Amyotrophic lateral sclerosis, ALS, is a progressive fatal neurodegenerative disorder affecting motor neurones in motor cortex, brain stem and spinal cord. This inevitably leads to paralysis, respiratory failure and death. In about 5% of patients with ALS there is an association with mutations in gene for the abundant intracellular scavenging enzyme superoxide dismutase1, SOD1. The noxious property of SOD1 is proposed to be due to gain of function. In familial cases the inheritance is most commonly dominant. This study focus on two disparate SOD1 mutations occurring in Scandinavia. The recessive D90A mutation which has properties similar to that of the normal wild-type human SOD1. The dominantly inherited G127insTGGG mutation, G127X, causes a C-terminal truncation of the last 21 amino acids and is a highly unstable protein. Transgenic mice were created expressing D90A and G127X mutated human SOD1. Results from studies of tissue from the central nervous system of patients carrying either of these mutations were compared with similar tissue collected from transgenic mice generated with the same mutations. Tissue from the mice were also compared to central nervous tissue from several other transgenic mouse strains expressing human wild type SOD1 as well as other ALS associated human SOD1 mutations. The transgenic mice expressing D90A respectively G127X mutated human SOD1 develop motor neurone disease. Microscopic studies of central nervous tissues from G127X transgenic mice reveals inclusions of aggregated misfolded SOD1 in motor neurones and adjacent supporting cells. These inclusions are composed of detergent resistant aggregates and preceded by accumulations of minute quantities of detergent-soluble aggregates. The inclusions mimic those found in G127X patients. In D90A transgenic mice the progression, as in the humans, was slower and the mice, as the patients, showed bladder disturbance. In the D90A patients, the SOD1 inclusions mimic those found in sporadic ALS patients. Aggregation of SOD1 in central nervous tissue appears to be related to severity of disease. Degenerative features as vacuolization and gliosis precedes phenotypic alterations. Changes are seen not only in motor areas but also in higher centres of the telencephalon.

Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.

Amyotrophic lateral sclerosis (ALS) is a devastating disease that affects people in their late mid-life, with fatal outcome usually within a few years. The progressive degeneration of neurons responsible for muscle movement (motor neurons) throughout the central nervous system (CNS) leads to muscle wasting and paralysis, and eventually affects respiratory function. Most cases have no familial background (sporadic) whereas about 10% of cases have relatives affected by the disease. A substantial number of familial cases are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). Since the initial discovery of this relationship about 17 years ago, numerous workers have tried to identify the pathogenicity of mutant SOD1 but without any final agreement or consensus regarding mechanism. The experiments in this thesis have been aimed at finding common pathogenic mechanisms by analyzing transgenic mouse models expressing mutant SOD1s with widely different properties.     Mitochondrial pathology and dysfunction have been reported in both ALS patients and murine models. We used density gradient ultracentrifugation for comparison of mitochondrial partitioning of SOD1 in our transgenic models. It was found that models with high levels of mutant protein, overloaded mitochondria with high levels of SOD1-protein whereas models with wild type-like levels of mutant protein did not. No significant association of the truncation mutant G127X with mitochondria was found. Thus, if mitochondrial dysfunction and pathology are fundamental for ALS pathogenesis this is unlikely to be caused by physical association of mutant SOD1 with mitochondria.     Density gradient ultracentrifugation was used to study SOD1 inclusions in tissues from an ALS patient with a mutant SOD1 (G127X). We found large amounts in the ventral horns of the spinal cord but also in the liver and kidney, although at lower levels. This showed that such signs of the disease can also be found outside the CNS.     This method was used further to characterize SOD1 inclusions with regard to the properties of mutant SOD1 and the presence of other proteins. The inclusions were found to be complex detergent-sensitive structures with mutant SOD1 reduced at disulfide C57-C146 being the major inclusion protein, constituting at least 50% of the protein content. Ten co-aggregating proteins were isolated, some of which were already known to be present in cellular inclusions. Of great interest was the presence of several proteins that normally reside in the endoplasmic reticulum (ER), which is in accordance with recent data suggesting that the unfolded protein response (UPR) has a role in ALS.     To obtain unbiased information on the pathogenesis of mutant SOD1, we performed a total proteome study on spinal cords from ALS transgenic mice. By multivariate analysis of the 1,800 protein spots detected, 420 (23%) were found to significantly contribute to the difference between transgenic and control mice. From 53 proteins finally identified, we found pathways such as mitochondrial function, oxidative stress, and protein degradation to be affected by the disease. We also identified a previously uncharacterized covalent SOD1 dimer.    In conclusion, the work described in this thesis suggests that mutant SOD1 affects the function of mitochondria, but not mainly through direct accumulation of SOD1 protein. It also suggests that SOD1 inclusions, present in both the CNS and peripheral tissues, mainly consist of SOD1 but they also trap proteins involved in the UPR. This might be deleterious as motor neurons, unable to renew themselves, are dependent on proper protein folding and degradation.

No description available.

Protein aggregation is deleterious to human health and detrimental to therapeutic shelf-life. The physical processes that induce aggregation are the same processes that drive productive folding reactions. As such, protein aggregation is a non-productive form of protein folding. To gain insight into the steps that serve as a partition between the folding and aggregation reactions, the folding mechanisms of several β-rich proteins with links to human disease or medicine were examined. In the ALS-linked protein, SOD1, a subpopulation of the unfolded ensemble is found to be a common source of both nonnative structure and frustrated folding. These behaviors are only observed upon the reduction of the intrinsic disulfide bond, indicating that this covalent interaction wards against aggregation. The nonnative structure presents an attractive target for the development of new therapeutic agents. In VH domains from therapeutic mAbs, the intramolecular disulfide bond protects against aggregation. However, it can also introduce complexity to the folding mechanism. This complexity is linked to the formation of a strained orientation of the disulfide bond. This strained orientation of the disulfide in certain VH domains is energetically unfavorable enough to disrupt the formation of the disulfide in the full length mAbs. The novel relationship observed between disulfide orientation, folding complexity, and incomplete oxidation warrants further examination in other Ig domains. Overall, these results demonstrate that mapping the folding free energy landscape for proteins with roles in human disease or therapeutics can provide valuable insights for developing and improving treatment options.

Final publication is available at http://www.sciencedirect.com/science/article/pii/S1873506115001191 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19579号 / 医博第4086号 / 新制||医||1013(附属図書館) / 32615 / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Dysregulation of the autonomic cardiovascular control is a complication of diseases including diabetes, hypertension, sleep apnea, and aging. A common factor in these conditions is an increase in reactive oxygen species (ROS) in neural, cardiac, and endothelial tissues. Cu/Zn superoxide dismutase (SOD1) is an intracellular anti-oxidant enzyme that catalyzes dismutation of the superoxide anion (O2.-) to hydrogen peroxide (H2O2). Expression and function of this enzyme are diminished in pathologies that impair cardiovascular autonomic control. This study employed mice overexpressing a transgene for human SOD1 (hSOD1) to determine if its overexpression would alter autonomic regulation of BP, HR, and BRS in healthy animals, and if this animal line (C57B6SJL-Tg (SOD1)2 Gur/J) could be used in future studies to determine if hSOD1 overexpression can preserve cardiac autonomic function in disease models. To accomplish this aim, using anesthetized SOD1 and C57 (control) mice, we recorded HR, and aortic depressor nerve (ADN) activity changes in response to pharmacologically-induced BP changes in order to measure baroreflex and baroreceptor sensitivity, respectively. In order to identify any alterations in central, efferent, and cardiac components of the baroreflex arc, we electrically stimulated the left ADN and left cervical vagus and compared the reductions in BP and HR between the C57 and SOD1 mice. Time- and frequency-domain analysis of heart rate variability (HRV) was performed using pulse pressure recordings prior to pharmacologic or surgical procedures. We found that hSOD1 overexpression in the SOD1 mouse line, in comparison to C57 controls did not significantly affect resting HR (C57: 558 ± 8 vs. SOD1:553 ± 13 beats-per-minute) or blood pressure (C57: 88.8 ± 2.9 vs.SOD1: 85.8 ± 2.1 mmHg). hSOD1 overexpression did not affect the decrease in average mean arterial pressure (MAP) following injection of sodium nitroprusside (SNP) (C57: 38.7 ± 1.4 vs. SOD1: 39.5 ± 1.3 mmHg) or increase in average MAP (C57: 135.8 ± 3.1 vs. SOD1: 136.6 ± 3.5 mmHg) following injection of phenylephrine (PE). BRS, as measured by the averaged regression lines for ΔHR/ΔMAP for the SNP-induced tachycardic baroreflex (C57: 0.57 ± 0.06 bpm/mmHg, SOD1: 0.61 ± 0.08 bpm/mmHg)) and the PE-induced bradycardic baroreflex (C57: -2.9 ± 0.57 bmp/mmHg, SOD1: -4.3 ± 0.84 bpm/mmHg) are not significantly different between C57 and SOD1. Baroreceptor activation showed a significant increase in gain (C57: 5.4 ± 0.3 vs. SOD1: 7.4 ± 0.5 %/mmHg, P < 0.01) in the SOD1 transgenic mice. Heart rate depression in response to electrical stimulation of the left ADN and cervical vagus was comparable between C57 and SOD1, though MAP reduction in response to ADN stimulation is slightly, but significantly increased at 50 Hz in SOD1 animals. Time- domain analysis of HRV did not reveal any significant difference in beat-to-beat variability between SOD1 and C57 (SDNN: C57: 2.78 ± 0.20, SOD1: 2.89 ± 0.27), although frequency-domain analysis uncovered a significant reduction in the low-frequency power component of the HRV power spectral distribution (C57: 1.19 ± 0.11, SOD1: 0.35 ± 0.06, P < 0.001). This study shows that although hSOD1 overexpression does not affect overall baroreflex function, it does potentiate baroreceptor sensitivity and brain stem control of arterial pressure, and reduces low-frequency beat-to-beat variations in HR, without affecting total HRV.

No description available.

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