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 1.Main cause of familial ALS is SOD1 mutations with are gain of function 2.Bunina bodies are inclusions found in ALS motor neurons 3.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.