1,720,979 research outputs found
Role of Chitin in Alzheimer’s disease: a new cytotoxic pathway
Get PDFLa patogenesi della malattia di Alzheimer (AD) è generalmente attribuita ad un’anomala produzione e accumulo di β-amiloide, in associazione con formazioni neurofibrillari (NTF). Questo accumulo di β-amiloide in cervelli AD culmina in tossicità neuronale sia in maniera diretta che attraverso l’attivazione della microglia che, producendo mediatori infiammatori, contribuisce al danno neuronale.
Negli ultimi anni la comunità scientifica ha sollevato dubbi circa l’esclusivo ruolo patologico dell’ amiloide. L’AD familiare, dove si suppone sia la deposizione di sostanza amiloide a svolgere un ruolo patogenetico prevalente, rappresenta una condizione in grado di confermare questa ipotesi, ma la maggior parte dei casi di AD sono sporadici e lo scenario è complicato dal ruolo di diversi componenti aggiuntivi. Infatti, una vasta gamma di molecole sono presenti nelle placche AD il cui significato non è stato ancora precisamente chiarito. Tra queste, studi precedenti hanno identificato la chitina, un polimero insolubile di N-acetil-glucosamina, presente in stretta associazione con la β-amiloide nei cervelli autoptici di AD sporadici. La presenza di chitina è stata rilevata tramite colorazione con Calcofluor sia nelle placche di amiloide che nel citoplasma della microglia circostante.
Lo scopo di questo studio è stato quello di verificare il ruolo patogenetico della chitina nell’ AD valutando i suoi effetti biologici su neuroni e microglia.
Prima di tutto, abbiamo trovato depositi di chitina solo nei casi di AD sporadico, ma non nei casi familiari né con sindrome di Down, sottolineando la complessità della patologia sporadica. Successivamente sono stati eseguiti esperimenti in vitro, in cui culture microgliali sottoposte all'esposizione di chitina hanno mostrato come le cellule erano in grado di fagocitare piccole particelle di chitina ed il processo risultava significativamente inibito da β-amiloide. Analogamente a quanto descritto per la proteina β-amiloide, la fagocitosi della chitina attiva le cellule microgliali.
Inoltre, esperimenti con colture neuronali hanno mostrato un significativo effetto citotossico indotto dalla chitina paragonabile a quello ottenuto a seguito del trattamento con β-amiloide.
Un punto centrale di questa ricerca ha riguardato la produzione di chitina da parte delle cellule di mammifero, che mancano di chitina sintasi. Nell’AD sporadico il metabolismo del glucosio è spesso compromesso con attivazione della via dell’esosamina e conseguente produzione di N-acetil-glucosamina. Studi precedenti hanno suggerito che, in tale condizione, l'assenza di un enzima in grado di sintetizzare chitina possa essere superata da ialuronicosintasi-1 (HA-1), che è stato dimostrato poter convertire in vitro UDP-N-acetil-glucosamina in chito-oligosaccaridi.
Abbiamo dimostrato che in presenza di UDP-N-acetil-glucosamina, sia microglia che neuroni sono in grado di produrre depositi chitino-simili che l’utilizzo di HPLC-MS ha confermato essere “composti chitino-simili appena formati”. Tale trattamento porta all’ attivazione della microglia così come ad una significativa citotossicità neuronale, mimando l'effetto della chitina esogena.
I nostri risultati indicano che, in particolari condizioni di alterato metabolismo del glucosio, sia microglia che neuroni producono polimeri di chitina, in grado di scatenare un effetto neurotossico sia diretto che attraverso l'attivazione della microglia. Inoltre, esperimenti preliminari, suggeriscono che anche la trasmissione sinaptica risulta influenzata in culture di fette murine ippocampali trattate con UDP-N-acetil-glucosamina. Questi risultati suggeriscono un ruolo citotossico delle molecole chitino-simili in AD offrendo nuove prospettive nella comprensione della complessa patogenesi dell’AD.The pathogenesis of Alzheimer’s disease (AD) is generally attributed to the abnormal production and accumulation of β-amyloid protein, in association with neurofibrillary tangle (NTF) formation. The production and subsequently accumulation of β-amyloid protein in AD brains finally results in direct neuronal toxicity and in microglial activation which, through the production of inflammatory mediators, contributes to neuronal damage.
In recent years the scientific community has raised doubts regarding the exclusive pathological role of amyloid. Familiar AD, where amyloid deposition is supposed to play a prevalent pathogenetic role, represents a condition confirming this hypothesis, but the vast majority of AD cases are sporadic and in this condition the scenario is complicated by the possible role of additional components/pathways involved. In fact, a wide range of molecules are present in AD plaques, whose significance has not been clearly characterized. Among these, previous studies have identified chitin, an insoluble polymer of N-acetyl-glucosamine, in close association with β-amyloid in autoptic sporadic AD brains. Chitin was detected by Calcofluor staining both in amyloid plaques and within the cytoplasm of surrounding microglia.
The aim of this study was to investigate whether chitin has a pathogenetic role in AD by assessing its biological effects on two important players: neurons and microglia.
First of all, we have found chitin deposits only in sporadic AD but not in familiar AD and Down syndrome, emphasizing the complexity of amyloid-related pathology. Then we performed in vitro experiments, in which the exposure of microglial cultures to chitin showed that the cells were able to phagocyte small chitin particles, and the process was significantly inhibited by β-amyloid. Similarly to what described with β-amyloid, phagocytosis of chitin activated microglial cells.
In addition, experiments with neuronal cultures clearly showed a significant cytotoxic effect induced by chitin on neurons to levels comparable to β-amyloid.
A central point of this research concerned the production of chitin by mammalian cells, which lack chitin synthase. In sporadic AD glucose metabolism is frequently impaired with activation of the exosamine pathway with consequent production of N-acetyl-glucosamine. Previous studies suggested that, under such condition, the absence of a chitin synthesizing enzyme may be overcome by hyaluronan synthase-1 (HAS-1), that has been shown to convert UDP-N-acetyl-glucosamine to chito-oligosaccharides in vitro.
We demonstrated that in the presence of UDP-N-acetyl-glucosamine, both microglia and neurons are able to produce chitin-like deposits that HPLC-MS analysis confirmed to be “new-formed” chitin-like compounds. Such treatment leads to activation of microglia as well as significant neuronal cytotoxicity, mimicking the effect of exogenous chitin.
Our results indicate that in particular conditions of altered glucose metabolism both microglia and neurons produce chitin-like polymers, which may trigger a neurotoxic effect either by direct neuronal toxicity and by microglia activation. Moreover, preliminary experiments suggest that synaptic transmission is affected in murine hippocampal slice cultures treated with UDP-N-acetyl-glucosamine.
Taken together, these results suggest a cytotoxic role of chitin-like molecules in AD and offer new insights in the understanding the complex pathogenesis of AD
Extracellular Vesicles from Mesenchymal Stem Cells: Towards Novel Therapeutic Strategies for Neurodegenerative Diseases
Get PDFNeurodegenerative diseases are fatal disorders of the central nervous system (CNS) which currently lack effective treatments. The application of mesenchymal stem cells (MSCs) represents a new promising approach for treating these incurable disorders. Growing evidence suggest that the therapeutic effects of MSCs are due to the secretion of neurotrophic molecules through extracellular vesicles. The extracellular vesicles produced by MSCs (MSC-EVs) have valuable innate properties deriving from parental cells and could be exploited as cell-free treatments for many neurological diseases. In particular, thanks to their small size, they are able to overcome biological barriers and reach lesion sites inside the CNS. They have a considerable pharmacokinetic and safety profile, avoiding the critical issues related to the fate of cells following transplantation. This review discusses the therapeutic potential of MSC-EVs in the treatment of neurodegenerative diseases, focusing on the strategies to further enhance their beneficial effects such as tracking methods, bioengineering applications, with particular attention to intranasal delivery as a feasible strategy to deliver MSC-EVs directly to the CNS in an effective and minimally invasive way. Current progresses and limiting issues to the extent of the use of MSC-EVs treatment for human neurodegenerative diseases will be also revised
Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches
No full textBACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASC) are known to promote neuroprotection and neuroregeneration in vitro and in vivo. These biological effects are probably mediated by paracrine mechanisms. In recent years, nanovesicles (NV) and microvesicles (MV) have been shown to play a major role in cell-to-cell communication. We tested the efficacy of NV and MV obtained from ASC in mediating neuroprotection and neuroregeneration in vitro.METHODS: We exposed neuronal cells (both cell line and primary cultures) to oxidative stress in the presence or not of NV or MV.RESULTS: In this experimental setting, we found that low doses of NV or MV protected neurons from apoptotic cell death. We then assessed the neuroregenerative effect of NV/MV in cerebellar slice cultures demyelinated with lysophosphatidylcholine. We observed that low but not higher doses of NV and MV increased the process of remyelination and activated nestin-positive oligodendroglial precursors.CONCLUSIONS: Taken together, our data in vitro support the relevance of ASC vesicles as a source of protecting and regenerating factors that might modulate the microenvironment in neuro-inflammatory as well as in neurodegenerative disorders. The present findings may suggest that stromal cell-derived vesicles might represent a potential therapeutic tool, enabling the safe administration of stromal cell effector factors, avoiding the cellular counterpart
Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy
Get PDFBackground: Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. Methods: In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 μg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10. Results: The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. Conclusions: Our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells
Adipose mesenchymal stem cells-derived extracellular vesicles exert their preferential action in damaged central sites of SOD1 mice rather than peripherally
Get PDFAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models
A cross-talk between the androgen receptor and the epidermal growth factor re-ceptor leads to p38MAPK-dependent activation of mTOR and cyclinD1 expression in prostate and lung cancer cells.
No full textIn androgen sensitive LNCaP prostate cancer cells, the proliferation induced by the epidermal growth factor (EGF) involves a cross-talk between the EGF receptor (EGFR) and the androgen receptor (AR). In lung cancer the role of the EGF–EGFR transduction pathway has been documented, whereas androgen activity has received less attention. Here we demonstrate that in LNCaP and A549 non-small cell lung cancer (NSCLC), AR and EGFR are required for either 5α-dihydrotestosterone (DHT) or EGF-stimulated cell growth. Only EGF activated ERK signaling and up-regulated early gene expression, while DHT triggered the expression of classical AR-responsive genes with the exception of the EGF-induced PSA transcript in A549 cells. DHT and EGF up-regulated cyclinD1 (CD1) at both mRNA and protein levels in A549 cells, while in LNCaP cells each mitogen increased only CD1 protein expression. In both cell contexts, CD1 up-regulation was prevented by selective inhibitors as well as by knock-down of either AR or EGFR and also inhibiting p38MAPK and the mammalian target of rapamycin (mTOR) pathways. Interestingly, p38MAPK and mTOR repression prevented the activation of the mTOR target ribosomal p70S6 kinase induced by DHT and EGF, indicating that p38MAPK acts as an upstream mTOR regulator. In addition, the proliferative effects promoted by both DHT and EGF in LNCaP and A549 cancer cells were no longer observed blocking either p38MAPK or mTOR activity. Hence, our data suggest that p38MAPK-dependent activation of the mTOR/CD1 pathway may represent a mechanism through which AR and EGFR cross-talk contributes to prostate and lung cancer progression
Beneficial and sexually dimorphic response to combined HDAC inhibitor valproate and AMPK/SIRT1 pathway activator resveratrol in the treatment of ALS mice
Get PDFAmyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder. There is no cure and current treatments fail to slow the progression of the disease. Epigenetic modulation in the acetylation state of NF-kB RelA and the histone 3 (H3) protein, involved in the development of neurodegeneration, is a drugable target for the class-I histone deacetylases (HDAC) inhibitors, entinostat or valproate, and the AMP-activated kinase (AMPK)-sirtuin 1 pathway activator, resveratrol. In this study, we demonstrated that the combination of valproate and resveratrol can restore the normal acetylation state of RelA in the SOD1(G93A) murine model of ALS, in order to obtain the neuroprotective form of NF-kB. We also investigated the sexually dimorphic development of the disease, as well as the sex-sensibility to the treatment administered. We showed that the combined drugs, which rescued AMPK activation, RelA and the histone 3 acetylation state, reduced the motor deficit and the disease pathology associated with motor neuron loss and microglial reactivity, Brain-Derived Neurotrophic Factor (BDNF) and B-cell lymphoma-extra large (Bcl-xL) level decline. Specifically, vehicle-administered males showed earlier onset and slower progression of the disease when compared to females. The treatment, administered at 50 days of life, postponed the time of onset in the male by 22 days, but not in a significant way in females. Nevertheless, in females, the drugs significantly reduced symptom severity of the later phase of the disease and prolonged the mice's survival. Only minor beneficial effects were produced in the latter stage in males. Overall, this study shows a beneficial and sexually dimorphic response to valproate and resveratrol treatment in ALS mice
Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis
Get PDFBackground aims: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. Methods: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. Results: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. Conclusions: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS
Nanovesicles from adipose-derived mesenchymal stem cells inhibit T lymphocyte trafficking and ameliorate chronic experimental autoimmune encephalomyelitis
Get PDFCell based-therapies represent promising strategies for the treatment of neurological diseases. We have previously shown that adipose stem cells (ASC) ameliorate chronic experimental autoimmune encephalomyelitis (EAE). Recent evidence indicates that most ASC paracrine effects are mediated by extracellular vesicles, i.e. micro- and nanovesicles (MVs and NVs). We show that preventive intravenous administration of NVs isolated from ASC (ASC-NVs) before disease onset significantly reduces the severity of EAE and decreases spinal cord inflammation and demyelination, whereas therapeutic treatment with ASC-NVs does not ameliorate established EAE. This treatment marginally inhibits antigen-specific T cell activation, while reducing microglial activation and demyelination in the spinal cord. Importantly, ASC-NVs inhibited integrin-dependent adhesion of encephalitogenic T cells in vitro, with no effect on adhesion molecule expression. In addition, intravital microscopy showed that encephalitogenic T cells treated with ASC NVs display a significantly reduced rolling and firm adhesion in inflamed spinal cord vessels compared to untreated cells. Our results show that ASC-NVs ameliorate EAE pathogenesis mainly by inhibiting T cell extravasation in the inflamed CNS, suggesting that NVs may represent a novel therapeutic approach in neuro-inflammatory diseases, enabling the safe administration of ASC effector factors
ASC-exosomes ameliorate the disease progression in SOD1(G93A) murine model underlining their potential therapeutic use in human ALS
Get PDFAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motoneurons. To date, there is no effective treatment available. Exosomes are extracellular vesicles that play important roles in intercellular communication, recapitulating the effect of origin cells. In this study, we tested the potential neuroprotective effect of exosomes isolated from adipose-derived stem cells (ASC-exosomes) on the in vivo model most widely used to study ALS, the human SOD1 gene with a G93A mutation (SOD1(G93A)) mouse. Moreover, we compared the effect of two different routes of exosomes administration, intravenous and intranasal. The effect of exosomes administration on disease progression was monitored by motor tests and analysis of lumbar motoneurons and glial cells, neuromuscular junction, and muscle. Our results demonstrated that repeated administration of ASC-exosomes improved the motor performance; protected lumbar motoneurons, the neuromuscular junction, and muscle; and decreased the glial cells activation in treated SOD1(G93A) mice. Moreover, exosomes have the ability to home to lesioned ALS regions of the animal brain. These data contribute by providing additional knowledge for the promising use of ASC-exosomes as a therapy in human ALS
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