Generation and differentiation of dopaminergic neurons from mouse multipotent and human induced pluripotent stem cells to study Parkinson´s disease

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Department of Molecular Biology Faculty of Sciences

Universidad Autónoma de Madrid

PhD THESIS

Generation and differentiation of dopaminergic neurons from mouse multipotent and human

induced pluripotent stem cells to study Parkinson’s disease

EVA RODRÍGUEZ TRAVER, MSc Molecular Medicine Thesis Director: CARLOS VICARIO ABEJÓN, PhD

Madrid, 2017

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Memoria presentada por Eva Rodríguez Traver para optar al grado de DOCTORA POR LA UNIVERSIDAD AUTÓNOMA DE MADRID EN EL PROGRAMA DE DOCTORADO EN BIOCIENCIAS MOLECULARES

CARLOS VICARIO ABEJÓN, Doctor en Farmacia e Investigador Científico del CSIC,

AUTORIZA la presentación de la Tesis Doctoral titulada “Generation and differentiation of dopaminergic neurons from mouse multipotent and human induced pluripotent stem cells to study Parkinson’s disease”

de la que es autora Eva Rodríguez Traver, Master en Medicina Molecular, y que ha sido realizada bajo mi dirección en el Instituto Cajal del CSIC, Madrid.

Y para que así conste a los efectos oportunos, firmo el presente certificado en Madrid, el día 14 de Junio de 2017.

Fdo: Carlos Vicario Abejón Instituto Cajal, CSIC

Madrid

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5

ACKNOWLEDGEMENTS

Tengo mucho que agradecer en estos años de tesis y son muchísimas las personas que me han acompañado en este camino en el que ha habido momentos mejores y peores o más difíciles, pero todos importantes y que me han hecho crecer, madurar, y convertirme en la persona que soy a día de hoy. Primero de todo, quisiera agradecer a Carlos, mi director de tesis, por estar siempre ahí cuando lo he necesitado, sin importar la hora o el día de la semana, por enseñarme y sobre todo por confiar en mí y apoyarme en los momentos difíciles para que esto saliese adelante, muchas gracias.

Quiero agradecer enormemente a mis compañeros de laboratorio, porque sin duda son los mejores compañeros que podría haber soñado y tengo la suerte de poder decir que se han convertido en mucho más que compañeros de trabajo si no en grandes amigos para mí. Gracias Jaime, por enseñarme tanto cuando llegué, por dedicarme tu tiempo y tener paciencia con el pajarillo, que siempre estaba revoloteando y preguntando cómo se hacían las cosas. Eva Díaz muchas gracias por enseñarme taaantas cosas, porque gracias a tí soy un poco más perfeccionista, porque todo se pega, y también gracias por ser una buena amiga en la que confiar siempre, gracias por estar ahí cuando lo he necesitado. Vane, muchas gracias a ti también por las risas en el laboratorio, por siempre tener una palabra agradable y siempre estar dispuesta a ayudar. A Maria José por ser la voz sensata del laboratorio, por siempre siempre ser tan agradable y ayudarnos tanto. A Maku, gracias por ser tan agradable y siempre tener una sonrisa con la que alegrarnos. Daniela, aunque ya no estás por aquí, gracias por ser una de mis mejores amigas el tiempo que coincidimos en Madrid, guardo momentos inolvidables contigo (para ti para siempre). A Elenita, porque eres un Sol, porque llegaste y fuiste mi angelito, gracias por las canciones, las risas, pero ya sabes…cuidado que te pica xD. A Pablito, mi pollito jeje, otro solecito, muy listo además, gracias por tener siempre una palabra agradable para mí y por sufrirme siempre sin queja. A las personas con las que aunque coincidí menos pude compartir muy buenos momentos: Carlitos (por deslumbrarme sobre todo al principio xD), Eva Vergaño (por tu alegría incomparable), Inma, Alonso, Manu.

Pero sobre todo, te quiero agradecer a ti mi Çaglita, mi gemelita, mi compi de piso y en la vida, porque estamos casadas como siempre decimos (tenemos casa), por compartirlo todo (hasta a Berto, R.I.P.), por hacerme la vida fácil, por cuidarme, porque sin ti me habría costado mucho más superar tantas cosas, por estar siempre a mi lado pase lo que pase, por nuestras locuras y risas que duran años…y por nuestras canciones (¡¡que nadie nos pare!!).

También agradezco a la gente de otros laboratorios, en especial a Rosario Moratalla y su laboratorio, el B-01, y al c-17 por ser nuestros laboratorios hermanos. Especialmente gracias a Patri, mi Patri, por ser una de mis mejores amigas en Madrid, por estar siempre a

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mi lado, por ayudarme y escucharme siempre que lo he necesitado, porque sé que siempre seremos amigas. Y a Rubén (Rubyyy), mi hermano en Madrid, que aunque te encante meterte conmigo (en el fondo me gusta que lo hagas jeje) te tengo muchísimo cariño, gracias por todos los recuerdos a tu lado y por los bailes. También gracias a Irene, por tu cariño; Liliana, por los momentos en las comidas y el tiempo que estuviste aquí, me llevo una gran amiga; Lula, por tu ayuda con la electro, ánimos y paciencia explicándome, a Guille, Samu, Lorena, Óscar, Miguel, Adri, Bea, Juan…Y del c-17 a mi ESTI, que es una persona increíble, de las que no existen, porque solo tengo palabras bonitas para ella, por ser el GRAN descubrimiento de esta etapa final, porque has sido y estás siendo mi gran apoyo en esta recta final, porque me llevo una amiga para toda la vida, espero. A Iris, otra persona increíble, siempre dispuesta a ayudarme cuando lo he necesitado, millones de gracias por todo. Y a Chaska, por ayudarme a cambiar la rueda del coche jeje y por estar siempre dispuesto también a ayudarme en lo que sea. También agradezco a mucha otra gente de otros laboratorios, a Víctor, por compartir tantas cosas y momentos juntos y por los que nos quedan. A mi Manu! porque es simplemente genial, uno de los mejores fichajes del Cajal, mi compi de parrandas y aventuras, porque cada día me caes mejor. A Paloma, porque eres única, por nuestras conversaciones, cervezas, fiestas…A Raquel, porque te tengo mucho cariño. A Marieta, por ser una Marco Aldanier única que le cortó un dedo a un cliente, por nuestros momentos, viajes, conversaciones…y los que nos quedan también (Tailandia nos espera!). A Natalia, por alegrarme cada vez que la veo con esa sonrisa siempre y por los astrocitos!!. Por supuesto, a Ali, que ha hecho posible junto con Lula registrar mis neuronitas y porque es un amor de persona. Y a mi compi de biblioteca, Carol, por hacer de las tardes en la biblioteca algo agradable, porque las penas compartidas son menos penas y porque estoy descubriendo a una persona genial con la que espero compartir muchos momentos fuera de la biblioteca!!! Y a tantas otras personas, que están por aquí o han pasado por el Cajal dejando una bonita huella en mí. A Paula, que me ha ayudado a contar mis celulitas siempre con una sonrisa. A Angelito, que es único, un solecito que me alegra muchísimo cada vez que veo. A Simona, con la que he compartido tantas veces el P2.

A Ramiro, porque guardo momentos muy especiales y bonitos a tu lado y por darle vidilla al Cajal mientras estuviste por estas tierras. A Irina, porque te echo de menos en las comidas.

A François, por ser uno de mis mejores amigos en el tiempo que compartimos en el Cajal, porque son infinitos los momentos y todos buenos (por las comidas, los bailes, Fallas…). A Dieguito por ser un bicho único, porque sin ti hubiese seguido dando la vuelta a la rotonda, por el swing, por regalarme momentos espectaculares dentro y fuera del Cajal.

Por supuesto, quiero agradecer a todos los servicios del Cajal, porque hacéis del Cajal no un centro de trabajo, sino un hogar y por salvarme la vida tantas veces. Quiero hacer una

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7 grandísimas profesionales siempre dispuestas a enseñar y ayudar, son unas personas

maravillosas, gracias por los momentos de terapia en confocal, por vuestro cariño, por preguntarme siempre cómo estoy, por los consejos y ayuda siempre. Muchas gracias también a Sonia y Silvia, por ser tan agradables conmigo y estar para todo siempre. A la gente de administración, por facilitar todo lo que pueden los papeleos y darme cambio para café siempre con una sonrisa. A la gente del almacén, Fernando y Jesús, por estar siempre dispuestos a ayudar con vuestra mejor sonrisa. A los informáticos Ángel y Juan, por salvarme la vida con mi ordenador. A la gente de recepción, en especial a Aurelio aunque ya no está, porque es una persona encantadora que desde el minuto uno ya sabía mi nombre. A la gente de mantenimiento, a Jose y Raúl, porque siempre tienen un gesto agradable hacia mí y a Javi, aunque ya no está, por ser un amigo, por ayudarme siempre.

Quiero agradecer también al resto de colaboradores en esta tesis, siempre dispuestos a ayudar: Jaime Kulisevsky, Fabian Arenas, Marcos Araúzo, María Orera y su equipo, Carlos Crespo e Inés Quintela.

A mis amigos fuera del Cajal. A mi hermanita Berta, que la quiero con locura, por aguantarme toda la vida, por nuestros momentos en Madrid pero también en Teruel, Valencia, Zaragoza…viajes…por ser mi gran apoyo y familia fuera del Cajal y en Madrid, siempre disponible. Al resto de mis chicas de Madrid: Elena, Nuria, Gema, Desi, Maca, Ceci y Jaci, porque la desconexión con vosotras está garantizada, sois lo más. A la persona que haciendo honor a su nombre, Ángel, ha sido mi ángel de la guarda tantas veces y me ha ayudado tanto desde que llegué a Madrid. A mi Mara, mi loqui favorita, por ser alguien tan especial para mí, aunque estés lejos, siempre estás cerca de mí, te quiero. A mis maridas: Robert (mi mejor amigo como siempre digo, porque cada vez que pienso en ti, solo puedo sonreir, porque a tu lado, todo es felicidad), a Cesc (porque eres una marida más), a Marta (porque la distancia no hace el olvido, porque tu dulzura me llega a Madrid y me llena de fuerzas) y a mi Sarita (porque sin ti no podría existir, porque te tengo un cariño infinito, por estar SIEMPRE conmigo, por venir a verme, por llamarme, porque te adoro). A mis amigas de toda la vida desde los 4 años, Elena, Anabel, Elvira, porque sois más que unas amigas, sois mi familia. A Lorena, porque aunque haga mucho que no nos veamos, te llevo en mi corazoncito. A mis biotecnólogos y bioquímicos, porque la aventura de la ciencia comenzó con vosotros y porque conservo amigos muy buenos de los años de universidad. Y como no, a mi Vero de Teruel, que me llena de alegría y amor cada vez que la veo, que siempre tiene palabras bonitas para mí, que me sube la autoestima y a la que admiro y adoro y ya le debo unas cuantas noches de despiporre, jeje. También gracias al resto de amigos y amigas de Teruel, en especial a mis chicas: Patri, Isa, Lola, Roci y Elenita, porque cada vez que voy a Teruel me hacéis sentir en

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casa y porque lo pasamos muy bien juntas siempre. Y a Santi, por tu amistad y por sacar siempre un rato para tomar algo conmigo.

También quiero agradecer a mi otra gran pasión, el flamenco, porque es mi gran vía de desconexión y porque me llena el alma. Especialmente quiero agradecer a mi profe Amelia Vega, que en este tiempo no ha hecho más que regalarme cosas buenas, gracias por enseñarme tanto y no solo flamenco, gracias por cuidarnos siempre, por preocuparte por nosotras, por querernos y demostrárnoslo, eres una persona espectacular a la cual me alegro muchísimo de haber conocido en estos años y que me ha dado fuerzas en los momentos que más lo he necesitado, muchas gracias. Y gracias a mis compis, que ahora son grandísimas amigas, especialmente, gracias a Mari Jose, Bianca y Paulita, porque solo veros en clase me saca una sonrisa, gracias por compartir tantos momentos maravillosos conmigo, os adoro.

Por encima de todo quiero agradecer a mi familia, porque sois lo mejor que tengo en la vida. A mis padres, porque todo lo que soy os lo debo a vosotros, por vuestra generosidad, por enseñarme a ser cada día mejor persona y a no dejarme vencer nunca. Porque sois mi ejemplo en la vida, y no puede haber un ejemplo mejor, porque tengo unos padres maravillosos que no solo han sabido quererme y darme todo su amor, sino que me han guiado en la vida y lo siguen haciendo. A mi hermano, por ser mi bro, porque no se puede tener un hermano mejor, por ser mi compañero de aventuras, porque aunque me hagas rabiar de vez en cuando, sé que me quieres, jeje, y yo a ti más. Quisiera agradecer especialmente a mis abuelos, en especial a mi abuela Josefa, porque vivir su enfermedad a su lado impulsó mis ganas de descubrir más a cerca de esta enfermedad en la que está basada gran parte de mi tesis, lo cual me ha dado siempre fuerzas para seguir con este trabajo adelante. Sé que nos vas a cuidar siempre desde el cielo, como lo hiciste cuando estuviste a nuestro lado.

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PRESENTATION

Parkinson’s disease (PD) is mainly characterized by the degeneration of DA neurons of the substantia nigra pars compacta, of largely unknow etiology although mutations in the NURR1 and GBA1 genes, among others, are genetic risk factors for this human disease. Thus, studying the dopaminergic (DA) system and establishing cellular models to study this disease might be very useful to understand its etiology and for therapeutic purposes. Consequently, in this thesis we have generated two cellular models, a mouse model based on NURR1 overexpression in neural stem cells, and a human model based on induced pluripotent cell (iPSC) generated by reprogramming fibroblasts isolated from PD patients with GBA1 mutations and controls. The human model has allowed us to study cellular, molecular and functional phenotypes related with this disease.

The transcription factor NURR1 is involved in progenitor cell differentiation, and the maturation, survival and maintenance of DA neurons. Its influence on the generation of DA neurons in cells isolated from the olfactory bulb region remained unclear. Thus, in this thesis we have overexpressed NURR1 in embryonic mouse olfactory bulb stem cells (eOBSCs) and we have found a marked inhibition of cell proliferation concomitant with an upregulation of the dopaminergic transcripts Th and Dat; and of the fibroblast growth factor receptor-2 (Fgfr2), suggesting a role of this receptor in regulating DA neuron generation. Moreover, NURR1 overexpression triggered the generation of two populations of DA neurons: a major mesencephalic-like DA population and an OB-like DA subpopulation. Some of the neurons obtained expressed molecular markers of mature DA neurons, synaptic proteins, responded to dopaminergic stimulation, and released dopamine indicating that they acquired functional properties.

Mutations in the glucocerebrosidase1 (GBA1) in heterozygosis are considered the strongest genetic risk factor for PD development. In this thesis we have established a cellular model to study PD associated with GBA1 mutations. Fibroblasts isolated from control subjects and PD patients with N370S/wt and L444P/wt GBA1 mutations have been reprogrammed to human iPSCs, using Sendai viral vectors, which have been differentiated into DA neurons.

After 17-18 days of differentiation, the hiPSC-derived cells started to express mesencephalic dopaminergic markers. Later, we have detected dopamine release after 33-59 days and electrophysiological properties after 80-92 days of differentiation. Notably, we have found effects of the GBA1 mutations on the expression of the chaperone CRYAB, dopamine release and excitability of the neurons generated.

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PRESENTACIÓN

La enfermedad de Parkinson (EP) se caracteriza principalmente por la degeneración de las neuronas dopaminérgicas de la substantia nigra pars compacta, debida generalmente a causas desconocidas, aunque las mutaciones en los genes NURR1 y la glucocerebrosidasa1 (GBA1), entre otros, son factores de riesgo genético para la misma. De este modo, el estudio del sistema dopaminérgico (DA) y el establecimiento de modelos celulares podrían ser muy útiles para discernir su etiología y con fines terapéuticos.

Consecuentemente, en esta tesis hemos generado dos modelos celulares, un modelo de ratón basado en la sobreexpresión de NURR1 en células madre neurales, y un modelo humano basado en la reprogramación a células madre inducidas pluripotentes (iPSC) de fibroblastos de pacientes con EP y mutaciones en GBA1 y controles. En el modelo humano hemos estudiado fenotipos celulares, moleculares y funcionales relacionados con esta enfermedad.

El factor de transcripción NURR1 está implicado en la diferenciación de células progenitoras, y en la maduración, supervivencia y mantenimiento de las neuronas DA. Su influencia en la generación de neuronas DA a partir de células aisladas del bulbo olfatorio no era conocida. En esta tesis, hemos sobre expresado NURR1 en células madre embrionarias de BO (CMBOe) de ratón. Hemos encontrando una inhibición marcada de la proliferación celular junto a un incremento de algunos transcritos dopaminérgicos (Th y Dat) y del receptor 2 del factor de crecimiento de fibroblastos (Fgfr2), lo que sugiere una función de este receptor en la regulación de la generación de neuronas DA. Además, la sobre expresión de NURR1 promovió la generación de dos poblaciones de neuronas DA: una población mayoritaria de neuronas DA tipo mesencefálica y una población de neuronas DA-GABAérgicas tipo BO.

Algunas de las neuronas obtenidas, expresaron marcadores moleculares de neurona DA madura, proteínas sinápticas, respondieron a estimulación dopaminérgica y liberaron dopamina, demostrándose la adquisición de algunas propiedades funcionales.

Las mutaciones en heterocigosis en GBA1 se consideran el mayor factor de riesgo genético para el desarrollo de EP. En esta tesis, hemos establecido un modelo celular para su estudiar la EP. Se han obtenido iPSCs, mediante la reprogramación de fibroblastos con vectores virales Sendai de sujetos control y pacientes con EP portadores de las mutaciones N370S/wt y L444P/wt en GBA1. Estas iPSCs se han diferenciado a neuronas DA. Tras 17-18 días en diferenciación, las neuronas comenzaron a expresar marcadores de neurona DA mesencefálica. Más adelante se detectó la liberación de dopamina tras 33-59 días y propiedades electrofisiológicas después de 80-92 días de diferenciación. Notablemente, hemos encontrado efectos de las mutaciones en GBA1 en la expresión de la chaperona

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ABBREVIATIONS

o A83: A83-01, 3-(6-Methyl-2-pyridinyl)- N-phenyl-4-(4-quinolinyl)-1H-pyrazole- 1-carbothioamide

o AA: ascorbic acid

o AADC: L-aromatic amino acid decarboxylase

o aCSF: artificial cerebrospinal fluid o AFP: alpha fetoprotein

o Amyg: amygdala o AP: action potentials o AP: alkaline phosphatase

o AraC: cytosine-β-D-arabinofuranoside o ASCL1: achaete-scute family bHLH

transcription factor 1

o ATP: adenosine triphosphate o Bax: BCL2-Associated X Protein o BCIP: 5-bromo-4-chloro-3-indolyl

phosphate

o Bcl2: B-cell lymphoma 2

o BDNF: brain-derived neurotrophic factor

o BMPs: bone morphogenetic proteins o BrdU: 5’-bromo-2-deoxyuridine o CA: catecholamine

o Calb: Calbindin

o cAMP: cyclic adenosine monophosphate o cDNA: complementary

deoxyribonucleic acid o CFU: colony forming units o CHIR: CHIR99021, 6-[[2-[[4-(2,4-

dichlorophenyl)-5-(5-methyl-1H- imidazol-2-yl)-2

pyrimidinyl]amino]ethyl] amino]-3- pyridinecarbonitrile

o Cm: membrane capacitance

o CMA: chaperone-mediated autophagy

o CNS: central nervous system o CNVs: copy number variations

o c-RET: protein tyrosine kinase receptor o CRYAB: crystallin alpha B

o Ct: control

o D1R: Dopamine class 1 receptor o D2R: Dopamine class 2 receptor o DA: Dopamine

o DA: Dopaminergic

o DAPT: gamma-secretase inhibitor o DAT: dopamine transporter o dbcAMP: Dibutyryl-cAMP o ddNTPs: dideoxynucleotides o DIV: Days in vitro

o DMEMF-12/N2 medium: Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture plus N2 supplement

o DMSO: dimethyl sulfoxide o DNA: deoxyribonucleic acid

o DPBS: Dulbecco's Phosphate-Buffered Saline

o DTT: Dithiothreitol

o E13.5: embryonic day 13.5 o EBs: embryoid bodies

o EDS: excessive daytime sleepiness o EGF: Epidermal Growth Factor o EGFP: enhanced green fluorescent

protein

o EN1: Engrailed 1 o EN2: Engrailed 2

o eOBSCs: embryonic olfactory bulb stem cells

o EPL: external plexiform layer o EPSC: excitatory postsynaptic

potential current

o ER: endoplasmic reticulum

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20 o ERAD: endoplasmic reticulum-

associated degradation o ESC: Embryonic stem cells o ET: large external tufted cells o F: Fusion protein

o FACs: fluorescence-activated cell sorting

o FBS: fetal bovine serum

o FGF-2: fibroblast growth factor 2 o FGF8: fibroblast growth factor family

member 8

o Fgfr2: fibroblast growth factor receptor 2

o FGFs: fibroblast growth factors o FN: fibronectin

o FOXA2: forkhead box A2 o FP: floor plate

o GABA: gamma-aminobutyric acid o GAD: glutamic acid decarboxylase o GADPH: glyceraldehyde-3-phosphate

dehydrogenase

o GBA1: glucocerebrosidase 1 o GBA1-PD: GBA1-associated PD o G-banding: Giemsa banding karyotype o Gcase1: Glucocerebrosidase 1

o GD: Gaucher’s disease o GDNF: glial cell line-derived

neurotrophic factor

o GFAP: glial fibrillary acidic protein o GFP: green fluorescent protein o GIRK2: G-protein-regulated inward-

rectifier potassium channel 2 o GlcCer: glucosylceramide or

glucocerebroside o GP: globus pallidus

o GSK3: Glycogen Synthase Kinase 3 Beta

o HAT: histone acetyltransferase o HBSS: Hank's Balanced Salt Solution

o hiPSC: human induced pluripotent stem cell

o HN: Hemagglutinin-Neuraminidase o Hp: hippocampus

o HSC: hematopoietic stem cell o HSP20: small heat shock protein 20 o I-V: Intensity-Voltage

o KCl: Potassium chloride o KLF4: Krüppel-like factor 4

o KSR: Knockout Serum Replacement o L: Large protein

o LacZ-EGFP: pRV-LacZ-IRES-EGFP o Lam: laminin

o LDN: LDN-193189, 4-(6-(4-(piperazin- 1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3- yl)quinoline hydrochloride

o LINGO2: leucine rich repeat and Ig domain containing 2

o LMX1A: LIM homeobox transcription factor 1 alpha

o LMX1B: LIM homeobox transcription factor 1 beta

o LOH: loss of heterozygosity

o LRRK2: leucine rich repeat kinase 2 o LSD: lysosomal storage disorder o MALDI-TOF: Matrix-Assisted Laser

Desorption/Ionization-Time of flight o MAP2ab: Microtubule-Associated

Protein 2

o MAPD: median absolute pairwise difference

o MCI: mild cognitive impairment o MEFs: mouse embryonic fibroblasts o Mes: mesencephalic

o mesDA: mesencephalic DA o MHB: mid-/hindbrain boundary o MLV: Moloney murine leukemia virus o MOI: multiplicity of infection

o MSDS: Material Safety Data Sheet o N Acc: nucleus accumbens

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o NBRE: NGFI-B response element o NBT: nitro blue tetrazolium o NEAA: Non-Essential Amino acids o NGN2: neurogenin 2

o NP: Nucleocapsid protein o NPC: neural progenitor cells o NuFFs: Newborn Human Foreskin

Fibroblasts

o NURR1: NR4A2, nuclear receptor subfamily 4, group A, member 2 o Nurr1-EGFP: pRV-Nurr1-IRES-EGFP o O. Tub: olfactory tubercle

o OB: olfactory bulb

o OBSCs: olfactory bulb stem cells o OCT4: octamer-binding transcription

factor 4 (also known as POU5F1) o ORF: Open Reading Frame o OSKM: Oct4/Sox2/Klf4/Myc

combination o P: Phosphoprotein

o PAG: pluripotency-associated genes o PAX6: Paired Box 6

o PB: phosphate buffer

o PCR: Polymerase Chain Reaction o PD: Parkinson’s disease

o PD0325901: MAP kinase/ERK kinase o PFA: paraformaldehyde

o PG: periglomerular cells o Pit: pituitary

o PITX3: paired like homeodomain 3 o PLO or PO: polyornithine

o pRV-Nurr1-EGFP: retroviral particles expressing Nurr1 and enhanced green fluorescent protein

o PSC: pluripotent stem cells o Pur: Purmorphamine

o PVDF: polyvinylidene difluoride o RBD: REM sleep behavior disorder o Rm: membrane resistance

o RNA: ribonucleic acid

o ROS: reactive oxygen species

o RPMI: Roswell Park Memorial Institute medium

o RT: Reverse Transcription o RT-qPCR: Real-Time Quantitative

Reverse Transcription Polymerase Chain Reaction

o RXR: retinoid X receptor

o S.E.M: standard error of the mean o S.D: standard deviation

o SAG: smoothened agonist o SB: SB431542, 4-[4-(3,4-

Methylenedioxyphenyl)-5-(2-pyridyl)- 1H-imidazol-2-yl]benzamide, Dihydrate o SeV: Sendai virus

o SHH: Sonic hedgehog

o SHH-C24II: human sonic hedgehog (C24II) N-terminus

o SHH-C25II: mouse sonic hedgehog (C25II) N-terminus

o SMAD: Contraction of Sma and Mad (Mothers against decapentaplegic) o SN: substantia nigra

o SNARE: SNAP (Soluble NSF Attachment Protein) Receptor o SNARE: soluble N-ethylmaleimide

sensitive fusion protein attachment protein receptor

o SNCA: synuclein alpha

o SNpc: substantia nigra pars compacta o SNPQC: single nucleotide

polymorphism quality control o SNPs: single nucleotide

polymorphisms o SOX2: SRY-box 2

o SSEA: Stage-specific embryonic antigen

o Str: striatum

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22 o SVZ: subventricular zone

o TF: transcription factor

o TGFβ-3: transforming growth factor- beta 3

o TH: tyrosine hydroxylase o Thal: thalamus

o TMB: 3, 3', 5, 5' - Tetramethylbenzidine

o UPR: unfolded protein response o VGAT: vesicular GABA transporter

o VMAT2: vesicular monoamine transporter 2.

o vmDA: ventral midbrain DA o VTA; ventral tegmental area o Wt: wildtype

o Y-27: Y-27632 dihydrochloride, trans- 4-[(1R)-1-Aminoethyl]-N-4-

pyridinylcyclohexanecarboxamide dihydrochloride

o α-syn: alpha synuclein

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INTRODUCTION

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1. The dopaminergic system

1.1 Types of dopaminergic (DA) neurons and their projections

During the early 1960s, the development of the formaldehyde histofluorescence method allowed the study of catecholamine (CA) neurons in the brain, and twelve groups of CA cells (A1-A12) were described from the medulla oblongata to the hypothalamus (Dahlstroem and Fuxe, 1964). A13-A17 CA groups, located in the diencephalon, olfactory bulb (OB) and retina, together with C1-C3 groups (adrenaline-containing cell groups), were identified later (Bjorklund and Dunnett, 2007, Prakash and Wurst, 2006). Depending on the cell body localization, DA neurons (a subtype of CA neurons) can be divided into several groups. From caudal to rostral regions, we can distinguish: mesencephalic (mes) DA neurons also called midbrain DA neurons (A8-A10); diencephalic DA neurons (A11-A15); and telencephalic DA neurons (A16-A17), as described in Figure 1 and Table 1 (A17 group is not shown).

Ventral midbrain DA neurons (Table 1) are involved in the voluntary movement control, body posture and the modulation of cognitive and emotional/rewarding behaviors. Recent findings also suggest that A9 and A10 neurons could regulate adult neurogenesis. These neurons degenerate in Parkinson’s disease (PD), being the DA neurons located in the substantia nigra pars compacta (SNpc, A9 DA neurons) the most affected ones (Prakash and Wurst, 2006, Bjorklund and Dunnett, 2007, Freundlieb et al., 2006, Yetnikoff et al., 2014).

Figure 1. Localization and axonal projections of dopaminergic cell groups in the mammalian adult brain. Dopaminergic neurons are arranged in 10 groups from the mesencephalon to the olfactory bulb: A8-A16. The A17 (retinal group) is not shown. The main axonal projections are depicted as arrows.

Amyg: amygdala; DA: Dopaminergic neurons; GP: globus pallidus; Hp: hippocampus; N Acc: nucleus accumbens; OB: olfactory bulb; O. Tub: olfactory tubercle; Pit: pituitary; Str: striatum; SVZ:

subventricular zone; Thal: thalamus. This figure is based on previous studies (reviewed and discussed in Rodriguez-Traver et al., 2016).

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Otherwise, OB DA neurons (Table 1), are thought to be involved in in odor information processing, having also an impact in odor-driven behavior. Moreover, olfactory dysfunction is associated to pathological states affecting the DA system, such as in PD (Bonzano et al., 2016).

Table 1. Molecular markers of mesDA and OB DA neurons.

Group Molecular markers Ref.

Ventral midbrain DA neurons (A8-A10)

FOXA2, LMX1A, LMX1B, NURR1, TH, AADC, calretinin, calbindin, DAT, VMAT2, GIRK2 (in soma and processes).

Ferri et al., 2007; Laguna et al., 2015;

Riddle and Pollock, 2003; Rodríguez- Traver et al., 2016; Smidt et al., 2003;

Vergaño-Vera et al., 2015; Yi et al., 2014

Olfactory bulb neurons (A16)

NURR1, TH, AADC, DAT, GABA, GAD, VGAT, GIRK2 (in processes).

Kiyokage et al., 2010; Kosaka and Kosaka, 2011; Rodríguez-Traver et al., 2016; Schein et al., 1998; Vergaño-Vera et al., 2015.

1.2 Regulation of mesencephalic (mes) DA and olfactory bulb (OB) DA neuron development

1.2.1 Neural induction

In vertebrates, the neural plate induction from the embryonic ectoderm takes place right after the onset of gastrulation. The default model, proposed for neural induction in the 1990s, establishes that unless exposed to epidermis inducing bone morphogenetic proteins (BMPS), all ectodermal cells will become neural (De Robertis et al., 2000, Munoz-Sanjuan and Brivanlou, 2002, Stern, 2006). Thus, the absence of BMP signals (by default) or the inhibition of the BMP signaling pathway induces neural fates (Kiecker and Lumsden, 2009, Levine and Brivanlou, 2007). However, some authors suggest a more complex model, where other molecules such as fibroblast growth factors (FGFs) might play a role for the induction of the neural fate (Stern, 2005). Within the neural plate, a crude pattern is established during gastrulation by gradients of signaling factors which determine anteroposterior polarity (FGFs, retinoic acid, signaling proteins of the Wnt family) and mediolateral polarity (BMPs and members of the Hedgehog family) by inducing the expression of region-specific transcription factors in a dose-dependent manner (De Robertis et al., 2000, Niehrs, 2004, Rhinn et al., 2006, Stern et al., 2006, Wilson and Houart, 2004). A regional interface occasionally becomes a cell- tight boundary confining cells to lineage-restricted compartments. Besides stabilizing emerging regionalization, boundaries function as local organizers that secrete molecular signals influencing the development of their flanking regions (Rhinn et al., 2006, Echevarria et al.,

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27 1.2.2 Regulation of mesDA neuron development

Sonic hedgehog (SHH) secretion by ventral midline cells of the neural tube or the floor plate (FP) and the activity of the fibroblast growth factor family member 8 (FGF8), released by the cells of the mid-/hindbrain boundary (MHB) or the so-called isthmic organizer, determine the DA neural induction in stem/progenitor cells (Hynes et al., 1995b, Hynes et al., 1995a, Hynes and Rosenthal, 1999). These two factors are required for the specification of the DA phenotype in the ventral midbrain and forebrain at initial stages of neural development (Ye et al., 1998). Neuronal differentiation and subtype specification are also mediated by other factors such as neurogenin 2 (NGN2), achaete-scute family bHLH transcription factor 1 (ASCL1) and forkhead box A2 (FOXA2). The proneural factors, NGN2 and ASCL1 mediate initial neurogenesis (Kele et al., 2006) and the specification of different neurotransmitter identities (Bertrand et al., 2002). Neurogenins can also promote a neuronal fate by repressing the glial fate (Tomita et al., 2000, Sun et al., 2001). FOXA2 positively regulates NGN2 expression, and subsequently it regulates the nuclear receptor subfamily 4, group A, member 2 (NR4A2 also called NURR1) and Engrailed 1 (EN1) expression in immature neurons. Additionally, FOXA2 cooperate with NURR1 through epigenetic mechanisms to activate the DA phenotype, including tyrosine hydroxylase (TH) and L-aromatic amino acid decarboxylase (AADC) expression. Furthermore, at late embryonic stages, it plays a role in the maintenance of dopaminergic properties of ventral midbrain neurons (Yi et al., 2014, Ferri et al., 2007, Kittappa et al., 2007, Stott et al., 2013).

The engrailed (EN1 and EN2) genes and LIM homeobox transcription factor 1 alpha and beta (LMX1A and LMX1B) genes are involved in early and late development of mesDA neurons. The isthmic organizer secretes EN1 and EN2 and regulates the expression of FGF8 mediating the induction of mesDA neurons. In addition, the expression of engrailed genes is needed for neuronal survival in mesDA neurons, (Alavian et al., 2014). LMX1A and LMX1B are key transcription factors (TFs) involved in the early specification of ventral midbrain DA neurons, which are also expressed in postmitotic differentiating midbrain DA neurons although their role after the specification of proliferating progenitors remains unknown. Remarkably, LMX1B is required for a proper autophagic-lysosomal function as well as for maintaining DA nerve terminals and the long-term survival of the midbrain DA neurons (Laguna et al., 2015).

There are other TFs such as NURR1 and paired like homeodomain 3 (PITX3), which are not involved in the initial specification of multipotent stem cells´ becoming mesDA neural progenitors, but are needed for progenitor differentiation, maturation and survival of postmitotic DA neurons (Smidt et al., 2004, van den Munckhof et al., 2003, Nunes et al., 2003, Zetterstrom et al., 1997, Jacobs et al., 2009a, Alavian et al., 2014, Saijo et al., 2009). Both NURR1 and PITX3 are implicated in neuronal patterning, axon outgrowth and terminal differentiation through gene expression regulation (Jacobs et al., 2009a). PITX3 is expressed after NURR1

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expression as some typical DA markers such as TH and dopamine transporter (DAT). Indeed, NURR1 has been demonstrated to control PITX3 expression by binding to a non-canonical NBRE consensus sequence, which promotes its translation (Volpicelli et al., 2012).

Finally, terminal DA differentiation and maturation require the expression of all the enzymes, receptors and transporters such as TH, AADC, vesicular monoamine trasporter 2 (VMAT2), Dopamine class 2 receptor (D2R) and DAT, for the synthesis, storage, release and reuptake of dopamine. Moreover, DA neurons have to reach, innervate and establish regulated synaptic contacts in their corresponding target areas of the brain (Riddle and Pollock, 2003, Smidt et al., 2003, Vergaño-Vera et al., 2015, Le Grand et al., 2015, Rodríguez-Traver et al., 2016).

1.2.3 Regulation of OB DA neuron development

Studies regarding the development of OB DA neurons are scarcer than those about mesDA neurons. There are two types of OB DA neurons, which are different in size and morphology: the small periglomerular neurons (PGNs), positioned in the glomerular layer and representing the large majority of OB DA cells, and the large external tufted cells (ET), located mostly at the boundary between the glomerular layer and the external plexiform layer (EPL) and rarely found within the EPL (Bonzano et al., 2016, Pignatelli and Belluzzi, 2017). OB DA neurons are thought to be initially specified by SHH and FGF8 as is the case of mesDA neurons (Prakash and Wurst, 2006). Although NURR1 is expressed in PGNs (Backman et al., 1999, Saino-Saito et al., 2004, Diaz-Guerra et al., 2013), its role and influence on DA generation from OB stem cells (OBSCs) is not well defined. Notwithstanding, recent data from our laboratory indicate that NURR1 induces the differentiation of olfactory bulb stem cells (OBSCs) into mesencephalic-like DA neurons and OB-like neurons through mechanisms possibly involving the regulation of the fibroblast growth factor receptor 2 (Fgfr2) (Diaz-Guerra et al., 2013, Vergaño-Vera et al., 2015).

A16 or OB DA neurons present AADC and dopamine transporter (DAT) expression.

Nevertheless, studies dealing with the expression of the vesicular monoamine transporter 2 (VMAT2) (needed for vesicle storage and release) in these neurons remain controversial.

Although VMAT2 expression could not be detected by immunocytochemistry in previous studies (Peter et al., 1995, Weihe et al., 2006), Vmat2 mRNA has been detected in OB DA neurons (Borisovska et al., 2013, Cave et al., 2010), Additionally, G-protein-regulated inward- rectifier potassium channel 2 (GIRK2) expression has been reported in dendrites of PGNs in the OB (Schein et al., 1998). In contrast, calbindin and calretinin are not expressed in OB DA neurons (Hurtado-Chong et al., 2009, Parrish-Aungst et al., 2007) while they are present in midbrain DA neurons (Gonzalez-Hernandez and Rodriguez, 2000, Vergaño-Vera et al., 2015).

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29 Interestingly, OB DA neurons co-express gamma-aminobutyric acid (GABA) and glutamic

acid decarboxylase (GAD), an enzyme involved in GABA synthesis; along with dopamine. This fact suggests that these neurons might use more than one neurotransmitter (Kiyokage et al., 2010, Kosaka and Kosaka, 2011). The release of GABA has also been reported from midbrain DA neurons (Tritsch et al., 2012). Nevertheless, most of mesDA neurons are not GABAergic (Rodriguez and Gonzalez-Hernandez, 1999).

2. The transcription factor NURR1

2.1 Expression and function of NURR1 during the development and maintenance of DA neurons

The transcription factor NURR1 belongs to the orphan nuclear receptor family 4 (NR4A) whose members are Immediate early genes (Diaz-Guerra et al., 2013). Members of NR4A family share a common structure consisting of a weakly conserved amino-terminal A/B region, which presents the activation function-1 transactivation domain, and two highly conserved domains: a DNA-binding domain and a carboxy-terminal ligand-binding domain. Transcription can be activated by the DNA-binding domain after binding to an NGFI-B response element (NBRE). NURR1 can form heterodimers with retinoid X receptor (RXR) and bind to RXR response element or it can activate transcription as homodimers or monomers. Ligands for NR4A have not been identified and their activity seems to be regulated by ligand-independent mechanisms at the level of gene expression, protein stability and protein-protein interactions (Hawk and Abel, 2011, Diaz-Guerra et al., 2013, Paulsen et al., 1995, Kurakula et al., 2014, Renaud et al., 1995, Ichinose et al., 1999).

NURR1 expression is restricted to the nucleus of neurons distributed along the central nervous system (CNS), the highest expression is detected in the substantia nigra (SN), ventral tegmental area (VTA) and retrorubral field of the midbrain and in limbic areas (Zetterström et al., 1996, Backman et al., 1999, Rodríguez-Traver et al., 2016). Additionally, NURR1 is present in the OB, where it appears to be expressed in a synaptic activity-dependent manner (Saino- Saito et al., 2004), as it happens in the dopaminergic phenotype in the SN DA neurons (Aumann and Horne, 2012), temporal cortex, hippocampus, posterior hypothalamus, striatum, subiculum, cerebellum and habenular nuclei (Saucedo-Cárdenas and Conneely, 1996, Xiao et al., 1996). Modest NURR1 expression was found in TH immunoreactive neurons from the paraventricular and periventricular hypothalamic nucleus, while it could not be detected in the noradrenergic neurons of the brainstem. The fact that NURR1 expression could not be observed in all CNS catecholaminergic neurons, but is highly confined to OB periglomerular and midbrain DA neurons suggests a possible specific role for this TF in the regulation of DA functions in the OB and midbrain (Backman et al., 1999, Diaz-Guerra et al., 2013).

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Different studies have provided evidence that NURR1 is essential for the development, maintenance and survival of dopaminergic neurons (Saucedo-Cardenas et al., 1998, Zetterstrom et al., 1997, Alavian et al., 2014, Kadkhodaei et al., 2009). The expression of NURR1 in the murine CNS begins around embryonic day (E) 10.5, after FOXA2 and just before PITX3 and TH (E11.5) can be detected. Although, NURR1 expression is reduced in postnatal animals, DA neurons express NURR1 throughout life. This fact suggests a role for NURR1 not only in an early stage of DA neuron differentiation, but also in cell maintenance and acquisition of neurotransmitter identity promoting the acquisition of DA marker expression such as TH, DAT, AADC and VMAT2 (Saucedo-Cardenas et al., 1998, Smits et al., 2003, Law et al., 1992, Zetterstrom et al., 1997, Le et al., 1999, Kadkhodaei et al., 2009, Kaestner et al., 1994, Alavian et al., 2014). Moreover, BDNF has been reported to be a downstream target of NURR1, which may have implications in the maturation of DA neurons (Volpicelli et al., 2007). Furthermore, this TF is involved in the maintenance of neuronal fiber integrity and nuclear-encoded mitochondrial gene expression (Kadkhodaei et al., 2013). NURR1 also prevents mitochondrial impairment, which has been described in PD (Lee et al., 2002, Zhang et al., 2009). Moreover, in microglia and astrocytes, NURR1 shows anti-inflammatory effects by inhibiting neurotoxic mediators, which protects against DA neuronal loss in PD (Saijo et al., 2009).

Due to the important role of NURR1 in the DA system, mutations in Nurr1 gene and alterations in its expression have been related with PD and several psychiatric disorders such as manic behaviour, schizophrenia and predisposition to cocaine abuse (Buervenich et al., 2000, Bannon et al., 2002, Xu et al., 2002, Vuillermot et al., 2011, Zheng et al., 2003, Le et al., 2003).

2.2 In vitro studies using NURR1 for the generation and transplantation of DA neurons

As mentioned previously, NURR1 is essential for the differentiation of DA progenitor cells to mature DA neurons and for the maintenance and survival of these neurons.

Consequently, the regulation of NURR1 expression levels is being investigated as a tool for the generation of DA neurons in vitro (Rodríguez-Traver et al., 2016). This could be used for the study of NURR1 function and its mechanisms of action within the DA system, as well as a potential source of neurons for both transplantations in PD and the establishment of a cellular model of PD to study mechanisms of degeneration.

2.2.1 NURR1 overexpression in neural progenitors and neural stem cells

Several approaches have been used for the generation of DA neurons in vitro using NURR1 overexpression. Nevertheless, the generation of immature DA neurons or non-

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31 overexpressed, implying that the neuronal phenotype of the cells expressing TH should be

confirmed (Hong et al., 2014, Park et al., 2006a, Sakurada et al., 1999, Wagner et al., 1999).

In 1999, Sakurada et al. showed that overexpressing NURR1 in adult hippocampal precursors induced TH expression by direct binding of NURR1 to the Th promoter. However, the expression of other DA markers such as AADC, the protein tyrosine kinase receptor c-RET, PITX3, D2R or VMAT2, was not affected suggesting that NURR1 did not stimulate fully neuronal differentiation (Sakurada et al., 1999). Additionally, Wagner et al., (1999) overexpressing NURR1 in a cerebellum derived immortalized cell line C17.2 obtained mature DA neurons only when they were co-cultured with ventral mesencephalic type I astrocytes (Wagner et al., 1999).

In subsequent studies, neural progenitor cells (NPCs) isolated from different developmental stages and regions of origin, have been differentiated in cell culture into mature and functional DA neurons although a proportion of NURR1-overexpressing neurons in the culture remained immature (Kim et al., 2003b). NURR1-derived DA neurons obtained, displayed spontaneous and potassium-evoked dopamine release and expressed dopaminergic markers as Th, Aadc, DAT, Vmat and Pitx-3 detected by RT-PCR. However, when these cells were grafted in 6-hydroxydopamine-lesioned rats Parkinsonian symptoms could not be restored. Furthermore, we have obtained DA neuronal mature phenotypes in vitro by overexpressing NURR1 in mouse embryonic OBSCs (eOBSCs, Vergaño-Vera et al., 2015).

As explained along this thesis, NURR1 overexpression in eOBSCs inhibited cell proliferation and gave rise to the generation of immature neurons, which under long-term culture conditions generated mature-like mesDA neurons, and a subpopulation of DA-GABAergic neurons.

3. Parkinson’s disease (PD)

PD is the most common multifactorial neurodegenerative movement disorder affecting around 1% of the population aged 65 or above, and increasing to 4% above 85 years of age (Kalia and Lang, 2015, Surmeier et al., 2017a, Przedborski, 2017). This disease presents with loss of the substantia nigra pars compacta DA neurons (A9), which project to the striatum, and the presence of Lewy bodies, containing α-synuclein (α-syn) and ubiquitin (Kalia and Lang, 2015, Michel et al., 2016, Moore et al., 2005). The symptoms resulting from this loss of DA neurons include motor deficits such as rigidity, slowness in movement (bradykinesia), postural instability, gait impairment and a characteristic tremor at rest (Parkinson, 1817). Besides the classical PD motor symptoms, other non-motor features (Figure 2) are related with PD as olfactory dysfunction, cognitive impairment, psychiatric symptoms, sleep disorders, autonomic dysfunction, pain, and fatigue (Kalia and Lang, 2015, Lees et al., 2009, Przedborski, 2017).

Several mechanisms, that will be discussed below, are implicated in the degeneration

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of SN DA neurons in PD including misfolding and aggregation of the synaptic protein α-syn, disruption of the autophagy-lysosome system, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, or dysregulation of calcium homeostasis (Michel et al., 2016).

Despite the historically consideration of PD as a sporadic disorder of unknown etiology, nowadays a remarkable proportion of cases (around 5-10%) have been related with familial genetic mutations (Lin and Farrer, 2014). Thus, mutations in different genes, which can act as Mendelian factors directly causing PD, such as the mutations in α-SYN (SNCA1) or in leucine rich repeat kinase 2 (LRRK-2) genes; risk factors, like the mutations in the glucocerebrosidase1 (GBA1) gene; or mutations associated with the disease, such as mutations in NURR1 (NR4A2); have shown the importance of genetic predisposition as causal factor of PD (Lees et al., 2009, Le et al., 2003, Delamarre and Meissner, 2017, Lill, 2016).

3.1 PD and GBA1 mutations

The GBA1 gene encodes the lysosomal enzyme β-glucocerebrosidase (GCase1), which catalyzes the hydrolysis of the β-glucosidic linkage of the glucosylceramide (GlcCer), also known as glucocerebroside, to ceramide and glucose (Figure 3). Heterozygous mutations in GBA1 are considered the strongest genetic risk factor for PD and PD-associated Dementia with Lewy bodies (Cilia et al., 2016, Sidransky and Lopez, 2012). Homozygous GBA1 mutations causes Gaucher’s disease (GD), the most prevalent lysosomal storage disorder (LSD) includes a decreased GCase1 activity and subsequent GlcCer accumulation in several tissues including the brain (Zhao and Grabowski, 2002, Aflaki et al., 2017). GBA1 mutations have been estimated to increase the risk of PD development 20-30-fold and 7-10% of PD Figure 2. Time course and clinical symptoms of Parkinson’s disease (PD) progression. Although a pre-motor or prodromal phase can appear 20 years before the manifestation of the motor symptoms and the search of prodromal biomarkers is increasing (Muller et al., 2016, Delenclos et al., 2016), PD diagnosis takes place once motor symptoms arise (time 0 years). Following disease progression, other non-motor symptoms can appear, causing clinically significant disability. Long-term complications as a result of dopaminergic therapy, including fluctuations, dyskinesia, and psychosis, also contribute to disability. EDS: excessive daytime sleepiness; MCI: mild cognitive impairment; RBD: REM sleep behaviour disorder (Kalia and Lang, 2015).

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33 idiopathic PD, but in GBA1-PD, the age of onset is slightly reduced and there is a tendency for

more cognitive impairment and dementia (Schapira, 2015, Abeliovich and Gitler, 2016, Seto- Salvia et al., 2012, Mata et al., 2016).

3.1.1 Pathology of GBA1-PD and effects of GBA1 mutations

The molecular mechanisms linking a decrease in GCase1 activity as a consequence of GBA1 mutations with an increase of the risk of PD are still not fully understood.

Figure 3. Glucocerebrosidase 1 reaction. Glucocerebrosidase 1 (Gcase1) catalizes the hydrolysis of the β-glucosidic linkage of the glucocerebroside/ glucosylceramide (GlcCer), giving rise to a ceramide and a glucose.

Figure 4. The impact of GBA1 mutations on cellular homeostasis. GBA1 mutations have an effect in the endoplasmic reticulum (ER) functioning. Misfolded GCase1 retained in the ER triggers an increase in the ubiquitin–proteasome system (UPS) and ER stress. ER stress leads to the unfolded protein response (UPR) and/or endoplasmic reticulum-associated degradation (ERAD). The continuous activation of UPR and ERAD subsequently gives rise to increased apoptosis. Misfolded GCase1 in the lysosomes and a reduction in wild-type GCase levels give rise to a retardation of alpha-synuclein degradation via chaperone-mediated autophagy (CMA) and an accumulation of glucocerebroside (GlcCer) which cannot be degraded, leading to alpha-synuclein accumulation and aggregation. Impaired lysosomal function decreases autophagosome clearance with a subsequent accumulation of them.

GBA1 mutations affect normal mitochondria functioning by increasing generation of free radical species (ROS) and decreasing adenosine triphosphate (ATP) production, oxygen consumption, and membrane potential. Moreover, GBA1 mutations trigger an accumulation of dysfunctional and fragmented mitochondria and a deregulation of calcium homeostasis, which could result in basal mitochondrial stress and affect neuronal survival. Modified from (Migdalska-Richards and Schapira, 2016).

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Nevertheless, the published studies point to a deregulation of cellular homeostasis coursing with endoplasmic reticulum stress, lysosome dysfunction, alpha-synuclein accumulation, autophagic impairment, mitochondrial dysfunction, reactive oxygen species (ROS) production and defects in calcium homeostasis, as mechanisms linked to GCase deficiency that contribute to cell death during GBA1-PD development and progression (Abeliovich and Gitler, 2016, Schapira and Tolosa, 2010, Migdalska-Richards and Schapira, 2016, Magalhaes et al., 2016, Mazzulli et al., 2016, McNeill et al., 2014, Schöndorf et al., 2014, Aflaki et al., 2017, Fernandes et al., 2016) (Figure 4).

Both, autosomal recessive (associated with loss of function of the mutated protein) and autosomal dominant (associated with gain of function of the mutated protein) modes of inheritance have been proposed for GBA1-PD (Sidransky, 2012, Westbroek et al., 2011).

Around 300 different mutations of the GBA1 gene have been reported, but the N370S and L444P are the most frequent in both GD and PD (Beavan and Schapira, 2013).

Most GBA1 mutations do not directly affect the catalytic site of the enzyme; but destabilize its native structure giving rise to misfolded protein accumulation, as mentioned above (Ron and Horowitz, 2005, Fernandes et al., 2016, Sanchez-Martinez et al., 2016).

Crystal structure of GCase1 was obtained in 2003, revealing three non-continuous domains (Figure 5).

The N370S mutation, located at the interface of domain II and III, is thought to cause relatively minor changes in the enzyme structure and does not directly affect the catalytic Figure 5. X-ray structure of glucocerebrosidase1 (GCase1). Domain I, shown in magenta, presents two disulphide bridges, their sulphur atoms are depicted as green balls. The glycosylation site at N19 is shown using a ball-and-stick model. Domain II, shown in green, is an immunoglobulin-like domain.

Domain III, shown in blue is a TIM barrel and is the catalytic domain, containing the active-site residues E235 and E340, shown as ball-and-stick models. The six most common GCase1mutations are shown as balls, with those that cause predisposition to severe Gaucher’s disease (types 2 and 3) and mild (type 1) disease in red and yellow, respectively. NAG, N-acetylglucosamine (Dvir et al., 2003).

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35 the enzyme activity has been reported and it might cause retention of this enzyme in the ER

triggering ER stress and UPR (Dvir et al., 2003, Garcia-Sanz et al., 2017). The L444P mutation, placed at the hydrophobic core of domain II, triggers conformational changes to the core and to domain II, which is postulated to generate an unstable GCase1 and had also lead to a reduction in the enzyme activity (Dvir et al., 2003).

3.1.1.1 Glucocerebrosidase1 (GCase1) alterations and dysfunctions in cellular homeostasis.

GlcCer accumulation as a consequence of a deficiency in GCase1 might lead to an overactivation of ERAD pathway (Abeliovich and Gitler, 2016, Schöndorf et al., 2014).

Misfolded proteins are recognized by the ER quality control system and retained for being refolded and if they are not properly refolded they are degraded by the ERAD. There are some studies reporting the implication of ER stress in PD pathogenesis (Imai et al., 2001, Imai et al., 2000, Ryu et al., 2002). For GBA1 N370S and L444P mutations, ER stress and UPR response could be activated due to the prolonged accumulation of misfolded GCase1 in the ER.

Retention of misfolded GCase in the ER and ERAD has been reported in GD fibroblasts (Bendikov-Bar et al., 2011, Ron and Horowitz, 2005, Garcia-Sanz et al., 2017), and UPR markers have shown to be increased in putamen samples from PD-GBA1 (Gegg et al., 2015) as well as in neurons derived from hiPSCs of PD-GBA1 patients (Fernandes et al., 2016).

Alterations in trafficking to lysosomes and altered lysosomal function are thought to be involved in the loss of neurons in PD (Abeliovich and Gitler, 2016). Thus, deficiency in GCase1 can trigger neurodegeneration due to defects in endosome-lysosome fusion, impairment of the autophagic flux with consequences for autophagosome clearance, accumulation of lysosomes or general lysosome dysfunction (Schöndorf et al., 2014).

Autophagy is a lysosomal pathway in which long-lived, misfolded or aggregated cellular proteins (as α-syn), lipids, intracytoplasmic aggregates and damaged organelles such as endoplasmic reticulum or mitochondria are degraded (Migdalska-Richards and Schapira, 2016, Yang and Klionsky, 2010). Although molecular mechanisms linking authophagic impairment in the developing PD-GBA1 still need to be fully elucidated, recent data suggests their importance (see above). Lysosomal dysfunction leading to GlcCer accumulation has a relevant role in GD, as accumulation of sphingolipids has shown to induce cell death and reduce autophagosome clearance, altering autophagy (Tamboli et al., 2011, Migdalska- Richards and Schapira, 2016).

Regarding the link between autophagic dysfunction in GBA1-PD, there is some controversy about the role of GlcCer accumulation since some authors suggested the requirement of both GBA1 alleles to carry a mutation for this accumulation (Migdalska- Richards and Schapira, 2016). Discrepancies among GlcCer accumulation have been

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observed along different brain regions. Thus, no accumulation of GlcCer was reported in putamen and cerebellum samples from GBA1-PD patients (Gegg et al., 2015). However, a significant increase in GlCer levels was detected in the hippocampus and substantia nigra from idiopathic PD patients (Rocha et al., 2015). Nevertheless, in the putamen and cerebellum of idiopathic PD patients no changes in GlcCer levels were reported (Gegg et al., 2015, Rocha et al., 2015). Otherwise, in iPSC-derived neurons from GBA1-PD patients, a reduction in glucocerebrosidase activity was accompanied by an increase in GlcCer and alpha-synuclein levels. Moreover, autophagic and lysosomal defects; and a dysregulation of calcium homeostasis was reported (Schöndorf et al., 2014, Fernandes et al., 2016). Further studies are needed to elucidate the link between the reduction of GCase activity and the accumulation of GlcCer, as well as whether autophagic dysfunction is at least a partial consequence of GlcCer accumulation (Migdalska-Richards and Schapira, 2016). Moreover, autophagic impairment by GBA1 mutations in PD could also be explained taking into account the link between α-syn accumulation and mutant GCase1 (section 3.1.1.2).

The role of mitochondrial dysfunction in PD-GBA1 pathogenesis remains unclear and only few studies have tried to elucidate the effect of GBA1 mutations on mitochondrial structure (fragmentation) and function, which lead to oxidative stress (Cleeter et al., 2013, Osellame et al., 2013, Xu et al., 2014, Garcia-Sanz et al., 2017).

Defects in calcium homeostasis may also play a crucial role in the preferential loss of DA neurons in PD and especially of SN DA neurons (Michel et al., 2016). These neurons have broad action potentials and display a distinctive pacemaker phenotype accompanied by large intracellular Ca²⁺ oscillations that enter through Cav1 channels. They are also characterized by low Ca²⁺buffering capacity which is thought to trigger basal mitochondrial oxidant stress (Surmeier et al., 2017a, Surmeier et al., 2017b). Defects in calcium homeostasis may appear mainly as a consequence of changes in the discharge activity of DA neurons, but may also be linked with α-syn aggregation, mitochondrial deficits, or ER dysfunction (Michel et al., 2016).

Deregulation of calcium homeostasis has been reported in neurons derived from hiPSCs of GBA1-PD patients in which an increase of the neuronal calcium-binding protein 2 (NECAB2) was accompanied by an increased vulnerability to stress responses involving elevation of cytosolic calcium (Schöndorf et al., 2014).

3.1.1.2 GCase1 and alpha-synuclein

Alpha-synuclein is a small protein which should be enriched in the presynaptic compartment (Iwai et al., 1995) and might promote the formation of the SNARE complex.

Consequently, this protein is thought to be involved in the regulation of vesicle dynamics, trafficking and neurotransmitter release (Abeliovich and Gitler, 2016). Indeed, it is thought to