Molecular Biology and Genetics of Amyotrophic Lateral Sclerosis

1. Molecular Biology and Genetics of Amyotrophic Lateral Sclerosis Michael Sidel February 13, 2008

2. Goals • To have a basis for the understanding of the genetics involved in ALS • To understand the current theories and concepts that underlie the pathogenesis of ALS

3. Outline • Brief clinical overview of the disease • 1 slide • The genetics of ALS • 6 Slides • The pathology of ALS • 5 Slides • An understanding of the disease pathogenesis • Priceless (or 8 slides)

4. Some Clinical Factoids about ALS • A progressive neurodegenerative disease • Involvement of motor neurons at all levels • 50% mortality at 3 years • Several clinically related variants: • Primary lateral sclerosis • Primary muscular atrophy • Progressive bulbar palsy • 90% of the cases are considered “sporadic” • 10% of cases are considered familial

5. Some of the Genetics of ALS

6. Genetics of ALS • 10% of cases of ALS have history of first-degree relative with the disease • Suggests Autosomal Dominant inheritance • Research direction: • Isolating genes involved in clear familial disease • Isolating polymorphisms that are potentially risk factors in sporadic disease

7. Genetics of Familial ALS Mitchell and Borasio, Lancet2007; 369: 2031–41

8. Genetics of Familial ALS Mitchell and Borasio, Lancet2007; 369: 2031–41

9. Genetics of Familial ALS Mitchell and Borasio, Lancet2007; 369: 2031–41

10. Genetics of Familial ALS Mitchell and Borasio, Lancet2007; 369: 2031–41

11. Genetics of Familial ALS • Represents 10-20% of cases of familial ALS • Over 100 mutations to the gene are related to pathology • Gain of function mutation causes the disease pathology • From mouse model data

12. Genetics of Familial ALS Mitchell and Borasio, Lancet2007; 369: 2031–41

13. Genetics of Familial ALS Mitchell and Borasio, Lancet2007; 369: 2031–41

14. Genetics Associations in ALS • There are other genetic mutations that may alter the risk of developing ALS: • Examples: • Angiogenin (14q11.2) • Vascular endothelial growth factor (6p12) • Survival motor neuron (5q12.2-q13.3) • Neurofilament protein (22q12.2) • Multivesicular body protein 2B (2p11.2)

15. Pathology of ALS

16. Gross Pathology • Motor neuron degeneration and death with gliosis • Gliosis of the CST • Atrophy of ventral nerve roots

19. Microscopic Pathology • Intracellular inclusions • Bunina bodies • Ubiquinated inclusions (not tau)

20. Pathogenic mechanisms of ALS

21. Proposed Pathogenic Mechanisms in ALS • Oxidative Stress • Excitotoxicity • Abnormal Protein Precipitation and/or Aggregation • Cytoskeletal defects • Axonal transport • Neuroinflammation • Abnormalities in hypoxia-regulated genes • Apoptosis

22. Oxidative Stress • Motor neuron damage as a result of oxidative stress • Not necessarily linked to gene mutation • i.e. SOD1 • Post-mortem studies have shown evidence of increased oxidative by-products • Linked to other mechanisms

23. Excitotoxicity • Glutamate mediated • Possible mechanisms of disease include: • Production of free radicals • Increased intracellular calcium • Decreased EAAT2 functions has been described in some ALS post-mortem studies

24. More on Excitoxocity Goodall and Morrison Expert Reviews in Molecular Medicine. Vol. 8(11) 24 May 2006.

25. Abnormal Protein Precipitation and/or Aggregation • Abnormal protein aggregates, including Bunina bodies, ubiquitinated inclusions and neurofilament rich hyaline inclusions are pathological hallmarks of ALS • Unknown cause and effect relationship • SOD1 mutants can misfold and coprecipitate with other molecules as well

26. Cytoskeletal Defects • Neurofilament proteins are the most abundant structural protein in motor neurons, and aggregates of neurofilament proteins motor neurons are commonly seen in ALS • Can be found as part of inclusions • Overexpression of peripherin or alpha-internexin in mice can cause motor neuron disease

27. Axonal Transport • Axonal transport of materials is essential for neuronal function and survival • Dynactin is a protein involved in fast retrograde transport in the axon • Mutations in this gene have been associated with motor neuron disease

28. Neuroinflammation • Microglia have been found to be activated in parts of CNS affected by ALS • COX-2 Receptors and downstream prostaglandins are elevated in ALS brains

29. Apoptosis • Represents eneergy-dependent programmed cell death • In human ALS spinal cord tissue, increases in caspase-1 and -9 activation have been detected • Very unclear how much this plays a role in the pathogenesis of the disease

30. Goodall and Morrison. Expert Reviews in Molecular Medicine. Vol. 8(11) 24 May 2006.

31. Why Motor Neurons? • Extreme size of cells • High metabolic activity • Sensitivity to mitochondrial dysfunction • Elevated neurofilament content • Reduced capacity to buffer calcium

32. Conclusions • We do not understand ALS • Familial ALS is caused by genetic mutations and is rare • Sporadic ALS is caused by a combination of: • Genetic susceptibility • Environmental triggers • Motor neuron specific cellular damage

33. Key Points to Remember • Main cause of familial ALS is SOD1 mutations with are gain of function • Bunina bodies are inclusions found in ALS motor neurons • Excitoxicity and oxidative damage are likely major contributors to motor neuron death in ALS

34. References • Valdmanis, P.N. and Rouleau, G.A. Genetics of familial amyotrophic lateral sclerosis. Neurology. 2008;70:144-152. • Goodall, E.F. and Morrison, K.E. Amyotrophic lateral sclerosis: proposed mechanisms and pathways to treatment. Expert Reviews in Molecular Medicine. Vol. 8; Issue 11; 24 May 2006. • Mitchell, J.D. and Barasio, G.D. Amyoptrophic Lateral Sclerosis. Lancet 2007; 369: 2031-41.