In-Press Preview
Articles in this category appear as authors submitted them for publication, prior to copyediting and publication layout.
Abstract
Alzheimer’s disease (AD) is characterized by plaques and tangles, including calcium dysregulation and glycated products produced by reactive carbonyl compounds. AD brains have increased glyoxalase I (GLO1), a major scavenger of inflammatory carbonyl compounds, at early, but not later, stages of disease. Calcium dysregulation includes calcium leak from phosphorylated ryanodine receptor 2 (pS2808-RyR2), seen in aged macaques and AD mouse models, but the downstream consequences of calcium leak remain unclear. Here, we show that chronic calcium leak is associated with increased GLO1 expression and activity. In macaque, we found age-related increases in GLO1 expression in prefrontal cortex (PFC), correlating with pS2808-RyR2, and localized to dendrites and astrocytes. To examine the relationship between GLO1 and RyR2, we used S2808D-RyR2 mutant mice exhibiting chronic calcium leak through RyR2, and found increased GLO1 expression and activity in the PFC and hippocampus as early as 1-month and as late as 21-months of age, with a bell-shaped aging curve. These aged S2808D-RyR2 mice demonstrated impaired working memory. As with macaques, GLO1 was expressed in astrocytes and neurons. Proteomics data generated from S2808D-RyR2 synaptosomes confirmed GLO1 upregulation. Altogether, these data suggest potential association between GLO1 and chronic calcium leak, providing resilience in early stages of aging.
Authors
Elizabeth Woo, Dibyadeep Datta, Shveta Bathla, Hannah E. Beatty, Pinar B. Caglayan, Ashley Kristant Albizu, TuKiet T. Lam, Jean Kanyo, Mary Kate P. Joyce, Shannon N. Leslie, Stacy Uchendu, Jonathan H. DeLong, Qinyue Stacy Guan, Jiaxin Li, Efrat Abramson, Alison L. Herman, Dawson C. Cooper, Pawel Licznerski, Tamas L. Horvath, Elizabeth A. Jonas, Angus C. Nairn, Amy F.T. Arnsten, Lauren H. Sansing
Abstract
Long chain fatty acid oxidation disorders (LC FAODs) cause energy deficits in heart and skeletal muscle that are only partially corrected by current medium chain lipid therapies such as triheptanoin. We find that heart and muscle lack medium chain acyl CoA synthetases, limiting the capacity for β-oxidation of medium-chain fatty acids. Instead, heart and muscle mitochondria robustly respire on medium-chain acylcarnitines. The mitochondrial matrix enzyme carnitine acetyltransferase (CrAT) efficiently converts orally delivered octanoylcarnitine (C8 carnitine) to octanoyl CoA for energy generation. C8-carnitine exhibits twice the oral bioavailability of triheptanoin and distributes to muscle and heart. A single oral dose significantly enhances grip strength and treadmill endurance while attenuating lactic acidosis in two mouse models of LC-FAODs. Thus, medium chain acylcarnitines overcome a previously unrecognized metabolic bottleneck in LC FAOD muscle and may represent an alternative to triglyceride based therapies for bioenergetic disorders.
Authors
Keaton J. Solo, Yuxun Zhang, Sivakama S. Bharathi, Bob B. Zhang, Adam C. Richert, Alexandra V. Schmidt, Clinton Van't Land, Olivia D'Annibale, Timothy C. Wood, Eric S. Goetzman
Abstract
Macular edema (ME) can cause profound vision impairment and occurs in several prevalent retinal diseases, including diabetic retinopathy (DR), choroidal neovascularization (CNV), retinal vein occlusion, and uveitis. Retinal edema typically results from dysfunction of the blood-retina barrier (BRB), which is associated with increased retinal expression of complement components. It is unclear whether the classical complement pathway has detrimental or protective roles in the context of BRB dysfunction. Here, we characterized Tspan12 KODBM (Disrupted Barrier Maintenance) mice, a mouse model of cystoid edema generated by genetically and pharmacologically manipulating beta-catenin-dependent norrin/frizzled4 (FZD4) signaling. We assessed BRB function, cystoid edema, ERG, and microglia activation outcomes in an aging study with WT, C1qa KO, Tspan12 KODBM, and Tspan12 KODBM;C1qa KO compound mutant mice. Phenotypic analyses and cell-based experiments indicated that C1QA contributes to maintaining basal β-catenin-dependent signaling and that the absence of C1QA exacerbates BRB dysfunction, cystoid edema, and neuroinflammation in Tspan12 KODBM;C1qa compound mutant mice. Activation of β-catenin-dependent signaling by a FZD4/LRP5 agonist antibody modality achieved complete resolution of cystoid edema. This study shows that reducing or enhancing norrin/frizzled4 signaling can increase or decrease cystoid edema, respectively, underscoring its potential as a therapeutic target in ME. Furthermore, this study provides novel insights into the contribution of C1QA to BRB maintenance.
Authors
Lingling Zhang, Jacklyn Levey, Md. Abedin, Ha-Neul Jo, Emmanuel Odame, Miranda Howe, Kaia L. Douglas, Elise Thoreen, Scott W. McPherson, Heidi Roehrich, Somasekar Seshagiri, Stephane Angers, Zhe Chen, Harald J. Junge
Abstract
The MTM1 gene encodes myotubularin (MTM1), a phosphatidylinositol 3-phosphate (PI(3)P) lipid phosphatase. Loss-of-function mutations in MTM1 cause X-linked myotubular myopathy (XLMTM), a severe congenital myopathy with no available cure and a poorly understood pathomechanism. The importance of MTM1 enzymatic activity and its PI(3)P substrate in physiology under normal conditions and in XLMTM is unclear. We generated the Mtm1 KI C375S mice in which the endogenous MTM1 was converted to a phosphatase-dead protein. Mutant mice survived a median of 12 weeks and demonstrated progressively impaired motor skills. Observed muscle hypotrophy and reduced force production compared to their WT littermates (~3.9-fold reduction in absolute maximal force) were responsible for these severe phenotypes. A significantly higher level of PI(3)P was found in the muscle of Mtm1 KI C375S mice. Muscle histology and molecular characterization revealed XLMTM hallmarks, with alteration of the mTOR and autophagy pathways correlating with muscle hypotrophy, and abnormal myofiber intracellular organization correlating with impaired muscle force. Overall, this study reveals the importance of MTM1 phosphatase activity and related PI(3)P substrate for postnatal muscle maintenance, and highlights the significance of MTM1 phosphatase activity in the development of X-linked myotubular myopathy.
Authors
Foteini Moschovaki-Filippidou, Christine Kretz, David Reiss, Gaetan Chicanne, Bernard Payrastre, Jocelyn Laporte
Abstract
IDH1/2 mutations (IDHmut) increase methylation of DNA and histones in gliomas. IDHmut inhibitors are effective against low-grade IDHmut gliomas, but new strategies against high grade IDHmut gliomas are needed. Although histone deacetylase inhibitors (HDACi) are ineffective against IDHwt glioblastoma (GBM), their potential in IDHmut gliomas has not been extensively studied. We previously established that IDHmut gliomas are more sensitive to HDACi than IDHwt GBM. Here we show that IDHmut is associated with greater sensitivity to HDACi only in glioma, not in IDHmut chondrosarcoma or cholangiocarcinoma. While HDACi induced more histone acetylation and gene regulation in IDHmut glioma than in IDHwt GBM, such acetylation was mostly within gene deserts, whereas IDHmut glioma promoters paradoxically lost histone acetylation. Two mediators of HDACi resistance, YAP and TAZ, were methylated and suppressed in IDHmut gliomas, but not in other IDHmut cancers. Inducing YAP or TAZ expression in IDHmut gliomas conferred resistance to HDACi. Finally, belinostat extended in vivo survival only in IDHmut glioma models, not in IDHmut GBM models. Our findings provide a mechanistic rationale for further studies of HDACi in IDHmut glioma patients, as well as the potential use of YAP/TAZ as a biomarker of HDACi sensitivity in cancers.
Authors
Thomas K. Sears, Matthew McCord, Wenxia Wang, Alicia Steffens, Kathleen McCortney, Rahul Chaliparambil, Jann N. Sarkaria, Craig M. Horbinski
Abstract
Insulin resistance impairs benefits of lipid-lowering treatment as evidenced by higher cardiovascular risk in individuals with type 2 diabetes versus those without. Because platelet activity is higher in insulin-resistant patients and promotes atherosclerosis progression, we questioned whether platelets impair inflammation resolution in plaques during lipid-lowering. In mice with obesity and insulin resistance, we induced advanced plaques, then implemented lipid-lowering to promote atherosclerotic plaque inflammation-resolution. Concurrently, mice were treated with either platelet-depleting or control antibodies for 3 weeks. Platelet activation and insulin resistance were unaffected by lipid-lowering. Both antibody-treated groups showed reduced plaque macrophages, but plaque cellular and structural composition differed. In platelet-depleted mice, scRNA seq revealed dampened inflammatory gene expression in plaque macrophages and an expansion of a subset of Fcgr4+ macrophages having features of inflammation-resolving, phagocytic cells. Necrotic core size was smaller and collagen content greater, resembling stable human plaques. Consistent with the mouse results, clinical data showed that patients with lower platelet counts had decreased pro-inflammatory signaling pathways in circulating non-classical monocytes after lipid-lowering. These findings highlight that platelets hinder inflammation-resolution in atherosclerosis during lipid-lowering treatment. Identifying novel platelet-targeted therapies following lipid-lowering treatment in individuals with insulin resistance may be a promising therapeutic approach to promote atherosclerotic plaque inflammation-resolution.
Authors
Maria Laskou, Sofie Delbare, Michael Gildea, Ada Weinstock, Vitor De Moura Virginio, Maxwell La Forest, Franziska Krautter, Casey Donahoe, Letizia Amadori, Natalia Eberhardt, Tessa J. Barrett, Chiara Giannarelli, Jeffrey S. Berger, Edward A. Fisher
Abstract
Coronary artery disease (CAD) is the leading cause of mortality worldwide, with macrophages playing a central role in shaping the inflammatory environment through cytokines, chemokines, and other mediators. Long noncoding RNAs (lncRNAs) are emerging as key regulators of cellular processes due to their interactions with DNA, RNA, microRNAs, and proteins, positioning them as promising therapeutic targets. Through integrative transcriptomic analysis, we identified SPANXA2-OT1 as a primate-specific lncRNA with a potential role in macrophage-mediated inflammation in CAD. Functional studies in primary human macrophages demonstrated that SPANXA2-OT1 is induced by inflammatory stimulation, localized to the cytoplasm, and exerts regulatory effects on chemokine expression and macrophage chemotaxis. Mechanistically, SPANXA2-OT1 acts as a molecular sponge for microRNA-338, thereby influencing the expression of interleukin-8 (IL-8), a critical mediator of monocyte recruitment and inflammatory signaling. Collectively, these findings establish SPANXA2-OT1 as a human-specific regulator of inflammatory pathways in CAD and highlight its translational potential as both a biomarker and therapeutic target.
Authors
Prabhash Kumar Jha, Sarvesh Chelvanambi, Yuto Nakamura, Lucas Yuji Umesaki Itto, Aatira Vijay, Adrien Lupieri, Miguel Cantadori Barbeiro, Thanh-Dat Le, Caio Borges Nascimento, Taku Kasai, Mary C. Whelan, Daiki Hosokawa, Dakota Becker-Greene, Sasha A. Singh, Elena Aikawa, Shizuka Uchida, Masanori Aikawa
Abstract
Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.
Authors
Valentin J. Weber, Alice Reschigna, Maximilian J. Gerhardt, Thomas Heigl, Klara S. Hinrichsmeyer, Sander van den Engel, Dina Y. Otify, Zoran Gavrilov, Frank Blaser, Isabelle Meneau, Christian Betz, Hanno J. Bolz, Martin Biel, Stylianos Michalakis, Elvir Becirovic
Abstract
In the rheumatoid arthritis (RA) synovium, resident fibroblast-like synoviocytes (FLS) express MHC class II molecules (HLA-D) but lack the co-stimulatory signals typically required for T cell activation. Here, we demonstrate that antigen presentation by FLS induces a distinct T cell activation state characterized by high CD69, yet reduced CD25 and HLA-DR expression, suppressed proliferation, and decreased effector cytokine production compared to professional antigen presenting cells (APCs), such as macrophages. FLS were also capable of suppressing macrophage-induced T cell activation, underscoring their dominant immunomodulatory role in the synovial microenvironment. Mechanistically, we identify indoleamine 2,3-dioxygenase (IDO1)-mediated tryptophan depletion as the primary driver of FLS-induced T cell hypo-responsiveness. Spatial transcriptomics revealed colocalization of IDO1 and CD69 within ectopic lymphoid structures in RA synovium, further supporting the in vivo relevance of this pathway. These findings provide the groundwork for positioning FLS as critical T cell regulators in RA and highlight the importance of preserving their immunosuppressive properties when therapeutically targeting pathogenic FLS functions.
Authors
Melissa R. Romoff, Preethi K. Periyakoil, Edward F. DiCarlo, Daniel Ramirez, Susan M. Goodman, Christina S. Leslie, Alexander Y. Rudensky, Laura T. Donlin, Melanie H. Smith
Abstract
Atrial fibrillation (AF) is a prevalent arrhythmia with known detriments such as heart failure, stroke, and cognitive decline even in patients without prior stroke. The mechanisms by which AF leads to cognitive dysfunction are yet unknown and there is a lack of animal models to study this disease process. We previously developed a murine model of spontaneous and prolonged episodes of AF, a double transgenic mouse model with cardiac specific expression of a gain-of-function mutant voltage-gated sodium channel (DTG-AF mice). Herein, we show for the first time a murine model of AF without any cerebral infarcts exhibiting cognitive dysfunction, including impaired visual learning and cognitive flexibility on touchscreen testing. Mesenteric resistance arterial function of DTG-AF mice showed significant loss of myogenic tone, increased wall thickness and distensibility, and mitochondrial dysfunction. Brain pial arteries also showed increased wall thickness and mitochondrial enlargement. Furthermore, DTG-AF mice have decreased brain perfusion on laser speckle contrast imaging compared to controls. Cumulatively, these findings demonstrate AF leads to vascular structural and functional alterations necessary for dynamic cerebral autoregulation resulting in increased cerebral stress and cognitive dysfunction. Expression of mitochondrial catalase (mCAT) to reduce mitochondrial reactive oxygen species (ROS) was sufficient to prevent vascular dysfunction due to AF, restore perfusion, and improve cognitive flexibility.
Authors
Pavithran Guttipatti, Ruiping Ji, Najla Saadallah, Uma Mahesh R. Avula, Deniz Z. Sonmez, Albert Fang, Eric Li, Amar D. Desai, Samantha Parsons, Parmanand Dasrat, Christine Sison, Yanping Sun, Chris N. Goulbourne, Steven R. Reiken, Elaine Y. Wan
Abstract
Intestinal epithelial barrier-integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the epithelial cytoskeleton is critical for maintaining gut barrier-integrity, the role of septins- a family of GTP-binding, cytoskeletal proteins- is largely unknown. This highlights an important knowledge gap as dysfunction of septins, and specifically septin 9 (SEPT9), is associated with intestinal pathologies. We determined that SEPT9 localizes to the apical junctions of intestinal epithelial cells (IECs), overlapping with both tight and adherens junctions. IEC-specific ablation of SEPT9 in mice resulted in leaky gut, due to mislocalization of junctional proteins, and increased susceptibility to experimental colitis. Consistently, SEPT9 expression was significantly reduced in intestinal mucosa of inflammatory bowel disease (IBD) patients. Using affinity-purification mass spectrometry, super-resolution imaging, and genetic knockout, we determined that SEPT9 interacts with and is necessary to recruit non-muscle myosin IIC (NMIIC) to the IEC peri-junctional actomyosin belt. Loss of NMIIC also caused IEC barrier disruption. In summary, SEPT9 regulates intestinal barrier-integrity by supporting the assembly of tight and adherens junctions through NMIIC recruitment to the actomyosin belt. The septin cytoskeleton safeguards the intestinal mucosa during acute inflammation, and its disruption in IBD suggests a loss of this protective function.
Authors
Nayden G. Naydenov, Gaizun Hu, Dominik Robak, Atif Zafar, Khosiyat Makhmudova, Susana Lechuga, Yuta Ohno, Naseer Sangwan, Saikat Bandyopadhyay, Ryan Musich, Erin Jeffery, Lei Sun, Armando Marino-Melendez, Florian Rieder, Gloria Sheynkman, Andrei I. Ivanov, Seham Ebrahim
Abstract
Impairment of desmosomal cell-cell adhesion leads to life-threatening diseases such as the autoimmune skin blistering disorder pemphigus vulgaris (PV). Disease management strategies that stabilize intercellular adhesion, in addition to the existing immunosuppression therapies, may result in improved clinical outcomes. Previous findings showed that the serine protease inhibitor SERPINB5 promotes intercellular adhesion by binding to and regulating the localization of the desmosomal adapter molecule desmoplakin (DSP) at the plasma membrane. We here show that SERPINB5 overexpression prevents PV-IgG-mediated loss of cell-cell adhesion and DSP dissociation from the cell membrane. We mechanistically demonstrate that SERPINB5 loss deregulates TGF-β signalling, a pathway known to destabilize DSP in keratinocytes. TGF-β signalling was also activated in skin biopsies of PV patients and keratinocytes treated with PV autoantibodies, suggesting a contribution to disease. Inhibition of TGF-β signaling ameliorated PV-IgG-mediated loss of cell-cell adhesion, increased DSP membrane expression, and prevented PV-IgG-induced blister formation in a human ex-vivo skin model. Together, SERPINB5 modulates DSP and intercellular adhesion through the regulation of TGF-β signalling. Further, TGF-β signalling was identified as a potential target for pemphigus treatment.
Authors
Maitreyi Rathod, Mariam Petrosyan, Aude Zimmermann, Maike Märker, Tobias Gosau, Henriette Franz, Tomás Cunha, Dario Didona, Michael Hertl, Enno Schmidt, Volker Spindler
Abstract
Atherosclerotic cardiovascular disease is a major contributor to the global disease burden. Atherosclerosis initiation depends on cholesterol accumulation in subendothelial macrophages (Mφs). To clarify the role of Bmal1 in Mφ function and atherosclerosis, we used several global and myeloid-specific Bmal1 deficient mouse models. Myeloid-specific Bmal1 deficient mice had higher Mφ cholesterol and displayed greater atherosclerosis compared to controls. Bmal1-deficient Mφs exhibited: (1) elevated expression of Cd36 and uptake of oxLDL; (2) diminished expression of Abca1 and Abcg1, and decreased cholesterol efflux and reverse cholesterol transport; and (3) reduced Npc1 and Npc2 expression, and diminished cholesterol egress from lysosomes. Molecular studies revealed that Bmal1 directly regulates basal and cyclic expression of Npc1 and Npc2 by binding the E-boxes in their promoters and indirectly regulates the basal and temporal regulation of Cd36 and Abca1/Abcg1 involving Rev-erbα and Znf202 repressors, respectively. In conclusion, Mφ Bmal1 is a key regulator of the uptake of modified lipoproteins, cholesterol efflux, lysosomal cholesterol egress and atherosclerosis, and therefore may be a master regulator of cholesterol metabolism in Mφs. Restoration of Mφ Bmal1 expression or blocking of factors that decrease its activity may be effective in preventing atherosclerosis.
Authors
Xiaoyue Pan, John O'Hare, Cyrus Mowdawalla, Samantha Mota, Nan Wang, M. Mahmood Hussain
Abstract
BACKGROUND. Cardiotoxicity is a major complication of anti-cancer therapy (CTx); yet, the impact of CTx on the human microcirculation is not well defined. This study evaluated the impact of CTx on microvascular function in breast cancer patients. METHODS. Endothelial function and angiogenic potential were assessed in arterioles and adipose biopsies obtained from breast cancer patients before, during, and after CTx (longitudinal and cross-sectional) and in healthy arterioles exposed to doxorubicin (Dox), trastuzumab (TZM), or paclitaxel (PTX) ex vivo. Conditioned media containing VEGF-B protein was used to test feasibility of a targeted intervention. RESULTS. Patients treated with Dox and/or TZM in vivo developed profound microvascular endothelial dysfunction that persisted for ≥9 months after treatment cessation. Angiogenic potential was reduced during CTx and recovered within one month after cessation. Gene expression related to angiogenesis and inflammation changed over the course of clinical treatment. Isolated adipose arterioles from healthy donors developed endothelial dysfunction when exposed to Dox or TZM ex vivo. In contrast, paclitaxel (PTX), which poses minimal cardiovascular risk, had no impact on vasomotor function. Ex vivo exposure to Dox or PTX suppressed angiogenic potential, whereas TZM had no effect. Treatment with VEGF-B protein preserved endothelial function in healthy arterioles exposed to Dox or TZM ex vivo. CONCLUSION. Breast cancer patients undergoing treatment with Dox and/or TZM develop prolonged microvascular endothelial dysfunction that is recapitulated in healthy arterioles exposed to Dox or TZM ex vivo. Targeted intervention with VEGF-B protects against direct Dox- or TZM-induced vascular toxicity in human arterioles ex vivo. FUNDING. National Institutes of Health grant R01 HL133029, HL173549 (AMB). National Institutes of Health grant T32 HL134643 (JDT, STH). American Heart Association grant SFRN847970 (AMB, DDG). We Care Foundation Grant (AMB, ALK). Medical College of Wisconsin Cardiovascular Center Pre-PPG Grant (AMB). Advancing a Healthier Wisconsin – Redox Biology Grant (AMB). Jenny and Antti Wihuri Foundation (RMK).
Authors
Janée D. Terwoord, Laura E. Norwood Toro, Shelby N. Hader, Stephen T. Hammond, Joseph C. Hockenberry, Jasmine Linn, Ibrahim Y. Vazirabad, Amanda L. Kong, Alison J. Kriegel, Ziqing Liu, Riikka M. Kivelä, Gillian Murtagh, David D. Gutterman, Andreas M. Beyer
Abstract
Ischemic cardiomyopathy (ICM) is a leading cause of heart failure characterized by extensive remodeling of the cardiac extracellular matrix (ECM). While initially adaptive, ECM deposition following ischemic injury eventually turns maladaptive, promoting adverse cardiac remodeling. The strong link between the extent of fibrosis and adverse clinical outcomes has led to growing interest in ECM targeted therapies to prevent or reverse maladaptive cardiac remodeling in ICM; yet, the precise composition of the ECM in ICM remains poorly defined. In this study, we employed a sequential protein extraction enabled by the photocleavable surfactant Azo to enrich ECM proteins from left ventricular tissues of patients with end-stage ICM (n=16) and nonfailing donor hearts (n=16). High-resolution mass spectrometry-based quantitative proteomics identified and quantified over 6,000 unique protein groups, including 315 ECM proteins. We discovered significant upregulation of key ECM components, particularly glycoproteins, proteoglycans, collagens, and ECM regulators. Notably, LOXL1, FBLN1, and VCAN were among the most differentially expressed. Functional enrichment analyses revealed enhanced TGFβ signaling, integrin-mediated adhesion, and complement activation in ICM tissues, suggesting a feedback loop driving continued ECM deposition in the end-stage failing heart. Together, our findings provide a comprehensive proteomic landscape of ECM alterations in the end-stage ICM myocardium and identify promising molecular targets for therapeutic intervention.
Authors
Kevin M. Buck, Holden T. Rogers, Zachery R. Gregorich, Morgan W. Mann, Timothy J. Aballo, Zhan Gao, Emily A. Chapman, Andrew J. Perciaccante, Scott J. Price, Ienglam Lei, Paul C. Tang, Ying Ge
Abstract
Genetic diseases such as ion-channelopathies substantially burden human health. Existing treatments are limited and not genotype specific. Here, we report a two-step high-throughput approach to rapidly identify drug candidates for repurposing as genotype-specific therapy. We first screened 1,680 medicines using a new thallium-flux trafficking assay against KV11.1 gene variants causing Long QT Syndrome (LQTS), an ion-channelopathy associated with fatal cardiac arrhythmias. We identify evacetrapib as a suitable drug candidate that improves membrane trafficking and activates channels. We then use deep mutational scanning to prospectively identify all KV11.1 missense variants in a LQTS hotspot region responsive to treatment with evacetrapib. Combining high-throughput drug screens with deep mutational scanning establishes a new paradigm for mutation-specific drug discovery translatable to personalized treatment of patients with rare genetic disorders.
Authors
Christian L. Egly, Alex Shen, Tri Q. Do, Carlos Tellet Cabiya, Paxton A. Ritschel, Suah Woo, Matthew J. Ku, Brian P. Delisle, Brett Kroncke, Bjorn C. Knollmann
Abstract
Rheumatoid arthritis (RA) is a common systemic autoimmune disorder. Fibroblast-like synoviocytes (FLS) have emerged as an attractive target for non-immunosuppressive RA therapy, but there are no approved drugs targeting FLS. The receptor protein tyrosine phosphatase sigma (PTPRS) negatively regulates FLS migration and has been proposed as a target for FLS-directed RA therapy. Here we examined the impact of sequence variations on efficacy of an FLS-targeted biologic comprised of Fc-fused PTPRS immunoglobulin (IgG)-like domains Ig1 and Ig2 (Ig1&2-Fc). Engineering the linker and Fc tag improved effectiveness of human Ig1&2-Fc in assays of FLS migration and a mouse model of arthritis. Treatment of mice with Ig1&2-Fc over four months revealed no signs of toxicity or organ pathology. Finally, we show potential of Ig1&2-Fc co-administration in combination or as a bispecific fusion with a tumor necrosis factor alpha inhibitor (TNFi). Combination treatment of mouse tumor necrosis factor receptor 2 (mTnfr2) with Ig1&2-Fc resulted in increased efficacy in suppressing arthritis beyond single agent treatment. When administered as a dual-action bispecific, Ig1&2 fused to mTnfr2 proved more efficacious at suppressing arthritis than mTnfr2 alone. This study illustrates the potential of Ig1&2-Fc as a combination or bispecific therapy with DMARDs to improve patient outcomes in RA.
Authors
Sterling H. Ramsey, Zixuan Zhao, Megan C. Lee, Thales Hein da Rosa, Ava C. Schneider, Miriam Bollmann, Nour Dada, Katie Frizzi, May M. Han, Jaeyeon Kim, Martina Zoccheddu, Nigel A. Calcutt, Gary S. Firestein, James W. Bryson, Mattias N.D. Svensson, Eugenio Santelli, Stephanie M. Stanford, Nunzio Bottini
Abstract
Glioblastoma IDH-wildtype is the most common and aggressive primary brain tumor in adults, with poor prognosis despite current therapies. To identify new therapeutic vulnerabilities, we investigated the role of CDK12, a transcription-associated cyclin-dependent kinase, in glioblastoma. Genetic or pharmacologic inactivation of CDK12 impaired tumor growth in patientderived xenograft (PDX) models and enhanced the efficacy of temozolomide. Metabolic profiling using extracellular flux analysis and stable isotope tracing with U-¹³C-glucose and U-¹³Cglutamine showed that CDK12 inhibition disrupted mitochondrial respiration, resulting in energy depletion and apoptotic cell death characterized by caspase activation and Noxa induction. Mechanistically, we identified a direct interaction between CDK12 and GSK3β. CDK12 inhibition activated GSK3β, leading to downregulation of PPARD, a transcriptional regulator of oxidative metabolism. This CDK12–GSK3β–PPARD axis was required for glioblastoma cell proliferation and metabolic homeostasis. In vivo, CDK12 inhibition significantly extended survival without overt toxicity and induced complete tumor regression in a subset of animals. Strikingly, combined CDK12 inhibition and temozolomide treatment led to complete tumor eradication in all animals tested. These findings establish CDK12 as a key regulator of glioblastoma metabolism and survival, and provide strong preclinical rationale for its therapeutic targeting in combination with standard-of-care treatments.
Authors
Jeong-Yeon Mun, Chang Shu, Qiuqiang Gao, Zhe Zhu, Hasan O. Akman, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D. Siegelin
Abstract
Focal cortical dysplasia (FCD) is a major cause of refractory epilepsy and is associated with pathogenic variants in mTOR pathway genes, including DEPDC5, the most common cause of familial focal epilepsy. The mechanisms of epileptogenesis associated with FCD and hyperactive mTOR signaling remain unclear in DEPDC5-related epilepsy. To test whether DEPDC5 loss leading to seizures require in utero cortical developmental defects or if postnatal neuronal dysfunction of mTORC1 is sufficient to drive seizures, we developed a postnatal focal cortical Depdc5 knockout mouse model. Postnatal day 0-1 Depdc5-floxed mice received unilateral motor cortex injections of either AAV-Cre-GFP or control AAV-GFP. The AAV-Cre-GFP injected hemisphere had decreased DEPDC5 levels with hyperactivation of mTOR that increased with age compared to both the contralateral hemisphere and the AAV-GFP injected mice. Cortical lamination was not disrupted by postnatal DEPDC5 loss. Pathologic hallmarks of FCDs were identified in the Depdc5 knockout hemisphere, including increased SMI-311 neurofilament staining, hypomyelination, astrogliosis, and microglial activation. Mice with postnatal cortical DEPDC5 loss exhibited lower seizure thresholds, increased focal seizures, and increased rates of seizure-induced death compared to control mice. This study demonstrates that postnatal DEPDC5 loss and subsequent mTOR hyperactivation without disruption of cortical migration is sufficient to cause epilepsy.
Authors
Karenna J. Groff, Yini Liang, Christopher Morici, Jinita Modasia, Leena Mehendale, Nishtha Gupta, Angelica D'Amore, Yongho Choe, Mustafa Q. Hameed, Alexander Rotenberg, Mustafa Sahin, Christopher J. Yuskaitis
Abstract
Empirical data from survivors of Lassa fever and experimental disease modelling efforts, particularly those using mouse models, are at odds with respect to T cell-mediated pathogenesis. In mice, T cells have been shown to be imperative in disease progression and lethality, whereas in humans, an early and robust T cell responses has been associated with survival. Here, we assessed the role of CD4+ and CD8+ T cells on disease progression and severity of Lassa virus infection in a non-human primate model. Using an antibody-mediated T cell depletion strategy prior to and post-inoculation, we were able to examine Lassa virus infection in the absence of specific T cell responses. In animals depleted for either CD4+ or CD8+ T cells, Lassa virus infection remained uniformly lethal, with only a slight delay in disease progression observed in the CD4-depleted group when compared to non-depleted controls. Milder pulmonary pathology was noticed in the absence of CD4+ or CD8+ T cells. Overall, our findings suggest that T cells have a limited impact on the development of Lassa fever in non-human primates.
Authors
Jérémie Prévost, Nikesh Tailor, Geoff Soule, Jonathan Audet, Yvon Deschambault, Robert Vendramelli, Jessica Prado-Smith, Kevin Tierney, Kimberly Azaransky, Darwyn Kobasa, Chad S. Clancy, Heinz Feldmann, Kyle Rosenke, David Safronetz
