Overstimulation of N-methyl-D-aspartate receptors (NMDARs) is the leading cause of brain excitotoxicity and often contributes to neurodegenerative diseases such as Alzheimer's Disease (AD), the most common form of dementia. This study aimed to evaluate a new NMDA receptor antagonist (UB-ALT-EV) and memantine in 6-month-old female 5XFAD mice that were exposed orally to a chronic low-dose treatment. Behavioral and cognitive tests confirmed better cognitive performance in both treated groups. Calcium-dependent protein calpain-1 reduction was found after UB-ALT-EV treatment but not after memantine. Changes in spectrin breakdown products (SBDP) and the p25/p35 ratio confirmed diminished calpain-1 activity. Amyloid β (Aβ) production and deposition was evaluated in 5XFAD mice and demonstrated a robust effect of NMDAR antagonists on reducing Aβ deposition and the number and size of Thioflavin-S positive plaques. Furthermore, glycogen synthase kinase 3β (GSK3β) active form and phosphorylated tau (AT8) levels were diminished after UB-ALT-EV treatment, revealing tau pathology improvement. Because calpain-1 is involved in autophagy activation, autophagic proteins were studied. Strikingly, results showed changes in the protein levels of unc-51-like kinase (ULK-1), beclin-1, microtubule-associated protein 1A/1B-light chain 3(LC3B-II)/LC3B-I ratio, and lysosomal-associated membrane protein 1 (LAMP-1) after NMDAR antagonist treatments, suggesting an accumulation of autophagolysosomes in 5XFAD mice, reversed by UB-ALT-EV. Likewise, treatment with UB-ALT-EV recovered a WT mice profile in apoptosis markers Bcl-2, Bax, and caspase-3. In conclusion, our results revealed the potential neuroprotective effect of UB-ALT-EV by attenuating NMDA-mediated apoptosis and reducing Aβ deposition and deposition jointly with the autophagy rescue to finally reduce cognitive alterations in a mice model of familial AD.

Muscular dystrophy encompasses a large number of heterogeneous genetic disorders characterized by progressive and devastating muscle wasting. Cell-based replacement strategies aimed at promoting skeletal muscle regeneration represent a candidate therapeutic approach to treat muscular dystrophies. Due to the difficulties of obtaining large numbers of stem cells from a muscle biopsy as well as expanding these in vitro, pluripotent stem cells (PSCs) represent an attractive cell source for the generation of myogenic progenitors, given that PSCs can repeatedly produce large amounts of lineage-specific tissue, representing an unlimited source of cells for therapy. In this review, we focus on the progress to date on different methods for the generation of human PSC-derived myogenic progenitor cells, their regenerative capabilities upon transplantation, their potential for allogeneic and autologous transplantation, as well as the specific challenges to be considered for future therapeutic applications.

Spinocerebellar ataxia type 1 (SCA1) is one of nine polyglutamine (polyQ) diseases and is characterized as an adult late-onset, progressive, dominantly inherited genetic disease. SCA1 is caused by an increase in the number of CAG repeats in the ATXN1 gene leading to an expanded polyQ tract in the ATAXIN-1 protein. ATAXIN-1 is broadly expressed throughout the brain. However, until recently, SCA1 research has primarily centered on the cerebellum, given the characteristic cerebellar Purkinje cell loss observed in patients, as well as the progressive motor deficits, including gait and limb incoordination, that SCA1 patients present with. There are, however, also other symptoms such as respiratory problems, cognitive defects and memory impairment, anxiety, and depression observed in SCA1 patients and mouse models, which indicate that there are extra-cerebellar effects of SCA1 that cannot be explained solely through changes in the cerebellar region of the brain alone. The existing gap between human and mouse model studies of extra-cerebellar regions in SCA1 makes it difficult to answer many important questions in the field. This review will cover both the cerebellar and extra-cerebellar effects of SCA1 and highlight the need for further investigations into the impact of mutant ATXN1 expression in these regions. This review will also discuss implications of extra-cerebellar effects not only for SCA1 but other neurodegenerative diseases showing diverse pathology as well.

Osteoarthritis (OA) is mainly characterized by articular cartilage degeneration, synovial fibrosis, and inflammation. LncRNA CRNDE (colorectal neoplasia differentially expressed) has been reported to be down-regulated in age-related OA, but its role in injury-induced OA needs to be further explored. In this study, an OA rat model was established using anterior cruciate ligament transection, and the adenovirus-mediated CRNDE overexpression (Ad-CRNDE) or DACT1 (dapper antagonist of catenin-1) interference (sh-DACT1) vectors were administered by intraarticular injection. Moreover, chondrocyte‑like ATDC5 cells were treated with IL-1β (10 ng/mL) to simulate OA conditions in vitro. We found that overexpression of CRNDE alleviated cartilage damage and synovitis in OA rats, and suppressed IL-1β-induced apoptosis, inflammation, and extracellular matrix (ECM) degradation in chondrocyte‑like ATDC5 cells, while silencing DACT1 effectively antagonized the protective effect of CRNDE both in vivo and in vitro. Mechanism studies revealed that DACT1 could act as a downstream target of CRNDE. By recruiting p300, CRNDE promoted the enrichment of H3K27ac in the DACT1 promoter, thus promoting DACT1 transcription. In addition, CRNDE hindered the activation of the Wnt/β-catenin pathway in IL-1β-stimulated cells by inducing DACT1 expression. In conclusion, CRNDE promoted DACT1 expression through epigenetic modification and restrained the activation of Wnt/β-catenin signaling to impede the progression of OA.

Brown and beige adipose tissues dissipate chemical energy in the form of heat to maintain your body temperature in cold conditions. The impaired function of these tissues results in various metabolic diseases in humans and mice. By bioinformatical analyses, we identified a functional thermogenic regulator of adipose tissue, Androgen-dependent tissue factor pathway inhibitor [TFPI]-regulating protein (Adtrp), which was significantly overexpressed in and functionally activated the mature brown/beige adipocytes. Hereby, we knocked out Adtrp in mice which led to multiple abnormalities in thermogenesis, metabolism, and maturation of brown/beige adipocytes causing excess lipid accumulation in brown adipose tissue (BAT) and cold intolerance. The capability of thermogenesis in brown/beige adipose tissues could be recovered in Adtrp KO mice upon direct β3-adrenergic receptor (β3-AR) stimulation by CL316,243 treatment. Our mechanistic studies revealed that Adtrp by binding to S100 calcium-binding protein b (S100b) indirectly mediated the secretion of S100b, which in turn promoted the β3-AR mediated thermogenesis via sympathetic innervation. These results may provide a novel insight into Adtrp in metabolism via regulating the differentiation and thermogenesis of adipose tissues in mice.

Age-associated B cells (ABCs) are a transcriptionally and functionally unique B cell population. In addition to arising with age and following infection, ABCs are expanded during autoimmune disease, including those with systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis. The exact nature of how ABCs impact disease remains unclear. Here, we review what is known regarding ABC development and distribution during diseases including systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis. We discuss possible mechanisms by which ABCs could contribute to disease, including the production of cytokines and autoantibodies or stimulation of T cells. Finally, we speculate on how ABCs might act as mediators between sex, infection, and autoimmune disease, and discuss avenues for further research.

Endoplasmic reticulum (ER) stress initiates the unfolded protein response (UPR) and is decisive for tumor cell growth and tumor microenvironment (TME) maintenance. Tumor cells persistently undergo ER stress and could transmit it to the neighboring macrophages and surroundings. Tumor infiltrating macrophages can also adapt to the microenvironment variations to fulfill their highly energy-demanding and biological functions via ER stress. However, whether the different macrophage populations differentially sense ER stress and transmit ER stress to surrounding tumor cells has not yet been elucidated. Here, we aimed to investigate the role of transmissible ER stress, a novel regulator of intercellular communication in the TME. Murine bone marrow-derived macrophage (BMDM) can be polarized toward distinct functional endpoints termed classical (M1) and alternative (M2) activation, and their polarization status has been shown to be tightly correlated with their functional significance. We showed that tumor cells could receive the transmissible ER stress from two differentially polarized macrophage populations with different extent of ER stress activation. The proinflammatory M1-like macrophages respond to ER stress with less extent, however they could transmit more ER stress to tumor cells. Moreover, by analyzing the secreted components of two ER-stressed macrophage populations, we identified certain damage-associated molecular patterns (DAMPs), including S100A8 and S100A9, which are dominantly secreted by M1-like macrophages could lead to significant recipient tumor cells death in synergy with transferred ER stress.

Although combination antiretroviral therapy (ART) has reduced mortality and improved lifespan for people living with HIV, it does not provide a cure. Patients must be on ART for the rest of their lives and contend with side effects, unsustainable costs, and the development of drug resistance. A cure for HIV is, therefore, warranted to avoid the limitations of the current therapy and restore full health. However, this cure is difficult to find due to the persistence of latently infected HIV cellular reservoirs during suppressive ART. Approaches to HIV cure being investigated include boosting the host immune system, genetic approaches to disable co-receptors and the viral genome, purging cells harboring latent HIV with latency-reversing latency agents (LRAs) (shock and kill), intensifying ART as a cure, preventing replication of latent proviruses (block and lock) and boosting T cell turnover to reduce HIV-1 reservoirs (rinse and replace). Since most people living with HIV are in Africa, methods being developed for a cure must be amenable to clinical trials and deployment on the continent. This review discusses the current approaches to HIV cure and comments on their appropriateness for Africa.

Hematopoietic stem/progenitor cells (HSPCs) originate from endothelial cells (ECs) localized on the ventral side of the dorsal aorta (DA), and hemodynamic parameters may suffer sharp changes in DA at HSPCs development stage for intersegmental vessel formation. However, the temporal-spatial shear stress parameters and biomechanics mechanisms of HSPC budding remain unknown. Here, we found that the hematopoietic endothelium (HE) in the aorta-gonad-mesonephros was heterogeneous; that is, HEs were mainly distributed at the ventral side of the vascular bifurcation in zebrafish embryos, which was found to show low shear stress (LSS) through numerical simulation analysis. Furthermore, HSPCs localized in the posterior somite of aorta-gonad-mesonephros with slow velocity. On the temporal scale, there was a slow velocity and LSS during HE budding from 36 h post-fertilization and decreased shear stress with drug expanded HSPC numbers. Mechanistically, matrix metalloproteinase (MMP) expression and macrophage chemotaxis were significantly increased in HEs by RNA-seq. After treatment with an MMP13 inhibitor, HSPCs were significantly reduced in both the aorta-gonad-mesonephros and caudal hematopoietic tissue in embryos. Our results show that HSPC budding is heterogeneous, and the mechanism is that physiological LSS controls the emergence of HSPCs by promoting the accumulation of macrophages and subsequent MMP expression.

Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD.

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

Change in cell size may bring in profound impact to cell function and survival, hence the integrity of the organs consisting of those cells. Nevertheless, how cell size is regulated remains incompletely understood. We used the fluorescent zebrafish transgenic line Tg-GGH/LR that displays inducible folate deficiency (FD) and hepatomegaly upon FD induction as in vivo model. We found that FD caused hepatocytes enlargement and increased liver stiffness, which could not be prevented by nucleotides supplementations. Both in vitro and in vivo studies indicated that RIPK3/MLKL-dependent necroptotic pathway and Hippo signaling interactively participated in this FD-induced hepatocytic enlargement in a dual chronological and cooperative manner. FD also induced hepatic inflammation, which convenes a dialog of positive feedback loop between necroptotic and Hippo pathways. The increased MMP13 expression in response to FD elevated TNFα level and further aggravated the hepatocyte enlargement. Meanwhile, F-actin was circumferentially re-allocated at the edge under cell membrane in response to FD. Our results substantiate the interplay among intracellular folate status, pathways regulation, inflammatory responses, actin cytoskeleton and cell volume control, which can be best observed with in vivo platform. Our data also support the use of this Tg-GGH/LR transgenic line for the mechanistical and therapeutic research for the pathologic conditions related to cell size alteration.

In the course of atherogenesis, the spleen plays an important role in the regulation of extramedullary hematopoiesis, and in the control of circulating immune cells, which contributes to plaque progression. Here, we have investigated the role of splenic nucleotide-binding oligomerization domain 1 (NOD1) in the recruitment of circulating immune cells, as well as the involvement of this immune organ in extramedullary hematopoiesis in mice fed on a high-fat high-cholesterol diet (HFD). Under HFD conditions, the absence of NOD1 enhances the mobilization of immune cells, mainly neutrophils, from the bone marrow to the blood. To determine the effect of NOD1-dependent mobilization of immune cells under pro-atherogenic conditions, Apoe-/- and Apoe-/-Nod1-/- mice fed on HFD for 4 weeks were used. Splenic NOD1 from Apoe-/- mice was activated after feeding HFD as inferred by the phosphorylation of the NOD1 downstream targets RIPK2 and TAK1. Moreover, this activation was accompanied by the release of neutrophil extracellular traps (NETs), as determined by the increase in the expression of peptidyl arginine deiminase 4, and the identification of citrullinated histone H3 in this organ. This formation of NETs was significantly reduced in Apoe-/-Nod1-/- mice. Indeed, the presence of Ly6G+ cells and the lipidic content in the spleen of mice deficient in Apoe and Nod1 was reduced when compared to the Apoe-/- counterparts, which suggests that the mobilization and activation of circulating immune cells are altered in the absence of NOD1. Furthermore, confirming previous studies, Apoe-/-Nod1-/- mice showed a reduced atherogenic disease, and diminished recruitment of neutrophils in the spleen, compared to Apoe-/- mice. However, splenic artery ligation reduced the atherogenic burden in Apoe-/- mice an effect that, unexpectedly was lost in Apoe-/-Nod1-/- mice. Together, these results suggest that neutrophil accumulation and activity in the spleen are driven in part by NOD1 activation in mice fed on HFD, contributing in this way to regulating atherogenic progression.

Ceramides are a heterogeneous group of bioactive membrane sphingolipids that play specialized regulatory roles in cellular metabolism depending on their characteristic fatty acyl chain lengths and subcellular distribution. As obesity progresses, certain ceramide molecular species accumulate in metabolic tissues and cause cell-type-specific lipotoxic reactions that disrupt metabolic homeostasis and lead to the development of cardiometabolic diseases. Several mechanisms for ceramide action have been inferred from studies in vitro, but only recently have we begun to better understand the acyl chain length specificity of ceramide-mediated signaling in the context of physiology and disease in vivo. New discoveries show that specific ceramides affect various metabolic pathways and that global or tissue-specific reduction in selected ceramide pools in obese rodents is sufficient to improve metabolic health. Here, we review the tissue-specific regulation and functions of ceramides in obesity, thus highlighting the emerging concept of selectively inhibiting production or action of ceramides with specific acyl chain lengths as novel therapeutic strategies to ameliorate obesity-associated diseases.

Iron is an essential micro-element, involved in multiple biological activities in vertebrates. Excess iron accumulation has been identified as an important mediator of lipid deposition. However, the underlying mechanisms remain unknown. In the present study, we found that a high-iron diet significantly increased intestinal iron content and upregulated the mRNA expression of two iron transporters (zip14 and fpn1). Intestinal iron overload increased lipogenesis, reduced lipolysis and promoted oxidative stress and mitochondrial dysfunction. Iron-induced lipid accumulation was mediated by hypoxia-inducible factor-1 α (HIF1α), which was induced in response to mitochondrial oxidative stress following inhibition of prolyl hydroxylase 2 (PHD2). Mechanistically, iron promoted lipid deposition by enhancing the DNA binding capacity of HIF1α to the pparγ and fas promoters. Our results provide experimental evidence that oxidative stress, mitochondrial dysfunction and the HIF1α-PPARγ pathway are critical mediators of iron-induced lipid deposition.

Body fluid homeostasis is critical to survival. The integrity of the hypothalamo-neurohypophysial system (HNS) is an important basis of the precise regulation of body fluid metabolism and arginine vasopressin (AVP) hormone release. Clinically, some patients with central diabetes insipidus (CDI) due to HNS lesions can experience recovery compensation of body fluid metabolism. However, whether the hypothalamus has the potential for structural plasticity and self-repair under pathological conditions remains unclear. Here, we report the repair and reconstruction of a new neurohypophysis-like structure in the hypothalamic median eminence (ME) after pituitary stalk electrical lesion (PEL). We show that activated and proliferating adult neural progenitor cells differentiate into new mature neurons, which then integrate with remodeled AVP fibers to reconstruct the local AVP hormone release neural circuit in the ME after PEL. We found that the transcription factor of NK2 homeobox 1 (NKX2.1) and the sonic hedgehog signaling pathway, mediated by NKX2.1, are the key regulators of adult hypothalamic neurogenesis. Taken together, our study provides evidence that adult ME neurogenesis is involved in the structural reconstruction of the AVP release circuit and eventually restores body fluid metabolic homeostasis during hypothalamic self-repair.

The contribution of astrocytes to Alzheimer's disease (AD) is still ill defined. AD involves an abnormal accumulation of amyloid-β peptides (Aβ) and increased production of danger signals such as ATP. ATP can direct or indirectly, through its metabolism into adenosine, trigger adaptive astrocytic responses resulting from intracellular Ca2+ oscillations. AD also triggers an upregulation of astrocytic adenosine A2A receptors (A2AR), which blockade prevents memory dysfunction in AD. We now investigated how Aβ peptides affect ATP-mediated Ca2+ responses in astrocytes measured by fluorescence live-cell imaging and whether A2AR control astrocytic Ca2+ responses mediated by ATP receptors, mainly P2X7R and P2Y1R. In primary cultures of rat astrocytes exposed to Aβ1-42, ATP-evoked Ca2+ responses had a lower amplitude but a longer duration than in control astrocytes and involved P2X7R and P2Y1R, the former potentiating the later. Moreover, Aβ1-42 exposure increased protein levels of P2Y1R in astrocytes. A2AR antagonism with SCH58261 controlled in a protein kinase A-dependent manner both P2X7R- and P2Y1R-mediated Ca2+ responses in astrocytes. The interplay between these purinoceptors in astrocytes was blunted upon exposure to Aβ1-42. These findings uncover the ability of A2AR to regulate the inter-twinned P2X7R- and P2Y1R-mediated Ca2+ dynamics in astrocytes, which is disrupted in conditions of early AD.

During sepsis, the importance of alterations in cell metabolism is underappreciated. The cellular metabolism, which has a variable metabolic profile in different cells and disease stages, is largely responsible for the immune imbalance and organ failure associated with sepsis. Metabolic reprogramming, in which glycolysis replaces OXPHOS as the main energy-producing pathway, is both a requirement for immune cell activation and a cause of immunosuppression. Meanwhile, the metabolites produced by OXPHOS and glycolysis can act as signaling molecules to control the immune response during sepsis. Sepsis-induced "energy shortage" leads to stagnated cell function and even organ dysfunction. Metabolic reprogramming can alleviate the energy crisis to some extent, enhance host tolerance to maintain cell survival functions, and ultimately increase the adaptation of cells during sepsis. However, a switch from glycolysis to OXPHOS is essential for restoring cell function. This review summarized the crosstalk between metabolic reprogramming and immune cell activity as well as organ function during sepsis, discussed the benefits and drawbacks of metabolic reprogramming to show the contradictions of metabolic reprogramming during sepsis, and assessed the feasibility of treating sepsis through targeted metabolism. Using metabolic reprogramming to achieve metabolic homeostasis could be a viable therapy option for sepsis.

Neural progenitor cell (NPC) transplantation represents a promising treatment strategy for spinal cord injury (SCI); however, the underlying therapeutic mechanisms remain incompletely understood. We demonstrate that severe spinal contusion in adult rats causes transcriptional dysregulation, which persists from early subacute to chronic stages of SCI and affects nearly 20,000 genes in total tissue extracts. Functional analysis of this dysregulated transcriptome reveals the significant downregulation of cAMP signalling components immediately after SCI, involving genes such as EPAC2 (exchange protein directly activated by cAMP), PKA, BDNF, and CAMKK2. The ectopic transplantation of spinal cord-derived NPCs at acute or subacute stages of SCI induces a significant transcriptional impact in spinal tissue, as evidenced by the normalized expression of a large proportion of SCI-affected genes. The transcriptional modulation pattern driven by NPC transplantation includes the rescued expression of cAMP signalling genes, including EPAC2. We also explore how the sustained in vivo inhibition of EPAC2 downstream signalling via the intrathecal administration of ESI-05 for 1 week impacts therapeutic mechanisms involved in the NPC-mediated treatment of SCI. NPC transplantation in SCI rats in the presence and absence of ESI-05 administration prompts increased rostral cAMP levels; however, NPC and ESI-05 treated animals exhibit a significant reduction in EPAC2 mRNA levels compared to animals receiving only NPCs treatment. Compared with transplanted animals, NPCs + ESI-05 treatment increases the scar area (as shown by GFAP staining), polarizes microglia into an inflammatory phenotype, and increases the magnitude of the gap between NeuN + cells across the lesion. Overall, our results indicate that the NPC-associated therapeutic mechanisms in the context of SCI involve the cAMP pathway, which reduces inflammation and provides a more neuropermissive environment through an EPAC2-dependent mechanism.

Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal-cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.

BACKGROUND: Cisplatin is an effective chemotherapeutic drug, but it may induce both acute and chronic kidney problems. The pathogenesis of chronic kidney disease (CKD) associated with cisplatin chemotherapy remains largely unclear. METHODS: Mice and renal tubular cells were subjected to repeated low-dose cisplatin (RLDC) treatment to induce CKD and related pathological changes. The roles of endoplasmic reticulum (ER) stress, PERK, and protein kinase C-δ (PKCδ) were determined using pharmacological inhibitors and genetic manipulation. RESULTS: ER stress was induced by RLDC in kidney tubular cells in both in vivo and in vitro models. ER stress inhibitors given immediately after RLDC attenuated kidney dysfunction, tubular atrophy, kidney fibrosis, and inflammation in mice. In cultured renal proximal tubular cells, inhibitors of ER stress or its signaling kinase PERK also suppressed RLDC-induced fibrotic changes and the expression of inflammatory cytokines. Interestingly, RLDC-induced PKCδ activation, which was blocked by ER stress or PERK inhibitors, suggesting PKCδ may act downstream of PERK. Indeed, suppression of PKCδ with a kinase-dead PKCδ (PKCδ-KD) or Pkcδ-shRNA attenuated RLDC-induced fibrotic and inflammatory changes. Moreover, the expression of active PKCδ-catalytic fragment (PKCδ-CF) diminished the beneficial effects of PERK inhibitor in RLDC-treated cells. Co-immunoprecipitation assay further suggested PERK binding to PKCδ. CONCLUSION: These results indicate that ER stress contributes to chronic kidney pathologies following cisplatin chemotherapy via the PERK-PKCδ pathway.

Comment on Cell Mol Life Sci. 2022 Jul 11;79(8):410.

BACKGROUND: A rare coding variant, P522R, in the phospholipase C gamma 2 (PLCG2) gene has been identified as protective against late-onset Alzheimer's disease (AD), but the mechanism is unknown. PLCG2 is exclusively expressed in microglia within the central nervous system, and altered microglial function has been implicated in the progression of AD. METHODS: Healthy control hiPSCs were CRISPR edited to generate cells heterozygous and homozygous for the PLCG2P522R variant. Microglia derived from these hiPSC's were used to investigate the impact of PLCγ2P522R on disease relevant processes, specifically microglial capacity to take up amyloid beta (Aβ) and synapses. Targeted qPCR assessment was conducted to explore expression changes in core AD linked and microglial genes, and mitochondrial function was assessed using an Agilent Seahorse assay. RESULTS: Heterozygous expression of the P522R variant resulted in increased microglial clearance of Aβ, while preserving synapses. This was associated with the upregulation of a number of genes, including the anti-inflammatory cytokine Il-10, and the synapse-linked CX3CR1, as well as alterations in mitochondrial function, and increased cellular motility. The protective capacity of PLCγ2P522R appeared crucially dependent on (gene) 'dose', as cells homozygous for the variant showed reduced synapse preservation, and a differential gene expression profile relative to heterozygous cells. CONCLUSION: These findings suggest that PLCγ2P522R may result in increased surveillance by microglia, and prime them towards an anti-inflammatory state, with an increased capacity to respond to increasing energy demands, but highlights the delicate balance of this system, with increasing PLCγ2P522R 'dose' resulting in reduced beneficial impacts.

Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function principle is of equal importance as the classical sequence → structure → function paradigm. To highlight this point, we describe the IDPs and/or IDRs behind the discoveries associated with 17 Nobel Prizes, 11 in Physiology or Medicine and 6 in Chemistry. The Nobel Laureates themselves did not always mention that the proteins underlying the phenomena investigated in their award-winning studies are in fact IDPs or contain IDRs. In several cases, IDP- or IDR-based molecular functions have been elucidated, while in other instances, it is recognized that the respective protein(s) contain IDRs, but the specific IDR-based molecular functions have yet to be determined. To highlight the importance of IDPs and IDRs as general principle in biology, we present here illustrative examples of IDPs/IDRs in Nobel Prize-winning mechanisms and processes.

Parkinson's disease, Multiple System Atrophy, and Lewy Body Dementia are incurable diseases called α-synucleinopathies as they are mechanistically linked to the protein, α-synuclein (α-syn). α-syn exists in different structural forms which have been linked to clinical disease distinctions. However, sleeping disorders (SDs) are common in the prodromal phase of all three α-synucleinopathies, which suggests that sleep-controlling neurons are affected by multiple forms of α-syn. To determine whether a structure-independent neuronal impact of α-syn exists, we compared and contrasted the cellular effect of three different α-syn forms on neurotransmitter-defined cells of two sleep-controlling nuclei located in the brainstem: the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus. We utilized size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy and transmission electron microscopy to precisely characterize ​​timepoints in the α-syn aggregation process with three different dominating forms of this protein (monomeric, oligomeric and fibril) and we conducted an in-depth investigation of the underlying neuronal mechanism behind cellular effects of the different forms of the protein using electrophysiology, multiple-cell calcium imaging, single-cell calcium imaging and live-location tracking with fluorescently-tagged α-syn. Interestingly, α-syn altered membrane currents, enhanced firing, increased intracellular calcium and facilitated cell death in a structure-independent manner in sleep-controlling nuclei, and postsynaptic actions involved a G-protein-mediated mechanism. These data are novel as the sleep-controlling nuclei are the first brain regions reported to be affected by α-syn in this structure-independent manner. These regions may represent highly important targets for future neuroprotective therapy to modify or delay disease progression in α-synucleinopathies.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

Increasing evidence suggests different, not completely understood roles of microRNA biogenesis in the development and progression of lung cancer. The overexpression of the DNA repair protein apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an important cause of poor chemotherapeutic response in lung cancer and its involvement in onco-miRNAs biogenesis has been recently described. Whether APE1 regulates miRNAs acting as prognostic biomarkers of lung cancer has not been investigated, yet. In this study, we analyzed miRNAs differential expression upon APE1 depletion in the A549 lung cancer cell line using high-throughput methods. We defined a signature of 13 miRNAs that strongly correlate with APE1 expression in human lung cancer: miR-1246, miR-4488, miR-24, miR-183, miR-660, miR-130b, miR-543, miR-200c, miR-376c, miR-218, miR-146a, miR-92b and miR-33a. Functional enrichment analysis of this signature revealed its biological relevance in cancer cell proliferation and survival. We validated DICER1 as a direct functional target of the APE1-regulated miRNA-33a-5p and miR-130b-3p. Importantly, IHC analyses of different human tumors confirmed a negative correlation existing between APE1 and Dicer1 protein levels. DICER1 downregulation represents a prognostic marker of cancer development but the mechanisms at the basis of this phenomenon are still completely unknown. Our findings, suggesting that APE1 modulates DICER1 expression via miR-33a and miR-130b, reveal new mechanistic insights on DICER1 regulation, which are of relevance in lung cancer chemoresistance and cancer invasiveness.

The RS1 gene on Xp 22.13 encodes retinoschisin which is known to directly interact with the retinal Na/K-ATPase at the photoreceptor inner segments. Pathologic mutations in RS1 cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in young males. To further delineate the retinoschisin-Na/K-ATPase complex, co-immunoprecipitation was performed with porcine and murine retinal lysates targeting the ATP1A3 subunit. This identified the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2 as direct interaction partners of the retinal Na/K-ATPase. Colocalization of the individual components of the complex was demonstrated at the membrane of photoreceptor inner segments. We further show that retinoschisin-deficiency, a frequent consequence of molecular pathology in XLRS, causes mislocalization of the macromolecular complex during postnatal retinal development with a simultaneous reduction of Kv2.1 and Kv8.2 protein expression, while the level of retinal Na/K-ATPase expression remains unaffected. Patch-clamp analysis revealed no effect of retinoschisin-deficiency on Kv channel mediated potassium ion currents in vitro. Together, our data suggest that Kv2.1 and Kv8.2 together with retinoschisin and the retinal Na/K-ATPase are integral parts of a macromolecular complex at the photoreceptor inner segments. Defective compartmentalization of this complex due to retinoschisin-deficiency may be a crucial step in initial XLRS pathogenesis.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Spinal muscular atrophy (SMA) is a genetic disease resulting in the loss of α-motoneurons followed by muscle atrophy. It is caused by knock-out mutations in the survival of motor neuron 1 (SMN1) gene, which has an unaffected, but due to preferential exon 7 skipping, only partially functional human-specific SMN2 copy. We previously described a Drosophila-based screening of FDA-approved drugs that led us to discover moxifloxacin. We showed its positive effect on the SMN2 exon 7 splicing in SMA patient-derived skin cells and its ability to increase the SMN protein level. Here, we focus on moxifloxacin's therapeutic potential in additional SMA cellular and animal models. We demonstrate that moxifloxacin rescues the SMA-related molecular and phenotypical defects in muscle cells and motoneurons by improving the SMN2 splicing. The consequent increase of SMN levels was higher than in case of risdiplam, a potent exon 7 splicing modifier, and exceeded the threshold necessary for a survival improvement. We also demonstrate that daily subcutaneous injections of moxifloxacin in a severe SMA murine model reduces its characteristic neuroinflammation and increases the SMN levels in various tissues, leading to improved motor skills and extended lifespan. We show that moxifloxacin, originally used as an antibiotic, can be potentially repositioned for the SMA treatment.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe cardiac disease that leads to heart failure or sudden cardiac death (SCD). For the pathogenesis of ARVC, various mutations in at least eight different genes have been identified. A rare form of ARVC is associated with the mutation TMEM43 p.S358L, which is a fully penetrant variant in male carriers. TMEM43 p.S358 is homologous to CG8111 p.S333 in Drosophila melanogaster. We established CRISPR/Cas9-mediated CG8111 knock-out mutants in Drosophila, as well as transgenic fly lines carrying an overexpression construct of the CG8111 p.S333L substitution. Knock-out flies developed normally, whereas the overexpression of CG8111 p.S333L caused growth defects, loss of body weight, cardiac arrhythmias, and premature death. An evaluation of a series of model mutants that replaced S333 by selected amino acids proved that the conserved serine is critical for the physiological function of CG8111. Metabolomic and proteomic analyses revealed that the S333 in CG8111 is essential to proper energy homeostasis and lipid metabolism in the fly. Of note, metabolic impairments were also found in the murine Tmem43 disease model, and fibrofatty replacement is a hallmark of human ARVC5. These findings contribute to a more comprehensive understanding of the molecular functions of CG8111 in Drosophila, and can represent a valuable basis to assess the aetiology of the human TMEM43 p.S358L variant in more detail.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

Clostridioides difficile infection (CDI) causes nosocomial/antibiotic-associated gastrointestinal diseases with dramatically increasing global incidence and mortality rates. The main C. difficile virulence factors, toxins A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We demonstrated that TcdB induces caspase-dependent, mitochondria-independent enteric glial cell (EGC) apoptosis that is enhanced by the pro-inflammatory cytokines TNF-α and IFN-γ (CKs) by increasing caspase-3/7/9 and PARP activation. Because this cytotoxic synergism is important for CDI pathogenesis, we investigated the apoptotic pathways involved in TcdB- and TcdB + CK-induced apoptosis indepth. EGCs were pre-treated with the inhibitors BAF or Q-VD-OPh (pan-caspase), Z-DEVD-fmk (caspase-3/7), Z-IETD-fmk (caspase-8), PD150606 (calpains), and CA-074Me (cathepsin B) 1 h before TcdB exposure, while CKs were given 1.5 h after TcdB exposure, and assays were performed at 24 h. TcdB and TcdB + CKs induced apoptosis through three signalling pathways activated by calpains, caspases and cathepsins, which all are involved both in induction and execution apoptotic signalling under both conditions but to different degrees in TcdB and TcdB + CKs especially as regards to signal transduction mediated by these proteases towards downstream effects (apoptosis). Calpain activation by Ca2+ influx is the first pro-apoptotic event in TcdB- and TcdB + CK-induced EGC apoptosis and causes caspase-3, caspase-7 and PARP activation. PARP is also directly activated by calpains which are responsible of about 75% of apoptosis in TcdB and 62% in TcdB + CK which is both effector caspase-dependent and -independent. Initiator caspase-8 activation mediated by TcdB contributes to caspase-3/caspase-7 and PARP activation and is responsible of about 28% of apoptosis in both conditions. Caspase-3/caspase-7 activation is weakly responsible of apoptosis, indeed we found that it mediates 27% of apoptosis only in TcdB. Cathepsin B contributes to triggering pro-apoptotic signal and is responsible in both conditions of about 35% of apoptosis by a caspase-independent manner, and seems to regulate the caspase-3 and caspase-7 cleaved fragment levels, highlighting the complex interaction between these cysteine protease families activated during TcdB-induced apoptosis. Further a relevant difference between TcdB- and TcdB + CK-induced apoptosis is that TcdB-induced apoptosis increased slowly reaching at 72 h the value of 18.7%, while TcdB + CK-induced apoptosis increased strongly reaching at 72 h the value of 60.6%. Apoptotic signalling activation by TcdB + CKs is enriched by TNF-α-induced NF-κB signalling, inhibition of JNK activation and activation of AKT. In conclusion, the ability of C. difficile to activate three apoptotic pathways represents an important strategy to overcome resistance against its cytotoxic activity.

BACKGROUND: Solitary fibrous tumour (SFT) is a rare mesenchymal malignancy that lacks robust prognostic and predictive biomarkers. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier, associated with tumour progression, and with poor survival of SFT patients, as previous published by our group. Here, we describe the role of ISG15 in the biology of this rare tumour. METHODS: ISG15 expression was assessed by immunohistochemistry in tissue microarrays from SFT patients and tested for correlation with progression-free survival and overall survival (OS). The effects of ISG15 knockdown or induction were investigated for cancer stem cell (CSC) characteristics and for drug sensitivity in unique in vitro models of SFT. RESULTS: The prognostic value of ISG15 for OS was validated at protein level in malignant SFT patients, prospectively treated with pazopanib and enrolled in GEIS-32 trial. In SFT in vitro models, ISG15 knockdown lead to a decrease in the expression of CSC-related genes, including SOX2, NANOG, ALDH1A1, ABCB1 and ABCC1. Likewise, ISG15 downregulation decreased the clonogenic/ tumoursphere-forming ability of SFT cells, while enhancing apoptotic cell death after doxorubicin, pazopanib or trabectedin treatment in 3D cell cultures. The regulation of CSC-related genes by ISG15 was confirmed after inducing its expression with interferon-β1; ISG15 induction upregulated 1.28- to 451-fold the expression of CSC-associated genes. CONCLUSIONS: ISG15 is a prognostic factor in malignant SFT, regulating the expression of CSC-related genes and CSCs maintenance. Our results suggest that ISG15 could be a novel therapeutic target in SFT, which could improve the efficacy of the currently available treatments.

It is widely assumed that as connective tissue, the intervertebral disc (IVD) plays a crucial role in providing flexibility for the spinal column. The disc is comprised of three distinct tissues: the nucleus pulposus (NP), ligamentous annulus fibrous (AF) that surrounds the NP, and the hyaline cartilaginous endplates (CEP). Nucleus pulposus, composed of chondrocyte-like NP cells and its secreted gelatinous matrix, is critical for disc health and function. The NP matrix underwent dehydration accompanied by increasing fibrosis with age. The degeneration of matrix is almost impossible to repair, with the consequence of matrix stiffness and senescence of NP cells and intervertebral disc, suggesting the value of glycoproteins in extracellular matrix (ECM). Here, via database excavation and biological function screening, we investigated a C-type lectin protein, CLEC3A, which could support differentiation of chondrocytes as well as maintenance of NP cells and was essential to intervertebral disc homeostasis. Furthermore, mechanistic analysis revealed that CLEC3A could stimulate PI3K-AKT pathway to accelerate cell proliferation to further play part in NP cell regeneration.

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.

Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.

Previous studies suggested that anti-inflammatory microglia/macrophages (Mi/MΦ) play a role in "normal phagocytosis," which promoted the rapid clearance of necrotic substances and apoptotic cells. More recently, a few studies have found that Mi/MΦ also play a role in "pathological phagocytosis" in the form of excessive or reduced phagocytosis, thereby worsening damage induced by CNS diseases. However, the underlying mechanisms and the Mi/MΦ subtypes related to this pathological phagocytosis are still unknown. Salt-inducible kinase 3 (SIK3), a member of the 5' adenosine monophosphate-activated protein kinase (AMPK) family, has been shown to regulate inflammation in several peripheral diseases. Whether SIK3 also regulates the inflammatory response in CNS diseases is currently unknown. Therefore, in this study, we created a transgenic tamoxifen-induced Mi/MΦ-specific SIK3 conditional knockout (SIK3-cKO) mouse to examine SIK3's role in phagocytotic function induced by transient focal cerebral ischemia (tFCI). By single-cell RNA-seq, we found the pro-inflammatory Mi/MΦ phenotype performed an excessive phagocytotic function, but the anti-inflammatory Mi/MΦ phenotype performed a normal phagocytotic function. We found that SIK3-cKO caused Mi/MΦ heterogenization from the transitional phenotype to the anti-inflammatory phenotype after tFCI. This phenotypic shift corresponded with enhanced phagocytosis of both apoptotic and live neurons. Interestingly, SIK3-cKO enhanced normal phagocytosis of myelin debris but attenuating excessive phagocytosis of non-damaged myelin sheath, thereby protecting white matter integrity after tFCI. CD16, a pro-inflammation marker, was decreased significantly by SIK3-cKO and correlated with "excessive phagocytosis." SIK3-cKO promoted long-term recovery of white matter function and neurological function as assessed with electrophysiological compound action potential (CAPs) and behavioral analysis. This study is the first to show a role of SIK3 in Mi/MΦ phagocytosis in CNS diseases, and reveals that promoting Mi/MΦ anti-inflammatory heterogenization inhibits "excessive phagocytosis" of live cells and facilitates "normal phagocytosis" of apoptotic cells. Therefore, inhibition of SIK3 in Mi/MΦ may be a potential therapeutic target in stroke and other CNS diseases with accompanying white matter destruction. In the acute stage of tFCI, Mi/MΦ polarized into different phenotypes. The pro-inflammatory Mi/MΦ phenotype performed an excessive phagocytotic function. In contrast, the anti-inflammatory Mi/MΦ phenotype performed a normal phagocytotic function. After tFCI, SIK3-cKO promoted anti-inflammatory phenotypic heterogenization of Mi/MΦ. SIK3-cKO promoted Mi/MΦ phagocytosis of apoptotic (normal phagocytosis) and living neuronal cell bodies (excessive phagocytosis) in gray matter. Interestingly, SIK3-cKO specifically increased normal phagocytosis of myelin debris concurrent with an attenuation of excessive phagocytosis of myelin sheath in white matter. These changes induced by SIK3-cKO were associated with protection of white matter integrity and long-term neurofunctional recovery after tFCI.

OBJECTIVE: The molecular heterogeneity of prostate cancer (PCa) gives rise to distinct tumor subclasses based on epigenetic modification and gene expression signatures. Identification of clinically actionable molecular subtypes of PCa is key to improving patient outcome, and the balance between specific pathways may influence PCa outcome. It is also urgent to identify progression-related markers through cytosine-guanine (CpG) methylation in predicting metastasis for patients with PCa. METHODS: We performed bioinformatics analysis of transcriptomic, and clinical data in an integrated cohort of 551 prostate samples. The datasets included retrospective The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. Two algorithms, Least Absolute Shrinkage and Selector Operation and Support Vector Machine-Recursive Feature Elimination, were used to select significant CpGs. RESULTS: We found that PCa progression is more likely to occur after the third year through conditional survival (CS) analysis, and prostate-specific antigen (PSA) was a better predictor of Progression-free survival (PFS) than Gleason score (GS). Our study first demonstrated that PCa tumors have distinct expression profiles based on the expression of genes involved in androgen receptor (AR) and PI3K-AKT, which influence disease outcome. Our results also indicated that there are multiple phenotypes relevant to the AR-PI3K axis in PCa, where tumors with mixed phenotype may be more aggressive or have worse outcome than quiescent phenotype. In terms of epigenetics, we obtained CpG sites and their corresponding genes which have a good predictive value of PFS. However, various evidences showed that the predictive value of CpGs corresponding genes was much lower than GpG sites in Overall survival (OS) and PFS. CONCLUSIONS: PCa classification specific to AR and PI3K pathways provides novel biological insight into previously established PCa subtypes and may help develop personalized therapies. Our results support the potential clinical utility of DNA methylation signatures to distinguish tumor metastasis and to predict prognosis and outcomes.

In humans, cell fusion is restricted to only a few cell types under normal conditions. In the placenta, cell fusion is a critical process for generating syncytiotrophoblast: the giant multinucleated trophoblast lineage containing billions of nuclei within an interconnected cytoplasm that forms the primary interface separating maternal blood from fetal tissue. The unique morphology of syncytiotrophoblast ensures that nutrients and gases can be efficiently transferred between maternal and fetal tissue while simultaneously restricting entry of potentially damaging substances and maternal immune cells through intercellular junctions. To maintain integrity of the syncytiotrophoblast layer, underlying cytotrophoblast progenitor cells terminate their capability for self-renewal, upregulate expression of genes needed for differentiation, and then fuse into the overlying syncytium. These processes are disrupted in a variety of obstetric complications, underscoring the importance of proper syncytiotrophoblast formation for pregnancy health. Herein, an overview of key mechanisms underlying human trophoblast fusion and syncytiotrophoblast development is discussed.

PIK3CA mutations are amongst the most prevalent somatic mutations in cancer and are associated with resistance to first-line treatment along with low survival rates in a variety of malignancies. There is evidence that patients carrying PIK3CA mutations may benefit from treatment with acetylsalicylic acid, commonly known as aspirin, particularly in the setting of colorectal cancer. In this regard, it has been clarified that Class IA Phosphatidylinositol 3-kinases (PI3K), whose catalytic subunit p110α is encoded by the PIK3CA gene, are involved in signal transduction that regulates cell cycle, cell growth, and metabolism and, if disturbed, induces carcinogenic effects. Although PI3K is associated with pro-inflammatory cyclooxygenase-2 (COX-2) expression and signaling, and COX-2 is among the best-studied targets of aspirin, the mechanisms behind this clinically relevant phenomenon are still unclear. Indeed, there is further evidence that the protective, anti-carcinogenic effect of aspirin in this setting may be mediated in a COX-independent manner. However, until now the understanding of aspirin's prostaglandin-independent mode of action is poor. This review will provide an overview of the current literature on this topic and aims to analyze possible mechanisms and targets behind the aspirin sensitivity of PIK3CA-mutated cancers.

Small extracellular vesicle (sEV)-mediated intercellular communication regulates multiple aspects of growth and development in multicellular organisms. However, the mechanism underlying cargo recruitment into sEVs is currently unclear. We show that the key nucleo-cytoplasmic transport (NCT) protein-RanGTPase, in its GTP-bound form (RanGTP), is enriched in sEVs secreted by mammalian cells. This recruitment of RanGTP into sEVs depends on the export receptor CRM1 (also called XPO1). The recruitment of GAPDH, a candidate cargo protein, into sEVs is regulated by the RanGTP-CRM1axis in a nuclear export signal (NES)-dependent manner. Perturbation of NCT through overexpression or depletion of nuclear transport components affected the recruitment of Ran, CRM1 and GAPDH into sEVs. Our studies, thus, suggest a link between NCT, particularly the Ran-CRM1 axis, and recruitment of NES-containing cargoes into the sEVs. Collectively, these findings implicate RanGTPase as a link between NCT and sEV mediated intercellular communication.

Alzheimer's disease (AD) is the most common cause of dementia with limited treatment options affecting millions of people and the prevalence increasing with the aging population. The current knowledge on the role of the hypoxia/hypoxia-inducible factor (HIF) in the AD pathology is restricted and controversial. We hypothesized based on benefits of the genetic long-term inactivation of HIF prolyl 4-hydroxylase-2 (HIF-P4H-2) on metabolism, vasculature and inflammatory response that prolonged moderate activation of the hypoxia response could hinder AD pathology. We used an aging model to study potential spontaneous accumulation of amyloid-β (Aβ) in HIF-P4H-2-deficient mice and a transgenic APP/PSEN1 mouse model subjected to prolonged sustained environmental hypoxia (15% O2 for 6 weeks) at two different time points of the disease; at age of 4 and 10 months. In both settings, activation of the hypoxia response reduced brain protein aggregate levels and this associated with higher vascularity. In the senescent HIF-P4H-2-deficient mice metabolic reprogramming also contributed to less protein aggregates while in APP/PSEN1 mice lesser Aβ associated additionally with hypoxia-mediated favorable responses to neuroinflammation and amyloid precursor protein processing. In conclusion, continuous, non-full-scale activation of the HIF pathway appears to mediate protection against neurodegeneration via several mechanisms and should be studied as a treatment option for AD.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron (MN) disease characterized by protein misfolding and aggregation leading to cellular degeneration. So far neither biomarker, nor effective treatment has been found. ATP signaling and P2X4 receptors (P2X4) are upregulated in various neurodegenerative diseases. Here we show that several ALS-related misfolded proteins including mutants of SOD1 or TDP-43 lead to a significant increase in surface P2X4 receptor density and function in vitro. In addition, we demonstrate in the spinal the cord of SOD1-G93A (SOD1) mice that misfolded SOD1-G93A proteins directly interact with endocytic adaptor protein-2 (AP2); thus, acting as negative competitors for the interaction between AP2 and P2X4, impairing constitutive P2X4 endocytosis. The higher P2X4 surface density was particularly observed in peripheral macrophages of SOD1 mice before the onset and during the progression of ALS symptoms positioning P2X4 as a potential early biomarker for ALS. P2X4 expression was also upregulated in spinal microglia of SOD1 mice during ALS and affect microglial inflammatory responses. Importantly, we report using double transgenic SOD1 mice expressing internalization-defective P2X4mCherryIN knock-in gene or invalidated for the P2X4 gene that P2X4 is instrumental for motor symptoms, ALS progression and survival. This study highlights the role of P2X4 in the pathophysiology of ALS and thus its potential for the development of biomarkers and treatments. We also decipher the molecular mechanism by which misfolded proteins related to ALS impact P2X4 trafficking at early pathological stage in cells expressing-P2X4.

Most cells involved in atherosclerosis release extracellular vesicles (EVs), which can carry bioactive substances to downstream tissues via circulation. We hypothesized that EVs derived from atherosclerotic plaques could promote atherogenesis in remote locations, a mechanism that mimics the blood metastasis of cancer. Ldlr gene knockout (Ldlr KO) rats were fed on a high cholesterol diet and underwent partial carotid ligation to induce local atherosclerosis. EVs were separated from carotid artery tissues and downstream blood of carotid ligation by centrifugation. MiRNA sequencing and qPCR were then performed to detect miRNA differences in EVs from rats with and without induced carotid atherosclerosis. Biochemical analyses demonstrated that EVs derived from atherosclerosis could increase the expression of ICAM-1, VCAM-1, and E-selectin in endothelial cells in vitro. EVs derived from atherosclerosis contained a higher level of miR-23a-3p than those derived from controls. MiR-23a-3p could promote endothelial inflammation by targeting Dusp5 and maintaining ERK1/2 phosphorylation in vitro. Inhibiting EV release could attenuate atherogenesis and reduce macrophage infiltration in vivo. Intravenously administrating atherosclerotic plaque-derived EVs could induce intimal inflammation, arterial wall thickening and lumen narrowing in the carotids of Ldlr KO rats, while simultaneous injection of miR-23a-3p antagomir could reverse this reaction. The results suggested that EVs may transfer atherosclerosis to remote locations by carrying proinflammatory factors, particularly miR-23a-3p.

Tight control of gene regulation in dendritic cells (DCs) is important to mount pathogen specific immune responses. Apart from transcription factor binding, dynamic regulation of enhancer activity through global transcriptional repressors like Nuclear Receptor Co-repressor 1 (NCoR1) plays a major role in fine-tuning of DC responses. However, how NCoR1 regulates enhancer activity and gene expression in individual or multiple Toll-like receptor (TLR) activation in DCs is largely unknown. In this study, we did a comprehensive epigenomic analysis of murine conventional type-I DCs (cDC1) across different TLR ligation conditions. We profiled gene expression changes along with H3K27ac active enhancers and NCoR1 binding in the TLR9, TLR3 and combined TLR9 + TLR3 activated cDC1. We observed spatio-temporal activity of TLR9 and TLR3 specific enhancers regulating signal specific target genes. Interestingly, we found that NCoR1 differentially controls the TLR9 and TLR3-specific responses. NCoR1 depletion specifically enhanced TLR9 responses as evident from increased enhancer activity as well as TLR9-specific gene expression, whereas TLR3-mediated antiviral response genes were negatively regulated. We validated that NCoR1 KD cDC1 showed significantly decreased TLR3 specific antiviral responses through decreased IRF3 activation. In addition, decreased IRF3 binding was observed at selected ISGs leading to their decreased expression upon NCoR1 depletion. Consequently, the NCoR1 depleted cDC1 showed reduced Sendai Virus (SeV) clearance and cytotoxic potential of CD8+ T cells upon TLR3 activation. NCoR1 directly controls the majority of these TLR specific enhancer activity and the gene expression. Overall, for the first time, we revealed NCoR1 mediates transcriptional control towards TLR9 as compared to TLR3 in cDC1.

The citrate carrier (CIC) is an integral protein of the inner mitochondrial membrane which catalyzes the efflux of mitochondrial citrate (or other tricarboxylates) in exchange with a cytosolic anion represented by a tricarboxylate or a dicarboxylate or phosphoenolpyruvate. In this way, the CIC provides the cytosol with citrate which is involved in many metabolic reactions. Several studies have been carried out over the years on the structure, function and regulation of this metabolite carrier protein both in mammals and in many other organisms. A lot of data on the characteristics of this protein have therefore accumulated over time thereby leading to a complex framework of metabolic and physiological implications connected to the CIC function. In this review, we critically analyze these data starting from the multiple roles played by the mitochondrial CIC in many cellular processes and then examining the regulation of its activity in different nutritional and hormonal states. Finally, the metabolic significance of the citrate flux, mediated by the CIC, across distinct subcellular compartments is also discussed.

Cardiac fibroblast (CF) population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for CF biology and interventional strategies to influence injury response and aging-associated remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within CF population has not been addressed. An inducible reporter mouse detects c-Kit promoter activation with Enhanced Green Fluorescent Protein (EGFP) expression in cardiac cells. Coincidence of EGFP and c-Kit with the DDR2 fibroblast marker was confirmed using flow cytometry and immunohistochemistry. Subsequently, CFs expressing DDR2 with or without c-Kit was isolated and characterized. A subset of DDR2+ CFs also express c-Kit with coincidence in ~ 8% of total cardiac interstitial cells (CICs). Aging is associated with decreased number of c-Kit expressing DDR2+ CFs, whereas pathological injury induces c-Kit and DDR2 as well as the frequency of coincident expression in CICs. scRNA-Seq profiling reveals the transcriptome of c-Kit expressing CFs as cells with transitional phenotype. Cultured cardiac DDR2+ fibroblasts that are c-Kit+ exhibit morphological and functional characteristics consistent with youthful phenotypes compared to c-Kit- cells. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration, including phospho-ERK and pro-caspase 3. The phenotype of c-kit+ on DDR2+ CFs correlates with multiple characteristics of 'youthful' cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2+ CF population and provides a fundamental basis for future studies to influence myocardial biology, response to pathological injury and physiological aging.

Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15-30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry.

Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.

The epithelial-to-mesenchymal transition (EMT) is a reversible process that may interact with tumour immunity through multiple approaches. There is increasing evidence demonstrating the interconnections among EMT-related processes, the tumour microenvironment, and immune activity, as well as its potential influence on the immunotherapy response. Long non-coding RNAs (lncRNAs) are emerging as critical modulators of gene expression. They play fundamental roles in tumour immunity and act as promising biomarkers of immunotherapy response. However, the potential roles of lncRNA in the crosstalk of EMT and tumour immunity are still unclear in sarcoma. We obtained multi-omics profiling of 1440 pan-sarcoma patients from 19 datasets. Through an unsupervised consensus clustering approach, we categorised EMT molecular subtypes. We subsequently identified 26 EMT molecular subtype and tumour immune-related lncRNAs (EILncRNA) across pan-sarcoma types and developed an EILncRNA signature-based weighted scoring model (EILncSig). The EILncSig exhibited favourable performance in predicting the prognosis of sarcoma, and a high-EILncSig was associated with exclusive tumour microenvironment (TME) characteristics with desert-like infiltration of immune cells. Multiple altered pathways, somatically-mutated genes and recurrent CNV regions associated with EILncSig were identified. Notably, the EILncSig was associated with the efficacy of immune checkpoint inhibition (ICI) therapy. Using a computational drug-genomic approach, we identified compounds, such as Irinotecan that may have the potential to convert the EILncSig phenotype. By integrative analysis on multi-omics profiling, our findings provide a comprehensive resource for understanding the functional role of lncRNA-mediated immune regulation in sarcomas, which may advance the understanding of tumour immune response and the development of lncRNA-based immunotherapeutic strategies for sarcoma.

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

Microtubule dynamics ensure multiple cellular events during oocyte meiosis, which is critical for the fertilization and early embryo development. KIF15 (also termed Hklp2) is a member of kinesin-12 family motor proteins, which participates in Eg5-related bipolar spindle formation in mitosis. In present study, we explored the roles of KIF15 in mouse oocyte meiosis. KIF15 expressed during oocyte maturation and localized with microtubules. Depletion or inhibition of KIF15 disturbed meiotic cell cycle progression, and the oocytes which extruded the first polar body showed a high aneuploidy rate. Further analysis showed that disruption of KIF15 did not affect spindle morphology but resulted in chromosome misalignment. This might be due to the reduced stability of the K-fibers, which further induced the loss of kinetochore-microtubule attachment and activated spindle assembly checkpoint, showing with the failed release of Bub3 and BubR1. Based on mass spectroscopy analysis and coimmunoprecipitation data we showed that KIF15 was responsible for recruiting HDAC6, NAT10 and SIRT2 to maintain the acetylated tubulin level, which further affected tubulin acetylation for microtubule stability. Taken together, these results suggested that KIF15 was essential for the microtubule acetylation and cell cycle control during mouse oocyte meiosis.

The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

Tumor cells exhibit altered cholesterol content. However, cholesterol structural subcellular distribution and implication in cancer cell invasion are poorly understood mainly due to difficulties to investigate cholesterol both quantitatively and qualitatively and to compare isogenic cell models. Here, using the MCF10A cell line series (non-tumorigenic MCF10A, pre-malignant MCF10AT and malignant MCF10CAIa cells) as a model of breast cancer progression and the highly invasive MDA-MB-231 cell line which exhibits the common TP53 mutation, we investigated if cholesterol contributes to cancer cell invasion, whether the effects are specific to cancer cells and the underlying mechanism. We found that partial membrane cholesterol depletion specifically and reversibly decreased invasion of the malignant cell lines. Those cells exhibited dorsal surface cholesterol-enriched submicrometric domains and narrow ER-plasma membrane and ER-intracellular organelles contact sites. Dorsal cholesterol-enriched domains can be endocytosed and reach the cell ventral face where they were involved in invadopodia formation and extracellular matrix degradation. In contrast, non-malignant cells showed low cell invasion, low surface cholesterol exposure and cholesterol-dependent focal adhesions. The differential cholesterol distribution and role in breast cancer cell invasion provide new clues for the understanding of the molecular events underlying cellular mechanisms in breast cancer.

Magnesium (Mg2+) is the most prevalent divalent intracellular cation. As co-factor in many enzymatic reactions, Mg2+ is essential for protein synthesis, energy production, and DNA stability. Disturbances in intracellular Mg2+ concentrations, therefore, unequivocally result in delayed cell growth and metabolic defects. To maintain physiological Mg2+ levels, all organisms rely on balanced Mg2+ influx and efflux via Mg2+ channels and transporters. This review compares the structure and the function of prokaryotic Mg2+ transporters and their eukaryotic counterparts. In prokaryotes, cellular Mg2+ homeostasis is orchestrated via the CorA, MgtA/B, MgtE, and CorB/C Mg2+ transporters. For CorA, MgtE, and CorB/C, the motifs that form the selectivity pore are conserved during evolution. These findings suggest that CNNM proteins, the vertebrate orthologues of CorB/C, also have Mg2+ transport capacity. Whereas CorA and CorB/C proteins share the gross quaternary structure and functional properties with their respective orthologues, the MgtE channel only shares the selectivity pore with SLC41 Na+/Mg2+ transporters. In eukaryotes, TRPM6 and TRPM7 Mg2+ channels provide an additional Mg2+ transport mechanism, consisting of a fusion of channel with a kinase. The unique features these TRP channels allow the integration of hormonal, cellular, and transcriptional regulatory pathways that determine their Mg2+ transport capacity. Our review demonstrates that understanding the structure and function of prokaryotic magnesiotropic proteins aids in our basic understanding of Mg2+ transport.

Cancer immunotherapy is a rapidly developing and effective method for the treatment of a variety of malignancies in recent years. As a significant immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) play the most significant role in cancer immune escape and cancer immunotherapy. Though PD-L1 have become an important target for drug development and there have been various approved drugs and clinic trials targeting it, and various clinical response rate and adverse reactions prevent many patients from benefiting from it. In recent years, combination trials have become the main direction of PD-1/PD-L1 antibodies development. Here, we summarized PD-L1 biofunctions and key roles in various cancers along with the development of PD-L1 inhibitors. The regulators that are involved in controlling PD-L1 expression including post-translational modification, mRNA level regulation as well as degradation and exosome secretory pathway of PD-L1 were focused. This systematic summary may provide comprehensive understanding of different regulations on PD-L1 as well as a broad prospect for the search of the important regulator of PD-L1. The regulatory factors of PD-L1 can be potential targets for immunotherapy and increase strategies of immunotherapy in combination.

The blood-brain barrier (BBB) provides essential neuroprotection from environmental toxins and xenobiotics, through high expression of drug efflux transporters in endothelial cells of the cerebral capillaries. However, xenobiotic exposure, stress, and inflammatory stimuli have the potential to disrupt BBB permeability in fetal and post-natal life. Understanding the role and ability of the BBB in protecting the developing brain, particularly with respect to drug/toxin transport, is key to promoting long-term brain health. Drug transporters, particularly P-gp and BCRP are expressed in early gestation at the developing BBB and have a crucial role in developmental homeostasis and fetal brain protection. We have highlighted several factors that modulate drug transporters at the developing BBB, including synthetic glucocorticoid (sGC), cytokines, maternal infection, and growth factors. Some factors have the potential to increase expression and function of drug transporters and increase brain protection (e.g., sGC, transforming growth factor [TGF]-β). However, others inhibit drug transporters expression and function at the BBB, increasing brain exposure to xenobiotics (e.g., tumor necrosis factor [TNF], interleukin [IL]-6), negatively impacting brain development. This has implications for pregnant women and neonates, who represent a vulnerable population and may be exposed to drugs and environmental toxins, many of which are P-gp and BCRP substrates. Thus, alterations in regulated transport across the developing BBB may induce long-term changes in brain health and compromise pregnancy outcome. Furthermore, a large portion of neonatal adverse drug reactions are attributed to agents that target or access the nervous system, such as stimulants (e.g., caffeine), anesthetics (e.g., midazolam), analgesics (e.g., morphine) and antiretrovirals (e.g., Zidovudine); thus, understanding brain protection is key for the development of strategies to protect the fetal and neonatal brain.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex and debilitating disease with a substantial social and economic impact on individuals and their community. Despite its importance and deteriorating impact, progresses in diagnosis and treatment of ME/CFS is limited. This is due to the unclear pathophysiology of the disease and consequently lack of prognostic biomarkers. To investigate pathophysiology of ME/CFS, several potential pathologic hallmarks have been investigated; however, these studies have failed to report a consistent result. These failures in introducing the underlying reason for ME/CFS have stimulated considering other possible contributing mechanisms such as tryptophan (TRP) metabolism and in particular kynurenine pathway (KP). KP plays a central role in cellular energy production through the production of nicotinamide adenine dinucleotide (NADH). In addition, this pathway has been shown to mediate immune response and neuroinflammation through its metabolites. This review, we will discuss the pathology and management of ME/CFS and provide evidence pertaining KP abnormalities and symptoms that are classic characteristics of ME/CFS. Targeting the KP regulation may provide innovative approaches to the management of ME/CFS.

N6-methyladenosine (m6A) regulates many aspects of RNA metabolism and is involved in learning and memory processes. Yet, the impact of a dysregulation of post-transcriptional m6A editing on synaptic impairments in neurodegenerative disorders remains unknown. Here we investigated the m6A methylation pattern in the hippocampus of Huntington's disease (HD) mice and the potential role of the m6A RNA modification in HD cognitive symptomatology. m6A modifications were evaluated in HD mice subjected to a hippocampal cognitive training task through m6A immunoprecipitation sequencing (MeRIP-seq) and the relative levels of m6A-modifying proteins (FTO and METTL14) by subcellular fractionation and Western blot analysis. Stereotaxic CA1 hippocampal delivery of AAV-shFTO was performed to investigate the effect of RNA m6A dysregulation in HD memory deficits. Our results reveal a m6A hypermethylation in relevant HD and synaptic related genes in the hippocampal transcriptome of Hdh+/Q111 mice. Conversely, m6A is aberrantly regulated in an experience-dependent manner in the HD hippocampus leading to demethylation of important components of synapse organization. Notably, the levels of RNA demethylase (FTO) and methyltransferase (METTL14) were modulated after training in the hippocampus of WT mice but not in Hdh+/Q111 mice. Finally, inhibition of FTO expression in the hippocampal CA1 region restored memory disturbances in symptomatic Hdh+/Q111 mice. Altogether, our results suggest that a differential RNA methylation landscape contributes to HD cognitive symptoms and uncover a role of m6A as a novel hallmark of HD.

Comment in Cell Mol Life Sci. 2022 Jul 27;79(8):451.

The increasing antibiotic resistance among uropathogenic bacteria warrants alternative therapeutic strategies. We demonstrate the potential of the synthetic peptide CD4-PP, designed by dimerization and backbone cyclization of the shortest antimicrobial region of human cathelicidin, LL-37. CD4-PP is active against clinical and type strains of common uropathogens Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa at concentrations substantially below cellular cytotoxic levels and induced membrane deformation and leakage in E. coli and P. aeruginosa. Furthermore, CD4-PP treatment prevented the formation of new biofilm and dissolved mature biofilm created by E. coli and P. aeruginosa and targeted curli amyloid in E. coli biofilms. In addition, CD4-PP also induced production of LL-37 by uroepithelial cells and increased the expression of tight junction proteins claudin-14 and occludin. During uroepithelial cell infection, CD4-PP significantly reduced uropathogen survival when treatment was given at the start of infection. Low micromolar of CD4-PP treatment initiated after 2 h was successful with all tested species, except P. aeruginosa where CD4-PP was unable to reduce survival, which could be attributed by early biofilm formation. Finally, we demonstrated that urinary catheter pieces coated with saline fluid supplemented with CD4-PP reduced the attachment of E. coli, giving it a potential clinical application.

Helicobacter pylori-mediated gastric carcinogenesis involves upregulation of the E3 ubiquitin ligase Siah2 and its phosphorylation-mediated stabilization. This study elucidates a novel mechanism of oxidative stress regulation by phosphorylated Siah2 in H. pylori-infected gastric epithelial cancer cells (GECs). We identify that H. pylori-mediated Siah2 phosphorylation at the 6th serine residue (P-S6-Siah2) enhances proteasomal degradation of the 78-kDa glucose-regulated protein (GRP78) possessing antioxidant functions. S6 phosphorylation stabilizes Siah2 and P-S6-Siah2 potentiates H. pylori-mediated reactive oxygen species (ROS) generation. However, infected S6A phospho-null Siah2-expressing cells have decreased cellular GRP78 level as surprisingly these cells release GRP78 to a higher extent and accumulate significantly higher ROS than the wild type (WT) Siah2 construct-expressing cells. Ectopic expression of GRP78 prevents the loss of mitochondrial membrane potential and cellular ROS accumulation caused by H. pylori. H. pylori-induced mitochondrial damage and mitochondrial membrane potential loss are potentiated in Siah2-overexpressing cells but these effects are further enhanced in S6A-expressing cells. This study also confirms that while phosphorylation-mediated Siah2 stabilization optimally upregulates aggresome accumulation, it suppresses autophagosome formation, thus decreasing the dependency on the latter mechanism in regulating cellular protein abundance. Disruption of the phospho-Siah2-mediated aggresome formation impairs proliferation of infected GECs. Thus, Siah2 phosphorylation has diagnostic and therapeutic significance in H. pylori-mediated gastric cancer (GC).

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration.

There is a growing need to uncover biomarkers of ionizing radiation exposure that leads to a better understanding of how exposures take place, including dose type, rate, and time since exposure. As one of the first organs to be exposed to external sources of ionizing radiation, skin is uniquely positioned in terms of model systems for radiation exposure study. The simultaneous evolution of both MS-based -omics studies, as well as in vitro 3D skin models, has created the ability to develop a far more holistic understanding of how ionizing radiation affects the many interconnected biomolecular processes that occur in human skin. However, there are a limited number of studies describing the biomolecular consequences of low-dose ionizing radiation to the skin. This review will seek to explore the current state-of-the-art technology in terms of in vitro 3D skin models, as well as track the trajectory of MS-based -omics techniques and their application to ionizing radiation research, specifically, the search for biomarkers within the low-dose range.

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1.