NMR characterization of angiogenin variants and tRNAAla products impacting aberrant protein oligomerization

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation

Misfolded Mutant SOD1 Directly Inhibits VDAC1 Conductance in a Mouse Model of Inherited ALS

SummaryMutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. With conformation-specific antibodies, we now demonstrate that misfolded mutant SOD1 binds directly to the voltage-dependent anion channel (VDAC1), an integral membrane protein imbedded in the outer mitochondrial membrane. This interaction is found on isolated spinal cord mitochondria and can be reconstituted with purified components in vitro. ADP passage through the outer membrane is diminished in spinal mitochondria from mutant SOD1-expressing ALS rats. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of individual channels when reconstituted in a lipid bilayer. Reduction of VDAC1 activity with targeted gene disruption is shown to diminish survival by accelerating onset of fatal paralysis in mice expressing the ALS-causing mutation SOD1G37R. Taken together, our results establish a direct link between misfolded mutant SOD1 and mitochondrial dysfunction in this form of inherited ALS

Motor neurone disease twin study using death discordant twins

Using a novel methodology termed the death discordant twin method, it has been possible to carry out an epidemiological study into the possible causes of sporadic MND. The study population was the largest twin sample so far collected worldwide for this rare disease, and identified 75 twin pairs—24 monozygotic and 51 dizygotic. This involved a comprehensive and detailed search of the MND death certificate population for England and Wales between 1979–1989 inclusive. The twin sample was utilised for two different purposes: 1) The estimation of the genetic contribution to sporadic MND; and 2) the formation of matched pairs for a case-control study of environmental factors. An extensive review of germane hypotheses and research was made and is reported with reference to relevant papers. Following a critique of the methods and problems of many traditional twin studies, the advantages of this new method are discussed. The study results are analysed and detailed together with statistical evaluation, and the genetic contribution estimated. Four monozygotic probands from two concordant pairs were identified, producing a MZ proband concordance rate of 17.4%. This was reduced to 10% when two probands were determined to have had familial MND. No dizygotic concordant pairs were found, but a "coefficient of genetic determination" ('G') between 0.38–0.85 was derived, using the methods of Falconer 1965 and Smith 1974. This supports a multifactorial aetiology for MND, probably involving several genetic factors, i.e. a single gene defect is excluded. The environmental risk factors were assessed using Odds Ratios (OR) with 95% Confidence Intervals (Cl). The statistically significant factors which held true during conditional logistic regression modelling were 'regular vehicle maintenance' [OR = 7.0 (CI 1.3–89.9)], and 'occupational paint usage' [OR = 3.75 (CI 1.1–17.1)]. Other factors were of clinical interest. Many of the environmental factors identified in previous studies to be associated with increased risk for MND were not verified

Fragment based drug discovery for SOD1-ALS and solution X-ray scattering studies on the copper chaperone for SOD1 (hCCS)and its functional complexes with SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal disease of the nervous system. The majority of ALS cases are have no attributable genetic link, however approximately 10% are familial and 20 % of these result from mutations in the SOD1 gene. How SOD1 mutation manifests as the ALS phenotype is not clear however the enzyme does gain an aggregative property characterised by SOD1 inclusions in the brain and spinal cord. Drug treatment for sporadic and familial ALS is currently limited to palliatives and there is currently no specific treatment for SOD1 mediated ALS. In order to find molecules that may be of use in the development of SOD1 therapeutics a crystallographic screening pipeline was set up to assess binding of small molecules to both wild-type and SOD1 mutants. Using in silico studies and previous crystallographic work as a starting point, this method revealed several low molecular weight compounds (Mr 183 – 310 gMol-1) that have SOD1 binding activity. These molecules belong to three distinct classes: catecholamine, quinazoline and fluorouridine and occupy two distinct binding sites on the surface of the SOD1 β-barrel in an area known to be important in disease pathogenesis. The incorporation of copper into biological macromolecules such as SOD1 is essential for the viability of most organisms. However, copper is toxic and therefore the intracellular free copper concentration is kept to an absolute minimum. The Copper Chaperone for SOD1 (CCS) is the major pathway for SOD1 copper loading and transfer of an intrasubunit disulphide bond known to stabilise SOD1. Using small angle X-ray scattering combined with online size exclusion chromatography high quality data were acquired for both homodimeric hCCS and the functionally critical hCCS-SOD1 heterodimer. SAXS measurements were made of the hCCS complex with wild-type SOD1 and the disease relevant L38V and I113T SOD1 mutants. A rigid body modelling approach enabled exploration of the conformational dynamics of each species. Homodimeric hCCS is found to adopt positions that would facilitate initial copper acquisition and transfer from domain I to domain III. This domain III is also found in positions that would allow disulphide and copper transfer to SOD1 in the heterodimeric complex. The hCCS-I113T SOD1 complex has characteristics which are convergent with a view of ALS initiated by improperly matured SOD1

Molecular Expression of Neuroprotective and Neurodestructive Signaling Systems Following Axotomy-Induced Target Disconnection: Relevance to ALS

Amyotrophic Lateral Sclerosis (ALS) is the most common adult motoneuron (MN) degenerative disease. Discovery of a portion of familial cases with a mutation in the gene superoxide dismutase 1 (SOD1) gene led to the development of a transgenic mouse model. Pre-symptomatic SOD1 mice show no symptoms well into adulthood, however once symptom onset has occurred they display pathological hallmarks of ALS. The initial pathological event is loss of neuromuscular junctions in the lower limbs and therefore the die-back theory of ALS, suggests disconnection from the target musculature leads to MN degeneration. Our lab utilizes a peripheral nerve injury model to investigate the mechanisms of MN survival in the pre-symptomatic SOD1 mouse. We have shown SOD1 mice display enhanced facial MN (FMN) cell loss following a facial nerve axotomy, compared to wild-type (WT) mice. Analysis of gene expression revealed that MN regenerative genes were expressed to a similar extent in SOD1 and WT mice however, differences were seen among genes expressed by the neuropil, namely pro-inflammatory genes and suppression of the astrocytic response. Experiments within this dissertation evaluated whether axotomy-induced molecular response in pre-symptomatic SOD1 mice, resembles the disease-induced molecular response within the facial nucleus in symptomatic SOD1 mice. This dissertation identified that microglia show abnormal, suppressed responses to axonal injury. In addition, while increased expression of death receptor genes is a reaction to MN injury in both WT and SOD1 mice initially, however, Fas death receptor genes are dysregulated in the SOD1 axotomized facial nucleus at a time consistent with enhanced FMN loss. Most importantly, the increased mRNA expression seen in the SOD1 disease-affected nucleus during the symptomatic stage is consistent with the mRNA expression response after axotomy in pre-symptomatic SOD1 mice. The work presented within this dissertation concludes that the molecular response within the SOD1 facial nucleus is similar regardless of the method MN injury (axotomy/disease) and therefore, allows for axotomy to be used in the pre-symptomatic mouse as a model of disease progression

The establishment of potential cerebrospinal fluid biomarkers for canine degenerative myelopathy

Canine degenerative myelopathy(DM) is a late onset neurodegenerative disease that primarily affects German Shepherd dog (GSD), though a number of other specific breeds are also affected. The underlying cause of the disorder remains elusive, though recent advances have implicated a mutation of superoxide dismutase 1(Sod1) in the aetiology, also implying DM is a potential orthologue of human amyotrophic lateral sclerosis. The identification of the Sod1 mutation raises the index of suspicion for an individual animal, however it is not specifically diagnostic as a proportion of dogs homozygous for the Sod1 mutation do not develop DM. Therefore, there is a clinical need for the development of specific biomarker(s) for DM to support genetic test. The aim of this study was to establish potential biomarkers for DM by exploring canine cerebrospinal fluid (CSF). A dual strategy was adopted;1) Evaluation of potential ALS biomarkers in DM CSF, 2) Identification of novel biomarker(s) in DM CSF. The cases selected in this project had a presumptive diagnosis of DM and were homozygous for Sod1 mutation. Preliminary characterisation by Western blot and mass spectrometry identified four protein candidates in DM CSF, comprised of cystatin C, transthyretin (dimeric and monomeric TTR), haptoglobin and clusterin. Since the validity of these putative biomarkers may be influenced by pre-analytical variables that may arise from the clinical environment, we therefore assessed the impact of three potential sample handling practices on these four proteins. The results from these experiments demonstrate that dimeric TTR and clusterin were affected by sample handling conditions. Therefore, an appropriate protocol for CSF sample handling was established. Western blot analyses indicated that clusterin is the most viable biomarker candidate for DM. Clusterin was significantly elevated in DM CSF when compared to a range of neurological conditions. The second potential candidate for DM biomarker is TTR, which is potentially reduced, an observation similar to those found in ALS CSF. The relationship of these proteins in the pathogenic mechanisms that underpin DM is unclear. However, based on observations on ALS, it is reasonable to speculate that their alterations are associated with a toxic gain of function of the mutant SOD1 protein. The successful characterisation of clusterin and TTR in DM CSF may therefore represent components of a panel of emerging biomarkers that may combine to distinguish DM in the clinic and provide further insights into the disease mechanisms