Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the degeneration of cortical and spinal motor neurons. Animal models of ALS based on known ALS-causing mutations are instrumental in advancing our understanding of the pathophysiology of motor neuron degeneration. Recent identification of mutations in the genes encoding RNA-binding proteins TDP-43 and FUS has suggested that aberrant RNA processing may underlie common mechanisms of neurodegeneration in ALS and focused attention on the normal activities of TDP-43 and FUS. However, the role of the normal functions of RNA-binding proteins in ALS pathogenesis has not yet been established. In this thesis I present my work on novel FUS-based mouse lines aimed at clarifying the relationships between ALS-causing FUS mutations, normal FUS function and motor neuron degeneration. Experiments in mutant FUS knock-in mice show evidence of both loss- and gain-of-function effects as well as misfolding of mutant FUS protein. Characterization of mice expressing ALS-mutant human FUS cDNA in the nervous system reveals selective, early onset and slowly progressive motor neuron degeneration that is mutation dependent, involves both cell autonomous and non-cell autonomous mechanisms and models key aspects of ALS-FUS. Using a novel conditional FUS knockout mutant mouse, I also demonstrate that postnatal elimination of FUS selectively in motor neurons or more broadly in the nervous system has no effect on long-term motor neuron survival. Collectively, our findings suggest that a novel toxic function of mutant FUS, and not the loss of normal FUS function, is the primary mechanism of motor neuron degeneration in ALS-FUS.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Sydney, 2007. / Title from title screen (viewed Apr. 12, 2007). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Medicine. Includes bibliography. Also issued in print.

Maturation of spinal motoneurons in rodents is characterized by a period of cell loss in the embryo, but researchers have claimed that some cell death occurs postnatally. This form of cell death is called apoptosis and involves active participation of the cell. Apoptotic cells have certain recognizable morphological and molecular features. I have used a monoclonal antibody against ubiquitin, (a putative marker of apoptotic cells), to do immunochemistry on mouse spinal cords at various postnatal ages till early adulthood. Staining is seen in large amotoneurons in the ventral horn. Staining is intense till P28, and faint thereafter. Substantial proportions of motoneurons stain till P21, followed by a sharp decline in the number of immunopositive cells. None of the cells exhibit signs of apoptosis.

In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key developmental pathways can be re-activated to regulate neuroregeneration in cellular recovery, I addressed the following questions: 1) What is the role of Notch signalling during regenerative mechanisms in the lesioned spinal cord of the adult zebrafish? 2) What is the role of Notch overexpression in neurogenesis in the adult zebrafish retina? 3) Which additional signalling pathways are involved in the generation of motor neurons during spinal cord regeneration in adult zebrafish? 1) In the main part of my thesis I have investigated the role of Notch signalling during spinal cord regeneration. The Notch pathway has been shown to regulate neural progenitor maintenance and inhibit neuronal differentiation in the vertebrate nervous system. In the injured mammalian spinal cord, increased Notch signalling is held partly responsible for the low regenerative potential of endogenous progenitors to generate new neurons. However, this is difficult to test in an essentially non-regenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g. of motor neurons from endogenous spinal progenitor cells, the Notch pathway is also reactivated. I over-activated the Notch pathway by forced expression of a heat-shock inducible active domain of notch in spinal progenitor cells. I observed that although apparently compatible with functional regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. Thus in summary our work demonstrates that Notch is a negative signal for regenerative neurogenesis in the spinal cord. Importantly, we show for the first time that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signalling. 2) While in the lesioned spinal cord, over-activation of Notch attenuated neurogenesis, I observed that in the unlesioned retina the same manipulation led to strong proliferation of cells in the inner nuclear layer, presumable Müller glia cells which are the retinal progenitor cells. This coincided with an increase in eye size in adult zebrafish. These preliminary findings provide the first hint that the role of Notch may differ for different adult progenitor cell pools and will lead to future investigations of Notch induced neurogenesis in the retina. 3) We have evidence from previous studies that the dopamine and retinoic acid (RA) signalling pathways may be involved in the generation of motor neurons in the adult lesioned spinal cord. Using in situ hybridisation, I assessed the gene expression patterns a) for all D2-like receptors and b) candidate genes that relate to the RA pathway in the adult lesioned spinal cord to identify the signalling components. a) I found that only the D4a receptor was upregulated in spinal progenitor cells in the ventricular zone rostral to the lesion site, but not caudal to it. This correlates with other results showing that dopamine agonists increase motor neuron regeneration rostral, but not caudal to a spinal lesion site. b) I observed a strong increase in the expression of Cyp26a, a RA catabolising enzyme, in the ventricular progenitor zone caudal to the lesion site, in contrast to the weak expression rostrally. Crabp2a, a cellular retinoic acid binding protein, was also upregulated rostral and in close proximity to the lesion site in a subpopulation of neurons located ventrolaterally in the spinal cord. In summary, we show that the Notch pathway negatively regulates neurogenesis in the spinal cord in contrast to the retina and provide evidence that dopamine from the brain signals via the D4a receptor to promote the generation of motor neurons in addition to RA, which may also play a role in this process. These insights into adult neural progenitor cell activation in zebrafish may ultimately inform therapeutic strategies for spinal cord injury and neurodegenerative diseases such as motor neuron disease.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The emergence of limbs in vertebrates represents a significant evolutionary innovation. Limbs facilitate diverse motor behaviors, yet require spinal networks that can coordinate the activities of many individual muscles within the limb. Here I describe several efforts to characterize the specification of spinal motor neurons and assembly of spinal circuits in higher vertebrates. I discuss the formation of selective presynaptic sensory inputs to motor pools, a process which has long been thought to occur in an activity-independent manner. I demonstrate an as yet unappreciated role of activity-dependent refinement in patterning the set of sensory-motor connections that link motor pools with synergist function. I also explore the genetic specification of motor pools that project to defined muscle targets. I show that the motor pools that control digits engage distinct developmental genetic programs which reflect underlying differences in Hox and retinoic acid signaling. The divergent mechanisms underlying the specification of digit-innervating motor neurons may reflect the unique status of digit control in the evolution of motor behavior.

Amyotrophic Lateral Sclerosis (ALS) is a neurological disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have revealed that many ALS-associated genes are involved in autophagy, but the role of this pathway in motor neurons remains poorly understood. Here, we use the SOD1G93A mouse model to investigate the role of autophagy in ALS. We find neuronal subtype-specific regulation of autophagy over the course of disease progression. Vulnerable motor neurons form large GABARAPL1-positive autophagosomes that engulf ubiquitinated cargo recognized by the selective autophagy receptor p62. Other motor neurons and interneurons do not engulf cargo within GABARAPL1-positive autophagosomes and instead accumulate somatodendritic aggregates. To investigate whether motor neuron autophagy is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 is specifically disrupted in motor neurons. Phenotypic analysis of these mice revealed that autophagy is dispensable for motor neuron survival but plays a key role in regulating presynaptic structure and function. By crossing these mice to the SOD1G93A mouse model, we find that autophagy inhibition accelerates early neuromuscular denervation and neurological dysfunction. However, loss of autophagy in motor neurons eventually leads to an extension of lifespan, and this is associated with reduced pathology in interneurons and glial cells. These data suggest that vulnerable motor neurons rely on autophagy to maintain neuromuscular innervation early in disease. However, autophagy eventually acts in a non-cell autonomous manner to promote disease spread and neuroinflammation. Our results reveal counteracting roles for motor neuron autophagy early and late in ALS disease progression.

Gemin5 是運動神經元綜合體(SMN Complex)的其中一個組件，這綜合體的主要功能是控制小型胞核核糖核蛋白(UsnRNPs)的合成。這些小型胞核核糖核蛋白組合成剪接核糖核酸前體(pre-mRNA)的剪接體(Spliceosome)，使核糖核酸分子可以用來翻譯成蛋白質。失去運動神經元綜合體功能引致脊髓肌肉萎縮症。果蠅是其中一個用作研究人類疾病重要的生物。更重要的是，部分組成運動神經元綜合體的組件也存在於果蠅。是次研究是利用遺傳方式在果蠅內研究Gemin5 的同源基因-- rigor mortis (rig) 的作用。果蠅帶有rig 突變基因表現神經肌肉接頭(neuromuscular junction)上的缺陷和異常的運動行為。這表明，rig 的功能可能與神經退化性疾病有關。為了進一步了解rig 的功能途徑(functional pathway)，已進行了一個利用移除突變體的基因過濾實驗，研究鎖定了 12 個染色體部份可能和rig 有基因上的相互作用，進一步研究與rig 有相互作用的基因有助於了解rig 的功能及研究脊髓肌肉萎縮症的發病機制。 / Gemin5 is a component of the Survival of Motor Neuron (SMN) complex, which is a protein complex regulating biogenesis of various Uridine-enriched small nuclear ribonucleoproteins (UsnRNPs). These UsnRNPs form the molecular machinery spliceosome, which mediates pre-messenger RNA splicing, an important mechanism before an mRNA molecule can be used to translate proteins. Loss-of-function of the SMN complex is now known to cause the neurodegenerative disease, Spinal Muscular Atrophy. Drosophila is one of the well-characterized model organisms for studying human diseases. More importantly, components of the SMN complex are also found in Drosophila. Here, I studied the function of rigor mortis (rig), the Gemin5 orthologue, in Drosophila using a genetic approach. Drosophila carrying mutations in the rig gene show defects in the neuromuscular junction (NMJ) and display abnormal motor behavior. This suggests that the function of rig may link to the neurodegenerative disease. To further characterize the function of rig, a genetic screen was carried out. Twelve chromosomal regions encoding possible rig-interacting genes were identified. Further characterization of these rig-interacting genes may help us better understand the function of rig. / Detailed summary in vernacular field only. / Cheng, Yat Pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 120-125). / Abstracts also in Chinese. / ABSTRACT --- p.i / ABSTRACT IN CHINESE --- p.ii / ACKNOWLEDGEMENT --- p.iii / LIST OF ABBREVIATIONS --- p.iv / LIST OF FIGURES --- p.v / LIST OF TABLES --- p.vi / TABLE OF CONTENTS --- p.vii / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Introduction of rigor mortis / Chapter 1.1.1 --- Orthologue of Gemin5 in Drosophila --- p.1 / Chapter 1.1.2 --- Published Phenotypic Analyses of rig Mutants --- p.1 / Chapter 1.2 --- Introduction of Gemin5 / Chapter 1.2.1 --- Introduction of Gemins --- p.4 / Chapter 1.2.2 --- Structural Properties of Gemin5 --- p.4 / Chapter 1.2.3 --- Gemin5-interacting partners --- p.7 / Chapter 1.2.4 --- Gemin5 as a Component of the Survival of Motor Neuron (SMN) Complex --- p.7 / Chapter 1.2.5 --- Function of the SMN Complex and Spinal Muscular Atrophy --- p.11 / Chapter 1.3 --- Drosophila as a Model Organism / Chapter 1.3.1 --- Advantages of Using Drosophila as a Model Organism --- p.11 / Chapter 1.3.2 --- Neuromuscular Junction of Drosophila --- p.15 / Chapter 1.4 --- Aim of the Present Study --- p.19 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila Culture / Chapter 2.1.1 --- Culture Medium --- p.20 / Chapter 2.1.2 --- Drosophila Stocks and Crosses Maintenance --- p.20 / Chapter 2.1.3 --- Larvae Collection --- p.21 / Chapter 2.1.3.1 --- Reagents --- p.21 / Chapter 2.1.3.2 --- Procedures --- p.21 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Reagents --- p.23 / Chapter 2.2.2 --- Drosophila S2R⁺ Cell Culture --- p.24 / Chapter 2.2.3 --- Establishment of Stable S2R⁺ Cells --- p.24 / Chapter 2.3 --- Genomic Polymerase Chain Reaction (PCR) / Chapter 2.3.1 --- Reagents --- p.25 / Chapter 2.3.2 --- Genomic DNA Extraction from a Single Larva --- p.26 / Chapter 2.3.3 --- Primer Design --- p.26 / Chapter 2.3.4 --- Polymerase Chain Reaction (PCR) --- p.27 / Chapter 2.4 --- Behavioral Assay / Chapter 2.4.1 --- Stable S2R⁺ Cell Staining --- p.29 / Chapter 2.4.1.1 --- Reagents --- p.29 / Chapter 2.4.1.2 --- Procedures --- p.30 / Chapter 2.4.2 --- Larvae Staining --- p.31 / Chapter 2.4.2.1 --- Reagents --- p.31 / Chapter 2.4.2.2 --- Larvae Dissection --- p.32 / Chapter 2.4.2.3 --- Larval Muscle Staining --- p.33 / Chapter 2.4.2.4 --- Larval Neuromuscular Junction Staining --- p.33 / Chapter 2.5 --- Microscopy / Chapter 2.5.1 --- Light Microscopy --- p.34 / Chapter 2.5.1.1 --- Microscopic Observation of Larval Movement --- p.34 / Chapter 2.5.1.2 --- Quantification of Larval Contraction Rate --- p.34 / Chapter 2.5.1.3 --- Quantification of Larval Travelling Distance --- p.34 / Chapter 2.5.2 --- Fluorescence Microscopy --- p.35 / Chapter 2.5.2.1 --- Microscopic Observation of Larval Muscle --- p.35 / Chapter 2.5.2.2 --- Microscopic Observation of Stable S2R⁺ Cells --- p.35 / Chapter 2.5.3 --- Confocal Microscopy --- p.36 / Chapter 2.5.3.1 --- Microscopic Observation of Larval Neuromuscular Junction --- p.36 / Chapter 2.5.3.2 --- Quantification of Larval Neuromuscular Junction --- p.36 / Chapter 2.6 --- Generation of transgenic fly lines expressing rig transgene / Chapter 2.6.1 --- Polymerase Chain Reaction --- p.36 / Chapter 2.6.2 --- Agarose Gel Electrophoresis --- p.38 / Chapter 2.6.2.1 --- Reagents --- p.38 / Chapter 2.6.2.2 --- Procedures --- p.39 / Chapter 2.6.3 --- Restriction Digestion --- p.39 / Chapter 2.6.4 --- Ligation Reaction --- p.39 / Chapter 2.6.5 --- Bacterial Transformation --- p.40 / Chapter 2.6.5.1 --- Reagents --- p.40 / Chapter 2.6.5.2 --- Procedures --- p.40 / Chapter 2.6.6 --- Bacterial Glycerol Stock for Long-term Storage --- p.41 / Chapter 2.7 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting / Chapter 2.7.1 --- Reagents --- p.41 / Chapter 2.7.2 --- Lysate Preparation of Stable S2R⁺ Cells, Adult Fly Heads and Larvae --- p.44 / Chapter 2.7.2.1 --- Stable S2R+ Cells --- p.44 / Chapter 2.7.2.2 --- Adult Fly Heads --- p.44 / Chapter 2.7.2.3 --- Larvae --- p.45 / Chapter 2.7.3 --- SDS-Polyacrylamide Gel Electrophoresis --- p.45 / Chapter 2.7.4 --- Immunoblotting --- p.45 / Chapter CHAPTER 3. --- PHENOTYPIC CHARACTERIZATION OF RIG MUTANT / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Re-balancing of rig Mutant Fly Lines Over the Cy; Tb Compound Balancer for Genotype Identification --- p.48 / Chapter 3.3 --- Verification of Model Genotype --- p.49 / Chapter 3.4 --- rig Mutant Larvae Displayed Abnormal Motor Behavior / Chapter 3.4.1 --- Contraction Rate of rig Mutant Larvae --- p.54 / Chapter 3.4.2 --- Traveling Distance of rig Mutant Larvae --- p.56 / Chapter 3.5 --- rig Mutant Larvae Showed Normal Body Wall Musculature --- p.58 / Chapter 3.6 --- rig Mutant Larvae Displayed Defects in the Neuromuscular Junction / Chapter 3.6.1 --- rig Mutant Larvae Showed Branching Defects --- p.60 / Chapter 3.6.2 --- rig Mutant Larvae Showed Fewer Boutons Number --- p.62 / Chapter 3.7 --- rig Mutant Larvae Showed Normal Active Zone Pattern --- p.64 / Chapter 3.8 --- Discussion --- p.66 / Chapter CHAPTER 4. --- A GENETIC SCREEN TO IDENTIFY GENES THAT INTERACT GENETICALLY WITH RIG / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Candidates and Design of the Screen --- p.72 / Chapter 4.3 --- Re-balancing of Deletion Lines Over the Cy; Tb Compound Balancer --- p.75 / Chapter 4.4 --- Identification of Chromosomal Regions That Genetically Interact With rig --- p.75 / Chapter 4.5 --- Identification of NMJ Genes That Genetically Interact With rig --- p.80 / Chapter 4.6 --- Discussion --- p.83 / Chapter CHAPTER 5. --- ATTEMPTS TO INVESTIGATE RIG FUNCTION IN PRE-AND POST-SYNAPTIC REGIONS OF THE NMJ / Chapter 5.1 --- Introduction --- p.89 / Chapter 5.2 --- Transgenic Rescue Experiment by Transgenic Expression of rig in rig Mutant / Chapter 5.2.1 --- Design of the Rescue Experiment --- p.90 / Chapter 5.2.2 --- Construct of pUAST-rig-FLAG --- p.93 / Chapter 5.2.3 --- Construct of the pUAST-myc-rig --- p.98 / Chapter 5.3 --- Tissue Specific Knockdown of rig expression --- p.102 / Chapter 5.4 --- Discussion --- p.105 / Chapter CHAPTER 6. --- ESTABLISHMENT OF AN INDUCIBLE S2R⁺ CELL MODEL FOR RIG OVEREXPRESSION / Chapter 6.1 --- Introduction --- p.108 / Chapter 6.2 --- Detection of Rig Protein in S2R⁺ Cells by Immunoblotting --- p.111 / Chapter 6.3 --- Detection of Rig Protein in S2R⁺ Cells by Immunostaining --- p.111 / Chapter 6.4 --- Detection of RNA in Immunopurified Rig Protein --- p.113 / Chapter 6.5 --- Discussion --- p.115 / Chapter CHAPTER 7. --- GENERAL DISCUSSION --- p.117 / References --- p.120 / Appendices --- p.126