The molecular pathology of the neurodegenerative disease spinal muscular atrophy

Institute of Neuroanatomy, Hannover Medical School Center for Systems Neuroscience Hannover

The molecular pathology of the

neurodegenerative disease Spinal Muscular Atrophy – role of nuclear complexes and

nuclear body regulation

DISSERTATION

Submitted for the degree - Doctor rerum naturalium -

( Dr. rer. nat. )

awarded by the University of Veterinary Medicine Hannover

by

Benjamin Förthmann Hannover

Hannover, Germany 2013

Supervisor: Prof. Dr. Peter Claus

Supervisor group: Prof. Dr. Peter Claus

Prof. Dr. Evgeni Ponimaskin

Prof. Dr. Anaclet Ngezahayo

1^st Evaluation: Prof. Dr. Peter Claus Institute of Neuroanatomy

Medical School Hannover, Germany

Prof. Dr. Evgeni Ponimaskin Institute for Neurophysiology

Medical School Hannover, Germany

Prof. Dr. Anaclet Ngezahayo Institute of Biophysics,

Leibniz University Hannover, Germany

2^nd Evaluation: Prof. Dr. Lars Klimaschewski Division of Neuroanatomy

Innsbruck Medical University, Austria

Parts of the thesis have been published previously in:

1. Förthmann B, Brinkmann H, Ratzka A, Stachowiak MK, Grothe C, Claus P (2013):

Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions. Cell Mol Life Sci. 70(14): 2555–2568.

2. Förthmann B, van Bergeijk J, Lee YW, Lübben V, Schill Y, Brinkmann H, Ratzka A, Stachowiak MK, Hebert M, Grothe C, Claus P: Regulation of neuronal differentiation by proteins associated with nuclear bodies. PLoS One.

3. Baron O, Förthmann B, Lee YW, Terranova C, Ratzka A, Stachowiak EK, Grothe C, Claus P, Stachowiak MK (2012): Cooperation of nuclear fibroblast growth factor receptor 1 and Nurr1 offers new interactive mechanism in postmitotic development of mesencephalic dopaminergic neurons. J Biol Chem. 287(24): 19827–19840.

Oral and poster presentations:

1. Benjamin Förthmann, Yu-Wei Lee, Jeroen van Bergeijk, Hella Brinkmann, Claudia Grothe, Michal K. Stachowiak and Peter Claus (2011): The intranuclear mobility and neuronal differentiation capacity of the survival of motoneuron protein is regulated by FGF-2. 8th UK SMA Researchers’ Conference, Oxford, Oct. 3 – Oct. 4, 2011.

2. Benjamin Förthmann (2011): Nuclear Fibroblast growth factor – 2 forms an inhibitory complex with the survival of motoneuron (SMN) protein. 4th Graduate School Day, Bad Salzdetfurth, Nov. 25 – Nov. 26, 2011.

3. Benjamin Förthmann, Hella Brinkmann, Claudia Grothe, Michal K. Stachowiak and Peter Claus (2012): Fibroblast growth factor – 2 affects intranuclear mobility and functions of the survival of motoneuron (SMN) protein. 16th Annual SMA Research Group Meeting, Minneapolis, June 21 – June 24, 2012.

4. Benjamin Förthmann, Hella Brinkmann, Claudia Grothe, Michal K. Stachowiak and Peter Claus (2012): Molecular dynamics of the survival of motoneuron (SMN) protein in the nucleus – mobilization of SMN in Cajal bodies. 5th Graduate School Day, Hannover, Nov. 23 – Nov. 24, 2012.

5. Benjamin Förthmann, Hella Brinkmann, Claudia Grothe, Michal K. Stachowiak and Peter Claus (2012): Molecular dynamics of the survival of motoneuron (SMN) protein in the nucleus – mobilization of SMN in Cajal bodies. 2012 ASCB Annual Meeting, San Francisco, Dec. 15 – Dec. 19, 2012.

6. Benjamin Förthmann, Hella Brinkmann, Claudia Grothe and Peter Claus (2013):

The function and dynamics of the survival of motoneuron (SMN) protein is regulated by a nuclear growth factor. 21. Kongress des Wissenschaftlichen Beirates der Deutschen Gesellschaft für Muskelkranke e.V., Aachen, Feb. 28 – Mar. 02, 2013.

Awards/ sponsorship:

1. First Poster prize

8th UK SMA Researchers’ Conference, Oxford, Oct. 3 – Oct. 4, 2011.

2. Best Poster Award

5th Graduate School Day, Hannover, Nov. 23 – Nov. 24, 2012.

3. Completion scholarship

from the Tierärztliche Hochschule Hannover, July 01 – Sept. 30, 2013.

Index

Index

Index... I Abbreviations ...II List of figures ... V

Summary ... 1

Zusammenfassung ... 3

Introduction ... 5

The nucleus ... 5

Cajal bodies ... 8

Coilin ... 10

Spinal muscular atrophy... 11

Fibroblast growth factor - 2... 14

Chapter I Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions... 19

Chapter II Regulation of neuronal differentiation by the survival of motoneuron (SMN) protein interaction partners FGF-2 and coilin... 21

Chapter III Cooperation of nuclear fibroblast growth factor receptor 1 and Nurr1 offers new interactive mechanism in postmitotic development of mesencephalic dopaminergic neurons... 23

Discussion ... 25

References ... 33

Affidavit ... 42

Acknowledgement... 43

Abbreviations

Abbreviations

AP-1 Activator protein – 1

a-SMN axonal-SMN

BAMC Bovine adrenal medullary cells

°C Degree Celsius

CB Cajal body

CBP CREB-binding protein

CNS Central nervous system

CREB cAMP response element-binding protein

CRM1 Chromosomal maintenance 1

DMEM Dulbecco´s modified eagle´s medium

DNA Deoxyribonucleic acid

E Embryonic day

EGFP Enhanced green fluorescent protein ERK Extracellular signal-regulated kinase

EtOH Ethanol

FGF-2¹⁸ Fibroblast growth factor – 2 (18 kDa) FGF-2²³ Fibroblast growth factor – 2 (23 kDa) FGFR1 Fibroblast growth factor receptor 1

FHF Fibroblast growth factor homologous factor

FIF FGF – 2 interacting factor

FL Full-length

FRAP Fluorescence recovery after photobleaching

gem Gemini body

h Hours

HEK HEK293T human embryonic kidney cell line

Abbreviations

kDa Kilo Dalton

LSm LikeSm or Sm-like

mDA Mesencephalic dopaminergic

NB SK-N-BE(2) human neuroblastoma cell line

NBRE Nurr1 monomer binding responsive element

NGF Nerve growth factor

NIH 3T3 Mouse embryonic fibroblast cell line 3T3 "3-day transfer, inoculum 3 x 10^5 cells."

NLS Nuclear localization sequence

NPAT Nuclear protein of the ataxia telangiectasia mutated locus

NPC Neuronal progenitor cell

NSC34 Mouse motoneuron-like hybrid cell line

PBS Phosphate buffered saline

PC12 Cell line derived from a pheochromocytoma of the rat adrenal medulla

PcG Polycomb group

PFA Paraformaldehyde

PIASy Protein inhibitor of activated STAT Y

PI 3 Phosphatidylinositol 3

PML Promyelocytic leukaemia oncoprotein

pre-mRNA Precursor mRNA

RA Retinoic acid

RAR Retinoic acid receptor

Ras Rat sarcoma protein

RG box Stretch of arginine and glycin dipeptide residues

RNA Ribonucleic acid

RNase Ribonuclease

RTK Receptor tyrosine kinase

RSK1 Ribosomal S6 kinase 1

RXR Retinoid X receptor

SAM-68 Src-associated in mitosis, 68 kDa

SART3 Squamous cell carcinoma antigen recognized by T cells 3

Abbreviations

scaRNA Small Cajal body-specific RNA

scr Scrambled

siRNA Small interfering RNA

SMA Spinal muscular atrophy

SMN Survival of motoneuron protein

snoRNA small nucleolar RNA

snRNA Small nuclear RNA

snRNP Small nuclear ribonucleoprotein particle

Tgs1 Trimethylguanosine synthase 1

TH Tyrosine hydroxylase

TMG 2,2,7-trimethylguanosine

UBF Upstream binding factor

VM Ventral midbrain

YFP Yellow fluorescent protein

ZPR1 Zinc finger protein 1

List of figures

List of figures

Introduction

Fig. 1 Hierarchically and stochastic nuclear body assembly models………..7 Fig. 2 Low levels of full length SMN protein is produced by the SMN2 gene……....…...…..12 Fig. 3 Transcription and functions of different FGF-2 isoforms...……….………....16 .

Discussion

Fig. 1 Model for fine tuning of neuronal differentiation by FGF-2²³ and CB

proteins SMN and coilin……….29

Summary

Benjamin Förthmann

The molecular pathology of the neurodegenerative disease Spinal Muscular Atrophy – role of nuclear complexes and nuclear body regulation

Summary

Spinal muscular atrophy (SMA) is a neurodegenerative disease, caused by reduced levels of the survival of motoneuron (SMN) protein. How SMN is involved in maintaining motoneuron integrity is unknown, so far. In the nucleus, SMN is associated to two compartments called nuclear gems and Cajal bodies (CBs). The binding of SMN to Cajal bodies occurs by a direct interaction with the CB marker protein coilin. Although the major amount of coilin is located diffuse in the nucleus outside of CBs, SMN and coilin are essential for maturation of small nuclear ribonucleoprotein particles (snRNPs) inside Cajal bodies. There, three single snRNPs are assembled to one tri-snRNP, a subunit of the spliceosom. Another direct binding partner of nuclear SMN is the 23 kDa isoform of fibroblast growth factor – 2 (FGF-2²³). The FGF- 2²³/SMN interaction is already known to destabilize nuclear gems, the second nuclear compartment SMN is associated to. By using fluorescence recovery after photobleaching (FRAP) and photoconversion experiments in the present study of Förthmann et al., 2013 (Chapter I), we have discovered that FGF-2²³/SMN complex formation leads to a release of immobile SMN from CBs, followed by snRNP accumulation. Thus, tri-snRNP assembly at Cajal bodies seems to be associated with immobile SMN. In an unpublished study of Förthmann et al. (Chapter II), we show that co-expression of FGF-2²³ and SMN inhibits both FGF-2²³- dependent transcription and SMN promoted neurite outgrowth. Hence, we propose a model in which FGF-2²³ and SMN form an inactive complex, antagonizing both protein functions. Moreover, there is evidence to suggest that neuronal differentiation is likewise affected by diffuse nucleoplasmic coilin. Endogenous coilin levels decrease during differentiation of human neuroblastoma cells (NB), as it is already pointed out for murine PC12 cells. Expression of coilin reduces neurite outgrowth, indicating a new function of this protein in inhibition of neuronal differentiation. While SMN has coilin and FGF-2²³ as interaction partners, among others, FGF-2²³ has another nuclear binding partner beside SMN:

the fibroblast growth factor receptor 1 (FGFR1). FGF-2²³/FGFR1 interaction heightens neuronal differentiation by inducing a pathway called integrative nuclear FGFR1 signaling

Summary

(INFS). The INFS activates the tyrosine hydroxylase (TH) gene. During the development of TH-positive cells in the ventral midbrain (VM) of embryonic mice, the FGFR1 builds a nuclear complex with the orphan nuclear receptor Nurr1, as we could demonstrate in the study of Baron et al., 2012 (Chapter III). FRAP experiments reveal a dynamic change:

FGFR1 is slowed down by interaction with Nurr1 into a chromatin-bound fraction. The TH gene promoter is activated by FGFR1/Nurr1 complex formation, similar to the FGFR1/FGF- 2²³ binding.

In conclusion, the regulation of the presented nuclear body and protein complexes influences neuronal differentiation and neurite outgrowth in vitro and in vivo. We assume the nuclear architecture to be relevant in differentiation processes of developing neuronal cells and therefore to be a possible target in neurodegenerative and neurodevelopmental disorders.

Zusammenfassung

Benjamin Förthmann

Die molekulare Pathologie der neurodegenerativen Erkrankung Spinale Muskelatrophie – die Rolle von nukleären Komplexen und Kernkörperregulation

Zusammenfassung

Die Spinale Muskelatrophie (SMA), eine neurodegenerative Erkrankung, wird durch eine reduzierte Menge des Survival of Motoneuron (SMN) Proteins hervorgerufen. Welchen Einfluss das SMN Protein auf das Überleben von Motoneuronen hat, ist bislang unbekannt.

Im Zellkern konzentriert sich SMN in zwei Kernkörpern: Gems und Cajal Bodies (CBs). Für die Anlagerung von SMN an Cajal Bodies ist Coilin verantwortlich, das SMN direkt bindet und ein Markerprotein von CBs darstellt. Es kommt im Zellkern vorwiegend diffus außerhalb von Cajal Bodies vor. Innerhalb der CBs sind SMN und Coilin für die Reifung kleiner nukleärer Ribonukleoproteinpartikel (snRNPs) verantwortlich. Eine zentrale Aufgabe von Cajal Bodies ist der Aufbau eines Tri-snRNPs aus drei einzelnen snRNPs, einer Untereinheit des Spliceosoms. Neben Coilin hat das SMN Protein einen weiteren Bindungspartner im Zellkern: die 23 kDa schwere Isoform des Fibroblasten-Wachstumsfaktors – 2 (FGF-2²³). Die Wechselwirkung von FGF-2²³ und SMN führt zur Destabilisierung von Gems, des zweiten Typus von Kernkörpern in denen SMN lokalisiert ist. In der vorliegenden Studie von Förthmann et al., 2013 (Chapter I) wird anhand von Fluorescence recovery after photobleaching (FRAP)- und Photokonversions- Experimenten gezeigt, dass die FGF- 2²³/SMN Komplexbildung zu einer Freisetzung von immobilem SMN an Cajal Bodies führt.

Daraus resultiert eine Anhäufung von snRNPs an Cajal Bodies. Wir nehmen daher an, dass immobiles SMN eine wesentliche Funktion bei der Assemblierung von Tri-snRNPs in den CBs erfüllt. Eine bisher unveröffentlichte Studie von Förthmann et al. (Chapter II) konkretisiert, dass die Co-Expression von FGF-2²³ und SMN einen hemmenden Einfluss auf die FGF-2²³ abhängige Transkription und auf gesteigertes Neuritenwachstum durch SMN ausübt. Infolgedessen schlagen wir ein Modell vor, in dem die Bindung von FGF-2²³ an SMN zu einem inaktiven Komplex führt, der beide Proteinfunktionen antagonisiert. Des Weiteren gibt es deutliche Hinweise darauf, dass die neuronale Entwicklung ebenfalls durch das diffuse Coilin im Zellkern beeinflusst wird. Während der Differenzierung verringert sich die Menge von endogenem Coilin in humanen Neuroblastoma Zellen, in Übereinstimmung mit früheren

Zusammenfassung

Experimenten mit murinen PC12 Zellen. Die Überexpression von Coilin bedingt kürzere Neuriten, womit sich eine neue Funktion dieses Proteins in der Hemmung neuronaler Differenzierung andeutet. Während u.a. Coilin und FGF-2²³ Bindungspartner von SMN sind, hat FGF-2²³ neben SMN einen weiteren Interaktionspartner im Zellkern: den Fibroblasten- Wachstumsfaktor-Rezeptor 1 (FGFR1). Die Bindung von FGF-2²³ an FGFR1 verstärkt die neuronale Differenzierung, indem sie den nukleären FGFR1 Signalweg (INFS) initiiert.

Dieser Signalweg aktiviert das Tyrosinhydroxylase (TH) Gen. In der Entwicklung von TH- positiven Zellen im ventralen Mittelhirn (VM) embryonaler Mäuse bildet sich aus FGFR1 und dem nukleären Rezeptor Nurr1 ein neuer Proteinkomplex, den wir in einer weiteren Studie, von Baron et al., 2012 (Chapter III) zeigen konnten. FRAP Experimente verdeutlichen den Einfluss von Nurr1 auf die Mobilität von FGFR1. In Anwesenheit von Nurr1 vergrößert sich der Anteil von langsamen, Chromatin-gebundenem FGFR1. Der TH-Promotor wird durch den FGFR1/Nurr1 Komplex aktiviert, ähnlich der Auswirkung von FGFR1/FGF-2²³.

Zusammenfassend lassen sich Einflüsse von nukleären Proteinkomplexen und Kernkörpern auf die neuronale Differenzierung und das Neuritenwachstum in vivo und in vitro feststellen.

Anscheinend spielt die Kernstruktur eine Rolle für die Entwicklung und Differenzierung von Nervenzellen. Diese könnte einen möglichen Ansatzpunkt zur weiteren Analyse neurodegenerativer Erkrankungen und von Entwicklungsstörungen des Nervensystems darstellen.

Introduction

Introduction

The nucleus

The first described cell organelle is discovered by Franz Bauer in 1802 and published later by Robert Brown who has named it the nucleus (Harris 2000). It is the most undeformable and mechanically resistant structure in the cell (Weiss et al. 1991).

The close examination of the nuclear substructure and architecture is possible with the use of fluorescence microscopy. This is feasible since the 1980s with the potential to visualize particular proteins in fixed cells (Spector 1993). This method allows to distinguish several intranuclear structures morphologically and facilitates the investigation of nuclear functions (Lamond and Earnshaw 1998). With the use of fluorescent tags, proteins and mRNA in living cells can be observed by quantitative analysis (Politz and Pederson 2000; Misteli 2001), illustrating that the nucleus is highly dynamic (Dundr and Misteli 2001).

Kinetic analysis of proteins in the nucleus of living cells with fluorescence recovery after photobleaching (FRAP) experiments demonstrate that proteins rapidly move throughout the nucleus due to a passive diffusion process and appear to associate and dissociate with nuclear compartments (Phair and Misteli 2000). High mobility is suggested to be a general feature of nuclear proteins, whereas nuclear compartments, also referred to as nuclear bodies, are stable structures, although they show a continuous and rapid exchange of proteins with the nucleoplasm (Dundr and Misteli 2001). The size of nuclear bodies is presumably affected by a balance between protein association (on-rate) and protein dissociation (off-rate) from nuclear complexes (Dundr and Misteli 2010).

Many nuclear macromolecules are segregated in distinct compartments, called nuclear bodies, including the chromosomes (Visser et al. 2000), the nucleolus (Pederson 1998), the speckle compartment (Lamond and Spector 2003), factories for replication and transcription (Cook 1999) and a growing family of small dot-like nuclear bodies like promyelocytic leukaemia oncoprotein (PML) bodies, histone locus bodies (HLBs), Cajal bodies (CBs), Gemini bodies (gems), polycomb group (PcG) bodies, SAM-68 bodies, heat shock factor 1 (HSF1) foci and GATA-1 foci (reviewed in Matera et al. 2009). Bodies are composed of multiple protein and RNA types, not enclosed by lipid membranes and appear as irregular foci

Introduction

(Machyna et al. 2013). During the cell cycle, the nucleus is reorganized, the disassembled nuclear domains like the nucleolus and nuclear bodies reassemble after mitosis in a coordinated event, crucial for the organization and function of the nucleus (Muro et al. 2010).

The mechanisms of nuclear body biogenesis are poorly understood by now. It is assumed that an initial nucleation event is required to start the formation of nuclear bodies, which serves as a template by immobilizing freely diffusing key components (Dundr and Misteli 2010). Hence, Cajal bodies (CBs) have often been used as a paradigm for nuclear body assembly (Matera et al. 2009; Dundr and Misteli 2010). CBs are found associated with small nuclear (sn)RNA genes that are proposed to be “CB organizers” (Frey and Matera 1995;

Gao et al. 1997), indicating an ordered mechanism of nuclear body formation (Matera et al.

2009). In agreement with this perception, the association of Cajal bodies with snRNA genes is dependent on active snRNA transcription (Frey et al. 1999; Frey and Matera 2001). During transcription, nascent RNA transcripts could act like immobilized templates, recruiting RNA- interacting proteins and starting nuclear body formation (Dundr and Misteli 2010; Carmo- Fonseca and Rino 2011). In opposite, large CBs can be observed in transcriptionally inactive pronuclei in vitro, using sperm nuclei placed in Xenopus egg extracts (Bauer et al. 1994), illustrating in this case that Cajal bodies can be formed independently of these proposed “CB organizers” (Matera et al. 2009). Another aspect against ordered nuclear body assembly is de novo formation (synthesis of complex structures out of simple molecules), which is observed for different nuclear bodies (Dundr and Misteli 2010). Immobilization of single proteins of Cajal bodies or PML bodies leads to de novo formation of the respective body in the nucleus of HeLa cells. Furthermore, de novo formed CBs show similar size and component dissociation kinetics as endogenous Cajal bodies (Dundr et al. 2004; Kaiser et al. 2008), supporting a model of stochastic assembly devoid of a hierarchical assembly pathway (Matera et al. 2009) (Fig. 1).

Introduction

Fig. 1 Hierarchically and stochastic nuclear body assembly models.

In both models, a nucleation event initiates nuclear body formation (Dundr and Misteli 2010). In a hierarchical model the assembly proceeds in an ordered, step-wise manner after immobilization of a template (Matera et al.

2009). In contrast, in a stochastic model, immobilization of a nuclear body component initiates nuclear body assembly in an unsystematic manner and is powered by interactions between single elements (Dundr and Misteli 2010). In this model, different NB components can serve as equal templates, resulting in similar nuclear bodies (Matera et al. 2009) (modified from Matera et al. 2009).

There is still a controversy about the formation and localization of the nuclear compartments (Pederson 2000; Nickerson 2001). Both could result either from macromolecular crowding caused by high protein concentrations in the nucleus or from the nuclear matrix, a model of a fibrous network, which is proposed to structure the nuclear interior (Hancock 2004). Macromolecular crowding could facilitate the assembly of nuclear complexes due to the finding that biochemical reaction rates are accelerated already by increased relative protein concentrations in the nucleus (Richter et al. 2008; Zhou et al. 2008).

Introduction

Cajal bodies

Nuclear bodies that include the survival of motoneuron (SMN) protein complex are distinguished in Cajal bodies and nuclear gems, which are near or associated to each other, akin in number and size (Liu and Dreyfuss 1996). It is suggested that CBs are inseparable from gems in most tissues. In the HeLa strain ATCC, nuclear gems and Cajal bodies cannot be distinguished while in HeLa strain PV distinct foci for gems beside CBs can be found (Matera and Frey 1998). In fetal tissues, these nuclear bodies appear as detached structures but during ontogenesis their colocalization increases (Young et al. 2001). Furthermore, in neuroblastoma cells as well as in murine and human central nervous systems (CNS) several intranuclear CBs and gems can be identified (Francis et al. 1998).

Cajal bodies are primary termed “nucleolar accessory bodies”, because of their closeness to the nucleolus, by Santiago Ramon y Cajal who has discovered these nuclear compartments in 1903 (Cajal 1903; Gall 2000). The size of CBs ranges from less than 0.2 µm up to 2 µm and even larger (Cioce and Lamond 2005). Similar to other nuclear structures, Cajal bodies seem to be very dynamic: they split into smaller bodies or fuse together, differ in size and number during the cell cycle and in specific composition and putative biological roles (Platani et al. 2000; Sleeman et al. 2001; Morris 2008). CBs disassemble during mitosis and reassemble in the G1 phase after the formation of the nucleolus and resumption of transcription (Carmo-Fonseca et al. 1993; Sleeman et al. 2003; Muro et al. 2010).

Cajal bodies share some proteins with other nuclear bodies: SMN and gemins 2,3,4,6,7 and 8 are also concentrated in gems (Morris 2008), FLASH and NPAT in HLBs (Ma et al.

2000; Barcaroli et al. 2006; White et al. 2011), Nopp140 and fibrillarin in the nucleolus (Bohmann et al. 1995), further PIASy and SUMO-1 in PML bodies (Sun et al. 2005;

Navascues et al. 2008), illustrating complex dynamic protein exchanges between different nuclear compartments. Moreover, the localization of Nopp140 to CBs correlates with the severity of the neurodegenerative disease spinal muscular atrophy (Renvoise et al. 2009) and

Introduction

nominating the respective snRNP as: U1, U2, U3, U4, U5 or U6 (Will and Luhrmann 1997;

Kambach et al. 1999). Cajal bodies participate in the transport and maturation of these snRNPs (Sleeman and Lamond 1999). Newly transcribed snRNAs by RNA polymerase II can be observed to enter the CB, to load proteins like PHAX and CRM1 and to be subsequently carried to the cytoplasm (Uguen and Murphy 2003; Suzuki et al. 2010). In the cytoplasm, the SMN protein complex induces the assembly of Sm proteins into a stable ring-shaped Sm core domain around the snRNAs (Meister et al. 2002). RNA methyltransferase (Tgs1) binds and hypermethylates the m⁷G cap to form a 2,2,7-trimethylguanosine (TMG) cap, the Sm ring and the tri-methyl cap together serve as a nuclear import signal (Mouaikel et al. 2002). After importin-β mediated transport into the nucleus (Palacios et al. 1997) the snRNPs are once again directed to CBs where final maturation steps like attachment of snRNP-specific proteins (Nesic et al. 2004) and modifications of snRNAs by guide RNAs (small Cajal body-specific RNAs; scaRNAs) take place, in particular 2'-O-methylation and pseudouridylation (Darzacq et al. 2002; Jady et al. 2003). Compared to this maturation pathway, U6 is the only snRNA transcribed by RNA polymerase III and does not receive a m⁷G cap but is methylated at the guanosine gamma phosphate at the 5'-end (Singh and Reddy 1989). U6 snRNA is not leaving the nucleus for maturation, rather a ring of seven LSm proteins is formed around the U6 snRNA in the nucleolus, analogue to the Sm core (Achsel et al. 1999) and modifications are guided by small nucleolar guide RNAs (snoRNAs) (Ganot et al. 1999).

Cajal bodies are assumed to be important for the intermediate U4/U6 di-snRNP and mature U4/U6•U5 tri-snRNP assembly (Stanek and Neugebauer 2006). The mature U4/U6•U5 tri-snRNP is a subunit of the spliceosome, which is composed outside of the CB by attachment of U1, U2 and U3 snRNPs and numerous other proteins, formed by ordered interaction (Will and Luhrmann 1997). During splicing, U4/U6 di-snRNPs and U4/U6•U5 tri- snRNPs are rearranged and require regeneration that is proposed to occur in CBs, which are suggested to play a crucial role in the spliceosome cycle (Stanek and Neugebauer 2006). For the formation of this spliceosome subunit, U4 and U6 snRNPs are targeted to the CBs independently and then assemble to the U4/U6 di-snRNP (Stanek et al. 2003; Stanek and Neugebauer 2004). An 11-fold enhanced U4/U6 di-snRNP assembly in presence of four Cajal bodies, compared to a nucleoplasm without CBs, can be shown in a mathematical model that is based on a 20-fold increased snRNP concentration in Cajal bodies versus nucleoplasm in HeLa cells (Klingauf et al. 2006). The protein SART3 is supposed to provide the scaffolding

Introduction

for the di-snRNP formation but leaves the di-snRNP afterwards (Bell et al. 2002), so the U5 snRNP is added to the complex to form the mature U4/U6•U5 tri-snRNP (Makarova et al.

2002).

The mono-snRNPs enter the Cajal body with SMN, which is suggested to target the snRNP to the CB (Frey and Matera 1995; Xu et al. 2005) but then hands the snRNP over to a protein called coilin, which is a marker protein for CBs (Stanek and Neugebauer 2006).

Coilin

In 1991, the well known marker protein for Cajal bodies p80 coilin is discovered in sera of patients with autoimmune features (Andrade et al. 1991; Raska et al. 1991). The protein comprises 576 amino acid residues (Toyota et al. 2010) and includes a highly conserved N-terminal region, promoting selfinteraction (Hebert and Matera 2000; Shpargel et al. 2003), two motifs that closely match the nuclear localization sequence (NLS) (Bohmann et al. 1995), a C-terminal domain, necessary for proper binding of Sm proteins (Xu et al. 2005) and a sequence called RG box, important for the association to the SMN protein (Hebert et al.

2001).

Coilin is shown to be essential for accurate formation of Cajal bodies. Cells of coilin knock-out mice display residual CBs with deficient recruitment of snRNPs and SMN (Tucker et al. 2001). Cajal body function is assumed to be dependent on both coilin and SMN expression (Buhler et al. 1999; Hebert et al. 2001; Sleeman et al. 2001; Tucker et al. 2001).

Loss of coilin is semi-lethal, homozygous knock-out of coilin causes prenatal death of almost 50% of Coil -/- mice between E13.5 and birth. Surviving mice display fecundity defects, illustrating that coilin has an influence on viability but is not an essential protein (Toyota et al. 2010).

Cajal bodies disassemble during mitoses, but the amount of coilin remains constant

Introduction

homologous DNA end-joining (Velma et al. 2010). Coilin has RNase activity and is proposed to have a role in U snRNP processing (Broome and Hebert 2012).

In particular in Cajal bodies, the interaction of SMN and coilin has been intensively investigated (Hebert et al. 2001; Tucker et al. 2001). Methylation of the RG box and/or phosphorylations at the C-terminal domain of coilin exhibit a strong impact on its residence in the nucleus (Tapia et al. 2010; Carrero et al. 2011). Symmetrical dimethylation of arginine residues in the coilin RG box increases the binding to SMN (Hebert et al. 2002). However, phosphorylation of the amino acid residues S571-572 and T573 decreases the binding to SMN (Toyota et al. 2010). Besides, some proteins seem to be essential for the colocalization of coilin and SMN: the WRAP 53 protein (Mahmoudi et al. 2010), the zinc-finger protein ZPR1 (Gangwani et al. 2001) and an integrator complex subunit called INTS4 (Takata et al. 2012).

The coilin interaction partner SMN is suggested to act as a master assembler of macromolecular, nuclear complexes and is associated in transcription, pre-mRNA splicing and ribosome production (Clelland et al. 2009). Certainly the loss of SMN causes a neurodegenerative disease called spinal muscular atrophy (SMA), one of the most frequent reasons of infant mortality (Lefebvre et al. 1995).

Spinal muscular atrophy

With a carrier frequency of one in 35 to one in 54 and an incidence between one in 6000 and one in 10000 in different studies, spinal muscular atrophy is the second most common autosomal recessive disorder in humans after cystic fibrosis (Monani 2005;

Sugarman et al. 2012). SMA affects lower motoneurons in the anterior horn of the spinal cord and results in atrophy in particular of the proximal muscles of the trunk and limbs (Monani 2005; Burghes and Beattie 2009).

SMA results from low levels of SMN (Helmken et al. 2003). Complete loss of this 38 kDa protein (Burlet et al. 1998) is shown to be lethal (Schrank et al. 1997). The SMN gene consists of nine exons and eight introns (Burglen et al. 1996). Only in humans two distinct copies of the SMN gene are distinguished, SMN1 (telomeric copy) and SMN2 (centomeric copy) (Burghes and Beattie 2009). Merely one copy of SMN is found in all other species (Rochette et al. 2001). The two copies of the SMN gene are nearly identical except differences in two base-pair positions in the sequences of exon 7 and 8 in the centromeric copy, also

Introduction

referred to as CBCD541, cenSMN, SMNC or SMN2 (Hahnen et al. 1996; Parsons et al. 1996;

van der Steege et al. 1996; Gavrilov et al. 1998). The critical distinction between SMN1 and SMN2 is a single base change C→T 6 bp inside exon 7 of SMN2 that alone is sufficient to alter processing of SMN mRNA severely, resulting mainly in the loss of exon 7 and a truncated protein SMN∆7 (Lorson et al. 1999). The oligomerization efficiency and stability of SMN∆7 is severely decreased compared to full-length SMN (Lorson et al. 1998; Lorson and Androphy 2000), followed by a fast ubiquitination and degradation of the truncated protein (Burnett et al. 2009) (Fig. 2).

Fig. 2 Low levels of full length SMN protein are produced by the SMN2 gene.

SMN1 and SMN2 differ in a relevant C→T base change in exon 7, mRNA resulting of SMN2 is mainly spliced alternatively and is accordingly lacking exon 7 (Lorson et al. 1999). SMN2 is therefore producing only low amounts of full length SMN but high amounts of SMN∆7 with decreased oligomerization efficiency and stability (Lorson et al. 1998; Lorson and Androphy 2000; Burnett et al. 2009) (modified from Burghes and Beattie 2010).

Introduction

function, compared to SMN1 (Burghes and Beattie 2009). From the SMN2 gene only a very low level of the SMN mRNA is spliced into the full-length transcript resulting in low amounts of full-length protein (Monani et al. 1999).

A further isoform of SMN is reported, the so-called axonal-SMN (a-SMN), a protein of approximately 19 kDa coming from the SMN1 gene but comprising only the first three exons of SMN (Setola et al. 2007). A-SMN is mainly located in axons of developing motoneurons in the spinal cords of rats and stimulates cell motility and axon outgrowth in NSC34 cells (Setola et al. 2007; Locatelli et al. 2012).

Based on the severity, SMA is classified into three distinct forms (Pearn 1980). About 60% of all newly diagnosed SMA cases belong to the most severe form of SMA, the Werdnig-Hoffmann disease or SMA Type I (Wernig 1891; Hoffmann 1893; Coady and Lorson 2011). Affected patients are never able to sit unassistedly (Monani 2005) and have a life expectancy of less than 2 years without supportive care and intense intervention (Oskoui et al. 2007). With a later onset of symptoms before 18 month of age, in the intermediate SMA Type II, patients sit unaided but are never able to walk (Dubowitz 1964; Coovert et al. 1997).

SMA Type III (Kugelberg-Welander disease) is an alleviated form of SMA with an emergence of symptoms typically after 18 months of age. Patients can walk unassisted and have a normal lifespan (Kugelberg and Welander 1956; Monani 2005).

The phenotype of SMA does not only correlate with the level of the SMN protein, but also with the number of SMN containing gems. Severe phenotypes display less gems (Coovert et al. 1997). A very similar nuclear phenotype is observed after expression of the 23 kDa isoform of the fibroblast growth factor - 2 (FGF-2²³) in vitro in HEK293 cells and in vivo in motoneurons of FGF-2 transgenic mice (Bruns et al. 2009). FGF-2²³ co-localizes and directly interacts with SMN (Claus et al. 2003; Claus et al. 2004) and competes with Gemin2 for binding to SMN and therefore destabilizes nuclear gems (Bruns et al. 2009).

Introduction

Fibroblast growth factor - 2

Growth factors are proteins that are decisive for cell proliferation, tissue differentiation and organogenesis (Cotton et al. 2008). The fibroblast growth factors comprise a family of 22 members (Ornitz and Itoh 2001), coded in 22 genes (Itoh and Ornitz 2004) and are firstly identified and purified from bovine pituitary glands (Gospodarowicz et al. 1974). FGFs have a strong attraction to heparin, facilitating efficient purification via heparin affinity chromatography (Shing et al. 1984). FGF signaling molecules bind and activate four distinct fibroblast growth factor receptors (FGFR1-4) which belong to the family of receptor tyrosine kinases (RTKs). A receptor called FGFR5, also referred to as fibroblast growth factor receptor like 1 (FGFRL1), is lacking a tyrosine kinase domain (Powers et al. 2000;

Schlessinger 2000; Wiedemann and Trueb 2000). Binding of FGFs to FGFRs and activation of the receptors 1 - 4 is heparin-dependent (Ornitz et al. 1992; Spivak-Kroizman et al. 1994).

Like other RTKs the FGFRs1-4 are transmembrane proteins, containing an extracellular ligand-binding domain and a cytoplasmic catalytic domain (Johnson and Williams 1993;

Hunter 2000). FGF binding provokes dimerization and initiation of FGFRs by autophosphorylation of several tyrosine residues in their cytoplasmic domain (Eswarakumar et al. 2005). Due to alternative splicing, multiple isoforms of FGFRs exist that differ in the carboxy-terminal of the immunoglobulin (Ig)-like domain III, producing IIIb and IIIc isoforms and thus modify the binding affinities for different FGFs (Ornitz et al. 1996; Zhang et al. 2006). FGF signaling is cell type specific and in addition depending upon the cell cycle phase, the differentiation stage as well as the duration and intensity of FGF stimulation (Murakami et al. 2008).

FGF signaling exhibits important functions in embryonic limb and skeletal development, cell proliferation, tissue differentiation and cell motility, and later especially in wound healing and tissue repair (Ornitz and Itoh 2001; Ornitz and Marie 2002; Grose and

Introduction

subsequently fine tuned by positive and negative feedback loops which evolve dynamic signal modulation (Bhalla and Iyengar 1999; Freeman 2000).

Not all fibroblast growth factors are produced with a conventional signal sequence and either persist after synthesis in the cytoplasm or are imported into the nucleus like the high molecular isoforms of FGF-2 and FGF-3 and the fibroblast growth factor homologous factor (FHF) isoforms FHF1a, FHF2a and FHF4a (Kiefer et al. 1994; Ornitz and Itoh 2001;

Goldfarb 2005). Furthermore, after treatment of NIH 3T3 cells with exogenous FGF-1, FGFR1 is translocated from the cell surface to the nucleus retaining enzymatic activity (Prudovsky et al. 1994; Jans and Hassan 1998). In the nuclei of bovine adrenal medullary cells (BAMC), FGFR1 binds FGF-2 revealing nuclear kinase activity (Stachowiak et al.

1996).

FGF-2 also named as basic fibroblast growth factor (bFGF) is translated from a single mRNA into different molecular isoforms (Delrieu 2000). In frame upstream to a conventional Kozak AUG start codon for translation of a 18 kDa FGF-2, several CUG codons in humans exist, giving rise to 22, 22.5, 24 and 34 kDa protein isoforms (Florkiewicz and Sommer 1989;

Prats et al. 1989; Arnaud et al. 1999; Sørensen et al. 2006) (Fig. 3).

FGF-2¹⁸ is secreted via exocytosis in an energy-dependent mechanism autonomous of the Golgi/ER pathway, though it is lacking a conventional signal peptide sequence (Mignatti et al. 1992; Florkiewicz et al. 1995). After secretion, FGF-2¹⁸ can either act in an endocrine mode directly on the producer cell or affect other cells in a paracrine mode with the capability to induce proliferation or migration (Bikfalvi et al. 1995; Joy et al. 1997; Dono et al. 1998).

Beside different signaling pathways, interaction of FGF-2 with cell surface receptors leads to internalization of the growth factor – receptor complex, resulting in translocation into the nucleus and N-terminal cleavage to a 16 kDa form (Bouche et al. 1987; Malecki et al. 2004).

Murine FGF-2 contains a C-terminal nuclear localization sequence (NLS), consisting of arginine 116 and 118, targeting the 18 kDa and 23 kDa isoforms to the nucleus (Claus et al.

2003). Endogenous or internalized FGF-2¹⁸, in the nucleus and nucleolus, interacts and directly regulates the upstream binding factor (UBF), a fundamental factor for rRNA transcription (Sheng et al. 2005) (Fig. 3).

Introduction

Fig. 3 Transcription and functions of different FGF-2 isoforms.

Human FGF-2 mRNA gives rise to 5 isoforms of FGF-2 proteins, initiated by one AUG start codon (18 kDa isoform) and four CUG start codons (22-34 kDa isoforms) (Florkiewicz and Sommer 1989; Prats et al. 1989;

Arnaud et al. 1999; Sørensen et al. 2006). Different nuclear localization sequences promote nuclear import, in particular of high molecular weight FGF-2 (Bugler et al. 1991; Arnaud et al. 1999; Claus et al. 2003). FGF-2¹⁸ is the only isoform secreted from cells independent of Golgi/ER secretion (Mignatti et al. 1992; Florkiewicz et al.

1995). Secreted FGF-2 can act endocrine or paracrine, binds to FGFRs, resulting in either signaling cascades like Ras/MAPK (Kouhara et al. 1997), PI3/Akt (Bottcher and Niehrs 2005) and PLCγ/Ca²⁺ (Mohammadi et al. 1992;

Peters et al. 1992) or endocytosis, followed by N-terminal cleavage to a 16 kDa FGF-2 and nuclear import (Bouche et al. 1987; Malecki et al. 2004). In the nucleus high molecular weight FGF-2 isoforms bind to L6/TAXREB107

Introduction

Rev protein (Arnaud et al. 1999). Nuclear FGF-2²³ affects preribosomal assembly by associating to the ribosomal protein L6/TAXREB107 (Shen et al. 1998) and has an influence on stress response by the antiapoptotic protein FGF-2 interacting factor (FIF) (Van den Berghe et al. 2000). Moreover, FGF-2²³ directly binds to the survival of motoneuron protein and destabilizes nuclear gems, causing a similar nuclear phenotype like in the spinal muscular atrophy disease (Bruns et al. 2009) (Fig. 2).

Nuclear import of FGF-2 occurs together with FGFR1 and is mediated by importin-β (Reilly and Maher 2001). In the nucleus, interaction of FGFR1 and FGF-2 induces a pathway referred to as integrative nuclear FGFR1 signaling (INFS), which promotes neuronal differentiation by activation of the TH gene (Peng et al. 2002; Stachowiak et al. 2003;

Stachowiak et al. 2007). FGFR1 signaling in the nucleus includes association and interrelation of the N-terminus of FGFR1 with CREB-binding protein (CBP), a transcriptional coactivator and the FGFR1 C-terminus binds to ribosomal S6 kinase 1 (RSK1). Both interactions result in amplified CBP mediated transcription (Fang et al. 2005). Binding of FGFR1 to CBP, RSK1, FGF-2²³ and chromatin slow down the protein in the nucleus (Dunham-Ems et al. 2009) and finally induces the tyrosine hydroxylase (TH) gene promoter (Peng et al. 2002).

A second protein, the orphan nuclear receptor Nurr1 is involved in the control of the maintenance, maturation and differentiation of mesencephalic dopaminergic (mDA) neurons, similar to FGFR1 (Law et al. 1992; Klejbor et al. 2006; Smidt and Burbach 2009). Nurr1 is an essential transcription factor for expression of the TH-gene and involved in synthesis and function of dopamine. The transcriptional activity of Nurr1 is affected by growth factors and hormones (Wallen and Perlmann 2003; Perlmann and Wallen-Mackenzie 2004; Jacobs et al.

2009). Disruption of either FGFR1 signaling or Nurr1 gene impairs severely development of TH-expressing mDA neurons (Zetterstrom et al. 1997; Klejbor et al. 2006).

FGF-2²³ binds SMN in the nucleus, but relatively little is known about SMNs nuclear functions. Therefore we have analyzed the effects of FGF-2²³ on the localization and dynamics of the SMN protein at Cajal bodies to explore its role in this nuclear body and implications of FGF-2²³ expression on CB functions.

Furthermore, we have investigated the relationship between nuclear architecture, nuclear body regulation and neuronal differentiation. CBs have been used to elucidate their putative role as regulators of neurite outgrowth and neuronal differentiation. Thus, Cajal

Introduction

bodies and their core proteins coilin and SMN as well as SMNs binding partner FGF-2²³ have been examined for their effects on differentiating neuronal cells.

Beside FGF-2²³ binding to SMN, it interacts with FGFR1 in the nucleus. Concerning this, we have aimed to understand its role in neuronal development and nuclear complex formation to detect cross-talks of integrative nuclear FGFR1 signaling (INFS) with nuclear proteins, affecting development and neuronal differentiation.

Chapter I

Chapter I Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions

Abstract

Reduced levels of survival of motoneuron (SMN) protein lead to Spinal Muscular Atrophy but it is still unknown how SMN protects motoneurons in the spinal cord against degeneration. In the nucleus, SMN is associated with two types of nuclear bodies denoted as gems and Cajal bodies (CBs). The 23 kDa isoform of Fibroblast growth factor-2 (FGF-2²³) is a nuclear protein which binds to SMN and destabilizes the SMN-Gemin2 complex. In the present study, we show that FGF-2²³ depletes SMN from CBs without affecting their general structure. FRAP analysis of SMN-EGFP in CBs demonstrated that the majority of SMN in CBs remained mobile and allowed quantification of fast, slow and immobile nuclear SMN populations. The potential for SMN release was confirmed by in vivo photoconversion of SMN-Dendra2, indicating that CBs concentrate immobile SMN that could have a specialized function in CBs. FGF-2²³ accelerated SMN release from CBs, accompanied by a conversion of immobile SMN into a mobile population. Furthermore, FGF-2²³ caused snRNP accumulation in CBs. We propose a model in which Cajal bodies store immobile SMN that can be mobilized by its nuclear interaction partner FGF-2²³, leading to U4 snRNP accumulation in CBs, indicating a role for immobile SMN in tri-snRNP assembly.

Benjamin Förthmann has performed the major part of the laboratory work in each experiment, has been involved in the scientific design and in the evaluation and has written the manuscript.

Cell Mol Life Sci. 2013 January; 70(14): 2555–2568 DOI 10.1007/s00018-012-1242-8

http://www.ncbi.nlm.nih.gov/pubmed/23334184

Chapter II

Chapter II Regulation of neuronal differentiation by proteins associated with nuclear bodies

Abstract

Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN) protein localizes to Cajal bodies (CBs) and nuclear gems.

Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA). How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2²³) is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs). Here we demonstrate that FGF-2²³ blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2²³- dependent transcription. Our results indicate that FGF-2²³ and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs). In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs). The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation.

Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation.

Benjamin Förthmann has performed all neurite outgrowth experiments with coilin and coilin mutants, has been involved in the scientific design, in the evaluation and has written the manuscript.

PLoS One 2013

doi: 10.1371/journal.pone.0082871

Chapter III

Chapter III Cooperation of nuclear fibroblast growth factor receptor 1 and Nurr1 offers new interactive mechanism in postmitotic development of mesencephalic dopaminergic neurons.

Abstract

Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation.

Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1- EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23- kDa FGF-2 ligand (FGF-2²³) enhances Nurr1-dependent activation of the TH gene promoter.

Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.

Benjamin Förthmann has performed the FRAP experiments, has been involved in the evaluation and has contributed to the manuscript.

J Biol Chem. 2012 Jun; 287(24):19827-40 doi: 10.1074/jbc.M112.347831

http://www.ncbi.nlm.nih.gov/pubmed/22514272

Discussion

Discussion

The role of nuclear complexes for the pathology of spinal muscular atrophy is analyzed in this thesis. Therefore, Cajal body core proteins and proteins with a direct influence on nuclear complexes are examined for functions in neuronal cells. There is scant information about the role of nuclear complex regulation and nuclear architecture in the context of neuronal differentiation and the neurodegenerative disease spinal muscular atrophy.

We could show that two Cajal body proteins, SMN and the CB marker protein coilin, exhibit particular influences on neuronal differentiation. Nuclear SMN functions are regulated by the 23 kDa isoform of the fibroblast growth factor – 2 (FGF-2²³) and vice versa. All three proteins are involved in fine tuning of neurite outgrowth. Furthermore, we have discovered and analyzed the nuclear FGFR1/Nurr1 complex displaying specific functions in neuronal differentiation.

In chapter one, we employ fluorescence recovery after photobleaching (FRAP) and photoconversion experiments to investigate the mobility of SMN at Cajal bodies (Förthmann et al. 2013). We have detected three different SMN populations in HEK cells: a tightly bound immobile, a slow and a fast mobile population. The flux of Cajal body proteins, like SMN and coilin, in and out of CBs is shown earlier in FRAP studies. In HeLa cells, after bleaching of YFP-coilin, full signal recovery is already achieved after one minute whereas only 50% of the pre-bleach fluorescence intensity of GFP-SMN is restored one hour after bleaching (Sleeman et al. 2003). However, FRAP experiments with YFP-tagged proteins have to be handled with care, because YFP is bleached irreversibly only to 75%; 25% of YFP is bleached reversibly and recovers by photoactivation (McAnaney et al. 2005). Additionally, three kinetic coilin populations are detected in Cajal bodies of Xenopus oocytes with detention times of several seconds up to 30 minutes and longer (Handwerger et al. 2003). With inverse FRAP in HeLa cells, several CB proteins are classified into three kinetically distinct groups with different detention times in the Cajal body. SMN and coilin belong to the group with the longest residence time (Dundr et al. 2004). The dynamic equilibrium of proteins between the nucleoplasm and CBs indicates complex interactions of various proteins within the Cajal body (Sleeman et al. 2003).

By using FRAP and photoconversion experiments, it becomes obvious that coilin/SMN interactions can be modified by FGF-2²³. Overexpression of this growth factor

Discussion

causes a release of immobile SMN from CBs into a fast SMN fraction. This confirms a model of complex protein interactions inside the Cajal body. Furthermore, the influence of FGF-2²³ modifies the molecular composition of the CB. Structural differences could change the possible biological role of the Cajal body (Cioce and Lamond 2005) and argues against hierarchical assembly pathways for this compartment, but supports a model of stochastic accumulation (Matera et al. 2009). Another finding, the competition of the snRNP protein SmB with coilin for binding to SMN and subsequently possible alterations in the molecular CB composition support this model as well (Hebert et al. 2001). The direct SMN/FGF-2²³ interaction shifts the equilibrium of SMN from CB-bound/immobile to fast/mobile in the nucleoplasm (Fig. 1).

Previous studies have already shown influences of different proteins on the localization of SMN at CBs: WRAP 53 is essential (Mahmoudi et al. 2010), as well as the zinc-finger protein ZPR1 including a proposed protective role in spinal muscular atrophy (Gangwani et al. 2001; Gangwani et al. 2005; Ahmad et al. 2012). Likewise, the integrator complex subunit INTS4 is required for this colocalization (Takata et al. 2012). Apparently, Cajal bodies contain a reservoir of immobile SMN that can be mobilized by FGF-2²³, antagonistic to the function of the mentioned proteins WRAP 53, ZPR1 and the integrator complex subunit INTS4. Subsequently, we have analyzed the functional consequences of depleting SMN from CBs by this interaction to gain a more detailed view of SMN functions.

Although the formation of a FGF-2²³/SMN complex destabilizes gems (Bruns et al.

2009), loss of SMN does not change the stability of the Cajal body. The total number of CBs remains constant like it is already shown for transgenic FGF-2 mice (Bruns et al. 2009). SMN targets snRNPs to Cajal bodies and passes them to coilin (Sleeman and Lamond 1999; Dundr and Misteli 2001; Stanek and Neugebauer 2006). We propose a direct function of immobile SMN in tri-snRNP assembly. Mobilization of SMN by FGF-2²³ results in an accumulation of U4 snRNPs at CBs (Fig. 1), hinting towards an impaired export of mature tri-snRNPs from the CB (Stanek and Neugebauer 2006). Similar disregulations at Cajal bodies are recognized

Discussion

In chapter two we have focused on the role of FGF-2²³ and the CB proteins coilin and SMN in neuronal differentiation. Decreasing amounts of diffuse coilin in the nucleus of differentiating NB cells are found, whereas the total number of CBs remains constant. The retinoic acid (RA) treatment of NB cells therefore displays similarities to previous work with PC12 cells, where coilin amounts decrease during nerve growth factor (NGF) stimulation (van Bergeijk et al. 2007). RA is crossing the cell membrane and immediately enters the cell, binds retinoic acid receptors in the cyto- and nucleoplasm and directly influences neuronal differentiation by promoting gene expression (Chambon 1996; Rohwedel et al. 1999). In contrast, NGF acts outside the cell where it binds cell surface receptors, stimulates ERK and Ras signaling cascades and thereby activates gene transcription (Vaudry et al. 2002). NGF and RA induction of neuronal differentiation result in decreased coilin levels after 72 hours, indicating a general connection of neurite outgrowth and coilin protein amounts (Fig. 1).

Furthermore, increasing coilin protein levels exhibit a negative regulating role in neuronal differentiation of PC12 cells. SMN binding is not affecting this influence. After expression of coilin mutants with a high (coilin AAA) and with a low affinity to SMN (coilin DDE) (Toyota et al. 2010) a significantly decreased neurite outgrowth is observed, as well as after expression of wild-type coilin. Coilin comprises specific and DNA-dependent RNase activity and interacts with double- (Broome and Hebert 2012) and single-stranded DNA (Bellini and Gall 1998), which could account for its arrestive role in this process.

Concentration dependent RNase activity for total HeLa RNA can be detected after purification of coilin (Broome and Hebert 2012) thus implying a function for coilin independent of SMN.

The nuclear architecture changes during neuronal differentiation (Takizawa and Meshorer 2008). Colocalization of coilin and SMN increases during RA induction of the neuroblastoma cell line SH-SY5Y (Clelland et al. 2009), contrary to different localization patterns in embryonic development (Young et al. 2001). During neuritogenesis, SMN is recruited to CBs from the nuclear pool (Navascues et al. 2004). In contrast to this findings, we do not observe increased colocalization of this proteins during RA differentiation of SK- N-BE(2) neuroblastoma cells, but coilin amounts decrease dramatically in this process. In PC12 cells, Cajal bodies reside near to nucleoli upon NGF treatment (Janevski et al. 1997). In rat hippocampus neurons, Cajal bodies are redistributed during differentiation (Santama et al.

Discussion

1996). In summary, multiple changes of coilin distribution and protein levels during neuronal differentiation indicate specific roles of coilin during this process.

Besides the role of SMN at Cajal bodies in tri-snRNP maturation, previous experiments reveal another function of nuclear SMN, the facilitation of neuronal differentiation by increasing neurite outgrowth (Rossoll et al. 2003; van Bergeijk et al. 2007).

Our experiments illustrate that this SMN feature again is blocked by its interaction partner FGF-2²³, indicating a general inhibition of SMN functions. Expression of FGF-2²³ shows no effect on differentiating PC12 cells, but expression of FGF-2¹⁸ increases the neurite length of these cells. Consistent with this data, FGF-2¹⁸ expression is sufficient to initiate neurite outgrowth of PC12 cells in the absence of NGF. In contrast, FGF-2²³ expression results in the stabilization of the endocrine phenotype (Grothe et al. 1998). Accordingly, the intracellular expression of diverging FGF-2 isoforms influences the differentiation process unequally. The SMN/FGF-2²³ complex seems to generally inhibit SMN functions in the nucleus.

Furthermore, not only SMN functions are antagonized by the SMN/FGF-2²³ interaction, nuclear functions of FGF-2²³ are constrained as well. Transcription of Nurr1 dependent genes, essential for neuronal differentiation of dopaminergic neurons, is amplified by FGF-2²³, but SMN antagonizes this effect. Truncated SMN is insufficient to bind FGF-2²³ and is not able to interfere with this growth factor function. By controlling the expression of FGF-2²³ dependent genes, SMN could therefore affect cell development.

SMN/FGF-2²³ contact appears to result in a formation of an inactive complex, both proteins loose specific nuclear abilities by interaction with each other (Fig. 1). Thus the physiological role of FGF-2²³ could be modified. All FGF-2 isoforms are expressed in several tissues including peripheral nerves and dorsal root ganglia (Grothe and Nikkhah 2001).

Endogenous FGF-2 is upregulated in vivo after lesion of the sciatic nerve (Grothe et al. 2001).

FGF-2²³ is particularly upregulated by reason of unilateral hypoglossal nerve transaction (Huber et al. 1997) and during regeneration after peripheral nerve lesion in the spinal ganglia (Meisinger and Grothe 1997). FGF-2 is therefore suggested to exhibit a specific role in early

Discussion

potential regulatory role of the FGF-2²³/SMN interaction in peripheral nerve regeneration needs to be revealed in further studies.

Fig.1 Model for fine tuning of neuronal differentiation by FGF-2²³ and CB proteins SMN and coilin.

In Cajal bodies, coilin and immobile SMN have essential functions in tri-snRNP assembly. Both CB proteins are in equilibrium with diffuse mobile proteins in the nucleoplasm. Diffuse nucleoplasmic SMN increases and per contra diffuse coilin inhibits neurite outgrowth. SMN/FGF-2²³ complex formation leads to a mobilization of SMN from CBs and to a dysfunction in tri-snRNP assembly, which we propose to be dependent on less immobile SMN. Diffuse FGF-2²³ promotes neuronal differentiation by Nurr1 dependent transcription. Binding of SMN and FGF-2²³ results in an inactive complex, inhibiting FGF-2²³ promoted neuronal differentiation as well as SMN promoted neurite outgrowth. Neuronal differentiation appears to be fine tuned by expression levels and interactions of Cajal body proteins coilin and SMN as well as by the interacting nuclear growth factor FGF-2²³ (adapted from Förthmann et al. 2013).

Discussion

SMN is not the only binding partner for FGF-2²³ in the nucleus. FGF-2²³ interacts with fibroblast growth factor receptor 1 (FGFR1) and initiates the integrative nuclear FGFR1 signaling (INFS) (Peng et al. 2002; Stachowiak et al. 2003; Stachowiak et al. 2007).

In chapter three we have found and examined a further nuclear complex formation, relevant for maturation of TH-expressing neurons and postmitotic precursors (Baron et al.

2012). The FGF-2 binding partner FGFR1 shows a nuclear localization in postmitotic ventral midbrain (VM) neurons and a complex formation with the nuclear factor Nurr1. Both proteins are integrators of different developmental signals (Stachowiak et al. 2007; Smidt and Burbach 2009) and are assumed to be relevant for the development and maintenance of mesencephalic dopaminergic (mDA) neurons during postnatal development (Calo et al. 2005; Klejbor et al.

2006; Kadkhodaei et al. 2009). In the CNS, Nurr1 is expressed only in mDA neurons (Backman et al. 1999). FGFR1 is expressed all over the adult CNS and is supposed to regulate numerous processes in the development of the brain (Ozawa et al. 1996).

Although FGFR1 retains enzymatic activity in the nucleus by binding to FGF-2²³ (Prudovsky et al. 1994; Stachowiak et al. 1996; Jans and Hassan 1998), we have focused on a chromatin binding and gene regulating function by association to a nuclear complex with Nurr1. We have discovered a colocalization of both proteins in TH-expressing, maturating neurons and postmitotic precursors in mice at embryonic day 14.5 in the mDA area by fluorescence immunocytochemistry and immunoprecipitations. FGFR1 is already known to interact with the CREB-binding protein (CBP), a transcriptional coactivator, in the substantia nigra of rats (Fang et al. 2005). FGFR1 therefore has a tendency to build nuclear complexes, involved in gene regulation. Furthermore, cytoplasmic FGFR1 in proliferating neuronal progenitor cells translocates to the nucleus and promotes neuronal differentiation (Stachowiak et al. 2003). It supports our findings of cytoplasmic FGFR1 in primary mesencaphalic progenitor cells in vitro and in the subventricular zone in vivo. In mDA neurons, FGFR1 appears to act by transmembran signaling in mesencephalic progenitors and otherwise to promote neuronal differentiation in a nuclear complex with Nurr1.