Mestrado em Mestrado em

Biologia Celular e MolecularBiologia Celular e Molecular Com a colaboração do Centro de Neurociências e Biologia CelularCentro de Neurociências e Biologia Celular (CNC)

Seminário II e Seminário em Neurobiologia II Apresentação de Projectos

11 - 15 Julho 2011 Anfiteatro do CNC

Programa Livro de Resumos

July 11

Molecules and Cell Biology

11:30 – 12:00 h Mapping the Molecular Determinants of mGluR2 Positive Allosteric Modulators Ana Isabel Farinha Supervisor(s): Hilde Lavreysen (Janssen Pharmaceutica NV, Beerse, Belgium) 12:00 – 12:30 h Functional screening in vitro for the effects of miRs regulated by one psychotropic drug on neuronal survival, dendrite morphology and BDNF translation Marta Jordão Supervisor(s): Enrico Tongiorgi (Laboratory of Cellular and Molecular Neuroanatomy, University of Trieste, Italy) and Carlos Duarte (DCV and CNC, University of Coimbra) 12:30 – 13:00 h Role of the Apoptosis-Inducing Factor (AIF) and p66Shc on Doxorubicin-induced Cardiomyoblast Cell Death Susana Sampaio Supervisor(s): Paulo Oliveira and Sancha Santos (Group Mitochondrial Toxicology and Disease, CNC, University of Coimbra) Neurobiology of Disease (I)

14:30 – 15:00 h Role of Matrix Metalloproteinases and Aquaporins in Blood-Brain Barrier Dysfunction Induced by Methamphetamine Ricardo Leitão Supervisor(s): Ana Paula Silva (Laboratory of Pharmacology and Experimental Therapeutics, Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra) 15:00 – 15:30 h γ-secretase modulators for the treatment of Alzheimer’s disease Joana Ramalho Supervisor(s): Bianca Van Broeck and Marc Mercken (CNS Discovery, Neuroscience; J&J Pharmaceutical Research and Development) Academic Supervisor at DCV: Carlos Duarte 15:30 – 16:00 h The role of chaperones in the clearance of aggregated and hyperphosphorylated tau in vitro Sara Calafate Supervisor(s): Arjan Buist (Dept. of Neuroscience; Johnson & Johnson Pharmaceutical Research & Development, a division of Janssen Pharmaceutica N.V) 16:00 – 16:30 h Explore the role parkin in endoplasmic reticulum stress and ER-associated protein degradation: cellular models for juvenile PD Miguel Ferreira Supervisor(s): Isabel Alonso (UnIGENe, IBMC, University of Porto, Portugal)

1

July 12 Biology of Reproduction and Human Fertility

11:00 – 11:30 h Papel do Cromossoma Sexual nas Propriedades dos Espermatozóides Humanos. Patrícia Sofia Morais Supervisor(s): João Ramalho-Santos and Ana Paula Sousa (Group Biology of Reproduction and Human Fertility, CNC, University of Coimbra) 11:30 – 12:00 h Characterisation of the Ca2+ signal propagation between the store in the neck/midpiece and the flagellum of human sperm Leonor Fernandes Supervisor(s): Stephen Publicover (School of Biosciences, University of Birmingham, UK) Academic Supervisor at DCV: João Ramalho-Santos

Stem Cell Biology

12:00 – 12:30 h Control of migration of neural stem cells by calpain signaling Manuela Azevedo Supervisor(s): Inês Araújo (Group Neuroendocrinology and Neurogenesis, CNC) 12:30 – 13:00 h Regulation of ECM mimetics on oligodendrocyte differentiation in vitro Tânia Lourenço Supervisor(s): Mário Grãos (Unidade de Biologia Celular- Biocant)

2

July 13 Genomics

10:00 – 10:30 h Pharmacogenomics and individualization of isoniazid therapy Maria Teresa Carvalho Supervisor(s): Henriqueta Coimbra Silva e Luís Mesquita (Unidade de Genética Médica da Faculdade de Medicina da Universidade de Coimbra) 10:30 – 11:00 h Investigação bigenómica e do crosstalk genómico na neuropatia óptica hereditária de Leber Tânia Sousa Supervisor(s): Manuela Grazina (Laboratório de Bioquímica Genética, Centro de Neurociências e Biologia Celular) e António Portugal (DCV, University of Coimbra)

Molecular Biotechnology

11:00 – 11:30 h Isolation and characterization of novel antimicrobial peptides from skin secretions of hylid frog, Phrynohyas resinifictrix Sónia André Supervisor(s): Ali Ladram (Biogenèse des Signaux Peptidiques (BioSiPe) – ER3, Université Pierre et Marie Curie, Paris, France) Academic Supervisor at DCV: Paula Veríssimo Neurobiology of Disease (II)

14:30 – 15:00 h Changes in transcription factors related with mitochondrial biogenesis in Alzheimer’s disease models Gladys Caldeira Supervisor(s): I. Luísa Ferreira, Tatiana Rosenstock and Ana Cristina Rego (Mitochondrial Dysfunction and Signaling in Neurodegeneration Group, Center for Neuroscience and Cell Biology, University of Coimbra) 15:00 – 15:30 h Pyruvate dehydrogenase and mitochondrial function in Huntington’s disease models – influence of histone deacetylase inhibitors Joana Rodrigues Supervisor(s): Teresa Cunha Oliveira, I. Luísa Ferreira and Ana Cristina Rego (Mitochondrial Dysfunction and Signaling in Neurodegeneration, Center for Neuroscience and Cell Biology, University of Coimbra) 15:30 – 16:00 h Role of selective kinases in alpha-synuclein phosphorylation and its relevance in Parkinson's disease Paulo A. R. Santos Supervisor(s): Rita Perfeito and Ana Cristina Rego (Mitochondrial Dysfunction and Signaling in Neurodegeneration, Center for Neuroscience and Cell Biology, University of Coimbra) 16:00 – 16:30 h Comparative study of the effect of selected mutations on α-synuclein oligomerization in living cells using BiFC Eva Rodrigues Supervisor(s): Tiago Fleming Outeiro (Univerzitatsmedizin Gottingen Abteilung, NeuroDegeneration Department of Neurodegeneration and Restorative Research) Academic Supervisor at DCV: Emília Duarte

3

16:30 – 17:00 h Protein kinase LRKK2 in Parkinson's disease: role in endocytosis. Marco Guimarães Supervisor(s): Dieder Moechars, Kristof Von Kolen and Hamdy Shaban (J&J Pharmaceutical Research and Development) Academic Supervisor at DCV: Emília Duarte

July 15

Cancer Biology

14:00 – 14:30 h Identification and characterization of a cell population with stem-like properties in canine mammary cancer Daniela Pereira Supervisor(s): Joana Paredes (Cancer Biology group, IPATIMUP) and Adelina Gama (UTAD)

14:30 – 15:00 h The involvement of cancer stem cells in mammary carcinoma development Mariana Val Supervisor(s): Carmen Alpoim (Group Mitochondrial Toxicology and Disease, CNC, and DCV, University of Coimbra)

15:00 – 15:30 h Alterações Citogenéticas e Epigenéticas e Susceptibilidade a Cancro na Síndrome de Down Patrícia Guarino Supervisor(s): Ana Bela Sarmento (Laboratorio de Biologia Molecular Aplicada e Hematologia, FMUC /CIMAGO) and Isabel Marques Carreira (Laboratório de Citogenética do Instituto de Biologia Médica, FMUC/CIMAGO) Academic Supervisor at DCV: Maria Graça Vale

Abstracts

5

Molecules and Cell Biology

Mapping the Molecular Determinants of mGluR2 Positive Allosteric Modulators Ana Isabel Farinha Janssen Pharmaceutica, Belgium

Glutamate is the most important neurotransmitter in the brain, and it is

utilized by approximately 90% of neurons. Its dysfunction is involved in several CNS disorders, such as schizophrenia and anxiety.

Glutamate receptors are divided in two main categories: ionotropic and metabotropic receptors (mGluRs). mGluRs can be divided in three groups, according to their amino-acid sequence homology, pharmacology and the preferred signal transduction mechanisms they couple to when expressed in vitro: I (mGluR1/5), II (mGluR2/3) and III (mGluR4/6/7/8). mGluRs, particularly mGluR2, can be regarded as an opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner1. Their wide and heterogeneous distribution in the CNS may enable them to act as selective targets for the development of new treatment strategies for CNS disorders2.

Several mGluR2 agonists have been identified, but the tolerance associated to them and the lack of specificity of orthosteric antagonists led to the research on positive allosteric modulators (PAMs), which bind to a site other than that of glutamate. The identification of the molecular mechanisms of such compounds, including the characterization of the binding site of PAMs in the receptor, the receptor regions involved and the amino acids critical for that binding, is essential to understand how the compound will produce its effects and to guide further research on this field.

1 Rowe B. A., Schaffhauser H., Morales S., Lubbers L. S., Bonnefous C., Kamenecka T. M., McQuiston J., Daggett L. P. (2008) Transposition of three amino acids transforms the human metabotropic glutamate receptor (mGluR)-3 positive allosteric modulation site to mGluR2, and additional characterization of the mGluR2 positive allosteric modulation Site. J. Pharmacol. Exp. Ther. Apr 22. 2 Niswender CM, Conn PJ (2010) Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295-322

6

Molecules and Cell Biology

Functional screening in vitro for the effects of miRs regulated by one psychotropic drug on neuronal survival, dendrite morphology and BDNF translation Marta Jordão, Enrico Tongiorgi and Carlos Duarte Laboratory of Cellular and Molecular Neuroanatomy, University of Trieste, Italy

Many studies demonstrated that BDNF is decreased in patients with psychiatric disorders, such as schizophrenia and depression. This neurotransmitter is a growth factor that is crucial for development and neuronal plasticity in adulthood.

In fact, the BDNF signaling seems to have an important role in the mechanism of action of antidepressant drugs, but the role in pathophysiology of depression is less clear. These drugs increase the level of BDNF and, consequently, the trkB receptor activation and its signaling, replacing defective systems and enhancing neuronal plasticity. Thus, antidepressants may somehow affect posttranscriptional regulation of BDNF and the hypothesis of the involvement of miRNAs in this field seems to be reasonable.

The posttranscriptinal modifications regulated by miRNAs (miRs) have been seen as a mechanism by which can be determined the amount of expressed proteins. MiRs are a class of a small non-coding RNAs with a length of about 22 nucleotides that regulate the amount and timing of the protein expression, beyond of determine the location where the protein is synthesized. They have the capacity of binding to a target mRNA and induce their degradation or inhibition of translation. Many miRNAs are expressed in the CNS, being expressed through a temporally and/or spatially manner during development and being vital for development of nervous system and brain morphology. One miRNA can act on target mRNAs of hundreds of genes and thus miRNAs can regulate the expression of several transcripts. Therefore, if miRNA were presented in abnormal levels in the cell, the expression of several genes could be severily perturbed and this could be the origin of several pathologies.

As demonstrated by recently studies, the overexpression of a specific kind of miRNA can nullify the capacity of BDNF to stimulate neurite growth. This result could indicate that miRNA can interact at posttrancriptional level with BDNF signaling pathways that control neuronal morphology and function. These results strongly suggest the working hypothesis of a role for miR-dependent regulation of BDNF by antidepressants.

For this purpose, from a list a miRs that seem regulated by novel antidepressant since that are up- or downregulated by this drug, our objective will be identify miRNAs that are involved in neuronal survival and their effects on the development of hippocampal neurons and, lastly, the effects of miRs up or downregulation on BDNF translation. Thus, may be demonstrated that miRs are a key to understanding the features of the pathophysiology and being a possible target of therapeutics.

7

Molecules and Cell Biology

Role of the Apoptosis-inducing Factor (AIF) and p66Shc on Doxorubicin-induced Cardiomyoblast Cell Death Susana Sampaio, Maria Sancha Santos and Paulo Oliveira Mitochondrial Toxicology and Disease Group, Center for Neuroscience and Cell Biology, University of Coimbra Doxorubicin (DOX) is a broad-spectrum antineoplastic agent prescribed for the treatment of several types of cancer. Despite its effectiveness, long term therapy with DOX is associated with a cumulative and irreversible cardiomyopathy [1]. Several mechanisms for DOX cardiotoxicity have been proposed including production of reactive oxygen species, disruption of mitochondrial function and calcium homeostasis, as well as activation of p53/Bax pathway and caspase activation [2]. Nevertheless, caspase inhibitors do not entirely prevent DOX-induced cell death [3]. This suggests that both caspase-dependent and independent mechanisms appear to be involved in DOX-induced cell death. Mitochondria play an important role in apoptosis signaling pathways through the release of different proapototic proteins [4]. One protein released from mitochondria upon apoptotic stimuli is the apoptosis-inducing factor (AIF), which translocates to the nucleus promoting cell death independently of caspase activation [3]. In another context, the adapter protein p66Shc has been suggested to play a role in oxidative stress, apoptosis and ageing, besides being implicated in the toxicity of several agents through interfering with mitochondrial antioxidant defenses [5]. Since the toxicity of DOX in cardiac cell appears to be multifactorial, we can speculate that p66Shc may contribute to DOX-induced cardiotoxicity. Thus, for the present project, we hypothesize that DOX causes cell death in cardiac cells through both caspase-dependent and independent mechanism, the latter involving AIF release from mitochondria. Also, we hypothesize that DOX causes activation of p66Shc, contributing to mitochondrial degeneration. The first specific aim is to demonstrate that AIF is involved in DOX-induced caspase-independent apoptosis in a model for cardiomyocytes, the cell line H9c2. For this specific aim, we will block caspase activity by treating cells with a caspase inhibitor previously to DOX treatment in order to demonstrate a caspase-independent mechanism of cell death. After that, extraction of cytosolic, mitochondrial and nuclear fractions in order to assess the possible translocation of AIF from mitochondria to the nucleus will be performed. Finally, we will investigate whether AIF is released due to mitochondrial outer membrane rupture following induction of the permeability transition pore. For the second specific aim, we will focus on p66Shc and investigate whether the rate between the phosphorylated form and total protein increases after DOX treatment. Secondly, we will study the consequences of DOX-induced phosphorylated p66Shc increase in the altered mitochondrial phenotype by using p66Shc inhibitors. Also, we will clarify if activation of p66Shc by phosphorylation remains present for some time after washing out DOX from the cell media, which would mimic the persistent nature of DOX cardiotoxicity. The effects of cell passage number in DOX toxicity and phosphorylated p66Shc/p66Shc content will also be investigated. Keywords: Doxorubicin; H9c2; cardiotoxicity; AIF; p66Shc 1. Menna.(2008).ChemResToxicol.21(5):978-989 2. Wallace.(2007).CardiovascToxicol.7(2):101-107. 3. Susin SA.(1999).Nature.397(6718):441-446. 4. Zhang.(2009).ArchImmunolTherExp.57(6):435-445. 5. Pinton.(2008).Cell Cycle 7(3):304-308.

8

Neurobiology of Disease

Role of Matrix Metalloproteinases and Aquaporins in Blood-Brain Barrier Dysfunction Induced by Methamphetamine Ricardo Leitão and Ana Paula Silva Laboratory of Pharmacology and Experimental Therapeutics, Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Portugal Methamphetamine (METH) is a potent and highly addictive psychostimulant which consumption in Europe has been increased over the last years. Several reports have demonstrated that radical production and oxidative stress, excitotoxicity, inflammation and mitochondrial dysfunction are some of the neurotoxic features of METH. More recently, it was shown that METH can also compromise the blood-brain barrier (BBB) function. BBB is a structure responsible for protecting the brain from toxic compounds, but nevertheless allows the passage of nutrients and several important molecules into the brain. The barrier function is determined by the endothelial cells, which together with pericytes, astrocytes, basal lamina, microglia and neurons form the neurovascular unit. There are several molecules that can be involved in BBB disruption induced by METH. Matrix metalloproteinases (MMPs), specifically MMP-2 and -9, have already been related with BBB disruption in some neuropathologies. MMPs are proteolytic enzymes capable of degrading the extracellular matrix present in basal lamina of the BBB or even the tight junction proteins that are responsible for limiting the paracellular transport across BBB. Additionally, large water fluxes continuously take place between the different compartments of the brain as well as between the brain parenchyma and the blood. Disturbances in this well-regulated water homeostasis may have deleterious effects on brain function. Aquaporins (AQPs) are water channels that contribute to water transport across BBB, being AQP4 one of the most important at the Central Nervous System (CNS). AQP4 is express on astrocytic end-feet in contact with brain vessels. Moreover, alterations in AQPs can originate cerebral edema due to abnormally increased water content and consequent brain swelling. Indeed, brain edema has been observed in several neuropathologies, including under conditions of METH consumption. Thus, the aim of my project is to clarify the possible involvement of MMPs or/and AQPs in BBB dysfunction induced by METH. For that, I will use male C57BL/6J wild-type mice with 3-4 months that will be injected intraperitoneally (i.p.) with METH (30mg/kg) and then sacrificed at 24h post-injection. After that, microvasculature will be isolated from mice brain. Protein expression of MMP-2 and -9, as well as AQP4 will be evaluated by Western Blot and immunochemistry. Also, MMPs activity will be analysed by gel zymography. If I observed changes in MMPs or AQPS, I will use inhibitors (30mg/kg BB-94 or 200 mg/kg, respectively) as follows: 1) 15 minutes prior METH; 2) 1 hour after METH exposure. Furthermore, knowing that astrocytes provide a structural barrier that helps to promote vascular integrity, and that express MMP-2 and -9, as well as AQP4, I will also use primary cultures of mouse astrocytes that will be exposed to non-toxic METH concentrations (1, 50, and 100 µM) for 24 hours. The direct effect of METH on astrocytes regarding the expression of MMPs and AQPs proteins will be evaluated as previously described. With the present work I expect to clarify the possible role of MMPs and AQP4, which could allow a new approach to prevent, or at least to diminish, the BBB dysfunction induced by METH.

9

Neurobiology of Disease γ-secretase modulators for the treatment of Alzheimer’s disease Joana Ramalho, Bianca Van Broeck, Marc Mercken, Carlos Duarte. Drug Discovery, Neuroscience Department, Janssen Research & Development, Belgium Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia in the elderly population, accounting for 50-60% of all dementia patients. (Blennow, K., et al, 2006) AD is an age-related condition characterized by progressive memory impairment, cognitive deficits, and behavioral changes. The principal neuropathological hallmarks of AD are extracellular deposition of the amyloid β (Aβ) peptide in senile plaques and intraneuronal accumulation of hyperphosphorylated tau protein in neurofibrillary tangles. As a result, there is a profound synaptic and neuronal loss located in the areas of the brain that are involved in memory consolidation and these pathological changes ultimately lead to dementia and death. (Van Broeck, B., et al, 2007) According to the amyloid cascade hypothesis the cerebral Aβ accumulation is the primary initiating factor of AD pathogenesis and that the rest of the disease process results from an imbalance between Aβ production and Aβ clearance. (Selkoe, 2004) Aβ present in biological samples has a wide heterogeneity of N- and C-terminals; therefore development of monoclonal antibodies that are specific to different Aβ species is a possible approach that can be used as a diagnostic tool or to monitor the effect of treatments. The production of Aβ peptide results from the sequential cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases turning them into the top targets for the development of Alzheimer therapeutics. (De Strooper, 2010) Currently available drugs can only improve cognitive symptoms temporarily, and no treatment can slow, stop, or even reverse the neurodegenerative process in these patients. (Chopra, et al, 2011) Thus, numerous disease-modifying strategies targeting the production and clearance of Aβ as an attempt to counter the progression of the disease are currently in clinical trials. The use of γ-secretase inhibitors (GSI) for that purpose has been investigated and some drugs have reached a Phase III trial but there were no significant cognitive improvements in the patients. Also, it is recognized that γ-secretase participates in the Notch signaling pathway, which plays a role in a variety of cell differentiation events from embryogenesis until adulthood and is responsible for releasing the Notch intracellular domain (NICD). Therefore, the use of GSI not only targets the proteolytic processing of APP but, certainly impairs Notch signaling with harmful cellular consequences. (De Strooper, Vassar, et al, 2010) Thus, γ-secretase modulators (GSMs) are preferential, since they modulate the preferred site of γ-secretase cleavage leading to a selective decrease in the production of Aβ42, the putative toxic Aβ species, and simultaneously they increase the production of shorter and less toxic Aβ peptides without interfering with the cleavage of other substrates like Notch. (De Strooper, Vassar, et al, 2010) With increasing life expectancy across the world, AD is a rapidly growing socio-economic and medical problem, and it is imperative to develop effective and safe therapeutic approaches for AD pathology that are capable to slow, stop or even reverse the progression of the disease.

10

Neurobiology of Disease

The role of chaperones in the clearance of aggregated and hyperphosphorylated Tau in vitro Sara Calafate and Arjan Buist Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium AD is a progressive neurodegenerative disorder characterized by global cognitive decline. One of the hallmarks of Alzheimer's disease is the presence of amyloid plaques and neurofibrillary tangles (NFTs) that are made of misfolded proteins. NFTs are inclusions comprised of microtubule-associated protein Tau (also known as MAPT) which is hyperphosphorylated. Tau post-translation modifications are believed to play a role inducing conformation impairment. The NFT density correlates well with cognitive decline in Alzheimer's disease suggesting the idea that Tau protein abnormalities play a critical role in the disease cascade. It has been hypothesized that when misfolded Tau is released from microtubules, it aggregates, the microtubules disintegrate, and compromises the normal function of microtubules network. This may result first in malfunctions in biochemical communication between neurons and later in the death of the cells. Many other neurodegenerative diseases like ALS, Huntington’s disease, and Parkinson’s disease are pathologically associated with misfolded proteins. Within these diseases heat shock proteins (HSPs) provide the first line of defense. HSPs are molecular chaperones that recognize misfolded client proteins and normally preserve their normal conformation. Overexpression of HSPs has been shown to reduce aggregation of a variety of neurodegenerative disease-associated proteins and their related toxicity both in vivo and in cellular models. One of the chaperones that can recognize misfolded Tau is Hsp90. Hsp90 is one of the main chaperones and can form multimeric complexes with co-chaperones. These co-chaperones control the activity of complex probably deciding the fate of client proteins between protein refolding and degradation pathway. Manipulating this complex it is possible to force degradation of Hsp90 substrates. Blocking Hsp90 mediated client refolding by interfering in the formation of the mature Hsp90 refolding complex has been shown to stimulate degradation of hyperphosphorylated Tau. The main aim of the project is to study the role of chaperones in tau degradation, analyzing the effects of Hsp90 inhibitors in tau hyperphosphorylation, aggregation and degradation. For this study a developing neuroblastoma cell model and hippocampal neurons expressing mutant tau will be used.

11

Neurobiology of Disease Explore the role parkin in endoplasmic reticulum stress and ER-associated protein degradation: cellular models for juvenile PD. Miguel Alves Ferreira and Isabel Alonso UnIGENe, IBMC – University of Porto Parkinson disease (PD) is the second most common neurodegenerative disorder and is mainly characterized by motor dysfunction, as the result of selective loss of dopaminergic neurons. Although the aetiology and pathogenesis in PD are still poorly understood, central players causing rare genetic forms of PD should prove useful to understand idiopathic PD. Among these is an Autosomal Recessive-Juvenile Parkinson’s disease (AR-JP) form, showing symptoms resembling idiopathic PD: resting tremor, rigidity, postural instability and bradykinesia, although being characterized by a young age-at-onset (usually under 40). This disorder is caused by mutations in the PARK2 gene which encodes parkin, an E3-ubiquitin ligase. Parkin misfolding seems to be the major mechanism for parkin inactivation. Sustained accumulation of unfolded or misfolded proteins results in the failure of the endoplasmic reticulum (ER) to cope with the excess of protein load, named ER stress. The unfolded protein response (UPR), leading to the activation of three protective cellular pathways, is required for the cells to cope with stressful conditions and results in the upregulation of ER stress genes, including binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP). Recent observations support the existence of escorting machinery, which connects the ubiquitylated substrates at the ER-membrane with the 26S proteasome, in a process known as ER-associated degradation (ERAD). Also, it has been shown that misfolded proteins and aggregates, even when involving cytosolic proteins, are able to induce ER stress, probably by suppressing proteasome function, an essential component of the ERAD machinery. Our aim is to clarify the role of different cellular response mechanisms in the formation and clearance of parkin aggregates, including the endoplasmic reticulum stress and endoplasmic reticulum (ER)-associated protein degradation. We will study a wide-range of mutations, located in different parkin domains, including the p.N52MfsX29 (c.155delA) and p.R402C (c.1204C>T), the most frequent mutations in Portuguese patients with juvenile Parkinson disease (our unpublished results). First, we will characterize the cellular models developed for juvenile PD, regarding aggregate type and number, as well as, cell viability. Then, we will explore the activation of an ER stress-response by the different parkin mutants, assessing UPR protein levels. Also, we will evaluate the role of ERAD in parkin cellular models, by potentiation or suppressing ERAD in the different mutants. As a result, this work will provide further clues about the effect of parkin mutations on ER function, which will yield additional insights into ER stress in Parkinson disease. Also, we will gain a broader knowledge regarding the crosstalk between the ER and UPS, through ERAD, in these disorders.

12

Biology of Reproduction and Human Fertility

Papel do Cromossoma Sexual nas Propriedades dos Espermatozóides Humanos Sofia Alçada Morais, Ana Paula Sousa e João Ramalho-Santos. Grupo de Biologia da Reprodução e Fertilidade Humana, CNC.

A procura de um método que permita seleccionar o sexo de um organismo iniciou-se há décadas. Em humanos, essa determinação está dependente da célula reprodutora masculina na medida em que, consoante o cromossoma sexual transportado pelo espermatozóide, resultarão indivíduos de um sexo ou de outro.

Até à data, o recurso à citometria de fluxo e cell sorting mostrou ser o único método verdadeiramente eficaz na separação de espermatozóides X e Y. Neste sistema de medição os espermatozóides marcados passam alinhada e individualmente por um fluxo contínuo, de modo a que a sonda utilizada possa ser excitada por um comprimento de onda específico. A fluorescência individual emitida pelas partículas vai ser detectada e convertida num sinal numérico para o computador, responsável por tratar a informação de acordo com diferentes intensidades de fluorescência. É esta possibilidade de detecção de diferentes fluorescências que permite então distinguir o diferente conteúdo de ADN existente em espermatozóides portadores de diferentes cromossomas sexuais. A diferença no conteúdo de ADN deve-se essencialmente à diferença de tamanho entre cromossomas X e Y.

Estão descritas actualmente várias aplicações para as quais o uso de técnicas de separação celular pode ser uma solução. Para o Homem, o desejo de determinar o sexo da descendência prende-se essencialmente com valores sociais ou com a tentativa de prevenir o aparecimento de doenças associadas ao sexo. No que respeita a outras espécies prevalecem os interesses económicos, nomeadamente através do melhoramento de explorações animais. O recurso a este método é ainda comum no que toca à conservação de espécies em vias de extinção.

Com efeito, o presente estudo visa a caracterização de espermatozóides humanos, tendo em conta o cromossoma sexual de que são portadores. A avaliação será feita considerando diversos parâmetros seminais, desde os mais clássicos a alguns mais funcionais.

Palavras-chave: Cromossomas Sexuais / Espermatozóides Humanos / Parâmetros

Espermáticos / Separação Celular

13

Biology of Reproduction and Human Fertility

Characterisation of the Ca2+ signal propagation between the store in the neck/midpiece and the flagellum of human sperm Leonor Cruz Fernandes, Stephen Publicover e João Ramalho-Santos School of Biosciences, University of Birmingham, UK The male gamete or sperm is a haploid, highly motile cell responsible for carrying the male genetic information of a species. In the female reproductive tract, sperm moves towards the female gamete – the oocyte or egg – in order to fertilise it. When facing the oocyte, sperm must get through two barriers: on the outside, layers of cumulus cells embedded in matrix and on the inside, the zona pellucida – a strong protein coat. [1] To achieve this, its flagellar motility needs to change to a state of hyperactivation that will alllow figure-of-eight movements due to asymmetrical bending in the proximal midpiece but little forward power. [2] The transition of swimming pattern, essential to fertilisation, is controlled by a rise in intracellular Ca2+ which can come from two sources: an extracellular one, mediated by plasma membrane Ca2+ channels and an intracellular one due to the opening of Ca2+-release channels in the store situated at the neck/midpiece region of the sperm. [3] It is believed that this store contributes to the regulation of hyperactivation [4]; however, its interaction with CatSpers (sperm-specific plasma membrane channels in the flagellum) is not very clear. Therefore, the main aim of this project is to clarify this intercommunication and the effects of store mobilisation on flagellar Ca2+. To study the Ca2+ signal propagation, fluorescent imaging, localised uncaging of Ca2+ and Ca2+ buffers will be used in loosely-tethered cells as well as inducers of CatSpers an hyperactivation such as alkaline pH, NH4Cl and others. Flagellar motility will be analised by computer assisted semen analysis (CASA). [1] Alberts B, Johnson A, Lewis J, Rafi M, Roberts K and Walter P (2007) Molecular Biology of the Cell, Fifth Edition. Garland Science Chapter 21, 1269-1304 [2] Ho HC and Suarez SS (2001) Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction 122, 519–526 [3] Jimenez-Gonzalez C, Michelangeli F, Harper CV, Barratt CL, Publicover SJ (2006) Calcium signalling in human spermatozoa: A specialized ‘toolkit’ of channels, transporters and stores. Hum Reprod Update 12 (3), 253–267 [4] Marquez B, Ignotz G and Suarez SS (2007) Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm. Dev Biol 303, 214–221

14

Stem Cell Biology

Control of migration of neural stem cells by calpain signaling Maria Manuela Azevedo and Inês Araújo Neuroendocrinology and Neurogenesis Group, Center for Neuroscience and Cell Biology, University of Coimbra

Calpains are ubiquitous calcium-dependent cysteine proteases. These proteases play an important role in physiological conditions, particularly in cell motility and migration, regulation of cell cycle and cell death [1]. There are two main calpains present in mammalian tissue, µ- and m-calpains (also known as calpain-1 and calpain-2, respectively), which are constitutively expressed. Calpains are formed by a large subunit, encoded by the genes Capn1 or Capn2, and by a common small subunit encoded by the gene Capn4. The small subunit is a regulatory subunit, and the disruption of Capn4 eliminates both µ- and m-calpain activities, showing that the small subunit is essential for the stability and activity of calpain[2]. The disruption of Capn4 seems to be closely related to early embryonic lethality, suggesting that calpains have an important role in development [3].

Neurogenesis occurs throughout life [4]. The new neurons are formed mainly in two well-defined regions of the adult mammalian CNS. Adult neurogenesis has consistently been found in the subventricular zone (SVZ) of the lateral ventricle and in the hippocampal subgranular zone. In humans, has also been demonstrated the occurrence of neurogenesis in dentate gyrus of the hippocampus [5]. Preliminary results from our group show that small molecule calpain inhibitors enhance the migration of neural stem cells, implying that calpains control migration of neural stem cells.

The aim of this study is to identify which signaling pathways are used by calpains to control migration of neural stem cells, using in vitro migration assays (scratch assays) in neural stem cells isolated from the mouse subventricular zone. We will investigate the activation of Rho, Rac, Cdc42 and the participation of integrins in calpain signaling during migration of neural stem cells, using selective inhibitors for each pathway. Furthermore, cell migration will be analysed in brain sections from wild-type and calpastatin-deficient mice, by looking at the distribution of the tracer 5-bromo-2’-deoxyuridine (BrdU), incorporated by dividing cells during DNA synthesis. The distance travelled by BrdU-positive cells from the SVZ towards the olfactory bulb will allow us to determine the migration rate. Key Words: Calpastatin; intracellular signaling; migration; neural stem cells; neurogenesis __________________________________________________________________ 1. Stifanese R, Averna M, Salamino F, Cantoni C, Mingari MC, Prato C,

Pontremoli S, and Melloni E.(2006) Arch Biochem Biophys. 456(1): 48-57. 2. Shimada M, Greer PA, McMahon AP, Bouxsein ML, and Schipani E.(2008) J

Biol Chem. 283(30): 21002-10. 3. Arthur JS, Elce JS, Hegadorn C, Williams K, and Greer PA.(2000) Mol Cell

Biol. 20(12): 4474-81. 4. Altman J and Das GD.(1965) J Comp Neurol. 124(3): 319-35. 5. Ming GL and Song H.(2005) Annu Rev Neurosci. 28: 223-50.

15

Stem Cell Biology

Regulation of ECM mimetics during oligodendrocytes differentiation in vitro Tânia Milene Pires Lourenço and Mário Grãos Cell Biology Lab, Biocant Oligodendrocytes (OLs) are the myelin-producing cells of the central nervous system (CNS). They provide the insulation of axons and facilitate the conduction of action potentials. Loss of the myelin sheath leads to anomalous nerve transmission and neuronal cell death, as in multiple sclerosis. Oligodendrocytes differentiation undergoes different stages: neural progenitors, oligodendrocyte precursor cells (OPCs), pre-OLs, immature OLs, non-myelinating mature OLs and myelinating mature OLs. The failure in remyelination and inhibition of oligodendrocytes differentiation observed in neurodegenerative diseases may be caused by modifications in the extracellular matrix (ECM) composition and rigidity occurring in the lesioned areas, which may result in loss of balance between intracellular and extracellular forces. Extracellular matrix composition and its mechanical properties play a key role in cell differentiation. OLs differentiation is modulated by ECM proteins like laminin, vitronectin and fibronectin. These ECM components bind to and activate integrins, triggering multiple signalling events between the extracellular environment and several intracellular signal transduction proteins, in which the actin cytoskeleton seems to play a key role. For example, laminin-2 is known to bind α6β1-integrins and promote oligodendrocytes differentiation and myelination. Additionally, this differentiation process seems to be directly affected by the substrate elasticity, as shown recently.

Tissues of mammalian organisms have different rigidities, ranging from 0.1 to 30,000,000 kPa. Cells can sense the rigidity of their environment and when cultured in vitro, seem to respond better when in substrates that mimic the stiffness of their native microenvironment. Depending on the substrate elasticity, neural stem cells either become neuronal, when cultured on soft substrates (0,01 to 0,5 KPa); or glial, when cultured on harder substrates (1 to 10 KPa). This effect has been suggested to recapitulate the development of the CNS, since neurons develop first (and glial cells later), in a softer environment, hence their preference for softer matrices. Other cell types respond to substrate rigidity, such as mesenchymal stem cells, which acquire myogenic, neurogenic or osteogenic phenotype when cultured on substrates whose stiffness is compliant with muscle, brain or bone, respectively.

Hydrogels functionalized with covalently-linked ECM proteins may be used to mimic tissues rigidity and ECM repertoire in vitro. We will use polyacrylamide hydrogels obtained using different percentages of acrylamide and bis–acrylamide, to obtain elastic moduli ranging from 4 to 20 kPa (characterized by rheometry). These gels will be covalently functionalized with ECM proteins (e.g.: laminin, fibronectin and poly-L-lysine as control), mimicking distinct ECM conditions. The human oligodendroglioma cell line (HOG) is considered to be at the OPCs stage and will be used as a model for OLs differentiation. HOG cells will be seeded on functionalized-hydrogels and induced to differentiate with appropriate differentiation media.

During OLs differentiation, the lineage markers A2B5 (for OPCs), GalC and myelin basic protein (MBP) (for OLs), are sequentially expressed. These will be evaluated by immunocytochemistry and fluorescence microscopy, as well as morphological changes that occur along the differentiation experiments. Activation of integrins-dependent signalling pathways relevant for OLs differentiation will also be studied by assessing the phosphorylation of Lyn and Fyn by immunocytochemistry and western-blot.

16

Genomics

Pharmacogenomics and individualization of isoniazid therapy Maria Teresa Carvalho, Henriqueta Coimbra Silva e Luís Mesquita Unidade de Genética Médica da Faculdade de Medicina da Universidade de Coimbra

Isoniazid is a pivotal agent in the treatment of tuberculosis, but it is also the most prevalent cause of drug-induced hepatotoxicity. Isoniazid plasma concentrations are highly dependent on metabolism via acetylation by the polymorfic enzyme N-acetyltransferase 2, and individuals can be classified as “fast acetylators”, intermediate acetylators or “slow acetylators”, according to their acetylation capacity. Recent genotyping methodologies enable the identification of NAT2 gene SNPs associated with the different acetylation phenotypes. In traditional Caucasian Portuguese population, SA amount to 64%. In the last decade, Portuguese population has changed due to continuous flow of migration with predictable repercussions on allele frequencies that need to be revaluated.

The most serious side effect of INH treatment is hepatoxicity, a complication with an incidence ranging from 1-36% that may be fatal or impose liver transplant. The risk factors for hepatoxicity include previous liver injury like hepatitis and genetic susceptibility. NAT2 polymorfisms determining a slow acetylator status have been the most frequently genotype implicated. Polymorphisms in genes encoding other metabolizing enzymes, like CYP2E1 and GSTM1, have also been associated with increased risk for INH-induced hepatotoxicity but reported studies have shown even more inconsistent results. According to some authors, INH-induced hepatotoxicity may also occur due to delayed hypersensitivity reactions. This could explain the difficulty to establish a cause-effect relation with acetylation efficiency. The diagnosis can be made in the presence of a positive lymphocyte transformation test (LTT), an in vitro assay that has the advantage of not exposing the patient to the sensitizing drug. In spite the supporting evidence that NAT2 genotype may significantly affect the incidence of isoniazid adverse reactions, NAT2 genotyping has not been incorporated into clinical practice. There is urgent need for prospective studies evaluating the repercussion of the characterization of patient acetylators’ status and individualization of therapy in clinical outcome. The cost of individualization of INH doses by genotyping or pharmacokinetics studies seems worth while when we have to consider the costs of a liver transplant and cyclosporine monitoring or even face a fatal outcome.

The project will enclude: a retrospective study, pharmacokinetic studies and a prospective study. The retrospective study aims to determine the frequencies of slow, intermediate and fast acetylators among tuberculosis patients, by genotyping 200 patients that have already complete their treatment, and to evaluate the association between acetylator status with Isoniazid-induced hepatotoxicity. The pharmacokinetic study will allow to establishing an adequate new standard dose of isoniazid, specific for slow-acetylators, by pharmacokinetic studies of a group of volunteers previously genotyped. The new standard dose will then be tested in a prospective random assay of 100 slow-acetylators patients (50 treated with the new dose and 50 with the standard dose). Clinical outcome, including hepatotoxicity, therapeutic success and emergency of resistance will be compared between slow-acetylators under isoniazid current standard dose and slow-acetylators under Isoniazid individualized dose. Simultaneously, patients that developed hepatitis and a similar number that didn’t developed hepatitis, recruited from retrospective and prospective studies, will be submitted to a LTT assay to evaluate hypersensitivity to Isoniazid.

17

Genomics

Investigação bigenómica e do crosstalk genómico na neuropatia óptica hereditária de Leber Tânia Sousa, António Portugal, Manuela Grazina Laboratório de Bioquímica Genética, Centro de Neurociências e Biologia Celular

A neuropatia óptica hereditária de Leber (LHON) é uma doença genética mitocondrial que se caracteriza pela morte de células ganglionares da retina, levando à atrofia do nervo óptico. A LHON afecta predominantemente indivíduos do sexo masculino em idade jovem (15-30 anos de idade), sendo a sua principal manifestação clínica a perda súbita de visão central, com evolução rapidamente progressiva. A maioria dos doentes, 90 a 95%, possuem uma das três mutações patogénicas primárias do DNA mitocondrial (mtDNA) previamente associadas à doença: 3460G>A, 11778G>A e 14484T>C. Estas variações, localizadas em genes que codificam subunidades do complexo I da cadeia respiratória mitocondrial, afectam a actividade do mesmo. No entanto, a LHON exibe penetrância incompleta, sugerindo que outros factores genéticos, mitocondriais ou nucleares, poderão estar envolvidos na etiologia da doença. Embora frequentemente esta doença se restrinja a anomalias oftalmológicas, existem casos de LHON-plus, com fenótipos mais severos que são agravados por sintomas neurológicos adicionais.

Em 2007, foi publicado um caso verdadeiramente atípico de LHON-plus no que respeita à idade de início dos sintomas, género, severidade e progressão das anomalias neurológicas. A doente em causa apresenta a mutação pontual 11778G>A do mtDNA, em diversos tecidos, sendo portadores da mesma alteração outros membros da família da linhagem materna, nomeadamente a mãe, avó, tias e tios. No entanto, apesar de alterações ligeiras de fenótipo, nenhum destes familiares desenvolveu a doença.

A maioria das proteínas mitocondriais é codificada pelo núcleo, sintetizada no citosol e posteriormente importada para a mitocôndria. Assim, os genes nucleares podem estar associados a doenças mitocondriais, nomeadamente os genes OPA1 e OPA3. Ambos codificam proteínas da mitocôndria e estão relacionados com outros tipos de atrofia óptica. Enquanto a função da proteína OPA3 permanece ainda desconhecida, a proteína OPA1 sabe-se estar envolvida na manutenção das cristas mitocondriais, na fusão mitocondrial, na regulação da apoptose e ainda na manutenção do mtDNA. Um estudo publicado no ano 2010 revelou que a expressão do gene OPA1 se encontra diminuída em doentes com LHON, sugerindo que este gene poderá constituir um factor que contribui para a patogénese da doença. Polimorfismos no mtDNA, em certas combinações, podem também actuar em sinergia com as mutações da LHON, aumentando o risco de expressão da doença ou causando uma evolução clínica mais grave.

Assim, o objectivo deste projecto é analisar a sequência completa do mtDNA e de genes nucleares que codificam proteínas mitocondriais, previamente associados a atrofia óptica, OPA1 e OPA3.

O projecto em questão será realizado através da sequenciação total do mtDNA e das regiões codificantes dos genes OPA1 e OPA3 para todos os membros da referida família. A amplificação do DNA será feita por PCR (polymerase chain reaction) seguida de electroforese em gel de agarose. A sequenciação será realizada pelo método de sequenciação automática, com análise posterior das sequências recorrendo ao software Seqscape v.2.5. Todas as alterações encontradas serão submetidas a análise in silico para avaliação de patogenicidade.

Esperamos encontrar variações genéticas que possam explicar a penetrância incompleta da doença nesta família bem como o fenótipo atípico verificado no caso index.

18

Molecular Biotechnology

Identification and characterization of novel antimicrobial peptides from skin secretions of the Amazon Milk Frog, Trachycephalus resinifictrix Sónia Maria Costa André, Ali Ladram and Paula Veríssimo Laboratory “Biogenesis of peptide signals (BioSiPe)”, ER3 UPMC, Pierre and Marie Curie University, France

The growing problem of resistance to conventional antibiotics and the need to develop new compounds with original modes of action has stimulated interest in antimicrobial peptides (AMPs) as substitutable pharmaceuticals. AMPs are key components of the innate immune system of several organisms (from microorganisms to vertebrates), acting as the first line of host defense against pathogens. Amphibian skin secretions represent one of the richest natural sources of AMPs. To date, approximately 37% of these peptides can be found in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php). These Gene-encoded AMPs are synthesized in frog serous glands as inactive precursors that are post-translationally modified to yield mature bioactive products. The precursors of the dermaseptin superfamily share structural similarities, i.e. a highly conserved N-terminal preproregion and a hypervariable antimicrobial C-terminal domain. Mature amphibian AMPs are hydrophobic, cationic, and adopt mainly an amphipathic α-helical structure with apolar and polar faces. These characteristics enable these peptides to interact with, and insert into the anionic outer leaflet of bacterial cytoplasmic membrane, thereby provoking membrane permeabilization and/or disruption and leading to cell death. This original membrane-lytic mechanism, in addition to the potent and broad-spectrum activity (bacteria, yeast, fungi, protozoa, viruses) of AMPs, makes it very difficult for microorganisms to modify their membrane composition and/or organization in order to develop resistance. Therefore, AMPs are promising candidates with therapeutic potential as a novel class of anti-infective agents.

Our study aims to analyze the AMPs content of the frog Trachycephalus resinifictrix, a South American tree frog of the family Hylidae (subfamily Hylinae) also referred to as Amazon Milk Frog because of its milky and poisonous secretions when threatened. The family Hylidae comprises 901 species and is divided into three subfamilies: Phyllomedusinae (58 sp.), Pelodryadinae (197 sp.) and Hylinae (646 sp.) (http://research.amnh.org/vz/herpetology/amphibia/index.php). Many different AMPs have already been characterized from frogs belonging to the two first subfamilies. Nevertheless, for the third subfamily Hylinae, which have a much higher number of species, only very few AMPs were identified and no information on the precursor sequences is available. T. resinifictrix is a frog of the subfamily Hylinae that has not yet been studied. Through bioguided fractionation of its skin secretions and biochemical and biophysical studies, we propose to isolate novel AMPs and determine their structure and mechanism of action. We plan also to characterize the precursors of these AMPs using molecular biology approaches.

Keywords: Antimicrobial peptides, family Hylidae, subfamily Hylinae, Trachycephalus resinifictrix, frog skin, bioguided fractionation, structure, mechanism of action.

19

Neurobiology of Disease Changes in transcription factors related with mitochondrial biogenesis in Alzheimer’s disease models Gladys Caldeira, I. Luísa Ferreira, Tatiana Rosenstock and A. Cristina Rego Mitochondrial Dysfunction and Signaling in Neurodegeneration Group, Center for Neuroscience and Cell Biology, University of Coimbra Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and assails about 10% of the world’s population over 65 years of age and 50% of the population over 85 years of age. Neurofibrillary tangles composed by hyperphosphorylated tau and extracellular amyloid β (Aβ) peptide accumulation are hallmarks of the disease. Intraneuronal Aβ has been observed in AD patient brains and interacts with several cellular compartments including the mitochondria. Interestingly, some transcription factors that have been implicated in mitochondrial biogenesis might be altered in AD, such as Nrf2 (nuclear factor erythroid derived 2-related factor 2), PGC-1α (peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1 α) and Tfam (mitochondrial transcription factor). Nrf2 plays a central role in the regulation of cellular redox state and is significantly reduced in AD brains, whereas Tfam is needed for mitochondrial DNA maintenance and transcriptional initiation possibly through the interaction with Nrf2. Moreover, PGC-1α co-activates the transcription of genes involved in mitochondrial biogenesis, and increases the activity of Tfam and the expression of Nrf1 and 2 in muscle. In the context of AD, elevated levels of Aβ1-42 coincide with low levels of PGC-1α in AD transgenic mice cortico-hippocampal neurons and the expression of PGC-1α in those neurons attenuated FoxO3a (forkhead transcription factor), activating the non-amyloidogenic processing of amyloid precursor protein. CREB (cAMP response-element binding protein) and CBP (CREB binding protein) which interact with PGC-1α have also been linked to the control of mitochondrial functions. Despite evidence of changes in transcription activity in AD, it is not known if Aβ is translocated to the nucleus and if it can modulate gene expression through interaction with transcription factors. Therefore, we aim to determine if Aβ peptide can be found in the nucleus, and if the nuclear protein levels of PGC-1α, Nrf2, Tfam, CREB and/or CBP as well as mRNA levels of candidate proteins regulated by these transcription factors are altered in samples from cortical neurons of 3xTg-AD mice and in cortical neurons exposed to oligomeric Aβ1-42. Considering our recent findings that oligomers of Aβ1-42 increase intracellular Ca2+ and affect mitochondrial function through the activation of N-methyl-D-aspartate receptors, this pathway will be also explored as a modifier of transcriptional changes. The protein expression levels of these transcription factors will be also evaluated in cortex, liver and muscle (considering that AD patients suffer severe metabolic derangements and muscle wasting) of 3xTg-AD versus wild-type mice, and in lymphocytes from AD patients versus age-matched controls. According with the results, we may also explore if these transcription factors are post-translationally modified in nuclear extracts from cultured neurons and brain tissue. Moreover, we will assess whether selective transcriptional modifications underlie differential expression and activity of proteins important for ROS detoxification and mitochondrial ATP generation, which may influence the decrease in mitochondrial density/activity and cellular dysfunction in AD.

20

Neurobiology of Disease  

Pyruvate dehydrogenase and mitochondrial function in Huntington’s disease models – influence of histone deacetylase inhibitors Joana Rodrigues, Teresa Oliveira, I. Luisa Ferreira, A. Cristina Rego Mitochondrial Dysfunction and Signaling in Neurodegeneration, Center for Neuroscience and Cell Biology, University of Coimbra

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease, characterized by selective neuronal loss of striatal neurons. It is marked by involuntary body movements (e.g. chorea), slurred speech, emotional disturbances and dementia. HD is caused by a mutation (CAG expansion) in the IT15 gene (or HD gene), which encodes the protein huntingtin (Htt). In HD patients, mutant Htt (mHtt) presents an increased number of polyglutamines in its N-terminal portion. Among other things, mHtt interferes with the ubiquitin-proteasome system (UPS), apoptosis mechanisms, autophagy, transcription and also mitochondrial homeostasis. Transcriptional deregulation involves decreased histone acetylation and thus the use of histone deacetylase (HDACs) inhibitors has shown to have a protective effect. In the mitochondria, mHtt is known to affect membrane potential, calcium buffering capacity, oxidative phosphorylation (OXPHOS) and production of reactive oxygen species (ROS), mitochondrial trafficking and dynamics, DNA integrity, and also transcriptional regulation. Cytoplasmic hybrids (cybrids), produced by fusing wild-type rho-zero cells (without functional mitochondria) with platelets from HD patients, have been used to study mitochondrial dysfunction in HD. Other HD models, such as lymphoblasts from HD patients, striatal cells from mice expressing the human mHtt (Q111 vs the wild type, Q7) or the YAC128 cells (from mice bearing the yeast artificial chromosome expressing human mHtt gene with 128 CAGs repeats) have been used to recapitulate the mechanisms underlying the human pathology. Recent studies in our group have shown that pyruvate dehydrogenase (PDH) activity and expression were decreased in HD cybrids, resulting in changes in mitochondrial bioenergetics in these cells. However, since HD cybrids do not actively express mHtt, we will study PDH levels and activity in other HD models (lymphoblasts, Q111, YAC128 vs corresponding wild-type models). This will hopefully allow a better understanding on the importance of PDH dysfunction in HD, and its relation with mHtt. The relation between the levels of PDH, ubiquitin proteasome system activity and autophagy will also be assessed in the previously mentioned models, by inhibiting those pathways and measuring the PDH levels over time. Similarly, we will evaluate the effect of HDAC inhibitors in the apoptosis levels and proliferation of the HD lymphoblasts. In this context, we will also measure PDH levels and the levels of mitochondrial transcription factors like PCG-1α (peroxisome proliferation-activated-γ (PPARγ) coactivator 1α) and mitochondrial transcription factor A (TFAM), as well as measure oxygen consumption and ATP production. In order to elucidate the role of the nuclear mutation in the development of mitochondrial pathogenesis, it would be interesting to create a new type of cybrids, using rho-zero cells obtained from HD patient’s lymphoblasts (with normal mitochondria), and characterize this model (for example, for the presence of mHtt, mitochondrial integrity, oxygen consumption, activity and integrity of mitochondrial respiratory chain complexes, or PDH levels). These cybrids would be of extreme importance in the understanding of mitochondrial-nuclear dynamics in HD, particularly the role of nuclear transcription in mitochondrial function. With this work, we hope to contribute to a better understanding of mitochondrial dysfunction in HD and to better characterize the protective role of HDAC inhibitors.

21

Neurobiology of Disease Role of selective kinases in alpha-synuclein phosphorylation and its relevance in Parkinson's disease Paulo A. R. Santos, Rita Perfeito and A. Cristina Rego Center for Neuroscience and Cell Biology and Faculty of Medicine, University of Coimbra, Coimbra

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder which pathology is characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. The neuropathological evidence of the disease are the progressive and intense loss of neuromelanin containing dopaminergic neurons in the substantia nigra pars compacta (SNpc) with presence of Lewy bodies (LB). Although, neuronal loss in SNpc is unquestionable, there is generalized neurodegeneration in the central nervous system (CNS). Clinical manifestations of this neurodegenerative disease include motor impairments involving postural instability, bradykinesia, resting tremor, gait difficulties and rigidity[1]. Aggregation of α-synuclein (α-syn), an abundant and conserved presynaptic protein, is involved in several neurodegenerative diseases known as synucleinopathies, including PD[2]. Emerging evidences suggest that phosphorylation may play an important role in the oligomerization and fibrillogenesis, Lewy body formation and neurotoxicity of α-syn in vivo [3]. Immunohistochemical and biochemical studies revealed that, the majority of α-syn within inclusions (isolated from Parkinsonian patients and other patients with synucleinopathies) is phosphorylated at Serine129 (S129-P). It remains unclear which kinases and phosphatases are responsible for regulating α-syn phosphorylation at S129 in vivo. Nevertheless, a series of in vitro and cell culture-based essays have identified several kinases, which phosphorylate α-syn at S129 and/or S87, including casein kinases (CKI and CKII) and the G protein-coupled receptor kinases (GRKs 1, 2, 5, and 6), leucine-rich repeat kinase 2 (LRRK2), and polo-like kinases (PLKs)[4]. Taking this into account, our project aims to clarify which kinases are involved in regulating the phosphorylation of α-syn. Thus, the role of LRRK2, CK2, PLK1, PLK2 and GRK5 in phosphorylating α-syn at serines 87 and 129 will be assessed in neuroblastoma (SH-SY5Y) cells conditionally expressing wild-type α-syn, in the absence or presence of glial-derived neurotrophic factor (GDNF, a neurotrophic factor largely described to promote the survival of dopaminergic neurons [5]) With this purpose, the effect of pharmacological kinase inhibitors and knockdown (using siRNAs) of selective kinases will be tested in order to assess the most important group of kinases involved in the phosphorylation of each of these α-syn serine residues. Furthermore, we will explore whether modifying S87-P and/or S129-P may have implications in the formation of Lewy body-like α-syn aggregates and toxicity in differentiated cells overexpressing α-syn, a model of PD pathogenesis. References: [1] Thomas B.and Beal M. F. (2007) Parkinson’s disease. Human Molecular Genetics, Vol. 16, Review Issue 2. [2] Uversky V. N. (2007) Neuropathology, biochemistry, and biophysics of α-synuclein aggregation. Journal of Neurochemistry, 103, 17–37. [3] Mbefo M. K., Paleologou K. E., Boucharaba A., Oueslati A. , Schell H. , Fournier M., Olschewski D., Yin G., Zweckstetter M., Masliah E., Kahle P. J., Hirling H. and Lashuel H. A. (2010) Phosphorylation of Synucleins by Members of the Polo-like Kinase Family. The journal of Biological Chemistry, 285, 2807–2822. [4] Paleologou K. E., Oueslati A., Shakked G., Rospigliosi C. C., Kim Hai-Young, et al. (2010) Phosphorylation at S87 Is Enhanced in Synucleinopathies, Inhibits α-Synuclein Oligomerization, and Influences Synuclein-Membrane Interactions. The Journal of Neuroscience, 30, 3184 –3198. [5] Airaksinen M, Saarma M (2002). "The GDNF family: signalling, biological functions and therapeutic value". Nat Rev Neurosci 3 (5): 383–394.

22

Neurobiology of Disease Comparative study of the effect of selected mutations on alpha-synuclein oligomerization in living cells using BiFC Eva Sofia Ferreira Rodrigues and Tiago Fleming Outeiro Department of NeuroDegeneration and Restorative Research, University Medizin Gottingen, Gottingen, Germany Alpha-synuclein (α-syn) plays a central role in pathogenesis of both idiopathic and familial Parkinson’s disease (PD) cases and is the major component of protein inclusions called Lewy bodies (LBs), recognized as the pathological hallmark of this disorder. α-Syn appears to aggregate into fibrillar inclusions in LBs through the formation of intermediate species, generally referred to as oligomeric species. Altogether, these different species are thought to lead to different degrees of toxicity causing neurodegeneration. PD-associated α-syn mutations (A53T, A30P and E46K) and several post-translational modifications (PTMs) have been described to modulate the propensity of a-syn to aggregate. Little is still known about the molecular determinants of oligomerization in neurodegenerative diseases. For this purpose, we will generate selected mutations on α-syn and investigate their behavior in living cells through biomolecular fluorescence complementation (BiFC). This assay is based on the association between two non-fluorescent fragments of a fluorescent protein, which are fused with the protein of interest. When the protein of interest dimerizes it facilitates the association between the protein fragments and forms a bimolecular fluorescent complex. Thus, the BiFC assay will allow us to determine the effect different mutations play on the oligomerization of α-syn, shedding light into the molecular mechanisms underlying neurodegeneration.

23

Neurobiology of Disease Protein kinase LRRK2 in Parkinson’s Disease ‐ role in endocytosis Marco Rafael Machado Guimarães Johnson & Johnson Pharmaceutical Research and Development (Beerse, Belgium) Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by severe motor symptoms (tremor, postural imbalance, slowness of movement and rigidity). Nevertheless cognitive and psychiatric symptoms such as depression and dementia are also common [1]. The typical hallmarks of PD, in post-mortem brain tissue, are selective loss of dopaminergic (DAergic) neurons and the presence of Lewy bodies (LBs) [1]. The pathogenesis of PD is not fully understood, but aging, environmental factors and genetic susceptibility are suggested to play an important role in PD [2]. The majority is sporadic PD but many loci/gene have been linked to PD such as PARK1 (α-synuclein); PARK2 (Parkin); PARK5 [Ubiquitin C-terminal hydrolase L1 (UCH-L1)]; PARK6 [PTEN-induced putative kinase 1 (PINK1)], PARK7 (DJ-1) and PARK8 [Leucine rich repeat kinase 2 (LRRK2)] [2]. LRRK2 is a large, complex, multidomain protein containing kinase and GTPase enzymatic activities and multiple protein–protein interaction domains. Mutations in LRRK2 cause autosomal dominant PD, far the most common cause of familial PD. The best characterized mutation, G2019S, leads to increased kinase activity, and mutations in the GTPase domain, such as R1441C/G, have also been reported to influence kinase activity [1]. The role of LRRK2 however remains to be established, but several implications have been described in decreased neurite outgrowth, mitochondrial dysfunction, increased protein translation, altered endocytosis and autophagy [1]. Impairments in endocytosis lead to problems in synaptic transmission in the long run, especially during intense neuronal activity, where the vesicle replenishment from the endosomal compartments is crucial for effective neurotransmitter secretion [3, 4]. In this project different primary neuronal cultures of wild type and LRRK2 knock out mouse and rat models as well as wild type and LRRK2 transgenic mouse models will be established. Next electrophysiological studies (patch clamp) combined with microscopic studies will be performed on such primary cultures to evaluate the effect of LRRK2 ablation/overexpression on synaptic vesicle endocytosis. Keywords: Parkinson’s Disease, LRRK2, endocytosis, synaptic vesicles

1. Berwick DC, Harvey K: LRRK2 signaling pathways: the key to unlocking neurodegeneration? Trends in cell biology 2011, 21(5):257-265. 2. Nuytemans K, Theuns J, Cruts M, Van Broeckhoven C: Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update. Hum Mutat 2010, 31(7):763-780. 3. Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, De Astis S, Meixner A, Sarioglu H, Vogt-Weisenhorn DM, Wurst W et al: LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool. The Journal of neuroscience : the official journal of the Society for Neuroscience 2011, 31(6):2225-2237. 4. Shin N, Jeong H, Kwon J, Heo HY, Kwon JJ, Yun HJ, Kim C-H, Han BS, Tong Y, Shen J et al: LRRK2 regulates synaptic vesicle endocytosis. Experimental Cell Research 2008, 314(10):2055-2065.

24

Cancer Biology

Identification and characterization of a cell population with stem-like properties in canine mammary cancer Daniela Pereira, Joana Paredes, Adelina Gama IPATIMUP, Porto; CECAV-UTAD, Vila Real Mammary gland tumours represent the most common neoplasia in dogs. As in humans, mammary cancer constitutes an important disease in the veterinary setting, with dogs being suggested as a model for human breast cancer. Currently, one of the most motivating concepts that are being explored in the cancer research field is the cancer stem cell hypothesis, which states that a minority of transformed stem cells, or progenitors with acquired self-renewal properties, are the source of tumour cell renewal and thereby determine tumour behaviour. In canine mammary cancer, a reduced number of studies focused on the existence of cancer stem cells. Non-adherent spheres have been generated from normal and neoplastic mammary tissue, which were enriched in progenitor/stem cells and exhibited tumour-initiating potential. Recently, spheres derived from a canine mammary adenocarcinoma cell line were characterized, showing high expression of stem-cell related genes, confirming sphere system as a useful tool for the identification of cancer stem cells in canine mammary cancer. If stem/progenitor cells are really the targets for transforming events in canine mammary gland, as proposed for human breast cancer, the elucidation of molecular pathways, which regulate self-renewal activity of cancer stem cells and their interaction with the environmental niche, will provide potential therapeutic targets for this disease. Recent observations imply that there is a crosstalk between epithelial-mesenchymal transition (EMT) and cancer stem cell properties. EMT is a multistep process in which cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. This process is ultimately thought to promote cancer cell progression and invasion. A hallmark of EMT is the loss of E-cadherin expression, a cell adhesion molecule that participates in homotypic adherent junctions. Recently, it was also showed that activation of β-catenin pathway is required for the maintenance of EMT-associated stem cell-like properties. The main goal of this project is to identify, isolate and characterize cancer stem cells from canine mammary cancer. With this purpose, we defined the following aims: 1. To investigate the presence of a stem cell subpopulation in canine mammary carcinomas by analysing ALDH1, CD44 and CD24 expression; 2. To investigate the ability of canine mammary carcinoma cell lines to grow in anchorage independent conditions as spheroids. 3. To access the differentiation and self-renewal potential of cells grown as spheroids; 4. To access the migratory and invasive potential of spheroid derived cells, in comparison to the respective parental cell lines. 5. To characterize spheroid derived cells and the respective parental stem cell phenotype, by analysing ALDH1, CD44 and CD24 expression. 6. To investigate a possible link between EMT and stem cell phenotype, by analysing the expression of vimentin, N-cadherin, E-cadherin and β-catenin in spheroid derived cells and parental cell lines. With this study we intend to expand the knowledge on cancer stem cells in canine mammary cancer, hopefully providing insights into canine mammary carcinogenesis, namely in the identification of therapeutic targets.

25

Cancer Biology

The involvement of cancer stem cells in mammary carcinoma development Mariana Monteiro Loureiro Val and Maria Carmen Martins de Carvalho Alpoim CNC - Grupo de Toxicologia

Breast cancer, the most common type of cancer in women, encompasses a

wide spectrum of diseases with diverse histological types ranging from low-grade lesions, that are not life threatening, to high-grade lesions that may harbor foci of invasive breast cancer.

Breast tumors display a high degree of intra-tumor heterogeneity that drives the tumor evolution responsible for therapeutic resistance, recurrence, and tumor progression. Cancer cells with stem cell–like properties in particular have been proposed to play a critical role in metastatic progression and resistance to commonly used cancer treatment. These cells can be identified by various functional assays (e.g., tumor spheres and xenograft assays) and using specific cell-surface markers. For example, the CD44+CD24– breast cancer cell population has been shown to be enriched for tumor-initiating and chemotherapy-resistant.

The exact origin of CSCs is still controversial. It is currently accepted that they may derive from tissues’ normal stem cells, immature progenitor cells or from differentiated cells which undergo de-differentiation and re-acquire stem cell-like features. These cells have also been recently blamed for the observed resistance to the conventional therapies.

Aiming to understand the relationship between CSCs phenotype and their tumorigenic and metastatic potential, we propose to look for CSCs in two different cellular systems of human breast carcinoma. Using the sphere-forming assay, CSCs will be isolated and subsequently characterized by flow-cytometry, western blot, immunocytochemistry and G-banding karyotyping. It is expected to understand: i) the contribution of CSCs for the breast carcinoma development; ii) their tumorigenic and metastatic potential; iii) their involvement in the development of resistance to conventional therapies; iv) the meaning of their cytogenetic individual signature. The use of primary cultures, established from patients’ breast cancer biopsies, will terminally confirm the achieved in vitro results and will enhance our knowledge on breast cancer development.

26

Cancer Biology

Alterações Citogenéticas e Epigenéticas e Susceptibilidade a Cancro na Síndrome de Down Patrícia Guarino, Ana Bela Sarmento Ribeiro e Isabel Marques Carreira Laboratório de Biologia Molecular Aplicada e Hematologia e Laboratório de Citogenética do Instituto de Biologia Médica (FMUC/CIMAGO e CNC/UC).

A Síndrome de Down (SD) é a forma mais comum de aneuploidia

constitucional, afectando cerca de 1 em 700 nascimentos. Está associada à presença de uma cópia adicional do cromossoma 21, sendo também denominada de trissomia 21. Esta síndrome apresenta características morfológicas típicas que parecem resultar da dosagem anormal de certos genes presentes na cópia adicional. As crianças com SD têm um risco de desenvolver cancro, 10 a 20 vezes mais elevado, relativamente a crianças sem SD, nomeadamente Leucemia Linfoblástica Aguda (LLA) e Leucemia Mieloblástica Aguda (LMA), mais especificamente o subtipo Leucemia Megacarioblástica Aguda (LMCA). Pelo contrário, verifica-se baixa incidência de tumores sólidos nas crianças com SD. Alguns genes localizados na banda 21q22 estão associados ao aumento da incidência de leucemia na SD, nomeadamente os genes ERG, ETS2 e RUNX1. Em crianças com SD, a LMCA pode ser precedida por uma Doença Mieloproliferativa Transitória (DMT), e em alguns dos casos esta pode desaparecer espontaneamente sem intervenção terapêutica. Tanto a DMT como a LMCA, associadas à SD, são caracterizadas por uma mutação que ocorre in útero, e que atinge um factor de transcrição localizado no cromossoma X. Esta alteração genética conduz à expressão da isoforma pequena de GATA1, a GATA1s, uma proteína que promove a proliferação incontrolada dos megacariócitos. GATA1s pode cooperar com oncogenes localizados no cromossoma 21, como RUNX1, ERG, ETS2 e miR-125, levando ao desenvolvimento de LMCA na SD.

Por outro lado, foi identificada uma mutação no gene que codifica a Cinase Janus 2 (JAK2) em doentes com LLA associada à SD. Além disso, durante a gravidez, a deficiência em folato in útero, pode ser um factor de risco para a SD e para o desenvolvimento de LLA. De facto, o folato é um dador de unidades de um carbono, e a sua deficiência pode levar à hipometilação do ADN, expressão génica aberrante e instabilidade cromossómica. Isto sugere uma relação entre a sua deficiência e algumas doenças como a SD. Recentemente foram identificados alguns polimorfismos em genes que codificam enzimas envolvidas nas vias de metilação, e que podem contribuir para o desenvolvido de várias doenças. Por outro lado, a SD é causada por uma falha na separação dos cromossomas durante a meiose, designada não-disjunção cromossómica. Cerca de 90% dos casos de não-disjunção do cromossoma 21 devem-se a erros meióticos maternos que ocorrem maioritariamente durante a Meiose I (MI), sendo mais frequente na idade materna avançada. A não-disjunção paterna ocorre apenas em 5-10% dos casos com SD, não sendo a idade paterna um factor de risco. Neste caso, a maioria dos erros ocorrem durante a Meiose II (MII). Este projecto tem como objectivos estudar os níveis de expressão de determinados genes localizados no cromossoma 21 e a sua relação com a modulação epigenética, de modo a identificar o seu papel no desenvolvimento de cancro/leucemias na SD; e analisar a origem do erro da não-disjunção meiótica que ocorre nestes doentes.

Assim, espera-se contribuir para o aumento do conhecimento sobre os mecanismos envolvidos no desenvolvimento de leucemia em doentes com SD.