Amelioration of BPSD-like phenotype and cognitive decline in SAMP8 mice model accompanied by molecular changes after treatment with I2-imidazoline receptor ligand MCR5

Behavioural and Psychological Symptoms of Dementia (BPSD), including fear-anxiety- and depressive-like behaviour, are present in Alzheimer's disease (AD), together with memory decline. I2-imidazoline receptors (I2-IRs) have been associated with neuropsychiatric and neurodegenerative disorders, further, I2-IR ligands have demonstrated a neuroprotective role in the central nervous system (CNS). In this study, we assessed the effect of the I2-IR ligand MCR5 on both cognitive and non-cognitive symptoms in the Senescence accelerated mice prone 8 (SAMP8) mouse model. Oral administration of I2-IR ligand MCR5 (5mg/kg/day for four weeks) in 10-month SAMP8 mice ameliorated both BPSD-like phenotype and cognitive decline by attenuating depressive-like behaviour, reducing fear-anxiety-like behaviour and improving cognitive performance using different tasks. Interaction of I2-IR ligand MCR5 with serotoninergic system did not account for behavioral or cognitive improvement, although changes in molecular pathways underlying depression and anxiety phenotype were observed. MCR5 increased levels of p-AKT, phosphorylated Glycogen synthase kinase 3 β (GSK3β) at Ser9 and phosphorylated mammalian target of rapamycin complex 1 (mTORC1) levels in SAMP8 treated mice compared to SAMP8 control. Moreover, MCR5 treatment altered NMDA2B phosphorylation, and decreased the protein levels of phosphorylated Cyclin-Dependent Kinase 5 (p-CDK5) and dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa phosphorylated at Thr75 (p-DARPP32), with a parallel increase in PKA and p-CREB levels. Consistent with these changes MCR5 attenuated neuroinflammation by decreasing expression of pro-inflammatory markers such as Tumor necrosis factor-alpha (Tnf-α), Interleukin 1β (Il-1β), Interleukin 6 (Il-6), and promoted synaptic plasticity by increasing levels of Postsynaptic density protein 95 (PSD95) as well as ameliorating Tropomyosin-related kinase B (TrkB) and Nerve growth factor receptor (NGFR) signalling. Collectively, these results increase the potential of highly selective I2-IR ligands as therapeutic agents in age-related BPSD and cognitive alterations

I2 imidazoline receptor modulation protects aged SAMP8 mice against cognitive decline by suppressing the calcineurin pathway

Brain aging and dementia are current problems that must be solved. The levels of imidazoline 2 receptors (I2-IRs) are increased in the brain in Alzheimer's disease (AD) and other neurodegenerative diseases. We tested the action of the specific and selective I2-IR ligand B06 in a mouse model of accelerated aging and AD, the senescence-accelerated mouse prone 8 (SAMP8) model. Oral administration of B06 for 4 weeks improved SAMP8 mouse behavior and cognition and reduced AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Likewise, B06 regulated glial excitatory amino acid transporter 2 and N-methyl-D aspartate 2A and 2B receptor subunit protein levels. Calcineurin (CaN) is a phosphatase that controls the phosphorylation levels of cAMP response element-binding (CREB), apoptotic mediator BCL-2-associated agonist of cell death (BAD) and GSK3β, among other molecules. Interestingly, B06 was able to reduce the levels of the CaN active form (CaN A). Likewise, CREB phosphorylation, BAD gene expression, and other factors were modified after B06 treatment. Moreover, phosphorylation of a target of CaN, nuclear factor of activated Tcells, cytoplasmic 1 (NFATC1), was increased in B06- treated mice, impeding the transcription of genes related to neuroinflammation and neural plasticity. In summary, this I2 imidazoline ligand can exert its beneficial effects on age-related conditions by modulating CaN pathway action and affecting several molecular pathways, playing a neuroprotective role in SAMP

Cognitive Decline and BPSD Are Concomitant with Autophagic and Synaptic Deficits Associated with G9a Alterations in Aged SAMP8 Mice

Behavioural and psychological symptoms of dementia (BPSD) are presented in 95% of Alzheimer's Disease (AD) patients and are also associated with neurotrophin deficits. The molecular mechanisms leading to age-related diseases are still unclear; however, emerging evidence has suggested that epigenetic modulation is a key pathophysiological basis of ageing and neurodegeneration. In particular, it has been suggested that G9a methyltransferase and its repressive histone mark (H3K9me2) are important in shaping learning and memory by modulating autophagic activity and synaptic plasticity. This work deepens our understanding of the epigenetic mechanisms underlying the loss of cognitive function and BPSD in AD. For this purpose, several tasks were performed to evaluate the parameters of sociability (three-chamber test), aggressiveness (resident intruder), anxiety (elevated plus maze and open field) and memory (novel object recognition test) in mice, followed by the evaluation of epigenetic, autophagy and synaptic plasticity markers at the molecular level. The behavioural alterations presented by senescence-accelerated mice prone 8 (SAMP8) of 12 months of age compared with their senescence-accelerated mouse resistant mice (SAMR1), the healthy control strain was accompanied by age-related cognitive deficits and alterations in epigenetic markers. Increased levels of G9a are concomitant to the dysregulation of the JNK pathway in aged SAMP8, driving a failure in autophagosome formation. Furthermore, lower expression of the genes involved in the memory-consolidation process modulated by ERK was observed in the aged male SAMP8 model, suggesting the implication of G9a. In any case, two of the most important neurotrophins, namely brain-derived neurotrophic factor (Bdnf) and neurotrophin-3 (NT3), were found to be reduced, along with a decrease in the levels of dendritic branching and spine density presented by SAMP8 mice. Thus, the present study characterizes and provides information regarding the non-cognitive and cognitive states, as well as molecular alterations, in aged SAMP8, demonstrating the AD-like symptoms presented by this model. In any case, our results indicate that higher levels of G9a are associated with autophagic deficits and alterations in synaptic plasticity, which could further explain the BPSD and cognitive decline exhibited by the model

A bicyclic α-iminophosphonate improves cognitive decline in 5xFAD murine model of neurodegeneration

I2 receptors (I2-IR) are widely distributed in the central nervous system. I2-IR ligands are associated with a neuroprotective effect but, as I2-IR structure remains unknown, the discovery of better and more selective ligands is necessary to understand the pharmacological and molecular implications of I2-IR. Recently, we described a new imidazoline-structure family which showed high affinity and selectivity for I2-IR. In vivo studies in mice indicated a neuroprotective role and revealed beneficial effects in behaviour and cognition with a murine model of neurodegeneration, senescence-accelerated prone mouse (SAMP8). Herein, we report a novel non-imidazoline-structure of bicyclic α-iminophosphonates family with high affinities for I2-IR. In vivo studies in 5X-FAD mice (a transgenic representative model of AD) and SAMP8 mice (a model of neurodegeneration linked to aging) showed an improvement in behaviour and cognition, a reduction of AD hallmarks and of neuroinflammation markers for the mice treated with the lead compound B06. After evaluating several pathways associated with neurodegeneration, we demonstrated that CaN pathway plays a critical role on the neuroprotective effects of I2-IR ligands on SAMP8 mice model. To rule out warnings of the novel family, we calculated DMPK and physicochemical properties for the novel bicyclic α-iminophosphonates. As well, we carried out drug metabolism, safety studies and in vivo pharmacokinetics for lead compound B06. In summary, we present a novel family of I2-IR ligands, its effectiveness in in vivo models and the possible neuroprotective molecular mechanism mediated by them. This highlights that the modulation of I2-IR by bicyclic α-iminophosphonates may open a new therapeutic venue for unmet neurodegenerative conditions

Bicyclic alfa-iminophosphonates as high affinity imidazoline I2 receptor ligands for Alzheimer's disease

Imidazoline I2 receptors (I2-IR), widely distributed in the CNS and altered in patients that suffered from neurodegenerative disorders, are orphan from the structural point of view and new I2-IR ligands are urgently required for improving their pharmacological characterization. We report the synthesis and 3D-QSAR studies of a new family of bicyclic α-iminophosphonates endowed with relevant affinities for human brain I2-IR. Acute treatment in mice with a selected compound significantly decreased the FADD protein in the hippocampus, a key marker in neuroprotective actions. Additionally, in vivo studies in the familial Alzheimer's disease 5xFAD murine model revealed beneficial effects in behavior and cognition. These results are supported by changes in molecular pathways related to cognitive decline and Alzheimer's disease. Therefore bicyclic α-iminophosphonates are tools that may open new therapeutic avenues for I2-IR, particularly for unmet neurodegenerative conditions

Behavioral and Cognitive Improvement Induced by Novel Imidazoline I2 Receptor Ligands in Female SAMP8 Mice

As populations increase their life expectancy, age-related neurodegenerative disorders such as Alzheimer's disease have become more common. I2-Imidazoline receptors (I2-IR) are widely distributed in the central nervous system, and dysregulation of I2-IR in patients with neurodegenerative diseases has been reported, suggesting their implication in cognitive impairment. This evidence indicates that high-affinity selective I2-IR ligands potentially contribute to the delay of neurodegeneration. In vivo studies in the female senescence accelerated mouse-prone 8 mice have shown that treatment with I2-IR ligands, MCR5 and MCR9, produce beneficial effects in behavior and cognition. Changes in molecular pathways implicated in oxidative stress, inflammation, synaptic plasticity, and apoptotic cell death were also studied. Furthermore, treatments with these I2-IR ligands diminished the amyloid precursor protein processing pathway and increased Aβ degrading enzymes in the hippocampus of SAMP8 mice. These results collectively demonstrate the neuroprotective role of these new I2-IR ligands in a mouse model of brain aging through specific pathways and suggest their potential as therapeutic agents in brain disorders and age-related neurodegenerative diseases. Keywords Imidazoline I2 receptors (2-imidazolin-4-yl)phosphonates Behavior Cognition Neurodegeneration Neuroprotection Agin

Targeting the I(2) Imidazoline Receptors: the neuroprotective role of selective ligands in Alzheimer’s Disease

[eng] The identification of imidazoline receptors (IR) as a novel class of receptors has posed manifold research questions regarding their pharmacological and potential therapeutic properties. Among them, I2-IR receptors are widely distributed in the central nervous system (CNS) with predominant localization in glial cells. Remarkably, their activation has been shown to exhibit neuroprotective properties, yet through not fully understood molecular mechanisms. Furthermore, alterations of I2-IR density have been detected in patients with different brain disorders, including Alzheimer’s Disease (AD), suggesting a potential therapeutic value of I2 -IR in AD. The latter suggestion is of special clinical significance since AD is an irreversible neu- rodegenerative disease and the most common form of dementia that affects millions of people worldwide. The consequences of AD range from detrimental outcomes for both the patients and their caregivers to a wide socioeconomic footprint, and unfortunately, there is still no cure for the disease. At the same time, AD prevalence is rapidly growing due to an increase in life expectancy in developed countries, further highlighting the urgency to identify new targets for halting disease progression and providing effective treatment. In this context, this doctoral dissertation aims to contribute to the scientific knowledge of I2 -IR pharmacological possibilities in neurodegenerative diseases with unmet medical needs, such as AD. More concretely, during this thesis, we evaluate the neuroprotective properties of both well-established and structurally novel selective I2 -IR ligands in late- onset AD (LOAD) and early-onset AD (EOAD) mouse models, with the aim to point out I2 -IR as a novel potential therapeutic target for AD. In summary, we demonstrate that chronic treatment with potent and highly selective I2-IR ligands prevents cognitive decline and ameliorates behavioural and psychological symptoms of dementia (BPSD) -related phenotypes such as anxiety-, depressive-like behaviours and social deficits in the AD mouse models used. Furthermore, the prevention of the generalized cognitive downfall of the mice delivered by I2-IR treatments is consistent with favourable alterations at a molecular level. Notably, we provide evidence for beneficial effects on the AD classical hallmarks, namely Aβ pathology and tau hyperphosphorylation, as well as mitigation of neuroinflammation and oxidative damage, processes with a pivotal role in AD initiation and progression. Remarkably, we detected alterations in Ca2+ related key enzymes induced by selective I2 -IR treatment, providing insights into processes involved in the mechanism of neuroprotective action of I2 -IR ligands. Ultimately, in our hands, I2-IR treatment exerted greater beneficial effects than donepezil under the neurodegenerative process. We conclude that I2-IR modulation by selective ligands can be a novel therapeutic strategy for AD therapy.[spa] La identificación de los receptores imidazolínicos (IR) como una nueva clase de receptores ha planteado múltiples cuestiones sobre sus propiedades farmacológicas potenciales y terapéuticas. Los receptores I2-IR están ampliamente distribuidos en el sistema nervioso central con una localización predominante en las células gliales. Se ha demostrado que su activación tiene propiedades neuroprotectoras, aunque los mecanismos moleculares por los cuales se produce no se no son totalmente conocidos. Además, en pacientes con diferentes trastornos cerebrales, incluida la enfermedad de Alzheimer (EA) se han detectado alteraciones en la densidad de I2-IR, lo que sugiere un valor terapéutico potencial de los I2-IR en la EA. Esta última sugerencia es de especial importancia clínica, ya que la EA es una enfermedad neurodegenerativa irreversible además de ser la forma más común de demencia, que afecta a millones de personas en todo el mundo. Las consecuencias de la EA abarcan desde los efectos perjudiciales tanto para los pacientes como para sus cuidadores hasta un impacto socioeconómico y, por desgracia, aún no existe cura para la enfermedad. Al mismo tiempo, la prevalencia de la EA está creciendo rápidamente debido al aumento de la esperanza de vida en los países desarrollados, lo que pone de manifiesto la urgencia de identificar nuevas dianas terapéuticas para detener la progresión de la enfermedad y proporcionar un tratamiento eficaz. En este contexto, esta tesis doctoral pretende contribuir al conocimiento científico de las posibilidades farmacológicas de los I2 -IR en enfermedades neurodegenerativas con necesidades médicas no cubiertas, como la EA. Más concretamente, durante esta tesis, evaluamos las propiedades neuroprotectoras de ligandos selectivos de los I2-IR, que están bien caracterizados además de ser estructuralmente novedosos, en modelos de ratón de EA de inicio tardío y de inicio temprano, con el objetivo de proponer a los I2-IR como una nueva diana terapéutica para la EA. En resumen, demostramos que el tratamiento crónico con ligandos potentes y altamente selectivos para los I2-IR previene el deterioro cognitivo y mejora los fenotipos relacionados con los síntomas conductuales y psicológicos de la demencia, como los comportamientos ansiosos y depresivos además de los déficits sociales en los modelos de ratón de EA utiliza- dos. Además, la prevención del declive cognitivo generalizado de los ratones tratados con los diferentes ligandos I2 -IR es coherente con los cambios positivos a nivel molecular. En particular, aportamos pruebas de los efectos beneficiosos sobre las principales característi- cas de la EA como la patología Aβ y la hiperfosforilación de Tau, además de la mitigación de la neuroinflamación y el daño oxidativo, procesos con un papel fundamental en el inicio y la progresión de la EA. Notablemente, hemos detectado alteraciones en las enzimas clave relacionadas con la señalización de Ca2+ inducidas por el tratamiento con ligandos selectivos de los I2-IR, lo que proporciona información sobre los procesos implicados en el mecanismo de acción neuroprotectora. Finalmente, el tratamiento con ligandos I2-IR ejerció mayores efectos beneficiosos que el donepezilo en el proceso neurodegenerativo. Concluimos, por lo tanto, que la modulación del I2-IR mediante ligandos selectivos puede ser una estrategia terapéutica novedosa para la EA

Μελέτη των παραγόντων κινδύνου ιώσεων του κατώτερου αναπνευστικού σε παιδιά προσχολικής ηλικίας

Εισαγωγή: Είναι γνωστό ότι στα παιδιά προσχολικής ηλικίας είναι ιδιαίτερα συχνές οι λοιμώξεις του κατώτερου αναπνευστικού. Διάφοροι ιοί έχουν ενοχοποιηθεί και διάφορες μελέτες έχουν γίνει για να διαπιστωθεί αν υπάρχουν παράγοντες είτε περιβαλλοντικοί είτε γενετικοί οι οποίοι καθιστούν τα παιδιά πιο ευάλωτα ώστε να βιώνουν τη συγκεκριμένη κατάσταση. Σκοπός: Στη συγκεκριμένη εργασία μελετώνται παράγοντες κινδύνου για λοιμώξεις αναπνευστικού στα παιδιά προσχολικής ηλικίας όπως το κάπνισμα των γονέων, το μορφωτικό τους επίπεδο αλλά και η περιοχή στην οποία βρίσκεται ο παιδικός σταθμός στον οποίο φοιτούν. Υλικό-Μέθοδοι: Δείγμα της μελέτης αποτέλεσαν 233 παιδιά δημόσιων παιδικών σταθμών από αστική, ημιαστική και αγροτική περιοχή 2-4 ετών όπως και ένας από τους γονείς τους. Πρόκειται για μία προοπτική μελέτη κοορτής τα στοιχεία της οποίας συλλέχθηκαν ύστερα από συμπλήρωση ερωτηματολογίου SICET (Smoking in Children ́s Environment Test) και της μέτρησης της κοτινίνης ούρων των παιδιών σε δύο χρονικές περιόδους το Δεκέμβριο και το Μάιο. Ακολούθησε διετές follow up των παιδιών με καταγραφή των λοιμώξεων κατώτερου αναπνευστικού που παρουσίασαν μέσα σε δύο χρόνια από την καταγραφή των πρώτων πληροφοριών. Αποτελέσματα: Δεν παρατηρήθηκε στατιστικά σημαντική συσχέτιση των υπό μελέτη παραγόντων κινδύνου με την πιθανότητα εμφάνισης λοιμώξεων του κατώτερου αναπνευστικού με εξαίρεση την ταξινόμηση των παιδικών σταθμών (p<0,05). Φάνηκε πως τα παιδιά που πηγαίνουν σε αγροτικούς και ημιαστικούς παιδικούς σταθμούς έχουν 2,5 (p=0,039) και 42 (p=0,001) φορές αντίστοιχα μεγαλύτερη πιθανότητα εμφάνισης λοιμώξεων του κατώτερου αναπνευστικού σε σχέση με τα παιδιά που πηγαίνουν σε αστικούς παιδικούς σταθμούς. Ακόμα βρέθηκε στατιστικά σημαντική και θετική συσχέτιση της κοτινίνης ούρων με το κάπνισμα του πατέρα (p=0,021) και οριακά σημαντική και αρνητική συσχέτιση (p=0,053) της κοτινίνης ούρων με την εκπαίδευση του πατέρα. Τέλος τα αποτελέσματα που προέκυψαν έδειξαν ότι η ατοπία του παιδιού σχετίζεται σημαντικά με την ταξινόμηση των παιδικών σταθμών και συγκεκριμένα βρέθηκε στατιστικά σημαντική και θετική συσχέτιση του ημιαστικού σε σχέση με τον αστικό παιδικό σταθμό (p=0,003) δηλαδή τα παιδιά που πηγαίνουν σε ημιαστικό παιδικό σταθμό έχουν 3,9 φορές μεγαλύτερη πιθανότητα εμφάνισης ατοπίας συγκριτικά με τα παιδιά που πηγαίνουν σε αστικούς παιδικούς σταθμούς. Συμπεράσματα: Φαίνεται ότι τα παιδιά που διέμεναν στην επαρχία είχαν αυξημένες πιθανότητες εμφάνισης λοιμώξεων αναπνευστικού. Αυτό μπορεί να σχετίζεται και με άλλους παράγοντες όπως είναι η θερμοκρασία περιβάλλοντος, τα θερμαντικά σώματα, η συντήρηση των κτιρίων των παιδικών σταθμών αλλά και η παρουσία μεγαλύτερων αδελφών που παρακολουθούν σχολείο. Σίγουρα βάσει και της προϋπάρχουσας βιβλιογραφίας θα μπορούσαν να διενεργηθούν μελέτες με μεγάλο αριθμό συμμετεχόντων ώστε να διαπιστωθεί αν όντως το κάπνισμα και ο τόπος διαβίωσης μπορεί να συντελέσουν στην αυξημένη εμφάνιση λοιμώξεων του κατώτερου αναπνευστικού.Introduction: Lower respiratory tract infections are known to be particularly common in preschool children. Various viruses have been blamed and various studies have been conducted to determine if there are either environmental or genetic factors that make children more vulnerable to experiencing this condition. Aim: In this study, risk factors for respiratory tract infections in preschool children are studied, such as the parent’s smoking, their educational level and the area in which the kindergarten they attend is located. Material-Methods: The sample of the study consisted of 233 children of public kindergartens from urban, semi-urban and rural areas 2-4 years old as well as one of their parents. This is a prospective cohort study, the data of which were collected after completing the SICET questionnaire (Smoking in Children's Environment Test) and measuring the urinary cotinine of children in two time periods in December and May. This was followed by a two-year follow-up of the children with the recording of the lower respiratory tract infections that they presented within two years from the recording of the first information. Results: There was no statistically significant correlation between the risk factors under study and the likelihood of lower respiratory tract infections with the exception of kindergarten classification (p <0.05). It turned out that children who attend to rural and semi-rural kindergartens are 2.5 (p = 0.039) and 42 (p = 0.001) times respectively more likely to develop lower respiratory infections than children who attend to urban kindergartens. It was also found a statistically significant and positive correlation of urinary cotinine with the father's smoking (p = 0.021) and a marginally significant and negative correlation (p = 0.053) of urinary cotinine with the father's education. Finally, the results showed that the atopy of the child is significantly related to the classification of kindergartens and in particular a statistically significant and positive correlation was found in relation to the urban kindergarten (p = 0.003) ie children attending a kindergarten are 3.9 times more likely to develop atopy than children attending urban daycare. Conclusions: It seems that children living in the province had an increased chance of developing respiratory infections. This may be related to other factors such as ambient temperature, radiators, maintenance of kindergarten buildings and the presence of older siblings attending school. Certainly, based on the pre-existing literature, studies with a large number of participants could be conducted to determine whether smoking and place of residence can indeed contribute to the increased incidence of lower respiratory infections

Microarray Analysis Revealed Inflammatory Transcriptomic Changes after LSL60101 Treatment in 5XFAD Mice Model

I2-IR have been found dysregulated in patients with neurodegenerative diseases, such as Alzheimer's disease (AD), in which the importance of neuroinflammation in the establishment and maintenance of cognitive decline is well-documented. To research the implication of I2-IR in neuroinflammatory pathways altered in AD, we determined the expression profile of genes associated with inflammation in the 5XFAD model treated with LSL60101, a well-established I2-IR ligand. Thus, we performed a qPCR array containing 84 inflammation-related genes. Hierarchical clustering analysis revealed three gene clusters, suggesting that treatment with LSL60101 affects the gene expression associated with inflammation in the 5XFAD model. Furthermore, we evaluated the functions of the three clusters; thereby performing a pathway enrichment analysis using the GO database. As we expected, clusters 2 and 3 showed alterations in the inflammatory response, chemotaxis and the chemokine-mediated signaling pathway, among others. To validate previous results from the gene profiling analysis, the expression levels of a representative subset of mRNAs were selected according to the intensity of the observed changes and their biological relevance. Interestingly, changes induced by LSL60101 in the 5XFAD model were validated for several genes. These results suggest that treatment with LSL60101 in the 5XFAD model reverses the inflammatory process during the development of AD