Multiscale modelling and structure-guided development of small molecules tackling neuroinflammation and inflammageing

Ph. D. ThesisEarly diagnosis and treatment of neurodegenerative diseases has gained attention, given their increasing prevalence. Multiple proteins are currently being investigated as novel targets for drug development, including α7 nicotinic receptors and mitochondrial translocator protein TSPO. These proteins play an important role in neuroinflammation, which makes them attractive for development of drugs and diagnostics. However, small molecule development has been hampered by existence of a human-specific dupa7 isoform, and an A147T-TSPO polymorph, which present a challenge for development of potent and selective ligands. In this work, I characterised the structure and dynamics of the most plausible functional a7 pentamers bearing dupα7 subunits. The receptors have been modelled and assessed using multiscale molecular dynamics (MD) simulations and enhanced sampling techniques. The energy landscapes of the pentamers with different stoichiometries showed that receptors with a low ratio of dupα7/α7 remained functional. Sensitivity of dupa7 receptors to an antagonist (α- BTX) and amyloid Aβ42 has also been assessed. Further, putative “druggable” binding sites at dupα7 receptors were mapped, and interactions between dupα7 and small molecules were explored using a combination of solvent mapping, MD simulations, and molecular docking. Results indicated that neither established orthosteric agonist nor allosteric ligands can bind to dupα7/dupα7 interfaces, however, α7/dupα7 interfaces remain “druggable”. In addition, several novel allosteric sites were detected on α7/dupα7 receptors. The final part of this work focused on development of novel tracers for A147T-TSPO variant. Using a combination of molecular modelling, MD simulations, and structure-guided drug design, I have evaluated plausible binding modes of established TSPO ligands to A147TTSPO. Results explain the origins of diminished affinity of some established TSPO ligands to A147T-TSPO. Moreover, I have identified the position of fluorine atom, which is a derivative of DPA-714 compound to bind to A147T-TSPO with sub-nanomolar affinity. The compound, denoted as MKD, is feasible for the radiosynthesis

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

C

Evaluation of the role of miR-9 and miR-29 in amyloid pathway of Alzheimer's disease

Alzheimer’s disease (AD) is a neurodegenerative disorder which occupies the 3rd place of the diseases that cause disability and death in the elderly, but their causes are yet in need of a better understanding. Also, the search of the etiopathological path leading from a healthy state to full-blown dementia could help with the establishment of more effective treatments. MicroRNAs (miRs or miRNAs) are small regulatory non-coding RNAs (sncRNA), which are involved in post-transcriptional gene expression regulation. Diverse studies have shown that the expression levels of several miRNAs are decreased in AD patients and, there is also evidence that certain miRNAs can control the pathologic hallmarks related to the biomarkers of AD. The main biomarkers of AD are extracellular deposits of amyloid-ß peptide (Aß) along with an accumulation of intraneuronal hyperphosphorylated form of the microtubule-associated protein Tau. The aim of this dissertation is to study the role of two under-expressed miRNAs in AD, miR-29 and miR-9, that are related to the regulation of messenger RNA (mRNAs) that encode proteins involved in various pathological pathways of AD. The selection of miRNAs with decreased levels was based on the possibility of performing its delivery into the cells, in order to contradict the pathological environment of AD. To accomplish this issue, synthetic miRNAs and precursors of miRNAs (pre-miRNAs) produced by two different methods (chemical and enzymatic synthesis, respectively) were used, in order to evaluate if the preparation method had influence in the biological activity. Thus, the pre-miRNAs were produced in the laboratory through in vitro transcription using a plasmid DNA (pDNA) as a template and were further subjected to a purification step. To evaluate the biological activity of the miRNAs, N2a695 cells (mutated APP expressing neuroblastoma mouse cell line that mimics the pathological pathway of AD) were used as in vitro model. Two different methods were also used for cells transfection, Lipofectamine and chitosan-nanoparticles for RNA encapsulation and delivery. After RNA extraction, the mRNA expression levels were evaluated for the APP, BACE1 and PS1 proteins. The findings demonstrated a silencing effect on the expression of APP mRNA by pre-miR-9 and of the BACE mRNA by miR-29b and pre-miR-29b. In turn, PS1 mRNA levels were decreased after cells transfection with all types of miRNAs/pre-miRNAs used in this study, being the most prominent result obtained by RT-qPCR. Western blot assays were also performed, in order to quantify the expression levels of the proteins in study. In conclusion, a better understanding of how miRNAs act and what are their specific targets may have a major impact on the pharmaceutical industry, because being regulators in the pathologic pathways in certain diseases, such as AD, they can be used as new therapeutic approaches to regulate altered proteins expression in this pathological environments.A doença de Alzheimer (DA) é uma doença neurodegenerativa e a forma mais comum de demência, ocupando o terceiro lugar das patologias que causam incapacidade e morte nos idosos. Apesar do seu impacto, as causas desta doença são, ainda hoje, pouco compreendidas. Entender as vias e mecanismos que conduzem a uma transição entre um estado de saúde até à demência pode ajudar a desenvolver e estabelecer novos tratamentos mais efetivos. A DA caracteriza-se pela expressão desregulada de diversas proteínas envolvidas na formação de péptidos ß-amiloide, nomeadamente a proteína precursora amiloide (PPA), a proteína ß-secretase (BACE1) e a proteína presenilina 1 (PS1), sendo detetados níveis elevados nesta condição. Como estas proteínas fazem parte da complexa via patológica da DA, têm vindo a ser intensamente estudadas como alvos terapêuticos. No entanto, as terapias que utilizam inibidores dessas proteínas têm mostrado efeitos adversos e são muitas vezes descontinuadas, o que torna difícil, mas urgente, a criação de novas estratégias terapêuticas para controlar a progressão da DA. Os microRNAs (miRs ou miRNAs) são moléculas de RNA não codificante, de baixo peso molecular, que têm como principal função regular o processo de tradução, com ação sobre o RNA mensageiro, permitindo uma diminuição dos níveis de expressão da proteína codificada. Os trabalhos de investigação utilizando miRNAs como estratégias terapêuticas para a doença de Alzheimer (DA) têm vindo a aumentar ao longo dos últimos anos. A ideia de utilizar os miRNAs como uma nova abordagem terapêutica nesta doença foi motivada essencialmente por dois fatores. Em primeiro lugar, estes miRNAs podem regular os RNA mensageiro (mRNAs) pós-transcricionalmente, e, consequentemente, controlar a expressão das proteínas responsáveis pelo surgimento da DA e, em segundo lugar, porque já foi amplamente descrito que os níveis de determinados miRNAs se encontram também desregulados, ocorrendo tendencialmente uma diminuição da sua expressão, na DA. Deste modo, os estudos que preveem o estabelecimento de uma relação entre os miRNAs e os seus mRNAs alvo, começaram a surgir e mostraram que certos miRNAs podem de facto controlar as principais proteínas relacionadas com os biomarcadores da DA. Os principais biomarcadores são os depósitos extracelulares de péptidos ß-amiloide juntamente com a acumulação intracelular da forma hiperfosforilada da proteína Tau associada aos microtúbulos. Apesar de alguns estudos mostrarem a regulação de proteínas pelos miRNAs na DA, esta é uma área vasta onde existem muito alvos ainda por descobrir, devido às múltiplas vias e funções que um só miRNA pode desempenhar. Assim, o objetivo deste trabalho é estudar a nível celular, o papel de miRNAs cuja expressão se encontra diminuída na DA, dos quais foram selecionados o miR-29 previamente utilizado no grupo de investigação e o miR-9 que foi também relacionado com a regulação de vários mRNAs que codificam proteínas envolvidas em diferentes vias patológicas da DA. O foco foi neste caso, nos miRNAs que apresentam níveis diminuídos na DA, considerando a possibilidade de entrega destes miRNAs às células, reestabelecendo a sua função e consequentemente contrariando assim o ambiente patológico existente na doença. Para isso foram utilizados miRNAs produzidos por métodos diferentes (síntese química ou enzimática), e apresentando a sua forma madura ou a forma precursora (pre-miRNAs), de modo a verificar a existência de um efeito biológico distinto entre eles. Os pre-miRNAs foram produzidos em laboratório através de transcrição in vitro utilizando um DNA plasmídico (pDNA) como template. Adicionalmente, foi também estudada a vantagem ou possível influência na atividade biológica, da introdução de um processo de purificação dos pre-miRNAs produzidos. De forma a avaliar a sua atividade biológica, as células N2A695 (linha celular de neuroblastoma de rato que expressa APP mutada, de maneira a replicar a via patológica da DA) foram transfetadas com os pre-miRNAs produzidos ou os miRNAs sintéticos, recorrendo à utilização de dois agentes de transfeção, como a Lipofectamina e nanopartículas de quitosano, que permitem igualmente a comparação destes sistemas. Após extração do RNA das células foi feita a avaliação dos níveis de expressão dos mRNAs que codificam as proteínas APP, BACE1 e PS1. Foram globalmente verificados efeitos de silenciamento para a APP pelo pre-miR-9 e para a BACE pelo miR-29b e pelo pre-mir-29b. A PS1 foi a proteína para a qual se verificou um efeito mais predominante, em termos de silenciamento, uma vez que todos os tipos de miRNAs/pre-miRNAs utilizados conduziram ao seu silenciamento. Em suma, um melhor entendimento de como os miRNAs atuam e quais os seus principais alvos, poderá ter um grande impacto na indústria farmacêutica, pois em patologias onde estes são reguladores das vias patológicas, como é o caso da DA, poderão ser utilizados como uma nova abordagem terapêutica, de forma a regular a expressão de proteínas alteradas nesse ambiente patológico

Aβ plaques

Aβ plaques are one of the two lesions in the brain that define the neuropathological diagnosis of Alzheimer’s disease. Plaques are highly diverse structures; many of them include massed, fibrillar polymers of the Aβ protein referred to as Aβ-amyloid, but some lack the defining features of amyloid. Cellular elements in ‘classical’ plaques include abnormal neuronal processes and reactive glial cells, but these are not present in all plaques. Plaques have been given various names since their discovery in 1892, including senile plaques, amyloid plaques, and neuritic plaques. However, with the identification in the 1980s of Aβ as the obligatory and universal component of plaques, the term ‘Aβ plaques’ has become a unifying term for these heterogeneous formations. Tauopathy, the second essential lesion of the Alzheimer’s disease diagnostic dyad, is downstream of Aβ-proteopathy, but it is critically important for the manifestation of dementia. The etiologic link between Aβ-proteopathy and tauopathy in Alzheimer’s disease remains largely undefined. Aβ plaques develop and propagate via the misfolding, self-assembly and spread of Aβ by the prion-like mechanism of seeded protein aggregation. Partially overlapping sets of risk factors and sequelae, including inflammation, genetic variations, and various environmental triggers have been linked to plaque development and idiopathic Alzheimer’s disease, but no single factor has emerged as a requisite cause. The value of Aβ plaques per se as therapeutic targets is uncertain; although some plaques are sites of focal gliosis and inflammation, the complexity of inflammatory biology presents challenges to glia-directed intervention. Small, soluble, oligomeric assemblies of Aβ are enriched in the vicinity of plaques, and these probably contribute to the toxic impact of Aβ aggregation on the brain. Measures designed to reduce the production or seeded self-assembly of Aβ can impede the formation of Aβ plaques and oligomers, along with their accompanying abnormalities; given the apparent long timecourse of the emergence, maturation and proliferation of Aβ plaques in humans, such therapies are likely to be most effective when begun early in the pathogenic process, before significant damage has been done to the brain. Since their discovery in the late 19th century, Aβ plaques have, time and again, illuminated fundamental mechanisms driving neurodegeneration, and they should remain at the forefront of efforts to understand, and therefore treat, Alzheimer’s disease

Role Of Astrocytes On Neuronal Health In Models Of Alzheimer’s Disease

Alzheimer’s disease (AD), one of the major forms of dementia in elderly population, is characterized by amyloid beta (Aβ) plaque along with commonly procured tau hyperphosphorylation. Here, we set to recognize the molecules which are secreted by Aβ1-42 treated astrocytes at very early stage and their probable role in conferring neuroprotection along with improvement in cognitive behavior and pathogenesis of AD. Astrocyte activation has been marked by various specific marker proteins like GFAP, Vimentin, S100β. The conditioned medium obtained from the astrocytes cultured from neonatal pups has been used to check the differential expression of various cytokines in Aβ42 treated astrocytes as compared to control cells. From there soluble intercellular adhesion molecule-1 (sICAM-1) has been noted as a potential candidate. We observed that sICAM-1 protects the cortical neurons from death influenced by Aβ42 oligomers. It attenuates the PARP cleavage caused by Aβ42 and increases the amount of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, along with decrease in the amount of pro-apoptotic protein Bim. TUNEL assay performed both in cortex and hippocampus of Aβ42 infused rat brain and 5xFAD mice brain showed that rrICAM-1 treatment reduced the number of TUNEL positive cells in vivo. Several behavioral experiments namely open field test, contextual and cue dependent fear conditioning, passive avoidance tests, novel object recognition and elevated plus maze that can be related to multiple kinds of memory and learning have displayed that rat recombinant ICAM-1 on being injected within the system intraperitoneally, restored the malfunctioning in the rat behavior caused due to bilateral Aβ42 infusion. We took further interest in checking the underlying disease modifying mechanism rendered by ICAM-1 both in vitro and in vivo systems which might be considered being a therapeutic target for neuroprotection. Mechanistically we observed that ICAM-1 administration decreases NF-κB protein level in AD pathology suggesting that ICAM-1 might play a crucial role in manipulating the stability of NF-κB through multiple degradation process. Also PDTC mediated inhibition of NF-κB protein in 5xFAD transgenic mice brain showed an improvement in cognitive behaviors including learning and memory in mice. Therapeutic approach towards prevalent neurodegenerative diseases like AD is one of the major concerns in the scientific world. Our work suggests that ICAM-1 protein could be said as a potential therapeutic agent that promotes neuronal protection along with recovery in cognitive functioning complemented with clearance of Aβ in Aβ42 infused rats and 5xFAD mice model of AD

Analysis of Mating Circuitry in The Fungal Pathogen Candida albicans

It has been proposed that the ancestral fungus was mating competent and homothallic. However, many mating competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating, the essentially obligate diploid ascomycete pathogen Candida albicans has to homozygose its mating type locus from MTLa/α to MTLa/a or MTLα/α, and then undergo an environmentally controlled epigenetic switch from the white form to the mating competent opaque form (white opaque switch). These requirements greatly reduce the potential for C. albicans mating. In this thesis, I used a mutant library to screen genes involved in white to opaque switching in the MTLa/α background, which allowed us to focus on the barriers of mating in C. albicans (Chapter 2 to 5). Construction of the mutant library is described as an appendix. Heterotrimeric G proteins are an important class of eukaryotic signaling molecules that have been identified as central elements in the pheromone response pathways of many fungi. As well, I did genetic studies on G proteins of the mating pheromone pathway in C. albicans (Chapter 6). Chapter 2 discusses how deletion of the YciI domain gene OFR1 allows the bypass of the need for C. albicans cells to homozygose the mating type locus prior to switching to the opaque form and mating, and allows homothallic mating of MTL heterozygous strains. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry, Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans, and functions in part to maintain the cryptic mating phenotype of the pathogen. Chapter 3 describes the gene termed OFR2, which affects the formation of lipid droplets, white opaque switching and mating. Chapter 4 discusses how the different transcripts of ORF19.7060 in white and opaque states play a role in white opaque switching and mating. Chapter 5 describes how the F1-ATPase chaperon ATP12 regulates white opaque switching and carbon metabolism. In Chapter 6, disruption of the STE18 gene which encodes a potential γ subunit of a heterotrimeric G protein was shown to cause sterility of MTLa mating cells and to block pheromone-induced gene expression and shmoo formation; deletion of just the C-terminal CAAX box residues is sufficient to inactivate Ste18 function in the mating process. Intriguingly, ectopic expression behind the strong ACT1 promoter of either the Gα or the Gβ subunit of the heterotrimeric G protein is able to suppress the mating defect caused by deletion of the Gγ subunit and restore both pheromone-induced gene expression and morphology changes

Ubiquitin and Autophagy

This book is a collection of articles from the Cells Special Issue on “Ubiquitin and Autophagy”. It contains an Editorial and 13 articles at the intersection of ubiquitin- and autophagy-related processes. Ubiquitin is a small protein modifier that is widely used to tag proteins, organelles, and pathogens for their degradation by the ubiquitin–proteasome system and/or autophagy–lysosomal pathway. Interestingly, several ubiquitin-like proteins are at a core of the autophagy mechanism. This book dedicates a lot of attention to the crosstalk between the ubiquitin–proteasome system and autophagy and serves as a good starting point for the readers interested in the current state of the knowledge on ubiquitin and autophagy

Rôle du transporteur neuronal Potassium/Chlore KCC2 dans la plasticité des synapses glutamatergiques

The polarity and efficacy of GABAergic synaptic transmission are both influenced by the intra-neuronal chloride concentration. In mature neurons, chloride extrusion through the neuronal K/Cl cotransporter KCC2 allows an inhibitory influx of chloride upon activation of GABAA receptors. Nevertheless, KCC2 is enriched in the vicinity of excitatory synapses within the dendritic spines that are actin-rich protrusions emerging from dendritic shafts. While it has become clear that KCC2 suppression alters chloride homeostasis and GABA signaling, little is known on its impact on glutamatergic transmission. In the laboratory, we have previously demonstrated that KCC2 suppression in mature neurons leads to decreased glutamatergic transmission efficacy through an ion-transport independent function of KCC2. During my PhD, I have explored how KCC2 may also impact LTP of glutamatergic synapses. My work reveals that KCC2 suppression compromises both functional and structural LTP at these synapses. This effect is associated with inhibition of the actin-severing protein cofilin and enhanced mobilization of F-actin in dendritic spines. Since LTP can be rescued by preventing cofilin inhibition upon KCC2 suppression, I suggest KCC2 might influence LTP through altered actin cytoskeleton dynamics. My results demonstrate that KCC2 function extends beyond the mere control of neuronal chloride homoeostasis and suggest regulation of KCC2 membrane stability may act as a metaplastic switch to gate long term plasticity at excitatory synapses in cortical neurons.L'efficacité de la transmission synaptique GABAergique est influencée par la concentration intracellulaire en ions chlorure. Dans les neurones matures, l'extrusion de ces ions par le transporteur neuronal potassium chlore de type 2 (KCC2) permet l'influx d'ions chlorure lors de l'activation des récepteurs du GABA de type A. Néanmoins, KCC2 est aussi enrichi à proximité des synapses excitatrices portées par les épines dendritiques qui correspondent à des protrusions dendritiques enrichies en actine. Alors que l'effet d'une suppression de KCC2 sur l'homéostasie des ions chlorure et la transmission GABAergique est largement documenté, peu de choses sont connues sur l'impact qu'une telle suppression peut avoir sur la transmission glutamatergique. Lors de ma thèse, j'ai exploré le rôle de KCC2 dans la potentialisation à long terme (LTP) de la transmission glutamatergique à l'origine des phénomènes d'apprentissage et de mémorisation. Ce travail a révélé que la suppression de KCC2 compromet les modifications fonctionnelles et structurales sous-tendant la LTP. Cet effet est associé à une inhibition de la cofilin, protéine responsable de la dépolymérisation de l'actine, qui corrèle avec une augmentation de la quantité d'actine filamenteuse dans les épines dendritiques. En empêchant l'inhibition de la cofilin liée à l'absence de KCC2, il m'a alors été possible de restaurer la LTP suggérant que KCC2 pourrait influencer cette forme de plasticité en régulant la dynamique de polymérisation du cytosquelette d'actine. Mes résultats démontrent que la fonction de KCC2 va au-delà du contrôle de l'homéostasie des ions chlorure et influence les mécanismes de plasticité de la synapse excitatrice