Nose-to-brain delivery of enveloped RNA - cell permeating peptide nanocomplexes for the treatment of neurodegenerative diseases

This document is the preprint manuscript version of a published work that appeared in final form in Biomaterials, © 2019 Elsevier Ltd. after peer review and technical editing by the publisher. To access the final edited and published work see: https://doi.org/10.1016/j.biomaterials.2019.119657Direct nose-to-brain (N-to-B) delivery enables the rapid transport of drugs to the brain, while minimizing systemic exposure. The objective of this work was to engineer a nanocarrier intended to enhance N-to-B delivery of RNA and to explore its potential utility for the treatment of neurological disorders. Our approach involved the formation of electrostatically driven nanocomplexes between a hydrophobic derivative of octaarginine (r8), chemically conjugated with lauric acid (C12), and the RNA of interest. Subsequently, these cationic nanocomplexes were enveloped (enveloped nanocomplexes, ENCPs) with different protective polymers, i.e. polyethyleneglycol - polyglutamic acid (PEG-PGA) or hyaluronic acid (HA), intended to enhance their stability and mucodiffusion across the olfactory nasal mucosa. These rationally designed ENCPs were produced in bulk format and also using a microfluidics-based technique. This technique enabled the production of a scalable nanoformulation, exhibiting; (i) a unimodal size distribution with a tunable mean size, (ii) the capacity to highly associate (100%) and protect RNA from degradation, (iii) the ability to preserve its physicochemical properties in biorelevant media and prevent the premature RNA release. Moreover, in vitro cell culture studies showed the capacity of ENCPs to interact and be efficiently taken-up by CHO cells. Finally, in vivo experiments in a mouse model of Alzheimer's disease provided evidence of a statistically significant increase of a potentially therapeutic miRNA mimic in the hippocampus area and its further effect on two mRNA targets, following its intranasal administration. Overall, these findings stress the value of the rational design of nanocarriers towards overcoming the biological barriers associated to N-to-B RNA delivery and reveal their potential value as therapeutic strategies in Alzheimer's diseaseThe work was supported by the European B-Smart Consortium, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 721058S

Evaluation of the Possible Transmission of BSE and Scrapie to Gilthead Sea Bream (Sparus aurata)

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). While the precise mechanism of the PrPC to PrPSc conversion is not understood, it is clear that host PrPC expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrPSc, or of a prion disease developing in farmed fish is alarming and requires further evaluation

MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer’s disease

Neuronal cell loss is a defining feature of Alzheimer’s disease (AD), but the underlying mechanisms remain unclear. We xenografted human or mouse neurons into the brain of a mouse model of AD. Only human neurons displayed tangles, Gallyas silver staining, granulovacuolar neurodegeneration (GVD), phosphorylated tau blood biomarkers, and considerable neuronal cell loss. The long noncoding RNA MEG3 was strongly up-regulated in human neurons. This neuron-specific long noncoding RNA is also up-regulated in AD patients. MEG3 expression alone was sufficient to induce necroptosis in human neurons in vitro. Down-regulation of MEG3 and inhibition of necroptosis using pharmacological or genetic manipulation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, or mixed lineage kinase domain-like protein (MLKL) rescued neuronal cell loss in xenografted human neurons. This model suggests potential therapeutic approaches for AD and reveals a human-specific vulnerability to AD

NACC2, a molecular effector of miR-132 regulation at the interface between adult neurogenesis and Alzheimer’s disease

The generation of new neurons at the hippocampal neurogenic niche, known as adult hippocampal neurogenesis (AHN), and its impairment, have been implicated in Alzheimer’s disease (AD). MicroRNA-132 (miR-132), the most consistently downregulated microRNA (miRNA) in AD, was recently identified as a potent regulator of AHN, exerting multilayered proneurogenic effects in adult neural stem cells (NSCs) and their progeny. Supplementing miR-132 in AD mouse brain restores AHN and relevant memory deficits, yet the exact mechanisms involved are still unknown. Here, we identify NACC2 as a novel miR-132 target implicated in both AHN and AD. miR-132 deficiency in mouse hippocampus induces Nacc2 expression and inflammatory signaling in adult NSCs. We show that miR-132-dependent regulation of NACC2 is involved in the initial stages of human NSC differentiation towards astrocytes and neurons. Later, NACC2 function in astrocytic maturation becomes uncoupled from miR-132. We demonstrate that NACC2 is present in reactive astrocytes surrounding amyloid plaques in mouse and human AD hippocampus, and that there is an anticorrelation between miR-132 and NACC2 levels in AD and upon induction of inflammation. Unraveling the molecular mechanisms by which miR-132 regulates neurogenesis and cellular reactivity in AD, will provide valuable insights towards its possible application as a therapeutic target

Assessing Proteinase K Resistance of Fish Prion Proteins in a Scrapie-Infected Mouse Neuroblastoma Cell Line

The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrPC, into an aberrant and partially proteinase K resistant isoform, PrPSc. Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK), as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs

Study of the possible transmission of spongiform encephalopathies to novel species and development of new experimental models on diagnosis and therapy of these diseases

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). While the precise mechanism of the PrPC to PrPSc conversion is not understood, it is clear that host PrPC expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. The current study aimed not only at evaluating the possible transmission of prion diseases to fish species, but also at gaining further understanding on the nature and the role of PrP proteins in fish. Thus, some general conclusions could be drawn concerning the function of prion proteins in health and disease. Molecular analysis techniques and the application of anti-fish PrP specific antibodies, suggested that these proteins become C-terminally glycosylated by the addition of N-acetylglusamine groups, whereas they display a similar to the mammalian prion proteins tissue distribution, exhibiting the highest expression levels in the CNS. These similarities, in combination with the conserved prion protein motifs of primary and secondary structure, render these proteins useful models for studying their orthologues’ function in mammals, whereas at the same time they raise concerns about a possible transmission of prion diseases to fish. In order to investigate such a possibility, we employed both in vitro and in vivo approaches. Our in vitro system included the introduction of the proteins SaurPrP-1 (Sparus aurata), ZebPrP-1 and ZebPrP-2 (Danio rerio) into mouse neuroblastoma cells (N2a), infected with the 22L mouse-adapted scrapie strain. Despite the fact that the fish PrPs followed common biosynthetic pathways with their mammalian relatives, they didn’t display resistance to proteinase K treatment. This finding suggests that this specific scrapie strain is not capable of converting the fish PrP proteins into a potentially pathological isoform. For the in vivo study, we orally challenged sea bass and sea bream with BSE and scrapie infectious brain material. Fish tissue samples were tested at regular time points post inoculation by a variety of techniques. Despite the fact that none of the TSE-inoculated groups developed clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in those challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrPSc is alarming and requires further evaluation.Στις Μεταδιδόμενες Σπογγώδεις Εγκεφαλοπάθειες, οι οποίες αποτελούν μία ομάδα ανίατων νευροεκφυλιστικών νόσων που προσβάλλουν πολλά είδη, καθοριστικό ρόλο κατά την παθογένεση διαδραματίζει η συσσώρευση της μη φυσιολογικής στερεοχημικής ισομορφής (PrPSc), μίας φυσιολογικής ενδογενούς πρωτεΐνης (PrPC). Παρά το γεγονός ότι ο ακριβής μηχανισμός της μετατροπής της PrPC προς PrPSc δεν είναι γνωστός, θεωρείται δεδομένο ότι η έκφραση της PrPC στον ξενιστή είναι απαραίτητη προϋπόθεση για την εκδήλωση μόλυνσης prion. Μολονότι έχουν γίνει πάρα πολλές μελέτες όσον αφορά στις Σπογγώδεις Εγκεφαλοπάθειες στα θηλαστικά, η υπάρχουσα γνώση σχετικά με την παθογένεση prion στους ιχθύες είναι περιορισμένη. Στο πλαίσιο της παρούσας διατριβής επιδιώχθηκε η μελέτη της πιθανής μετάδοσης των νόσων prion σε είδη ιχθυών, καθώς και η καλύτερη κατανόηση της φύσης και του ρόλου των PrP πρωτεϊνών στους ιχθύες, προκειμένου να εξαχθούν συμπεράσματα σχετικά με τη γενικότερη δράση των prion πρωτεϊνών σε φυσιολογικές και παθολογικές καταστάσεις. Με την εφαρμογή μοριακών τεχνικών ανάλυσης και τη χρήση αντισωμάτων εκλεκτικών για τις PrP πρωτεΐνες ιχθύων, διαπιστώθηκε ότι οι πρωτεΐνες αυτές, φέρουν όπως και στα θηλαστικά ομάδες Ν-ακετυλογλυκοζαμίνης στο καρβοξυτελικό άκρο, ενώ παράλληλα παρουσιάζουν όμοια ιστική κατανομή, εμφανίζοντας υψηλότερα επίπεδα στο ΚΝΣ. Οι άνωθεν ομοιότητες σε συνδυασμό με τα διατηρημένα μοτίβα πρωτοταγούς και δευτεροταγούς διαμόρφωσης, χαρακτηριστικά των πρωτεϊνών prion, καταρχήν καθιστούν τις πρωτεΐνες αυτές χρήσιμα μοντέλα για τη μελέτη της δράσης των ορθόλογων μορίων τους στα θηλαστικά, και κατά δεύτερον διεγείρουν ανησυχίες σχετικά με μία πιθανή μετάδοση των νόσων prion στους ιχθύες. Προκειμένου να διερευνηθεί μία τέτοια πιθανότητα, ακολουθήθηκαν τόσο in vitro, όσο και in vivo προσεγγίσεις. Το in vitro σύστημα μελέτης περιέλαμβανε την εισαγωγή των πρωτεϊνών SaurPrP-1 (Sparus aurata), ZebPrP-1 και ZebPrP-2 (Danio rerio) σε κύτταρα νευροβλαστώματος μυός (Ν2a), μολυσμένα με το στέλεχος scrapie μυός 22L. Παρά το γεγονός ότι οι εξωγενείς PrP πρωτεΐνες των ιχθύων φαίνεται πως ακολούθησαν κοινά βιοσυνθετικά μονοπάτια με τις αντίστοιχες των θηλαστικών, δεν εμφάνισαν ανθεκτικότητα στην κατεργασία με πρωτεϊνάση Κ. Η παρατήρηση αυτή οδηγεί στο συμπέρασμα ότι το συγκεκριμένο στέλεχος scrapie δεν είναι σε θέση να μετατρέψει τις PrP πρωτεΐνες των ιχθύων που χρησιμοποιήθηκαν στη μελέτη προς κάποια δυνητικά παθολογική ισομορφή. Κατά την in vivo μελέτη, χορηγήθηκε από του στόματος μολυσματικό εγκεφαλικό υλικό BSE και scrapie σε λαβράκια και τσιπούρες, δείγματα των οποίων αναλύθηκαν ανά τακτά χρονικά διαστήματα με ένα πλήθος τεχνικών. Παρά το γεγονός ότι καμία από τις ομάδες των ιχθύων δεν ανέπτυξε κλινική νόσο, οι εγκέφαλοι από τσιπούρες που έλαβαν TSE ομογενοποίημα, δύο χρόνια μετά το πέρας του ενοφθαλμισμού παρουσίασαν ίχνη νευροεκφύλισης και συσσώρευσης εναποθέσεων. Ειδικά οι εναποθέσεις στον εγκέφαλο ιχθύων που έλαβαν BSE εγκεφαλικό ιστό, παρουσίασαν μερική ανθεκτικότητα στην κατεργασία με ΡΚ, κονγκοφιλία και διπλοθλαστικότητα στο πολωμένο φως, στοιχείο που συνάδει με την ύπαρξη αμυλοειδούς δομής. Η παρουσία νευροεκφύλισης και μη φυσιολογικής εναπόθεσης στους εγκεφάλους των ιχθύων που έλαβαν TSE μολυσματικό υλικό, εγείρει ορισμένους προβληματισμούς για τη δημόσια υγεία. Καθώς η ιχθυοκαλλιέργεια αποτελεί μία οικονομικά σημαντική βιομηχανία, παρέχοντας προϊόντα υψηλής διατροφικής αξίας για τον άνθρωπο και άλλα θηλαστικά, το ενδεχόμενο πιθανής μόλυνσης των καλλιεργούμενων ιχθύων με μολυσματική PrPSc ζωικής προέλευσης αποτελούν ιδιαίτερα ανησυχητικά ζητήματα, τα οποία χρήζουν περαιτέρω μελέτης

Non-coding RNAs with essential roles in neurodegenerative disorders

The importance of various classes of regulatory non-protein-coding RNA molecules (ncRNAs) in the normal functioning of the CNS is becoming increasingly evident. ncRNAs are involved in neuronal cell specification and patterning during development, but also in higher cognitive processes, such as structural plasticity and memory formation in the adult brain. We discuss advances in understanding of the function of ncRNAs in the CNS, with a focus on the potential involvement of specific species, such as microRNAs, endogenous small interfering RNAs, long intergenic non-coding RNAs, and natural antisense transcripts, in various neurodegenerative disorders. This emerging field is anticipated to profoundly affect clinical research, diagnosis, and therapy in neurology.status: publishe

microRNA-132: a key noncoding RNA operating in the cellular phase of Alzheimer's disease

With the consideration of the broad involvement of microRNAs (miRNAs) in the regulation of molecular networks in the brain, it is not surprising that miRNA dysregulation causes neurodegeneration in animal models. miRNA profiling in the human brain has revealed miR-132 as one of the most severely down-regulated miRNAs at the intermediate and late Braak stages of Alzheimer's disease (AD), as well as in other neurodegenerative disorders. Suppression of miR-132 aggravates multiple layers of pathology at the molecular and functional level. We describe the potential therapeutic implications of these findings and suggest miRNA targeting or replacement as a realistic multi-hit, therapeutic strategy for AD. Salta, E., De Strooper, B. microRNA-132: a key noncoding RNA operating in the cellular phase of Alzheimer's disease.status: publishe

Noncoding RNAs in neurodegeneration

The emerging complexity of the transcriptional landscape poses great challenges to our conventional preconceptions of how the genome regulates brain function and dysfunction. Non-protein-coding RNAs (ncRNAs) confer a high level of intricate and dynamic regulation of various molecular processes in the CNS and they have been implicated in neurodevelopment and brain ageing, as well as in synapse function and cognitive performance, in both health and disease. ncRNA-mediated processes may be involved in various aspects of the pathogenesis of neurodegenerative disorders. Understanding these events may help to develop novel diagnostic and therapeutic tools. Here, we provide an overview of the complex mechanisms that are affected by the diverse ncRNA classes that have been implicated in neurodegeneration.status: publishe

miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer's brain

microRNA-132 (miR-132) is involved in prosurvival, anti-inflammatory and memory-promoting functions in the nervous system and has been found consistently downregulated in Alzheimer's disease (AD). Whether and how miR-132 deficiency impacts AD pathology remains, however, unaddressed. We show here that miR-132 loss exacerbates both amyloid and TAU pathology via inositol 1,4,5-trisphosphate 3-kinase B (ITPKB) upregulation in an AD mouse model. This leads to increased ERK1/2 and BACE1 activity and elevated TAU phosphorylation. We confirm downregulation of miR-132 and upregulation of ITPKB in three distinct human AD patient cohorts, indicating the pathological relevance of this pathway in AD.status: publishe