Cortical malformations

Cortical Malformations

  • Can be classified based on:
    • Abnormal neuronal and glial differentiation/proliferation.
    • Abnormal cortical organization.
  • Examples:
    • Tuberous Sclerosis.
    • Focal Cortical Dysplasia (FCD).
    • Hemimegaloencephaly.
    • Lysencephaly.
    • Periventricular nodular heterotopia.
    • Subcortical band heterotopia.
    • Polimicrogyria.
    • Schizencephaly.

mTORopathies Classification

  • A group of cortical malformations caused by hyperactivation of the mTOR pathway.
  • Examples:
    • Tuberous Sclerosis.
    • Focal Cortical Dysplasia.
    • Hemimegaloencephaly.

mTOR Pathway

  • mTOR stands for mammalian Target Of Rapamycin.
  • Involved in:
    • Protein turnover.
    • Lipid and glucose metabolism.
    • Cellular growth and proliferation.
    • Cytoskeleton organization.
    • Ribosome biogenesis.
    • Autophagy.
  • Dysregulation can lead to severe disorders.

Focal Cortical Dysplasia (FCD)

  • An mTORopathy caused by somatic or mosaic mutations in neuronal progenitors.
  • Types: FCD I, FCD II, FCD III, and white matter mMCD with oligodendroglial hyperplasia in epilepsy (MOGHE).
  • FCD II is the most common structural brain lesion in children with drug-resistant epilepsy.
  • 5-10% of epilepsy patients have FCD.
  • Mean age of onset is 6.3 years.
  • 38% of FCD patients suffer recurring seizures after surgery resection.

FCD Classification

  • FCD I:
    • Ia: Abnormal cortical lamination.
    • Ib: Abnormal radial cortical lamination.
    • Ic: White matter mMCD with oligodendroglial hyperplasia in epilepsy (MOGHE).
  • FCD II:
    • IIa: Dysmorphic neurons. Loss of cortical lamination, blurred gray-white matter junction
    • IIb: Balloon cells and dysmorphic neurons. Loss of cortical lamination, blurred gray-white matter junction
  • FCD III:
    • IIIa: Combined features of FCD Ia and IIb with Acquired lesion (Vascular malformation or glial scars).
    • IIIb: Cellular and architectural abnormalities also present in type I & II with Glial/ neuroglial tumour.
    • IIIc: MTLE. Cellular and architectural abnormalities also present in type I & II
    • IIId: Small immature neurons & hypertrophic pyramidal cells

FCD Type II Characteristics

  • Astrocytes and Activated microglia.
  • Hypertrophic pyramidal neurons.
  • Dysmorphic neurons.
  • Excessive cellular gliosis.
  • Hypomyelination of white matter and indistinct grey-white matter boundaries.
  • Balloon cells (Nestin +).
  • Neurofilament+ dysmorphic neurons.

FCD II Pathology Mechanism

  • Disease-causing mutations lead to unknown pathology mechanisms.
  • This leads to pathology hallmarks, seizures, and drug-resistant epilepsy/cognitive impairments.

FCD II Animal Models

How to Model FCD II in Rodents

  • Induce a somatic mutation in a specific cell cluster at a specific time-point using:
    • In-utero electroporation. For targeting neuronal progenitors.
    • Cre/lox system.

Examples of FCDII Animal Models

  • RhebCA:
    • Constitutively activated form by in-utero electroporation.
    • 93.3% of mice show spontaneous seizures.
    • Displays dysmorphic & hypertrophic neurons and cortical dyslamination.
  • mTOR:
    • Constitutively activated form by in-utero electroporation.
  • TSC1 & TSC2:
    • Inhibition by CRISPR-Cas9 via in-utero electroporation.
  • DEPCD5 cKO:
    • Cre/lox system under Synapsin promoter.
  • PIK3CA:
    • Cre/lox system under Emx promoter.

RhebCA Mouse Model

  • Generated by in-utero electroporation of a constitutively active form of Rheb at E14.5-15.5.
  • Validation:
    • Histological phenotype: Cortical dyslamination, disorganized neuronal placement, hypertrophic neurons, heterotopic neurons in the corpus callosum, mTORC1 hyperactivation (pS6 staining).
    • Behavioral phenotype: Generalized spontaneous seizures, cognitive impairments (impaired working memory and social odour recognition). Along a 15 day continuous recording the number of seizures/day is of 1.4 on average and highly variable among mice.
    • Response to treatment: Reduced seizure frequency and growth with rapamycin (mTORC1 inhibitor).

FCD II animal model characterization

  • Face validity: Histology assays - Macroscopic and microscopic hallmarks

  • Predictive validity: Behavioural assays and EEG recordings after substance administration i.e. rapamycin

  • Construct validity: List of reported causal mutations in patients. Literature search. Mouse model generation. Mutation induction at a specific timepoint and location

  • Biomolecular assays: Gene and protein quantification

  • EEG assays: Seizure frequency and inter-ictal spike analysis

  • Behavioural assays: Cognitive and behavioural assessment

Remarks on Animal Models

  • The development of a translatable therapy needs an animal model with face, predictive, and construct validity.
  • For modeling FCD II, mutation generation needs to target cortical neuronal progenitors.

Designing a Gene Therapy for FCD II

  • Main challenge: the lack of connection between the brain insult and the causality of seizures.
  • Different target genes can be chosen.
  • The therapy can:
    • Restore the physiological activity of the impaired signaling pathway.
    • Rescue protein levels impaired in FCD.

Restore Physiological Activity of the Impaired Signalling Pathway

  • Pros:
    • The impaired pathway needs to be well-known.
    • Allows for easier fine-tuning of the therapeutic effect.
  • Cons:
    • The therapy effectiveness depends on the mutation.
    • The mutated gene can be specifically targeted.

Rescue Protein Levels Impaired in FCD

  • Pros:
    • Targets the direct cause of the symptoms.
    • Is effective independently of the causing mutation.
  • Cons:
    • Harder to predict the potential secondary effects.
    • Compensatory mechanisms might affect the long-term effectiveness of the therapy.

Examples of Therapeutic Strategies

Targeting altered protein expression:

  1. Rescuing levels the Kv1.1 reduces seizure frequency in FCD II
  2. Targeting FLNA reduces seizure frequency in FCD II

Targeting the altered signalling pathway:

  1. RAPTOR expression inhibition
  2. 4EBP1 overexpression in-utero

Targeting mTORC1 Pathway

*   Challenges:
    *   Off-target effects due to interconnectivity with other key signalling pathways.
    *   Effectiveness depends on the mutation causing mTORC1 hyperactivity.
    *   The therapy needs fine-tuning, as total inhibition is detrimental.
*   Downstream:
    *   Inhibit mTORC1 activity.
  • Upstream:
    • Difficult to predict therapy effect.
    • Compensatory signalling mechanisms might overwrite the therapy.

Kv1.1 & mTOR

  • Kv1.1 expression is reduced in animals electroporated with RhebCA.
  • In an inactive synapse with low mTORC1 activation, Kv1.1 is translated with the initiation of the HuD protein.
  • In an active synapse with mTORC1 activation, Kv1.1 translation is hindered by the transcription of the miRNA 129, which interferes with the HuD binding to the Kv1.1 miRNA, inhibiting its translation

Kv1. 1 overexpression:
A gene therapy based on the overexpression of Kv1.1 reduces the excitability of the neurons in the epileptic focus and potentially rescues the depleted Kv1.1 levels caused by mTORC1 hyperactivation

Filamin A inhibition: PTI-125 binds to FILNA and changes its conformation
A gene therapy based on restoring the increased levels of Filamin A due to mTOR hyperactivation, would decrease seizure frequency. In this article, they used PTI-125 a compound that binds to Filamin A, challenging its signalling action by changing its comformation.

  • RPTOR inhibition (upstream):
    • A gene therapy based on inhibiting the activation of mTORC1 by the reduction of RPTOR levels reduces seizure frequency when injected in the dysplastic focus.
  • 4E-BP1 overactivation (downstream):
    • The prevention of the decrease in translation through the hyper inhibition of 4EBP caused by the hyperactivation of mTORC1, leads to the restoration of several disease hallmarks as cortical dyslamination, increased dendritic arborisation and reduced spine count. Therefore, a gene therapy that increases 4EBP activation is a good disease modifying candidate.

Summary of therapeutic strategies:

  1. Restoring protein levels affected in FCD: EKC gene therapy, PTI-125 therapy
  2. Restoring mTORC1 signalling: RPTOR shRNA therapy, 4EBPCA therapy