ALS Flashcards
Describe ALS
Rapidly progressive and fatal neurodegenerative disease of death of MNs
ALS is most common MND
Develops at ages 40-70
What are the 3 classifications of ALS/MND?
Sporadic; 90-95% of cases. Random, no cause
Familial; inherited, 5-10%
Guamanian; high incidence of ALS was observed in guam in 1950s
Describe the onset of ALS
Limbs; weakening of grip, decreased dexterity, foot drop, leg stiffness, tripping
Throat; slurred speech, difficulty chewing or swallowing
Describe the progression of ALS
Limbs; unable to hold objects, write, feed or toilet, unable to walk, stand or turn in bed
Throat; impaired swallow
Breathing; breathless with exertion or lying flatt
Cognition; dementia rare but subtle deficits
What is the average life expectancy of ALS from diagnosis?
Average of 22 months
What is a motor neuron?
Specialised nerve cells in brain and spinal cord which transmit electrical signals to muscle for generation of movement
What are the 2 different types of motor neurons?
UMN; motor cortex and travel down spinal cord to connect with LMNs
LMNs; travel out with spinal cord to connect to muscle
Describe the clinical signs of MND in respect to the location of degeneration
Pre-motor; difficulty planning and initiating movement
UMN; modest weakness, stiffness, spasticity, hyperreflexia, extensor plantar response
LMN; major weakness, muscle wasting, fasciculation
Cognition; subtle deficits in 30% of patients including verbal fluency, executive function
Spares; eye movement, bladder and bowel control
How is MND/ALS diagnosed?
No simple diagnostic test
Diagnosis of exclusion
EMG/ nerve conduction utilised
Average of 12 months after symptom initiation
Describe the pathological changes within neurons seen in MND?
Protein inclusions within motor neurons and surrounding astrocytes
Why do people with MND develop paralysis?
Pyramidal motor neurons in frontal lobe degenerate and die causing severe spasticity and mild weakness of muscle groups
Motor neurons in spinal cord degenerate and die causing wasting and major weakness
Results in complete muscle paralysis; no linkage between motor neurons and muscles
What are 2 key proteins that have been identified as having mutations linked to MND?
TDP-43
FUS
Describe the most common gene defects in MND?
C9orf72 (24% of all gene targets in MND)
SOD1 (20%)
FUS (5%)
TDP43 (5%)
How does gene testing help MND patients?
Why the disease occurred
Excludes the presence of gene mutations - if family history
IVF and gene testing
Why do we focus on familial MND when the population effect is small?
Huge biological impact
Only 1 in 10 people with MND have a FMHx of MND
MND-causing gene mutations can be used to model disease
Allows studying of disease mechanisms and development of novel therapies
In terms of genetic abnormalities; why do the motor neurons degenerate?
TDP-43 accumulates in the cell body of motor neurons in 95% of all genetically linked MND cases
TDP-43 resides in nucleus where is binds to DNA and involved in transcription
In ALS; TDP-43 aberrantly accumulated in the cytoplasm where it forms aggregates
Changes from a nuclear protein to a cytoplasmic protein
What animal model has been utilised to model TDP-43 toxicity in ALS?
Chick spinal neurons
Expression of WT and TDP-43 mutant proteins causes toxicity in chick embryonic motor neurons in vivo
What do neurons that express TDP-43 mutant forms display?
Cytoplasmic aggregates
Reduction in axonal length
Cellular toxicity
Which animal models are used in ALS?
Zebrafish - swimming abnormalities
Drosophila - flying abnormalities
Mouse - abnormal stature, can look at motor evoked potentials within the muscle (reduced in TDP-43 mutant mice)
Describe SOD1 in its pathogenesis of ALS
Protein Cu/Zn superoxide dismutase
Most common is alanine-valanine of SOD1 (A4V)
Aggressive course; survival of 12 months
Mice and rats expressing mutant SOD1 develop MND
Mutant SOD1 is a key component of protein that aggregates
Describe the molecular mechanisms by which SOD-1 results in ALS
Converts superoxide radials to hydrogen peroxide and oxygen
Metalloprotein (protein with metal ion co-factor) and is a key enzyme in anti-oxidation
Where is SOD-1 found in the neuron?
Cell cytosol, nucleus and mitochondrial membrane
What metal ions are assoc with SOD-1?
Copper
Zinc
How many mutations in SOD1 have been identified in ALS?
More than 140
What is the role of mutated SOD1 in ALS?
Early studies of mutant SOD1 indicated that the disease process is NOT due to enzymatic loss
-/- SOD1 mice = no ALS
THEREFORE
Mutations to SOD1 that result in ALS must gain a toxic property
What are the potential cellular mechanisms underlying ALS (using SOD1 models)
Oxidative damage Accumulation of intracellular aggregates Mitochondrial dysfunction Glutamate excitotoxicity Growth factor deficiency Glial cell pathology Defective axonal transport Ca2+ dysregulation
What is the role of oxidative damage in ALS?
Mutations in SOD1 = structural changes to expose the Cu site on SOD1
SOD1 mutations catalyse Cu-mediated breakdown of hydrogen peroxide to hydroxyl radicals = oxidative damage
Peroxynitrite can become a substrate = nitration of tyrosine residues in target proteins of cells
Hydrogen peroxide and peroxynitrite mediated oxidative damage both involved Cu
Cu mediated oxidative damage may play a role in ALS pathology
Describe how SOD1 results in abnormal aggregation of proteins?
Intracellular cytoplasmic inclusions are evident in MNs and glial cells in mouse ALS models and human ALS
Several studies have shown that protein aggregates contain misfolded SOD1
Mutated SOD1 has an increased propensity for aggregation
Is there a role for mitochondrial dysfunction in ALS pathogenesis?
Histological studies = swelling of mitochondria in ALS (human and mouse)
Sporadic ALS = impairment in mitochondrial electron transport chain function
Mutant SOD1 directly disrupts mitochondrial function
Aggregates of mutant SOD1 accumulate within mitochondria
Mutant SOD1 disrupts cytochrome C - interferes with inner mitochondrial membrane functionality and respiration
Disturbed mitochondrial function = ROS production
Is there a role for excitotoxicity in ALS?
In MNs; glutamate is cleared from synapses by glial transporters; EAAT2 (GLT1)
In sporadic ALS; glutamate levels are elevated in the CSF suggesting abnormal glutamate handling
In ALS; glutamate transport is markedly reduced in affected brain regions, due to pronounced loss of EAAT2
Lowering of EAAT2 levels with antisense oligonucleotides (i.e. loss of transport activity) induces motor neuron death
Mutant SOD1 transgenic strains, EAAT2 protein levels are markedly reduced
Mutant SOD1 selectively inactivates the glial glutamate transporter EAAT2
Describe the role of deficient growth factors in ALS
Targeted deletion of VEGF causes motor deficits and ALS pathology = suggest putative vole of VEGF in ALS
Onset and progression of MN disease is delayed in mutant SOD1 after; overexpression of VEGF, ICV administration of VEGF, IM delivery of VEGF expressing lentiviral vectors
IGF-1 and GDNF also delay disease onset and progression in mutant SOD-1 mice
What is the role of glial cells in ALS?
Astrocytic inclusions are an indicator of SOD1 mutant toxicity
EAAT2 receptors on glial cells reuptake glutamate (lack thereof excitotoxicity )
Astrocytes expressing mutant SOD1 secrete toxic factors such as TNF-alpha
Describe the role of defective axonal transport in ALS
Neurofilaments are the most common cytoskeletal protein in MNs that play a key role in axonal growth
Abnormal accumulation of neurofilaments in the soma and axons of MNs = hallmark of ALS
Transgenic mice with mutations or overexpression of neurofilaments display MN dysfunction
Mutant SOD1 mice have defective axonal transport mechanisms
Why is there selective degeneration of MNs in ALS?
Only MNs degenerate in familial ALS despite expression of mutant SOD1 in every cell
Theories;
MNs are more susceptible to excitotoxicity
Spinal MNs;
Strong glutamergic input
Express Ca2+ permeable AMPARs (GluA2 lacking)
Low Ca2+ buffering capacity
Is calcium dysregulated in ALS?
MNs have a much lower calcium buffing capacity and high AMPAR calcium permeable receptors make MNs very vulnerable to excitotoxicity
Glutamate will stimulate calcium permeable AMPARs increasing intracellular calcium, which is in part taken up by the mitochondria
Astrocytic glutamate transporters remove excess glutamate from synapse. Factors release from astrocytes increase GluR2 expression in nearby MNs
Presence of mutant SOD1 within neurons and astrocytes interferes with the MN synapse
Balance shifted from normal glutamergic transmission to MN death
Does GluR2 play a role in calcium permeability of AMPARS
Link this with ALS
Yes; in most cells AMPARs are tetramers containing at least one GluR2 subunit and have a low Ca2+ permeability
But, GluR2 mRNA is much lower in MNs than other neurons suggesting GluR2 is regulated at a transcriptional level
The lack of GluR2 accelerates MN degeneration and shortens the life span in mutant SOD1 mice
Replacement of GluR2 in mutant SOD1 will increase the life span
What are 2 key features of ALS pathogenesis?
SOD1 mutation
Loss of glial glutamate transporter
This suggests an excess in extracellular glutamate triggers harmful calcium influx = neurotoxicity
What were some of the first therapies tested in rodent ALS models?
Agents that block glutamate excitotoxicity
E.g. antioxidant agents or antagonists for glutamate receptors
What is the only approved therapy for ALS (on NHS)?
Riluzole
What effect does Riluzole have on the life expectancy in ALS?
Extends survival by 2-3 months
Does not confer any lasting protection
What are the acute effects of riluzole?
Decreasing persistent voltage gated sodium currents
Potentiation of a calcium dependent potassium current
Inhibition of glutamate release
DECREASE MN excitability - limits excitotoxicity
Describe a novel therapy that targets glutamate transport in the tx of ALS?
Ceftriaxone (antibiotic) - increases glial mediated glutamate transport by stimulating expression of EAAT2
In animal models; it prolongs survival and upregulates EAAT2 mRNA
Describe a novel therapy that targets protein misfolding and aggregation as a tx for ALS
Heat shock proteins (HSPs) are involved in protein folding and degradation
Abnormalities in HSPs promote MN degeneration in ALS
Arimoclomol is an oral agent that increases expression of HSPs involved in neuroprotective mechanisms
Arimoclomol delays disease progression and extends life span in SOD1 transgenic ALS models
Recent studies indicate that the drug is safe for use in humans and penetrates CSF
Describe novel treatment targeting mitochondria in tx for ALS
Agents that improve mitochondrial function (creatine) has beneficial effects in SOD1 transgenic mice (disappointing in clinical trials)
Olesoxime; mitochondrial pore modulator; delays disease onset and extends survival in SOD1 mice
Dexpramipexole; lowers oxidative stress and maintains mitochondrial function; extends survival of SOD1 transgenic mice
Describe novel treatment using stem cells in ALS
Harvested from bone marrow or umbilical cord blood can be implanted into patient to replace damaged cells and stimulate site of trauma
In ALS, cells adjacent to MNs become damaged resulting in widespread cells death and a rapid decrease in normal functioning
Therefore, stem cells would need to be directed to the damaged area and provide the necessary growth factors to surrounding cells
What stem cells can be used to treat ALS?
Mouse embryonic stem cells treated with chemical cocktail (retinoic acid and sonic hedgehog)
2 weeks; take shape of MNs and become dependent on same growth factors as normal neurons
Transplanted into chick embryo spinal cord = migrate to anterior horn
Transplanted MNs axons make functional contacts with muscle cells
Embryonic stem cells generate motor neurons that have the ability to communicate with muscle fibres
What must stem cells be able to do to be effective in tx of ALS?
Must differentiate into MNs and replace dead MNs
Make connections to denervated muscle over 3 meters. This connection is vital to regeneration of activity
Avoid immuno-rejection
How is the usefulness of stem cells in ALS limited?
Rapid disease progression
Length of axon growth required to restore functionality
What have been some recent advances in stem cell use within ALS?
Use of neural progenitor cells which are modified to deliver specific genes to regenerate damaged MNs (e.g. glial cell line derived neurotrophic factor (GDNF))
Recent studies: cells that thrived in rat models involved 2 points of injection (i.e. MNs and affected muscle)
These rats displayed a slower disease progression and increased survival rate (no cure)
These results were due to increased production of nerve projections from MNs in the spinal cord to the muscles
Will stem cell therapy in ALS be curative?
Unlikely; more of a protective manner to MNs
Stem cells can differentiate into non-neuronal cells to release growth factors that reduce toxic damage
In transgenic ALS models, stem cell transplantation has powerful neuroprotective effects
Describe ACE-031 as a novel muscle targets in tx of ALS
Resp failure main cause of mortality in ALS == therefore improving diaphragm function can improve QOL and survival
Agents that strengthen the diaphragm have clinical benefit
ACE-031; protein that inhibits negative regulators (myostatin) of muscle growth
Animal models; ACE-031 promotes lean muscle growth and strength
S/c tx with ACE-031 = increases muscle mass and volume
Describe CK-2017357
Activates fast skeletal muscle troponin complex by increasing sensitivity to calcium
Increases muscle force
Well tolerated; improves pulmonary function and muscle strength compared to placebo
Describe novel targets in terms of RNA targets in ALS tx
Advances in gene therapy has provided new targets for familial forms of ALS
Use of antisense oligonucleotides and small inhibitory RNAs to lower mutant mRNA (e.g. mutant SOD1)
Will not help pts with sporadic ALS or familial forms of unknown genes
Describe the use of RNA targets towards mutant SOD1
ISIS 333611; antisense oligonucleotide intrathecally targets mutant SOD1
Antisense technique will silence SOD1 gene expression, thus reducing the SOD1 enzyme’s toxic gain of function
Currently in phase 1 trial
Can VEGF be used to treat ALS?
Mutations in VEGF lead to; 2x greater risk of developing ALS
Baseline VEGF levels in human csf are decreased in ALS
Potential of VEGF as a therapeutic target
What is the role of VEGF HuR in health and in ALS?
Health: mRNA stabiliser HuR binds to VEGF mRNA resulting in sufficient VEGF protein for neuroprotection and oxygen supply to MNs
ALS: mutant SOD1 competes with HuR for binding, compromising neuroprotection and perfusion = MN death
Describe the role of recombinant VEGF in ALS
Delivered intracerebroventricularly to prevent degeneration of MNs by restoring neurotrophic support and oxygen supply
