Research Article
Abstract
Radiotherapy (RT) remains a primary treatment modality for glioblastoma (GBM), but it induces cellular senescence and is strongly implicated in GBM progression and RT-related injury. Recently, eliminating senescent cells has emerged as a promising strategy for treating cancer and for mitigating radiation-induced brain injury (RBI). Here, we investigated the impact of all-trans retinoic acid (RA) on radiation-induced senescence. The findings of this study revealed that RA effectively eliminated astrocytes, which are particularly prone to senescence after radiation, and that the removal of senescence-associated secretory phenotype factor–producing astrocytes inhibited GBM cell proliferation in vitro. Moreover, RA-mediated clearance of senescent cells improved survival in GBM-bearing mice and alleviated radiation-induced cognitive impairment. Through RNA sequencing, we found that the AKT/mTOR/PPARγ/Plin4 signaling pathway is involved in RA-mediated clearance of senescent cells. In summary, these results suggest that RA could be a potential senolytic drug for preventing GBM progression and improving RBI.
Authors
Min Fu, Yiling Zhang, Bi Peng, Na Luo, Yuanyuan Zhang, Wenjun Zhu, Feng Yang, Ziqi Chen, Qiang Zhang, Qianxia Li, Xin Chen, Yuanhui Liu, Guoxian Long, Guangyuan Hu, Xiaohong Peng
Abstract
Radiation-induced lung injury (RILI) initiates radiation pneumonitis and progresses to fibrosis as the main side effect experienced by patients with lung cancer treated with radiotherapy. There is no effective drug for RILI. Sustained vascular activation is a major contributor to the establishment of chronic disease. Here, using a whole thoracic irradiation (WTI) mouse model, we investigated the mechanisms and effectiveness of thrombopoietin mimetic (TPOm) for preventing RILI. We demonstrated that administering TPOm 24 hours before irradiation decreased histologic lung injury score, apoptosis, vascular permeability, expression of proinflammatory cytokines, and neutrophil infiltration in the lungs of mice 2 weeks after WTI. We described the expression of c-MPL, a TPO receptor, in mouse primary pulmonary microvascular endothelial cells, showing that TPOm reduced endothelial cell–neutrophil adhesion by inhibiting ICAM-1 expression. Seven months after WTI, TPOm-treated lung exhibited less collagen deposition and expression of MMP-9, TIMP-1, IL-6, TGF-β, and p21. Moreover, TPOm improved lung vascular structure, lung density, and respiration rate, leading to a prolonged survival time after WTI. Single-cell RNA sequencing analysis of lungs 2 weeks after WTI revealed that TPOm shifted populations of capillary endothelial cells toward a less activated and more homeostatic phenotype. Taken together, TPOm is protective for RILI by inhibiting endothelial cell activation.
Authors
Jeb English, Sriya Dhanikonda, Kathryn E. Tanaka, Wade Koba, Gary Eichenbaum, Weng-Lang Yang, Chandan Guha
Abstract
Mucosal melanoma (MucM) is a rare cancer with a poor prognosis and low response rate to immune checkpoint inhibition (ICI) compared with cutaneous melanoma (CM). To explore the immune microenvironment and potential drivers of MucM’s relative resistance to ICI drugs, we characterized 101 MucM tumors (43 head and neck [H&N], 31 female urogenital, 13 male urogenital, 11 anorectal, and 3 other gastrointestinal) using bulk RNA-Seq and immunofluorescence. RNA-Seq data show that MucM has a significantly lower IFN-γ signature levels than CM. MucM tumors of the H&N region show a significantly greater abundance of CD8+ T cells, cytotoxic cells, and higher IFN-γ signature levels than MucM from lower body sites. In the subcohort of 35 patients with MucM treated with ICI, hierarchical clustering reveals clusters with a high and low degree of immune infiltration, with a differential ICI response rate. Immune-associated gene sets were enriched in responders. Signatures associated with cancer-associated fibroblasts, macrophages, and TGF-β signaling may be higher in immune-infiltrated, but ICI-unresponsive tumors, suggesting a role for these resistance mechanisms in MucM. Our data show organ region–specific differences in immune infiltration and IFN-γ signature levels in MucM, with H&N MucM displaying the most favorable immune profile. Our study might offer a starting point for developing more personalized treatment strategies for this disease.
Authors
Joris L. Vos, Joleen J.H. Traets, Xiaohang Qiao, Iris M. Seignette, Dennis Peters, Michel W.J.M. Wouters, Erik Hooijberg, Annegien Broeks, Jacqueline E. van der Wal, M. Baris Karakullukcu, W. Martin C. Klop, Arash Navran, Marc van Beurden, Oscar R. Brouwer, Luc G.T. Morris, Mariette I.E. van Poelgeest, Ellen Kapiteijn, John B.A.G. Haanen, Christian U. Blank, Charlotte L. Zuur
Abstract
The blood-brain barrier (BBB) is critical for maintaining brain homeostasis but is susceptible to inflammatory dysfunction. While transporter-dependent efflux of some lipophilic substrates across the BBB shows circadian variation due to rhythmic transporter expression, basal transporter–independent permeability and leakage is nonrhythmic. Whether daily timing influences BBB permeability in response to inflammation is unknown. Here, we induced systemic inflammation through repeated LPS injections either in the morning (ZT1) or evening (ZT13) under standard lighting conditions; we then examined BBB permeability to a polar molecule that is not a transporter substrate, sodium fluorescein. We observed clear diurnal variation in inflammatory BBB permeability, with a striking increase in paracellular leak across the BBB specifically following evening LPS injection. Evening LPS led to persisting glia activation as well as inflammation in the brain that was not observed in the periphery. The exaggerated evening neuroinflammation and BBB disruption were suppressed by microglial depletion or through keeping mice in constant darkness. Our data show that diurnal rhythms in microglial inflammatory responses to LPS drive daily variability in BBB breakdown and reveal time of day as a key regulator of inflammatory BBB disruption.
Authors
Jennifer H. Lawrence, Asha Patel, Melvin W. King, Collin J. Nadarajah, Richard Daneman, Erik S. Musiek
Abstract
Effective, reproducible, and safe delivery of therapeutics into the inner ear is required for the prevention and treatment of hearing loss. A commonly used delivery method is via the posterior semicircular canal (PSCC); however, its specific targeting within the cochlea remains unclear, impacting precision and reproducibility. To assess safety and target specificity, we conducted in vivo recordings of the pharmacological manipulations delivered through the PSCC. Measurements of auditory brainstem response (ABR), vibrometry, and vestibular behavioral and sensory-evoked potential (VsEP) revealed preserved hearing and vestibular functions after artificial perilymph injections. Injection of curare, a mechanoelectrical transducer (MET) channel blocker that affects hearing when in the endolymph, had no effect on ABR or VsEP thresholds. Conversely, injection of CNQX, an AMPA receptor blocker, or lidocaine, a Na+ channel blocker, which affects hearing when in the perilymph, significantly increased both thresholds, indicating that PSCC injections selectively target the perilymphatic space. In vivo tracking of gold nanoparticles confirmed their exclusive distribution in the perilymph during PSCC injection, supporting the pharmacological finding. Together, PSCC injection is a safe method for inner ear delivery, specifically targeting the perilymphatic space. Our findings will allow for precise delivery of therapeutics within the inner ear for therapeutic and research purposes.
Authors
Jinkyung Kim, Jesus Maldonado, Dorothy W. Pan, Patricia M. Quiñones, Samantha Zenteno, John S. Oghalai, Anthony J. Ricci
Abstract
The accumulation of mutant huntingtin protein aggregates in neurons is a pathological hallmark of Huntington’s disease (HD). The glymphatic system, a brain-wide perivascular network, facilitates the exchange of interstitial fluid and cerebrospinal fluid (CSF), supporting interstitial solute clearance of brain wastes. In this study, we employed dynamic glucose-enhanced (DGE) MRI to measure d-glucose clearance from CSF as a tool to predict glymphatic function in a mouse model of HD. We found significantly diminished CSF clearance efficiency in HD mice before phenotypic onset. The impairment of CSF clearance efficiency worsened with disease progression. These DGE MRI findings in compromised glymphatic function were further verified with fluorescence-based imaging of CSF tracer influx, suggesting an impaired glymphatic function in premanifest HD. Moreover, expression of the astroglial water channel aquaporin-4 in the perivascular compartment, a key mediator of glymphatic function, was significantly diminished in both HD mouse brain and human HD brain. Our data, acquired using a clinically translatable MRI, indicate a perturbed glymphatic network in the HD brain. Further validation of these findings in clinical studies will provide insights into the potential of glymphatic clearance as a therapeutic target as well as an early biomarker in HD.
Authors
Hongshuai Liu, Lin Chen, Chuangchuang Zhang, Chang Liu, Yuguo Li, Liam Cheng, Yuxiao Ouyang, Catherine Rutledge, John Anderson, Zhiliang Wei, Ziqin Zhang, Hanzhang Lu, Peter C.M. van Zijl, Jeffrey J. Iliff, Jiadi Xu, Wenzhen Duan
Abstract
The pathogenesis of the murine model of autoimmune pancreatitis associated with IgG4-related disease (AIP/IgG4-RD) induced by administration of polyinosinic-polycytidylic acid (poly[I:C]) is incompletely understood. While it is known that murine and human AIP/IgG4-RD is driven by plasmacytoid dendritic cells (pDCs) producing IFN-α, the origin of these cells and their relation to effector T cells is not known. Here, we show that murine AIP was initiated by TLR3-bearing conventional DCs in the uninflamed pancreas whose activation by the TLR3 ligand poly(I:C) caused IFN-α, CXCL9, and CXCL10 secretion. This, in turn, induced pancreatic recruitment of CXCR3+ T cells and these T cells, via their secretion of CCL25, facilitated migration of pDCs bearing CCR9 into the pancreas. This established a feedback loop anchored by the now dominant pDC production of IFN-α and the continued CXCR3+ T cell facilitation of pDC migration. Remarkably, the interaction between CXCR3+ T cells and pDCs also existed at the functional level since this interaction enhanced the production of CCL25 and IFN-α by CXCR3+ T cells and pDCs, respectively. Evidence presented here that a similar disease mechanism was present in human AIP/IgG4-RD creates new avenues of disease treatment.
Authors
Akane Hara, Tomohiro Watanabe, Kosuke Minaga, Tomoe Yoshikawa, Masayuki Kurimoto, Ikue Sekai, Yasuhiro Masuta, Ryutaro Takada, Yasuo Otsuka, Ken Kamata, Shiki Takamura, Masatoshi Kudo, Warren Strober
Abstract
Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart failure. Recessive mutations of EIF2AK4 gene (encoding general control nonderepressible 2 kinase, GCN2) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients but rarely in patients with PAH. The role of GCN2 kinase activation in the pathogenesis of PAH remains unclear. Here, we show that GCN2 was hyperphosphorylated and activated in pulmonary vascular endothelial cells (ECs) of hypoxic mice, monocrotaline-treated rats, and patients with idiopathic PAH. Unexpectedly, loss of GCN2 kinase activity in Eif2ak4–/– mice with genetic disruption of the kinase domain induced neither PVOD nor pulmonary hypertension (PH) but inhibited hypoxia-induced PH. RNA-sequencing analysis suggested endothelin-1 (Edn1) as a downstream target of GCN2. GCN2 mediated hypoxia-induced Edn1 expression in human lung ECs via HIF-2α. Restored Edn1 expression in ECs of Eif2ak4–/– mice partially reversed the reduced phenotype of hypoxia-induced PH. Furthermore, GCN2 kinase inhibitor A-92 treatment attenuated PAH in monocrotaline-treated rats. These studies demonstrate that GCN2 kinase activation mediates pulmonary vascular remodeling and PAH at least partially through Edn1. Thus, targeting GCN2 kinase activation is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss-of-function mutations.
Authors
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
Abstract
Spinal muscular atrophy (SMA) is a recessive developmental disorder caused by the genetic loss or mutation of the gene SMN1 (survival of motor neuron 1). SMA is characterized by neuromuscular symptoms and muscle weakness. Several years ago, SMA treatment underwent a radical transformation, with the approval of 3 different SMN-dependent disease-modifying therapies. This includes 2 SMN2 splicing therapies — risdiplam and nusinersen. One main challenge for type II SMA patients treated with these drugs is ongoing muscle fatigue, limited mobility, and other skeletal problems. To date, few molecular studies have been conducted on SMA patient–derived tissues after treatment, limiting our understanding of what targets remain unchanged after the spinal cord–targeted therapies are applied. Therefore, we collected paravertebral muscle from 8 type II patients undergoing spinal surgery for scoliosis and 7 controls. We used RNA-seq to characterize their transcriptional profiles and correlate these molecular changes with muscle histology. Despite the limited cohort size and heterogeneity, we observed a consistent loss of oxidative phosphorylation (OXPHOS) machinery of the mitochondria, a decrease in mitochondrial DNA copy number, and a correlation between signals of cellular stress, denervation, and increased fibrosis. This work provides new putative targets for combination therapies for type II SMA.
Authors
Fiorella Carla Grandi, Stéphanie Astord, Sonia Pezet, Elèna Gidaja, Sabrina Mazzucchi, Maud Chapart, Stéphane Vasseur, Kamel Mamchaoui, Piera Smeriglio
Abstract
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many patients with Mendelian disease. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here, we describe targeted long-read sequencing of Mendelian disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 individuals with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in 2 IRD cases with inconclusive testing, which uncovered noncoding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
Authors
Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi
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