Explore the vast range of titles available.

Chronic kidney disease (CKD) is a long-term kidney damage caused by gradual loss of essential kidney functions. A global health issue, CKD affects up to 16% of the population worldwide. Symptoms are often not apparent in the early stages, and if left untreated, CKD can progress to end-stage kidney disease (ESKD), also known as kidney failure, when the only possible treatments are dialysis and kidney transplantation. The end point of nearly all forms of CKD is kidney fibrosis, a process of unsuccessful wound-healing of kidney tissue. Detection of kidney fibrosis, therefore, often means detection of CKD. Renal biopsy remains the best test for renal scarring, despite being intrinsically limited by its invasiveness and sampling bias. Urine is a desirable source of fibrosis biomarkers as it can be easily obtained in a non-invasive way and in large volumes. Besides, urine contains biomolecules filtered through the glomeruli, mirroring the pathological state. There is, however, a problem of highly abundant urinary proteins that can mask rare disease biomarkers. Urinary extracellular vesicles (uEVs), which originate from renal cells and carry proteins, nucleic acids, and lipids, are an attractive source of potential rare CKD biomarkers. Their cargo consists of low-abundant proteins but highly concentrated in a nanosize-volume, as well as molecules too large to be filtered from plasma. Combining molecular profiling data (protein and miRNAs) of uEVs, isolated from patients affected by various forms of CKD, this review considers the possible diagnostic and prognostic value of uEVs biomarkers and their potential application in the translation of new experimental antifibrotic therapeutics.

Protein misfolding and aggregation is a central feature of several neurodegenerative disorders including Alzheimer’s disease (AD), in which assemblies of amyloid β (Aβ) peptides accumulate in the brain in the form of parenchymal and/or vascular amyloid. A widely accepted concept is that AD is characterized by distinct clinical and neuropathological phenotypes. Recent studies revealed that Aβ assemblies might have structural differences among AD brains and that such pleomorphic assemblies can correlate with distinct disease phenotypes. We found that in both sporadic and inherited forms of AD, amyloid aggregates differ in the biochemical composition of Aβ species. These differences affect the physicochemical properties of Aβ assemblies including aggregation kinetics, resistance to degradation by proteases and seeding ability. Aβ-amyloidosis can be induced and propagated in animal models by inoculation of brain extracts containing aggregated Aβ. We found that brain homogenates from AD patients with different molecular profiles of Aβ are able to induce distinct patterns of Aβ-amyloidosis when injected into mice. Overall these data suggest that the assembly of mixtures of Aβ peptides into different Aβ seeds leads to the formation of distinct subtypes of amyloid having distinctive physicochemical and biological properties which result in the generation of distinct AD molecular subgroups.

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments’ quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications.

The rat sub-total nephrectomy (SNx) is a functional model of general chronic kidney disease (CKD) where the main pathological driver is glomerular hypertension representative of several subtypes of CKD. Comprehensive transcriptomics and proteomics analyses on the SNx rats were performed to identify biomarkers in plasma or urine that correlate with kidney disease and functional kidney loss. Kidneys were subjected to collagen I and III staining for fibrosis scoring, SWATH-MS proteomics and bulk RNA-sequencing transcriptomics, with SWATH-MS also performed on plasma and urine. Differential expression analysis demonstrated significant dysregulation of genes and proteins involved in fibrosis, metabolism, and immune response in the SNx rats compared to controls. Gene ontology analysis of the intersecting genes and proteins from both studies demonstrated common biology between animal cohorts that reached the predefined kidney disease thresholds (serum creatinine > two-fold or proteinuria > three-fold increase over sham-operated). Thirteen significantly differential molecules were detected with consistent directional changes in both omics datasets. These molecules were detected independently in kidney (both RNA and protein) and urine (protein only), but not in plasma. Bioinformatics analysis enabled the identification of mechanistic CKD biomarkers including lumican and collagen alpha-1(III) chain, whose co-expression has previously been both implicated in fibrosis and detected in urine in CKD patients.

In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed \"pollensomes\", are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. To do so, pollensomes were isolated from hydrated and germinated kiwi ( Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles.

Androgen independency is associated with poor prostate cancer (PCa) survival. Here we report that silencing of transglutaminase-2 (TG2) expression by CRISPR-Cas9 is associated with upregulation of androgen receptor (AR) transcription in PCa cell lines. Knockout of TG2 reversed the migratory potential and anchorage independency of PC3 and DU145 cells and revealed a reduced level of mucin-1 (MUC1) RNA transcript through unbiased multi-omics profiling, which was restored by selective add-back of the truncated TG2 isoform (TGM2_v2). Silencing of AR resulted into increased MUC1 in TG2KO PC3 cells showing that TG2 affects transcriptional regulation of MUC1 via repressing AR expression. Treatment of PC3 WT cell line with TG2 inhibitor ZDON led to a significant increase in AR expression and decrease in MUC1. ZDON also blocked the formation of MUC1-multimers labelled with TG amine-donor substrates in reducing conditions, revealing for the first time a role for TG2, which we show to be externalised via extracellular vesicles, in MUC1 stabilisation via calcium-dependent transamidation. A specific antibody towards TGM2_v2 revealed its restricted nuclear location compared to the canonical long form of TG2 (TGM2_v1), which is predominantly cytosolic, suggesting that this form contributes to the previously suggested TG2-mediated NF-κB activation and AR transcriptional repression. As TGM2_v2 transcription was increased in biopsies of early-stage prostate adenocarcinoma (PRAD) patients compared to subjects presenting inflammatory prostatitis, and total TG2 protein expression significantly increased in PRAD versus normal tissue, the role of TG2 and its truncated form as a prostate malignancy marker is suggested. In conclusion, this investigation has provided the first unbiased discovery of a novel pathway mediated by TG2 via MUC1, which is shown to contribute to androgen insensitivity and malignancy of PCa cells and be upregulated in PCa biopsies, with potential relevance to cancer immune evasion.

Amyloid-beta (Aβ) deposition in the brain is closely linked with the development of Alzheimer’s disease (AD). Unfortunately, therapies specifically targeting Aβ deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2−/−) mice. We found that absence of TG2 had no (statistically) significant effect on Aβ pathology, soluble brain levels of Aβ1–40 and Aβ1–42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2−/− brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

Background We recently demonstrated that large extracellular vesicles (EVs) released by Aβ‐loaded microglia and carrying Aβ (Aβ‐EVs) propagate synaptic dysfunction in the mouse brain by moving at the axon surface (Gabrielli et al., Brain, 2022; Falcicchia et al., Brain Commun, 2023). Compared to ctrl‐EVs, not carrying Aβ, a higher number of Aβ‐EVs move along axons and travel longer distances through a yet undefined mechanism. Our previous work indicates that large EVs motion can be passively driven by neurons, via EV interaction with a neuronal receptor linked to the cytoskeleton, or may happen autonomously when EVs contain actin filaments and ATP in their lumen. To get insights into the mechanisms underlying higher extracellular Aβ‐EVs motion, we here analysed microglial EVs proteome. Method Aβ‐EVs were obtained from primary microglia exposed to Aβ42. High‐resolution accurate‐mass spectrometry SWATH™‐MS followed by DIA‐NN quantitative analysis was employed to identify differentially expressed proteins in Aβ‐ vs. ctrl‐EVs, which may justify Aβ‐EVs empowered motility and describe the molecular mechanisms underlying EVs extracellular motion. Western blot and optical manipulation coupled to time‐lapse imaging were also employed to explore the involvement of additional candidate molecules, such as transglutaminase 2 (TG2). Result A total of 148 proteins were upregulated in Aβ‐ compared to ctrl‐EVs, while 169 were downregulated. Panther GO analysis showed that terms enriched in large Aβ‐EVs include cytoskeletal protein binding (GO:0008092) and ATP hydrolysis activity (GO:0016887), suggesting that a higher fraction of Aβ‐EVs may undergo independent motion. STRING analysis revealed a high protein‐protein interaction (PPI) among Aβ‐EVs enriched molecules, some of which are differentially expressed also in EVs from AD/MCI patients or AD mouse models, validating the relevance of our findings. Conclusion Our data suggest that Aβ‐EVs higher motility and capability to propagate synaptic dysfunction may be due to their augmented capacity to undergo active motion, and identify novel proteins potentially involved in EVs motion. Further analysis will be necessary to validate these findings in AD. Fundings: 2018‐AARF‐588984, Horizon2020#874721PREMSTEM.

We recently demonstrated that large extracellular vesicles (EVs) released by Aβ-loaded microglia and carrying Aβ (Aβ-EVs) propagate synaptic dysfunction in the mouse brain by moving at the axon surface (Gabrielli et al., Brain, 2022; Falcicchia et al., Brain Commun, 2023). Compared to ctrl-EVs, not carrying Aβ, a higher number of Aβ-EVs move along axons and travel longer distances through a yet undefined mechanism. Our previous work indicates that large EVs motion can be passively driven by neurons, via EV interaction with a neuronal receptor linked to the cytoskeleton, or may happen autonomously when EVs contain actin filaments and ATP in their lumen. To get insights into the mechanisms underlying higher extracellular Aβ-EVs motion, we here analysed microglial EVs proteome. Aβ-EVs were obtained from primary microglia exposed to Aβ High-resolution accurate-mass spectrometry SWATH™-MS followed by DIA-NN quantitative analysis was employed to identify differentially expressed proteins in Aβ- vs. ctrl-EVs, which may justify Aβ-EVs empowered motility and describe the molecular mechanisms underlying EVs extracellular motion. Western blot and optical manipulation coupled to time-lapse imaging were also employed to explore the involvement of additional candidate molecules, such as transglutaminase 2 (TG2). A total of 148 proteins were upregulated in Aβ- compared to ctrl-EVs, while 169 were downregulated. Panther GO analysis showed that terms enriched in large Aβ-EVs include cytoskeletal protein binding (GO:0008092) and ATP hydrolysis activity (GO:0016887), suggesting that a higher fraction of Aβ-EVs may undergo independent motion. STRING analysis revealed a high protein-protein interaction (PPI) among Aβ-EVs enriched molecules, some of which are differentially expressed also in EVs from AD/MCI patients or AD mouse models, validating the relevance of our findings. Our data suggest that Aβ-EVs higher motility and capability to propagate synaptic dysfunction may be due to their augmented capacity to undergo active motion, and identify novel proteins potentially involved in EVs motion. Further analysis will be necessary to validate these findings in AD. 2018-AARF-588984, Horizon2020#874721PREMSTEM.

Transglutaminase 2 (TG2) is a calcium-dependent protein crosslinking enzyme activated in misfolding diseases and it is implicated in multiple disorders linked to calcium dysregulation, including neurodegeneration. In vitro, TG2 has been involved in the generation of toxic amyloid-β (Aβ) oligomers by post-translational modification (PTM), and literature data suggest that TG2 is activated in disease, e.g. the early stages of Alzheimer’s disease (AD). TG2 is also involved in cell-matrix dynamics and has been suggested to be a cargo of extracellular vesicles (EVs) in cancer and tissue fibrosis. EVs have been implicated in the spreading of pathogenic proteins in neurodegenerative diseases (e.g. Aβ and tau) and represent a new field of research in dementia. The aims of this study are to: i. investigate the role of extracellular TG2 in neuron-glia cross-talk in the context of neurodegeneration; ii. explore substrates of TG2 PTM in a cell model simulating AD; iii. evaluate TG2 as a potential marker of dementia. To this purpose, both primary cells (embryonic rat brain cells) and biological samples from dementia patients were analysed. We found that when raised at levels compatible with inflammatory states, extracellular TG2 consistently increased basal calcium concentration ([Ca2+]i) in hippocampal neurons, affecting calcium homeostasis, which is at the basis of neuronal functions. This effect was mediated by TG2- driven membrane depolarisation, which may be caused by the interaction of TG2 with plasma membrane ionic channels [i.e. Voltage Operated Calcium Channels (VOCCs) and Na+ /Ca2+ exchanger (NCX)]. We confirmed previous evidence showing that astrocytes are a rich source of extracellular TG2 in brain and showed for the first time that TG2 is released as a cargo of astrocytic EVs. Simulation of AD pathology in primary hippocampal neurons stimulated with Aβ1-42 led to the identification of 11 TG2 substrates (TG2 transamidome) using a global quantitative proteomic approach (SWATH™-MS/MS proteomics). These included proteins involved in ion transport [Plasma Membrane Ca2+ Transporting ATPase 2 (AT2B2) and Transmembrane Channel-like Protein 5 (TMC)], which could be involved in TG2-mediated alteration of calcium homeostasis in pathology. We also found that a number of neurotrophic proteins involved in neuronal growth and apoptosis were significantly decreased upon TG2 inhibition, suggesting that TG2 might play a dual role in neuronal survival during neurodegeneration. Finally, analysis of plasma from 45 dementia patients and healthy controls by an optimised ELISA assay revealed no significant changes in TG2 between the study groups. Our preliminary data suggest that quantitation of TG2 should be performed in plasma-derived EVs for a more accurate and sensitive evaluation.