A novel 3D bilayer hydrogel tri-culture system for studying functional motor units.

BACKGROUND: A motor unit (MU) is formed by a single alpha motor neuron (MN) and the muscle fibers it innervates. The MU is essential for all voluntary movements. Functional deficits in the MU result in neuromuscular disorders (NMDs). The pathological mechanisms underlying most NMDs remain poorly understood, in part due to the lack of in vitro models that can comprehensively recapitulate multistage intercellular interactions and physiological function of the MU. RESULTS: We have designed a novel three-dimensional (3D) bilayer hydrogel tri-culture system where architecturally organized MUs can form in vitro. A sequential co-culture procedure using the three cell types of a MU, MN, myoblast, and Schwann cell was designed to construct a co-differentiating tri-culture on a bilayer hydrogel matrix. We utilized a µ-molded hydrogel with an additional Matrigel layer to form the bilayer hydrogel device. The µ-molded hydrogel layer provides the topological cues for myoblast differentiation. The Matrigel layer, with embedded Schwann cells, not only separates the MNs from myoblasts but also provides a proper micro-environment for MU development. The completed model shows key MU features including an organized MU structure, myelinated nerves, aligned myotubes innervated on clustered neuromuscular junctions (NMJs), MN-driven myotube contractions, and increases in cytosolic Ca2+ upon stimulation. CONCLUSIONS: This organized and functional in vitro MU model provides an opportunity to study pathological events involved in NMDs and peripheral neuropathies, and can serve as a platform for physiological and pharmacological studies such as modeling and drug screening. Technically, the rational of this 3D bilayer hydrogel co-culture system exploits multiple distinct properties of hydrogels, facilitating effective and efficient co-culturing of diverse cell types for tissue engineering.

Targeting Cellular Retinoic Acid Binding Protein 1 with Retinoic Acid-like Compounds to Mitigate Motor Neuron Degeneration.

All-trans-retinoic Acid (atRA) is the principal active metabolite of Vitamin A, essential for various biological processes. The activities of atRA are mediated by nuclear RA receptors (RARs) to alter gene expression (canonical activities) or by cellular retinoic acid binding protein 1 (CRABP1) to rapidly (minutes) modulate cytosolic kinase signaling, including calcium calmodulin-activated kinase 2 (CaMKII) (non-canonical activities). Clinically, atRA-like compounds have been extensively studied for therapeutic applications; however, RAR-mediated toxicity severely hindered the progress. It is highly desirable to identify CRABP1-binding ligands that lack RAR activity. Studies of CRABP1 knockout (CKO) mice revealed CRABP1 to be a new therapeutic target, especially for motor neuron (MN) degenerative diseases where CaMKII signaling in MN is critical. This study reports a P19-MN differentiation system, enabling studies of CRABP1 ligands in various stages of MN differentiation, and identifies a new CRABP1-binding ligand C32. Using the P19-MN differentiation system, the study establishes C32 and previously reported C4 as CRABP1 ligands that can modulate CaMKII activation in the P19-MN differentiation process. Further, in committed MN cells, elevating CRABP1 reduces excitotoxicity-triggered MN death, supporting a protective role for CRABP1 signaling in MN survival. C32 and C4 CRABP1 ligands were also protective against excitotoxicity-triggered MN death. The results provide insight into the potential of signaling pathway-selective, CRABP1-binding, atRA-like ligands in mitigating MN degenerative diseases.

Modulation of adipose inflammation by cellular retinoic acid-binding protein 1.

OBJECTIVES: Obesity, a metabolic syndrome, is known to be related to inflammation, especially adipose tissue inflammation. Cellular interactions within the expanded white adipose tissue (WAT) in obesity contribute to inflammation and studies have suggested that inflammation is triggered by inflamed adipocytes that recruit M1 macrophages into WAT. What causes accumulation of unhealthy adipocytes is an important topic of investigation. This study aims to understand the action of Cellular Retinoic Acid Binding Protein 1 (CRABP1) in WAT inflammation. METHODS: Eight weeks-old wild type (WT) and Crabp1 knockout (CKO) mice were fed with a normal diet (ND) or high-fat diet (HFD) for 8 weeks. Body weight and food intake were monitored. WATs and serum were collected for cellular and molecular analyses to determine affected signaling pathways. In cell culture studies, primary adipocyte differentiation and bone marrow-derived macrophages (BMDM) were used to examine adipocytes' effects, mediated by CRABP1, in macrophage polarization. The 3T3L1-adipocyte was used to validate relevant signaling pathways. RESULTS: CKO mice developed an obese phenotype, more severely under high-fat diet (HFD) feeding. Further, CKO's WAT exhibited a more severe inflammatory state as compared to wild type (WT) WAT, with a significantly expanded M1-like macrophage population. However, this was not caused by intrinsic defects of CKO macrophages. Rather, CKO adipocytes produced a significantly reduced level of adiponectin and had significantly lowered mitochondrial DNA content. CKO adipocyte-conditioned medium, compared to WT control, inhibited M2-like (CD206+) macrophage polarization. Mechanistically, defects in CKO adipocytes involved the ERK1/2 signaling pathway that could be modulated by CRABP1. CONCLUSIONS: This study shows that CRABP1 plays a protective role against HFD-induced WAT inflammation through, in part, its regulation of adiponectin production and mitochondrial homeostasis in adipocytes, thereby modulating macrophage polarization in WAT to control its inflammatory potential.

CRABP1-CaMKII-Agrn regulates the maintenance of neuromuscular junction in spinal motor neuron.

Cellular retinoic acid-binding protein 1 (CRABP1) binds retinoic acid (RA) specifically in the cytoplasm with unclear functions. CRABP1 is highly and specifically expressed in spinal motor neurons (MNs). Clinical and pre-clinical data reveal a potential link between CRABP1 and MN diseases, including the amyotrophic lateral sclerosis (ALS). We established a sequenced MN-muscle co-differentiation system to engineer an in vitro functional 3D NMJ model for molecular studies and demonstrated that CRABP1 in MNs contributes to NMJ formation and maintenance. Consistently, Crabp1 knockout (CKO) mice exhibited an adult-onset ALS-like phenotype with progressively deteriorated NMJs, characterized with behavioral, EchoMRI, electrophysiological, histological, and immunohistochemical studies at 2-20-months old. Mechanistically, CRABP1 suppresses CaMKII activation to regulate neural Agrn expression and downstream muscle LRP4-MuSK signaling, thereby maintaining NMJ. A proof-of-concept was provided by specific re-expression of CRABP1 to rescue Agrn expression and the phenotype. This study identifies CRABP1-CaMKII-Agrn signaling as a physiological pre-synaptic regulator in the NMJ. This study also highlights a potential protective role of CRABP1 in the progression of NMJ deficits in MN diseases.

Crabp1 Modulates HPA Axis Homeostasis and Anxiety-like Behaviors by Altering FKBP5 Expression.

Retinoic acid (RA), the principal active metabolite of vitamin A, is known to be involved in stress-related disorders. However, its mechanism of action in this regard remains unclear. This study reports that, in mice, endogenous cellular RA binding protein 1 (Crabp1) is highly expressed in the hypothalamus and pituitary glands. Crabp1 knockout (CKO) mice exhibit reduced anxiety-like behaviors accompanied by a lowered stress induced-corticosterone level. Furthermore, CRH/DEX tests show an increased sensitivity (hypersensitivity) of their feedback inhibition in the hypothalamic-pituitary-adrenal (HPA) axis. Gene expression studies show reduced FKBP5 expression in CKO mice; this would decrease the suppression of glucocorticoid receptor (GR) signaling thereby enhancing their feedback inhibition, consistent with their dampened corticosterone level and anxiety-like behaviors upon stress induction. In AtT20, a pituitary gland adenoma cell line elevating or reducing Crabp1 level correspondingly increases or decreases FKBP5 expression, and its endogenous Crabp1 level is elevated by GR agonist dexamethasone or RA treatment. This study shows, for the first time, that Crabp1 regulates feedback inhibition of the the HPA axis by modulating FKBP5 expression. Furthermore, RA and stress can increase Crabp1 level, which would up-regulate FKBP5 thereby de-sensitizing feedback inhibition of HPA axis (by decreasing GR signaling) and increasing the risk of stress-related disorders.

Regulation of exosome secretion by cellular retinoic acid binding protein 1 contributes to systemic anti-inflammation.

BACKGROUND: Intercellular communications are important for maintaining normal physiological processes. An important intercellular communication is mediated by the exchange of membrane-enclosed extracellular vesicles. Among various vesicles, exosomes can be detected in a wide variety of biological systems, but the regulation and biological implication of exosome secretion/uptake remains largely unclear. METHODS: Cellular retinoic acid (RA) binding protein 1 (Crabp1) knockout (CKO) mice were used for in vivo studies. Extracellular exosomes were monitored in CKO mice and relevant cell cultures including embryonic stem cell (CJ7), macrophage (Raw 264.7) and hippocampal cell (HT22) using Western blot and flow cytometry. Receptor Interacting Protein 140 (RIP140) was depleted by Crispr/Cas9-mediated gene editing. Anti-inflammatory maker was analyzed using qRT-PCR. Clinical relevance was accessed by mining multiple clinical datasets. RESULTS: This study uncovers Crabp1 as a negative regulator of exosome secretion from neurons. Specifically, RIP140, a pro-inflammatory regulator, can be transferred from neurons, via Crabp1-regulated exosome secretion, into macrophages to promote their inflammatory polarization. Consistently, CKO mice, defected in the negative control of exosome secretion, have significantly elevated RIP140-containing exosomes in their blood and cerebrospinal fluid, and exhibit an increased vulnerability to systemic inflammation. Clinical relevance of this pathway is supported by patients' data of multiple inflammatory diseases. Further, the action of Crabp1 in regulating exosome secretion involves its ligand and is mediated by its downstream target, the MAPK signaling pathway. CONCLUSIONS: This study presents the first evidence for the regulation of exosome secretion, which mediates intercellular communication, by RA-Crabp1 signaling. This novel mechanism can contribute to the control of systemic inflammation by transferring an inflammatory regulator, RIP140, between cells. This represents a new mechanism of vitamin A action that can modulate the homeostasis of system-wide innate immunity without involving gene regulation. Video Abstract.

Upregulation of RelB in the miR-122 knockout mice contributes to increased levels of proinflammatory chemokines/cytokines in the liver and macrophages.

OBJECTIVE: MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and it plays an important role in regulating liver metabolism and tumor formation. Previous studies also reveal an anti-inflammatory function of miR-122; however, relatively little is known about the mechanisms by which miR-122 suppresses inflammation. This study aims to search the effect of miR-122 on proinflammatory chemokines/cytokines production in mice. METHODS: Quantitative real-time PCR, Western blot analysis, and ELISA were performed to examine gene expression. TargetScan, miRanda, and microT v3.0 were used to search for possible miR-122 target sites in the 3'-untranslated regions (3'-UTR) of candidate genes. Luciferase reporter assay and site-directed mutagenesis were applied to verify miR-122 target sequences. LPS was applied to peritoneal macrophages and mice to evaluate inflammatory response. RESULTS: The expression of proinflammatory chemokines, including Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10, and Relb in the livers of miR-122 knockout (KO) mice was increased. We identified Relb as a direct miR-122 target. Overexpressing RelB in the mouse liver increased the expression of Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10. Peritoneal macrophages from miR-122 KO mice had a higher level of RelB, and they showed a stronger NF-κB activation and more TNF-α and IL-6 secretion after LPS stimulation. Overexpression of RelB in a macrophage cell line augmented LPS-induced TNF-α and IL-6 production. miR-122 KO mice showed a greatly increased mortality rate and generated a stronger and lasting inflammatory response to LPS. CONCLUSIONS: Deletion of miR-122 caused an upregulation of proinflammatory chemokines and RelB in the liver. Increased RelB may contribute to increases in these chemokine in the liver. Intriguingly, deletion of miR-122 also enhanced the sensitivity of macrophages and mice to LPS. Our results reveal that reducing RelB expression is a new mechanism by which miR-122 regulates inflammation.

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

Equilibrative Nucleoside Transporter 3 Regulates T Cell Homeostasis by Coordinating Lysosomal Function with Nucleoside Availability.

T cells are a versatile immune cell population responding to challenges by differentiation and proliferation followed by contraction and memory formation. Dynamic metabolic reprogramming is essential for T cells to meet the biosynthetic needs and the reutilization of biomolecules, processes that require active participation of metabolite transporters. Here, we show that equilibrative nucleoside transporter 3 (ENT3) is highly expressed in peripheral T cells and has a key role in maintaining T cell homeostasis by supporting the proliferation and survival of T cells. ENT3 deficiency leads to an enlarged and disturbed lysosomal compartment, resulting in accumulation of surplus mitochondria, elevation of intracellular reactive oxygen species, and DNA damage in T cells. Our results identify ENT3 as a vital metabolite transporter that supports T cell homeostasis and activation by regulating lysosomal integrity and the availability of nucleosides. Moreover, we uncovered that T cell lysosomes are an important source of salvaged metabolites for survival and proliferation.