Role of Chitin in Alzheimer\u2019s disease: a new cytotoxic pathway

La patogenesi della malattia di Alzheimer (AD) \ue8 generalmente attribuita ad un\u2019anomala produzione e accumulo di \u3b2-amiloide, in associazione con formazioni neurofibrillari (NTF). Questo accumulo di \u3b2-amiloide in cervelli AD culmina in tossicit\ue0 neuronale sia in maniera diretta che attraverso l\u2019attivazione della microglia che, producendo mediatori infiammatori, contribuisce al danno neuronale. Negli ultimi anni la comunit\ue0 scientifica ha sollevato dubbi circa l\u2019esclusivo ruolo patologico dell\u2019 amiloide. L\u2019AD 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 \ue8 complicato dal ruolo di diversi componenti aggiuntivi. Infatti, una vasta gamma di molecole sono presenti nelle placche AD il cui significato non \ue8 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 \u3b2-amiloide nei cervelli autoptici di AD sporadici. La presenza di chitina \ue8 stata rilevata tramite colorazione con Calcofluor sia nelle placche di amiloide che nel citoplasma della microglia circostante. Lo scopo di questo studio \ue8 stato quello di verificare il ruolo patogenetico della chitina nell\u2019 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 n\ue9 con sindrome di Down, sottolineando la complessit\ue0 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 \u3b2-amiloide. Analogamente a quanto descritto per la proteina \u3b2-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 \u3b2-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\u2019AD sporadico il metabolismo del glucosio \ue8 spesso compromesso con attivazione della via dell\u2019esosamina 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 \ue8 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\u2019utilizzo di HPLC-MS ha confermato essere \u201ccomposti chitino-simili appena formati\u201d. Tale trattamento porta all\u2019 attivazione della microglia cos\uec come ad una significativa citotossicit\ue0 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\u2019AD.The pathogenesis of Alzheimer\u2019s disease (AD) is generally attributed to the abnormal production and accumulation of \u3b2-amyloid protein, in association with neurofibrillary tangle (NTF) formation. The production and subsequently accumulation of \u3b2-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 \u3b2-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 \u3b2-amyloid. Similarly to what described with \u3b2-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 \u3b2-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 \u201cnew-formed\u201d 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

Neurodegenerative 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

Beneficial and sexually dimorphic response to combined HDAC inhibitor valproate and AMPK/SIRT1 pathway activator resveratrol in the treatment of ALS mice

Amyotrophic 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

ASC-exosomes ameliorate the disease progression in SOD1(G93A) murine model underlining their potential therapeutic use in human ALS

Amyotrophic 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

Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis

Background 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

Immune Response after COVID-19 mRNA Vaccination in Multiple Sclerosis Patients Treated with DMTs

The impact of disease-modifying therapies (DMTs) on the immune response to coronavirus disease-2019 (COVID-19) vaccines in persons with multiple sclerosis (pwMS) needs further elucidation. We investigated BNT162b2 mRNA COVID-19 vaccine effects concerning antibody seroconversion, inflammatory mediators' level and immunophenotype assessment in pwMS treated with cladribine (c-pwMS, n = 29), fingolimod (f-pwMS, n = 15) and ocrelizumab (o-pwMS, n = 54). Anti-spike immunoglobulin (Ig)-G detection was performed by an enzyme immunoassay; molecular mediators (GrB, IFN-gamma and TNF-alpha) were quantified using the ELLA platform, and immunophenotype was assessed by flow cytometry. ANCOVA, Student's t-test and Pearson correlation analyses were applied. Only one o-pwMS showed a mild COVID-19 infection despite most o-pwMS lacking seroconversion and showing lower anti-spike IgG titers than c-pwMS and f-pwMS. No significant difference in cytokine production and lymphocyte count was observed in c-pwMS and f-pwMS. In contrast, in o-pwMS, a significant increase in GrB levels was detected after vaccination. Considering non-seroconverted o-pwMS, a significant increase in GrB serum levels and CD4+ T lymphocyte count was found after vaccination, and a negative correlation was observed between anti-spike IgG production and CD4+ T cells count. Differences in inflammatory mediators' production after BNT162b2 vaccination in o-pwMS, specifically in those lacking anti-spike IgG, suggest a protective cellular immune response

Extracellular Vesicles from Mesenchymal Stem Cells: Towards Novel Therapeutic Strategies for Neurodegenerative Diseases

Neurodegenerative 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

BACKGROUND 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

Neurotoxicity and synaptic plasticity impairment of N-acetylglucosamine polymers: implications for Alzheimer’s disease

Here we assessed whether polymers of GlcNAc have any pathogenetic role in AD. First, by using specific dyes we found deposits of polymers of GlcNAc in sporadic, but not in familial AD. We found that neurons and microglia exposed to GlcNAc and UDP-GlcNAc are able to form GlcNAc polymers, which display a significant neurotoxicity in vitro. Moreover, the exposure of organotypic hippocampal cultures to the same compounds led to synaptic impairment with decreased levels of syntaxin and synaptophysin. In addition, acute hippocampal slices treated with GlcNAc/UDP-GlcNAc showed a clear reduction of long-term potentiation of excitatory synapses. Finally, we demonstrated that microglial cells are able to phagocytose chitin particles and, when exposed to GlcNAc/UDP-GlcNAc, show cellular activation and intracellular deposition of GlcNAc polymers which are eventually released in the extracellular space. Taken together, our results indicate that both microglia and neurons produce GlcNAc polymers, which trigger neurotoxicity both directly and through microglia activation. GlcNAc polymer-driven neurotoxicity offers novel pathogenic insights in sporadic AD and new therapeutic options

Human Adipose-Derived Mesenchymal Stem Cells Systemically Injected Promote Peripheral Nerve Regeneration in the Mouse Model of Sciatic Crush

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach in nerve tissue engineering. To date, the local implantation of MSC in injured nerves has been the only route of administration used. In case of multiple sites of injury, the systemic administration of cells capable of reaching damaged nerves would be advisable. In this regard, we found that an intravenous administration of adipose-derived MSC (ASC) 1 week after sciatic nerve crush injury, a murine model of acute axonal damage, significantly accelerated the functional recovery. Sciatic nerves from ASC-treated mice showed the presence of a restricted number of undifferentiated ASC together with a significant improvement in fiber sprouting and the reduction of inflammatory infiltrates for up to 3 weeks. Besides the immune modulatory effect, our results show that ASC may contribute to peripheral nerve regeneration because of their ability to produce in culture neuroprotective factors such as insulin-like growth factor I, brain-derived neurotrophic factor, or basic fibroblast growth factor. In addition to this production in vitro, we interestingly found that the concentration of glial-derived neurotrophic factor (GDNF) was significantly increased in the sciatic nerves in mice treated with ASC. Since no detectable levels of GDNF were observed in ASC cultures, we hypothesize that ASC induced the local production of GDNF by Schwann cells. In conclusion, we show that systemically injected ASC have a clear therapeutic potential in an acute model of axonal damage. Among the possible mechanisms promoting nerve regeneration, our results rule out a process of trans-differentiation and rather suggest the relevance of a bystander effect, including the production of in situ molecules, which, directly or indirectly through a cross-talk with local glial cells, may modulate the local environment with the down-regulation of inflammation and the promotion of axonal regeneration