lec 11 Flashcards
What is ALS?
ALS - amyotrophic lateral sclerosis
Loss of stimulation leads to loss of muscle but only voluntary muscles.
What are the cells involved in ALS?
Schwann cells produce the myelin sheath
Astrocytes also known as glial cells support several motor neurons by protecting the synapse sites and produces glutamate, activating motor neurons.
Causes of ALS?
10% familial - mutations in C9orf72 (33%) (open reading mutation therefore mutation is not in the gene therefore the function is still unknown).
mutations in the superoxide dismutase 1 (SOD1) (20%) .
Some mutations in TDP-43, FUS and VCP.
90% sporadic and idiopathic
What was the old method of studying ALS?
previously before IPSCs motor neurons would be studied by taking the spinal cord post mortem, however, there would be no motor neurons left to study as the disease depletes them.
-Mouse models only cover the known mutation (familial) so… can’t cover the 90% of the sporadic types
What strategies can we use to treat ALS?
- Replace
Although motor neurons are the cells that are being destroyed, is it due to the effects of the glial cells not being able to do their job?
So do we replace neurons and glial cells?- Repair
Repair the gene mutation, but we need to know where the mutation is so only applies to the 10% - Regenerate
Not the easiest but potentially the most useful, can regenerate motor neurons without knowing the mutations.
- Repair
How do the mutations affect ALS patients?
C9orf72 mutation
- Forms RNA foci which are toxic by products, the function of C9orf72 is still unknown
SOD1 mutations
- The SOD1 forms aggregates which may sequester other protein components required for neuronal function
TDP-43 mutations
- TDP-43 aggregates form in motor neurons but not all
- It is thought that they cause cells to be more sensitive to cell stress
Mutations in astrocytes (glial cells)
- They cannot maintain the motor neurons in culture
- It is thought that they release toxic levels of glutamate. As they usually function as a controller for glutamate levels
How have IPSCs changed the field of ALS?
We can reprogramme cells from the patient into IPSCs therefore getting a disease specific IPSC (we use skin fibroblasts)
Reprogramming takes a month so for ALS patients that’s a long time.
Now trying to make it quicker by using mRNA and micro RNAs instead of viral vectors
We can differentiate the IPSC into the motor neurons
This will help develop cell therapy, human pathology models and drug discovery.
How do we form motor neurons from IPSCs
Push to an ectodermal fate first using: BMP, Wnt and FGF
Then use retinoic acid and sonic hedgehog to differentiate in to neural tube and then motor neurons
For astrocytes we use EGF and FGF after ectodermal fate has been set.
What do we see in a TDP-43 mutated astrocyte?
When we derive an astrocyte from a TDP-43 mutated ALS patient we can see that ALS astrocytes express significantly more TDP-43 in the cytoplasm whilst it is the same in the nucleus. This makes it toxic to itself and increasing risk of astrocyte death. Which would lead to subsequent motor neuron death due to inability of glutamate regulation.
What do we see in a TDP-43 mutated motor neuron?
Motor neurons from TDP 43 iPSCs show a short axon and are sensitive to oxidative stress, causing them to die more quickly.
Which drug was seen to protect TDP-43 mutated neurons?
4 drugs were tested to see if it could prevent motor neuron death due to oxidative stress. Anacardic acid was shown to protect against arsenite induced death but no effect on regeneration
How did we test replacement methods
In a murine model ALS:SOD-1 glial rich progenitors were differentiated from normal IPSCs and injected into spinal cord of mice, upon injection they differentiated into astrocytes.
There was no reversal of the disease or regeneration, but it did prolong life span. NO tumour formation.
So what is the future?
Take cells from patient Reprogramme in a short amount of time Can correct mutations Find the right drug for the patient Neural transplants
What is one of the big problems with ALS?
One mutation can also result in many phenotypes meaning there is an environmental factor
