T2

Investigating the nonlinear complex dynamics of the tuft cell-microbiome cross-talk: the impact of feedback loops on immune regulation, microbial modulation and response to tissue insults

Project Abstract Tuft cells (TCs) are specialized chemosensory epithelial cells that are emerging as critical regulators of intestinal homeostasis. Named over 70 years ago based on their distinct morphology, a defined function for TCs was only elucidated in the last decade. TCs in the small intestine sense succinate from helminths to initiate type 2 immune responses that mediate parasite expulsion. Recently, we discovered a novel physiologic function for TCs in the colon, where their role had been considered minimal. Succinate, a key microbial metabolite, is produced by colonic microbiota as both a precursor to other metabolites and a cross-feeding fuel source for pathogens. TCs respond to succinate by secreting interleukin-25 (IL-25), which activates type 2 cytokine- producing lymphocytes (T2Ls), amplifying TC expansion and reinforcing barrier function. We recently demonstrated that this SPB–TC–IL-25–T2L feedback loop is essential for protection against pathogen-induced colitis. Our preliminary data further suggest that TCs actively promote colonization by succinate-producing bacteria (SPBs), establishing positive feedback on TC-supporting microbes, while other epithelial cells such as goblet cells (GCs) and Paneth cells (PCs) may exert complementary or counterbalancing influences. Supported by new modeling insights, we hypothesize that these epithelial–immune–microbiome interactions form coordinated feedback loops that collectively optimize intestinal resilience. These loops may create a dynamic, multi-stable system that flexibly transitions between homeostatic and hyperplastic states, buffering against microbial fluctuations and pathogenic insults while preventing uncontrolled type 2 inflammation. Using a combination of mathematical modeling and experimental validation, we will develop a multi- layered systems framework to explore how epithelial–immune–microbial feedbacks shape resilience or breakdown in clinically relevant models of colonic infection and inflammation. Our three Aims will (1) develop, calibrate, and validate a mathematical model that integrates TCs, GCs, PCs, SPBs, and SCBs; (2) define the immunological circuits governing epithelial–microbiome equilibrium; and (3) determine how epithelial feedbacks regulate microbial community structure and resilience. In line with NIH’s new initiative to prioritize human-based research, our proposal combines computational modeling, human colonic organoids, and complementary mouse models. Organoid experiments will provide human-relevant data for model calibration, while in vivo studies validate systemic predictions, ensuring both rigor and translational relevance while minimizing reliance on animal models. This work will generate interoperable models that integrate epithelial, microbial, and immune networks, providing predictive insight into intestinal outcomes under homeostatic, infectious, and inflammatory conditions and informing therapeutic strategies for microbiome-targeted interventions.

Dosing and Deployment Trial: A Home-based Optokinetic Treatment for Ipsilesional Gaze Deviation

Stroke can have devastating consequences including ipsilesional gaze deviation (IGD), which directly impacts mobility and falls. IGD, a hallmark sign of spatial neglect (SN), is a major predictor of poor recovery and can persist after inpatient rehabilitation with targeted treatments. Our preliminary data show that more than half of stroke survivors who have SN at the time of admission to inpatient rehabilitation still have SN at time of discharge, even after treatment. Therefore, because of the challenges of the traditional rehabilitation paradigm we need to bring treatments into the home setting. We plan to examine the feasibility and deployment of Eyemove, an optokinetic stimulation treatment, which induces brain neural plasticity and improves spatial exploration, in turn reducing SN symptoms, including IGD. We hypothesize that by treating IGD, improvements in mobility and fall risk scores will occur, as participants can now interact with the space that was previously “neglected”. Here, we propose to test the following aims with 50 community-dwelling individuals with SN, by identifying the practical dosage associated with mobility improvement: Aim 1 will determine feasibility and acceptability of home deployment of Eyemove. We will collect qualitative information from stroke survivors and their care partners, to determine their pre-treatment and post-treatment perspectives of this home treatment. Aim 2 will determine whether Eyemove in the home is associated with improved mobility-related outcomes (including risk of falls) and to evaluate sufficient dosing. We will randomize participants into either 3 or 5 sessions of a 40-minute treatment given over a week-long intervention period. The primary outcome will be the Mobility Assessment Course and secondary outcomes will be the Stroke Assessment of Fall Risk and the Life Space Assessment. For Aim 1, we expect to learn practical suggestions for home implementation and obtain reports of post-experience enthusiasm and acceptability for specific aspects of the intervention. Our hypotheses for Aim 2 are: 1a-- After controlling for pre-treatment score changes (T2-T1), the intervention (T3) will lead to improved mobility/ fall risk compared to baseline (T1), regardless of treatment group; 1b-- The amount of mobility/ fall risk improvement (T3-T1) in the 3- session and 5-session groups will be different. The expected findings will provide critical insight into the use of Eyemove for spatial neglect remediation. Results from this research will be used to develop a subsequent R01 proposal that uses pragmatic, randomized clinical trial methods to determine the efficacy of Eyemove, in order to provide an effective, accessible treatment to remediate SN at home and improve individuals’ ability to move without spatial bias or risk of falls.

A novel MRI method for noninvasive imaging of bone quality in type 2 diabetes

ABSTRACT: Type 2 diabetes mellitus (T2DM) affects 500 million of the global population, which is expected to increase to 800 million in 20 years. One of the multiple complications involved with T2DM is the significantly increased bone fracture risk and post-fracture mortality. Dual-energy X-ray absorptiometry (DXA) scans are routinely performed to measure bone mineral density (BMD) and associated fracture risk. However, T2DM patients often show preserved or even elevated BMD despite the significantly increased fracture risk. This mismatch between the BMD measurement and actual fracture risk hampers the accurate assessment of fracture risk and the appropriate treatment of T2DM that considers patient bone health. The lack of an accurate fracture risk assessment tool also confounds the evaluation of the bone health effect of antidiabetic drugs, including recently highlighted glucagon-like peptide-1 receptor agonists (e.g., semaglutide) and sodium-glucose cotransporter-2 inhibitors. Previous studies have suggested that bone quality, rather than bone quantity, as represented by BMD, is a crucial factor contributing to fracture risk in T2DM settings. Collagen crosslinking via advanced glycation end-products (AGEs) in cortical bone has been identified as a distinctive bone quality characteristic of T2DM patients, which explains the increased bone fragility. Although this finding is highly promising for improving the bone health management of T2DM patients, currently, no non-invasive method can monitor collagen crosslinking in the bones. This proposal aims to develop an ultrashort echo time (UTE) MRI-based method for measuring the degree of bone collagen crosslinking by quantifying magnetization transfer between water and collagen in the bone. This method, termed UTE-quantitative magnetization transfer (UTE-qMT) MRI, measures not only the quantity of macromolecules (e.g., collagen) in the bone but also the rates of exchange between water and macromolecular protons, which are related to the degree of collagen crosslinking. The proposal will develop and optimize the accelerated UTE-qMT method for reliably measuring the exchange rate in Aim 1. The optimized technique will be validated by correlating exchange rates with AGE-driven collagen crosslinking and subsequent compromise of bone mechanical properties in Aim 2. Finally, the optimized UTE-qMT MRI method will be translated to animal and human studies to demonstrate its clinical feasibility for investigating the effect of antidiabetic drugs on bone health in patients with T2DM in Aim 3. The successful completion of these aims will enable rapid and accurate assessment of bone fracture risk in patients with T2DM. Furthermore, noninvasively probing bone quality can also accurately assess the effect of antidiabetic drugs on bone health and aid in screening novel T2DM therapeutics for their impact on bone health.

Characterizing adipocyte heterogeneity in response to metabolic stress

Project Summary Adipose tissue is a central player in metabolism, storing energy healthily under normal conditions but becoming dysfunctional when overloaded. This can lead to the development of metabolic disease, most notably insulin resistance and type 2 diabetes (T2D). Understanding the contribution of adipose tissue to these complications requires knowledge of the individual cell types within adipose tissue and how they respond to different metabolic conditions. My previous work used single nucleus RNA sequencing to profile the cell types in adipose tissue and identified a number of subpopulations of white adipocytes that are differentially associated with clinical characteristics such as body mass index. In this grant, I now aim to better understand how a diverse array of stimuli influences adipocyte development and specification, the role that intra-individual variation plays in the response to these stimuli, and a better understanding of the relationship of adipocyte state to the development of metabolic disease. To do this, I propose using a model in which I can study human adipocyte development and function in mice to perform experiments such as high fat diet and cold exposure that are well-characterized in mice but not in humans. By performing experiments using cells from humans with a range of starting clinical characteristics, I can determine what changes will happen in response to a stimuli in all individuals verses those that only occur in specific populations. The experience that I have in characterizing adipocytes and adipose tissue both at the bench and computationally make me uniquely positioned to answer these questions. Taken together, these studies can test the behavior of adipocyte subpopulations from different people and under different conditions, ultimately leading to a better understanding of how subpopulations develop and, eventually, how we can target these populations to treat metabolic disease.

Chromatin-Based Mechanisms Linking Transcriptional Dysregulation to Genome Instability in Neurodevelopmental Disorders.

PROJECT SUMMARY/ABSTRACT Neurons depend on a finely tuned interplay between chromatin regulation and genome maintenance, yet they are acutely vulnerable to DNA damage generated during activity-dependent transcription of long, synaptic genes. Disruption of this balance is increasingly recognized as a driver of neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD), intellectual disability, and epilepsy. High-confidence genetic studies converge on regulators of histone H3 lysine 4 (H3K4) methylation, such as the writers ASHIL and Klv1T2C and the eraser KDNISB, as recurrently mutated loci in NTIDs. The overarching goal of this study is to investigate how dysregulated H3K4 methylation compromises genome integrity in human neurons, thereby contributing to the pathogenesis of NDDs. The central, hypothesis is that coordinated II3K4 methylation safeguards neuronal genomes by maintaining an open chromatin architecture that permits the efficient detection and repair of transcription-coupled DNA lesions. The rationale/Or this study is to define the epigenetic control of DNA repair, which will illuminate a shared pathogenic hub across multiple ~I)D-linked genes. During the mentoredK99 phase, I will define how ASHIL, KMT2C, and KDM5B regulate chromatin structure and DNA repair at baseline and during transcriptional stress. Aim-1: I will use isogenic iPSC-derived cortical neurons with patient-relevant mutations or CRrSPRi knockdowns of these regulators, applying an integrated multi-omic pipeline: CUT&Tag and Micro-C to map H3K4 methylation and 3D chromatin topology. Aim-2: I will use Paired-Damage-seq, and CUT&RUN to chart oxidative lesions, repair synthesis, and recruitment of key repair factors; and RNA-seq to relate damage hotspots to altered gene expression. Aims l and 2 will be performed under the guidance of Dr. Lizarraga and Dr. Morrow, experts in the field of neurodevelopmental biology. My advisory team brings unique and complementary skills, enhancing my knowledge in 3D chromatin structure, transcription-coupled repair, gene editing, and multi-omics analysis. I will utilize these skills in the R00 phase (Aim 3), expanding the framework to include additional H3K4 regulators (e.g., LSD1, KMT2A) and broader neural lineages, thereby developing a comprehensive model. This study is innovative in its integration of single-cell D.NA damage mapping with chromatin topology and transcriptional profiling, enabling a direct and mechanistic connection between disrupted H3K4 methylation and genome instability. By uncovering how H3.K4 methylation prevents transcription-coupled genome instability in the developing brain, this research will address a critical gap in our understanding of NDD mechanisms. This award will enable me to launch an independent research program dedicated to determining mechanisms of chromatin-based processes that maintain genome stability in the developing human brain.

In vivo direct imaging of neuronal activity at high temporospatial resolution

Advanced noninvasive neuroimaging methods provide valuable information on the brain function, but they have obvious pros and cons in terms of temporal and spatial resolution. Functional magnetic resonance imaging (fMRI) using blood-oxygenation-level-dependent (BOLD) effect provides good spatial resolution in the order of millimeters, but has a poor temporal resolution in the order of seconds due to slow hemodynamic responses to neuronal activation, providing indirect information on neuronal activity. In contrast, electroencephalography (EEG) and magnetoencephalography (MEG) provide excellent temporal resolution in the millisecond range, but spatial information is limited to centimeter scales. Therefore, there has been a longstanding demand for noninvasive brain imaging methods capable of detecting neuronal activity at both high temporal and spatial resolution. In this talk, I will introduce a novel approach that enables Direct Imaging of Neuronal Activity (DIANA) using MRI that can dynamically image neuronal spiking activity in milliseconds precision, achieved by data acquisition scheme of rapid 2D line scan synchronized with periodically applied functional stimuli. DIANA was demonstrated through in vivo mouse brain imaging on a 9.4T animal scanner during electrical whisker-pad stimulation. DIANA with milliseconds temporal resolution had high correlations with neuronal spike activities, which could also be applied in capturing the sequential propagation of neuronal activity along the thalamocortical pathway of brain networks. In terms of the contrast mechanism, DIANA was almost unaffected by hemodynamic responses, but was subject to changes in membrane potential-associated tissue relaxation times such as T2 relaxation time. DIANA is expected to break new ground in brain science by providing an in-depth understanding of the hierarchical functional organization of the brain, including the spatiotemporal dynamics of neural networks.

Programmed axon death: from animal models into human disease

Programmed axon death is a widespread and completely preventable mechanism in injury and disease. Mouse and Drosophila studies define a molecular pathway involving activation of SARM1 NA Dase and its prevention by NAD synthesising enzyme NMNAT2 . Loss of axonal NMNAT2 causes its substrate, NMN , to accumulate and activate SARM1 , driving loss of NAD and changes in ATP , ROS and calcium. Animal models caused by genetic mutation, toxins, viruses or metabolic defects can be alleviated by blocking programmed axon death, for example models of CMT1B , chemotherapy-induced peripheral neuropathy (CIPN), rabies and diabetic peripheral neuropathy (DPN). The perinatal lethality of NMNAT2 null mice is completely rescued, restoring a normal, healthy lifespan. Animal models lack the genetic and environmental diversity present in human populations and this is problematic for modelling gene-environment combinations, for example in CIPN and DPN , and identifying rare, pathogenic mutations. Instead, by testing human gene variants in WGS datasets for loss- and gain-of-function, we identified enrichment of rare SARM1 gain-of-function variants in sporadic ALS , despite previous negative findings in SOD1 transgenic mice. We have shown in mice that heterozygous SARM1 loss-of-function is protective from a range of axonal stresses and that naturally-occurring SARM1 loss-of-function alleles are present in human populations. This enables new approaches to identify disorders where blocking SARM1 may be therapeutically useful, and the existence of two dominant negative human variants in healthy adults is some of the best evidence available that drugs blocking SARM1 are likely to be safe. Further loss- and gain-of-function variants in SARM1 and NMNAT2 are being identified and used to extend and strengthen the evidence of association with neurological disorders. We aim to identify diseases, and specific patients, in whom SARM1 -blocking drugs are most likely to be effective.

The circadian clock and neural circuits maintaining body fluid homeostasis

Neurons in the suprachiasmatic nucleus (SCN, the brain’s master circadian clock) display a 24 hour cycle in the their rate of action potential discharge whereby firing rates are high during the light phase and lower during the dark phase. Although it is generally agreed that this cycle of activity is a key mediator of the clock’s neural and humoral output, surprisingly little is known about how changes in clock electrical activity can mediate scheduled physiological changes at different times of day. Using opto- and chemogenetic approaches in mice we have shown that the onset of electrical activity in vasopressin releasing SCN neurons near Zeitgeber time 22 (ZT22) activates glutamatergic thirst-promoting neurons in the OVLT (organum vasculosum lamina terminalis) to promote water intake prior to sleep. This effect is mediated by activity-dependent release of vasopressin from the axon terminals of SCN neurons which acts as a neurotransmitter on OVLT neurons. More recently we found that the clock receives excitatory input from a different subset of sodium sensing neurons in the OVLT. Activation of these neurons by a systemic salt load delivered at ZT19 stimulated the electrical activity of SCN neurons which are normally silent at this time. Remarkably, this effect induced an acute reduction in non-shivering thermogenesis and body temperature, which is an adaptive response to the salt load. These findings provide information regarding the mechanisms by which the SCN promotes scheduled physiological rhythms and indicates that the clock’s output circuitry can also be recruited to mediate an unscheduled homeostatic response.

Keeping axons alive after injury: Inhibiting programmed axon death

Activation of pro-degenerative protein SARM1 in response to diverse physical and disease-relevant injuries triggers programmed axon death. Original studies indicated substantially decreased levels of SARM1 were required for neuroprotection. However, we demonstrate that lowering SARM1 levels by 50% in Sarm1 haploinsufficient mice delays axon degeneration in vivo (after sciatic nerve transection), in vitro (in response to diverse traumatic, neurotoxic, and genetic triggers), and partially prevents neurite outgrowth defects in mice lacking pro-survival factor NMNAT2. We also demonstrate the capacity for Sarm1 antisense oligonucleotides to decrease SARM1 levels by more than 50% which delays or prevents programmed axon degeneration in vitro. Combining Sarm1 haploinsufficiency with antisense oligonucleotides further decreases SARM1 levels and prolongs protection after neurotoxic injuries. These data demonstrate that axon protection occurs in a Sarm1 gene-dose responsive manner and that SARM1 lowering agents have therapeutic potential. Thus, antisense oligonucleotide targeting of Sarm1 is a promising therapeutic strategy against diverse triggers of axon degeneration.

Targeting the brain to improve obesity and type 2 diabetes

The increasing prevalence of obesity and type 2 diabetes (T2D) and associated morbidity and mortality emphasizes the need for a more complete understanding of the mechanisms mediating energy homeostasis to accelerate the identification of new medications. Recent reports indicate that obesity medication, 5-hydroxytryptamine (5-HT, serotonin)2C receptor (5-HT2CR) agonist lorcaserin improves glycemic control in association with weight loss in obese patients with T2D. We examined whether lorcaserin has a direct effect on insulin sensitivity and how this effect is achieved. We clarify that lorcaserin dose-dependently improves glycemic control in a mouse model of T2D without altering body weight. Examining the mechanism of this effect, we reveal a necessary and sufficient neurochemical mediator of lorcaserin’s glucoregulatory effects, via activation of brain pro-opiomelanocortin (POMC) peptides. We observed that lorcaserin reduces hepatic glucose production and improves insulin sensitivity. These data suggest that lorcaserin’s action within the brain represents a mechanistically novel treatment for T2D: findings of significance to a prevalent global disease.

From function to cognition: New spectroscopic tools for studying brain neurochemistry in-vivo

In this seminar, I will present new methods in magnetic resonance spectroscopy (MRS) we’ve been working on in the lab. The talk will be divided into two parts. In the first, I will talk about neurochemical changes we observe in glutamate and GABA during various paradigms, including simple motors tasks and reinforcement learning. In the second part, I’ll present a new approach to MRS that focuses on measuring the relaxation times (T1, T2) of metabolites, which reflect changes to specific cellular microenvironments. I will explain why these can be exciting markers for studying several in-vivo pathologies, and also present some preliminary data from a cohort of mild cognitive impairment (MCI) patients, showing changes that correlate to cognitive decline.

Programmed Axon Death and its Roles in Human Disease

Axons degenerate before the neuronal soma in many neurodegenerative diseases. Programmed axon death (Wallerian degeneration) is a widely-occurring mechanism of axon loss that is well understood and preventable in animals. Its aberrant activation by mutation of the pro-survival gene Nmnat2 directly causes axonopathy in mice with severity ranging from mild polyneuropathy to perinatal lethality. Rare biallelic mutations in the homologous human gene cause related phenotypes in patients. NMNAT2 is a negative regulator of the prodegenerative NADase SARM1. Constitutive activation of SARM1 is cytotoxic and the human SARM1 locus is significantly associated with sporadic ALS. Another negative regulator, STMN2, has also been implicated in ALS, where it is commonly depleted downstream of TDP-43. In mice, programmed axon death can be robustly blocked by deletion of Sarm1, or by overexpression, axonal targeting and/or stabilization of various NMNAT isoforms. This alleviates models of many human disorders including some forms of peripheral neuropathy, motor neuron diseases, glaucoma, Parkinson’s disease and traumatic brain injury, and it confers lifelong rescue on the lethal Nmnat2 null phenotype and other conditions. Drug discovery programs now aim to achieve similar outcomes in human disease. In order to optimize the use of such drugs, we have characterized a range of human NMNAT2 and SARM1 functional variants that underlie a spectrum of axon vulnerability in the human population. Individuals at the vulnerable end of this spectrum are those most likely to benefit from drugs blocking programmed axon death, and disorders associated with these genotypes are promising indications in which to apply them.

Chemogenetic activation of vGluT2-expressing neurons in the nodose ganglion of the left vagus nerve suppresses rapid-eye-movement sleep in mice

Constitutive 5-HT2C receptor knock-out facilitates fear extinction through altered activity of a dorsal raphe–bed nucleus of the stria terminalis pathway

Different modulatory effects of serotonin and 5-HT2A receptor subtype activation on sensorimotor and medial prefrontal basal ganglia circuits

Dmrt2 splicing regulation in sex and development of the nervous system

Effects of a psychedelic 5-HT2A receptor agonist on anxiety-related behavior and fear processing in mice

N-glycosylation of induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from a person with Down Syndrome (DS) caused by Trisomy 21 (T21)

Sulfiredoxin 1 ameliorates oxidative stress in HT22 cells and ischemic damage in gerbils

FENS Forum 2024

Regulation of the DNA damage response by E2F4 phosphorylation in its T249/T251 conserved motif and Alzheimer’s disease

Role of TET2 in neural stem cell maintenance and differentiation

Single-cell RNA sequencing reveals senescent-like neurons in the injured mouse brain and treatment with senolytic drug ABT263 improves injury-induced cognitive impairment: is there therapeutic potential?

Striatal dysfunction in the novel DYT25-GNAL dystonia knockout rat model

Structural 3D modeling of VMAT2 mutants to improve diagnostic and therapeutic treatment of infantile Parkinsonism

Structure-function relationships in the interaction of Proline-rich transmembrane protein 2 (PRRT2) with voltage gated Na+ channels

SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes

Antidepressant-like effects of psychedelics in a chronic despair mouse model: Is the 5-HT2A receptor the unique player?

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CD8 T cells play a major role in CNS inflammation and brain atrophy in type I interferon-mediated neuroinflammation of RNaseT2-deficient mice

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Effects of 5-HT2AR-mGluR2-based interventions on electrophysiological biomarkers in a rat model of alcohol addiction

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Is GABA a substrate for the vesicular monoamine transporter VMAT2?

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Heterodimerization and interaction of the serotonin receptors 5-HT1A and 5-HT2C

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Inhibition of p38MAPK-dependent phosphorylation of E2F4 in its T249/T251 motif prevents DNA damage-induced death in N2a-derived neurons

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New insights from modelling neurons in PRRT2 patients

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Mitigation of polyglutamine-induced toxicity through depletion of Trmt2a in an MJD/SCA3 mouse model

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A role for interoceptive vGluT2-expressing neurons in the jugular-nodose ganglion of the left vagus nerve in the regulation of sleep architecture and spectral composition

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Selective effects of psilocin on cortico-amygdalar neurons mediated by 5-HT2A and 5-HT1A receptors

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SGLT2 and DPP4 inhibitors improve Alzheimer’s disease–like pathology and cognitive function through distinct mechanisms in a T2D–AD mouse model

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Synaptic phosphoproteome signature evoked by hallucinogenic agonist stimulation of the 5-HT2A receptor

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TET2-mediated regulation of genomic imprinting in adult neural stem cells

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Transient dopamine depletion increases vesicular glutamate transporter (VGLUT2) expression in midbrain dopamine neurons – implications for Parkinson’s disease

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Unraveling pyroptosis in microglia: Lessons from Rnaset2-/- mice

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