Insights

Session 9A: Modulating SOD1 toxicity


The first known ALS causing mutations were found in the gene SOD1 in 1993 [1] and the study of mutant SOD1 would underpin most ALS research for the next two decades. However, the precise mechanism by which SOD1 causes motor neuron death is still unknown. The pathological effects are not thought to result from loss or inhibition of dismutase activity but rather from gain-of-function effects through which the protein acquires one or more toxic properties [2]. Currently, over 170 mutations are associated with familial ALS. Many of these mutations in SOD1 result in a destabilized conformation of the SOD1 protein meaning the prevailing hypothesis suggests that mutation-induced conformational changes lead to SOD1 misfolding and subsequent aggregation leading to neuron dysfunction and subsequent death [3-5].

SOD1 mice have 23 copies of human mutant G93A and have extremely high levels of human G93A SOD1 in motor neurons. Prof Joe Beckman, Oregon State University, has crossed the mice overexpressing the human copper chaperone (hCCS) and human G93A mice and finds that their lifespan is extremely shortened. SOD1 has one of the highest affinities for copper of any protein in the proteome and the high levels of SOD1 require significant amounts of copper such that in combination with overexpression of the copper chaperone the depletion of copper is catastrophic. One hypothesis for why the specific chaperone for human SOD1 decreases the lifespan of SOD1 mice is that it further adds to the depletion of copper from the mitochondria. Prof Beckman reports that the drug Copper ATSM (CuATSM) is known to deliver copper only to cells with damaged mitochondria [6] and that treatment of hCCS/hG93ASOD1 mice with CuATSM starting from birth was shown to indefinitely rescue these mice (mice currently over 400 days). Upon removal of the treatment the mice begin to decline, which again can be halted by further delivery of the drug. In addition, the treatment of the original G93A SOD1 mice with CuATSM extends the lifespan of these mice by 16% when treated from 50 days.

SOD1 in the absence of metals is thought to be largely unfolded or misfolded that may result in aggregation or inappropriate interactions with other cellular machinery. Accordingly the treatment of SOD1 ALS with molecules that could inhibit the aggregation are attractive. Two very different approaches to this problem were presented in the session. Firstly, small molecules that may suppress the aggregation of SOD1 were identified from screens of NSC-34 cells transfected with G93A and treated with thapsigargin by Dr Arie Gruzman, Bar-Ilan University. Dr Gruzman identified compounds that reduced the amount of protein aggregates and prevented ER stress and apoptosis. Regardless of Dr Gruzman’s self confessed “Organic Chemistry Hands” his group was able to show that the lead compound reduced total protein content in GFP tagged SOD1 models and when G93A mice were treated from day 40 increases in body weight were observed. However, there was no significant effect on lifespan.
In addition, Les Grad from the University of British Columbia presented his work around identifying small molecule inhibition of cell-to-cell SOD1 misfolding transmission. Previous work had showed that expression of misfolded SOD1 in human cells (HEKs) converted endogenous human SOD1 to the misfolded form [7]. When expressed in mouse cells this transmission does not occur. One specific difference between human and mouse SOD1 is Trp at position 32 (Ser in mouse SOD1). Using the model generated by Dr Grad where misfolded SOD1 can be propagated to naïve cultures of human SOD1 expressing cells [8]. Dr Grad presented work to show that conversion of wt to misfolded SOD1 is reliant on Trp at position 32 and importantly analogues of 5-Fluorouradine including uridine, but not unrelated small molecule folinic acid, blocks transmission of SOD1 misfolding in a dose dependant fashion. Dr Grad hypothesized that previous studies [9] that had identified uridine as a molecule that is protective in the G93A mouse model may be due to its ability to inhibit aggregation. Dr Grad and others in the laboratory of Prof Neil Cashman are now developing Small molecule screen adapted from their SOD1 transmission assay.

Regardless of the mode of SOD1 toxicity an attractive treatment strategy is to lower SOD1 levels and a number of systems of reduction of SOD1 expression levels have already been proposed. Lorelei Stoica from the University of Massachusetts Medical School presented her work on the high dose treatment of Adeno‐Associated Virus (AAV)-microRNA strategy. AAV9 was chosen because of its safety and effectiveness for gene therapy in the CNS. Dr Stoica used miRNA to target exon2 in human but not mouse SOD1 and delivered the AAV9-miRNA to lateral ventricle of neonatal mice. This treatment resulted in a 50% reduction in spinal cord SOD1 in 4 week old animals. It was clearly demonstrated that the AAV9 was able to transduce motor neurons in the ventral horn of spinal cord are motor cortex. This single treatment increase the mean lifespan of G93A mice from 137 to 206 days in treated animals. Moreover, the number of motor neurons, motor units and muscle innervation was preserved.

Taken together the papers presented in the session paint a picture of a positive future for potential treatment for SOD1 associated ALS and it was pointed out during the session that there is potential for these strategies to work in sporadic ALS where misfolding of SOD1 has also been identified.

References

  1. Rosen, D.R., et al., Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 1993. 362(6415): p. 59-62.
  2. Benmohamed, R., et al., Identification of compounds protective against G93A-SOD1 toxicity for the treatment of amyotrophic lateral sclerosis. Informa Healthcare, 2011. 12: p. 87-96.
  3. Bruijn, L.I., et al., Aggregation and Motor Neuron Toxicity of an ALS-Linked SOD1 Mutant Independent from Wild-Type SOD1. Science, 1998. 281(5384): p. 1851-1854.
  4. Furukawa, Y. and T.V. O’Halloran, Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo- and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation. The Journal of Biological Chemistry, 2005. 280(17): p. 17266-17274.
  5. Ilieva, H., Magdalini Polymenidou, and D.W. Cleveland, Non–cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. Journal of Cell Biology, 2009. 187(6): p. 761-772.
  6. Donnelly, P.S., et al., An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copperII. Proc Natl Acad Sci U S A, 2012. 109(1): p. 47-52.
  7. Grad, L.I., et al., Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc Natl Acad Sci U S A, 2011. 108(39): p. 16398-403.
  8. Grad, L.I., et al., Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms. Proc Natl Acad Sci U S A, 2014. 111(9): p. 3620-5.
  9. Amante, D.J., et al., Uridine ameliorates the pathological phenotype in transgenic G93A-ALS mice. Amyotroph Lateral Scler, 2010. 11(6): p. 520-30.
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