SCBM303. Amyotrophic lateral sclerosis (ALS) Laran T. Jensen. Department of Biochemistry Faculty of Science Mahidol University

Transcription

1 SCBM303 Amyotrophic lateral sclerosis (ALS) Laran T. Jensen Department of Biochemistry Faculty of Science Mahidol University 1

2 Amyotrophic lateral sclerosis (ALS) A-myo-trophic no-muscle-nourishment Refers to atrophy (progressive muscle loss) Lateral refers to involvement of the lateral corticospinal tracts Sclerosis refers to demyelination and buildup of scar tissue (sclerosis = scar) As the disease progresses eventually all muscle groups become involved The spread into all muscle groups is a defining characteristic of ALS 2

3 ALS has different names depending on location In the United States, Amyotrophic Lateral Sclerosis (ALS) is commonly known as Lou Gehrig s disease, named after the legendary Yankees baseball player who died from ALS in 1941 In the United Kingdom and other parts of the world, ALS is often called Motor Neuron Disease (MND) Lou Gehrig Famous Baseball Player Stephen Hawking Physicist/Author In many countries where Spanish is spoken, it is called Esclerosis Lateral Amiotrófica (ELA) Jon Stone Creator of Sesame Street/Muppets

4 ALS was first described in 1869 as a disease of the nervous system Amyotrophic Lateral Sclerosis (ALS) is the most common form of motor neuron disease and effects both the upper and lower motor neurons Dr. Jean-Martin Charcot Key Facts ALS has no cure The exact causes of ALS remain unknown ALS results in the death of motor neurons in the brain and spinal cord Although ALS can affect anyone, it is more common in whites, males, and people over 60 years of age

5 Frequency and patterns of ALS EPIDEMIOLGY 1-2/ 100,000 Males > females 2: % sporadic 5-10% inherited Onset >40 years Increase with age AETIOLOGY Unknown Multifactorial Genetic Viral Autoimmune Neurotoxicity hypothesis RISK FACTORS Trauma Long bone fracture Manual work Occupational exposure Solvents Foods

6 ALS is a progressive neurodegenerative disease A progressive spread of symptoms or signs within a region or to other regions Progresses motor function with complete disability within 10 years 20% of patients survive more than 5 years Familial and juvenile onset patients survive years after diagnosis Martin S, Al Khleifat A and Al-Chalabi A. What causes amyotrophic lateral sclerosis? [version 1]. F1000Research 2017, 6:371 (doi: /f1000research )

7 Symptoms of ALS The onset of ALS can be so subtle that the symptoms are overlooked Gradually these symptoms develop into more obvious weakness or atrophy that may cause a physician to suspect ALS Some of the early symptoms: Muscle twitches in the arm, leg, shoulder, or tongue Muscle cramps Tight and stiff muscles (spasticity) Muscle weakness affecting an arm, a leg, neck or diaphragm Slurred and nasal speech Difficulty chewing or swallowing

8 Symptoms of ALS are due to motor neuron damage By the time the first symptoms of ALS are noticeable, as much as 80% of neuron damage has already occurred

9 Damage to motor neurons but not other neuron types occurs in ALS For many people, there are certain functions ALS does not affect Most people maintain: A sharp and alert mind Senses of sight, touch, taste, hearing, and smell Control of eye muscles, bladder, and bowel functions assessingpsyche.wordpress.com

10 Sensory neurons vs. motor neurons The somatic nervous system consists of sensory neurons and motor neurons Sensory neurons are responsible for relaying sensation from the body to the central nervous system Motor neurons are responsible for sending out commands from the CNS to the body, stimulating muscle contraction

11 Sensory neurons vs. motor neurons Sensory neuron: Dendrite: Cell body: Axon: Motor neuron: Motor end plate: Myelin sheath: Receptor (free nerve ending): Nerve cord carrying sensory stimuli to the brain Cytoplasmic extension of a nerve cell Part of the nerve cell between the axon and the dendrite Extension of a neuron Nerve that produces muscular activity Muscular organ that becomes active in response to stimuli Envelope of phosphorylated fats Receiver of nervous stimuli

12 Upper vs. Lower motor neurons Both upper and lower motor neurons are damaged in ALS backyardbrains.com

13 Many factors may contribute to motor neuron damage Motor neurodegeneration eventually causes weakness of all voluntary muscles and progressive paralysis ALS gradually spreads to muscles involved in speaking, swallowing, and breathing. The leading cause of death in ALS is respiratory failure

14 Why are motor neurons susceptible to damage? The precise answer is not known but several possible reasons have been proposed Motor neuron cells may be more sensitive to damage due to: Extreme size of motor neuron processes High metabolic activity Sensitivity to mitochondrial dysfunction Elevated neurofilamentcontent Red Blood Cells Sperm Egg Reduced capacity to buffer calcium Cone Cell Motor neuron

15 Both genetics and environment play a role in the development of ALS The cause of ALS is not known but both heredity and environment contribute ALS? Copyright American Heart Association, Inc.

16 Few environmental factors for ALS are known Possible Environmental Factors Heavy metals (lead, cadmium, etc.) Cyanotoxins Pesticides Head trauma Clusters of ALS can be found in small geographical pockets An Extremely high incidence of ALS has been observed in Chamarro people of Guam Incidence ranging cases / 100,000 (normal rate 1/100,000) Recent theory is that this was due to eating of bats resulting in exposure to to beta-methylamino- L-alanine a potent neurotoxin

17 Two types of ALS: Familial and Sporadic Many factors can contribute to the loss of motor neurons in the brain These factors may increase the chances of someone developing ALS

18 The majority of ALS has no know genetic cause (Sporadic) Approximately 90% cases of ALS are called sporadic, meaning the cause or causes of the disease are unknown Approximately 10% of cases are due to genetic mutations and are inherited from a family member Several gene mutations have been identified both in familial and seemingly sporadic patients Classification of ALS as sporadic and familial is not are clear as it was originally thought It is likely that genetics plays a significant role in sporadic ALS Major genetic causes of familial ALS are mutations in SOD1 and C9orf72 repeat expansions 18

19 Discovery of genes associated with ALS SOD1 mutations are the best studied (20% of cases) and C9orf72 variants are the most common gene (40% of cases) associated with familial ALS 19

20 Genetic factors for ALS: Familial ALS ALS mutations are typically Autosomal Dominant Autosomal means that whatever gene is involved is found on one of the first 22 chromosomes (called the autosomes) and not on the X or Y chromosome (the sex chromosomes)

21 Dominant inheritance in Familial ALS ALS mutations are typically Autosomal Dominant

22 Penetrance of ALS Figure mutations 2 is variable SOD1 mutations are highly penetrant and are strongly associated with familial ALS Hexanucleotide repeat expansions within the C9orf72 gene display reduced penetrance A multi-gene model for ALS has been proposed with coincidence C9orf72 repeats with TARDBP, FUS, and SQSTM1 mutations Low penetrance may also suggest an environmental component for ALS progression in association with some mutations Trends in Genetics , DOI: ( /j.tig )

23 Mechanisms of disease implicated in ALS

24 SOD1 is a major enzyme that protects cells against oxidative stress Cells lacking SOD1 are sensitized to environmental oxidants and mouse models show elevated rates of cancer incidence

25 SOD1 is a metalloenzyme and requires both Copper and Zinc for catalytic activity

26 SOD1 mutations in over 70 of its 153 amino acids have been associated with dominantly inherited ALS Mutant SOD1 acquire one or more toxic properties Mutations Thr54Arg Val87Met Asp90Ala Gly93Ala Gly93Asp Val97Met Ile113Phe Ile113Thr Leu144Phe Ile35Thr Leu67Val Criteria of choice Dimer interface, charge change to positive amino acid Amino acid with a propensity within b strand Amino acid at the protein surface Amino acid with a propensity just outside b strand Charge variation (to negative residue) on buried amino acid Amino acid with a propensity within b strand Dimer interface, still hydrophobic amino acid Dimer interface, change to hydrophilic amino acid Amino acid with decreased a propensity just outside b strand Non-ALS mutation located on the only SOD1 b strand without mutations; mutation from polar hydrophobic amino acid to non-polar hydrophilic amino acid Non-ALS mutation on a pathogenic site located on the zincbinding loop doi: /journal.pone t001

27 Multiple models have been proposed for motor neuron death in ALS from SOD1 mutations Mutations in the SOD1 gene explain about 1 in 5 cases of the inherited form of ALS But what's toxic? Complete absence of SOD1 in mice neither diminishes life span nor promotes ALS disease Expression of SOD1 mutants in mice yields selective killing of motor neurons culminating in fatal paralysis, despite unchanged or elevated SOD1 activity levels

28 Expression of human G93A SOD1 in mice produces ALS symptoms Presymptomatic Disease state evident Animals contain their endogenous copies of mouse SOD1 SOD1 is elevated in these mice due to the over-expression of G93A human SOD1

29 Model involving aberrant oxidative chemistry in SOD1 mutants linked to ALS The oxidative hypothesis ascribes toxicity to binding of SOD1 to aberrant substrates, such as peroxynitrite or hydrogen peroxide, These non native substrates gain access to the catalytic copper through mutantdependent loosening of the SOD1 protein conformation The in vivo evidence for oxidative damage remains mixed and highly controversial

30 Mitochondrial dysfunction and oxidative stress (OS) are tightly dependent on each other and are the basis of the redox dysregulation in ALS Increased production of ROS ER stress transcriptional dysregulation and abnormal RNA processing are all consequences of mitochondrial dysfunction and OS contributing to death of motor neurons

31 Model involving aggregation of mutant SOD1 Toxicity may occur by a loss of essential components through coprecipitation with mutant SOD1 aggregates Failure to correctly fold essential protein components due to reduction in molecular chaperone activity from chronic refolding of the abundant mutant SOD1 Inhibition of proteasome degradation by misfolded SOD1 mutants

32 The ALS associated A4V SOD1 has a change in structure at the dimer interface The A4V SOD1 promotes monomer formation instead of dimer seen in WT SOD by National Academy of Sciences Soumya S. Ray, and Peter T. Lansbury, Jr. PNAS 2004;101:16:

33 SOD1 dimer dissociation in A4V mutants may be the first step in ALS pathogenesis 2004 by National Academy of Sciences Soumya S. Ray, and Peter T. Lansbury, Jr. PNAS 2004;101:16:

34 SOD1 mutants that disrupt Cu or Zn binding also promote the formation of aggregates Figure 6. Mutants SOD1 aggregation. Formation of soluble oligomers occuring when apo WT SOD1 protein is kept close to physiological conditions for an extended period of time. In the absence of metal ions, SOD1 proteins form abnormal disulfide cross-links though the two free cysteines (Cys 6 and Cys 111) and noncovalent associations with other SOD1 monomers or dimers. doi: /journal.pone g006 34

35 Aggregation of mutant SOD1 has similarity with Prion disease The prion-like, templated conversion of a natively folded protein into a misfolded version of itself is now recognized as a prominent feature of the cell-to-cell spread of protein aggregates in neurodegenerative diseases Examples include α-synuclein templating in Parkinson's disease, amyloid-β aggregation in Alzheimer's disease and tau misfolding in chronic brain injury Evidence for similar templated toxicity has emerged for misfolded SOD1 with wildtype SOD1 exacerbating the toxicity of mutant SOD1 in mice Prion-like propagation and development of disease that is initiated focally has been shown to occur after the injection of lysates containing mutant SOD1 into mice that express mutant SOD1

36 Model for motor neuron death from mutant SOD1 Mutant SOD1 aggregates Saturate molecular chaperones leading to mis-folding of other essential proteins Limit degradation of aberrant proteins by the proteasome Block axonal transport of secretory vesicles

37 Penetrance of ALS Figure mutations 2 is variable C9orf72 mutations are not highly penetrant but strongly associated with Familial ALS Hexanucleotide repeat expansions within the C9orf72 gene display reduced penetrance A multi-gene model for ALS has been proposed with coincidence C9orf72 repeats with TARDBP, FUS, and SQSTM1 mutations Low penetrance may also suggest an environmental component for ALS progression in association with some mutations Trends in Genetics , DOI: ( /j.tig )

38 The C9orf72 gene product is a guanine nucleotide exchange factor (GEF) The C9orf72 gene product has been implicated in intra-cellular membrane trafficking A reduction in C9ORF72 protein levels has been found in ALS patients with the GGGGCC repeat expansion This finding supports the hypothesis that loss-of-protein function from C9orf72 repeat expansion contributes to ALS progression

39 The C9orf72 gene product functions at several steps in membrane trafficking C9ORF72 interacts with several Rabs, and could be contributing to membrane trafficking to the nascent phagophore (with Rab11) and with the fusion of autophagosome with the lysosome (with Rab7)

40 Nucleotide Repeat Expansion as a Pathogenic Mechanism of Neurodegeneration Expansion of non-coding GGGGCC repeats in a gene named Chromosome 9 open reading frame 72 (C9orf72) is associated with 40% of familial ALS The non-coding repeat expansion can reduce protein expression Toxic gain-of-function also appears to contribute to pathogenesis from C9orf72 mutations

41 Possible mechanisms of toxicity from C9orf72 repeat expansion 1. Reduced C9orf72 protein 2. Toxicity from production of C9orf72 repeat-containing RNAs 3. Toxicity from the synthesis of dipeptide repeat proteins These and other factors may work together to determine if, when, and how ALS, appears in people harboring repeat expansions in the C9orf72 gene

42 Toxicity due to down-regulation of C9orf72 gene expression The GGGGCC-repeat region lies between two alternatively spliced noncoding exons of the C9orf72 gene Reduced activity of C9orf72 protein can impair membrane trafficking resulting in fragmentation of the Golgi apparatus Failure of the Golgi to produce transport vesicles prevents axonal secretory trafficking leading to loss of ER and Golgi outposts in the axon Loss of axonal ER and Golgi outposts can lead to cell death in motor neurons Axonal transport of materials is essential for neuronal function and survival

43 C9orf72 RNA Foci and Cellular Protein Sequestration C9orf72 RNA containing GGGGCC repeats has been reported to form RNA foci (DeJesus-Hernandez et al., 2011; An inverse correlation between RNA foci and age-at-onset of ALS has been found. Biochemical and microscopic investigations showed that C9orf72 RNA is capable of recruiting cellular proteins to RNA foci Among these proteins are RNA-binding proteins such as hnrnps and RNA export factors (Sareen et al., 2013; Cooper-Knock et al., 2014) The recruitment of these proteins to C9orf72 RNA foci compromises their cellular functions and may lead to ALS pathologies

44 Bi-Directional Transcription and Repeat-Associated Non- ATG Translation of C9orf72 Although GGGGCC repeat expansion is located in non-coding exon of C9orf72 the GGGGCC repeats are transcribed bi-directionally Both the sense and anti-sense GGGGCC C9orf72 transcripts have been reported to generate five different dipeptide repeat (DPR)-containing proteins composed of GA, GP, GR, PR and AP amino acid repeats

45 Dipeptide repeat (DPR)-containing proteins form aggregates that may produce cellular toxicity DPR protein aggregates in ALS patients and co-localize with other proteins The neurotoxicity of DPR proteins is determined by their subcellular localization and by the sequestration of cellular proteins to DPR aggregates Nucleolar localization of DPR aggregates impairs pre-rrna biogenesis and causes cell death Cytosolic DPR aggregates impair the ubiquitin-proteasome system (UPS) and induce ER stress

46 Summary Both Familial and Sporadic forms of ALS occur Several genes have been found to be associated with Familial forms SOD1, C9orf72, etc. Gain of function mutations (Dominant) are common in Familial ALS Motor neuron damage is a primary cause of ALS Protein aggregation is a major event in pathogenesis of ALS