Despite improvements in extracranial therapy, survival rate for patients suffering from brain metastases remains very poor. This is coupled with the incidence of brain metastases continuing to rise. In this review, we focus on core contributions of the blood-brain barrier to the origin of brain metastases. We first provide an overview of the structure and function of the blood-brain barrier under physiological conditions. Next, we discuss the emerging idea of a pre-metastatic niche, namely that secreted factors and extracellular vesicles from a primary tumor site are able to travel through the circulation and prime the neurovasculature for metastatic invasion. We then consider the neurotropic mechanisms that circulating tumor cells possess or develop that facilitate disruption of the blood-brain barrier and survival in the brain's parenchyma. Finally, we compare and contrast brain metastases at the blood-brain barrier to the primary brain tumor, glioma, examining the process of vessel co-option that favors the survival and outgrowth of brain malignancies.

The receptor interacting protein kinases (RIPK) are a family of serine/threonine kinases that are involved in the integration of various stress signals. In response to several extracellular and/or intracellular stimuli, RIP kinases engage signaling cascades leading to the activation of NF-κB and mitogen-activated protein kinases, cell death, inflammation, differentiation and Wnt signaling and can have kinase-dependent and kinase-independent functions. Although it was previously suggested that seven RIPKs are part of the RIPK family, phylogenetic analysis indicates that there are only five genuine RIPKs. RIPK1 and RIPK3 are mainly involved in controlling and executing necroptosis in keratinocytes, while RIPK4 controls proliferation and differentiation of keratinocytes and thereby can act as a tumor suppressor in skin. Therefore, in this review we summarize and discuss the functions of RIPKs in skin homeostasis as well as the signaling pathways involved.

Human carbonic anhydrase II catalyzes the reversible reaction of carbon dioxide and water to form bicarbonate and a proton. His64-mediated proton shuttling between the active site and the bulk solvent is rate limiting. Here we investigate the protonation behavior of His64 as well as its structural and dynamic features in a pH dependent way. We derive two pKa values for His64, 6.25 and 7.60, that we were able to assign to its inward and outward conformation. Furthermore, we show that His64 exists in both conformations equally, independent of pH. Both conformations display an equal distribution of their two neutral tautomeric states. The life time of each conformation is short and both states display high flexibility within their orientation. Therefore, His64 is never static, but rather poised to change conformation. These findings support an energetic, dynamic and solution ensemble-based framework for the high enzymatic activity of human carbonic anhydrase II.

Dendritic cells (DCs) can mediate immune responses or immune tolerance depending on their immunophenotype and functional status. Remodeling of DCs' immune functions can develop proper therapeutic regimens for different immune-mediated diseases. In the immunopathology of autoimmune diseases (ADs), activated DCs notably promote effector T-cell polarization and exacerbate the disease. Recent evidence indicates that metformin can attenuate the clinical symptoms of ADs due to its anti-inflammatory properties. Whether and how the therapeutic effects of metformin on ADs are associated with DCs remain unknown. In this study, metformin was added to a culture system of LPS-induced DC maturation. The results revealed that metformin shifted DC into a tolerant phenotype, resulting in reduced surface expression of MHC-II, costimulatory molecules and CCR7, decreased levels of proinflammatory cytokines (TNF-α and IFN-γ), increased level of IL-10, upregulated immunomodulatory molecules (ICOSL and PD-L) and an enhanced capacity to promote regulatory T-cell (Treg) differentiation. Further results demonstrated that the anti-inflammatory effects of metformin in vivo were closely related to remodeling the immunophenotype of DCs. Mechanistically, metformin could mediate the metabolic reprogramming of DCs through FoxO3a signaling pathways, including disturbing the balance of fatty acid synthesis (FAS) and fatty acid oxidation (FAO), increasing glycolysis but inhibiting the tricarboxylic acid cycle (TAC) and pentose phosphate pathway (PPP), which resulted in the accumulation of fatty acids (FAs) and lactic acid, as well as low anabolism in DCs. Our findings indicated that metformin could induce tolerance in DCs by reprogramming their metabolic patterns and play anti-inflammatory roles in vitro and in vivo.

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks.

Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1-NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure-activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

Nuclear Cyclin D1 (Ccnd1) is a main regulator of cell cycle progression and cell proliferation. Interestingly, Ccnd1 moves to the cytoplasm at the onset of differentiation in neuronal precursors. However, cytoplasmic functions and targets of Ccnd1 in post-mitotic neurons are unknown. Here we identify the α4 subunit of gamma-aminobutyric acid (GABA) type A receptors (GABAARs) as an interactor and target of Ccnd1-Cdk4. Ccnd1 binds to an intracellular loop in α4 and, together with Cdk4, phosphorylates the α4 subunit at threonine 423 and serine 431. These modifications upregulate α4 surface levels, increasing the response of α4-containing GABAARs, measured in whole-cell patch-clamp recordings. In agreement with this role of Ccnd1-Cdk4 in neuronal signalling, inhibition of Cdk4 or expression of the non-phosphorylatable α4 decreases synaptic and extra-synaptic currents in the hippocampus of newborn rats. Moreover, according to α4 functions in synaptic pruning, CCND1 knockout mice display an altered pattern of dendritic spines that is rescued by the phosphomimetic α4. Overall, our findings molecularly link Ccnd1-Cdk4 to GABAARs activity in the central nervous system and highlight a novel role for this G1 cyclin in neuronal signalling.

Human papillomavirus (HPV) encoded E7 oncoprotein plays an important role in supporting the viral productive cycle and inducing cancer phenotypes. The ability of E7 to exercise these functions, partly, depends upon its steady-state level. HPV manipulates the host de-ubiquitination pathway to maintain the stability of its viral proteins. In this study, we uncovered that HPV interacts with the host ubiquitin specific protease 7 (USP7), a universal de-ubiquitinating enzyme, leading to the stabilization of E7 oncoprotein. We observed that HPV16E7 complexes with USP7 via the E7-CR3 domain, and this E7-USP7 complex exists predominantly in the nucleus. Our results showed that USP7 stabilizes and prolongs the half-life of HPV16E7 by antagonizing ubiquitination and proteasomal degradation. Consistently, when we inhibited USP7 activity using HBX 19818, HPV16E7 protein level was reduced and its turnover was increased. We also provide evidence that HBX 19818-induced USP7 inhibition can halt HPV-mediated carcinogenesis, including cell proliferation, invasion, migration and transformation. These findings indicate that USP7 plays an essential role in stabilizing E7. The specific and potent inhibitory effects of HBX 19818 on HPV-induced carcinogenesis provide a molecular insight, suggesting the potential of targeting USP7 as a new therapeutic approach for the treatment of HPV-associated cancers.

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11β splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11βki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11βki/- males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11β and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy.

BACKGROUND: The tightly controlled balance between osteogenic and adipogenic differentiation of human bone marrow-derived stromal cells (BMSCs) is critical to maintain bone homeostasis. Age-related osteoporosis is characterized by low bone mass with excessive infiltration of adipose tissue in the bone marrow compartment. The shift of BMSC differentiation from osteoblasts to adipocytes could result in bone loss and adiposity. METHODS: TNS3 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of TNS3 was used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining at multiple time points. The role of TNS3 and its domain function in osteogenic differentiation were evaluated by ALP activity, calcium assay, and Alizarin Red S staining. The expression of Rho-GTP was determined using the RhoA pull-down activation assay. RESULTS: Loss of TNS3 impaired osteogenic differentiation of BMSCs but promoted adipogenic differentiation. Conversely, TNS3 overexpression hampered adipogenesis while enhancing osteogenesis. The expression level of TNS3 determined cell shape and cytoskeletal reorganization during osteogenic differentiation. TNS3 truncation experiments revealed that for optimal osteogenesis to occur, all domains proved essential. Pull-down and immunocytochemical experiments suggested that TNS3 mediates osteogenic differentiation through RhoA. CONCLUSIONS: Here, we identify TNS3 to be involved in BMSC fate decision. Our study links the domain structure in TNS3 to RhoA activity via actin dynamics and implicates an important role for TNS3 in regulating osteogenesis and adipogenesis from BMSCs. Furthermore, it supports the critical involvement of cytoskeletal reorganization in BMSC differentiation.

Major depressive disorder (MDD) is a pervasive and devastating mental disease. Broad spectrum histone deacetylase (HDAC) inhibitors are considered to have potential for the treatment of depressive phenotype in mice. However, due to its non-specific inhibition, it has extensive side effects and can not be used in clinical treatment of MDD. Therefore, finding specific HDAC subtypes that play a major role in the etiology of MDD is the key to develop corresponding specific inhibitors as antidepressants in the future. Copy number variation in HDAC9 gene is thought to be associated with the etiology of some psychiatric disorders. Herein, we found that HDAC9 was highly expressed in the hippocampus of chronic restraint stress (CRS) mouse model of depression. Upregulation of HDAC9 expression in hippocampal neurons of mice induced depression-like phenotypes, including anhedonia, helplessness, decreased dendritic spine density, and neuronal hypoexcitability. Moreover, knockdown or knockout of HDAC9 in hippocampal neurons alleviated depression-like phenotypes caused by chronic restraint stress (CRS) in WT mice. Importantly, using immunoprecipitation-mass spectrometry (IP-MS), we further found that Annexin A2 (ANXA2) was coupled to and deacetylated by HDAC9. This coupling resulted in the inhibition of ubiquitinated ANXA2 degradation and then mediates depression-like behavior. Overall, we discovered a previously unrecognized role for HDAC9 in hippocampal neurons in the pathogenesis of depression, indicating that inhibition of HDAC9 might be a promising clinical strategy for the treatment of depressive disorders.