Evaluating cortical transcriptomic differences between Alzheimer’s disease PSEN1- mutated mouse models and human patients, and their implications in drug development

Tese de mestrado, Ciências Biofarmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.A doença de Alzheimer (AD) é uma doença progressiva e irreversível do sistema nervoso central, sendo atualmente a demência mais prevalente a nível mundial, cuja incidência aumenta com o avançar da idade. Esta patologia caracteriza-se pela acumulação extracelular de placas insolúveis de péptido amiloide-beta (A), e pela acumulação intracelular de proteína tau irregularmente hiperfosforilada sob a forma de agregados fibrilares. Outras características patofisiológicas incluem morte neuronal e perda de sinapses, exacerbação do sistema imunitário e inflamação crónica, e atrofia cerebral. O presente rápido envelhecimento da população mundial prevê, com o aumento da proporção de população envelhecida, um igual aumento da prevalência e incidência de doenças neurodegenerativas associadas à idade, como é o caso das demências, categoria em que se inclui a AD. Atualmente não existe um tratamento eficaz que abrande ou impeça a progressão desta doença, o que, simultaneamente com a escassez de aprovação de novos medicamentos que se tem sentido na última década, constitui uma preocupação social, económica e de saúde pública. Adicionalmente, a maioria dos ensaios clínicos em doenças neurodegenerativas, inclusive em AD, apresenta elevadas taxas de insucesso, especialmente a nível dos ensaios de toxicidade e eficácia. O insucesso nesta fase dos ensaios reflete as dificuldades de transpor os resultados obtidos através dos modelos animais durante os estudos pré-clínicos para a doença humana, sugerindo que esta não será bem representada por estes modelos. Neste sentido, é imperativo avaliar as diferenças moleculares que distinguem os modelos animais e os doentes com Alzheimer em termos da fisiopatologia da doença, e também desenvolver diferentes abordagens que possam auxiliar a descoberta de modelos animais mais representativos da AD. Nesse sentido, este projeto propõe avaliar os perfis de alteração de expressão génica (também referenciadas como alterações transcritómicas) entre amostras de cérebro de controlos e doentes com AD, tanto para amostras humanas como para amostras obtidas a partir de modelos animais, sendo que em ambas as espécies as amostras relativas aos portadores de um fenótipo de doença apresentam mutações no gene da presenilina 1 (PSEN1). Com esta abordagem pretendeu-se comparar os perfis de alterações transcritómicas induzidos pela AD no cérebro obtidos para cada uma das espécies através de modelação linear de dados de microarrays, sendo que para essa análise foi considerada, para além da condição (controlo versus doente), outra informação sobre as amostras como a idade do dador. A interpretação biológica dessas alterações transcritómicas foi feita por análise de alguns genes encontrados diferencialmente expressos, e também com recurso a Gene Set Enrichment Analysis (GSEA), um método que identifica as vias metabólicas mais desreguladas, tendo por base o perfil das alterações transcritómicas entre as condições em estudo. As amostras de ratinhos com AD consideradas no presente estudo dividem-se em três categorias: (1) animais com mutações exclusivamente a nível do PSEN1, e animais que adicionalmente são portadores de mutações no gene APP, podendo estes ser (2) heterozigóticos ou (3) homozigóticos. Os três modelos mostraram inexistente ou fraca correlação com a doença humana, aquando da comparação dos perfis de alteração transcritómica. Adicionalmente, os dois primeiros modelos não apresentaram diferenças de expressão significativas entre as amostras controlo e as amostras doentes. Considerando que o modelo homozigótico com mutações em PSEN1 e APP foi o único a apresentar alterações a nível do perfil transcritómico, todas as análises e comparações descritas consideraram apenas estes ratinhos. Os resultados mostram que tanto em humano como em ratinho portadores de doença de Alzheimer existe uma sobre-expressão dos genes envolvidos nos mecanismos de regulação do sistema imunitário, nomeadamente a nível de inflamação crónica, e uma diminuição do transporte de neurotransmissores. As restantes vias mais alteradas com a AD diferem entre humano e ratinho, embora a maioria esteja alinhada com a bibliografia existente sobre a patologia. Algumas vias metabólicas também surgiram inversamente desreguladas entre as duas espécies. Genes envolvidos em vias metabólicas de diferenciação, proliferação e apoptose celular, processamento de DNA e RNA, e mecanismos relacionados com o sistema cardiovascular surgiram sobre-expressos na doença humana; enquanto genes envolvidos em vias associadas com atividade sináptica e neuronal, canais de transporte de membrana, e com a diabetes surgiram sub-expressos. Contrariamente, no caso do ratinho, verificou-se um exacerbar da diabetes, juntamente com o de vias metabólicas relacionadas com a colesterol e interações celulares; e sub-expressão de genes envolvidos na atividade mitocondrial e respiração celular, e em mecanismos de expressão génica, nomeadamente a nível do spliceossoma. Realizou-se também uma análise conjunta dos dados de humano e ratinho, com a qual se observou uma maior variância de expressão génica entre os controlos humanos e os indivíduos doentes humanos, comparativamente com os ratinhos controlo e doentes, reforçando a possibilidade de que o desenvolvimento e a progressão da doença em ratinho não sejam demarcados o suficiente para que, a nível do transcritoma, exista uma explícita diferenciação entre as condições de doença e de não-doença. As diferenças de expressão génica para os dados conjuntos foram modeladas linearmente, incorporando nos modelos, como variáveis, informação não só sobre a idade e condição das amostras, mas também sobre a espécie a que pertencem. Deste modo, foi possível isolar o efeito doença do efeito espécie e obter as diferenças transcritómicas ocorridas mais preponderantemente em humano e em ratinho, bem como as diferenças comuns às duas espécies, ou seja, independentes da espécie. Esta análise revelou que a diminuição da atividade neuronal e sináptica está associada à AD, mas que surge menos afetada nos modelos de ratinho comparativamente aos doentes humanos. Quanto aos genes envolvidos nos mecanismos de regulação do sistema imunitário que também se revelam sobre-expressos na doença geral, encontraram-se mais sobre-expressos na doença humana do que no modelo de ratinho considerado. Estas subtis diferenças entre a informação transcritómica do humano e do ratinho sugerem que as dinâmicas associadas à AD possam ser específicas da espécie, reforçando a necessidade de ajustar os modelos animais para que simulem mais eficientemente a patologia humana. Este projeto teve ainda como objetivo encontrar compostos e perturbações genéticas (knockdowns ou sobre-expressões) com potencial de recapitular as diferenças de expressão mais específicas da AD humana, para que possam ser administrados/manipulados em modelos de ratinho com o intuito de melhorar modelos já existentes ou encontrar um novo e aperfeiçoado modelo animal que replique de forma mais fidedigna as alterações decorridas da doença humana. Adicionalmente, também serão de interesse perturbações químicas e genéticas com capacidade de replicar perfis transcritómicos antagónicos daquele encontrado para a generalidade da doença, que possam ser utilizados como novas terapêuticas ou como objetos de estudo dos mecanismos associados à AD. Para o propósito mencionado acima, recorreu-se à base de dados do Connectivity Map, que inclui informação transcritómica para diversas linhas celulares, antes e após lhes serem administrados diferentes compostos ou alterações genéticas. Usando um software desenvolvido no nosso laboratório, cTRAP, podemos, a partir dos perfis de alteração de expressão genética encontrados no nosso estudo, obter as perturbações químicas e genéticas que recapitulem as alterações transcritómicas do nosso interesse. No tempo do estudo, analisou-se apenas as perturbações químicas, fazendo uma separação entre dois grupos de compostos: (1) aqueles com indicações para doenças do foro neurológico e (2) os com indicação de foro não neurológico. Para cada uma das duas categorias foram selecionados os 10 compostos mais relevantes, isto é, aqueles que estatisticamente estão mais correlacionados com as alterações transcritómicas de interesse. Apenas compostos em fase III de desenvolvimento clínico ou disponíveis no mercado foram considerados. A análise similar das perturbações genéticas fica então referenciada para trabalho a desenvolver no futuro. Adicionalmente, dada a complexidade celular do sistema nervoso central em termos de heterogeneidade e proporção celular, a qual é afetada em estados de doença, e especificamente neste caso de doenças neurodegenerativas, seria de interesse acrescentar uma assinatura que distinga os vários tipos celulares à informação proporcionada ao modelo linear utilizado para derivar os perfis de alteração transcritómica. Desta forma, seria possível distinguir alterações de expressão genética associadas a mudanças na composição celular daquelas relacionadas com mecanismos específicos da AD. O objetivo final do projeto será testar perturbações químicas e genéticas escolhidas cuidadosamente, em linhas celulares e em modelos de ratinho, e testar a sua capacidade em gerar um modelo animal cujo desenvolvimento e progressão da AD seja mais similar ao observado em condições de doença humana; bem como o potencial dos mesmos em reverter características desta patologia.Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disease of the central nervous system, being nowadays considered the most prevalent age-related dementia worldwide. AD pathology is characterized by the extracellular deposition of insoluble amyloidbeta plaques, and the intracellular accumulation of abnormally phosphorylated tau protein into neurofibrillary tangles. Other hallmarks include neuronal death, exacerbation of the immune system and chronic inflammation, synaptic loss, and brain atrophy. The world population is rapidly aging, and an increase in the older population is foreseen, as well as in the prevalence of dementias such as AD. Currently, there is no effective treatment to neither decrease nor cease the damage of this disease, which, allied with the lack of new approved medicines since 2003, comprises a social, economic and health burden. Moreover, clinical trials have been exhibiting high failure rates, especially during toxicity and efficacy assessments, which implies a poor representation of the actual human disease in preclinical animal models. Thus, it is vital to evaluate what molecularly distinguishes them in terms of disease pathophysiology, and how can they be improved to better represent the human disease. On this note, this project purposes to assess the dissimilarities between the AD-induced gene expression (i.e. transcriptomic) alterations between preclinical AD mouse models and human AD patients, both carrying mutations in the PSEN1 gene. For this purpose, microarray data was used for both species, and gene expression differences between AD and non-AD conditions were assessed through linear modelling for each specie. To unveil the biological meaning behind this changes, gene set enrichment analyses (GSEA) were performed. Mechanisms associated with the immune system, namely with the inflammatory response, appear up-regulated in both human AD patients and mouse models, whereas neurotransmitter trafficking processes appear down-regulated in both. The majority of the other most strikingly disrupted pathways varied between human and mouse, but were often in accordance with prior scientific knowledge on AD. However, a few of them appeared differently altered between species, such as diabetes mellitus associated pathways, that appeared down-regulated in human patients and up-regulated in AD mouse models. The analysis of the joint dataset (resulting of merging the human and mouse datasets) unveiled synaptic and neuronal activity -related pathways as down-regulated in the disease common to both species, but less so in mouse AD compared to human patients. On the other hand, immune system genes and pathways were commonly up-regulated in the disease but more so in the human patients. These subtle variations between human and mouse transcriptomic information suggest that disease dynamics are potentially species-specific and reinforce the need to generate models that are able to more effectively replicate the human disease. Additionally, we also identified compounds able to induce GE alterations opposite to those observed for the species-common component of the disease, as well as those capable of emulating human-specific AD-induced transcriptomic alterations. Those candidate compounds can be further explored as therapeutics to combat AD or as a vehicle to obtain novel and innovative mouse models that more effectively replicate the transcriptomic signature of the actual human disease. Two groups of compounds were considered: those with prescription information for neurology-related conditions and those prescribed for other conditions. Moreover, only compounds positioned at the phase III of clinical trials or already available in the market were considered. For future work, it would be possible to perform a similar analysis but to assess genetic perturbations (i.e. knockdowns or overexpression) rather than compounds, which could, likewise, be able to induce an opposite transcriptomic profile to that of the species-common disease, and of those that could promote the development of a human AD signature in a mouse model. Moreover, given the complexity of the brain in terms of cell type composition and interactions between cell types, and the consequences of a neurodegenerative disease upon these, it would be interesting to incorporate brain cell-type-specific signatures as explanatory variables in the linear model used to estimate GE changes, in order to decouple AD-associated cell-type-specific and systemic GE alterations from brain cellular composition changes (namely neuronal loss). The end goal of the present project would be to evaluate the effects of carefully selected genetic perturbations and compounds in cell lines and mouse models, in order to obtain a model able to more accurately develop the human AD.Com o patrocínio do Instituto de Medicina Molecular (iMM) João Lobo Antunes

Prosystemin coordinates multiple responses in tomato.

In Solanaceae, a family of peptide hormones called Systemins are involved in the activation of defense genes in response to wounding and herbivory. In tomato, Systemin (Sys) is an 18-aa oligopeptide released from a cytosolic precursor protein of 200 aa called prosystemin (ProSys). ProSys involvement in tomato response to chewing insects has long been described, but little is known about its role in modulating defense responses to phloem-feeding insects or other biotic agents such as pathogenic fungi. The aim of this research activity is to shed more light on ProSys involvement in tomato responses against several stress conditions and to evaluate a possible use of this plant hormone as a broad-spectrum defence instrument. For this reason, tomato cv. \u201cRed Setter\u201d was chosen to over-express ProSys gene since this tomato cultivar lacks the dominant allel at Mi locus, a gene involved in resistance against aphids, nematodes and white flies (Rossi et al., 1998). Solanum lycopersicum cv. \u201cRed Setter\u201d was genetically transformed via A. tumefaciens containing the pMZ vector carrying 35S2:prosystemin, already described by Rocco and collaborators (2008). ProSys conferred-resistance to aphids and fungi was underlined by bioassays. Tomato responses were further investigated by time-course transcriptional analysis of untransformed \u201cRed Setter\u201d plants challenged by aphids or fungi. The comparison with the expression profiles of the same genes in ProSys over-expressing plants underlined that ProSys induces most of the genes associated to tomato responses against these pests. In order to get a global vision of ProSys impact on tomato transcriptome, a microarray analysis was carried out underlining many defence-related differentially expressed genes, most of them supporting these biological evidences. The functional analyses of differentially espressed genes indicated that prosystemin affects genes involved in JA-, SA-, ET and auxin pathways. Molecular data were further investigated in silico to predict networks that highlight protein-protein interactions active in the signaling cascade activated by ProSys gene. This work was carried out at the Bioinformatic Department of \u201cCentro de Investigation Principe Felipe\u201d (CIPF, Valencia). The resulted network contained 195 proteins and highlighted some possible novel interactions such as between the ProSys-regulated ERD4 (early responsive to dehydration) and FPS (pharnesyl pyrophosphate synthase), a promising molecular link between abiotic and biotic response

Pubertal mouse mammary gland development - transcriptome analysis and the investigation of Fbln2 expression and function

Mouse mammary gland morphogenesis at puberty is a complex developmental process, regulated by systemic hormones, local growth factors and dependent on the epithelial/epithelial and epithelial/stromal interactions. TEBs which invade the fat pad are important in laying down the epithelial framework of the gland at this time point. The objective of this thesis was to use a combination of ‘pathway-‘ and ‘candidate gene analysis’ of the transcriptome of isolated TEBs and ducts and associated stroma, combined with detailed analyses of selected proteins, to further define the key proteins and processes involved at puberty. Using GeneChip® Mouse Exon 1.0 ST Arrays we identified the epithelial-, epithelial-stromal- and stromal transcriptomes of TEBs and ducts and defined the major functional pathways/biological processes in each compartment. By ranking the transcripts according to their expression levels and known functions in other systems, we identified genes of potential importance for pubertal mammary morphogenesis. We focused our study on Upk3a and Fbln2 and their protein products. Upk3a could only be detected at mRNA level and thus further analysis was based on Fbln2. We demonstrated that Fbln2 V1 and Fbln2 protein are predominantly expressed in the epithelium and stroma of TEBs. Using hormone primed mice we demonstrated that Fbln2 expression and localisation in the mouse mammary gland is positively regulated by E2 and P. Furthermore, by a combination of further in silico analysis, in vitro functional assays, IHC and IF we identified Vcan, Lama1, Fbn1, ColVIαIII, ColIVαI, ColXVIIIαI, Eln, Per, Acan, Nid, Itgb3 and Itga5 as potential binding partners of Fbln2 in mammary gland. Finally, we reported lack of an obvious mammary phenotype in Fbln2 KO-/- mice at puberty but demonstrated that this may be attributed to the over-compensation by Fbln1. This thesis demonstrates the benefit of DNA microarray analysis in studying pubertal development of mouse mammary gland. It identifies Fbln2 as a potential pubertal mammary regulator which by interacting with various ECM proteins at different sites of mammary milieu may contribute to an array of structural and migratory functions during mammary morphogenesis. These data substantially add to the understanding of the development of mammary gland at puberty and reveal many potential avenues for further investigations

Investigation of molecular and cellular events associated with beta cell function and elucidation of extracellular RNAs as potential biomarker for diabetes

Diabetes is a chronic disorder of glucose metabolism and a major cause of premature mortality. The potential use of replacement beta cells as therapy for diabetes requires an ability to culture such cells while maintaining their functional status. Glucose stimulated insulin secretion (GSIS) is lost in long-term cultured MIN6 heterogeneous cells. MIN6 B1, a clonal sub-line derived from MIN6, has been described as highly glucose-responsive. This study aimed to investigate the GSIS function, changes in gene expression and, subsequently, to develop possible experimental approaches to overcome this loss. Understanding the molecular basis for loss of GSIS may contribute to better culture conditions for islets in transplantation programmes; it may also add to our understanding of beta cell insensitivity to high blood glucose in Type 2 diabetes. Whole genome microarray analysis on biological triplicate samples of glucoseresponsive low passage (p19) (MIN6 B1(GSIS)) and non glucose-responsive high passage (p23) (MIN6 B1(Non-GSIS)) was performed. 111 differentially-regulated genes were identified and 16 gene transcripts were selected based on p-value <0.05; fold change 1.2; difference 100 and validated using qRT-PCR including Txnip, Gcg and Pcsk9. Txnip was up-regulated whereas Gcg and Pcsk9 were down-regulated in MIN6 B1(Non-GSIS) compared to MIN6 B1(GSIS). siRNA and shRNA silencing of Txnip in MIN6(H) (non-responsive) significantly increased the GSIS. Over-expression of Txnip cDNA in MIN6(L) (glucose-responsive) caused significant loss of GSIS. siRNA silencing of Gcg and Pcsk9 caused a significant loss of GSIS in MIN6(L) compared to scrambled-transfected cells. Over-expression of Gcg cDNA in MIN6(H) increased GSIS. In parallel, in an attempt to identify more reliable biomarkers for diabetes, we also investigated if extracellular mRNAs are reproducibly detectable in conditioned medium (CM) from a range of insulin-producing cell types and in serum specimens from Type 2 diabetes and controls. Pdx1, Npy, Egr1, Pld1, Chgb, Ins1, Ins2, and betaactin from MIN6(L), MIN6(H), and MIN6 B1 cells and their CM suggests that beta cells transcribe and release these mRNAs into their culture environment.This study was subsequently translated to analysis of serum from people with Type 2 diabetes and controls to help establish the clinical relevance of these findings. The result from this clinical phase of the study indicated Txnip to be up-regulated and Egr1 to be down-regulated in Type 2 diabetes compared to controls

Novel nanocarriers for invasive glioma

The invasive nature of glioblastoma (GBM) represents a significant challenge to the standard of care and contributes to poor clinical outcomes. Invasion of tumors into healthy brain restricts chemotherapeutic access and complicates surgical resection. The central hypothesis of the thesis is that an effective anti-invasive agent can enhance the standard chemotherapeutic response in invasive brain tumors. Through a screen of novel compounds, a new anti-invasive small molecule, Imipramine Blue (IB), was identified. This triphenylmethane compound inhibits invasion of highly invasive glioma in vitro and in vivo. To elicit a response in vivo, Imipramine Blue was liposomally encapsulated to yield better delivery to tumor. Using this formulation, it is shown that IB attenuates invasion of glioma in vivo leading to a more compact tumor in an aggressively invasive rodent glioma model. Further, it is shown that this novel compound binds NADPH oxidases and alters expression of actin regulatory elements to elicit this anti-invasive activity. To test our hypothesis that anti-invasive therapy coupled with chemotherapy will enhance efficacy, nano-IB therapy was followed by liposomally encapsulated doxorubicin (DXR) chemotherapy. Additionally, a co-encapsulated formulation of IB and DXR was developed and tested in vivo. This combination therapy significantly enhanced survival compared to IB or DXR alone, resulting in long-term survival in the syngeneic invasive rat astrocytoma model RT2. It was seen that sequential treatment was more effective than the co-encapsulated treatment indicating a benefit of pre-treating the tumor with the anti-invasive. This thesis demonstrates that novel anti-invasive IB mediated 'containment' of diffuse glioma significantly enhances the efficacy of DXR chemotherapy compared to chemotherapy or anti-invasive therapy alone.Ph.D.Committee Chair: Bellamkonda, Ravi; Committee Member: Arbiser, Jack; Committee Member: Barker, Thomas; Committee Member: Brat, Daniel; Committee Member: Lu, Hang; Committee Member: Taite, Lakeshi