In vivo liposomes, like other types of nanoparticles, acquire a totally new 'biological identity' due to the formation of a biomolecular coating known as the protein corona that depends on and ...modifies the liposomes' synthetic identity. The liposome-protein corona is a dynamic interface that regulates the interaction of liposomes with the physiological environment. Here we show that the biological identity of liposomes is clearly linked to their sequestration from peripheral blood mononuclear cells (PBMCs) of healthy donors that ultimately leads to removal from the bloodstream. Pre-coating liposomes with an artificial corona made of human plasma proteins drastically reduces capture by circulating leukocytes in whole blood and may be an effective strategy to enable prolonged circulation in vivo. We conclude with a critical assessment of the key concepts of liposome technology that need to be reviewed for its definitive clinical translation.
Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion ...and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.
The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we ...report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS
mutation associated with ALS.
Alzheimer's disease (AD) is the most common cause of dementia in the elderly. In the pathogenesis of AD a pivotal role is played by two neurotoxic proteins that aggregate and accumulate in the ...central nervous system: amyloid beta and hyper-phosphorylated tau. Accumulation of extracellular amyloid beta plaques and intracellular hyper-phosphorylated tau tangles, and consequent neuronal loss begins 10-15 years before any cognitive impairment. In addition to cognitive and behavioral deficits, sensorial abnormalities have been described in AD patients and in some AD transgenic mouse models. Retina can be considered a simple model of the brain, as some pathological changes and therapeutic strategies from the brain may be observed or applicable to the retina. Here we propose new retinal biomarkers that could anticipate the AD diagnosis and help the beginning and the follow-up of possible future treatments. We analyzed retinal tissue of triple-transgenic AD mouse model (3xTg-AD) for the presence of pathological hallmarks during disease progression. We found the presence of amyloid beta plaques, tau tangles, neurodegeneration, and astrogliosis in the retinal ganglion cell layer of 3xTg-AD mice, already at pre-symptomatic stage. Moreover, retinal microglia in pre-symptomatic mice showed a ramified, anti-inflammatory phenotype which, during disease progression, switches to a pro-inflammatory, less ramified one, becoming neurotoxic. We hypothesize retina as a window through which monitor AD-related neurodegeneration process.
Abstract
Increasing evidence suggests that in Amyotrophic Lateral Sclerosis (ALS) mutated RNA binding proteins acquire aberrant functions, leading to altered RNA metabolism with significant impact on ...encoded protein levels. Here, by taking advantage of a human induced pluripotent stem cell-based model, we aimed to gain insights on the impact of ALS mutant FUS on the motoneuron proteome. Label-free proteomics analysis by mass-spectrometry revealed upregulation of proteins involved in catabolic processes and oxidation–reduction, and downregulation of cytoskeletal proteins and factors directing neuron projection. Mechanistically, proteome alteration does not correlate with transcriptome changes. Rather, we observed a strong correlation with selective binding of mutant FUS to target mRNAs in their 3′UTR. Novel validated targets, selectively bound by mutant FUS, include genes previously involved in familial or sporadic ALS, such as
VCP
, and regulators of membrane trafficking and cytoskeleton remodeling, such as
ASAP1
. These findings unveil a novel mechanism by which mutant FUS might intersect other pathogenic pathways in ALS patients’ motoneurons.
Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell ...apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network–derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.
•LAMP1 silencing inhibits cytotoxicity of human NK cells.•LAMP1 is important for perforin trafficking to the lytic granules and granule movement.
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•Ultrasound irradiation of microbubbles may alter membrane permeability.•High-fidelity microvessels-on-chip allows to perform reliable in vitro studies on drug delivery.•Combining in ...vitro platforms and cavitation is crucial for improving targeted drug delivery.
Traditional drug delivery systems, where pharmaceutical agents are conveyed to the target tissue through the blood circulation, suffer of poor therapeutic efficiency and limited selectivity largely due to the low permeability of the highly specialised biological interface represented by the endothelial layer. Examples concern cancer therapeutics or degenerative disorders where drug delivery is inhibited by the blood-brain barrier (BBB). Microbubbles injected into the bloodstream undergo volume oscillations under localised ultrasound irradiation and possibly collapse near the site of interest, with no effect on the rest of the endothelium. The resulting mechanical action induces a transient increase of the inter-cellular spaces and facilitates drug extravasation. This approach, already pursed in in vivo animal models, is extremely expensive and time-consuming. On the other hand in vitro studies using different kinds of microfluidic networks are firmly established in the pharmaceutical industry for drug delivery testing. The combination of the in vitro approach with ultrasound used to control microbubbles oscillations is expected to provide crucial information for developing cavitation enhanced drug delivery protocols and for screening the properties of the biological interface in presence of healthy or diseased tissues. Purpose of the present review is providing the state of the art in this rapidly growing field where cavitation is exploited as a viable technology to transiently modify the permeability of the biological interface. After describing current in vivo studies, particular emphasis will be placed on illustrating characteristics of micro-devices, biological functionalisation, properties of the artificial endothelium and ultrasound irradiation techniques.
Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cell (MuSC) activity. This ability requires the proper balance between MuSC expansion and ...differentiation, which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated long noncoding RNA (lncRNA), Lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, Lnc-Rewind acts as an epigenetic regulator of MuSC proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct Lnc-Rewind target. We show that Lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs.
In the CNS, the chemokine CX3CL1 (fractalkine) is expressed on neurons while its specific receptor CX3CR1 is expressed on microglia and macrophages. Microglia play an important role in health and ...disease through CX3CL1/CX3CR1 signaling, and in many neurodegenerative disorders, microglia dysregulation has been associated with neuro-inflammation. We have previously shown that CX3CL1 has neuroprotective effects against cerebral ischemia injury. Here, we investigated the involvement of CX3CL1 in the modulation of microglia phenotype and the underlying neuroprotective effect on ischemia injury. The expression profiles of anti- and pro-inflammatory genes showed that CX3CL1 markedly inhibited microglial activation both in vitro and in vivo after permanent middle cerebral artery occlusion (pMCAO), accompanied by an increase in the expression of anti-inflammatory genes. Moreover, CX3CL1 induces a metabolic switch in microglial cells with an increase in the expression of genes related to the oxidative pathway and a reduction in those related to glycolytic pathway, which is the metabolic state associated to the pro-inflammatory phenotype for energy production. The data reported in this paper suggest that CX3CL1 protects against cerebral ischemia modulating the activation state of microglia and its metabolism in order to restrain inflammation and organize a neuroprotective response against the ischemic insult.
Amyotrophic lateral sclerosis (ALS) has been genetically linked to mutations in RNA-binding proteins (RBPs), including FUS. Here, we report the RNA interactome of wild-type and mutant FUS in human ...motor neurons (MNs). This analysis identified a number of RNA targets. Whereas the wild-type protein preferentially binds introns, the ALS mutation causes a shift toward 3′ UTRs. Neural ELAV-like RBPs are among mutant FUS targets. As a result, ELAVL4 protein levels are increased in mutant MNs. ELAVL4 and mutant FUS interact and co-localize in cytoplasmic speckles with altered biomechanical properties. Upon oxidative stress, ELAVL4 and mutant FUS are engaged in stress granules. In the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. We propose that pathological mutations in FUS trigger an aberrant crosstalk with ELAVL4 with implications for ALS.
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•We report the RNA interactome of wild-type and mutant FUS in human motor neurons•Mutant FUS binds the mRNA 3′ UTR of other RNA-binding proteins, including ELAVL4•ELAVL4, expressed at increased levels, interacts with mutant FUS in the cytoplasm•ELAVL4 proteinopathy occurs in both FUS ALS and in sporadic ALS patients
De Santis et al. show that the mutant RNA-binding protein FUS, linked to amyotrophic lateral sclerosis (ALS), targets other RNA-binding proteins, such as ELAVL4, in human motor neurons. This triggers aberrant crosstalk between mutant FUS and ELAVL4, which is found in pathological inclusions of ALS patients’ motor neurons.
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