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Hippocampal Output to the Septum Regulates Locomotion and Exploration
Get PDFThe cognitive maps are established in the dorsal hippocampus (DH) when the animal explores the environment. During exploration, the DH combined extrinsic inputs from multiple sensory and motor-related structures to generate locomotion-dependent activity; however, the hippocampal mechanisms of locomotion and exploratory behavior remain controversial and unclear. By using optogenetics and chemogenetics, we selectively manipulated the DH pan-inhibitory interneurons (INs) and revealed that disrupting the DH inhibitory circuit is sufficient to acutely induce hyperlocomotion. Furthermore, we found that the DH-septum inhibitory pathway is critical for regulation of locomotion and exploratory behavior. Activation of the DH inhibitory output to the medial septum complex (MSc) decreased animals' distance traveled and increased immobility in the open field. Alternatively, inhibition of the DH inhibitory output to the MSc increased maximum speed and exploratory behavior. Moreover, activation of the septum-projecting DH INs is sufficient to decrease locomotion and exploratory behavior. On the other hand, activation of a subset of retrosplenial cortex-specific projecting DH INs or the entorhinal cortex-projecting DH neurons did not change animal locomotion. For the clinical application, targeting the DH-MSc inhibitory pathway can help us to determine the neuronal mechanisms of locomotion in animal models. Therefore, we may be able to ameliorate spatial processing deficits and motor symptoms in patients with memory impairment or psychosis and find therapeutic treatments for them
On Sulfur Sensing in Saccharomyces cerevisiae
Get PDFThe unique chemistry available to sulfur compared to oxygen, such as the ability to exist in numerous oxidation states and greater nucleophilicity, makes many of the biochemical reactions requisite for cellular life possible. As a result of this critical importance, organisms have developed several mechanisms for sensing and maintaining levels of sulfur-containing metabolites. In the yeast Saccharomyces cerevisiae, regulation of sulfur metabolism can be distilled down to the actions of two proteins; the F-box protein Met30, and the transcriptional coactivator Met4. Met30 belongs to the family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases, and negatively regulates the transcriptional activity of the master transcriptional activator of sulfur metabolism genes, Met4, via oligo-ubiquitination when sulfur metabolite levels are high. When yeast are starved of sulfur, Met30 ceases to ubiquitinate Met4, releasing it to be deubiquitinated and transcriptionally active to boost levels of a network of sulfur metabolic genes known as the MET regulon to restore sulfur metabolite levels. While the molecular activities of both Met30 and Met4 have been extensively studied over the last two decades, the biochemical basis for sulfur-sensing by the Met30 E3 ligase has remained unknown. Herein, I reveal the biochemical details by which Met30, the master regulator of sulfur metabolism, senses the availability of sulfur metabolites to modulate its E3 ligase activity to regulate sulfur metabolism in yeast. Utilizing a combination of yeast genetics and biochemical assays, I show that Met30 uses redox-active cysteine residues in its C-terminal WD-40 repeat region to modulate binding between itself and its substrate Met4 in accordance with the availability of sulfur metabolites. These insights represent significant advances in the understanding of sulfur metabolic regulation in yeast
AXL Targeting Restores PD-1 Blockade Sensitivity of STK11/LKB1 Mutant NSCLC Through Expansion of TCF1+ CD8 T Cells
No full textMutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB) for unknown reasons. We found that introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically, this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. We found that systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expands tumor-associated TCF1+ PD-1+ CD8 T cells, restoring therapeutic response to PD-1 ICB for KPL tumors. This effect was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. Anecdotal NSCLC patients with STK11/LKB1 mutant tumors also demonstrated responses to the combination of AXL inhibitor bemcentinib and pembrolizumab. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC
Health equity grands rounds: exploring some complexities of bias & health equity
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern's Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Parkland's Global Diabetes Program: 10 years of collaboration, growth, and success
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern's Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Iridium-Catalyzed Enantioselective Allylation of Alkenyl Boronates
No full textOrganoboronic esters are highly functionalizable synthetic intermediates owing to their unique reactivity that allows for pre-complexation with organometallic reagents to form boronates, which can then engage in bimolecular reactions. Furthermore, incorporation of boronic esters into their corresponding products allows for subsequent diversification through a wide variety of synthetically useful transformations. Some of the most valuable transformations of organoboronic esters involve 1,2-metalate shifts from anionic "ate" complexes. First described are select methodologies that showcase the use of this 1,2-metalate rearrangement from organoboron compounds over the past several decades. This includes 1,2-metalate shifts onto sp3 and sp2-hybridized adjacent carbons through intramolecular expulsion of the leaving group, as well as stoichiometric use of external electrophiles. Select catalyst-promoted 1,2-metalate rearrangements will be discussed that engage alkenyl boronates and in some cases render the migration stereoselective. Additionally, select examples of iridium-catalyzed nucleophilic substitution reactions will be examined. The second chapter describes the reaction of alkenyl boronates with allylic carbonates to generate tertiary bis-homoallyl boronic esters with high enantioselectivity and 1,3-diastereocontrol. The three-component coupling features an Ir(phosphoramidite) complex, which catalyzes a kinetic resolution of secondary allylic carbonates. Alkenyl boronate addition to an Ir(π-allyl) intermediate and a 1,2-metalate shift provides the observed products. Synthetic transformations of the tertiary boronic ester provide access to quaternary stereocenters in a diastereoselective manner. An extension to trisubstituted olefins sets three-contiguous stereocenters and provides initial insights into the diastereoselectivity of the reaction through a conserved syn-addition pathway. The final chapter provides detailed mechanistic investigations that outline the overall catalytic cycle and reveal trends in reactivity and selectivity. Analysis of relative stereochemistry in a variety of 1,1-disubtituted alkenyl boronates provides insight into the transition state of the addition and indicates a concerted pathway. Kinetic analysis of the reaction revealed the kinetic order dependence in boronate, catalyst, and both the slow- and fast-reacting enantiomer of allylic carbonate as well as the turnover-limiting step of the reaction. Hammett studies explored substituent effects in both aryl-derived alkenyl boronates and aryl carbonates. Nucleophile-specific parameters N and sN for the alkenyl boronate complex were determined and compared to other classes of compounds. Initial investigations into the migratory selectivity of the 1,2-metalate shift were also examined using (bis)alkenyl boronates
Ghosts in the Making
Get PDFThe author submitted this entry in the Open Verse Poetry category (Amateur division) for the 2024 On My Own Time (OMOT) Art Show.This piece is inspired by someone who was and still is, but will never be
Investigation of Accessory Olfactory Interneuron Physiology and Function After Experience-Dependent Plasticity
Get PDFThe accessory olfactory system (AOS) is critical for the development and expression of social behavior. Through the detection of non-volatile chemosignals, the AOS has been specifically implicated in the display of sex-typical behavior, such as mating, maternal behavior, and territorial aggression. The proper display of these sex-typical behaviors is essential for rodents to successfully navigate through their environments. The first dedicated circuit in the AOS, the accessory olfactory bulb (AOB), exhibits cellular and network plasticity in male and female mice after social experience. In the AOB, interneurons called internal granule cells (IGCs) express the plasticity-associated immediate-early gene Arc following intermale aggression or mating. Here, I sought to understand how Arc-expressing IGCs shape AOB information processing and social behavior in the context of territorial aggression over multiple days. I used "ArcTRAP" (Arc-CreERT2) transgenic mice to selectively and permanently label Arc-expressing IGCs following intermale resident-intruder interactions. Using whole-cell patch clamp electrophysiology, I found that Arc-expressing IGCs display increased intrinsic excitability for several days after a single resident-intruder interaction. Further, I found that Arc-expressing IGCs maintain this increased excitability across repeated resident-intruder interactions, during which resident mice increase or "ramp" their aggression. I tested the hypothesis that Arc-expressing IGCs participate in ramping aggression. Using a combination of ArcTRAP mice and chemogenetics (Cre-dependent hM4D(Gi)-mCherry AAV injections), I found that disrupting Arc-expressing IGC activity during repeated resident-intruder interactions abolished the ramping aggression exhibited by resident male mice. This work shows that Arc-expressing AOB IGC ensembles are activated by specific chemosensory environments, and that regulation of their activity plays an integral role in the establishment and expression of sex-typical social behavior. These studies identify a population of plastic interneurons in an early chemosensory circuit that display physiological features consistent with simple memory formation, increasing our understanding of central chemosensory processing and mammalian social behavior
Carbon Starvation Metabolically Regulates Chromatin for Transcriptome Rewiring
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Cells robustly rewire their transcriptomes to survive under stress conditions. Yet, how does such reprogramming of gene expression occur? Under favorable nutrient conditions, acetyl-CoA normally promotes histone acetylation to activate genes required for cell growth. However, glucose starvation significantly reduces the availability of acetyl-CoA. And it is unclear how such a change impacts genome-wide histone acetylation and gene expression. In this study, I set up a robust glucose starvation model in budding yeast to discover a mechanism by which cells preserve acetyl-CoA, a key intermediate in energy metabolism, in order to sustain histone acetylation for gene activation even under stress conditions.
I demonstrate a dramatic redistribution of histone acetylation upon glucose starvation. Mechanistically, I determined that a major histone deacetylase (HDAC) releases acetyl groups from histones at growth-promoting genes, which can subsequently be used to acetylate histones at a distinctive set of stress-responsive genes. Strikingly, bioinformatic analysis revealed these genes to be required for gluconeogenic and fat metabolism, which are metabolic pathways that generate acetyl-CoA for oxidation and ATP synthesis. Genetic deletion of histone modifiers mediating this reallocation, including the key HDAC or histone acetyltransferase (HAT), disrupts proper transcriptome rewiring for survival. Given the importance of acetate for recycling the acetyl- group, I next characterize acetyl-CoA synthetases (Acs), metabolic enzymes that convert acetate to acetyl-CoA. I demonstrate that Acs2 is required for maintaining global histone acetylation, yet its nuclear localization appears to be dispensable for such regulation. I observe that the catalytic activity of Acs2 governs the intracellular acetyl-CoA level and global histone acetylation amounts. Compromising its activity leads to up-regulation of ergosterol biosynthetic pathways in addition to gluconeogenic and fat metabolism genes upon glucose starvation.
In summary, I reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting. I have elucidated how transcriptome rewiring is driven by reallocation of histone acetylation. My findings present a mechanism by which cells recycle acetyl groups to differentially acetylate histones for activation of key genes required for metabolism and survival
Analysis of Interrelationships Among NAD+, PARP1, ADP-Ribosylation, and Splicing in Murine Embryonic Stem Cells
Get PDFThe differentiation of embryonic stem cells (ESC) into a lineage-committed state is a dynamic process involving changes in epigenetic modifications, gene expression, RNA processing, and cellular metabolism. Previous studies have implicated poly(ADP-ribose) polymerase 1 (PARP1), an abundant nuclear enzyme that plays key roles in a variety of nuclear processes, in ESC self-renewal and lineage commitment. Given the diverse molecular functions of PARP1, I sought to determine the potential regulatory role of PARP1 in determining ESC state. PARP1 functions both as an enzyme, through its NAD+-dependent ADP-ribosyltransferase catalytic activity, and as a structural protein, through its NAD+-independent nucleic acid binding activity. I observed a dramatic induction of PARP1 catalytic activity during the early stages of mESC differentiation (e.g., within 12 hours of LIF removal) leading me to query the regulation and outcome of PARP1-mediated ADP-ribosylation in mESCs.
NAD+ is synthesized through three main pathways - De novo, Salvage, and Preiss-Handler - and is constrained within cellular compartments. I found that both pathway usage and subcellular localization were dynamic during differentiation in a PARP1-dependent manner, with transition from De novo to Salvage pathway usage and increases in nuclear NAD+ levels upon differentiation feeding PARP1 catalytic activity. Using an NAD+ analog-sensitive PARP (asPARP) chemical biology approach, I characterized the PARP1-mediated ADP-ribosylated proteome during mESC differentiation. PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. In addition, I observed a genome-wide dysregulation of splicing events upon loss of PARP1 in transcriptomic analysis. These results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC cell state, specifically in the splicing program during differentiation