Chemokines and Their Role in Dopaminergic Development

The main pathological hallmark in Parkinson’s disease (PD) is the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), affecting movements such as walking, talking and writing. Today, there is no cure available for PD and existing treatments only provide symptomatic relief, but the prospect of using stem cell based cell replacement therapies for patients with PD have provided new optimism. In order to improve the efficiency of DA neuron production, engraftment and function, a solid understanding of the molecular mechanisms that govern DA neurogenesis is crucial. In recent years, morphogens and intrinsic DA neuron determinants, that enable stem and progenitor cells to establish proper identity, have been identified. However, the whole story has yet to be uncovered. In order to improve the efficiency of DA neuron production, engraftment and function, a solid understanding of the molecular mechanisms that govern DA neurogenesis and differentiation is crucial. This thesis examines the expression and function of chemokines during DA neurogenesis. In paper I we demonstrate that three α-chemokines, CXCL1, -6 and -8, increase the number of DA neurons in ventral midbrain (VM) precursor and neurosphere cultures by diverse mechanisms. CXCL6 does so by promoting the differentiation of Nurr1+ precursors into DA neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in homo sapiens, enhanced progenitor cell division, neurogenesis and Tyrosine hydroxylase positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited similar, but not identical activities to CXCL8. In paper II we identify two β-chemokines, CCL2 and CCL7, as novel regulators of DA neurogenesis. CCL2 and CCL7 were found to promote the development of DA neurons by selectively enhancing the differentiation of Nurr1+ precursors into TH+ DA neurons. Moreover, both CCL2 and CCL7 were found to increase neuritogenesis in TH+ neurons in VM precursor and neurosphere cultures. In paper III we report that CXCL12/CXCR4 signaling regulates migration of A9-A10 DA neurons, neuritogenesis and the initial orientation of their processes. Taken together, our data provides evidence that chemokines are highly involved in DA neuron development and suggest that they may be useful to enhance DA cell preparations for cell replacement therapy as well as for drug discovery in PD. LIST OF PUBLICATIONS I. Linda C Edman, Helena Mira, Alejandro Erices, Seth Malmersjö, Emma R Andersson, Per Uhlén and Ernest Arenas. α-chemokines regulate proliferation, neurogenesis and dopaminergic differentiation of ventral midbrain precursors and neurospheres. (2008) Stem Cells. II. Linda C Edman, Helena Mira and Ernest Arenas. The β-chemokines CCL2 and CCL7 are two novel differentiation factors for midbrain dopaminergic precursors and neurons. (2008) Exp. Cell Res. III. Linda C Edman, Juan Antonio Sánchez-Alcañiz, Nicolas Fritz, Sonia Bonilla, Jonathan Hecht, Per Uhlén, Samuel J. Pleasure, Oscar Marin and Ernest Arenas. CXCL12/CXCR4 signaling controls the migration and the initial process orientation of A9-A10 dopaminergic neurons. Manuscript. LIST OF ABBREVIATIONS ADHD Attention deficit disorder AHD2 Aldehyde dehydrogenase 2 ALS Amyotrophic lateral sclerosis A-P Anterior-posterior axes BBB Blood brain barrier BMP Bone morphogenic protein BrdU Bromodeoxyuridine bHLH Basic helix-loop-helix Ca Calcium CNS Central Nervous System CRT Cell replacement therapy DA Dopaminergic DM Dorsal Midbrain DAG Diacylglycerol DAT Dopamine transporter DBS Deep brain stimulation D-V Dorsalventral axes E Embryonic EGF Epidermal Growth Factor ESC Embryonic Stem cell ERK-1/2 Extracelllar Signal-Regulated Kinase EGL External Granule Layer FP Floor Plate FGF8 Fibroblast Growth Factor 8 FACS Fluorescence-activated cell sorting Gbx Gastrulation brain homoebox GPCR G protein-coupled receptors IP3 Inositol Trisphosphate IZ Intermediate Zone ICM Inner cell mass JAK/STAT Janus Kinase-Signal Transducers and Activators of Transcription Pathway m Mouse MZ Marginal Zone MAPK Mitogen-Activated Protein Kinase mDA Midbrain dopaminergic MHB MidHindbrain barrier MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mES Mouse embryonic stem Ngn Neurogenin NSC Neural Stem Cell Nurr1 Nuclear receptor related 1 Otx Orthodenticle homologue PKC Protein Kinase C PI3K Phosphatidyl Inositol-3-OH Kinase PLC Phospholipase C PD Parkinson’s Disease Pitx3 Paired-like homeodomain transcription factor 3 PTX Pertussis toxin RA Retinoic acid RNAi Ribonucleic acid interference RP Roof Plate Sox Sex determining region Y box two Shh Sonic hedgehog SVZ Subventricular Zone SNpc Substantia Nigra pars compacta SN Substantia Nigra TH Tyrosine hydroxylase TUJ1 β Tubulin-III 7TMR Seven Transmembrane Receptors TFs Transcription factors VM Ventral Midbrain VZ Venticular Zone VTA Ventral Tegmental Area VMAT Vesicular monoamine transporter wt Wild type