Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer's and Parkinson's diseases and indirectly affected in Huntington's disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction

Circadian cycle-dependent MeCP2 and brain chromatin changes

Abstract Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome

Neurosphere based differentiation of human iPSC improves astrocyte differentiation

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes

Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG- binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763

A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect

Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.Seventh Framework Programme (European Commission) (Grant HEALTH-F5-2010-258236-SYSCOL)Seventh Framework Programme (European Commission) (Grant HEALTH-F2-2011-259015-COLTHERES)Cellex FoundationOlga Torres FoundationEuropean Research Council (EPINORC Project Grant Agreement 268626)Spain. Ministerio de Economia y Competividad (MINECO Project SAF2011-22803)Institute of Health Carlos III (RTICC Grant RD12/0036/0039

A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect

Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis

Identification of novel therapeutic targets and evaluation of pharmacological treatments in epigenetic and chromatin diseases- the case of Rett syndrome

[spa] INTRODUCCIÓN: En 1966, el síndrome de Rett (RTT, OMIM#312750) fue por primera vez descrito como un problema clínico por el pediatra austriaco Andreas Rett. Observó síntomas similares en 22 pacientes. Pocos años después, Hagberg y colaboradores describieron el síndrome en 35 niñas más. El síndrome de Rett causa retraso mental en 1 de cada 10000 niñas, lo que hace que sea la segunda causa de retraso mental en niñas. En 1999 en el laboratorio de Huda Zoghbi descubrieron las bases genéticas de la enfermedad. El 95% de los casos de Rett clásico se produce por mutaciones en MeCP2. MeCP2 es una proteína nuclear, que se expresa en diferentes tejidos, pero es especialmente abundante en neuronas del sistema nervioso maduro. Pocos años antes Bird y colaboradores habían identificado MeCP2 como proteína con capacidad para unirse a dinucleótidos CpG. Aunque la función de MeCP2 todavía no se conoce con exactitud, se considera que probablemente actúa como regulador de la expresión génica, tanto mediante el silenciamiento o activación de genes específicos como actuando de manera más global sobre la transcripción. El síndrome de Rett fue la primera enfermedad del desarrollo neuronal relacionada con la epigenética. OBJETIVOS DEL ESTUDIO Hipótesis: El principal objetivo de esta tesis es realizar la evaluación preclínica de fármacos que actúan sobre las diferentes rutas alteradas en el síndrome de Rett. Es necesario también investigar nuevos mecanismos asociados al desarrollo de la enfermedad, con el fin de descubrir nuevas rutas que puedan estar relacionadas con la patología y que sean susceptibles de ser manipuladas mediante la utilización de fármacos específicos. Objetivos: 1. Determinar qué tests pueden reflejar las diferencias tanto a nivel conductual como molecular entre ratones knockout de MeCP2 y salvajes de la misma camada. 2. Optimización de protocolos de evaluación de tratamientos farmacológicos in vivo. 3. Identificación de fármacos dirigidos a dianas específicas para la mejora del curso de la enfermedad, con la finalidad de revertir la sintomatología, aumentar la supervivencia o mejorar disfunciones relacionadas con mecanismos neuronales o inflamación. 4. Determinar el potencial de las nuevas terapias desarrolladas para las rutas que se descubran alteradas en el síndrome de Rett. Métodos: El knockout de MeCP2 es un modelo murino bien establecido que mimetiza el síndrome de Rett humano. Este es un excelente modelo para el estudio de las consecuencias relacionadas con la pérdida de MeCP2 en las funciones neuronales. Una vez conocida la dosis que se ha de administrar, se utilizaron dos grupos experimetales, el grupo de tratados y no tratados. El tratamiento se inicia cuando los animales tienen 4 semanas y empiezan a mostras síntomas característicos del modelo: movilidad reducida, retracción de las patas traseras, temblores, pelo encrespado y anormalidades respiratorias, dificultas al andar, retracción de las patas delanteras. Durante el test, los ratones serán evaluados según los síntomas derivados de la deficiencia de MeCP2. Resultados: Parte 1 • Hemos realizado una serie de experimentos investigando las diferencias entre ratones MeCP2 KO y sus hermanos de camada salvajes, tanto a nivel de comportamiento como molecular. Posteriormente, se evaluó la eficacia de una serie de tratamientos. Parte 2 • La administración combinada de Levodopa y un inhibidor de la Dopa decarboxylasa en ratones con síndrome de Rett fue bien tolerada por los animales, disminuyendo los síntomas asociados al síndrome y aumentando la supervivencia. • El uso de L-Dopa + Ddci en el los ratones Mecp2 KO indujo el crecimiento dendrítico mediado por neuronas dopaminérgicas. • El grupo tratado con L-Dopa + Ddci muestra exhibió un aumento en los niveles de expression Th y pTh y de los niveles de dopamina en comparación con el grupo tratado con el vehículo. Parte 3 • Los resultados presentados revelan que la relación entre Mecp2 y la ruta de señalización de GSK3 juega un papel importante en el síndrome de Rett. • El inhibidor de GSK3, SB216763, mejoró la supervivencia y redujo la gravedad de los síntomas, así como la deficiencia motora ,de los ratones Mecp2 KO • La inhibición de GSK3 es una posible vía de estimulación del crecimiento de las neuronas dendríticas, avalado por los elevados niveles del receptor D2 encontrados tras la administración de la droga. • El tratamiento con SB216763 disminuyó la inflamación y reforzó las defensas antioxidantes en el cerebro de los ratones Mecp2 KO. Parte 4 • El tratamiento con copaxona, un análogo de BDNF dio lugar a una menor mejora de los ratones Mecp2 KO, mostrando además una gran variabilidad entre los ratones estudiados. • El tratamiento con dexamethasona, un ejemplo de tratamiento con glucocorticoides, mostró una pequeña mejora, aproximadamente un 20% cuando se comparan los ratones tratados con los no tratados. • El tratamiento con Ropirinol confirmó que la vía dopaminérgica está alterada en Rett. Tanto la supervivencia como la sintomatología mejoró tras la administración de la droga. Sin embrago, la eficiencia fue menor a la obtenida con la tratamiento combinado de Dopa + Ddci. • Las inyecciones de bromperidol, un modulador de la serotonina, resultaron ser tóxicas en ratones Mecp2. Incluso aunque las dosis administradas fueran muy bajas, los ratones sufrían pérdida de peso acusado. • Resultados previos describiendo un efecto positive en los ratones Mecp2 tras la administración de cysteamina no pudieron ser reproducidos en nuestro laboratorio (Roux et al., 2012). • Gabapentin, un modulador de GABA, mejoró los síntomas pero no tuvo ningún efecto en la supervivencia de los animales Mecp2. • El tratamiento con TDZD8 confirmó resultados los previos describiendo el efecto positivo de la inhibición de GSK3 en Rett. Se mejoró tanto la supervivencia como la sintomatología. Sin embrago, la eficiencia fue menor a la obtenida con SB216763. • Los antioxidantes estudiados en esta tesis mostraron una mejora del 30% en la sintomatología de Rett, siendo algunos también eficaces en la mejora de la supervivencia. • Las drogas epigenéticas evaluadas en esta tesis no mostraron ninguna mejora significativa en el tratamiento del síndrome de Rett. Conclusiones: El tratamiento con L-Dopa + Ddci es muy prometedor para sobreponer los defectos dopaminérgicos observados en el modelo preclínico del síndrome de Rett utilizado en esta tesis (Szczesna et al., 2014). Los nuevos datos indican que el inhibidor de GSK3, SB216763, es un posible fármaco para el tratamiento del síndrome de Rett. La droga ha mostrado propiedades neuroprotectoras mediante la mejora de la plasticidad sináptica y la reducción del daño oxidativo e inflamación, así como la mejora de las disfunciones motoras (Szczesna et al, artículo en preparación).[eng] INTRODUCTION: In 1966, Rett syndrome (RTT, OMIM#312750) was for the first time described as a clinical issue by Dr. Andreas Rett, an Austrian pediatrician. He has observed in 22 patients similar unique symptoms. A few years later Hagberg and colleagues described further the syndrome in 35 girls. Rett syndrome is the cause of mental retardation that affects 1 in 10.000 female births, which makes it the second cause of mental retardation in girls. In 1999 Zoghbi lab found out the genetic basis of Rett disease. Mutation in MeCP2 is in 95% cases the reason of classical Rett. MeCP2 is a nuclear protein, expressed widely in different tissues, but is most abundant in neurons of the mature nervous system. A few years earlier Bird and coworkers indentified MeCP2 as a new protein that binds to the methylated CpG dinucleotides. Although the function of the MeCP2 is still unknown, it is considered likely to regulate gene expression, either through the silencing or activation of the specific genes or by more global regulation of transcriptional processes. RTT was the first neurodevelopmental disorder related to epigenetics. AIM OF THE STUDY Hypotesis: The main aim of this thesis is to perform preclinical evaluation of drugs that are known to target pathways which are altered in Rett syndrome. It is also necessary to investigate new mechanisms associated with the development of Rett syndrome, aiming to find new pathways related to Rett phenotype that can be manipulated through the pharmacological approach. Aims: 1. Determine tests that can reflect the difference at the behavioral and molecular levels between the Mecp2 KO and WT littermate mice. 2. Optimize study design protocol for evaluation of in vivo drug treatments. 3. Identify candidate drugs against selected targets in order to improve Rett disease, with the goal to reverse the symptoms, prolong the life span or ameliorate dysfunctions based on inflammation and neural mechanisms. 4. Determine the potential effects of novel therapeutic approaches for newly discovered pathways dysregulated in Rett syndrome. Methods: Animals knockout (KO) to the MeCP2 gene are a well-established murine model that mimics RTT human disease. This model is an excellent tool for the study of the consequences of the loss of MeCP2 in neuronal function. Once we know the dose administered, use two experimental groups of treated and untreated animals. Treatment is initiated when the animals are 4 weeks old and start showing symptoms characteristic of this model: reduced mobility, retraction of the hind legs, tremors, hair frizzing and respiratory abnormalities, difficulty walking, retraction of the rear legs. During the test, the mice will be evaluated according to a set of symptoms resulting from MeCP2 deficiency. Results: Part 1 • We performed a comprehensive panel of experiments investigating the differences between Mecp2 KO and wt littermate mice at the behavioral and molecular levels. This was further extended to evaluate the specific drug treatments efficacy. Part 2 • Combined administration of Levodopa and a Dopa decarboxylase inhibitor in Rett syndrome mouse models was well tolerated, diminished Rett syndrome-associated symptoms and increased lifespan. • The use of L-Dopa + Ddci in the Mecp2 KO mice induced dendritic growth mediated by dopaminergic neurons. • L-Dopa + Ddci-treated group exhibited higher Th and pTh expression and dopamine levels in comparison to the vehicle treated group. Part 3 • The results presented here reveal an important role for the relationship between Mecp2 and GSK3 signaling in Rett syndrome disease • Inhibitor of GSK3, SB216763, improved life span and reduced single and total symptoms scores, as well as motor deficiency in Mecp2 KO mice • Inhibition of GSK3 is a possible line of stimulation of neuronal dendritic growth, supported by elevated levels of D2- receptor after drug administration. • Treatment with SB216763 decreased inflammation and strengthened antioxidant defense in the brain of Mecp2KO mice Part 4 • Treatment with copaxone, a BDNF analogue reflect rather low improvement in Mecp2 KO studied mice, displaying also very variable results between studied animals. • Treatment with dexamethasone, an example of glucocorticoids intervention display rather low improvement – approximately 20% when compared to untreated mice. • Ropirinol treatment confirmed that dopminergic pathway is dysregulated in Rett. Both life span and the phenotype were improved. However, the efficiency was lower compared to L-dopa + Ddci treatment. • Injections of bromperidol, a serotonin modulator, were toxic for Mecp2 KO mice. Even though the selected doses were very low, they caused significant reduction of body weight in the KO treated group. • Previously described positive effect of cysteamine was not reproduced in our laboratory (Roux et al., 2012). • Gabapentin, one of GABA modulators, improved the phenotype, but not life span of Mecp2 KO animals, being more efficient with the low dose. • TDZD8 treatment confirmed previously a described role of GSK3 inhibition in Rett. Both life span and phenotype were improved. However, the efficacy was lower than after SB216763 treatment. • Antioxidants studied in this thesis displayed improvement of 30% in Rett syndrome phenotype and some of them prolonged the life span by 25% when compared to the vehicle group. • Epigenetics drugs evaluated in this thesis, even though some of them were suggested to be relevant for Rett syndrome, did not show any specific improvement. Conclusions: The treatment with L-Dopa + Ddci is promising to overcome the dopaminergic defects observed in the preclinical model of Rett syndrome (Szczesna et al., 2014) The new data indicate selected inhibitor, SB216763, as a being potential drug treatment for Rett disease. The drug can be adressed to have neuroprotection properties via the improvement of synaptic plasticity, reduced oxidant and inflammation damage or partial rescue of mobility dysfunctions (Szczesna et al in preparation)

Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer's and Parkinson's diseases and indirectly affected in Huntington's disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction

Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer’s and Parkinson’s diseases and indirectly affected in Huntington’s disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction