Impact of normal ageing and cerebral hypoperfusion on myelinated axons and its relation to the development of Alzheimer’s disease

Cerebral hypoperfusion can occur in normal ageing and is proposed to underlie white matter disturbances observed in the ageing brain. Moreover, cerebral hypoperfusion and white matter attenuation are early events in the progression of Alzheimer’s disease (AD). White matter mostly consists of myelinated axons which have distinct protein architecture, segregated into defined regions; the axon initial segment (AIS), the node of Ranvier, paranode, juxtaparanode, and internode. These sites are essential for action potential initiation and/or propagation and subsequently effective brain function. At the outset of the studies in the thesis there was evidence that the different regions within the myelinated axons are vulnerable to injury and disease. Thus it is hypothesised that in response to normal ageing and/or cerebral hypoperfusion these structures are altered and associated with cognitive impairment and that these effects are exacerbated in a transgenic mouse model (APPSw,Ind, J9 line) which develops age-dependent amyloid-β (Αβ) pathology. The first study aims to investigate the effect of normal ageing and Aβ deposition on myelinated axons and on learning and memory. To address this, the effects of normal ageing on the integrity of the AIS, nodes of Ranvier, myelin, axons, synapses and spatial working memory are examined in young and aged wild-type and TgAPPSw,Ind mice. A significant reduction in the length of nodes of Ranvier is demonstrated in aged wild-type and TgAPPSw,Ind mice. In addition, the length of AIS, is significantly reduced in the aged wild-type animals while the young TgAPPSw,Ind have significantly shorter AIS than the young wild-type mice. These effects are not influenced by the presence of Aβ. Myelin integrity is affected by age but this is more prominent in the wild-type animals whilst axonal integrity is intact. Moreover, there is an age-related decrease of presynaptic boutons only in the TgAPPSw,Ind mice. Contrary to the original hypothesis, working memory performance is not altered with age or influenced by increasing Aβ levels. The second study aims to examine the effects of cerebral hypoperfusion in combination with Αβ pathology and/or ageing on cognitive performance and the structure of myelinated axons. To address this, the effects of surgically induced cerebral hypoperfusion on the integrity of the nodes of Ranvier, paranodes, myelin, axons and spatial working memory performance are investigated in young and aged wild-type and TgAPPSw,Ind mice. A decrease in nodal length is observed in response to hypoperfusion in young and aged animals. This effect is shown to be exacerbated in the young TgAPPSw,Ind animals. Moreover, the disruption of the nodal domain is shown to occur without any gross alterations in myelin and axonal integrity. It is also demonstrated that in response to hypoperfusion, spatial working memory performance is defected in young and aged animals of both genotypes. This deficit is exacerbated in the young TgAPPSw,Ind. The observed changes in the nodal structure are associated with poor working memory performance indicating functional implication for the nodal changes. These data highlight that structures within myelinated axons are vulnerable to ageing and cerebral hypoperfusion. Therefore, the development of strategies that minimize injury or drive repair to these regions is necessary together with therapeutic approaches against the vascular insults that induce hypoperfusion and lead to white matter attenuation and cognitive decline. In the future, it would be interesting to investigate how alterations at the AIS/nodes of Ranvier affect neuronal excitability

Neurosteroids as regulators of neuroinflammation

Neuroinflammation is a physiological protective response in the context of infection and injury. However, neuroinflammation, especially if chronic, may also drive neurodegeneration. Neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD) and traumatic brain injury (TBI), display inflammatory activation of microglia and astrocytes. Intriguingly, the central nervous system (CNS) is a highly steroidogenic environment synthesizing steroids de novo, as well as metabolizing steroids deriving from the circulation. Neurosteroid synthesis can be substantially affected by neuroinflammation, while, in turn, several steroids, such as 17β-estradiol, dehydroepiandrosterone (DHEA) and allopregnanolone, can regulate neuroinflammatory responses. Here, we review the role of neurosteroids in neuroinflammation in the context of MS, AD, PD and TBI and describe underlying molecular mechanisms. Moreover, we introduce the concept that synthetic neurosteroid analogues could be potentially utilized for the treatment of neurodegenerative diseases in the future

Επίδραση της φυσιολογικής γήρανσης και της εγκεφαλικής υποαιμάτωσης στους εμμύελους άξονες για η σχέση τους με την ανάπτυξη της νόσου Αλτσχάιμερ

Cerebral hypoperfusion can occur in normal ageing and is proposed to underlie white matter disturbances observed in the ageing brain. Moreover, cerebral hypoperfusion and white matter attenuation are early events in the progression of Alzheimer’s disease (AD). White matter mostly consists of myelinated axons which have distinct protein architecture, segregated into defined regions; the axon initial segment (AIS), the node of Ranvier, paranode, juxtaparanode, and internode. These sites are essential for action potential initiation and/or propagation and subsequently effective brain function. At the outset of the studies in the thesis there was evidence that the different regions within the myelinated axons are vulnerable to injury and disease. Thus it is hypothesised that in response to normal ageing and cerebral hypoperfusion these structures are altered and associated with cognitive impairment and that these effects are exacerbated in a transgenic mouse model (APPSw,Ind, J9 line) which develops age-dependent amyloid-β (Αβ) pathology. The first study aims to investigate the effect of normal ageing and Aβ deposition on myelinated axons and on learning and memory. To address this, the effects of normal ageing on the integrity of the AIS, nodes of Ranvier, myelin, axons, synapses and spatial working memory are examined in young and aged wild-type and TgAPPSw,Ind mice. A significant reduction in the length of nodes of Ranvier is demonstrated in aged wild-type and TgAPPSw,Ind mice. In addition, the length of AIS, is significantly reduced in the aged wild-type animals while the young TgAPPSw,Ind have significantly shorter AIS than the young wild-type mice. These effects are not influenced by the presence of Aβ. Myelin integrity is affected by age but this is more prominent in the wild-type animals whilst axonal integrity is intact. Moreover, there is an age-related decrease of presynaptic boutons only in the TgAPPSw,Ind mice. Contrary to the original hypothesis, working memory performance is not altered with age or influenced by increasing Aβ levels. The second study aims to examine the effects of cerebral hypoperfusion in combination with Αβ pathology and/or ageing on cognitive performance and the structure of myelinated axons. To address this, the effects of surgically induced cerebral hypoperfusion on the integrity of the nodes of Ranvier, paranodes, myelin, axons and spatial working memory performance are investigated in young and aged wild-type and TgAPPSw,Ind mice. A decrease in nodal length is observed in response to hypoperfusion in young and aged animals. This effect is shown to be exacerbated in the young TgAPPSw,Ind animals. Moreover, the disruption of the nodal domain is shown to occur without any gross alterations in myelin and axonal integrity. It is also demonstrated that in response to hypoperfusion, spatial working memory performance is defected in young and aged animals of both genotypes. This deficit is exacerbated in the young TgAPPSw,Ind. The observed changes in the nodal structure are associated with poor working memory performance indicating functional implication for the nodal changes. These data highlight that structures within myelinated axons are vulnerable to ageing and cerebral hypoperfusion. Therefore, the development of strategies that minimize injury or drive repair to these regions is necessary together with therapeutic approaches against the vascular insults that induce hypoperfusion and lead to white matter attenuation and cognitive decline. In the future, it would be interesting to investigate how alterations at the AIS and the nodes of Ranvier affect neuronal excitability.Η εγκεφαλική υποαιμάτωση εμφανίζεται στη φυσιολογική γήρανση και προτείνεται ότι προκαλεί διαταραχές στη λευκή ουσία οι οποίες παρατηρούνται στους γερασμένους εγκεφάλους. Επιπλέον, η εγκεφαλική υποαιμάτωση και οι αλλοιώσεις της λευκής ουσίας είναι πρώιμα συμβάντα στην εξέλιξη της νόσου του Αλτσχάιμερ (AD). Η λευκή ουσία αποτελείται κυρίως από μυελινωμένους άξονες που έχουν μία ιδιαίτερη πρωτεϊνική αρχιτεκτονική καθώς διαχωρίζονται σε συγκεκριμένες περιοχές οι οποίες είναι το αρχικό τμήμα του άξονα (AIS), ο κόμβος του Ranvier, ο paranode, ο juxtaparanode, και ο internode. Αυτές οι διαφορετικές περιοχές είναι απαραίτητες για την έναρξη και τη διάδοση του δυναμικού δράσης και κατά συνέπεια για την αποτελεσματική λειτουργία του εγκεφάλου. Ένα εύρος δεδομένων συνηγορούν ότι οι διαφορετικές περιοχές εντός των εμμύελων αξόνων είναι ευάλωτες σε περιπτώσεις τραυματισμού ή ασθένειας. Για αυτό υποθέτουμε ότι ως απόκριση στη φυσιολογική γήρανση και στην εγκεφαλική υποαιμάτωση αυτές οι περιοχές θα αλλοιώνονται οδηγώντας σε γνωστική εξασθένηση και ότι αυτές οι επιδράσεις θα επιδεινώνονται σε ένα διαγονιδιακό μοντέλο ποντικού (σειρά APPSw, Ind, J9) που αναπτύσσει παθολογία β-αμυλοειδoύς (Αβ) εξαρτώμενη από την ηλικία. Η πρώτη μελέτη στοχεύει στη διερεύνηση της επίδρασης της φυσιολογικής γήρανσης και της εναπόθεσης του Αβ στους μυελινωμένους άξονες καθώς και στη μνήμη και τη μάθηση. Για αυτό, οι επιδράσεις της φυσιολογικής γήρανσης στην ακεραιότητα του AIS, των κόμβων του Ranvier, της μυελίνης, των αξόνων, των συνάψεων και της χωρικής μνήμης εργασίας εξετάστηκαν σε νεαρα και ηλικιωμένα wild-type και TgAPPSw,Ind ποντίκια. Παρατηρήθηκε σημαντική μείωση του μήκους των κόμβων του Ranvier στα ηλικιωμένα wild-type και TgAPPSw,Ind ποντίκια. Επιπλέον, το μήκος του AIS μειώνεται σημαντικά στα ηλικιωμένα wild-type ζώα, ενώ τα νεαρά TgAPPSw,Ind έχουν σημαντικά μικρότερο AIS από τα νεαρά wild-type ποντίκια. Αυτές οι επιδράσεις δεν επηρεάζονται από την παρουσία του Αβ. Η ακεραιότητα της μυελίνης επηρεάζεται από την ηλικία, αλλά αυτό είναι πιο εμφανές στα wild-type ζώα, ενώ η ακεραιότητα των νευραξόνων είναι ανέπαφη. Επιπλέον, υπάρχει μια σχετιζόμενη με την ηλικία μείωση των προσυναπτικών buttons μόνο στα TgAPPSw,Ind ποντίκια. Σε αντίθεση με την αρχική υπόθεση, η μνήμη εργασία δεν μεταβάλλεται με την ηλικία ούτε επηρεάζεται από την αύξηση των επιπέδων Αβ. Η δεύτερη μελέτη στοχεύει να εξετάσει τις επιδράσεις της εγκεφαλικής υποαιμάτωσης σε συνδυασμό με την παθολογία του Αβ και τη γήρανση στις γνωστικές λειτουργίες και στη δομή των μυελινωμένων αξόνων. Για αυτό, διερευνήθηκαν τα αποτελέσματα της χειρουργικά επαγόμενης εγκεφαλικής υποαιμάτωσης στην ακεραιότητα των κόμβων του Ranvier, των paranodes, της μυελίνης, των αξόνων και στη χωρική μνήμη εργασίας σε νεαρά και ηλικιωμένα wild-type και TgAPPSw,Ind ποντίκια. Παρατηρείται μείωση του μήκους των κόμβων του Ranvier ως απόκριση στην υποαιμάτωση σε νεαρά και ηλικιωμένα ζώα. Αυτή η επίδραση φαίνεται να επιδεινώνεται στα νεαρά TgAPPSw,Ind ζώα. Επιπλέον, η διαταραχή της περιοχής του κόμβου φαίνεται να συμβαίνει χωρίς μεγάλες αλλαγές στη μυελίνη ή στην ακεραιότητα του νευράξονα. Αποδεικνύεται επίσης ότι ως απόκριση στην υποαιμάτωση, η χωρική μνήμη εργασίας είναι ελαττωματική τόσο στα νεαρά όσο και στα ηλικιωμένα ζώα και των δύο γονότυπων. Αυτό η βλάβη επιδεινώνεται στα νεαρά TgAPPSw,Ind ζώα. Οι παρατηρούμενες αλλαγές στη δομή του κόμβου του Ranvier συσχετίζονται με την μείωση της μνήμης εργασίας, υποδεικνύοντας τις λειτουργικές επιπτώσεις στις αλλαγές της δομής του κόμβου του Ranvier. Αυτά τα δεδομένα υπογραμμίζουν ότι οι δομές εντός των μυελινωμένων αξόνων είναι ευάλωτες στη γήρανση και στην εγκεφαλική υποαιμάτωση. Επομένως, η ανάπτυξη στρατηγικών που ελαχιστοποιούν τον τραυματισμό ή οδηγούν στην αποκατάσταση αυτών των περιοχών είναι απαραίτητη μαζί με θεραπευτικές προσεγγίσεις κατά των αγγειακών προσβολών που προκαλούν υποαιμάτωση και οδηγούν σε αλλοίωση της λευκής ουσίας και γνωστική έκπτωση. Στο μέλλον, θα ήταν ενδιαφέρον να διερευνηθεί πώς οι αλλαγές στο AIS και τους κόμβους του Ranvier επηρεάζουν τη νευρωνική διεγερσιμότητα

Microneurotrophin BNN27 Reduces Astrogliosis and Increases Density of Neurons and Implanted Neural Stem Cell-Derived Cells after Spinal Cord Injury

Microneurotrophins, small-molecule mimetics of endogenous neurotrophins, have demonstrated significant therapeutic effects on various animal models of neurological diseases. Nevertheless, their effects on central nervous system injuries remain unknown. Herein, we evaluate the effects of microneurotrophin BNN27, an NGF analog, in the mouse dorsal column crush spinal cord injury (SCI) model. BNN27 was delivered systemically either by itself or combined with neural stem cell (NSC)-seeded collagen-based scaffold grafts, demonstrated recently to improve locomotion performance in the same SCI model. Data validate the ability of NSC-seeded grafts to enhance locomotion recovery, neuronal cell integration with surrounding tissues, axonal elongation and angiogenesis. Our findings also show that systemic administration of BNN27 significantly reduced astrogliosis and increased neuron density in mice SCI lesion sites at 12 weeks post injury. Furthermore, when BNN27 administration was combined with NSC-seeded PCS grafts, BNN27 increased the density of survived implanted NSC-derived cells, possibly addressing a major challenge of NSC-based SCI treatments. In conclusion, this study provides evidence that small-molecule mimetics of endogenous neurotrophins can contribute to effective combinatorial treatments for SCI, by simultaneously regulating key events of SCI and supporting grafted cell therapies in the lesion site

Development and Comparative In Vitro and In Vivo Study of BNN27 Mucoadhesive Liposomes and Nanoemulsions for Nose-to-Brain Delivery

Intranasal administration offers an alternative and promising approach for direct nose-to-brain delivery. Herein, we developed two chitosan (CHT)-coated (and uncoated) nanoformulations of BNN27 (a synthetic C-17-spiro-dehydroepiandrosterone analogue), liposomes (LIPs), and nanoemulsions (NEs), and compared their properties and brain disposition (in vitro and in vivo). LIPs were formulated by thin film hydration and coated with CHT by dropwise addition. BNN27-loaded NEs (BNEs) were developed by spontaneous emulsification and optimized for stability and mucoadhesive properties. Mucoadhesive properties were evaluated by mucin adherence. Negatively charged CHT-coated LIPs (with 0.1% CHT/lipid) demonstrated the highest coating efficiency and mucoadhesion. BNEs containing 10% w/w Capmul-MCM and 0.3% w/w CHT demonstrated the optimal properties. Transport of LIP or NE-associated rhodamine-lipid across the blood–brain barrier (in vitro) was significantly higher for NEs compared to LIPs, and the CHT coating demonstrated a negative effect on transport. However, the CHT-coated BNEs demonstrated higher and faster in vivo brain disposition following intranasal administration compared to CHT-LIPs. For both BNEs and LIPs, CHT-coating resulted in the increased (in vivo) brain disposition of BNN27. Current results prove that CHT-coated NEs consisting of compatible nasal administration ingredients succeeded in to delivering more BNN27 to the brain (and faster) compared to the CHT-coated LIPs

Biocompatible polymeric electrospun matrices: Micro-nanotopography effect on cell behaviour

This work reports a preliminary biological study performed on nanofibrous biocompatible polylactidecopolycaprolactone (PLA-PCL) scaffolds intended for tissue regeneration. The aim was to evaluate how matrix surface topography affects cell adhesion and proliferation. Scaffolds prepared by electrospinning either equipped with plane or rotating mandrel collectors, were characterized for their surface topography and nanofiber size. Cell culture studies were carried out using mouse embryonic fibroblast cells lines (NIH-3T3), as model for skin, murine neuroblastoma neuro-2α cell line, as model for neuronal tissue, and mouse mesenchymal stem cells (MSCs), because of their differentiation ability. Imaging analysis by scanning electron microscope and laser scanning confocal microscopy together with cell viability (MTT, L 3-(4,5-dymethiltiazol-2-y)-2,5 diphenyltetrazolium bromide) test, were performed on cell cultures at fixed time laps. The results showed that electrospun nanofibers supported growth and proliferation of the tested cell lines, but electrospun matrices obtained with rotating mandrel showed significantly higher cell viability that follows the orientation of electrospun nanofibers

Development and Comparative In Vitro and In Vivo Study of BNN27 Mucoadhesive Liposomes and Nanoemulsions for Nose-to-Brain Delivery

Intranasal administration offers an alternative and promising approach for direct nose-to-brain delivery. Herein, we developed two chitosan (CHT)-coated (and uncoated) nanoformulations of BNN27 (a synthetic C-17-spiro-dehydroepiandrosterone analogue), liposomes (LIPs), and nanoemulsions (NEs), and compared their properties and brain disposition (in vitro and in vivo). LIPs were formulated by thin film hydration and coated with CHT by dropwise addition. BNN27-loaded NEs (BNEs) were developed by spontaneous emulsification and optimized for stability and mucoadhesive properties. Mucoadhesive properties were evaluated by mucin adherence. Negatively charged CHT-coated LIPs (with 0.1% CHT/lipid) demonstrated the highest coating efficiency and mucoadhesion. BNEs containing 10% w/w Capmul-MCM and 0.3% w/w CHT demonstrated the optimal properties. Transport of LIP or NE-associated rhodamine-lipid across the blood–brain barrier (in vitro) was significantly higher for NEs compared to LIPs, and the CHT coating demonstrated a negative effect on transport. However, the CHT-coated BNEs demonstrated higher and faster in vivo brain disposition following intranasal administration compared to CHT-LIPs. For both BNEs and LIPs, CHT-coating resulted in the increased (in vivo) brain disposition of BNN27. Current results prove that CHT-coated NEs consisting of compatible nasal administration ingredients succeeded in to delivering more BNN27 to the brain (and faster) compared to the CHT-coated LIPs

Biocompatible polymeric electrospun matrices: Micro–nanotopography effect on cell behavior

This work reports a preliminary biological study performed on nanofibrous biocompatible polylactidecopolycaprolactone (PLA-PCL) scaffolds intended for tissue regeneration. The aim was to evaluate how matrix surface topography affects cell adhesion and proliferation. Scaffolds prepared by electrospinning either equipped with plane or rotating mandrel collectors, were characterized for their surface topography and nanofiber size. Cell culture studies were carried out using mouse embryonic fibroblast cells lines (NIH-3T3), as model for skin, murine neuroblastoma neuro-2α cell line, as model for neuronal tissue, and mouse mesenchymal stem cells (MSCs), because of their differentiation ability. Imaging analysis by scanning electron microscope and laser scanning confocal microscopy together with cell viability (MTT, L 3-(4,5-dymethiltiazol-2-y)-2,5 diphenyltetrazolium bromide) test, were performed on cell cultures at fixed time laps. The results showed that electrospun nanofibers supported growth and proliferation of the tested cell lines, but electrospun matrices obtained with rotating mandrel showed significantly higher cell viability that follows the orientation of electrospun nanofibers

Neural stem cell delivery via porous collagen scaffolds promotes neuronal differentiation and locomotion recovery in spinal cord injury

Neural stem cell (NSC) grafts have demonstrated significant effects in animal models of spinal cord injury (SCI), yet their clinical translation remains challenging. Significant evidence suggests that the supporting matrix of NSC grafts has a crucial role in regulating NSC effects. Here we demonstrate that grafts based on porous collagen-based scaffolds (PCSs), similar to biomaterials utilized clinically in induced regeneration, can deliver and protect embryonic NSCs at SCI sites, leading to significant improvement in locomotion recovery in an experimental mouse SCI model, so that 12 weeks post-injury locomotion performance of implanted animals does not statistically differ from that of uninjured control animals. NSC-seeded PCS grafts can modulate key processes required to induce regeneration in SCI lesions including enhancing NSC neuronal differentiation and functional integration in vivo, enabling robust axonal elongation, and reducing astrogliosis. Our findings suggest that the efficacy and translational potential of emerging NSC-based SCI therapies could be enhanced by delivering NSC via scaffolds derived from well-characterized clinically proven PCS