Intravitreal AAV2.COMP-Ang1 prevents neurovascular degeneration in a murine model of diabetic retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the United States. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable to non-diabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally-delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Characterization of PPP1 interacting proteins in male reproduction

A fosforilação reversível de proteínas é um importante mecanismo de controlo em eucariotas. A fosfoproteína fosfatase 1 (PPP1) é uma fosfatase de serina/treonina envolvida em vários processos celulares. Existem três isoformas da subunidade catalítica (α/CA, δ/β/CB e γ/CC) com pequenas diferenças nos terminais amino e carboxílico. O gene PPP1CC sofre ainda splicing alternativo para produzir duas isoformas, a PPP1CC1 ubíqua e a PPP1CC2 enriquecida em testículo e específica de esperma. A localização e especificidade de substratos da PPP1 está dependente da formação de complexos oligoméricos com proteínas que interagem com a PPP1 (PIPs). O objetivo principal desta tese foi estudar novas PIPs, específicas de testículo e esperma, a fim de melhor caracterizar o papel desta fosfatase e dos respetivos complexos na reprodução em mamíferos. Com este fim, estudou-se a presença, localização e possíveis funções de uma PIP previamente conhecida, PPP1R2, e de duas novas PIPs, PPP1R2P3 e Tctex1d4. PPP1R2 e PPP1R2P3 estão presentes em esperma humano colocalizando com a PPP1CC2, na cabeça e na cauda. A hipótese é que as holoenzimas localizadas na cabeça terão um papel na reação acrossómica, enquanto que as holoenzimas presentes no axonema são relevantes para o controlo da motilidade flagelar. De seguida foram estudados os pseudogenes da PPP1R2, em termos de história evolutiva e de possíveis funções. Na espécie humana, a PPP1R2 tem 10 pseudogenes, 7 deles específicos de primatas. Estudos de bioinformática e dados de expressão mostram que os PPP1R2P1/P3/P9 são os pseudogenes com maior probabilidade de serem transcritos e traduzidos. Também identificámos o PPP1R2P9 em esperma humano e mostrámos que alguns pseudogenes poderão estar associados a estados fisiopatológicos. Isto indica que o processo de evolução poderá estar ligado á formação de novos genes ou ao controlo do mRNA da PPP1R2. A sobre-expressão da PPP1R2 ou PPP1R2P3 em testículo de ratinho também foi realizada, para caracterizar os mecanismos envolvidas na função dos complexos PPP1R2/PPP1R2P3-PPP1CC2 na espermatogénese e fisiologia dos espermatozoides. A dineína de cadeia leve, Tctex1d4, foi encontrada como interagindo com a PPP1C e como estando presente em testículo de ratinho e em esperma humano. Demonstrámos que a Tctex1d4 e a PPP1 colocalizam no centro organizador de microtúbulos e nos microtúbulos e que o motivo de ligação à PPP1 presente na Tctex1d4 parece ser importante para manter a PPP1 no centro organizador de microtúbulos e/ou para disromper ou atrasar o seu movimento ao longo dos microtúbulos emergentes. Estes resultados abrem novos caminhos para os possíveis papéis do complexo Tctex1d4-PPP1 na dinâmica dos microtúbulos, motilidade do esperma, reação acrossómica e na regulação da barreira hemato-testicular, provavelmente, através da via de sinalização do TGFß. A análise do motivo de ligação à PPP1 mostra que este é altamente conservado entre os mamíferos, com exceção das Pikas, sugerindo que esta perda aconteceu antes da radiação das Pikas, há 6-20 milhões de anos atrás. Através de um rastreio por mutações demonstrámos que a capacidade da Tctex1d4 se ligar à PPP1 é mantida nas Pikas, embora o motivo de ligação à PPP1 esteja disrompido. Este estudo abre portas para novas descobertas na área da reprodução mostrando o papel da PPP1CC2 na espermatogénese e fisiologia do esperma.

Synthesis of new Aβ-ligands useful in diagnosis of Alzheimer's disease

Alzheimer’s disease is a chronic progressive neurodegenerative disease and is the most common form of dementia (estimated 50−60% of all cases), associated with loss of memory (in particular episodic memory), cognitive decline, and behavioural and physical disability, ultimately leading to death. Alzheimer’s disease is a complex disease, mostly occurring sporadically with no apparent inheritance and being the age the main risk factor. The production and accumulation of amyloid-beta peptide in the central nervous system is a key event in the development of Alzheimer’s disease. This project is devoted to the synthesis of amyloid-beta ligands, fluorophores and blood brain barrier-transporters for diagnosis and therapy of Alzheimer’s disease. Different amyloid-beta ligands will be synthesized and their ability to interact with amyloid-beta plaques will be studied with nuclear magnetic resonance techniques and a process of lead optimization will be performed. Many natural and synthetic compounds able to interact as amyloid-beta ligands have been identified. Among them, a set of small molecules in which aromatic moieties seem to play a key role to inhibit amyloid-beta aggregation, in particular heteroaromatic polycyclic compounds such as tetracyclines. Nevertheless tetracyclines suffer from chemical instability, low water solubility and possess, in this contest, undesired anti-bacterial activity. In order to overcome these limitations, one of our goals is to synthesize tetracyclines analogues bearing a polycyclic structure with improved chemical stability and water solubility, possibly lacking antibacterial activity but conserving the ability to interact with amyloid-beta peptides. Known tetracyclines have in common a fourth cycle without an aromatic character and with different functionalisations. We aim to synthesize derivatives in which this cycle is represented by a sugar moiety, thus bearing different derivatisable positions or create derivatives in which we will increase or decrease the number of fused rings. In order to generate a potential drug-tool candidate, these molecules should also possess the correct chemical-physical characteristics. The glycidic moiety, not being directly involved in the binding, it assures further possible derivatizations, such as conjugation to others molecular entities (nanoparticles, polymeric supports, etc.), and functionalization with chemical groups able to modulate the hydro/lipophilicity. In order to be useful such compounds should perform their action within the brain, therefore they have to be able to cross the blood brain barrier, and to be somehow detected for diagnostic purposes.

Péptidos para a detecção de agregados de alfa-sinucleína

Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2013

Kyotorphin derived peptides in the relationship between analgesia and alzheimer’s disease

Tese de doutoramento, Ciências Biomédicas (Bioquímica Médica), Universidade de Lisboa, Faculdade de Medicina, 2014

Neuro-glia-vascular alterations by common neonatal clinical conditions

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

Characterization of adenosinergic system in Rett syndrome

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2015

Methamphetamine promotes a-tubulin deacetylation in endothelial cells: The protective role of acetyl-L-carnitine

Methamphetamine (METH) is a powerful psychostimulant drug used worldwide for its reinforcing properties. In addition to the classic long-lasting monoaminergic-disrupting effects extensively described in the literature, METH has been consistently reported to increase blood brain barrier (BBB) permeability, both in vivo and in vitro, as a result of tight junction and cytoskeleton disarrangement. Microtubules play a critical role in cell stability, which relies on post-translational modifications such as a-tubulin acetylation. As there is evidence that psychostimulants drugs modulate the expression of histone deacetylases (HDACs), we hypothesized that in endothelial cells METH-mediation of cytoplasmatic HDAC6 activity could affect tubulin acetylation and further contribute to BBB dysfunction. To validate our hypothesis, we exposed the bEnd.3 endothelial cells to increasing doses of METH and verified that itleads to an extensivea-tubulin deacetylation mediated by HDACs activation. Furthermore, since we recently reported that acetyl-L-carnitine (ALC), a natural occurring compound, prevents BBB structural loss in a context of METH exposure, we reasoned that ALC could also preserve the acetylation of microtubules under METH action. The present results confirm that ALC is able to prevent METH-induced deacetylation providing effective protection on microtubule acetylation. Although further investigation is still needed, HDACs regulation may become a new therapeutic target for ALC.

Acetyl-L-Carnitine Prevents Methamphetamine-Induced Structural Damage on Endothelial Cells via ILK-Related MMP-9 Activity

Methamphetamine (METH) is a potent psychostimulant highly used worldwide. Recent studies evidenced the involvement of METH in the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised function. The involvement of the matrix metalloproteinases (MMPs) in the degradation of the neurovascular matrix components and tight junctions (TJs) is one of the most recent findings in METH-induced toxicity. As BBB dysfunction is a pathological feature of many neurological conditions, unveiling new protective agents in this field is of major relevance. AcetylL-carnitine (ALC) has been described to protect the BBB function in different paradigms, but the mechanisms underling its action remain mostly unknown. Here, the immortalized bEnd.3 cell line was used to evaluate the neuroprotective features of ALC in METH-induced damage. Cells were exposed to ranging concentrations of METH, and the protective effect of ALC 1 mM was assessed 24 h after treatment. F-actin rearrangement, TJ expression and distribution, and MMPs activity were evaluated. Integrin-linked kinase (ILK) knockdown cells were used to assess role of ALC in ILK mediated METHtriggered MMPs’ activity. Our results show that METH led to disruption of the actin filaments concomitant with claudin-5 translocation to the cytoplasm. These events were mediated by MMP-9 activation in association with ILK overexpression. Pretreatment with ALC prevented METH-induced activation of MMP-9, preserving claudin-5 location and the structural arrangement of the actin filaments. The present results support the potential of ALC in preserving BBB integrity, highlighting ILK as a new target for the ALC therapeutic use.

Uncovering the role of IFNAR1 in Experimental Cerebral malaria

Dissertation presented the Ph.D degree in Biology

Long-term citalopram administration reduces responsiveness of HPA axis in patients with major depression: relationship with S-citalopram concentrations in plasma and cerebrospinal fluid (CSF) and clinical response.

RATIONALE: A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a well-documented neurobiological finding in major depression. Moreover, clinically effective therapy with antidepressant drugs may normalize the HPA axis activity. OBJECTIVE: The aim of this study was to test whether citalopram (R/S-CIT) affects the function of the HPA axis in patients with major depression (DSM IV). METHODS: Twenty depressed patients (11 women and 9 men) were challenged with a combined dexamethasone (DEX) suppression and corticotropin-releasing hormone (CRH) stimulation test (DEX/CRH test) following a placebo week and after 2, 4, and 16 weeks of 40 mg/day R/S-CIT treatment. RESULTS: The results show a time-dependent reduction of adrenocorticotrophic hormone (ACTH) and cortisol response during the DEX/CRH test both in treatment responders and nonresponders within 16 weeks. There was a significant relationship between post-DEX baseline cortisol levels (measured before administration of CRH) and severity of depression at pretreatment baseline. Multiple linear regression analyses were performed to identify the impact of psychopathology and hormonal stress responsiveness and R/S-CIT concentrations in plasma and cerebrospinal fluid (CSF). The magnitude of decrease in cortisol responsivity from pretreatment baseline to week 4 on drug [delta-area under the curve (AUC) cortisol] was a significant predictor (p<0.0001) of the degree of symptom improvement following 16 weeks on drug (i.e., decrease in HAM-D21 total score). The model demonstrated that the interaction of CSF S-CIT concentrations and clinical improvement was the most powerful predictor of AUC cortisol responsiveness. CONCLUSION: The present study shows that decreased AUC cortisol was highly associated with S-CIT concentrations in plasma and CSF. Therefore, our data suggest that the CSF or plasma S-CIT concentrations rather than the R/S-CIT dose should be considered as an indicator of the selective serotonergic reuptake inhibitors (SSRIs) effect on HPA axis responsiveness as measured by AUC cortisol response.

Stem cell transplantation in brain tumors: a new field for molecular imaging?

Neural stem cells have been proposed as a new and promising treatment modality in various pathologies of the central nervous system, including malignant brain tumors. However, the underlying mechanism by which neural stem cells target tumor areas remains elusive. Monitoring of these cells is currently done by use of various modes of molecular imaging, such as optical imaging, magnetic resonance imaging and positron emission tomography, which is a novel technology for visualizing metabolism and signal transduction to gene expression. In this new context, the microenvironment of (malignant) brain tumors and the blood-brain barrier gains increased interest. The authors of this review give a unique overview of the current molecular-imaging techniques used in different therapeutic experimental brain tumor models in relation to neural stem cells. Such methods for molecular imaging of gene-engineered neural stem/progenitor cells are currently used to trace the location and temporal level of expression of therapeutic and endogenous genes in malignant brain tumors, closing the gap between in vitro and in vivo integrative biology of disease in neural stem cell transplantation.

Humoral immunity in brain aging and Alzheimer's disease.

Although the contribution of inflammatory processes in the etiology of late-onset Alzheimer's disease (AD) has been suspected for years, most studies were confined to the analysis of cell-mediated immunological reactions thought to represent an epiphenomenon of AD lesion development. Based on the traditional view of the "immunological privilege" of the brain, which excludes a direct access of human immunoglobulins (Ig) to the central nervous system under normal conditions, little attention has been paid to a possible role of humoral immunity in AD pathogenesis. In the first part of this review, we summarize evidences for a blood-brain barrier (BBB) dysfunction in this disorder and critically comment on earlier observations supporting the presence of anti-brain autoantibodies and immunoglobulins (Ig) in AD brains. Current concepts regarding the Ig turnover in the central nervous system and the mechanisms of glial and neuronal Fc receptors activation are also discussed. In the second part, we present new ex vivo and in vitro data suggesting that human immunoglobulins can interact with tau protein and alter both the dynamics and structural organization of microtubules. Subsequent experiments needed to test this new working hypothesis are addressed at the end of the review.

Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

Les transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 contribuent au maintien de l’homéostasie du cholestérol intratesticulaire

Le testicule assure la production des spermatozoïdes et la sécrétion de la testostérone. Chaque fonction est assumée par un compartiment cellulaire distinct: l’épithélium séminifère et le tissu interstitiel. Le cholestérol, présent dans les deux compartiments, est un composé indispensable aux membranes cellulaires et un précurseur essentiel de la testostérone. Dans le compartiment interstitiel, environ 40 % du cholestérol utilisé pour la production hormonale est importé du sang à partir des lipoprotéines HDL et/ou LDL. Dans l’épithélium séminifère, la cellule de Sertoli assure le contrôle et le maintien de la spermatogenèse. Elle a la capacité de synthétiser du cholestérol à partir de l’acétate in vitro, néanmoins, il n’y a pas d’évidence qu’elle le fait in vivo. De plus il existe, au niveau des tubules séminifères, une barrière hémato-testiculaire qui empêche le libre passage de plusieurs composés sanguins, y compris le cholestérol. Nous avons testé l’hypothèse qu’il existe des moyens d’importation du cholestérol sanguin, mais aussi l’exportation du cholestérol intra-tissulaire, qui contourneraient cette barrière et qui contribueraient au maintien du taux intratubulaire du cholestérol compatible avec le bon déroulement de la spermatogenèse. Nous avons comparé les taux de variation de l’expression de l’ARNm et de la protéine des transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 aux taux de variation du cholestérol libre et estérifié au cours de la spermatogenèse chez les souris normales durant le développement postnatal. Afin de mieux apprécier le niveau d’implication de chacun de ces récepteurs, nous avons examiné comment la suppression du gène d’une enzyme comme la lypase hormono-sensible (HSL) ou de celui d’un transporteur de cholestérol comme SR-BI, CD36 ou NPC1 était compensée et comment cette suppression affectait le taux de cholestérol libre et estérifié dans chacun des deux compartiments cellulaires du testicule. Nous avons dans un premier temps mis au point une nouvelle technique d’isolation des testicules en fraction enrichie en tissu interstitiel (ITf) et en tubules séminifères (STf) qui a l’avantage de mieux préserver l’intégrité des formes phosphorylées et glycosylées des protéines comparée aux techniques préexistantes. Les résultats de nos analyses ont montré que l’expression de SR-BI et CD36 étaient maximales dans les ITf au moment où les souris ont complété leur maturité sexuelle et où le niveau de synthèse de la testostérone était maximal. Dans les tubules séminifères, l’expression maximale de SR-BI et le taux le plus élevé de cholestérol estérifié étaient mesurés de façon concomitante à 35 jours après la naissance, au moment où la première vague de l’activité spermatogénétique était complétée. L’expression de l’ABCA1 était maximale au moment où le taux de cholestérol était élevé et minimale au moment où le taux de cholestérol était le plus bas, alors que le niveau d’expression de CD36 était maximal chez l’adulte au moment où le taux de spermiation était le plus élevé. L’expression de SR-BII variait peu dans les deux compartiments cellulaires durant le développement. La suppression génétique de la HSL et de NPC1, qui cause une infertilité chez les souris mâles, était accompagnée d’une accumulation de cholestérol libre et estérifié dans les tubules séminifères. Par contre, la suppression génétique de SR-BI et CD36, qui ne causent pas d’infertilité chez les souris mâles était sans impact significatif sur le taux de cholestérol intratubulaire. Nous avons montré que l’invalidation génétique d’un transporteur sélectif ou d’une enzyme du métabolisme du cholestérol était accompagnée d’un ensemble de mécanismes de compensation visant à maintenir le taux de cholestérol libre aux niveaux semblables à ceux mesurés dans les fractions tissulaires de souris normales. Ensemble, nos résultats ont montré que l’expression des transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 variait en fonction de la spermatogenèse et du taux intratesticulaire du cholestérol suggérant leur contribution au maintien de l’homéostasie du cholestérol intratesticulaire.