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

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

Trends in the Molecular Pathogenesis and Clinical Therapeutics of Common Neurodegenerative Disorders

The term neurodegenerative disorders, encompasses a variety of underlying conditions, sporadic and/or familial and are characterized by the persistent loss of neuronal subtypes. These disorders can disrupt molecular pathways, synapses, neuronal subpopulations and local circuits in specific brain regions, as well as higher-order neural networks. Abnormal network activities may result in a vicious cycle, further impairing the integrity and functions of neurons and synapses, for example, through aberrant excitation or inhibition. The most common neurodegenerative disorders are Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis and Huntington’s disease. The molecular features of these disorders have been extensively researched and various unique neurotherapeutic interventions have been developed. However, there is an enormous coercion to integrate the existing knowledge in order to intensify the reliability with which neurodegenerative disorders can be diagnosed and treated. The objective of this review article is therefore to assimilate these disorders’ in terms of their neuropathology, neurogenetics, etiology, trends in pharmacological treatment, clinical management, and the use of innovative neurotherapeutic interventions

Group I metabotropic glutamatergic receptors regulating glutamate release and microglia phenotype in a murine model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the death of upper and lower motor neurons. Although the aetiology of the disease is still unclear, glutamate (Glu)-mediated excitotoxicity is a major cause. Our previous studies demonstrated that presynaptic Group-I metabotropic Glu receptors (mGluR1 and mGluR5) are over-expressed in spinal cord synaptosomes of 120-day-old SOD1G93A mice, that represent the late stage of the disease, and that their activation by the selective mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) produced abnormal Glu release. The aim of the present study was to investigate whether mGluR1 and mGluR5 also affect Glu release during the pre- and early-symptomatic time-course of the pathlogy (30, 60 and 90 days), in the same animal model. Our results showed that the mGluR1/5 agonist 3,5-DHPG evoked the release of glutamate in a concentration-dependent way and the effects were almost superimposable between 30/60-day-old WT and SOD1G93A mice. At variance, 0.3 \u3bcM 3,5-DHPG significantly increased Glu release (25%, p<0.05) in 90-day-old SOD1G93A mice but not in WT aged controls. The involvement of both metabotropic glutamate receptor subtypes was demonstrated using mGluR1 and mGluR5 selective antagonists/negative allosteric modulators (LY367385, MPEP, respectively). The analysis of the molecular mechanisms underlying the 3,5-DHPG-evoked Glu release revealed that it was of vesicular origin and induced by Ca2+ released from intra terminal stores. Confocal imaging confirmed that both mGluR1 and mGluR5 were co-localized onto glutamatergic nerve terminals and their expression was increased in SOD1G93A mice at the onset of the disease. We have also set up a method to isolate extracellular vesicles enriched in exosomes to investigate whether EVs derived from cultured activated astrocytes, treated with a mGluR5 antagonist, were able to change the the inflammatory pattern of microglia

Drosophila as a tool to identify genes and mechanisms involved in Amyotrophic Lateral Sclerosis

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions (NMJs). Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human disease including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis. To elucidate the patho-physiology underlying motor neuron degeneration in humans, we also generated a Drosophila model of ALS8 in the adult eye. Targeted expression of mutant DVAP-33A in the Drosophila compound eye causes a degenerative phenotype characterized by a smaller eye containing missing or aberrantly oriented bristles and fused ommatidia. In a F1 deficiency screen, we performed a genome-wide survey aimed at identifying enhancers and suppressors of the degenerative eye phenotype. Several interacting regions have been found and the identification of these interacting genes will shed new light on the molecular mechanisms underlying VAP-induced ALS

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