Effects of matrix metalloproteinase 9 (MMP-9) on insulin survival pathways in primary neuronal cultures from wild type and transgenic mice models of Alzheimer's disease

A hallmark of Alzheimer’s disease (AD) is the accumulation of oligomeric amyloid-β (Αβ) peptide, which may be primarily responsible for neuronal dysfunction. Among others, insulin survival pathway is impaired due to the presence of Αβ-oligomers. On the contrary, unperturbed insulin signaling provides a defense mechanism against neuronal loss. Previous studies have shown that matrix metalloproteinase-9 (MMP-9), an enzyme of the extracellular matrix which is implicated in synaptic plasticity, may have a neuroprotective role against AD, by decreasing the formation of Αβ oligomers. In the present study, the role of MMP-9 on the insulin survival pathway was examined in two different experimental systems; in primary hippocampal cultures and hippocampal tissue extracts from 3 month-old wild type mice. Four different mouse genotypes were used; mice model for AD (5XFAD), MMP-9-overexpressing (TgMMP-9) mice, double transgenic mice (5XFAD/TgMMP-9) and wild type mice (WT). The data demonstrate that the insulin pathway was compromised in samples from 5XFAD mice, in both experimental systems, when compared to the wild type and MMP-9 overexpressing mice. This was due to enhanced phosphorylation of IRS1 at Serine 636, which renders IRS1 inactive and prevents insulin-mediated signaling. On the contrary, in 5XFAD/TgMMP-9 samples, the insulin survival pathway was rescued through enhanced activation by phosphorylation of IRS1 at Tyrosine 465, followed by increased levels of BDNF and TrkB activation. Increased phosphorylation of Akt and GSK-3β, and decreased phosphorylation of JNK kinase was observed. Finally, decreased apoptosis, as well as decreased oligomeric Αβ levels, were also observed in 5XFAD/TgMMP-9 mice in vitro and in vivo respectively, compared to 5XFAD mice. In conclusion, our findings indicate that overexpression of MMP-9 is able to rescue insulin survival signaling in vitro and in early stages in the 5XFAD model of AD. Thus, insulin-mediated survival enhancement, via targeted MMP-9 expression, could possibly be used as a novel promising therapeutic strategy.Ένα από τα κύρια χαρακτηριστικά της νόσου Alzheimer (ΝΑ) αποτελεί η συσσώρευση του ολιγομερούς Αβ πεπτιδίου (Αβ), το οποίο πιθανώς αποτελεί την κύρια αιτία για τη νευρική δυσλειτουργία που παρατηρείται στη νόσο. Ένα από τα κυτταρικά μονοπάτια που πλήττεται από τη δράση των Αβ ολιγομερών αφορά το μονοπάτι κυτταρικής επιβίωσης της ινσουλίνης. Αντιθέτως, η ορθή κυτταρική σηματοδότηση της ινσουλίνης αποτελεί έναν από τους αμυντικούς μηχανισμούς έναντι της συναπτικής απώλειας. Έχει αποδειχθεί από προηγούμενες μελέτες ότι η μεταλλοπρωτεϊνάση-9 (MMP-9), ένα ένζυμο της εξωκυττάριας ουσίας, στενά συνδεδεμένο με τη συναπτική πλαστικότητα, πιθανόν να έχει νευροπροστατευτικές ιδιότητες στη ΝΑ, καθώς έχει την ιδιότητα να μειώνει τη δημιουργία των Αβ ολιγομερών. Στην παρούσα διατριβή μελετήθηκε ο ρόλος της MMP-9 στο ινσουλινο-εξαρτώμενο μονοπάτι επιβίωσης σε δύο διαφορετικά συστήματα: σε πρωτογενείς νευρώνες ιππόκαμπου και σε ιστούς ιππόκαμπου νεαρών μυών 3 μηνών. Μελετήθηκαν 4 διαφορετικοί γονότυποι: μυς μοντέλα της NA (5XFAD), μυς που υπερεκφράζουν την MMP-9 (TgMMP-9), διπλά διαγονιδιακοί μυς (5XFAD/TgMMP-9) και μυς άγριου τύπου (WT). Από τα αποτελέσματα προκύπτει ότι το ινσουλινο-εξαρτώμενο μονοπάτι επιβίωσης διαταράσσεται στα δείγματα από τους 5XFAD μυς και στα 2 πειραματικά συστήματα σε σύγκριση με τους μυς που υπερεκφράζουν την MMP-9 ή τους μυς άγριου τύπου. Συγκεκριμένα, ανιχνεύθηκε αυξημένη φωσφορυλίωση του IRS1 στη σερίνη 636, η οποία προκαλεί την απενεργοποίηση του και οδηγεί στην αναστολή της σηματοδότησης της ινσουλίνης. Αντιθέτως, το γεγονός αυτό απετράπη στους 5XFAD/TgMMP-9 μυς, λόγω της αυξημένης φωσφορυλίωσης του IRS1 στη τυροσίνη 465, η οποία συνοδεύτηκε από αυξημένα επίπεδα του παράγοντα BDNF και αυξημένη ενεργοποίηση του υποδοχέα TrkB. Παράλληλα, ανιχνεύθηκε αυξημένη φωσφορυλίωση των Akt και GSK-3β κινασών, καθώς και μειωμένη φωσφορυλίωση της JNK κινάσης. Τέλος, στους ίδιους μυς παρατηρήθηκε μικρότερο ποσοστό κυτταρικής απόπτωσης στις πρωτογενείς κυτταροκαλλιέργειες, καθώς και μειωμένα επίπεδα Αβ ολιγομερών στους ενήλικες μυς σε σχέση με τους 5XFAD μυς. Συνολικά, τα ευρήματα της παρούσας μελέτης συνηγορούν στο ότι η υπερέκφραση της MMP-9 επαναφέρει την κυτταρική σηματοδότηση της ινσουλίνης τόσο in vitro, όσο και σε πρώιμα στάδια της νόσου στο 5XFAD μοντέλο της ΝΑ. Συνεπώς, η ενίσχυση της ινσουλινο-εξαρτώμενης οδού επιβίωσης, μέσω της ελεγχόμενης έκφρασης της MMP-9, θα μπορούσε πιθανώς να αποτελέσει μια καινοτόμο και ελπιδοφόρο θεραπευτική στρατηγική

Carbon Dots–Biomembrane Interactions and Their Implications for Cellular Drug Delivery

The effect of carbon dots (CDs) on a model blayer membrane was studied as a means of comprehending their ability to affect cell membranes. Initially, the interaction of N-doped carbon dots with a biophysical liposomal cell membrane model was investigated by dynamic light scattering, z-potential, temperature-modulated differential scanning calorimetry, and membrane permeability. CDs with a slightly positive charge interacted with the surface of the negative-charged liposomes and evidence indicated that the association of CDs with the membrane affects the structural and thermodynamic properties of the bilayer; most importantly, it enhances the bilayer’s permeability against doxorubicin, a well-known anticancer drug. The results, like those of similar studies that surveyed the interaction of proteins with lipid membranes, suggest that carbon dots are partially embedded in the bilayer. In vitro experiments employing breast cancer cell lines and human healthy dermal cells corroborated the findings, as it was shown that the presence of CDs in the culture medium selectively enhanced cell internalization of doxorubicin and, subsequently, increased its cytotoxicity, acting as a drug sensitizer

Engineering Mitochondriotropic Carbon Dots for Targeting Cancer Cells

Aiming to understand and enhance the capacity of carbon dots (CDs) to transport through cell membranes and target subcellular organelles—in particular, mitochondria—a series of nitrogen-doped CDs were prepared by the one-step microwave-assisted pyrolysis of citric acid and ethylenediamine. Following optimization of the reaction conditions for maximum fluorescence, functionalization at various degrees with alkylated triphenylphosphonium functional groups of two different alkyl chain lengths afforded a series of functionalized CDs that exhibited either lysosome or mitochondria subcellular localization. Further functionalization with rhodamine B enabled enhanced fluorescence imaging capabilities in the visible spectrum and allowed the use of low quantities of CDs in relevant experiments. It was thus possible, by the appropriate selection of the alkyl chain length and degree of functionalization, to attain successful mitochondrial targeting, while preserving non-toxicity and biocompatibility. In vitro cell experiments performed on normal as well as cancer cell lines proved their non-cytotoxic character and imaging potential, even at very low concentrations, by fluorescence microscopy. Precise targeting of mitochondria is feasible with carefully designed CDs that, furthermore, are specifically internalized in cells and cell mitochondria of high transmembrane potential and thus exhibit selective uptake in malignant cells compared to normal cells

Synthesis of Hydroxyapatite, β-Tricalcium Phosphate and Biphasic Calcium Phosphate Particles to Act as Local Delivery Carriers of Curcumin: Loading, Release and In Vitro Studies

The successful synthesis of hydroxyapatite (HA), β-Tricalcium phosphate (β-TCP) and two biphasic mixtures (BCPs) of the two was performed by means of wet precipitation. The resulting crystals were characterized and the BCP composition was analyzed and identified as 13% HA—87% TCP and 41% HA—59% TCP. All samples were treated with curcumin solutions, and the degree of curcumin loading and release was found to be proportional to the TCP content of the ceramic. No further cytotoxicity was observed upon MG-63 treatment with the curcumin-loaded ceramics. Finally, the alkaline phosphatase activity of the cells was found to increase with increasing content of TCP, which provides an encouraging proof of concept for the use of curcumin-loaded synthetic biomaterials in bone remodeling