and the efficacy of GLP-1RA-based anti-obesity therapy.

biochemistry↗

bioRxiv2025

Mitochondria-derived PEP licenses glycerolipid synthesis via SLC25A35

Mitochondria provide a variety of metabolites, in addition to ATP, to meet cell-specific needs. One such metabolite is phosphoenolpyruvate (PEP), which contains a higher-energy phosphate bond than ATP and has diverse biological functions. However, how mitochondria-generated PEP is delivered to the cytosol and fulfills cell-specific requirements remains elusive. Here, we show that SLC25A35 regulates mitochondrial PEP efflux and glyceroneogenesis in lipogenic cells that utilize the pyruvate-to-PEP bypass. Reconstitution and structural studies demonstrated PEP transport by SLC25A35 in a pH gradient-dependent manner. Loss of SLC25A35 in adipocytes impaired the conversion of mitochondrial PEP into glycerol-3-phosphate, thereby reducing glycerolipid synthesis. Significantly, hepatic inhibition of SLC25A35 in obese mice alleviated steatosis and improved systemic glucose homeostasis. Together, these results suggest that mitochondria facilitate glycerolipid synthesis by providing PEP via SLC25A35, offering lipogenic mitochondria as a target to limit glycerolipid synthesis, a pivotal step in the pathogenesis of hepatic steatosis and Type 2 diabetes.

cell biology↗

bioRxiv2025

Derepression of transposable elements in mouse prefrontal cortex disrupts social behavior

Here, we present a synthetic biology approach to assess the social behavioral consequences of altered function of the Kruppel-associated box zinc finger protein (KZFP) interacting protein TRIM28 within the prefrontal cortex (PFC) of male and female mice. We reprogrammed natural TRIM28WT by replacing the transcriptionally repressive domain with an enhanced transcriptional activation domain VP64-p65-Rta (TRIM28VPR), or by excising the transcriptional regulatory domain (TRIM28NFD). In vitro validation confirmed that TRIM28WT represses, and TRIM28VPR activates, the expression of a KZFP-regulated luciferase reporter gene. Upon intra-PFC viral-mediated delivery of TRIM28 variants, we observed that inversion of TRIM28 transcriptional control via HSV-TRIM28VPR reduced the salience of novel social interaction for male and female mice while not affecting non-social behaviors. RNA-sequencing revealed HSV-TRIM28VPR promoted transcriptional escape of all classes of TEs, particularly those located within intronic and distal enhancer regions of downregulated immune genes. HSV-TRIM28VPR-driven social deficits were reversible by intra-PFC repletion of interferon cytokines. These novel data point to PFC KZFP-TRIM28 interactions as necessary to stabilize TEs to enable cis-regulation of key immune gene expression and enhance organismal capacity for complex, pro-social behaviors.

neuroscience↗

bioRxiv2024

Regulation of transposons within medium spiny neurons enables molecular and behavioral responses to cocaine

A more complete understanding of the molecular mechanisms by which substance use is encoded in the brain could illuminate novel strategies to treat substance use disorders, including cocaine use disorder (CUD). We have previously discovered that Zfp189, which encodes a Kruppel-associated box zinc finger protein (KZFP) transcription factor (TF), differentially accumulates in nucleus accumbens (NAc) Drd1+ and Drd2+ medium spiny neurons (MSNs) over the course of cocaine exposure and is causal in producing MSN functional and behavioral changes to cocaine1. Here, we aimed to illuminate the brain cell-type specific molecular mechanisms through which this KZFP TF produces CUD-related brain changes, with emphasis on investigating transposable elements (TEs), since KZFPs like ZFP189 are known regulators of TEs2-6. First, we annotated TEs in existing single nuclei RNA-sequencing (snRNAseq) datasets of rodents that were exposed to either acute or repeated cocaine. We discovered that expression of NAc TEs was dramatically altered by cocaine experience, the most sensitive NAc cell-type was MSNs, and TEs in Drd1+ MSNs were considerably more dynamic over the course of cocaine exposure than TEs in Drd2+ MSNs. To determine the causality of this TE dysregulation within NAc MSNs in cocaine-induced brain changes, we virally delivered conditional synthetic ZFP189 TFs of our own design to Drd1+ or Drd2+ MSNs. These synthetic ZFP189 TFs are capable of directly activating (ZFP189VPR) or repressing (ZFP189WT) brain TEs2. We discover that behavioral and cell morphological adaptations to cocaine are produced by activating TEs with ZFP189VPR in Drd1+ MSNs or stabilizing TEs with ZFP189WT in Drd2+ MSNs, revealing a persistent opponent process balanced across MSN subtypes and weighted by TE stability and consequent gene expression within MSN subtype. We next performed snRNAseq of the whole NAc virally manipulated with ZFP189 TFs. We observed that, relative to ZFP189WT, NAc manipulated with ZFP189VPR impeded cocaine-induced gene expression in NAc cell-types, including both Drd1+ and Drd2+ MSNs. Within either MSN subtype, the consequence of normal ZFP189 function was to enhance immune-related gene expression, and ZFP189VPR impeded these gene expression profiles. We finally performed cocaine intravenous self-administration to determine the consequence of NAc ZFP189-mediated transcriptional control on cocaine use behaviors. We observed that ZFP189VPR impeded any increases in active lever responses following a period forced cocaine abstinence. This research demonstrates that KZFP-mediated transcriptional repression of TEs within NAc MSNs is a causal molecular step in enabling gene expression and subsequent cellular and behavioral responses to cocaine use, and the use of ZFP189VPR in this work demonstrates cell-type specific mechanistic strategies to block CUD-related brain adaptations, which may inform future CUD treatments.

neuroscience↗

bioRxiv2024

Localized K63 ubiquitin signaling is regulated by VCP/p97 during oxidative stress

Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote these concerted response mechanisms remain understudied. Here, we show that K63-linked ubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 10-fold the pool of proteins known to be ubiquitinated during arsenite stress (2,046) and revealed their involvement in pathways related to immune signaling and translation control. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by cellular exposure to reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.

cell biology↗

bioRxiv2023

Histone H1x in mouse ventral hippocampus correlates with, but does not cause behavioral adaptations to stress

Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrate that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8-10-week-old male C57BL/6J mice were randomly assigned to stressed and unstressed groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced experimental mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or unstressed mice. In sum, although we confirm vHipp protein levels of H1x correlate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are correlated with, but are not singularly sufficient to drive development of behavioral adaptations to stress.

neuroscience↗

bioRxiv2023

A zinc finger transcription factor tunes social behaviors by controlling transposable elements and immune response in prefrontal cortex

The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in the rodent prefrontal cortex was previously determined to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189VPR, which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT. Upon viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, dysregulation of ZFP189-mediated transcription in this brain area, achieved by delivery of synthetic ZFP189VPR, precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. By performing RNA sequencing in virally manipulated prefrontal cortex tissues, we discover that ZFP189 transcription factors of opposing regulatory function have opposite influence on the expression of genetic transposable elements as well as genes that participate in immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for social behaviors by binding transposable element-rich regions of DNA to regulate immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animals capacity for functioning in social groups.

neuroscience↗

bioRxiv2022

Effects of the Automatic Self-Transcending Meditation on cognition and mental states in the EEG, skin conductance and behavioral performance: a pilot study

The main purpose of this pilot study was to investigate the immediate effects of Automatic Self-Transcending (AST) meditation on the cognitive function, EEG activity and autonomic arousal (Study 1) and characterize the frontal EEG synchrony during resting state, cognitive activity and AST mental states in traditional and wireless EEGs (Study 2). We report the results of three healthy AST meditation volunteers in this case-report study (Case 1 - age = 26 years, meditative practice time = 2 months; Case 2 - age = 39 years, meditative practice time = 6 years; Case 3 - age = 59 years, meditative practice time = 40 years). In study 1, the volunteers performed a protocol with simultaneous recording of EEG and skin conductance while performing the Stroop test (T0), followed by 20 minutes of AST meditation and immediately the same protocol performed at T0 (T1). We analyzed P300 amplitude and latency, as well as test behavioral response and skin conductance activity before and immediately after a single session of AST. In study 2, the same volunteers performed three tasks with eyes closed on two EEG equipment (traditional and wireless): resting state, cognitive activity and one session of AST, each for 20 minutes. We measured the frontal interhemispheric coherence of alpha1 (8-10 Hz) and beta (13-30 Hz) for each condition and EEG type. Our main findings show that there is an immediate beneficial effect after AST meditation at the level of the same individual with different patterns of P300 and skin conductance activity and that AST meditation is marked by an overall increase in the frontal coherence of alpha1 and beta bands, when compared to other mental states. We conclude that 1) there is an immediate effect on cognition and executive control after AST meditation, 2) the frontal interhemispheric coherence of alpha1 and beta bands are increased during AST, and 3) wireless EEG exhibits the same characteristics observed in traditional EEG and therefore can be used to describe cortical dynamics during AST.

neuroscience↗

bioRxiv2021

Redox sensitive E2 Rad6 controls cellular response to oxidative stress via K63 ubiquitination of ribosomes.

Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Among its non-proteolytic functions, K63 ubiquitin accumulates in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63 ubiquitin conjugates accumulate because of redox inhibition of the deubiquitinating enzyme Ubp2, however, the role and regulation of ubiquitin conjugating enzymes in this pathway remained unclear. Here we found that the E2 Rad6 binds and modifies elongating ribosomes during oxidative stress. We elucidated a mechanism by which Rad6 and its human homolog UBE2A are redox-regulated by forming reversible disulfides with the E1 activating enzyme, Uba1. We further showed that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress. Finally, we showed that Rad6 is required to induce phosphorylation of the translation initiation factor eIF2, providing a novel link for K63 ubiquitin, elongation stalling, and the integrated stress response.

cell biology↗

bioRxiv2018

Site-specific K63 ubiquitinomics reveals post-initiation regulation of ribosomes under oxidative stress

During oxidative stress, K63-linked polyubiquitin chains accumulate in the cell and modify a variety of proteins including ribosomes. Knowledge of the precise sites of K63 ubiquitin is key to understanding its function during the response to stress. To identify the sites of K63 ubiquitin, we developed a new mass-spectrometry based method that quantified >1,100 K63 ubiquitination sites in yeast responding to oxidative stress induced by H2O2. We determined that under stress, K63 ubiquitin modified proteins involved in several cellular functions including ion transport, protein trafficking, and translation. The most abundant ubiquitination sites localized to the head of the 40S subunit of the ribosome, modified assembled polysomes, and affected the binding of translation factors. The results suggested a new pathway of post-initiation control of translation during oxidative stress and illustrated the importance of high-resolution mapping of noncanonical ubiquitination events.

molecular biology↗