Activation of Muscle-Specific Kinase (MuSK) Reduces Neuromuscular Defects in the Delta7 Mouse Model of Spinal Muscular Atrophy (SMA)

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.     

Molecular Mechanisms Underlying β-Adrenergic Receptor-Mediated Cross-Talk between Sympathetic Neurons and Immune Cells

Cross-talk between the sympathetic nervous system (SNS) and immune system is vital for health and well-being. Infection, tissue injury and inflammation raise firing rates of sympathetic nerves, increasing their release of norepinephrine (NE) in lymphoid organs and tissues. NE stimulation of β₂-adrenergic receptors (ARs) in immune cells activates the cAMP-protein kinase A (PKA) intracellular signaling pathway, a pathway that interfaces with other signaling pathways that regulate proliferation, differentiation, maturation and effector functions in immune cells. Immune–SNS cross-talk is required to maintain homeostasis under normal conditions, to develop an immune response of appropriate magnitude after injury or immune challenge, and subsequently restore homeostasis. Typically, β₂-AR-induced cAMP is immunosuppressive. However, many studies report actions of β₂-AR stimulation in immune cells that are inconsistent with typical cAMP–PKA signal transduction. Research during the last decade in non-immune organs, has unveiled novel alternative signaling mechanisms induced by β₂-AR activation, such as a signaling switch from cAMP–PKA to mitogen-activated protein kinase (MAPK) pathways. If alternative signaling occurs in immune cells, it may explain inconsistent findings of sympathetic regulation of immune function. Here, we review β₂-AR signaling, assess the available evidence for alternative signaling in immune cells, and provide insight into the circumstances necessary for “signal switching” in immune cells.     

Aberrant Notch Signaling Pathway as a Potential Mechanism of Central Precocious Puberty

The Notch signaling pathway is highly conserved during evolution. It has been well documented that Notch signaling regulates cell proliferation, migration, and death in the nervous, cardiac, and endocrine systems. The Notch pathway is relatively simple, but its activity is regulated by numerous complex mechanisms. Ligands bind to Notch receptors, inducing their activation and cleavage. Various post-translational processes regulate Notch signaling by affecting the synthesis, secretion, activation, and degradation of Notch pathway-related proteins. Through such post-translational regulatory processes, Notch signaling has versatile effects in many tissues, including the hypothalamus. Recently, several studies have reported that mutations in genes related to the Notch signaling pathway were found in patients with central precocious puberty (CPP). CPP is characterized by the early activation of the hypothalamus–pituitary–gonadal (HPG) axis. Although genetic factors play an important role in CPP development, few associated genetic variants have been identified. Aberrant Notch signaling may be associated with abnormal pubertal development. In this review, we discuss the current knowledge about the role of the Notch signaling pathway in puberty and consider the potential mechanisms underlying CPP.     

Running and Swimming Differently Adapt the BDNF/TrkB Pathway to a Slow Molecular Pattern at the NMJ

Physical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes’ fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools.     

Plant Responses to Herbivory,Wounding, and Infection

Plants have various self-defense mechanisms against biotic attacks, involving both physical and chemical barriers. Physical barriers include spines, trichomes, and cuticle layers, whereas chemical barriers include secondary metabolites (SMs) and volatile organic compounds (VOCs). Complex interactions between plants and herbivores occur. Plant responses to insect herbivory begin with the perception of physical stimuli, chemical compounds (orally secreted by insects and herbivore-induced VOCs) during feeding. Plant cell membranes then generate ion fluxes that create differences in plasma membrane potential (Vm), which provokes the initiation of signal transduction, the activation of various hormones (e.g., jasmonic acid, salicylic acid, and ethylene), and the release of VOCs and SMs. This review of recent studies of plant–herbivore–infection interactions focuses on early and late plant responses, including physical barriers, signal transduction, SM production as well as epigenetic regulation, and phytohormone responses.     

The Neuromuscular Junction: Roles in Aging and Neuromuscular Disease

The neuromuscular junction (NMJ) is a specialized synapse that bridges the motor neuron and the skeletal muscle fiber and is crucial for conversion of electrical impulses originating in the motor neuron to action potentials in the muscle fiber. The consideration of contributing factors to skeletal muscle injury, muscular dystrophy and sarcopenia cannot be restricted only to processes intrinsic to the muscle, as data show that these conditions incur denervation-like findings, such as fragmented NMJ morphology and corresponding functional changes in neuromuscular transmission. Primary defects in the NMJ also influence functional loss in motor neuron disease, congenital myasthenic syndromes and myasthenia gravis, resulting in skeletal muscle weakness and heightened fatigue. Such findings underscore the role that the NMJ plays in neuromuscular performance. Regardless of cause or effect, functional denervation is now an accepted consequence of sarcopenia and muscle disease. In this short review, we provide an overview of the pathologic etiology, symptoms, and therapeutic strategies related to the NMJ. In particular, we examine the role of the NMJ as a disease modifier and a potential therapeutic target in neuromuscular injury and disease.     

The Impact of Radiation-Induced DNA Damage on cGAS-STING-Mediated Immune Responses to Cancer

Radiotherapy is a major modality used to combat a wide range of cancers. Classical radiobiology principles categorize ionizing radiation (IR) as a direct cytocidal therapeutic agent against cancer; however, there is an emerging appreciation for additional antitumor immune responses generated by this modality. A more nuanced understanding of the immunological pathways induced by radiation could inform optimal therapeutic combinations to harness radiation-induced antitumor immunity and improve treatment outcomes of cancers refractory to current radiotherapy regimens. Here, we summarize how radiation-induced DNA damage leads to the activation of a cytosolic DNA sensing pathway mediated by cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING). The activation of cGAS–STING initiates innate immune signaling that facilitates adaptive immune responses to destroy cancer. In this way, cGAS–STING signaling bridges the DNA damaging capacity of IR with the activation of CD8+ cytotoxic T cell-mediated destruction of cancer—highlighting a molecular pathway radiotherapy can exploit to induce antitumor immune responses. In the context of radiotherapy, we further report on factors that enhance or inhibit cGAS–STING signaling, deleterious effects associated with cGAS–STING activation, and promising therapeutic candidates being investigated in combination with IR to bolster immune activation through engaging STING-signaling. A clearer understanding of how IR activates cGAS–STING signaling will inform immune-based treatment strategies to maximize the antitumor efficacy of radiotherapy, improving therapeutic outcomes.     

TGF-Beta as a Master Regulator of Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the most common complications in diabetes mellitus and the leading cause of end-stage renal disease. TGF-β is a pleiotropic cytokine and has been recognized as a key mediator of DN. However, anti-TGF-β treatment for DN remains controversial due to the diverse role of TGF-β1 in DN. Thus, understanding the regulatory role and mechanisms of TGF-β in the pathogenesis of DN is the initial step towards the development of anti-TGF-β treatment for DN. In this review, we first discuss the diverse roles and signaling mechanisms of TGF-β in DN by focusing on the latent versus active TGF-β1, the TGF-β receptors, and the downstream individual Smad signaling molecules including Smad2, Smad3, Smad4, and Smad7. Then, we dissect the regulatory mechanisms of TGF-β/Smad signaling in the development of DN by emphasizing Smad-dependent non-coding RNAs including microRNAs and long-non-coding RNAs. Finally, the potential therapeutic strategies for DN by targeting TGF-β signaling with various therapeutic approaches are discussed.     

Potentially Treatable Disorder Diagnosed Post Mortem by Exome Analysis in a Boy with Respiratory Distress

We highlight the importance of exome sequencing in solving a clinical case of a child who died at 14 months after a series of respiratory crises. He was the half-brother of a girl diagnosed at 7 years with the early-onset seizure variant of Rett syndrome due to CDKL5 mutation. We performed a test for CDKL5 in the boy, which came back negative. Driven by the mother’s compelling need for a diagnosis, we moved forward performing whole exome sequencing analysis. Surprisingly, two missense mutations in compound heterozygosity were identified in the RAPSN gene encoding a receptor-associated protein with a key role in clustering and anchoring nicotinic acetylcholine receptors at synaptic sites. This gene is responsible for a congenital form of myasthenic syndrome, a disease potentially treatable with cholinesterase inhibitors. Therefore, an earlier diagnosis in this boy would have led to a better clinical management and prognosis. Our study supports the key role of exome sequencing in achieving a definite diagnosis in severe perinatal diseases, an essential step especially when a specific therapy is available.     

Pharmacological Effects of Polyphenol Phytochemicals on the Intestinal Inflammation via Targeting TLR4/NF-κB Signaling Pathway

TLR4/NF-κB is a key inflammatory signaling transduction pathway, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory response. Toll like receptor 4 (TLR4), the first mammalian TLR to be characterized, is the innate immune receptor that plays a key role in inflammatory signal transductions. Nuclear factor kappa B (NF-κB), the TLR4 downstream, is the key to accounting for the expression of multiple genes involved in inflammatory responses, such as pro-inflammatory cytokines. Inflammatory bowel disease (IBD) in humans is a chronic inflammatory disease with high incidence and prevalence worldwide. Targeting the TLR4/NF-κB signaling pathway might be an effective strategy to alleviate intestinal inflammation. Polyphenol phytochemicals have shown noticeable alleviative effects by acting on the TLR4/NF-κB signaling pathway in intestinal inflammation. This review summarizes the pharmacological effects of more than 20 kinds of polyphenols on intestinal inflammation via targeting the TLR4/NF-κB signaling pathway. We expected that polyphenol phytochemicals targeting the TLR4/NF-κB signaling pathway might be an effective approach to treat IBD in future clinical research applications.     

Deregulation of Ca2+-Signaling Systems in White Adipocytes,Manifested as the Loss of Rhythmic Activity,Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca²⁺-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP₃-receptors (PLC/IP₃R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)–dependent Ca²⁺ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors’ interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca²⁺]_i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP₃R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5–15% vs. 30–80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca²⁺-signaling systems deregulation, and development of general hormonal resistance to obesity.     

Ang–Tie Angiogenic Pathway Is Distinctively Expressed in Benign and Malignant Adrenocortical Tumors

The differential diagnosis between adrenocortical adenomas (ACAs) and adrenocortical carcinomas (ACCs) relies on unspecific clinical, imaging and histological features, and, so far, no single molecular biomarker has proved to improve diagnostic accuracy. Similarly, prognostic factors have an insufficient capacity to predict the heterogeneity of ACC clinical outcomes, which consequently lead to inadequate treatment strategies. Angiogenesis is a biological process regulated by multiple signaling pathways, including VEGF and the Ang–Tie pathway. Many studies have stressed the importance of angiogenesis in cancer development and metastasis. In the present study, we evaluated the expression of VEGF and Ang–Tie pathway mediators in adrenocortical tumors (ACTs), with the ultimate goal of assessing whether these molecules could be useful biomarkers to improve diagnostic accuracy and/or prognosis prediction in ACC. The expression of the proteins involved in angiogenesis, namely CD34, VEGF, VEGF-R2, Ang1, Ang2, Tie1 and Tie2, was assessed by immunohistochemistry in ACC (n = 22), ACA with Cushing syndrome (n = 8) and non-functioning ACA (n = 13). ACC presented a significantly higher Ang1 and Ang2 expression when compared to ACA. Tie1 expression was higher in ACC with venous invasion and in patients with shorter overall survival. In conclusion, although none of these biomarkers showed to be useful for ACT diagnosis, the Ang–Tie pathway is active in ACT and may play a role in regulating ACT angiogenesis.     

The Tyrosine Phosphatase hPTPRβ Controls the Early Signals and Dopaminergic Cells Viability via the P2X7 Receptor

ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X₇ receptor (P2X₇R) and stimulates neuronal cell death. Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X₇R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRβ and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson’s Disease.     

The Crosstalk between FAK and Wnt Signaling Pathways in Cancer and Its Therapeutic Implication

Focal adhesion kinase (FAK) and Wnt signaling pathways are important contributors to tumorigenesis in several cancers. While most results come from studies investigating these pathways individually, there is increasing evidence of a functional crosstalk between both signaling pathways during development and tumor progression. A number of FAK–Wnt interactions are described, suggesting an intricate, context-specific, and cell type-dependent relationship. During development for instance, FAK acts mainly upstream of Wnt signaling; and although in intestinal homeostasis and mucosal regeneration Wnt seems to function upstream of FAK signaling, FAK activates the Wnt/β-catenin signaling pathway during APC-driven intestinal tumorigenesis. In breast, lung, and pancreatic cancers, FAK is reported to modulate the Wnt signaling pathway, while in prostate cancer, FAK is downstream of Wnt. In malignant mesothelioma, FAK and Wnt show an antagonistic relationship: Inhibiting FAK signaling activates the Wnt pathway and vice versa. As the identification of effective Wnt inhibitors to translate in the clinical setting remains an outstanding challenge, further understanding of the functional interaction between Wnt and FAK could reveal new therapeutic opportunities and approaches greatly needed in clinical oncology. In this review, we summarize some of the most relevant interactions between FAK and Wnt in different cancers, address the current landscape of Wnt- and FAK-targeted therapies in different clinical trials, and discuss the rationale for targeting the FAK–Wnt crosstalk, along with the possible translational implications.     

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G₁₆ protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα₁₆ limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα₁₆ mediated pathway more specifically. Our data demonstrated agonist-specific activation of G₁₆ pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα₁₆ pathway at the M₂ receptor and at the same time impaired Gα₁₆ signaling of iperoxo at M₅ receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.     

Identifying the miRNA Signature Association with Aging-Related Senescence in Glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor and its malignant phenotypic characteristics are classified as grade IV tumors. Molecular interactions, such as protein–protein, protein–ncRNA, and protein–peptide interactions are crucial to transfer the signaling communications in cellular signaling pathways. Evidences suggest that signaling pathways of stem cells are also activated, which helps the propagation of GBM. Hence, it is important to identify a common signaling pathway that could be visible from multiple GBM gene expression data. microRNA signaling is considered important in GBM signaling, which needs further validation. We performed a high-throughput analysis using micro array expression profiles from 574 samples to explore the role of non-coding RNAs in the disease progression and unique signaling communication in GBM. A series of computational methods involving miRNA expression, gene ontology (GO) based gene enrichment, pathway mapping, and annotation from metabolic pathways databases, and network analysis were used for the analysis. Our study revealed the physiological roles of many known and novel miRNAs in cancer signaling, especially concerning signaling in cancer progression and proliferation. Overall, the results revealed a strong connection with stress induced senescence, significant miRNA targets for cell cycle arrest, and many common signaling pathways to GBM in the network.     

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H_1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC₅₀ values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gα_q/11, Gα_s, Gα_12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH_1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gα_i/o, the Gα_q/11 protein was proven to contribute to the DMR response of hH_3,4Rs. Moreover, the Gα_12/13 was identified to be involved in the hH₂R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H_1–4Rs.     

Glutamate and GABA in Microglia-Neuron Cross-Talk in Alzheimer’s Disease

The physiological balance between excitation and inhibition in the brain is significantly affected in Alzheimer’s disease (AD). Several neuroactive compounds and their signaling pathways through various types of receptors are crucial in brain homeostasis, among them glutamate and γ-aminobutyric acid (GABA). Activation of microglial receptors regulates the immunological response of these cells, which in AD could be neuroprotective or neurotoxic. The novel research approaches revealed the complexity of microglial function, including the interplay with other cells during neuroinflammation and in the AD brain. The purpose of this review is to describe the role of several proteins and multiple receptors on microglia and neurons, and their involvement in a communication network between cells that could lead to different metabolic loops and cell death/survival. Our review is focused on the role of glutamatergic, GABAergic signaling in microglia–neuronal cross-talk in AD and neuroinflammation. Moreover, the significance of AD-related neurotoxic proteins in glutamate/GABA-mediated dialogue between microglia and neurons was analyzed in search of novel targets in neuroprotection, and advanced pharmacological approaches.