Ketogenic diet changes microglial morphology and the hippocampal lipidomic profile differently in stress susceptible versus resistant male mice upon repeated social defeat.

Psychological stress confers an increased risk for several diseases including psychiatric conditions. The susceptibility to psychological stress is modulated by various factors, many of them being modifiable lifestyle choices. The ketogenic diet (KD) has emerged as a dietary regime that offers positive outcomes on mood and health status. Psychological stress and elevated inflammation are common features of neuropsychiatric disorders such as certain types of major depressive disorder. KD has been attributed anti-inflammatory properties that could underlie its beneficial consequences on the brain and behavior. Microglia are the main drivers of inflammation in the central nervous system. They are known to respond to both dietary changes and psychological stress, notably by modifying their production of cytokines and relationships among the brain parenchyma. To assess the interactions between KD and the stress response, including effects on microglia, we examined adult male mice on control diet (CD) versus KD that underwent 10 days of repeated social defeat (RSD) or remained non-stressed (controls; CTRLs). Through a social interaction test, stressed mice were classified as susceptible (SUS) or resistant (RES) to RSD. The mouse population fed a KD tended to have a higher proportion of individuals classified as RES following RSD. Microglial morphology and ultrastructure were then analyzed in the ventral hippocampus CA1, a brain region known to present structural alterations as a response to psychological stress. Distinct changes in microglial soma and arborization linked to the KD, SUS and RES phenotypes were revealed. Ultrastructural analysis by electron microscopy showed a clear reduction of cellular stress markers in microglia from KD fed animals. Furthermore, ultrastructural analysis showed that microglial contacts with synaptic elements were reduced in the SUS compared to the RES and CTRL groups. Hippocampal lipidomic analyses lastly identified a distinct lipid profile in SUS animals compared to CTRLs. These key differences, combined with the distinct microglial responses to diet and stress, indicate that unique metabolic changes may underlie the stress susceptibility phenotypes. Altogether, our results reveal novel mechanisms by which a KD might improve the resistance to psychological stress.

Ketogenic diet alters microglial morphology and changes the hippocampal lipidomic profile distinctively in stress susceptible versus resistant male mice upon repeated social defeat.

Psychological stress confers an increased risk for several diseases including psychiatric conditions. The susceptibility to psychological stress is modulated by various factors, many of them being modifiable lifestyle choices. The ketogenic diet (KD) has emerged as a dietary regime that offers positive outcomes on mood and health status. Psychological stress and elevated inflammation are common features of neuropsychiatric disorders such as certain types of major depressive disorder. KD has been attributed anti-inflammatory properties that could underlie its beneficial consequences on the brain and behavior. Microglia are the main drivers of inflammation in the central nervous system. They are known to respond to both dietary changes and psychological stress, notably by modifying their production of cytokines and relationships among the brain parenchyma. To assess the interactions between KD and the stress response, including effects on microglia, we examined adult male mice on control diet (CD) versus KD that underwent 10 days of repeated social defeat (RSD) or remained non-stressed (controls; CTRLs). Through a social interaction test, stressed mice were classified as susceptible (SUS) or resistant (RES) to RSD. The mouse population fed a KD tended to have a higher proportion of individuals classified as RES following RSD. Microglial morphology and ultrastructure were then analyzed in the ventral hippocampus CA1, a brain region known to present structural alterations as a response to psychological stress. Distinct changes in microglial soma and arborization linked to the KD, SUS and RES phenotypes were revealed. Ultrastructural analysis by electron microscopy showed a clear reduction of cellular stress markers in microglia from KD fed animals. Furthermore, ultrastructural analysis showed that microglial contacts with synaptic elements were reduced in the SUS compared to the RES and CTRL groups. Hippocampal lipidomic analyses lastly identified a distinct lipid profile in SUS animals compared to CTRLs. These key differences, combined with the distinct microglial responses to diet and stress, indicate that unique metabolic changes may underlie the stress susceptibility phenotypes. Altogether, our results reveal novel mechanisms by which a KD might improve the resistance to psychological stress.

The structure and function of YTHDF epitranscriptomic m6A readers.

Specific RNA sequences modified by a methylated adenosine, N6-methyladenosine (m6A), contribute to the post-transcriptional regulation of gene expression. The quantity of m6A in RNA is orchestrated by enzymes that write and erase it, while its effects are mediated by proteins that bind to read this modification. Dysfunction of this post-transcriptional regulatory process has been linked to human disease. Although the initial focus has been on pharmacological targeting of the writer and eraser enzymes, interest in the reader proteins has been challenged by a lack of clear understanding of their functional roles and molecular mechanisms of action. Readers of m6A-modified RNA (m6A-RNA) - the YTH (YT521-B homology) domain-containing protein family paralogs 1-3 (YTHDF1-3, referred to here as DF1-DF3) - are emerging as therapeutic targets as their links to pathological processes such as cancer and inflammation and their roles in regulating m6A-RNA fate become clear. We provide an updated understanding of the modes of action of DF1-DF3 and review their structures to unlock insights into drug design approaches for DF paralog-selective inhibition.

Minocycline treatment improves cognitive and functional plasticity in a preclinical mouse model of major depressive disorder.

Major depressive disorder (MDD) is a chronic, recurring, and potentially life-threatening illness, which affects over 300 million people worldwide. MDD affects not only the emotional and social domains but also cognition. However, the currently available treatments targeting cognitive deficits in MDD are limited. Minocycline, an antibiotic with anti-inflammatory properties recently identified as a potential antidepressant, has been shown to attenuate learning and memory deficits in animal models of cognitive impairment. Here, we explored whether minocycline recovers the deficits in cognition in a mouse model of depression. C57BL6/J adult male mice were exposed to two weeks of chronic unpredictable mild stress to induce a depressive-like phenotype. Immediately afterward, mice received either vehicle or minocycline for three weeks in standard housing conditions. We measured anhedonia as a depressive-like response, and place learning to assess cognitive abilities. We also recorded long-term potentiation (LTP) as an index of hippocampal functional plasticity and ran immunohistochemical assays to assess microglial proportion and morphology. After one week of treatment, cognitive performance in the place learning test was significantly improved by minocycline, as treated mice displayed a higher number of correct responses when learning novel spatial configurations. Accordingly, minocycline-treated mice displayed higher LTP compared to controls. However, after three weeks of treatment, no difference between treated and control animals was found for behavior, neural plasticity, and microglial properties, suggesting that minocycline has a fast but short effect on cognition, without lasting effects on microglia. These findings together support the usefulness of minocycline as a potential treatment for cognitive impairment associated with MDD.

Integrative Organelle-Based Functional Proteomics: In Silico Prediction of Impaired Functional Annotations in SACS KO Cell Model.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS.

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease.

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5-/- mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.

Unveiling the Function of the Mitochondrial Filament-Forming Protein LACTB in Lipid Metabolism and Cancer.

LACTB is a relatively unknown mitochondrial protein structurally related to the bacterial penicillin-binding and beta-lactamase superfamily of serine proteases. LACTB has recently gained an increased interest due to its potential role in lipid metabolism and tumorigenesis. To date, around ninety studies pertaining to LACTB have been published, but the exact biochemical and cell biological function of LACTB still remain elusive. In this review, we summarise the current knowledge about LACTB with particular attention to the implications of the recently published study on the cryo-electron microscopy structure of the filamentous form of LACTB. From this and other studies, several specific properties of LACTB emerge, suggesting that the protein has distinct functions in different physiological settings. Resolving these issues by further research may ultimately lead to a unified model of LACTB's function in cell and organismal physiology. LACTB is the only member of its protein family in higher animals and LACTB may, therefore, be of particular interest for future drug targeting initiatives.

Isotope-labeled amyloid-ß does not transmit to the brain in a prion-like manner after peripheral administration.

Findings of early cerebral amyloid-ß deposition in mice after peripheral injection of amyloid-ß-containing brain extracts, and in humans following cadaveric human growth hormone treatment raised concerns that amyloid-ß aggregates and possibly Alzheimer's disease may be transmissible between individuals. Yet, proof that Aß actually reaches the brain from the peripheral injection site is lacking. Here, we use a proteomic approach combining stable isotope labeling of mammals and targeted mass spectrometry. Specifically, we generate 13 C-isotope-labeled brain extracts from mice expressing human amyloid-ß and track 13 C-lysine-labeled amyloid-ß after intraperitoneal administration into young amyloid precursor protein-transgenic mice. We detect injected amyloid-ß in the liver and lymphoid tissues for up to 100 days. In contrast, injected 13 C-lysine-labeled amyloid-ß is not detectable in the brain whereas the mice incorporate 13 C-lysine from the donor brain extracts into endogenous amyloid-ß. Using a highly sensitive and specific proteomic approach, we demonstrate that amyloid-ß does not reach the brain from the periphery. Our study argues against potential transmissibility of Alzheimer's disease while opening new avenues to uncover mechanisms of pathophysiological protein deposition.

Epitranscriptomics of Ischemic Heart Disease-The IHD-EPITRAN Study Design and Objectives.

Epitranscriptomic modifications in RNA can dramatically alter the way our genetic code is deciphered. Cells utilize these modifications not only to maintain physiological processes, but also to respond to extracellular cues and various stressors. Most often, adenosine residues in RNA are targeted, and result in modifications including methylation and deamination. Such modified residues as N-6-methyl-adenosine (m6A) and inosine, respectively, have been associated with cardiovascular diseases, and contribute to disease pathologies. The Ischemic Heart Disease Epitranscriptomics and Biomarkers (IHD-EPITRAN) study aims to provide a more comprehensive understanding to their nature and role in cardiovascular pathology. The study hypothesis is that pathological features of IHD are mirrored in the blood epitranscriptome. The IHD-EPITRAN study focuses on m6A and A-to-I modifications of RNA. Patients are recruited from four cohorts: (I) patients with IHD and myocardial infarction undergoing urgent revascularization; (II) patients with stable IHD undergoing coronary artery bypass grafting; (III) controls without coronary obstructions undergoing valve replacement due to aortic stenosis and (IV) controls with healthy coronaries verified by computed tomography. The abundance and distribution of m6A and A-to-I modifications in blood RNA are charted by quantitative and qualitative methods. Selected other modified nucleosides as well as IHD candidate protein and metabolic biomarkers are measured for reference. The results of the IHD-EPITRAN study can be expected to enable identification of epitranscriptomic IHD biomarker candidates and potential drug targets.

Proteomic Profiling of Leukocytes Reveals Dysregulation of Adhesion and Integrin Proteins in Chronic Kidney Disease-Related Atherosclerosis.

A progressive loss of functional nephrons defines chronic kidney disease (CKD). Complications related to cardiovascular disease (CVD) are the principal causes of mortality in CKD; however, the acceleration of CVD in CKD remains unresolved. Our study used a complementary proteomic approach to assess mild and advanced CKD patients with different atherosclerosis stages and two groups of patients with different classical CVD progression but without renal dysfunction. We utilized a label-free approach based on LC-MS/MS and functional bioinformatic analyses to profile CKD and CVD leukocyte proteins. We revealed dysregulation of proteins involved in different phases of leukocytes' diapedesis process that is very pronounced in CKD's advanced stage. We also showed an upregulation of apoptosis-related proteins in CKD as compared to CVD. The differential abundance of selected proteins was validated by multiple reaction monitoring, ELISA, Western blotting, and at the mRNA level by ddPCR. An increased rate of apoptosis was then functionally confirmed on the cellular level. Hence, we suggest that the disturbances in leukocyte extravasation proteins may alter cell integrity and trigger cell death, as demonstrated by flow cytometry and microscopy analyses. Our proteomics data set has been deposited to the ProteomeXchange Consortium via the PRIDE repository with the data set identifier PXD018596.

PGC-1α Signaling Increases GABA(A) Receptor Subunit α2 Expression, GABAergic Neurotransmission and Anxiety-Like Behavior in Mice.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondria biogenesis and cell stress playing a role in metabolic and degenerative diseases. In the brain PGC-1α expression has been localized mainly to GABAergic interneurons but its overall role is not fully understood. We observed here that the protein levels of γ-aminobutyric acid (GABA) type A receptor-α2 subunit (GABARα2) were increased in hippocampus and brain cortex in transgenic (Tg) mice overexpressing PGC-1α in neurons. Along with this, GABARα2 expression was enhanced in the hippocampus of the PGC-1α Tg mice, as shown by quantitative PCR. Double immunostaining revealed that GABARα2 co-localized with the synaptic protein gephyrin in higher amounts in the striatum radiatum layer of the hippocampal CA1 region in the Tg compared with Wt mice. Electrophysiology revealed that the frequency of spontaneous and miniature inhibitory postsynaptic currents (mIPSCs) was increased in the CA1 region in the Tg mice, indicative of an augmented GABAergic transmission. Behavioral tests revealed an increase for anxiety-like behavior in the PGC-1α Tg mice compared with controls. To study whether drugs acting on PPARγ can affect GABARα2, we employed pioglitazone that elevated GABARα2 expression in primary cultured neurons. Similar results were obtained using the specific PPARγ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino) ethyl]-L-tyrosine hydrate (GW1929). These results demonstrate that PGC-1α regulates GABARα2 subunits and GABAergic neurotransmission in the hippocampus with behavioral consequences. This indicates further that drugs like pioglitazone, widely used in the treatment of type 2 diabetes, can influence GABARα2 expression via the PPARγ/PGC-1α system.

A muscle growth-promoting treatment based on the attenuation of activin/myostatin signalling results in long-term testicular abnormalities.

Activin/myostatin signalling acts to induce skeletal muscle atrophy in adult mammals by inhibiting protein synthesis as well as promoting protein and organelle turnover. Numerous strategies have been successfully developed to attenuate the signalling properties of these molecules, which result in augmenting muscle growth. However, these molecules, in particular activin, play major roles in tissue homeostasis in numerous organs of the mammalian body. We have recently shown that although the attenuation of activin/myostatin results in robust muscle growth, it also has a detrimental impact on the testis. Here, we aimed to discover the long-term consequences of a brief period of exposure to muscle growth-promoting molecules in the testis. We demonstrate that muscle hypertrophy promoted by a soluble activin type IIB ligand trap (sActRIIB) is a short-lived phenomenon. In stark contrast, short-term treatment with sActRIIB results in immediate impact on the testis, which persists after the sessions of the intervention. Gene array analysis identified an expansion in aberrant gene expression over time in the testis, initiated by a brief exposure to muscle growth-promoting molecules. The impact on the testis results in decreased organ size as well as quantitative and qualitative impact on sperm. Finally, we have used a drug-repurposing strategy to exploit the gene expression data to identify a compound - N6-methyladenosine - that may protect the testis from the impact of the muscle growth-promoting regime. This work indicates the potential long-term harmful effects of strategies aimed at promoting muscle growth by attenuating activin/myostatin signalling. Furthermore, we have identified a molecule that could, in the future, be used to overcome the detrimental impact of sActRIIB treatment on the testis.

Inhibition of Activin/Myostatin signalling induces skeletal muscle hypertrophy but impairs mouse testicular development.

Numerous approaches are being developed to promote post-natal muscle growth based on attenuating Myostatin/Activin signalling for clinical uses such as the treatment neuromuscular diseases, cancer cachexia and sarcopenia. However there have been concerns about the effects of inhibiting Activin on tissues other than skeletal muscle. We intraperitoneally injected mice with the Activin ligand trap, sActRIIB, in young, adult and a progeric mouse model. Treatment at any stage in the life of the mouse rapidly increased muscle mass. However at all stages of life the treatment decreased the weights of the testis. Not only were the testis smaller, but they contained fewer sperm compared to untreated mice. We found that the hypertrophic muscle phenotype was lost after the cessation of sActRIIB treatment but abnormal testis phenotype persisted. In summary, attenuation of Myostatin/Activin signalling inhibited testis development. Future use of molecules based on a similar mode of action to promote muscle growth should be carefully profiled for adverse side-effects on the testis. However the effectiveness of sActRIIB as a modulator of Activin function provides a possible therapeutic strategy to alleviate testicular seminoma development.

Epicardial transplantation of atrial appendage micrograft patch salvages myocardium after infarction.

BACKGROUND: Ischemic heart disease remains the leading cause of mortality and morbidity worldwide despite improved possibilities in medical care. Alongside interventional therapies, such as coronary artery bypass grafting, adjuvant tissue-engineered and cell-based treatments can provide regenerative improvement. Unfortunately, most of these advanced approaches require multiple lengthy and costly preparation stages without delivering significant clinical benefits. METHODS: We evaluated the effect of epicardially delivered minute pieces of atrial appendage tissue material, defined as atrial appendage micrografts (AAMs), in a mouse myocardial infarction model. An extracellular matrix patch was used to cover and fix the AAMs onto the surface of the infarcted heart. RESULTS: The matrix-covered AAMs salvaged the heart from the infarction-induced loss of functional myocardium and attenuated scarring. Site-selective proteomics of injured ischemic and uninjured distal myocardium from AAMs-treated and -untreated tissue sections revealed increased expression of several cardiac regeneration-associated proteins (i.e., periostin, transglutaminases, and glutathione peroxidases) and activation of pathways responsible for angiogenesis and cardiogenesis in relation to AAMs therapy. CONCLUSIONS: Epicardial delivery of AAMs encased in an extracellular matrix patch scaffold salvages functional cardiac tissue from ischemic injury and restricts fibrosis after myocardial infarction. Our results support the use of AAMs as tissue-based therapy adjuvants for salvaging the ischemic myocardium.

Proteomic and functional analyses in disease models reveal CLN5 protein involvement in mitochondrial dysfunction.

CLN5 disease is a rare form of late-infantile neuronal ceroid lipofuscinosis (NCL) caused by mutations in the CLN5 gene that encodes a protein whose primary function and physiological roles remains unresolved. Emerging lines of evidence point to mitochondrial dysfunction in the onset and progression of several forms of NCL, offering new insights into putative biomarkers and shared biological processes. In this work, we employed cellular and murine models of the disease, in an effort to clarify disease pathways associated with CLN5 depletion. A mitochondria-focused quantitative proteomics approach followed by functional validations using cell biology and immunofluorescence assays revealed an impairment of mitochondrial functions in different CLN5 KO cell models and in Cln5 - /- cerebral cortex, which well correlated with disease progression. A visible impairment of autophagy machinery coupled with alterations of key parameters of mitophagy activation process functionally linked CLN5 protein to the process of neuronal injury. The functional link between impaired cellular respiration and activation of mitophagy pathways in the human CLN5 disease condition was corroborated by translating organelle-specific proteome findings to CLN5 patients' fibroblasts. Our study highlights the involvement of CLN5 in activation of mitophagy and mitochondrial homeostasis offering new insights into alternative strategies towards the CLN5 disease treatment.

Dynamic Interaction of USP14 with the Chaperone HSC70 Mediates Crosstalk between the Proteasome, ER Signaling, and Autophagy.

USP14 is a deubiquitinating enzyme associated with the proteasome important for protein degradation. Here we show that upon proteasome inhibition or expression of the mutant W58A-USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70, in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, and to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whereas inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or use of the mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons, and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways.

Electrophysiological Profile Remodeling via Selective Suppression of Voltage-Gated Currents by CLN1/PPT1 Overexpression in Human Neuronal-Like Cells.

CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.

Reactive oxygen species-regulating proteins peroxiredoxin 2 and thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase are differentially abundant in induced sputum from smokers with lung cancer or asbestos exposure.

Lung cancer is a deadly disease, typically caused by known risk factors, such as tobacco smoke and asbestos exposure. By triggering cellular oxidative stress and altering the antioxidant pathways eliminating reactive oxygen species (ROS), tobacco smoke and asbestos predispose to cancer. Despite easily recognizable high-risk individuals, lung cancer screening and its early detection are hampered by poor diagnostic tools including the absence of proper biomarkers. This study aimed to recognize potential lung cancer biomarkers using induced sputum noninvasively collected from the lungs of individuals in risk of contracting lung cancer. Study groups composed of current and former smokers, who either were significantly asbestos exposed, had lung cancer, or were unexposed and asymptomatic. Screening of potential biomarkers was performed with 52, and five differentially abundant proteins, peroxiredoxin 2 (PRDX2), thioredoxin (TXN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), extracellular matrix protein 1 (ECM1), and protein S100 A8 (S100A8), were chosen to undergo validation, for their previously known connection with oxidative stress or cancer. Results from the validation in 123 sputa showed that PRDX2, TXN, and GAPDH were differentially abundant in sputa from individuals with lung cancer. TXN had a negative correlation with asbestos exposure, yet a positive correlation with smoking and lung cancer. Thus, tobacco smoking, asbestos exposure, and lung carcinogenesis may disturb the cellular redox state in different ways. A strong correlation was found among PRDX2, TXN, GAPDH, and S100A8, suggesting that these proteins may present a diagnostic biomarker panel to aid recognizing individuals at high risk of contracting lung cancer.

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.

Simple 3D culture of dissociated kidney mesenchyme mimics nephron progenitor niche and facilitates nephrogenesis Wnt-independently.

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3ß inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and induced to differentiate in a simple culture system. They also imply that GSK3ß/WNT-independent pathways contribute to the maintenance and induction of mouse KM. The robust and easy 3D culture enables further characterization of NPs, and may facilitate disease modeling when applied to human cells.