京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13163号 / 論医博第2150号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation. / Funded by a biomedical research grant from the Motor Neurone Disease Association (ref Rattray/Apr15/837-791). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

Abnormal expression of the eukaryotic translation elongation factor 1A (eEF1A) has been implicated in disease states such as motor neuron degeneration and cancer. Two variants of eEF1A are found in mammals, named eEF1A1 and eEF1A2. These two variants are encoded by different genes, produce proteins which are 92% identical but have very different patterns of expression. eEF1A1 is almost ubiquitously expressed while eEF1A2 is expressed only in specialised cell types such as motor neurons and muscle. A spontaneous mutation in eEF1A2 results in the wasted mouse phenotype which shows similar characteristics in the mouse to those seen in human motor neuron degeneration. This mutation has been shown to be a 15.8kb deletion resulting in the complete loss of the promoter region and first non coding exon of eEF1A2 which completely abolishes protein expression. The main aim of this project was to further investigate the role of eEF1A2 in motor neuron degeneration. Firstly, although the wasted phenotype is considered to be caused by a recessive mutation, I established a cohort of aged heterozygote mice to evaluate whether any changes are seen later in life that might model late onset motor neuron degeneration. A combination of behavioural tests and pathology was used to compare wild type and heterozygous mice up to 21 months of age. Whilst results indicate that there is no significant difference between ageing heterozygotes and wildtype controls, there is an indication that female heterozygote mice perform slightly worse that wildtype controls on the rotarod (a behavioural test for motor function). Secondly, I aimed to investigate the primary cause of the wasted pathology by generating transgenic wasted mice expressing neuronal eEF1A2 only. This would complement previous experiments in the lab which studied transgenic wasted mice expressing eEF1A2 in muscle only. Unfortunately the expression of eEF1A2 in the transgenic animals was not neuronal specific. However a transgenic line with expression of eEF1A2 in neurons and skeletal muscle but not cardiac muscle has been generated which clearly warrants further investigation. Thirdly, I wished to assess whether eEF1A2 has any role in human motor neuron degeneration. To achieve this, eEF1A2 expression was investigated in spinal cords from human motor neuron disease (MND) patients. Preliminary data suggests that motor neurons from some MND patients express significantly less eEF1A2 than motor neurons of control samples. Further work is required to confirm these findings. Finally, I investigated the individual roles of eEF1A1 and eEF1A2 in the heat shock response. I used RNAi to ablate each variant separately in cells and subsequently measured the ability of each variant individually to mount a heat shock response. Results indicate a clear role for eEF1A1 but not eEF1A2 in the induction of heat shock. This may explain in part why motor neurons exhibit a poor heat shock response as they express eEF1A2 and not eEF1A1. These experiments shed light on our understanding of the role of eEF1A2 in motor neuron degeneration and uncover many new avenues of future investigation.

Trichostatin A (TSA) is a histone deacetylase inhibitor with beneficial effects in spinal muscular atrophy mouse models that carry the human SMN2 transgene. Whether TSA specifically targets the upregulation of the SMN2 gene or whether other genes respond to TSA and in turn provide neuroprotection in SMA mice is unclear. We have taken advantage of the Smn2B/- mouse model that does not harbor the human SMN2 transgene, to test the hypothesis that TSA has its beneficial effects through a non-Smn mediated pathway. Daily intraperitoneal injection of TSA from postnatal day 12 to 25 was performed in the Smn2B/- mice and littermate controls. Previous work from our laboratory demonstrated that treatment with TSA increased the median lifespan of Smn2B/- mice from twenty days to eight weeks. As well, there was a significant attenuation of weight loss and improved motor behavior. Pen test and righting reflex both showed significant improvement, and motor neurons in the spinal cord of Smn2B/-mice were protected from degeneration. Both the size and maturity of neuromuscular junctions were significantly improved in TSA treated Smn2B/- mice. Here, we have shown that TSA treatment does not increase the levels of Smn protein in mouse embryonic fibroblasts or myoblasts obtained from the Smn2B/- mice. Further, qPCR analysis revealed no changes in the level of Smn transcripts in the brain or spinal cord of TSA-treated SMA mice. Similarly, western blot analysis revealed no significant increase in Smn protein levels in the brain, spinal cord, hind limb muscle, heart muscle, or the liver of TSA treated Smn2B/- mice. However, TSA has beneficial effects in the muscles of Smn2B/- mice and improves motor behavior and myofiber size. TSA improves muscle development by enhancing the activity of myogenic regulatory factors independent of the Smn gene. The beneficial effect of TSA is therefore likely through an Smn-independent manner. Identification of these protective pathways will be of therapeutic value for the treatment of SMA.

Trichostatin A (TSA) is a histone deacetylase inhibitor with beneficial effects in spinal muscular atrophy mouse models that carry the human SMN2 transgene. Whether TSA specifically targets the upregulation of the SMN2 gene or whether other genes respond to TSA and in turn provide neuroprotection in SMA mice is unclear. We have taken advantage of the Smn2B/- mouse model that does not harbor the human SMN2 transgene, to test the hypothesis that TSA has its beneficial effects through a non-Smn mediated pathway. Daily intraperitoneal injection of TSA from postnatal day 12 to 25 was performed in the Smn2B/- mice and littermate controls. Previous work from our laboratory demonstrated that treatment with TSA increased the median lifespan of Smn2B/- mice from twenty days to eight weeks. As well, there was a significant attenuation of weight loss and improved motor behavior. Pen test and righting reflex both showed significant improvement, and motor neurons in the spinal cord of Smn2B/-mice were protected from degeneration. Both the size and maturity of neuromuscular junctions were significantly improved in TSA treated Smn2B/- mice. Here, we have shown that TSA treatment does not increase the levels of Smn protein in mouse embryonic fibroblasts or myoblasts obtained from the Smn2B/- mice. Further, qPCR analysis revealed no changes in the level of Smn transcripts in the brain or spinal cord of TSA-treated SMA mice. Similarly, western blot analysis revealed no significant increase in Smn protein levels in the brain, spinal cord, hind limb muscle, heart muscle, or the liver of TSA treated Smn2B/- mice. However, TSA has beneficial effects in the muscles of Smn2B/- mice and improves motor behavior and myofiber size. TSA improves muscle development by enhancing the activity of myogenic regulatory factors independent of the Smn gene. The beneficial effect of TSA is therefore likely through an Smn-independent manner. Identification of these protective pathways will be of therapeutic value for the treatment of SMA.

Amyotrophic Lateral Sclerosis is the most common type of motor neuron disease, characterized by progressive degeneration of the upper and lower motor neurons. Sufferers present with symptoms of muscle weakness and this quickly develops on to paralysis and finally death due to respiratory failure within 5 years of disease onset. Although the majority of cases are sporadic, about 10% are familial and it is hoped that through the investigation of these few cases a greater understanding of the disease process, the reasons for its delayed onset and vulnerability of motor neurons will be achieved. Recently a novel mutation linked to ALS was discovered in an evolutionary conserved protein named Vesicle associated membrane protein (VAMP) associated protein B (VAPB). VAPB is an integral type II membrane protein localised at the Endoplasmic Reticulum and thought to have a role in protein transport. The orthologue in Drosophila has been shown to be involved in the homeostatic regulation of bouton formation at the Neuromuscular Junction through an association with the microtubule network. To elucidate the mechanism through which this protein causes ALS, Pennetta et al have created a Drosophila model of the disease by expressing the mutated orthologue in the fly. To complement this model, I have undertaken a number of biochemical experiments to look for potential interactors of the VAP proteins. The yeast two hybrid system utilises the yeast GAL4 transcriptional activator to indicate a protein interaction within a yeast cell and can be used to test a cDNA library for interactors. Through this technique a number of interesting binding partners have been found that may play crucial roles in the progression of the disease.

No description available.

No description available.

Neurotrophins are a family of growth factors necessary for the development and maintenance of the nervous system. They produce their effects through receptor mediated signaling mechanisms that are highly regulated by sophisticated intracellular transport networks. The impairment of intracellular trafficking of neurotrophins in motor neurons has been identified as one possible factor in the development of motor neuron diseases, but remains inadequately studied. Aided by advances in imaging technology and the development of more powerful and sensitive detection tools for in-vitro studies, the dynamics of intracellular transport of neurotrophins are beginning to be unraveled. However, a primary limiting factor in the study of neurotrophin-transport dynamics in motor neurons has been the lack of alternative and easily available in-vitro systems able to substitute the often difficult and costly primary motor neuron cultures. The aim of this project was to develop a suitable motor neuron model using the NSC-34 cell line for the study of receptor mediated trafficking events through endosomal transport pathways. Successful evaluation and characterization of NSC-34 cells for motor neuron specific markers would result in the investigation of the p75 neurotrophin receptor (p75NTR) trafficking pathways in the presence of exogenous neurotrophins, with a variety of confocal imaging techniques. Chapter 3 describes the optimisation of NSC-34 cell culture conditions through media modification and the development of a suitable growth substrate matrix, which significantly improved cell adhesion, differentiation and the ability to culture the cells for extended time periods in serum free conditions. Quantitative measurements of cell proliferation, culture viability, cell-body size and neurite length are described to highlight the increased value of the cell line for long-term culture and experiments examining a broad range of issues relevant to motor neurons. In Chapter 4, multiple experimental approaches were used to extensively screen the NSC-34 cell line for the presence of motor neuron-specific markers, neurotrophin receptors and proteins involved in regulation of endosomal transport. This characterization established the presence of a developing motor neuron-like neurotrophin receptor profile (p75NTR, TrkB and TrkC), a genetic marker of developing motor neurons, cholinergic markers, proteins regulating transport within the endosomal pathway, and additional proteins previously shown to directly interact with neurotrophin receptors, including sortilin, and the lipid raft associated ganglioside GT1b. Furthermore, evidence is provided that NSC-34 cells undergo apoptosis in response to exogenous nerve growth factor (NGF) or neurotrophin-3 (NT-3), but not brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4). In addition characterization of mouse specific p75NTR antibodies is presented to establish their suitability for internalization studies without altering the binding of exogenous neurotrophins to the receptor. Subsequent confocal microscopy examination focusing on p75NTR trafficking in Chapter 5 revealed that internalization and intracellular transport of this receptor is regulated by exogenous neurotrophins at the cell surface where ligand binding and internalization occur, and in endosomal compartments where the bulk of receptors and ligands are targeted to their specific destinations. Evidence is provided showing that p75NTR internalization is altered in the presence of NGF, NT-3, or NT-4, but not BDNF, and the receptor is diverted into non-clathrin mediated endosomal pathways in response to NGF but not BDNF. Immunofluorescence confocal microscopy suggests that p75NTR recycles to the plasma membrane in a Rab4 GTPase dependent manner in the absence of neurotrophins. Addition of neurotrophins diverted p75NTR from the recycling Rab4 positive pathway, into EEA-1 positive sorting endosomes in the presence of NGF or NT-3, or lysosomal degradation in the presence of BDNF or NT-4. This study clearly demonstrates the suitability of the NSC-34 cell line as an alternate in-vitro system for the study of motor neuron biology, particularly the study of neurotrophin receptor trafficking. Taken together the results represented in this study suggest for the first time, that the fate of the p75NTR receptor depends on which neurotrophin is bound. These findings have important implications for understanding the dynamic mechanisms of action of p75NTR in normal neuronal function, and may also offer further insight into the potential role of neurotrophins in the treatment of neurodegenerative diseases.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition that affects corticospinal and spinal motor neurons and leads to death within 30 months of symptom onset in half of all cases. It remains incurable and treatment is supportive. The genetic and molecular understanding of ALS has gone through a rapid expansion in recent years, notably with the discoveries of TDP-43, a heterogeneous ribonucleoprotein as a major component of neuronal inclusions in ALS, as well as the discovery of the C9orf72 hexanucleotide expansion as the most common genetic cause of this disease. This first part of this thesis addresses the question of which of the various pathological hallmarks of the C9orf72 Hexanucleotide Repeat Expansion (HRE) in autopsy material correlates best with the clinical presentation. The main finding is that TDP-43 distribution, rather than C9orf72 RNA foci or dipeptide aggregation in the brain, corresponds best with the areas relevant to the clinical subtype of ALS-FTD. Subsequently the role of TDP-43 was investigated in induced pluripotent stem cell derived motor neurons, and no evidence of the hallmarks of TDP-43 dysfunction, were seen in this model of the disease. No mislocalisation is found on immunofluorescence, and biochemical analysis shows no differences in insoluble species between the patient and control cell lines. In the final section, RNA sequencing was used to study the transcriptome of a BAC transgenic mouse carrying a human M337V transgene expressed at low levels, to identify early presymptomatic differences in gene expression. Interestingly, no changes were found in genes known to be associated with ALS through mutations, and the constitutive nuclear functions of TDP-43 in the regulation of splicing was maintained, prior to the emergence of a clinical phenotype in the mouse. This favours a gain of function mechanism for TDP-43 mutations in ALS.