Niklas Dahl – Heritable epilepsies and early onset cognitive disorders: Identification of neuronal disease mechanisms and new candidate drugs

Decoding of the human genome has led to the identification of disease-causing gene variants in a large number of disorders affecting the central nervous system (CNS). Our focus is to unravel mechanisms that mediate the pathophysiology of heritable (genetic) forms of therapy resistant epilepsies as well as of some early onset forms of cognitive decline.

Our long-term objective is to rescue disease associated biomarkers and to identify candidate lead-compounds as a first step towards drug development.

Our specific aims are to:

Establish and develop neuronal cellular models from induced pluripotent cells (iPSC) of selected forms of therapy-resistent epilepsies and intellectual disabilities.
Analyse these models for disease-specific cellular and molecular mechanisms.
Establish conditions for rescue-screening of neuronal disease-specific biomarkers using small compound libraries (drug screening). I.e. identify lead compounds for pharma development.

A need for understanding disease mechanisms

Genetic epilepsies and early onset cognitive decline comprise heterogeneous groups of conditions for which treatment options are limited. The development of novel treatments and progress in understanding disease mechanisms are lagging. Main reasons are limited access of biological material and the lack of model systems to faithfully recapitulate human neuropathophysiology. The advent of high throughput methods, e.g. next generation sequencing, induced pluripotent stem cell (iPSC) technologies combined with CRISPR/Cas9 genome editing, provide new prerequisites to overcome the limitations.

Modelling nervous system disorders

To model neuronal disease-mechanisms, we use induced pluripotent stem cell (iPSC) cultures differentiated into specific neuronal cell-types in 2D and as well as in 3D (brain organoids; Fig 1). The iPSCs are derived from patients with defined gene-variants (that cause specific forms of therapy resistant epilepsies or early onset intellectual disability). iPSCs are also obtained from healthy individuals that has been gene edited using CRISPR/Cas to introduce desired gene variant and to obtain isogenic iPSC lines.

Microscope image of brain organoid — Fig 1. Brain organoid derived from iPSCs after 3 months culture. Organoids recapitulate formation of brain structures in 3D. Cortical structures (blue) are surrounded by meningeal cells (red). Ventricular zone structures are indicated with arrows.

Novel disease-associated biomarkers

The neuronal cell models are analysed using various techniques such as RNA-sequencing, immune-staining, image analysis, flow-sorting, electrophysiology etc. We have to date identified several novel disease-associated biomarkers in neural iPSC models for epilepsy-resistant Dravet syndrome and Neurochondrin deficiency, as well as for intellectual disability in Down syndrome and Mowat-Wilson syndrome (see reference list).

Drug-screen

We have established a screening pipe-line of our neuronal models based on cell-painting by using a combination of organell-specific stains (Fig). Stained neuronal cells are subsequently analysed by high through-put imaging and artificial intelligence (AI). The platform is up-running and has been adapted for iPSCs in collaboration with Chemical Biological Consortium Sweden (CBCS). Disease associated neuronal abnormalities detected by the platform (upon stain/imaging/AI) are used for rescue screening using available libraries of small chemical compounds.

Our goal is to define lead compounds that can rescue disease associated cellular abnormalities towards development of novel drugs.

Microscope image of interneurons derived from iPSC at day 67 of differentiation in 2D — Fig 2. Interneurons derived from iPSC at day 67 of differentiation in 2D. The cells are stained for image-based screening of biomarkers associated with Dravet disease (CBCS cell-paint platform, Uppsala Univ.). Blue: Nucleus; Red: Mitochondria; Green: Plasma membrane and Golgi.