ALS

Question 1

Describe ALS

Answer 1

Rapidly progressive and fatal neurodegenerative disease of death of MNs
ALS is most common MND
Develops at ages 40-70

Question 2

What are the 3 classifications of ALS/MND?

Answer 2

Sporadic; 90-95% of cases. Random, no cause

Familial; inherited, 5-10%

Guamanian; high incidence of ALS was observed in guam in 1950s

Question 3

Describe the onset of ALS

Answer 3

Limbs; weakening of grip, decreased dexterity, foot drop, leg stiffness, tripping
Throat; slurred speech, difficulty chewing or swallowing

Question 4

Describe the progression of ALS

Answer 4

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

Question 5

What is the average life expectancy of ALS from diagnosis?

Answer 5

Average of 22 months

Question 6

What is a motor neuron?

Answer 6

Specialised nerve cells in brain and spinal cord which transmit electrical signals to muscle for generation of movement

Question 7

What are the 2 different types of motor neurons?

Answer 7

UMN; motor cortex and travel down spinal cord to connect with LMNs

LMNs; travel out with spinal cord to connect to muscle

Question 8

Describe the clinical signs of MND in respect to the location of degeneration

Answer 8

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

Question 9

How is MND/ALS diagnosed?

Answer 9

No simple diagnostic test
Diagnosis of exclusion
EMG/ nerve conduction utilised
Average of 12 months after symptom initiation

Question 10

Describe the pathological changes within neurons seen in MND?

Answer 10

Protein inclusions within motor neurons and surrounding astrocytes

Question 11

Why do people with MND develop paralysis?

Answer 11

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

Question 12

What are 2 key proteins that have been identified as having mutations linked to MND?

Answer 12

TDP-43

FUS

Question 13

Describe the most common gene defects in MND?

Answer 13

C9orf72 (24% of all gene targets in MND)
SOD1 (20%)
FUS (5%)
TDP43 (5%)

Question 14

How does gene testing help MND patients?

Answer 14

Why the disease occurred
Excludes the presence of gene mutations - if family history
IVF and gene testing

Question 15

Why do we focus on familial MND when the population effect is small?

Answer 15

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

Question 16

In terms of genetic abnormalities; why do the motor neurons degenerate?

Answer 16

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

Question 17

What animal model has been utilised to model TDP-43 toxicity in ALS?

Answer 17

Chick spinal neurons

Expression of WT and TDP-43 mutant proteins causes toxicity in chick embryonic motor neurons in vivo

Question 18

What do neurons that express TDP-43 mutant forms display?

Answer 18

Cytoplasmic aggregates
Reduction in axonal length
Cellular toxicity

Question 19

Which animal models are used in ALS?

Answer 19

Zebrafish - swimming abnormalities
Drosophila - flying abnormalities
Mouse - abnormal stature, can look at motor evoked potentials within the muscle (reduced in TDP-43 mutant mice)

Question 20

Describe SOD1 in its pathogenesis of ALS

Answer 20

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

Question 21

Describe the molecular mechanisms by which SOD-1 results in ALS

Answer 21

Converts superoxide radials to hydrogen peroxide and oxygen

Metalloprotein (protein with metal ion co-factor) and is a key enzyme in anti-oxidation

Question 22

Where is SOD-1 found in the neuron?

Answer 22

Cell cytosol, nucleus and mitochondrial membrane

Question 23

What metal ions are assoc with SOD-1?

Answer 23

Copper

Zinc

Question 24

How many mutations in SOD1 have been identified in ALS?

Answer 24

More than 140

Question 25

What is the role of mutated SOD1 in ALS?

Answer 25

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

Question 26

What are the potential cellular mechanisms underlying ALS (using SOD1 models)

Answer 26

``` Oxidative damage Accumulation of intracellular aggregates Mitochondrial dysfunction Glutamate excitotoxicity Growth factor deficiency Glial cell pathology Defective axonal transport Ca2+ dysregulation ```

Question 27

What is the role of oxidative damage in ALS?

Answer 27

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

Question 28

Describe how SOD1 results in abnormal aggregation of proteins?

Answer 28

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

Question 29

Is there a role for mitochondrial dysfunction in ALS pathogenesis?

Answer 29

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

Question 30

Is there a role for excitotoxicity in ALS?

Answer 30

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

Question 31

Describe the role of deficient growth factors in ALS

Answer 31

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

Question 32

What is the role of glial cells in ALS?

Answer 32

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

Question 33

Describe the role of defective axonal transport in ALS

Answer 33

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

Question 34

Why is there selective degeneration of MNs in ALS?

Answer 34

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

Question 35

Is calcium dysregulated in ALS?

Answer 35

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

Question 36

Does GluR2 play a role in calcium permeability of AMPARS | Link this with ALS

Answer 36

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

Question 37

What are 2 key features of ALS pathogenesis?

Answer 37

SOD1 mutation Loss of glial glutamate transporter This suggests an excess in extracellular glutamate triggers harmful calcium influx = neurotoxicity

Question 38

What were some of the first therapies tested in rodent ALS models?

Answer 38

Agents that block glutamate excitotoxicity | E.g. antioxidant agents or antagonists for glutamate receptors

Question 39

What is the only approved therapy for ALS (on NHS)?

Answer 39

Riluzole

Question 40

What effect does Riluzole have on the life expectancy in ALS?

Answer 40

Extends survival by 2-3 months | Does not confer any lasting protection

Question 41

What are the acute effects of riluzole?

Answer 41

Decreasing persistent voltage gated sodium currents Potentiation of a calcium dependent potassium current Inhibition of glutamate release DECREASE MN excitability - limits excitotoxicity

Question 42

Describe a novel therapy that targets glutamate transport in the tx of ALS?

Answer 42

Ceftriaxone (antibiotic) - increases glial mediated glutamate transport by stimulating expression of EAAT2 In animal models; it prolongs survival and upregulates EAAT2 mRNA

Question 43

Describe a novel therapy that targets protein misfolding and aggregation as a tx for ALS

Answer 43

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

Question 44

Describe novel treatment targeting mitochondria in tx for ALS

Answer 44

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

Question 45

Describe novel treatment using stem cells in ALS

Answer 45

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

Question 46

What stem cells can be used to treat ALS?

Answer 46

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

Question 47

What must stem cells be able to do to be effective in tx of ALS?

Answer 47

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

Question 48

How is the usefulness of stem cells in ALS limited?

Answer 48

Rapid disease progression | Length of axon growth required to restore functionality

Question 49

What have been some recent advances in stem cell use within ALS?

Answer 49

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

Question 50

Will stem cell therapy in ALS be curative?

Answer 50

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

Question 51

Describe ACE-031 as a novel muscle targets in tx of ALS

Answer 51

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

Question 52

Describe CK-2017357

Answer 52

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

Question 53

Describe novel targets in terms of RNA targets in ALS tx

Answer 53

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

Question 54

Describe the use of RNA targets towards mutant SOD1

Answer 54

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

Question 55

Can VEGF be used to treat ALS?

Answer 55

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

Question 56

What is the role of VEGF HuR in health and in ALS?

Answer 56

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

Question 57

Describe the role of recombinant VEGF in ALS

Answer 57

Delivered intracerebroventricularly to prevent degeneration of MNs by restoring neurotrophic support and oxygen supply