Flashcards in ALS Deck (58):
is ALS a pure motor neuron disease?
why is ALS not considered as pure MN disease?
1. 15% ALS patients - FTD
2. 50% - subtle cognitive + behavioural dysfunction: emotional lability
3. mutated genes - like TDP43 and C9, found in both ALS and FTD
therefore, it is a spectrum with different range on scale with ALS and FTLD on the extreme ends and cases with both falling in between the range
Name four prominent genes on ALS?
what type of mutations are usually seen in ALS ?
In ALS, 90% of cases are sporadic and 10% are familial?
what type of mutation takes place in
1. SOD1 - dominant, missense (point mutation)
2. TDP43 - point mutation
3. C9 - repeat mutation
what is the role of SOD1?
- clears/removes all free radicals from the inter membrane space of mitochondria
- acts like a free radical scavenger
why are free radicals harmful for the cell?
free radicals can cause
1. DNA damage
2. Protein alterations
what happens to the mutant SOD1 in cytoplasm?
- SOD1 is a sticky protein
1. mutant SOD1 forms aggregates
2a. aggregates + normal protein = increased ER stress -> shut down of protein synthesis until the problem is fixed or if not fixed -> cell death
2b. the aggregates also interfere with normal protein (chaperones) folding resulting in decreased chaperone activity
3. due to this even WT SOD1 starts misfolding
what happens to the mutant SOD1 in mitochondria?
1. mutant SOD1 aggregate -> increased toxicity -> toxic interaction of mutant SOD1 with BCL2 resulting in
2. increase BCL2 levels
2a. interaction of Bcl2 and PTP = altered mitochondrial permeability (PTP) -> chemicals (CytC) leak out of the cell
2b. decreased protein input through TOM (translocase of outer membrane)
3. activation of apoptic pathway + cytochrome C -> cell death
which are the proteins SOD1 aggregates interact with?
1. chaperone proteins (cytoplasm)
2. Bcl2 (mitochondria)
what are the defects due to mutant SOD1?
explain the axonal defect due to SOD1 mutant?
Due to mtSOD1 NF form aggregate inclusions which impairs the axon transport
- It may also lead to neurite collapse
what is HO, CHAT and NF-H?
HO - nuclear stain
CHAT - motor neuron stain
NF-H - neurofilament heavy chain
these are these are markers used in immunohistochemistry to study the axonal defect due to mtSOD1 in mtSOD1 MN and healthy MN
what is the role of TDP43?
1. normal pre-mRNA splicing
2. microRNA processing and transport
where is SOD1 found?
inter membrane space of mitochondria
where is TDP43 found?
in the nucleus but also shuttles between nucleus and cytoplasm
what are the downstream effects of mutant TDP43?
1. pre-mRNA slicing - mutated TDP43 impairs the slicing function (unable to remove the introns)
2. microRNA process and transport - impairs the biogenesis of microRNA and found everywhere in MN
3. produces abnormal protein -> causes aggregation of stress granules and cellular contents -> sequestration of essential proteins-> cell death
what is microRNA ?
- very short RNA
- regulate RNA by binding 3'UTR and inhibit translation
what is found in C9 mutation of ALS?
A. Gain of function
B. Loss of function
Gain of function - toxic protein formation
Loss of function - inhibition of translation
what are the downstream effects of mutant C9?
1. decreased C9 translation -> decreased protein
2. Formation of toxic RNA species
3. Formation of toxic protein species
how many repeats of C9 are present in an healthy individual and ALS?
healthy individual - 25
ALS - 100-1000
how does mutant C9 cause nucleocytoplasmic transport defects?
C9 repeat expansion disrupt Ran gradient (GTP in nucleus to GDP in cytoplasm) and cause nucleocytoplasmic transport defects
If >80% of ALS cases are sporadic, why bother studying familial mutations?
1. there are few common genetic mutations in SALS and FALS
1a. phosphorylated TDP43 inclusions
1b. aggregation of cytoplasmic proteins
2. FALS can be easily detected by tasing family history
what are the advantages and disadvantages of ex vivo models (patient sample)?
1. actual disease - closest you can get to the presentation of ALS in humans
1. Post mortem changes
2. Expression affected by freezing conditions
3. detection - early detection of disease not possible
4. Cell type composition - may affect the results (the ratio of neurons differs between healthy and ALS individuals)
5. Genetic variation confounds results
what are the advantages and disadvantages of in vivo models (animal samples) ?
1. cell types - all relevant types are present
2. Possible to investigate adult neurons
1. multiple copies of mutant protein - required to generate a phenotype; copies can get lost over generations
2. Mutant gene mouse model - displays sudden death instead of progressive death (death due to bowel obstruction)
3. Many drugs proven to be effective in in-vivo models (mouse models) but fail in human clinical trials; due to poor study designs or species-specific differences
4. Species specific differences confound results
what are the advantages and disadvantages of in vitro models (cellular samples)?
1. Compatible with large scale screening
2. Model investigates human cells (so no species-specific differences)
3. iPSCs can be formed into any cell
1. Few cell types are included
2. iPSC derived neurons are largely foetal
What is non-cell-autonomous effects?
when a cell displays a phenotype due to the mutation in genome of another cell
Gives few examples of non-cell-autonomous effects
1. astrocytes secrete toxic factors due to mutation in SOD1 and kill the cell, microglia and motor neuron
2. SOD1 synthesis is silenced in microglia, astrocyte or oligodendrocytes
which cells have shown non-cell-autonomous effects to contribute to motor neuron degeneration?
what is the role of microglia and how does it exert its non-cell-autonomous effects in ALS?
1. microglia - the innate immune cells of the nervous system become activated in all instances of ALS
2a. synthesis of mutant SOD1 by microglia
2b. mutant SOD1 may drive microglia to produce high levels of extracellular superoxide
3. C9 inactivation -> abnormal microglia and age related neuroinflammation
what is the role of oligodendrocytes (ODC) and how does it exert its non-cell-autonomous effects in ALS?
1. the cells that myelinate the axons of upper MN and initial axonal segment of lower MN
2. reduced mutant SOD1 in ODC maturation -> delayed disease onset
what is the role of astrocytes and how does it exert its non-cell-autonomous effects in ALS?
1. provides nutrients, buffering ions, recycling of the neurotransmitter glutamate and the final layer of blood brain barrier
2. reduced mutant SOD1 in astrocytes -> delayed disease onset or progression
3. mutated SOD1 impairs astrocytes function of glutamate uptake -> excitotoxicity -> cell death
How does excitotoxicity play a role in motor neuron degeneration?
1. glutamate release at synaptic cleft
2. astrocytes take up these glutamate through GLAST and GLT1
3. due to mutation -> astrocytes convert into reactive astrocytes
4. reactive astrocytes -> block glutamate uptake -> increased levels of Ca2+, ROS, RNS -> excitotoxicity -> cell death
Autophagy is increased in ALS
is the increased autophagy beneficial or deleterious in ALS?
deleterious in ALS as it leads to the motor neuron degeneration
What are the current approved therapies for ALS?
Riluzole and Edaravone - proved by FDA
how does Riluzole work for ALS?
by suppressing excessive motor neuron firing (Every time neuron is fired the vesicles are released and Ach is released at the NMJ and that causes the muscle to contract)
how does Edaravone work for ALS?
by suppressing oxidative stress (induced by mtSOD1 aggregates by interacting with chaperone proteins and TDP43 interacting with stress granules)
what is cell-autonomous effect?
Cell displays a phenotype due to mutation in its own genome e.g. SOD1
what are the defects at NMJ in ALS?
1. reduction in pre and post-synaptic mitochondria compartments
2. reduction of ChAT (MN marker protein) and synaptic vesicles
3. Reduction in Acetylcholinesterase (AchE, breaks down Ach therefore, its reduction means more Ach resulting in excessive firing of MN)
what is the difference b/w dying forward Vs dying back theory of ALS?
1. Dying forward - motor cortex
2. Dying back - NMDA receptor
1. Dying forward - anterograde degeneration via glutamate excitoxicity
2. Dying back - retrograde transport due to deficiency of neurotrophic factors
what are the differences between (a) animal models and (b) iPSC?
1. (a) cells present in vivo conditions Vs (b) cells grown in 2D
2. (a) all relevant cells types present Vs (b) few cell types included
3. (a) adult neurone investigation possible Vs (b) iPSC largely foetal
4. (a) species-specific differences confound results Vs (b) model investigates human cells
5. (a) over expression of mutant protein Vs (b) physiological level expression of mutant protein
6. (a) not compatible for large scale screening Vs (b) compatible for large scale screening
what are the early clinical symptoms of ALS?
1. weakness in legs and hands
2. difficulties with fine movements
what are the late clinical symptoms of ALS?
2. swallowing problems
3. sensory symptoms ( may be due to lack of nutrition)
4. eye movements preserved (ocular MN intact)
5. autonomous functions maintained (HR, BR, urination etc)
what is final stage of ALS post the early and late clinical symptoms?
intercostal regions blocked -> hypoxic -> respiratory failure
what does the failure of upper and lower MN result in?
upper MN - muscle stiffness and spasticity
lower MN - spontaneous muscle twitching (fasciculations)
what are the four types of mutation and its consequences?
1. missense - incorrect AA may produce malfunctioning protein
2. non sense - incorrect sequence causes shortening of protein
3. insertion - incorrect AA may produce malfunctioning protein
4. repeat expansion - repeated trinucleotides adds a string of glutamines (gin) to the protein
what is the meaning of amyotrophic lateral sclerosis?
a-myo-trophic - no muscle nourishment
lateral sclerosis - scarring in the lateral column of spinal cord due to neurodegeneration
what is the role of NMJ in ALS?
Every time neuron is fired the vesicles (filled with Ach/Ca2+?) are released and Ach is released at the NMJ and that causes the muscle to contract
what causes contraction?
- signal from somatic MN to limbic system
- release of Ach at NMJ
where is upper and lower MN found?
upper MN - motor cortex
lower MN - brain stem + spinal cord
what are the other names of ALS?
1. Charcot disease
2. Lou-Gehrig's disease
what are the types of motor neuron disease?
there are 3 types:
1. Spinal and Bulbar Muscular Atrophy (SBMA)
2. Hereditary Spastic Paraplegia (HSP)
3. Spinal Muscular Atrophy (SMA)
what is the difference between autosomal dominant vs recessive?
autosomal dominant - one mutant allele is enough to cause the disease
autosomal recessive - both mutant alleles are needed to cause the disease
why there is decreased glutamate uptake in ALS syanptosome?
Decreased uptake of glutamate is due to the non cell autonomous activity of astrocyte as it fails to take up glutamate since it is inhibited via GLT and GLAST
how is glutamate measured in ALS synaptosome?
1. spinal cord tissue extracted -> homogenisation of synaptosome
2. syanptosome + radioactive labelled -> measurement of gluatamate inside the synaptosome