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Abstract Background Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. Methods Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. Results We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. Conclusions Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

Pancreatic malignancy is the fourth cause of cancer-related death in Western countries and is predicted to become the second leading cause of cancer-related mortality by 2030. The standard therapies (FOLFIRINOX and gemcitabine with nab-paclitaxel) are not resolutive because this type of cancer is also characterized by a high chemoresistance, due in part to the activity of the ATP Binding Cassette (ABC) pumps accounting for the reduction in the intracellular concentration of the drugs. In this work, we analyze the occurrence of single-nucleotide polymorphisms (SNPs) in the MDR-1 gene, in different pancreatic cancer cell lines, and in tissues from pancreatic cancer patients by DNA sequencing, as well as the expression levels of MDR-1 mRNA and protein, by qRT-PCR and Western Blot analysis. We found that gemcitabine-resistant cells, in conjunction with homozygosis of analyzed SNPs, showed high MDR-1 basal levels with further increases after gemcitabine treatment. Nevertheless, we did not observe in the human PDAC samples a correlation between the level of MDR-1 mRNA and protein expression and SNPs. Preliminary, we conclude that in our small cohort, these SNPs cannot be used as molecular markers for predicting the levels of MDR-1 mRNA/protein levels and drug responses in patients with PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.

Alterations in cellular signaling, chronic inflammation, and tissue remodeling contribute to hepatocellular carcinoma (HCC) development. The release of damage-associated molecular patterns (DAMPs) upon tissue injury and the ensuing sterile inflammation have also been attributed a role in HCC pathogenesis. Cargoes of extracellular vesicles (EVs) and/or EVs themselves have been listed among circulating DAMPs but only partially investigated in HCC. Mitochondria-derived vesicles (MDVs), a subpopulation of EVs, are another missing link in the comprehension of the molecular mechanisms underlying the onset and progression of HCC biology. EVs have been involved in HCC growth, dissemination, angiogenesis, and immunosurveillance escape. The contribution of MDVs to these processes is presently unclear. Pyroptosis triggers systemic inflammation through caspase-dependent apoptotic cell death and is implicated in tumor immunity. The analysis of this process, together with MDV characterization, may help capture the relationship among HCC development, mitochondrial quality control, and inflammation. The combination of immune checkpoint inhibitors (i.e., atezolizumab and bevacizumab) has been approved as a synergistic first-line systemic treatment for unresectable or advanced HCC. The lack of biomarkers that may allow prediction of treatment response and, therefore, patient selection, is a major unmet need. Herein, we overview the molecular mechanisms linking mitochondrial dysfunction, inflammation, and pyroptosis, and discuss how immunotherapy targets, at least partly, these routes.

Mitochondrial DNA (mtDNA) is as a double-stranded molecule existing in hundreds to thousands copies in cells depending on cell metabolism and exposure to endogenous and/or environmental stressors. The coordination of mtDNA replication and transcription regulates the pace of mitochondrial biogenesis to guarantee the minimum number of organelles per cell. mtDNA inheritance follows a maternal lineage, although bi-parental inheritance has been reported in some species and in the case of mitochondrial diseases in humans. mtDNA mutations (e.g., point mutations, deletions, copy number variations) have been identified in the setting of several human diseases. For instance, sporadic and inherited rare disorders involving the nervous system as well higher risk of developing cancer and neurodegenerative conditions, including Parkinson's and Alzheimer's disease, have been associated with polymorphic mtDNA variants. An accrual of mtDNA mutations has also been identified in several tissues and organs, including heart and muscle, of old experimental animals and humans, which may contribute to the development of aging phenotypes. The role played by mtDNA homeostasis and mtDNA quality control pathways in human health is actively investigated for the possibility of developing targeted therapeutics for a wide range of conditions.

The Rab7V162M mutation associated with Charcot Marie Tooth 2B peripheral neuropathy causes mitochondrial fragmentation in patient-derived fibroblasts and primary cultured dorsal root ganglion sensory neurons from E18 mouse embryos.

Abstract Background In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA. Methods A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers. Results The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis. Conclusions Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein–protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.

Extracellular vesicles have been identified as pivotal mediators of intercellular communication with critical roles in physiological and pathological conditions. Via this route, several molecules (e.g., nucleic acids, proteins, metabolites) can be transferred to proximal and distant targets to convey specific information. Extracellular vesicle-associated cargo molecules have been proposed as markers of several disease conditions for their potential of tracking down the generating cell. Indeed, circulating extracellular vesicles may represent biomarkers of dysfunctional cellular quality control systems especially in conditions characterized by the accrual of intracellular misfolded proteins. Furthermore, the identification of extracellular vesicles as tools for the delivery of nucleic acids or other cargo molecules to diseased tissues makes these circulating shuttles possible targets for therapeutic development. The increasing interest in the study of extracellular vesicles as biomarkers resides mainly in the fact that the identification of peripheral levels of extracellular vesicle-associated proteins might reflect molecular events occurring in hardly accessible tissues, such as the brain, thereby serving as a “brain liquid biopsy”. The exploitation of extracellular vesicles for diagnostic and therapeutic purposed might offer unprecedented opportunities to develop personalized approaches. Here, we discuss the bright and dark sides of extracellular vesicles in the setting of two main neurodegenerative diseases (i.e., Parkinson’s and Alzheimer’s diseases). A special focus will be placed on the possibility of using extracellular vesicles as biomarkers for the two conditions to enable disease tracking and treatment monitoring.

Mitophagy is crucial for maintaining mitochondrial quality. However, its assessment in vivo is challenging. The endosomal–lysosomal system is a more accessible pathway through which subtypes of extracellular vesicles (EVs), which also contain mitochondrial constituents, are released for disposal. The inclusion of mitochondrial components into EVs occurs in the setting of mild mitochondrial damage and during impairment of lysosomal function. By releasing mitochondrial-derived vesicles (MDVs), cells limit the unload of mitochondrial damage-associated molecular patterns with proinflammatory activity. Both positive and negative effects of EVs on recipient cells have been described. Whether this is due to the production of EVs other than those containing mitochondria, such as MDVs, holding specific biological functions is currently unknown. Evidence on the existence of different MDV subtypes has been produced. However, their characterization is not always pursued, which would be relevant to exploring the dynamics of mitochondrial quality control in health and disease. Furthermore, MDV classification may be instrumental in understanding their biological roles and promoting their implementation as biomarkers in clinical studies.

Abstract V-ATPase is a large multi-subunit complex that regulates acidity of intracellular compartments and of extracellular environment. V-ATPase consists of several subunits that drive specific regulatory mechanisms. The V1G1 subunit, a component of the peripheral stalk of the pump, controls localization and activation of the pump on late endosomes and lysosomes by interacting with RILP and RAB7. Deregulation of some subunits of the pump has been related to tumor invasion and metastasis formation in breast cancer. We observed a decrease of V1G1 and RAB7 in highly invasive breast cancer cells, suggesting a key role of these proteins in controlling cancer progression. Moreover, in MDA-MB-231 cells, modulation of V1G1 affected cell migration and matrix metalloproteinase activation in vitro, processes important for tumor formation and dissemination. In these cells, characterized by high expression of EGFR, we demonstrated that V1G1 modulates EGFR stability and the EGFR downstream signaling pathways that control several factors required for cell motility, among which RAC1 and cofilin. In addition, we showed a key role of V1G1 in the biogenesis of endosomes and lysosomes. Altogether, our data describe a new molecular mechanism, controlled by V1G1, required for cell motility and that promotes breast cancer tumorigenesis.

Intermediate filaments are the most heterogeneous class among cytoskeletal elements. While some of them have been well-characterized, little is known about peripherin. Peripherin is a class III intermediate filament protein with a specific expression in the peripheral nervous system. Epigenetic modifications are involved in this cell-type-specific expression. Peripherin has important roles in neurite outgrowth and stability, axonal transport, and axonal myelination. Moreover, peripherin interacts with proteins involved in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism, suggesting a role in all these processes. This review collects information regarding peripherin gene regulation, post-translational modifications, and functions and its involvement in the onset of a number of diseases.

Sterile inflammation develops as part of an innate immunity response to molecules released upon tissue injury and collectively indicated as damage-associated molecular patterns (DAMPs). While coordinating the clearance of potential harmful stimuli, promotion of tissue repair, and restoration of tissue homeostasis, a hyper-activation of such an inflammatory response may be detrimental. The complex regulatory pathways modulating DAMPs generation and trafficking are actively investigated for their potential to provide relevant insights into physiological and pathological conditions. Abnormal circulating extracellular vesicles (EVs) stemming from altered endosomal-lysosomal system have also been reported in several age-related conditions, including cancer and neurodegeneration, and indicated as a promising route for therapeutic purposes. Along this pathway, mitochondria may dispose altered components to preserve organelle homeostasis. However, whether a common thread exists between DAMPs and EVs generation is yet to be clarified. A deeper understanding of the highly complex, dynamic, and variable intracellular and extracellular trafficking of DAMPs and EVs, including those of mitochondrial origin, is needed to unveil relevant pathogenic pathways and novel targets for drug development. Herein, we describe the mechanisms of generation of EVs and mitochondrial-derived vesicles along the endocytic pathway and discuss the involvement of the endosomal-lysosomal in cancer and neurodegeneration (i.e., Alzheimer’s and Parkinson’s disease).

Mitochondria are primarily involved in cell bioenergetics, regulation of redox homeostasis, and cell death/survival signaling. An immunostimulatory property of mitochondria has also been recognized which is deployed through the extracellular release of entire or portioned organelle and/or mitochondrial DNA (mtDNA) unloading. Dynamic homo- and heterotypic interactions involving mitochondria have been described. Each type of connection has functional implications that eventually optimize mitochondrial activity according to the bioenergetic demands of a specific cell/tissue. Inter-organelle communications may also serve as molecular platforms for the extracellular release of mitochondrial components and subsequent ignition of systemic inflammation. Age-related chronic inflammation (inflamm-aging) has been associated with mitochondrial dysfunction and increased extracellular release of mitochondrial components—in particular, cell-free mtDNA. The close relationship between mitochondrial dysfunction and cellular senescence further supports the central role of mitochondria in the aging process and its related conditions. Here, we provide an overview of (1) the mitochondrial genetic system and the potential routes for generating and releasing mtDNA intermediates; (2) the pro-inflammatory pathways elicited by circulating mtDNA; (3) the participation of inter-organelle contacts to mtDNA homeostasis; and (4) the link of these processes with senescence and age-associated conditions.

Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by five mutations in the RAB7A gene. Autophagy and late endocytic trafficking were already characterized in CMT2B. Indeed, impairment of autophagy and an increase in lysosomal degradative activity were found in cells expressing the mutant proteins. Recently, we described a novel RAB7 mutation associated with predominantly motor CMT2 and impaired EGFR trafficking. With the aim to analyze the autophagy process and lysosomal activity in CMT2B fibroblasts carrying the p.K126R RAB7 novel mutation and to investigate further the causes of the different phenotype, we have performed Western blot, immunofluorescence and cytometric analyses monitoring autophagic markers and endocytic proteins. Moreover, we investigated lipophagy by analyzing accumulation of lipid droplets and their co-localization with endolysosomal degradative compartments. We found that cells expressing the RAB7K126R mutant protein were characterized by impairment of autophagy and lipophagy processes and by a moderate increase in lysosomal activity compared to the previously studied cells carrying the RAB7V162M mutation. Thus, we concluded that EGFR trafficking alterations and a moderate increase in lysosomal activity with concomitant impairment of autophagy could induce the specific predominantly motor phenotype observed in K126R patients.

Physical frailty and sarcopenia (PF&S) is a prototypical geriatric condition characterized by reduced physical function and low muscle mass. The multifaceted pathophysiology of this condition recapitulates all hallmarks of aging making the identification of specific biomarkers challenging. In the present study, we explored the relationship among three processes that are thought to be involved in PF&S (i.e., systemic inflammation, amino acid dysmetabolism, and mitochondrial dysfunction). We took advantage of the well-characterized cohort of older adults recruited in the “BIOmarkers associated with Sarcopenia and Physical frailty in EldeRly pErsons” (BIOSPHERE) study to preliminarily combine in a multi-platform analytical approach inflammatory biomolecules, amino acids and derivatives, and mitochondrial-derived vesicle (MDV) cargo molecules to evaluate their performance as possible biomarkers for PF&S. Eleven older adults aged 70 years and older with PF&S and 10 non-sarcopenic non-frail controls were included in the analysis based on the availability of the three categories of biomolecules. A sequential and orthogonalized covariance selection—linear discriminant analysis (SO-CovSel–LDA) approach was used for biomarkers selection. Of the 75 analytes assayed, 16 had concentrations below the detection limit. Within the remaining 59 biomolecules, So-CovSel–LDA selected a set comprising two amino acids (phosphoethanolamine and tryptophan), two cytokines (interleukin 1 receptor antagonist and macrophage inflammatory protein 1β), and MDV-derived nicotinamide adenine dinucleotide reduced form:ubiquinone oxidoreductase subunit S3 as the best predictors for discriminating older people with and without PF&S. The evaluation of these biomarkers in larger cohorts and their changes over time or in response to interventions may unveil specific pathogenetic pathways of PF&S and identify new biological targets for drug development.

Parkinson’s Disease (PD) is a highly prevalent neurodegenerative disease among older adults. PD neuropathology is marked by the progressive loss of the dopaminergic neurons of the substantia nigra pars compacta and the widespread accumulation of misfolded intracellular α-synuclein (α-syn). Genetic mutations and post-translational modifications, such as α-syn phosphorylation, have been identified among the multiple factors supporting α-syn accrual during PD. A decline in the clearance capacity of the ubiquitin-proteasome and the autophagy-lysosomal systems, together with mitochondrial dysfunction, have been indicated as major pathophysiological mechanisms of PD neurodegeneration. The accrual of misfolded α-syn aggregates into soluble oligomers, and the generation of insoluble fibrils composing the core of intraneuronal Lewy bodies and Lewy neurites observed during PD neurodegeneration, are ignited by the overproduction of reactive oxygen species (ROS). The ROS activate the α-syn aggregation cascade and, together with the Lewy bodies, promote neurodegeneration. However, the molecular pathways underlying the dynamic evolution of PD remain undeciphered. These gaps in knowledge, together with the clinical heterogeneity of PD, have hampered the identification of the biomarkers that may be used to assist in diagnosis, treatment monitoring, and prognostication. Herein, we illustrate the main pathways involved in PD pathogenesis and discuss their possible exploitation for biomarker discovery.

Pancreatic cancer (PC) is among the most devastating digestive tract cancers worldwide. This cancer is characterized by poor diagnostic detection, lack of therapy, and difficulty in predicting tumorigenesis progression. Although mutations of key oncogenes and oncosuppressor involved in tumor growth and in immunosurveillance escape are known, the underlying mechanisms that orchestrate PC initiation and progression are poorly understood or still under debate. In recent years, the attention of many researchers has been concentrated on the role of extracellular vesicles and of a particular subset of extracellular vesicles, known as exosomes. Literature data report that these nanovesicles are able to deliver their cargos to recipient cells playing key roles in the pathogenesis and progression of many pancreatic precancerous conditions. In this review, we have summarized and discussed principal cargos of extracellular vesicles characterized in PC, such as miRNAs, lncRNAs, and several proteins, to offer a systematic overview of their function in PC progression. The study of extracellular vesicles is allowing to understand that investigation of their secretion and analysis of their content might represent a new and potential diagnostic and prognostic tools for PC.

RAB7A is a small GTPase that controls the late endocytic pathway but also cell migration through RAC1 (Ras-related C3 botulinum toxin substrate 1) and vimentin. In fact, RAB7A regulates vimentin phosphorylation at different sites and vimentin assembly, and, in this study, we identified vimentin domains interacting with RAB7A. As several kinases could be responsible for vimentin phosphorylation, we investigated whether modulation of RAB7A expression affects the activity of these kinases. We discovered that RAB7A regulates AKT and PAK1, and we demonstrated that increased vimentin phosphorylation at Ser38 (Serine 38), observed upon RAB7A overexpression, is due to AKT activity. As AKT and PAK1 are key regulators of several cellular events, we investigated if RAB7A could have a role in these processes by modulating AKT and PAK1 activity. We found that RAB7A protein levels affected beta-catenin and caspase 9 expression. We also observed the downregulation of cofilin-1 and decreased matrix metalloproteinase 2 (MMP2) activity upon RAB7A silencing. Altogether these results demonstrate that RAB7A regulates AKT and PAK1 kinases, affecting their downstream effectors and the processes they regulate, suggesting that RAB7A could have a role in a number of cancer hallmarks.

Epidermal growth factor receptor (EGFR) is the first discovered member of the receptor tyrosine kinase superfamily and plays a fundamental role during embryogenesis and in adult tissues, being involved in growth, differentiation, maintenance and repair of various tissues and organs. The role of EGFR in the regulation of tissue development and homeostasis has been thoroughly investigated and it has also been demonstrated that EGFR is a driver of tumorigenesis. In the nervous system, other growth factors, and thus other receptors, are important for growth, differentiation and repair of the tissue, namely neurotrophins and neurotrophins receptors. For this reason, for a long time, the role of EGFR in the nervous system has been underestimated and poorly investigated. However, EGFR is expressed both in the central and peripheral nervous systems and it has been demonstrated to have specific important neurotrophic functions, in particular in the central nervous system. This review discusses the role of EGFR in regulating differentiation and functions of neurons and neuroglia. Furthermore, its involvement in regeneration after injury and in the onset of neurodegenerative diseases is examined.

Mitochondria are intracellular organelles involved in a myriad of activities. To safeguard their vital functions, mitochondrial quality control (MQC) systems are in place to support organelle plasticity as well as physical and functional connections with other cellular compartments. In particular, mitochondrial interactions with the endosomal compartment support the shuttle of ions and metabolites across organelles, while those with lysosomes ensure the recycling of obsolete materials. The extrusion of mitochondrial components via the generation and release of mitochondrial-derived vesicles (MDVs) has recently been described. MDV trafficking is now included among MQC pathways, possibly operating via mitochondrial–lysosomal contacts. Since mitochondrial dysfunction is acknowledged as a hallmark of aging and a major pathogenic factor of multiple age-associated conditions, the analysis of MDVs and, more generally, of extracellular vesicles (EVs) is recognized as a valuable research tool. The dissection of EV trafficking may help unravel new pathophysiological pathways of aging and diseases as well as novel biomarkers to be used in research and clinical settings. Here, we discuss (1) MQC pathways with a focus on mitophagy and MDV generation; (2) changes of MQC pathways during aging and their contribution to inflamm-aging and progeroid conditions; and (3) the relevance of MQC failure to several disorders, including neurodegenerative conditions (i.e., Parkinson’s disease, Alzheimer’s disease) and cardiovascular disease.

Mitochondrial dysfunction and systemic inflammation are major factors in the development of sarcopenia, but the molecular determinants linking the two mechanisms are only partially understood. The study of extracellular vesicle (EV) trafficking may provide insights into this relationship. Circulating small EVs (sEVs) from serum of 11 older adults with physical frailty and sarcopenia (PF&S) and 10 controls were purified and characterized. Protein levels of three tetraspanins (CD9, CD63, and CD81) and selected mitochondrial markers, including adenosine triphosphate 5A (ATP5A), mitochondrial cytochrome C oxidase subunit I (MTCOI), nicotinamide adenine dinucleotide reduced form (NADH):ubiquinone oxidoreductase subunit B8 (NDUFB8), NADH:ubiquinone oxidoreductase subunit S3 (NDUFS3), succinate dehydrogenase complex iron sulfur subunit B (SDHB), and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2) were quantified by Western immunoblotting. Participants with PF&S showed higher levels of circulating sEVs relative to controls. Protein levels of CD9 and CD63 were lower in the sEV fraction of PF&S older adults, while CD81 was unvaried between groups. In addition, circulating sEVs from PF&S participants had lower amounts of ATP5A, NDUFS3, and SDHB. No signal was detected for MTCOI, NDUFB8, or UQCRC2 in either participant group. Our findings indicate that, in spite of increased sEV secretion, lower amounts of mitochondrial components are discarded through EV in older adults with PF&S. In-depth analysis of EV trafficking might open new venues for biomarker discovery and treatment development for PF&S.

The rare autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) is associated with mutations in the RAB7A gene, involved in the late endocytic pathway. CMT2B is characterized by predominant sensory loss, ulceromutilating features, with lesser-to-absent motor deficits. We characterized clinically and genetically a family harboring a novel pathogenic RAB7A variant and performed structural and functional analysis of the mutant protein. A 39-year-old woman presented with early-onset walking difficulties, progressive distal muscle wasting and weakness in lower limbs and only mild sensory signs. Electrophysiology demonstrated an axonal sensorimotor neuropathy. Nerve biopsy showed a chronic axonal neuropathy with moderate loss of all caliber myelinated fibers. Next-generation sequencing (NGS) technology revealed in the proband and in her similarly affected father the novel c.377A>G (p.K126R) heterozygous variant predicted to be deleterious. The mutation affects the biochemical properties of RAB7 GTPase, causes altered interaction with peripherin, and inhibition of neurite outgrowth, as for previously reported CMT2B mutants. However, it also shows differences, particularly in the epidermal growth factor receptor degradation process. Altogether, our findings indicate that this RAB7A variant is pathogenic and widens the phenotypic spectrum of CMT2B to include predominantly motor CMT2. Alteration of the receptor degradation process might explain the different clinical presentations in this family.

Systemic inflammation and mitochondrial dysfunction are involved in neurodegeneration in Parkinson’s disease (PD). Extracellular vesicle (EV) trafficking may link inflammation and mitochondrial dysfunction. In the present study, circulating small EVs (sEVs) from 16 older adults with PD and 12 non-PD controls were purified and characterized. A panel of serum inflammatory biomolecules was measured by multiplex immunoassay. Protein levels of three tetraspanins (CD9, CD63, and CD81) and selected mitochondrial markers (adenosine triphosphate 5A (ATP5A), mitochondrial cytochrome C oxidase subunit I (MTCOI), nicotinamide adenine dinucleotide reduced form (NADH):ubiquinone oxidoreductase subunit B8 (NDUFB8), NADH:ubiquinone oxidoreductase subunit S3 (NDUFS3), succinate dehydrogenase complex iron sulfur subunit B (SDHB), and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2)) were quantified in purified sEVs by immunoblotting. Relative to controls, PD participants showed a greater amount of circulating sEVs. Levels of CD9 and CD63 were lower in the sEV fraction of PD participants, whereas those of CD81 were similar between groups. Lower levels of ATP5A, NDUFS3, and SDHB were detected in sEVs from PD participants. No signal was retrieved for UQCRC2, MTCOI, or NDUFB8 in either participant group. To identify a molecular signature in circulating sEVs in relationship to systemic inflammation, a low level-fused (multi-platform) partial least squares discriminant analysis was applied. The model correctly classified 94.2% ± 6.1% PD participants and 66.7% ± 5.4% controls, and identified seven biomolecules as relevant (CD9, NDUFS3, C-reactive protein, fibroblast growth factor 21, interleukin 9, macrophage inflammatory protein 1β, and tumor necrosis factor alpha). In conclusion, a mitochondrial signature was identified in circulating sEVs from older adults with PD, in association with a specific inflammatory profile. In-depth characterization of sEV trafficking may allow identifying new biomarkers for PD and possible targets for personalized interventions.

Epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells to assume mesenchymal features, endowing them with enhanced motility and invasiveness, thus enabling cancer dissemination and metastatic spread. The induction of EMT is orchestrated by EMT-inducing transcription factors that switch on the expression of “mesenchymal” genes and switch off the expression of “epithelial” genes. Mitochondrial dysfunction is a hallmark of cancer and has been associated with progression to a metastatic and drug-resistant phenotype. The mechanistic link between metastasis and mitochondrial dysfunction is gradually emerging. The discovery that mitochondrial dysfunction owing to deregulated mitophagy, depletion of the mitochondrial genome (mitochondrial DNA) or mutations in Krebs’ cycle enzymes, such as succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase, activate the EMT gene signature has provided evidence that mitochondrial dysfunction and EMT are interconnected. In this review, we provide an overview of the current knowledge on the role of different types of mitochondrial dysfunction in inducing EMT in cancer cells. We place emphasis on recent advances in the identification of signaling components in the mito-nuclear communication network initiated by dysfunctional mitochondria that promote cellular remodeling and EMT activation in cancer cells.

Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

β-catenin plays an important role as regulatory hub in several cellular processes including cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved by its dual role as structural component of cadherin-based adherens junctions, and as a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins are differentially regulated via β-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after β-catenin knockdown in the breast cancer cell line MCF-7. We used a label free analysis to compare trypsin-digested proteins from CTR (shCTR) and β-catenin knockout cells (shβcat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of β-catenin induced morphological changes and a significant modulation of the expression levels of proteins associated with primary metabolic processes. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas proteins associated to lipid metabolism were found up-regulated in shβcat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in shβcat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in shβcat cells. β-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, β-catenin knockout cells showed increased incorporation of [1-14C]acetate and decreased utilization of [U-14C]glucose for fatty acid synthesis. Our data highlight a role of β-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

Rab-interacting lysosomal protein (RILP) is a regulator of late stages of endocytosis. Recent work proved that depletion of RILP promotes migration of breast cancer cells in wound healing assay, whereas its overexpression influences re-arrangements of actin cytoskeleton. Here, we further characterized the role of RILP in cell migration by analyzing several aspects of this process. We showed that RILP is fundamental also for migration of lung cancer cells regulating cell velocity. RILP silencing did not affect Golgi apparatus nor microtubules reorientation during migration. However, both RILP over-expression and expression of its mutated form, RILP-C33, impair cell adhesion and spreading. In conclusion, our results demonstrate that RILP has important regulatory roles in cell motility affecting migration velocity but also in cell adhesion and cell spreading.

RAB7 is a small guanosine triphosphatase (GTPase) extensively studied as regulator of vesicular trafficking. Indeed, its role is fundamental in several steps of the late endocytic pathway, including endosome maturation, transport from early endosomes to late endosomes and lysosomes, clustering and fusion of late endosomes and lysosomes in the perinuclear region and lysosomal biogenesis. Besides endocytosis, RAB7 is important for a number of other cellular processes among which, autophagy, apoptosis, signaling, and cell migration. Given the importance of RAB7 in these cellular processes, the interest to study the role of RAB7 in cancer progression is widely grown. Here, we describe the current understanding of oncogenic and oncosuppressor functions of RAB7 analyzing cellular context and other environmental factors in which it elicits pro and/or antitumorigenic effects. We also discuss the role of RAB7 in cisplatin resistance associated with its ability to regulate the late endosomal pathway, lysosomal biogenesis and extracellular vesicle secretion. Finally, we examined the potential cancer therapeutic strategies targeting the different molecular events in which RAB7 is involved.

Crosstalk between lysosomes and mitochondria plays a central role in Parkinson’s Disease (PD). Lysosomal function may be influenced by mitochondrial quality control, dynamics and/or respiration, but whether dysfunction of endocytic or autophagic pathway is associated with mitochondrial impairment determining accumulation of defective mitochondria, is not yet understood. Here, we performed live imaging, western blotting analysis, sequencing of mitochondrial DNA (mtDNA) and senescence-associated beta-galactosidase activity assay on primary fibroblasts from a young patient affected by PD, her mother and a healthy control to analyze the occurrence of mtDNA mutations, lysosomal abundance, acidification and function, mitochondrial biogenesis activation and senescence. We showed synergistic alterations in lysosomal functions and mitochondrial biogenesis, likely associated with a mitochondrial genetic defect, with a consequent block of mitochondrial turnover and occurrence of premature cellular senescence in PARK2-PD fibroblasts, suggesting that these alterations represent potential mechanisms contributing to the loss of dopaminergic neurons.

The progressive loss of dopaminergic neurons in the nigro-striatal system is a major trait of Parkinson’s disease (PD), manifesting clinically as motor and non-motor symptoms. Mitochondrial dysfunction and oxidative stress are alleged pathogenic mechanisms underlying aggregation of misfolded α-synuclein that in turn triggers dopaminergic neurotoxicity. Peripheral processes, including inflammation, may precede and contribute to neurodegeneration. Whether mitochondrial dyshomeostasis in the central nervous system and systemic inflammation are linked to one another in PD is presently unclear. Extracellular vesicles (EVs) are delivery systems through which cells can communicate or unload noxious materials. EV trafficking also participates in mitochondrial quality control (MQC) by generating mitochondrial-derived vesicles to dispose damaged organelles. Disruption of MQC coupled with abnormal EV secretion may play a role in the pathogenesis of PD. Furthermore, due to its bacterial ancestry, circulating mitochondrial DNA can elicit an inflammatory response. Therefore, purification and characterisation of molecules packaged in, and secreted through, small EVs (sEVs)/exosomes in body fluids may provide meaningful insights into the association between mitochondrial dysfunction and systemic inflammation in PD. The EXosomes in PArkiNson Disease (EXPAND) study was designed to characterise the cargo of sEVs/exosomes isolated from the serum of PD patients and to identify candidate biomarkers for PD.

The small GTPases of the Rho family regulate many aspects of actin dynamics, but are functionally connected to many other cellular processes. Rac1, a member of this family, besides its known function in the regulation of actin cytoskeleton, plays a key role in the production of reactive oxygen species, in gene transcription, in DNA repair, and also has been proven to have specific roles in neurons. This review focuses on the cooperation between Rac1 and Rab proteins, analyzing how the coordination between these GTPases impact on cells and how alterations of their functions lead to disease.

The progressive decline of cell function and integrity, manifesting clinically as increased vulnerability to adverse outcomes and death, is core to biological aging. Mitochondrial dysfunction, oxidative stress, altered intercellular communication (including chronic low-grade inflammation), genomic instability, telomere attrition, loss of proteostasis, altered nutrient sensing, epigenetic alterations, and stem cell exhaustion have been proposed as hallmarks of aging. These “aging pillars„ are not mutually exclusive, making the matter intricate and leaving numerous unanswered questions. The characterization of circulating extracellular vesicles (EVs) has recently allowed specific secretory phenotypes associated with aging to be identified. As such, EVs may serve as novel biomarkers for capturing the complexity of aging. Besides the mitochondrial⁻lysosomal axis, EV trafficking has been proposed as an additional layer in mitochondrial quality control. Indeed, disruption of the mitochondrial⁻lysosomal axis coupled with abnormal EV secretion may play a role in the pathogenesis of aging and several disease conditions. Here, we discuss (1) the mechanisms of EV generation; (2) the relationship between the mitochondrial⁻lysosomal axis and EV trafficking in the setting of mitochondrial quality control; and (3) the prospect of using EVs as aging biomarkers and as delivery systems for therapeutics against age-related conditions.

Background: Cisplatin (CDDP) is widely used in treatment of cancer, yet patients often develop resistance with consequent therapeutical failure. In CDDP-resistant cells alterations of endocytosis and lysosomal functionality have been revealed, although their causes and contribution to therapy response are unclear. Methods: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Modulation of RAB7A expression was performed by transfection and RNA interference, while CDDP sensitivity and intracellular accumulation were evaluated by viability assays and chemical approaches, respectively. Also extracellular vesicles were purified and analyzed. Finally, correlations between RAB7A and chemotherapy response was investigated in human patient samples. Results: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases CDDP-resistance while RAB7A overexpression decreases it. In addition, increased production of extracellular vesicles is modulated by RAB7A expression levels and correlates with reduction of CDDP intracellular accumulation. Conclusions: We demonstrated, for the first time, that RAB7A regulates CDDP resistance determining alterations in late endocytic trafficking and drug efflux through extracellular vesicles.

Rab7 is a small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes.

Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway.

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.