Integrating quantitative proteomics and metabolomics in a cellular model of diabetic retinopathy

Diabetic retinopathy is the leading cause of visual loss in individuals under the age of 55. Most investigations into the pathogenesis of diabetic retinopathy have been concentrated on the neural retina since this is where clinical lesions are manifested. Recently, however, various abnormalities in the structural and secretory functions of retinal pigment epithelium that are essential for neuroretina survival, have been found in diabetic retinopathy. In this context, here we study the effect of hyperglycemic and hypoxic conditions on the metabolism of a human retinal pigment epithelial cell line (ARPE-19) by integrating quantitative proteomics using tandem mass tagging (TMT), untargeted metabolomics using MS and NMR, and 13C-glucose isotopic labeling for metabolic tracking. We observed a remarkable metabolic diversification under our simulated in vitro hyperglycemic conditions of diabetes, characterized increased flux through polyol pathways and inhibition of the Krebs cycle and oxidative phosphorylation. Importantly, under low oxygen supply RPE cells seem to consume rapidly glycogen storages and stimulate anaerobic glycolysis. Our results therefore pave the way to future scenarios involving new therapeutic strategies addressed to modulating RPE metabolic impairment, with the aim of regulating structural and secretory alterations of RPE. Finally, this study shows the importance of tackling biomedical problems by integrating metabolomic and proteomics results.

Dysregulation of REST-regulated coding and non-coding RNAs in a cellular model of Huntington's disease

Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington's disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brain-derived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity. We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down. We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse. These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.

Rescue of gene expression by modified REST decoy oligonucleotides in a cellular model of Huntington's disease

Transcriptional dysfunction is a prominent hallmark of Huntington's disease (HD). Several transcription factors have been implicated in the aetiology of HD progression and one of the most prominent is repressor element 1 (RE1) silencing transcription factor (REST). REST is a global repressor of neuronal gene expression and in the presence of mutant Huntingtin increased nuclear REST levels lead to elevated RE1 occupancy and a concomitant increase in target gene repression, including brain-derived neurotrophic factor. It is of great interest to devise strategies to reverse transcriptional dysregulation caused by increased nuclear REST and determine the consequences in HD. Thus far, such strategies have involved RNAi or mutant REST constructs. Decoys are double-stranded oligodeoxynucleotides corresponding to the DNA-binding element of a transcription factor and act to sequester it, thereby abrogating its transcriptional activity. Here, we report the use of a novel decoy strategy to rescue REST target gene expression in a cellular model of HD. We show that delivery of the decoy in cells expressing mutant Huntingtin leads to its specific interaction with REST, a reduction in REST occupancy of RE1s and rescue of target gene expression, including Bdnf. These data point to an alternative strategy for rebalancing the transcriptional dysregulation in HD.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multifactorial and remain unclear. Here we examined DNA damage;p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93) --> Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis. (C) 2010 Elsevier B.V. All rights reserved.

Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin.

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.

Development of a polyplexes-based miRNA delivery system in a 3D-bioplotted osteoarthritis cellular model.

L'osteoartrite (OA) è una patologia infiammatorio/degenerativa ossea per la quale non sono disponibili terapie causali efficaci ma solo approcci palliativi per la riduzione del dolore cronico. E’ quindi giustificato un investimento per individuare nuove strategie di trattamento. In quest’ottica, lo scopo di questa tesi è stato quello di indagare l’efficacia di polyplexi a base di chitosano o di PEI-g-PEG in un modello cellulare 3D in vitro basato su un hydrogel di Gellan Gum Metacrilato (GGMA) con a bordo condrociti in condizioni simulate di OA. Inizialmente sono state studiate la dimensione e il potenziale-Z di un pool di formulazioni di poliplexi. Quindi se ne è valutata la citocompatibilità utilizzando cellule staminali mesenchimali immortalizzate Y201. Infine, una miscela di GGMA, cellule e polyplexi è stata utilizzata per la stampa 3D di campioni che sono stati coltivati fino a 14 giorni. La condizione OA è stata simulata trattando le cellule con una miscela di citochine implicate nello sviluppo della malattia. Tutte le formulazioni a base di chitosano e due basate su PEI-g-PEG si sono dimostrate citocompatibili e sono hanno veicolato i miRNA nelle cellule (come mostrato dai risultati di analisi in fluorescenza). I risultati delle colorazioni H&E e AlcianBlue hanno confermato che il terreno condizionato ha ben ricreato le condizioni di OA. I polyplexi a base di chitosano e PEI-g-PEG hanno controbilanciato gli effetti delle citochine. Risultati incoraggianti, anche se da approfondire ulteriormente, provengono anche dall’analisi di espressione (RT-PCR) di cinque geni specifici della cartilagine. Concludendo, questo modello ha ben riprodotto le condizioni di OA in vitro; il chitosano ha mostrato di essere un adeguato veicolo per un trattamento a base di miRNA; il PEI-g-PEG si propone come un'alternativa più economica e ragionevolmente affidabile, sebbene il rischio di citotossicità alle concentrazioni più elevate richieda una più esteva validazione sperimentale.

A model of cellular dosimetry for macroscopic tumors in radiopharmaceutical therapy.

PURPOSE: In the radiopharmaceutical therapy approach to the fight against cancer, in particular when it comes to translating laboratory results to the clinical setting, modeling has served as an invaluable tool for guidance and for understanding the processes operating at the cellular level and how these relate to macroscopic observables. Tumor control probability (TCP) is the dosimetric end point quantity of choice which relates to experimental and clinical data: it requires knowledge of individual cellular absorbed doses since it depends on the assessment of the treatment's ability to kill each and every cell. Macroscopic tumors, seen in both clinical and experimental studies, contain too many cells to be modeled individually in Monte Carlo simulation; yet, in particular for low ratios of decays to cells, a cell-based model that does not smooth away statistical considerations associated with low activity is a necessity. The authors present here an adaptation of the simple sphere-based model from which cellular level dosimetry for macroscopic tumors and their end point quantities, such as TCP, may be extrapolated more reliably. METHODS: Ten homogenous spheres representing tumors of different sizes were constructed in GEANT4. The radionuclide 131I was randomly allowed to decay for each model size and for seven different ratios of number of decays to number of cells, N(r): 1000, 500, 200, 100, 50, 20, and 10 decays per cell. The deposited energy was collected in radial bins and divided by the bin mass to obtain the average bin absorbed dose. To simulate a cellular model, the number of cells present in each bin was calculated and an absorbed dose attributed to each cell equal to the bin average absorbed dose with a randomly determined adjustment based on a Gaussian probability distribution with a width equal to the statistical uncertainty consistent with the ratio of decays to cells, i.e., equal to Nr-1/2. From dose volume histograms the surviving fraction of cells, equivalent uniform dose (EUD), and TCP for the different scenarios were calculated. Comparably sized spherical models containing individual spherical cells (15 microm diameter) in hexagonal lattices were constructed, and Monte Carlo simulations were executed for all the same previous scenarios. The dosimetric quantities were calculated and compared to the adjusted simple sphere model results. The model was then applied to the Bortezomib-induced enzyme-targeted radiotherapy (BETR) strategy of targeting Epstein-Barr virus (EBV)-expressing cancers. RESULTS: The TCP values were comparable to within 2% between the adjusted simple sphere and full cellular models. Additionally, models were generated for a nonuniform distribution of activity, and results were compared between the adjusted spherical and cellular models with similar comparability. The TCP values from the experimental macroscopic tumor results were consistent with the experimental observations for BETR-treated 1 g EBV-expressing lymphoma tumors in mice. CONCLUSIONS: The adjusted spherical model presented here provides more accurate TCP values than simple spheres, on par with full cellular Monte Carlo simulations while maintaining the simplicity of the simple sphere model. This model provides a basis for complementing and understanding laboratory and clinical results pertaining to radiopharmaceutical therapy.

A model of cellular dosimetry for macroscopic tumors in radiopharmaceutical therapy.

PURPOSE: In the radiopharmaceutical therapy approach to the fight against cancer, in particular when it comes to translating laboratory results to the clinical setting, modeling has served as an invaluable tool for guidance and for understanding the processes operating at the cellular level and how these relate to macroscopic observables. Tumor control probability (TCP) is the dosimetric end point quantity of choice which relates to experimental and clinical data: it requires knowledge of individual cellular absorbed doses since it depends on the assessment of the treatment's ability to kill each and every cell. Macroscopic tumors, seen in both clinical and experimental studies, contain too many cells to be modeled individually in Monte Carlo simulation; yet, in particular for low ratios of decays to cells, a cell-based model that does not smooth away statistical considerations associated with low activity is a necessity. The authors present here an adaptation of the simple sphere-based model from which cellular level dosimetry for macroscopic tumors and their end point quantities, such as TCP, may be extrapolated more reliably. METHODS: Ten homogenous spheres representing tumors of different sizes were constructed in GEANT4. The radionuclide 131I was randomly allowed to decay for each model size and for seven different ratios of number of decays to number of cells, N(r): 1000, 500, 200, 100, 50, 20, and 10 decays per cell. The deposited energy was collected in radial bins and divided by the bin mass to obtain the average bin absorbed dose. To simulate a cellular model, the number of cells present in each bin was calculated and an absorbed dose attributed to each cell equal to the bin average absorbed dose with a randomly determined adjustment based on a Gaussian probability distribution with a width equal to the statistical uncertainty consistent with the ratio of decays to cells, i.e., equal to Nr-1/2. From dose volume histograms the surviving fraction of cells, equivalent uniform dose (EUD), and TCP for the different scenarios were calculated. Comparably sized spherical models containing individual spherical cells (15 microm diameter) in hexagonal lattices were constructed, and Monte Carlo simulations were executed for all the same previous scenarios. The dosimetric quantities were calculated and compared to the adjusted simple sphere model results. The model was then applied to the Bortezomib-induced enzyme-targeted radiotherapy (BETR) strategy of targeting Epstein-Barr virus (EBV)-expressing cancers. RESULTS: The TCP values were comparable to within 2% between the adjusted simple sphere and full cellular models. Additionally, models were generated for a nonuniform distribution of activity, and results were compared between the adjusted spherical and cellular models with similar comparability. The TCP values from the experimental macroscopic tumor results were consistent with the experimental observations for BETR-treated 1 g EBV-expressing lymphoma tumors in mice. CONCLUSIONS: The adjusted spherical model presented here provides more accurate TCP values than simple spheres, on par with full cellular Monte Carlo simulations while maintaining the simplicity of the simple sphere model. This model provides a basis for complementing and understanding laboratory and clinical results pertaining to radiopharmaceutical therapy.

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Cellular and molecular mechanisms underlying the effects of sleep loss on hippocampal synaptic plasticity

SUMMARY : The function of sleep for the organism is one of the most persistent and perplexing questions in biology. Current findings lead to the conclusion that sleep is primarily for the brain. In particular, a role for sleep in cognitive aspects of brain function is supported by behavioral evidence both in humans and animals. However, in spite of remarkable advancement in the understanding of the mechanisms underlying sleep generation and regulation, it has been proven difficult to determine the neurobiological mechanisms underlying the beneficial effect of sleep, and the detrimental impact of sleep loss, on learning and memory processes. In my thesis, I present results that lead to several critical steps forward in the link between sleep and cognitive function. My major result is the molecular identification and physiological analysis of a protein, the NR2A subunit of NMDA receptor (NMDAR), that confers sensitivity to sleep loss to the hippocampus, a brain structure classically involved in mnemonic processes. Specifically, I used a novel behavioral approach to achieve sleep deprivation in adult C57BL6/J mice, yet minimizing the impact of secondary factors associated with the procedure,.such as stress. By using in vitro electrophysiological analysis, I show, for the first time, that sleep loss dramatically affects bidirectional plasticity at CA3 to CA1 synapses in the hippocampus, a well established cellular model of learning and memory. 4-6 hours of sleep loss elevate the modification threshold for bidirectional synaptic plasticity (MT), thereby promoting long-term depression of CA3 to CA 1 synaptic strength after stimulation in the theta frequency range (5 Hz), and rendering long-term potentiation induction.more difficult. Remarkably, 3 hours of recovery sleep, after the deprivation, reset the MT at control values, thus re-establishing the normal proneness of synapses to undergo long-term plastic changes. At the molecular level, these functional changes are paralleled by a change in the NMDAR subunit composition. In particular, the expression of the NR2A subunit protein of NMDAR at CA3 to CA1 synapses is selectively and rapidly increased by sleep deprivation, whereas recovery sleep reset NR2A synaptic content to control levels. By using an array of genetic, pharmacological and computational approaches, I demonstrate here an obligatory role for NR2A-containing NMDARs in conveying the effect of sleep loss on CA3 to CAl MT. Moreover, I show that a genetic deletion of the NR2A subunit fully preserves hippocampal plasticity from the impact of sleep loss, whereas it does not alter sleepwake behavior and homeostatic response to sleep deprivation. As to the mechanism underlying the effects of the NR2A subunit on hippocampal synaptic plasticity, I show that the increased NR2A expression after sleep loss distinctly affects the contribution of synaptic and more slowly recruited NMDAR pools activated during plasticity-induction protocols. This study represents a major step forward in understanding the mechanistic basis underlying sleep's role for the brain. By showing that sleep and sleep loss affect neuronal plasticity by regulating the expression and function of a synaptic neurotransmitter receptor, I propose that an important aspect of sleep function could consist in maintaining and regulating protein redistribution and ion channel trafficking at central synapses. These findings provide a novel starting point for investigations into the connections between sleep and learning, and they may open novel ways for pharmacological control over hippocampal .function during periods of sleep restriction. RÉSUMÉ DU PROJET La fonction du sommeil pour l'organisme est une des questions les plus persistantes et difficiles dans la biologie. Les découvertes actuelles mènent à la conclusion que le sommeil est essentiel pour le cerveau. En particulier, le rôle du sommeil dans les aspects cognitifs est soutenu par des études comportementales tant chez les humains que chez les animaux. Cependant, malgré l'avancement remarquable dans la compréhension des mécanismes sous-tendant la génération et la régulation du sommeil, les mécanismes neurobiologiques qui pourraient expliquer l'effet favorable du sommeil sur l'apprentissage et la mémoire ne sont pas encore clairs. Dans ma thèse, je présente des résultats qui aident à clarifier le lien entre le sommeil et la fonction cognitive. Mon résultat le plus significatif est l'identification moléculaire et l'analyse physiologique d'une protéine, la sous-unité NR2A du récepteur NMDA, qui rend l'hippocampe sensible à la perte de sommeil. Dans cette étude, nous avons utilisé une nouvelle approche expérimentale qui nous a permis d'induire une privation de sommeil chez les souris C57BL6/J adultes, en minimisant l'impact de facteurs confondants comme, par exemple, le stress. En utilisant les techniques de l'électrophysiologie in vitro, j'ai démontré, pour la première fois, que la perte de sommeil est responsable d'affecter radicalement la plasticité bidirectionnelle au niveau des synapses CA3-CA1 de l'hippocampe. Cela correspond à un mécanisme cellulaire de l'apprentissage et de la mémoire bien établi. En particulier, 4-6 heures de privation de sommeil élèvent le seuil de modification pour la plasticité synaptique bidirectionnelle (SM). Comme conséquence, la dépression à long terme de la transmission synaptique est induite par la stimulation des fibres afférentes dans la bande de fréquences thêta (5 Hz), alors que la potentialisation à long terme devient plus difficile. D'autre part, 3 heures de sommeil de récupération sont suffisant pour rétablir le SM aux valeurs contrôles. Au niveau moléculaire, les changements de la plasticité synaptiques sont associés à une altération de la composition du récepteur NMDA. En particulier, l'expression synaptique de la protéine NR2A du récepteur NMDA est rapidement augmentée de manière sélective par la privation de sommeil, alors que le sommeil de récupération rétablit l'expression de la protéine au niveau contrôle. En utilisant des approches génétiques, pharmacologiques et computationnelles, j'ai démontré que les récepteurs NMDA qui expriment la sous-unité NR2A sont responsables de l'effet de la privation de sommeil sur le SM. De plus, nous avons prouvé qu'une délétion génétique de la sous-unité NR2A préserve complètement la plasticité synaptique hippocampale de l'impact de la perte de sommeil, alors que cette manipulation ne change pas les mécanismes de régulation homéostatique du sommeil. En ce qui concerne les mécanismes, j'ai .découvert que l'augmentation de l'expression de la sous-unité NR2A au niveau synaptique modifie les propriétés de la réponse du récepteur NMDA aux protocoles de stimulations utilisés pour induire la plasticité. Cette étude représente un pas en avant important dans la compréhension de la base mécaniste sous-tendant le rôle du sommeil pour le cerveau. En montrant que le sommeil et la perte de sommeil affectent la plasticité neuronale en régulant l'expression et la fonction d'un récepteur de la neurotransmission, je propose qu'un aspect important de la fonction du sommeil puisse être finalisé au règlement de la redistribution des protéines et du tracking des récepteurs aux synapses centraux. Ces découvertes fournissent un point de départ pour mieux comprendre les liens entre le sommeil et l'apprentissage, et d'ailleurs, ils peuvent ouvrir des voies pour des traitements pharmacologiques dans le .but de préserver la fonction hippocampale pendant les périodes de restriction de sommeil.

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein–protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

Human iPSC derived disease model of MERTK-associated retinitis pigmentosa

Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches.

Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity:an animal-free CNS cell culture model

Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

Anàlisi genètica i molecular de les malalties Niemann-Pick tipus A/B i tipus C. Estratègies terapèutiques.

Aquest treball es basa en l’estudi de dues malalties lisosòmiques: la malaltia de Niemann-Pick A/B (NPAB) i la malaltia de Niemann-Pick tipus C (NPC). En relació a la malaltia de NPAB, s’ha realitzat l’expressió in vitro d’algunes de les mutacions de canvi d’aminoàcid trobades en pacients espanyols per tal de detectar les activitats enzimàtiques residuals. Totes les mutacions presenten una activitat molt baixa, gairebé nul•la, excepte la p.L225P i la R608del que tenen un 11% i 20% d’activitat respectivament. Els resultats obtinguts són coherents amb la severitat del fenotip que presenten els pacients. D’altra banda, s’ha caracteritzat un al•lel amb una mutació que afecta a una posició poc conservada d’un donador de splicing i que produeix la generació de trànscrits aberrants corresponents a trànscrits minoritaris de SMPD1, prèviament descrits, que no codifiquen per proteïna funcional. Respecte a malaltia de NPC, s’ha realitzat una anàlisi molecular de pacients espanyols prèviament estudiats identificant, en la majoria dels casos, la segona mutació responsable de la patologia. S’ha descrit per primer cop per aquesta malaltia una gran deleció que inclou el gen NPC1 i altres gens flanquejants i s’ha estudiat l’efecte que tenen les mutacions de splicing trobades a nivell de RNA. Per una d’aquestes mutacions, c.1554-1009G&A, s’ha assajat amb èxit una estratègia terapèutica basada en la utilització d’oligonuclèotids antisentit. D’altra banda, s’està desenvolupant un model cel•lular neuronal de la malaltia de Niemann-Pick tipus C, basat en la utilització de RNAs d’interferència, sobre el qual es podran assajar possibles estratègies terapèutiques en un futur.