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Abstract
A single sub-anesthetic dose of ketamine, an NMDA receptor (NMDAR) antagonist, produces rapid and sustained antidepressant actions in depressed patients, addressing a major unmet need for the treatment of mood disorders. Ketamine produces a rapid increase in extracellular glutamate and synaptic formation in the prefrontal cortex, but the initial cellular trigger that initiates these and its behavioral actions has not been identified. To address this question, we used a combination of viral shRNA and conditional mutation to produce cell specific knockdown or deletion of a key NMDAR subunit, GluN2B, implicated in the actions of ketamine. The results demonstrate that the antidepressant actions of ketamine were blocked by GluN2B-NMDAR knockdown on GABA (Gad1) interneurons, as well as subtypes expressing somatostatin (Sst), or parvalbumin (Pvalb), but not glutamate principle neurons in the mPFC. Further analysis of GABA subtypes showed that cell specific knockdown or deletion of GluN2B in Sst interneurons blocked or occluded the antidepressant actions of ketamine and revealed sex-specific differences that are associated with excitatory postsynaptic currents on mPFC principle neurons. These findings demonstrate that GluN2B-NMDARs on GABA interneurons are the initial cellular trigger for the rapid antidepressant actions of ketamine and show sex-specific adaptive mechanisms to GluN2B modulation.
Authors
Danielle M. Gerhard, Santosh Pothula, Rong-Jian Liu, Min Wu, Xiao-Yuan Li, Matthew J. Girgenti, Seth R. Taylor, Catharine H. Duman, Eric Delpire, Marina Picciotto, Eric S. Wohleb, Ronald S. Duman
Abstract
Background: Ceramides are sphingolipids that play causative roles in diabetes and heart disease, with their serum levels measured clinically as biomarkers of cardiovascular disease (CVD). Methods: We performed targeted lipidomics on serum samples of individuals with familial coronary artery disease (CAD) (n = 462) and population-based controls (n = 212) to explore the relationship between serum sphingolipids and CAD, employing unbiased machine learning to identify sphingolipid species positively associated with CAD. Results: Nearly every sphingolipid measured (n = 30 of 32) was significantly elevated in subjects with CAD compared with population controls. We generated a novel Sphingolipid Inclusive CAD risk score, termed SIC, that demarcates CAD patients independently and more effectively than conventional clinical CVD biomarkers including LDL-cholesterol and serum triglycerides. This new metric comprises several minor lipids which likely serve as measures of flux through the ceramide biosynthesis pathway, rather than the abundant deleterious ceramide species that are incorporated in other ceramide-based scores. Conclusion: This study validates serum ceramides as candidate biomarkers of cardiovascular disease and suggests that comprehensive sphingolipid panels be considered as measures of CVD.
Authors
Annelise M. Poss, J. Alan Maschek, James E. Cox, Benedikt J. Hauner, Paul N. Hopkins, Steven C. Hunt, William L. Holland, Scott A. Summers, Mary C. Playdon
Abstract
Pattern recognition receptors (PRRs) are crucial for responses to infections and tissue damage, however, their role in autoimmunity is less clear. Herein we demonstrate that two C-type lectin receptors (CLRs), Mcl and Mincle, play an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS). Congenic rats expressing lower levels of Mcl and Mincle on myeloid cells exhibited a drastic reduction in EAE incidence. In vivo silencing of Mcl and Mincle or blockade of their endogenous ligand SAP130 revealed that receptors expression in the central nervous system is crucial for the T cell recruitment and reactivation into a pathogenic Th17/GM-CSF phenotype. Consistent with this, we uncovered MCL/MINCLE-expressing cells in brain lesions of MS patients and we further found an upregulation of the MCL/MINCLE signaling pathway and an increased response following MCL/MINCLE stimulation in peripheral blood mononuclear cells from MS patients. Together these data support a role for CLRs in autoimmunity and implicate the MCL/MINCLE pathway as a potential therapeutic target in MS.
Authors
Marie N'diaye, Susanna Brauner, Sevasti Flytzani, Lara Kular, Andreas Warnecke, Milena Z. Adzemovic, Eliane Piket, Jin-Hong Min, Will Edwards, Filia Mela, Hoi Ying Choi, Vera Magg, Tojo James, Magdalena Linden, Holger M. Reichardt, Michael R. Daws, Jack van Horssen, Ingrid Kockum, Robert A. Harris, Tomas Olsson, Andre O. Guerreiro-Cacais, Maja Jagodic
Abstract
Neuronal hyperexcitability and cytoplasmic mislocalization of the nuclear RNA binding proteinTDP43 are universal features in amyotrophic lateral sclerosis (ALS), but the relationship between these phenomena remains poorly defined. Here, we show that neuronal hyperexcitability drives TDP43 pathology by upregulating shortened (s)TDP43 splice variants missing the canonical C-terminus. sTDP43 isoforms preferentially accumulate in the cytoplasm,forming insoluble inclusions that sequester full-length TDP43 via preserved N-terminal interactions. Consistent with these findings, sTDP43 overexpression is highly toxic to mammalian neurons, suggesting that neurodegeneration results from complementary gain- and loss-of-function mechanisms. In humans and mice, sTDP43 transcripts are significantly enriched in vulnerable motor neurons, and we observed a striking accumulation of sTDP43 protein within neurons and glia of ALS patients. These studies uncover a hitherto unknown role of alternative TDP43 splice isoforms in ALS, and indicate that sTDP43 production may be a key contributor to the susceptibility of motor neurons in ALS.
Authors
Kaitlin Weskamp, Elizabeth M. Tank, Roberto Miguez, Jonathon P. McBride, Nicolás B. Gómez, Matthew White, Ziqiang Lin, Carmen Moreno Gonzalez, Andrea Serio, Jemeen Sreedharan, Sami J. Barmada
Abstract
Influenza A virus (IAV) is among the most common causes of pneumonia related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection results from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and non-infected cells triggered by the activation of type I interferon receptor and mediated by IRF1, which was maladaptive and contributed to hyper-inflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.
Authors
Patricia L. Brazee, Luisa Morales-Nebreda, Natalia D. Magnani, Joe G.N. Garcia, Alexander V. Misharin, Karen M. Ridge, G.R. Scott Budinger, Kazuhiro Iwai, Laura A. Dada, Jacob I. Sznajder
Abstract
Cancer immune evasion is achieved through multiple layers of immune tolerance mechanisms including immune editing, recruitment of tolerogenic immune cells, and secretion of immune suppressive cytokines. Recent success with immune checkpoint inhibitors in cancer immunotherapy suggests a dysfunctional immune synapse as a pivotal tolerogenic mechanism. Tumor cells express immune synapse proteins to suppress the immune system, which is often modulated by epigenetic mechanisms. When the methylation status of key immune synapse genes was interrogated, we observed disproportionately hyper-methylated co-stimulatory genes and hypo-methylation of immune checkpoint genes, which were negatively associated with functional T-cell recruitment to the tumor microenvironment. Therefore, the methylation status of immune synapse genes reflects tumor immunogenicity and correlates with survival.
Authors
Anders Berglund, Matthew Mills, Ryan M. Putney, Imène Hamaidi, James Mulé, Sungjune Kim
Abstract
Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and non-invasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to non-invasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate non-invasive cell therapy characterization can be achieved with QBAM and machine learning.
Authors
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon, Jr., Peter Bajcsy, Kapil Bharti
Abstract
Parkinson's disease (PD) is a neurodegenerative disorder associated with loss of striatal dopamine, secondary to degeneration of midbrain dopamine (mDA) neurons in the substantia nigra, rendering cell transplantation a promising therapeutic strategy. To establish human induced pluripotent stem cell (hiPSC)-based autologous cell therapy, we report a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, we developed a method to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, we established a “spotting”-based in vitro differentiation methodology to generate functional and healthy mDA cells in a scalable manner. Third, we developed a chemical method that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor dysfunction and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.
Authors
Bin Song, Young Cha, Sanghyeok Ko, Jeha Jeon, Nayeon Lee, Hyemyung Seo, Kyung-joon Park, In-Hee Lee, Claudia Lopes, Melissa Feitosa, María José Luna, Jin Hyuk Jung, Jisun Kim, Dabin Hwang, Bruce Cohen, Martin Teicher, Pierre Leblanc, Bob Carter, Jeffrey H. Kordower, Vadim Y. Bolshakov, Sek Won Kong, Jeffrey S. Schweitzer, Kwang-Soo Kim
Abstract
Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed which possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities especially for multi-antibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed globally impacting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dMAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dMAbs cassettes and studied their activity in small and large animals. Sera from animals administered dMAbs neutralized multiple HIV-1 isolates with similar activity to their parental recombinant MAbs. Delivery of multiple dMAbs to a single animal led to increased neutralization breadth. Two dMAbs, PGDM1400 and PGT121, were advanced into non-human primates for study. High peak circulating levels (between 6-34µg/ml) of these dMAbs were measured and the sera of all animals displayed broad neutralizing activity. The dMAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.
Authors
Megan C. Wise, Ziyang Xu, Edgar Tello-Ruiz, Charles Beck, Aspen Trautz, Ami Patel, Sarah T.C. Elliott, Neethu Chokkalingam, Sophie Kim, Melissa G. Kerkau, Kar Muthumani, Jingjing Jiang, Paul Fisher, Stephany J. Ramos, Trevor R.F. Smith, Janess Mendoza, Kate E. Broderick, David C. Montefiori, Guido Ferrari, Daniel W. Kulp, Laurent Humeau, David B. Weiner
Abstract
Background: Chronic HCV-infection is characterized by a severe impairment of HCV-specific CD4 T cell help that is driven by chronic antigen stimulation. We aimed to study the fate of HCV-specific CD4 T cells after viral elimination. Methods:HCV-specific CD4 T cell responses were longitudinally analyzed using MHC class II tetramer-technology, multicolor flow cytometry and RNA sequencing in a cohort of chronically HCV-infected patients undergoing therapy with direct-acting antivirals. In addition, HCV-specific neutralizing antibodies and CXCL13 levels were analyzed. Results: We observed that the frequency of HCV-specific CD4 T cells increased within two weeks after initiation of DAA therapy. Multicolor flow cytometry revealed a downregulation of exhaustion and activation markers and an upregulation of memory-associated markers. While cells with a Th1 phenotype were the predominant subset at baseline, cells with phenotypic and transcriptional characteristics of follicular T helper cells increasingly shaped the circulating HCV-specific CD4 T cell repertoire, suggesting antigen-independent survival of this subset. These changes were accompanied by a decline of HCV-specific neutralizing antibodies and the germinal center activity. Conclusion: We identified a population of HCV-specific CD4 T cells with a follicular T helper cell signature that is maintained after therapy-induced elimination of persistent infection and may constitute an important target population for vaccination efforts to prevent re-infection and immunotherapeutic approaches for persistent viral infections.
Authors
Maike Smits, Katharina Zoldan, Naveed Ishaque, Zuguang Gu, Katharina Jechow, Dominik Wieland, Christian Conrad, Roland Eils, Catherine Fauvelle, Thomas F. Baumert, Florian Emmerich, Bertram Bengsch, Christoph Neumann-Haefelin, Maike Hofmann, Robert Thimme, Tobias Boettler
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ISSN: 0021-9738 (print), 1558-8238 (online)