Proteomic profiling of human plasma extracellular vesicles identifies PF4 and C1R as novel biomarker in sarcopenia.

BACKGROUND: Sarcopenia, the gradual and generalized loss of muscle mass and function with ageing, is one of the major health problems in older adults, given its high prevalence and substantial socioeconomic implications. Despite the extensive efforts to reach consensus on definition and diagnostic tests and cut-offs for sarcopenia, there is an urgent and unmet need for non-invasive, specific and sensitive biomarkers for the disease. Extracellular vesicles (EVs) are present in different biofluids including plasma, whose cargo reflects cellular physiology. This work analysed EV proteome in sarcopenia and robust patients in the search for differentially contained proteins that can be used to diagnose the disease. METHODS: Plasma small EVs (sEVs) from a total of 29 robust controls (aged 73.4 ± 5.6 years; 11 men and 18 women) and 49 sarcopenic patients (aged 82.3 ± 5.4 years; 15 men and 34 women) aged 65 years and older were isolated and their cargo was analysed by proteomics. Proteins whose concentration in sEVs was different between sarcopenic and robust patients were further validated using ELISA. The concentration of these candidates was correlated to the EWGSOP2 sarcopenia tests for low muscle strength and low physical performance, and receiver operating characteristic (ROC) curve analyses were carried out to evaluate their diagnostic power, sensitivity and specificity. RESULTS: Proteomic analysis identified 157 sEVs proteins in both sarcopenic and robust samples. Among them, 48 proteins had never been reported in the ExoCarta nor Vesiclepedia databases. Statistical analysis revealed eight proteins whose concentration was significantly different between groups: PF4 (log2 FC = 4.806), OIT3 (log2 FC = -1.161), MMRN1 (log2 FC = -1.982), MASP1 (log2 FC = -0.627), C1R (log2 FC = 1.830), SVEP1 (log2 FC = 1.295), VCAN (FC = 0.937) and SPTB (log2 FC = 1.243). Among them, platelet factor 4 (PF4) showed the lowest concentration while Complement C1r subcomponent (C1R) increased the most in sarcopenic patients, being these results confirmed by ELISA (P = 1.07E-09 and P = 0.001287, respectively). The concentrations of candidate proteins significantly correlated with EWGSOP2 tests currently used. ROC curve analysis showed an area under the curve of 0.8921 and 0.7476 for PF4 and C1R, respectively. Choosing the optimal for PF4, 80% sensitivity and 85.71% specificity was reached while the optimal cut-off value of C1R would allow sarcopenia diagnosis with 75% sensitivity and 66.67% specificity. CONCLUSIONS: Our results support the determination of EV PF4 and C1R as plasma diagnostic biomarkers in sarcopenia and open the door to investigate the role of the content of these vesicles in the pathogeny of the disease.

PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets.

Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.

Motor skill learning modulates striatal extracellular vesicles' content in a mouse model of Huntington's disease.

Huntington's disease (HD) is a neurological disorder caused by a CAG expansion in the Huntingtin gene (HTT). HD pathology mostly affects striatal medium-sized spiny neurons and results in an altered cortico-striatal function. Recent studies report that motor skill learning, and cortico-striatal stimulation attenuate the neuropathology in HD, resulting in an amelioration of some motor and cognitive functions. During physical training, extracellular vesicles (EVs) are released in many tissues, including the brain, as a potential means for inter-tissue communication. To investigate how motor skill learning, involving acute physical training, modulates EVs crosstalk between cells in the striatum, we trained wild-type (WT) and R6/1 mice, the latter with motor and cognitive deficits, on the accelerating rotarod test, and we isolated their striatal EVs. EVs from R6/1 mice presented alterations in the small exosome population when compared to WT. Proteomic analyses revealed that striatal R6/1 EVs recapitulated signaling and energy deficiencies present in HD. Motor skill learning in R6/1 mice restored the amount of EVs and their protein content in comparison to naïve R6/1 mice. Furthermore, motor skill learning modulated crucial pathways in metabolism and neurodegeneration. All these data provide new insights into the pathogenesis of HD and put striatal EVs in the spotlight to understand the signaling and metabolic alterations in neurodegenerative diseases. Moreover, our results suggest that motor learning is a crucial modulator of cell-to-cell communication in the striatum.

Neuropilin-1 sex-dependently modulates inflammatory, angiogenic and osteogenic phenotypes in the calcifying valve interstitial cell.

The pathological mechanisms underlying the sex-dependent presentation of calcific aortic stenosis (AS) remain poorly understood. We aim to analyse sex-specific responses of valve interstitial cells (VICs) to calcific environments and to identify new pathological and potentially druggable targets. First, VICs from stenotic patients were modelled using pro-calcifying media (HP). Both male and female VICs were inflamed upon calcific HP challenge, although the inflammatory response was higher in female VICs. The osteogenic and calcification responses were higher in male VICs. To identify new players involved in the responses to HP, proteomics analyses were performed on additional calcifying VICs. Neuropilin-1 (NRP-1) was significantly up-regulated in male calcifying VICs and that was confirmed in aortic valves (AVs), especially nearby neovessels and calcifications. Regardless of the sex, NRP-1 expression was correlated to inflammation, angiogenesis and osteogenic markers, but with stronger associations in male AVs. To further evidence the role of NRP-1, in vitro experiments of silencing or supplementation with soluble NRP-1 (sNRP-1) were performed. NRP-1 silencing or addition of sNRP-1 reduced/mended the expression of any sex-specific response triggered by HP. Moreover, NRP-1 regulation contributed to significantly diminish the baseline enhanced expression of pro-inflammatory, pro-angiogenic and pro-osteogenic markers mainly in male VICs. Validation studies were conducted in stenotic AVs. In summary, pharmacologic targeting of NRP-1 could be used to target sex-specific phenotypes in AS as well as to exert protective effects by reducing the basal expression of pathogenic markers only in male VICs.

Drug-Induced Reorganisation of Lipid Metabolism Limits the Therapeutic Efficacy of Ponatinib in Glioma Stem Cells.

The standard of care for glioblastoma (GBM) involves surgery followed by adjuvant radio- and chemotherapy, but often within months, patients relapse, and this has been linked to glioma stem cells (GSCs), self-renewing cells with increased therapy resistance. The identification of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) as key players in gliomagenesis inspired the development of inhibitors targeting these tyrosine kinases (TKIs). However, results from clinical trials testing TKIs have been disappointing, and while the role of GSCs in conventional therapy resistance has been extensively studied, less is known about resistance of GSCs to TKIs. In this study, we have used compartmentalised proteomics to analyse the adaptive response of GSCs to ponatinib, a TKI with activity against PDGFR. The analysis of differentially expressed proteins revealed that GSCs respond to ponatinib by broadly rewiring lipid metabolism, involving fatty acid beta-oxidation, cholesterol synthesis, and sphingolipid degradation. Inhibiting each of these metabolic pathways overcame ponatinib adaptation of GSCs, but interrogation of patient data revealed sphingolipid degradation as the most relevant pathway in GBM. Our data highlight that targeting lipid metabolism, and particularly sphingolipid degradation in combinatorial therapies, could improve the outcome of TKI therapies using ponatinib in GBM.

Effects of Exercise Intervention for the Management of Delirium in Hospitalized Older Adults: A Randomized Clinical Trial.

OBJECTIVE: Delirium is a serious neuropsychiatric syndrome frequently occurring in hospitalized older adults, for which pharmacological treatments have shown limited effectiveness. Multicomponent physical exercise programs have demonstrated functional benefits; however, the impact of exercise on the course of delirium remains unexplored. The aim of this study was to investigate the effect of an individualized, multicomponent exercise intervention on the evolution of delirium and patient outcomes. DESIGN: A single-center, single-blind randomized controlled trial. SETTING AND PARTICIPANTS: Medical inpatients with delirium in an acute geriatric unit of a tertiary public hospital. METHODS: Thirty-six patients (mean age 87 years) were recruited and randomized into 2 groups. The control group received usual care and the intervention group received individualized physical exercise (1 daily session) for 3 consecutive days. Primary endpoints were the duration and severity of delirium (4-AT, Memorial Delirium Assessment Scale) and change in functional status [Barthel Index, Short Physical Performance Battery, Hierarchical Assessment of Balance and Mobility (HABAM), and handgrip strength]. Secondary endpoints included length of stay, falls, and health outcomes at 1- and 3-month follow-up. RESULTS: The intervention group showed more functional improvement at discharge (HABAM, P = .015) and follow-up (Barthel, P = .041; Lawton P = .027). Less cognitive decline was observed at 1 and 3 months (Informant Questionnaire on Cognitive Decline in the Elderly, P = .017). Exercise seemed to reduce delirium duration by 1 day and contribute to delirium resolution at discharge, although findings did not reach statistical significance. No exercise-related adverse events occurred. CONCLUSION AND IMPLICATIONS: Findings suggest that individualized exercise in acutely hospitalized older patients with delirium is safe, may improve delirium course and help preserve post-hospitalization function and cognition.

Involvement of Glucosamine 6 Phosphate Isomerase 2 (GNPDA2) Overproduction in ß-Amyloid- and Tau P301L-Driven Pathomechanisms.

Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.

Neuropathological stage-dependent proteome mapping of the olfactory tract in Alzheimer's disease: From early olfactory-related omics signatures to computational repurposing of drug candidates.

Alzheimer's disease (AD) is the most common form of dementia, characterized by an early olfactory dysfunction, progressive memory loss, and behavioral deterioration. Albeit substantial progress has been made in characterizing AD-associated molecular and cellular events, there is an unmet clinical need for new therapies. In this study, olfactory tract proteotyping performed in controls and AD subjects (n = 17/group) showed a Braak stage-dependent proteostatic impairment accompanied by the progressive modulation of amyloid precursor protein and tau functional interactomes. To implement a computational repurposing of drug candidates with the capacity to reverse early AD-related olfactory omics signatures (OMSs), we generated a consensual OMSs database compiling differential omics datasets obtained by mass-spectrometry or RNA-sequencing derived from initial AD across the olfactory axis. Using the Connectivity Map-based drug repurposing approach, PKC, EGFR, Aurora kinase, Glycogen synthase kinase, and CDK inhibitors were the top pharmacologic classes capable to restore multiple OMSs, whereas compounds with targeted activity to inhibit PI3K, Insulin-like growth factor 1 (IGF-1), microtubules, and Polo-like kinase (PLK) represented a family of drugs with detrimental potential to induce olfactory AD-associated gene expression changes. To validate the potential therapeutic effects of the proposed drugs, in vitro assays were performed. These validation experiments revealed that pretreatment of human neuron-like SH-SY5Y cells with the EGFR inhibitor AG-1478 showed a neuroprotective effect against hydrogen peroxide-induced damage while the pretreatment with the Aurora kinase inhibitor Reversine reduced amyloid-beta (Aß)-induced neurotoxicity. Taken together, our data pointed out that OMSs may be useful as substrates for drug repurposing to propose novel neuroprotective treatments against AD.

Special Issue "Deployment of Proteomics Approaches in Biomedical Research".

Many angles of personalized medicine, such as diagnostic improvements, systems biology [...].

Mapping the human brain proteome: opportunities, challenges, and clinical potential.

INTRODUCTION: Due to the segmented functions and complexity of the human brain, the characterization of molecular profiles within specific areas such as brain structures and biofluids is essential to unveil the molecular basis for structure specialization as well as the molecular imbalance associated with neurodegenerative and psychiatric diseases. AREAS COVERED: Much of our knowledge about brain functionality derives from neurophysiological, anatomical, and transcriptomic approaches. More recently, laser capture and imaging proteomics, technological and computational developments in LC-MS/MS, as well as antibody/aptamer-based platforms have allowed the generation of novel cellular, spatial, and posttranslational dimensions as well as innovative facets in biomarker validation and druggable target identification. EXPERT OPINION: Proteomics is a powerful toolbox to functionally characterize, quantify, and localize the extensive protein catalog of the human brain across physiological and pathological states. Brain function depends on multi-dimensional protein homeostasis, and its elucidation will help us to characterize biological pathways that are essential to properly maintain cognitive functions. In addition, comprehensive human brain pathological proteomes may be the basis in computational drug-repositioning methods as a strategy for unveiling potential new therapies in neurodegenerative and psychiatric disorders.

Association of postoperative delirium with serum and cerebrospinal fluid proteomic profiles: a prospective cohort study in older hip fracture patients.

Postoperative delirium (POD) is a common neuropsychiatric complication in geriatric inpatients after hip fracture surgery and its occurrence is associated with poor outcomes. The purpose of this study was to investigate the relationship between preoperative biomarkers in serum and cerebrospinal fluid (CSF) and the development of POD in older hip fracture patients, exploring the possibility of integrating objective methods into future predictive models of delirium. Sixty hip fracture patients were recruited. Blood and CSF samples were collected at the time of spinal anesthesia when none of the subjects had delirium. Patients were assessed daily using the 4AT scale, and based on these results, they were divided into POD and non-POD groups. The Olink® platform was used to analyze 45 cytokines. Twenty-one patients (35%) developed POD. In the subsample of 30 patients on whom proteomic analyses were performed, a proteomic profile was associated with the incidence of POD. Chemokine (C-X-C motif) ligand 9 (CXCL9) had the strongest correlation between serum and CSF samples in patients with POD (rho = 0.663; p < 0.05). Although several cytokines in serum and CSF were associated with POD after hip fracture surgery in older adults, there was a significant association with lower preoperative levels of CXCL9 in CSF and serum. Despite the small sample size, this study provides preliminary evidence of the potential role of molecular biomarkers in POD, which may provide a basis for the development of new delirium predictive models.

RTP801 mediates transneuronal toxicity in culture via extracellular vesicles.

Extracellular vesicles (EVs) play a crucial role in intercellular communication, participating in the paracrine trophic support or in the propagation of toxic molecules, including proteins. RTP801 is a stress-regulated protein, whose levels are elevated during neurodegeneration and induce neuron death. However, whether RTP801 toxicity is transferred trans-neuronally via EVs remains unknown. Hence, we overexpressed or silenced RTP801 protein in cultured cortical neurons, isolated their derived EVs (RTP801-EVs or shRTP801-EVs, respectively), and characterized EVs protein content by mass spectrometry (MS). RTP801-EVs toxicity was assessed by treating cultured neurons with these EVs and quantifying apoptotic neuron death and branching. We also tested shRTP801-EVs functionality in the pathologic in vitro model of 6-Hydroxydopamine (6-OHDA). Expression of RTP801 increased the number of EVs released by neurons. Moreover, RTP801 led to a distinct proteomic signature of neuron-derived EVs, containing more pro-apoptotic markers. Hence, we observed that RTP801-induced toxicity was transferred to neurons via EVs, activating apoptosis and impairing neuron morphology complexity. In contrast, shRTP801-EVs were able to increase the arborization in recipient neurons. The 6-OHDA neurotoxin elevated levels of RTP801 in EVs, and 6-OHDA-derived EVs lost the mTOR/Akt signalling activation via Akt and RPS6 downstream effectors. Interestingly, EVs derived from neurons where RTP801 was silenced prior to exposing them to 6-OHDA maintained Akt and RPS6 transactivation in recipient neurons. Taken together, these results suggest that RTP801-induced toxicity is transferred via EVs, and therefore, it could contribute to the progression of neurodegenerative diseases, in which RTP801 is involved.

Orosomucoid-1 Arises as a Shared Altered Protein in Two Models of Multiple Sclerosis.

Multiple sclerosis (MS) is a complex autoimmune and neurodegenerative disorder that affects the central nervous system (CNS). It is characterized by a heterogeneous disease course involving demyelination and inflammation. In this study, we utilized two distinct animal models, cuprizone (CPZ)-induced demyelination and experimental autoimmune encephalomyelitis (EAE), to replicate various aspects of the disease. We aimed to investigate the differential CNS responses by examining the proteomic profiles of EAE mice during the peak disease (15 days post-induction) and cuprizone-fed mice during the acute phase (38 days). Specifically, we focused on two different regions of the CNS: the dorsal cortex (Cx) and the entire spinal cord (SC). Our findings revealed varied glial, synaptic, dendritic, mitochondrial, and inflammatory responses within these regions for each model. Notably, we identified a single protein, Orosomucoid-1 (Orm1), also known as Alpha-1-acid glycoprotein 1 (AGP1), that consistently exhibited alterations in both models and regions. This study provides insights into the similarities and differences in the responses of these regions in two distinct demyelinating models.

Sex-specific role of galectin-3 in aortic stenosis.

BACKGROUND: Aortic stenosis (AS) is characterized by inflammation, fibrosis, osteogenesis and angiogenesis. Men and women develop these mechanisms differently. Galectin-3 (Gal-3) is a pro-inflammatory and pro-osteogenic lectin in AS. In this work, we aim to analyse a potential sex-differential role of Gal-3 in AS. METHODS: 226 patients (61.50% men) with severe AS undergoing surgical aortic valve (AV) replacement were recruited. In AVs, Gal-3 expression and its relationship with inflammatory, osteogenic and angiogenic markers was assessed. Valve interstitial cells (VICs) were primary cultured to perform in vitro experiments. RESULTS: Proteomic analysis revealed that intracellular Gal-3 was over-expressed in VICs of male AS patients. Gal-3 secretion was also higher in men's VICs as compared to women's. In human AVs, Gal-3 protein levels were significantly higher in men, with stronger immunostaining in VICs with myofibroblastic phenotype and valve endothelial cells. Gal-3 levels in AVs were positively correlated with inflammatory markers in both sexes. Gal-3 expression was also positively correlated with osteogenic markers mainly in men AVs, and with angiogenic molecules only in this sex. In vitro, Gal-3 treatment induced expression of inflammatory, osteogenic and angiogenic markers in male's VICs, while it only upregulated inflammatory and osteogenic molecules in women-derived cells. Gal-3 blockade with pharmacological inhibitors (modified citrus pectin and G3P-01) prevented the upregulation of inflammatory, osteogenic and angiogenic molecules. CONCLUSIONS: Gal-3 plays a sex-differential role in the setting of AS, and it could be a new sex-specific therapeutic target controlling pathological features of AS in VICs.

Aortic stenosis (AS) is a condition that affects the aortic valves (AVs) of the heart and leads to death if untreated. Males and females show clear differences in the onset of AS, both clinically and in valve deterioration. In this study we identified galectin-3 (Gal-3) as a molecule involved in the development of AS alterations with different effects in men and women. We analyzed AVs of 226 patients (139 male and 87 female) with severe AS who underwent surgical AV replacement to study the association of Gal-3 with markers of mechanisms related to AS, such as inflammation, calcification and blood vessels formation. We performed experiments in valvular interstitial cells (VICs) to evaluate the impact of Gal-3 in these cells and its potential use as a therapeutic target. Our results showed that Gal-3 was more expressed in AVs and VICs of men over women. In AVs, Gal-3 levels were associated with inflammatory markers either in male and female, while they correlated with osteogenic markers mainly in men and with angiogenic only in male. The treatment of VICs with Gal-3 produced increased levels of inflammatory and osteogenic molecules by cells of both sexes, but of angiogenic markers only in male's. Pharmacological inhibition of Gal-3 prevented the increase of these pathological markers in VICs. Overall, our study indicates that Gal-3 is a molecule implicated in the setting of AS in a sex-differential way and its targeting may lead to a new sex-specific therapeutic option for AS treatment.

Signature-driven repurposing of Midostaurin for combination with MEK1/2 and KRASG12C inhibitors in lung cancer.

Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.

Effect of immunology biomarkers associated with hip fracture and fracture risk in older adults.

Osteoporosis is a skeletal disease that can increase the risk of fractures, leading to adverse health and socioeconomic consequences. However, current clinical methods have limitations in accurately estimating fracture risk, particularly in older adults. Thus, new technologies are necessary to improve the accuracy of fracture risk estimation. In this observational study, we aimed to explore the association between serum cytokines and hip fracture status in older adults, and their associations with fracture risk using the FRAX reference tool. We investigated the use of a proximity extension assay (PEA) with Olink. We compared the characteristics of the population, functional status and detailed body composition (determined using densitometry) between groups. We enrolled 40 participants, including 20 with hip fracture and 20 without fracture, and studied 46 cytokines in their serum. After conducting a score plot and two unpaired t-tests using the Benjamini-Hochberg method, we found that Interleukin 6 (IL-6), Lymphotoxin-alpha (LT-α), Fms-related tyrosine kinase 3 ligand (FLT3LG), Colony stimulating factor 1 (CSF1), and Chemokine (C-C motif) ligand 7 (CCL7) were significantly different between fracture and non-fracture patients (p < 0.05). IL-6 had a moderate correlation with FRAX (R2 = 0.409, p < 0.001), while CSF1 and CCL7 had weak correlations with FRAX. LT-α and FLT3LG exhibited a negative correlation with the risk of fracture. Our results suggest that targeted proteomic tools have the capability to identify differentially regulated proteins and may serve as potential markers for estimating fracture risk. However, longitudinal studies will be necessary to validate these results and determine the temporal patterns of changes in cytokine profiles.

4R-Tau seeding activity unravels molecular subtypes in patients with Progressive Supranuclear Palsy.

Progressive Supranuclear palsy (PSP) is a 4-repeat (4-R) tauopathy. We hypothesized that the molecular diversity of tau could explain the heterogeneity seen in PSP disease progression. To test this hypothesis, we performed an extensive biochemical characterisation of the high molecular weight tau species (HMW-Tau) in 20 different brain regions of 25 PSP patients. We found a correlation between the HMW-Tau species and tau seeding capacity in the primary motor cortex, where we confirmed that an elevated 4R-Tau seeding activity correlates with a shorter disease duration. To identify factors that contribute to these differences, we performed proteomic and spatial transcriptomic analysis that revealed key mechanistic pathways, in particular those involving the immune system, that defined patients demonstrating high and low tau seeding capacity. These observations suggest that differences in the tau seeding activity may contribute to the considerable heterogeneity seen in disease progression of patients suffering from PSP.

Proteomic and functional characterisation of extracellular vesicles from collagen VI deficient human fibroblasts reveals a role in cell motility.

Extracellular vesicles (EVs) are key mediators of cell-to-cell communication. Their content reflects the state of diseased cells representing a window into disease progression. Collagen-VI Related Muscular Dystrophy (COL6-RD) is a multi-systemic disease involving different cell types. The role of EVs in this disease has not been explored. We compared by quantitative proteomics the protein cargo of EVs released from fibroblasts from patients with COL6-RD and controls. Isolated EVs contained a significant proportion of the most frequently reported proteins in EVs according to Exocarta and Vesiclepedia. We identified 67 differentially abundant proteins associated with vesicle transport and exocytosis, actin remodelling and the cytoskeleton, hemostasis and oxidative stress. Treatment of control fibroblasts with EVs from either patient or healthy fibroblasts altered significantly the motility of cells on a cell migration assay highlighting the functional relevance of EVs. In parallel, we analysed the secretome from the same cells and found a distinctly different set of 48 differentially abundant proteins related to extracellular matrix organisation and remodelling, growth factor response, RNA metabolism and the proteasome. The EVs and secretome sets of proteins only shared two identifiers indicating that the sorting of proteins towards EVs or the secretory pathway is tightly regulated for different functions.

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy.

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma.

Brain IGF-I regulates LTP, spatial memory, and sexual dimorphic behavior.

Insulin-like growth factor-I (IGF-I) exerts multiple actions, yet the role of IGF-I from different sources is poorly understood. Here, we explored the functional and behavioral consequences of the conditional deletion of Igf-I in the nervous system (Igf-I Δ/Δ), and demonstrated that long-term potentiation was impaired in hippocampal slices. Moreover, Igf-I Δ/Δ mice showed spatial memory deficits in the Morris water maze, and the significant sex-dependent differences displayed by Igf-I Ctrl/Ctrl mice disappeared in Igf-I Δ/Δ mice in the open field and rota-rod tests. Brain Igf-I deletion disorganized the granule cell layer of the dentate gyrus (DG), and it modified the relative expressions of GAD and VGLUT1, which are preferentially localized to inhibitory and excitatory presynaptic terminals. Furthermore, Igf-I deletion altered protein modules involved in receptor trafficking, synaptic proteins, and proteins that functionally interact with estrogen and androgen metabolism. Our findings indicate that brain IGF-I is crucial for long-term potentiation, and that it is involved in the regulation of spatial memory and sexual dimorphic behaviors, possibly by maintaining the granule cell layer structure and the stability of synaptic-related protein modules.