Biology subjects

Slusher, B. S.

Publications and source records attributed to Slusher, B. S..

14 recordsLinked to original sources

bioRxiv2026

N-Acetylaspartate Synthesis as a Thermodynamic Relief Mechanism for Mitochondrial Aspartate Aminotransferase

N-acetylaspartate (NAA) is the most abundant neuron-enriched acetylated metabolite in the mammalian brain, but its metabolic purpose remains unresolved. We developed a simplified kinetic model of mitochondrial aspartate metabolism to test whether NAA synthesis by aspartate N-acetyltransferase (ASPNAT) acts as a thermodynamic relief valve for mitochondrial aspartate aminotransferase (AAT) under the low-oxaloacetate (OAA) conditions expected in neuronal mitochondria. In the mitochondrial-compartment model, ASPNAT lowered steady-state mitochondrial aspartate from 141 to 105 M and increased net forward AAT flux by 30.9%. The relative AAT-relief effect was largest when OAA and aspartate-glutamate carrier 1 (AGC1/Aralar1)-mediated export were both low, whereas acetyl-CoA availability controlled the substrate-supported capacity for NAA synthesis. That places the relief effect in a narrow regime where product removal matters most. ASPNAT titration produced a graded, concentration-dependent response rather than a binary on/off switch. Energetic comparisons showed that the gain in AAT-linked support comes at a modest acetyl-CoA cost, which makes NAA synthesis easier to sustain in carbon-replete states than in carbon-poor ones. Some studies have suggested a secondary cytoplasmic site of NAA synthesis, and we therefore examined how the network response changed with a change in ASPNAT topology. Mitochondrial matrix ASPNAT increased forward AAT flux by 53.32%, whereas cytoplasmic ASPNAT decreased ASPNAT flux by 17.8%. Allowing OAA to vary preserved the positive ASPNAT-dependent relief of AAT flux, but because this simplified extension produced unrealistically low absolute fluxes, it is interpreted as a robustness check on the direction of the mechanism rather than as a prediction of physiological metabolic rates. These results suggest that ASPNAT can serve as an auxiliary matrix-aspartate sink when the AAT node is product-limited.

neuroscience↗

bioRxiv2026

BCG vaccination mitigates tau pathology and restores cognitive function in PS19 mice.

Retrospective studies in patients with non-muscle invasive bladder cancer (NMIBC) have reported a significant reduction in Alzheimers disease (AD) incidence (12-78%) among Bacillus Calmette-Guerin (BCG) recipients versus controls. To investigate the underlying mechanisms, we evaluated BCG in the PS19 mouse model of tauopathy. We found that BCG administration reduced hippocampal phospho-tau and microgliosis while preserving neuronal markers. In vivo volumetric T2-MRI demonstrated attenuation of brain atrophy accompanied by increased glutamate-weighted CEST-MRI signals. Functionally, BCG-treated mice showed improved performance in the novel object recognition test (NORT), as well as improved body-weight maintenance and survival. Transcriptomic profiling of the hippocampus revealed near complete normalization of the PS19 disease-associated gene expression signature towards that of healthy controls. Flow cytometric profiling of brain myeloid populations demonstrated a reduction in activated resident microglia, but total microglia cells remain elevated. Moreover, an increase of the co-stimulatory marker CD80 on the recruited peripheral myeloid cells ensues following BCG treatment. Consistent with this shift in myeloid state, primary brain myeloid cells from BCG-treated mice also exhibited enhanced phagocytosis of FITC-labeled tau fibrils and increased lactate production. Together, these findings indicate that BCG induces systemic and CNS myeloid cell reprogramming that limits neuroinflammation, enhances tau clearance, and rescues cognitive and neurodegenerative phenotypes in a tauopathy model. BCG is a safe, readily available therapy that merits consideration as a preventive agent against dementia. One sentence summaryBCG therapy prevents tauopathy in PS19 mouse model.

neuroscience↗

bioRxiv2026

Single-cell RNA sequencing reveals that host Glutamine Metabolism Inhibition Enhances Macrophage Phagocytosis of Mycobacterium tuberculosis

Macrophages are crucial for host defense against the pathogen. However, pathogens such as Mycobacterium tuberculosis (Mtb) have evolved mechanisms to alter macrophage physiology and exploit these cells as their primary niche. Mtb-infected macrophages upregulate several metabolic pathways including glutamine metabolism. We previously showed that inhibiting glutamine metabolism with the pleiotropic glutamine metabolism antagonist prodrug JHU083 has dual antibacterial and immunomodulatory effects in a mouse model of tuberculosis. In the present study, using single-cell RNA sequencing and LS-MS/MS metabolomics, we showed that JHU083-mediated glutamine metabolism inhibition increased the population of interstitial macrophages in Mtb-infected lungs. JHU083 treatment also increased inflammatory signatures while lowering immunosuppressive markers on these macrophages. Metabolically, these macrophages exhibited marked depletion of complex lipids, accumulation of free fatty acids, and increased expression of transcripts associated with the {beta}-oxidation pathway. Additionally, JHU083-treatment also improved phagocytic activity of macrophages, as measured by using fluorescent E. coli as a bait. In conclusion, JHU083-mediated glutamine metabolism inhibition metabolically reprograms macrophages, increasing both their lipid utilization as well as phagocytic activity, potentially driving their antimycobacterial activity that we had observed earlier.

microbiology↗

bioRxiv2025

Region-specific proteomic analysis of aging rhesus macaques following chronic glutamate-carboxypeptidase-II (GCPII) inhibition elucidates potential treatment strategies for sporadic Alzheimer's disease

Sporadic Alzheimers disease (sAD) lacks effective preventive therapies, underscoring the need to target pathogenic drivers. Aberrant calcium signaling is an established early event in sAD pathogenesis that is closely linked to neuroinflammation. Aged rhesus macaques are predominantly APOE-{varepsilon}4 homozygotes and naturally exhibit cognitive decline, calcium dysregulation, amyloid deposition, and tau pathology, which allows for a translationally relevant animal model. We previously identified an evolutionarily expanded role for postsynaptic type 3 metabotropic glutamate receptors (mGluR3) in dorsolateral prefrontal and entorhinal cortex, where they regulate cAMP- calcium opening of K channels to sustain neuronal firing and working memory. mGluR3 signaling is driven by N-acetylaspartylglutamate (NAAG) and constrained by glutamate carboxypeptidase II (GCPII), whose expression rises with age and inflammation. In prior work, chronic inhibition of GCPII with the orally bioavailable inhibitor 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) improved neuronal firing, working memory, and reduced pT217Tau pathology in aged macaques. Here, we employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to define the proteomic consequences of chronic 2-MPPA treatment in vulnerable (entorhinal cortex, dorsolateral prefrontal cortex) versus resilient (primary visual cortex) regions. We identified >2,400 proteins across experimental conditions, and label-free quantification revealed region-specific differential expression patterns paralleling known vulnerability gradients in sAD. Gene ontology enrichment of vulnerable regions implicated pathways governing protein deneddylation, amyloid and tau-associated processes, synaptic plasticity, mitochondrial homeostasis, and oxidative stress, revealing putative targets for therapeutic intervention in sAD. These findings demonstrate that GCPII inhibition engages distinct, region-selective molecular programs in the aging primate cortex, consistent with the protection of circuits most vulnerable to sAD. By mapping the proteomic shifts that occur with treatment, we reveal molecular signatures that not only serve as candidate biomarkers but also highlight novel mechanistic pathways contributing to calcium-driven degeneration in sAD. As such, more focused investigations into these pathways of therapeutic interest are warranted, in addition to the analysis of key post-translational modifications and their potential roles in sAD.

neuroscience↗

bioRxiv2024

CTPS1 inhibition synergizes with replication stress signaling inhibition in MYC-amplified Group 3 medulloblastoma

MYC-driven medulloblastomas (MBs) represent the most aggressive and deadly subgroup of MB, the most common malignant pediatric brain tumor. Direct targeting of MYC itself remains an unmet clinical need, therefore focusing on vulnerabilities driven by MYC may be a viable option for novel therapeutic interventions. Using whole-genome CRISPR screening, we identified the de novo pyrimidine synthesis enzyme CTP synthase (CTPS1) as a strong dependency in MYC-driven MB. CTPS1 is the final and rate-limiting step in the de novo pyrimidine synthesis pathway. Targeted inhibition of CTPS1 leads to decreased tumor cell proliferation and markedly reduces MYC expression in G3 MB models. Mechanistically, we demonstrate that single agent CTPS1 inhibition activates the replication stress signaling pathway mediated by ATM-CHK2 and ATR-CHK1. Blockade of CHK1 kinase activity increases sensitivity to CTPS1 inhibition and significantly impedes heterotopic MB tumor growth. CTPS1 enzymatic activity requires the amino acid glutamine, therefore we inhibited CTPS1 using the glutamine antagonists, JHU083 and JHU395. These compounds are prodrugs of 6-diazo-5-oxo-L-norleucine (DON) which were developed to exhibit better tumor targeting and enhanced blood-brain barrier penetrability. Combining JHU083 and CHK1 inhibition demonstrates potent synergy against patient-derived MB xenografts in vivo. Our findings strongly suggest that combining de novo pyrimidine synthesis and ATR-CHK1 inhibitors is a promising treatment for MYC-driven MBs. Key PointsO_LICTPS1 is a unique vulnerability in MYC-driven medulloblastoma C_LIO_LICTPS1 inhibition activates the ATR-CHK1 replication stress response pathway for cell survival C_LIO_LIBlockade of CTPS1 enzymatic activity synergizes with CHK1 inhibition in vitro and in vivo C_LI Importance of the StudyMYC hyperactivation in tumors drives multiple anabolic processes which contribute to tumor proliferation and aggressiveness in patients. We show that targeting de novo pyrimidine synthesis (via CTPS1) limits tumor growth and targets MYC itself through a feedback mechanism. CTPS1 inhibition potently combines with CHK1 blockade and enhances disease control in both heterotopic and orthotopic models of medulloblastoma (MB). Our results support the clinical evaluation of combined CTPS1 and CHK1 inhibition in patients with MYC-driven MB.

cancer biology↗

bioRxiv2024

Development of novel tools for dissection of central versus peripheral dopamine D2-like receptor signaling in dysglycemia

Dopamine (DA) D2-like receptors in both the central nervous system (CNS) and the periphery are key modulators of metabolism. Moreover, disruption of D2-like receptor signaling is implicated in dysglycemia. Yet, the respective metabolic contributions of CNS versus peripheral D2-like receptors including D2 (D2R) and D3 (D3R) receptors remain poorly understood. To address this, we developed new pharmacological tools, D2-like receptor agonists with diminished and delayed blood-brain barrier capability, to selectively manipulate D2R/D3R signaling in the periphery. We designated bromocriptine methiodide (BrMeI), a quaternary methiodide analogue of D2/3R agonist and diabetes drug bromocriptine, as our lead compound based on preservation of D2R/D3R binding and functional efficacy. We then used BrMeI and unmodified bromocriptine to dissect relative contributions of CNS versus peripheral D2R/D3R signaling in treating dysglycemia. Systemic administration of bromocriptine, with unrestricted access to CNS and peripheral targets, significantly improved both insulin sensitivity and glucose tolerance in obese, dysglycemic mice in vivo. In contrast, metabolic improvements were attenuated when access to bromocriptine was restricted either to the CNS through intracerebroventricular administration or delayed access to the CNS via BrMeI. Our findings demonstrate that the coordinated actions of both CNS and peripheral D2-like receptors are required for correcting dysglycemia. Ultimately, the development of a first-generation of drugs designed to selectively target the periphery provides a blueprint for dissecting mechanisms of central versus peripheral DA signaling and paves the way for novel strategies to treat dysglycemia.

neuroscience↗

bioRxiv2023

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a novel glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomics analysis of lungs from JHU083-treated Mtb-infected mice revealed reduced glutamine levels, citrulline accumulation suggesting elevated NOS activity, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. When tested in an immunocompromised mouse model of Mtb infection, JHU083 lost its therapeutic efficacy suggesting the drugs host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

microbiology↗

bioRxiv2022

The nuclear GAPDH-HMGB cascade in cortical microglia regulates cognitive flexibility

We report a mechanism that underlies stress-induced cognitive inflexibility at the molecular level. In a mouse model under subacute stress in which deficits in rule shifting tasks were elicited, the nuclear glyceraldehyde dehydrogenase (N-GAPDH) cascade was activated specifically in microglia in the prelimbic cortex. The cognitive deficits were normalized with a pharmacological intervention with a compound (the RR compound) that selectively blocked the initiation of N-GAPDH cascade without affecting glycolytic activity. The normalization was also observed with a microglia-specific genetic intervention targeting the N-GAPDH cascade. Furthermore, hyperactivation of the prelimbic layer 5 excitatory neurons, which are known to be a neuronal substrate of cognitive inflexibility, was also normalized by the pharmacological and microglia-specific genetic interventions. The RR compound may offer a mechanism-driven, translational opportunity against stress-induced cognitive inflexibility. Taken together, we show a pivotal role of cortical microglia and microglia-neuron interaction in stress-induced cognitive inflexibility. We underscore the N-GAPDH cascade in microglia, which causally mediates stress-induced cognitive alteration.

molecular biology↗

bioRxiv2021

High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

biochemistry↗

bioRxiv2020

Characterization of extracellular vesicles and artificial nanoparticles with four orthogonal single-particle analysis platforms

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

cell biology↗

bioRxiv2019

Nuclear GAPDH signaling mediates pathological cardiac hypertrophy

Here we report that stress-induced nuclear translocation of GAPDH mediates heart hypertrophy via Brahma-Related-Gene-1 (BRG1)-associated chromatin remodelling. In response to pressure overload elicited by transverse aortic constriction, we observed nuclear translocation of GAPDH in the mouse heart. We also demonstrated a robust nuclear localization of GAPDH in cardiomyocytes from patients with dilated hypertrophic cardiomyopathy, whereas negligible GAPDH in the nucleus in control subjects. This is the first demonstration of disease-associated nuclear GAPDH directly in living patients. Using immunohistochemical methods and a pharmacological way that selectively blocks GAPDH nuclear translocation (RR compound), we proved the causal involvement of GAPDH cysteine-150 modification in this translocation in Gq-induced cell model for heart hypertrophy. Accordingly, both pharmacological and genetic interventions proved that the same mechanism played a causal role for heart hypertrophy and dysfunction in vivo. We discovered that, upon nuclear translocation, GAPDH augmented the protein interaction of BRG1 and histone deacetylase 2 (HDAC2), which further facilitated the Myh7/Myh6 isoform ratio from the mature to immature status, an essential mechanism for heart hypertrophy. Beyond medical implications, we provide a novel mechanism of stress-induced reversion of a cellular phenotype from adult to fetal state, which is mediated by a "moonlighting" function of GAPDH.

molecular biology↗

bioRxiv2019

Inhibition of neutral sphingomyelinase-2 facilitates remyelination

For reasons that are not completely understood, remyelination is often incomplete, producing thin myelin sheaths with disorganized structure. We investigated the cellular basis for this altered myelin structure, and found that the response of oligodendrocyte progenitor cells (OPCs), and mature oligodendrocytes to TNF and IL-1{beta} is modified by the expression of the sphingomyelin hydrolase nSMase2. OPCs do not express nSMase2, and exhibit a protective response to these cytokines manifest by decreased ceramide, increased sphingosine 1-phosphate, and increased cell motility. Mature oligodendrocytes express nSMase2, and respond to TNF and IL-1{beta} with a stress phenotype, evidenced by increased ceramide, decreased sphingosine, and active caspase 3. Pharmacological inhibition or a targeted genetic deletion of nSMase2 in vivo increased myelin thickness, and enhanced myelin compaction. These results suggest that inhibition of nSMase2 improves the quality of new myelin by protecting maturing/myelinating oligodendrocytes. Pharmacological inhibition of nSMase2 following a demyelinating event could stabilize the structure of these newly formed myelin sheaths and protect them from secondary demyelination.

molecular biology↗

bioRxiv2019

Targeting glutamine metabolism enhances tumor specific immunity by inhibiting the generation of MDSCs and reprogramming tumor associated macrophages

Myeloid cells comprise a major component of the Tumor Microenvironment (TME) promoting tumor growth and immune evasion. By employing a novel small molecule inhibitor of glutamine metabolism not only were we able to inhibit tumor growth but we markedly inhibited the generation and recruitment of Myeloid Derived Suppressor Cells (MDSC). Targeting tumor glutamine metabolism led to a decrease in CSF-3 and hence recruitment of MDSC as well immunogenic cell death leading to an increase in inflammatory Tumor Associated Macrophages (TAMs). Alternatively, inhibiting glutamine metabolism of the MDSC themselves led to activation induced cell death and conversion of MDSC to inflammatory macrophages. Surprisingly, blocking glutamine metabolism also inhibited IDO expression of both the tumor and myeloid derived cells leading to a marked decrease in kynurenine levels. This in turn inhibited the development of metastasis and further enhanced anti-tumor immunity. Indeed, targeting glutamine metabolism rendered checkpoint blockade-resistant tumors susceptible to immunotherapy. Overall, our studies define an intimate interplay between the unique metabolism of tumors and the metabolism of suppressive immune cells.

immunology↗

bioRxiv2018

JHU-2545 Selectively Shields Salivary Glands and Kidneys during PSMA-Targeted Radiotherapy

PURPOSEProstate-specific membrane antigen (PSMA) radiotherapy is a promising treatment for metastatic castration-resistant prostate cancer (mCRPC) with several beta or alpha particle-emitting radionuclide-conjugated small molecules showing efficacy in late stage patients. However, PSMA is also expressed in kidneys and salivary glands where specific uptake causes dose-limiting xerostomia and potential for nephrotoxicity. The PSMA inhibitor 2- (phosphonomethyl)pentanedioic acid (2-PMPA) can prevent kidney uptake in mice, but also blocks tumor uptake, precluding its clinical utility. Selective delivery of 2-PMPA to non-malignant tissues could improve the therapeutic window of PSMA radiotherapy. EXPERIMENTAL DESIGNA tri-alkoxycarbonyloxy alkyl (TrisPOC) prodrug of 2-PMPA, JHU-2545, was synthesized to enhance 2-PMPA delivery to non-malignant tissues. Preclinical pharmacokinetic and imaging experiments were conducted prior to assessment in 3 mCRPC patients receiving PSMA PET and radiotherapy. RESULTSJHU-2545 resulted in 3- and 53-fold greater exposure of 2-PMPA in rodent salivary glands (18.0 {+/-} 0.97 h*nmol/g) and kidneys (359 {+/-} 4.16 h*nmol/g) versus prostate tumor xenograft (6.79 {+/-} 0.19 h*nmol/g). JHU-2545 also blocked rodent kidneys and salivary glands uptake of the PSMA PET tracers 68Ga-PSMA-11 and 18F-DCFPyL by up to 85% without effect on tumor. In a mCRPC patient, JHU-2545 treatment prior to 68Ga-PSMA-617 administration reduced kidney SUVmax by 76% without effect on metastatic lesions. When administered prior to injection of the beta emitter 177Lu-PSMA-617, JHU-2545 shielded both the salivary glands (72% Gy reduction) and kidneys (45% Gy reduction) without effect on metastases dose. CONCLUSIONSJHU-2545 pre-treatment raises the cumulative dose limit and improves the safety and efficacy profile of PSMA radiotherapy. STATEMENT OF TRANSLATIONAL RELEVANCEProstate Specific Membrane Antigen (PSMA) molecular radiotherapy has emerged as a promising treatment for metastatic castration-resistant prostate cancer (mCRPC), but endogenous expression of PSMA in kidneys and salivary glands causes uptake into these organs resulting in dose-limiting toxicities. We describe the discovery of JHU-2545, a PSMA inhibitor prodrug that selectively blocks kidney and salivary gland uptake of PSMA theranostics without altering tumor uptake in both preclinical models and in mCRPC patients. Pretreatment of JHU-2545 thereby improves the safety and efficacy profile of the multiple PSMA radiotherapies in development.

pharmacology and toxicology↗