Cortical malformations

Question 1

Two main types fo cortical malformations

Answer 1

1. abnormal neuronal and glial proliferation or differentiation
2. abnormal cortical organisation

Question 1

Abnormal types of neuronal and glial ploferiations (6)

Answer 1

1. Tuberous sclerosis
2. Focal cortical dysplasia
3. Hemimegalencephaly
4. Periventricular nodular heterotopia
5. Lysencephaly
6. Subcortical band heterotopia

Question 2

Abnormal cortical organisation types (2) :

Answer 2

1. polymicrogyria
2. Schizencephaly

Question 3

Types of mTORapthies (3):

Answer 3

1. tuberous sclerosis
2. focal cortical dysplasia
3. hemimegalencephaly

Question 4

What are mTORopathies?

Answer 4

neurological diseases caused by a mutation in the mTOR signalling pathway.

Question 5

Two divisions of mTORopathies:

Answer 5

1. mutations that produce DIFFUSE malformations of cortical development (MCD)
2. mutations that produce FOCAL MCD

Question 6

Pathological effects of mutations in the mTOR pathway (6)

Answer 6

1. Turnover of proteins
2. Lipid and glucose metabolism
3. Cellular growth and proliferation
4. Cytoskeleton organisation
5. Ribosome biogenesis
6. Autophagy.

Question 7

Focal cortical dysplasia

Answer 7

caused by somatic OR mosaic mutations in the neuronal progenitors.

• 5-10% of epilepsy patients have FCD.
• 6.3 years is the mean age of onset.
• Most common structural brain lesion in children with drug-resistant epilepsy
• 38% of FC patients suffer recurring seizures after surgery resection.

Question 8

FCD II

Answer 8

involves the loss of cortical lamination, blurred gray-white matter junction.

Question 9

What pathological alterations does FCD II introduce into the cortical layers (6)

Answer 9

1. Balloon cells – huge cells, highly spherical, no excitability or synaptic function.
2. Activated microglia – indicates inflammation
3. Hypertrophic pyramidal cells – neurofilaments, twice as big as healthy pyramidal cells.
4. Dysmorphic neurons
5. Excessive cellular gliosis – indicates inflammation
6. Hypomyelination of white matter and indistinct grey-white matter boundaries

Question 10

How many types of FCD?

Answer 10

Four

Question 10

Hallmarks of FCD I

Answer 10

Small immature neurons and hypertrophic pyramidal cells.

Heterotopic neurons

Question 10

Hallmarks of FCD III

Answer 10

abnormal cortical laminations.

Cellular and architectural abnormalities also seen present in type I and II.

Question 11

Animal model characterisation of FCD II - 3 steops

Answer 11

1. Face validity
2. Predictive validity
3. Construct validity

Question 12

Four steps of face validity

Answer 12

1. Histological assays
2. Biomolecular assays
3. EEG assays
4. Behavioural assays

Question 13

Cre/lox xystem

Answer 13

Cre/lox system: cre is a protein that is expressed in transgenic mice model or injected.

Main point – cre will recognise the sites and cut them – to cut out the gene and knock down the protein of interest. So, in this case knock down identified inhibitor mTOR – to hyperactive it.

Question 14

Two systems for mouse model development

Answer 14

1. In-utero electroporation
2. cre/lox system

Question 15

RHEB CA ANIMAL MODEL in FCD

Answer 15

• The RhebCA animal model shows a significant reduction of seizure frequency with rapamycin treatment > predictive validity
• The RhebCA animal model is generated by in-utero electroporation for targeting neuronal progenitors > construct validity
• The RhebCA animal model displays the histological and behavioural hallmarks of the disorder > face validity.

Question 16

options for gene therapy for FCD II (2)

Answer 16

1. Restore the physiological activity of the impaired signalling pathway
2. Rescue protein levels impaired in FCD.

Question 17

Pros (2) and cons (2) of restoring the physiological activity of the impaired signalling pathway - FCD-II

Answer 17

+ allows for easier fine-tuning of the therapeutic effect
+ the mutated gene can be specifically targetd

• the impaired pathway needs to be well-known
• the therapy effectiveness depends on the mutation

Question 18

pros (2) and cons (2) of rescuing protein levels impaired in FCD

Answer 18

+ targets the direct cause of the symptoms
+ is effective independently of the causing mutation

• harder to predict the potential secondary effects
• compensatory mechanisms may affect long-term effectivity of the therapy.

Question 19

Kv1.1 and excitability

Answer 19

reduces excitability of neurons – so it is probable that it is contributing to seizure occurrence in FCD.

Question 20

Kv1.1 and mTOR

Answer 20

• In an active synapse – activation of NMDA receptors leads to activation of mTOR pathway > which in turn leads to synthesis of micro-interference RNA-129.
• miRNA-129 – target Kv1.1. mRNA – induces degradation of this mRNA and therefore the preventing the translation fo the Kv1.1 channel.

mTOR activation hinders expression Kv1.1 by degrading its mRNA. So need to design a plasmid that is carrying the KCNA1 gene (that encodes for Kv1.1) that doesn’t have the region where miRNA is binding.

Question 21

Filamin A and gene therapy for FCD II

Answer 21

Filamin A inhibition reduces seizure activity in a mouse model of focal cortical malformations.

Question 22

challenges of upstream targeting the mTOR pathway

Answer 22

- Off target effects due to interconnectivity of the mTOR pathway to other key signalling pathways. - The effectiveness of the therapy depends on the mutation causing the mTORC1 hyperactivity. - The therapy needs to be fine-tuned, as the total inhibition of mTORC1 is detrimental.

Question 23

Challneges of downstream targeting the MTOR pathway

Answer 23

- Difficult to predict the effect of the therapy. - Compensatory signalling mechanism may overwrite your therapy.

Question 24

RAPTOR inhibition - in MTOR pathway

Answer 24

RAPTOR inhibition - upstream in the mTOR pathway. - a gene therapy based on inhibiting the activation of mTORC1 by the reudction of RPTOR levels. reduces seizure frequnecy when injected in the dysplastic focus.

Question 25

4E-BP1 overaction

Answer 25

Downstream targeting of the mTOR pathway. the prevention of the decrease in translation through the hyper inhibition of 4EBP caused by the hyperactivation of mTORC1, leads to restoration of disease hallmarks. therefore a gene therapy increasing 4EBP activation is good. e.g. 4EBP1 over-expression in-utero

Question 26

Four therapeutic strategies for FCD

Answer 26

1. restoring protein levels affected in FCD - EXC gene therapy - PT-125 therapy 2. restoring mTORC1 signalling - RPTOR shRNA therapy - 4EBPca therapy