Prof. Dr. Gaia Novarino

Project I - SLc7a5 in microcephaly (with Ecker and Mihovilovic)

Background: Disorders of brain overgrowth are a significant cause of epilepsy, intellectual disability and autism. In the last few years we have studied mutations in the gene SLC7A5 as a cause of a form of autism spectrum disorder. In addition to autism core symptoms, children carrying mutations in SLC7A5 present with a brain size below average (i.e. microcephaly). Similarly, mice lacking expression of Slc7a5 in specific cell types in the brain show reduced cortical thickness and neuronal cell number. Slc7a5-associated microcephaly is due to abnormal regulation of branched chain amino acid levels, however the exact underlying mechanisms are unknown.

Objectives: Understand how mutation or inhibition of the LAT1 (i.e. Slc7a5) transporter leads to microcephaly employing in vitro and in vivo models.

Methods:

• Generation of in vitro and in vivo models: We will use Slc7a5 mutant mouse models to study how mutations in Slc7a5 lead to microcephaly. In addition we will employ the CRISPR/Cas9 technology to introduce specific mutations in human embryonic stem cells and generate human brain organoids. Employing the aforementioned models we will study how somatic or germline loss of function mutations in Slc7a5 impact development and rescue of brain overgrowth.
• Pharmacological inhibition of Slc7a5 by known and novel compounds: employing our in vitro and in vivo models to screen known and novel compounds (molecules developed and synthetized within MolTag) inhibiting Slc7a5.

Project II - Studying novel epilepsy mutations in brain organdies (with Ernst)

Background: To study how mutations in epilepsy-associated genes affect the development and activity of the brain in a human genetic background, in the last funding period we invested a lot of effort to establish the culture of human stem cell-derived cerebral organoids, also called “mini brains”, in my laboratory (a paper employing cerebral organoids is currently in preparation). In addition, we utilized calcium imaging, histological analysis and RNA sequencing as tools to assess human-derived mutations in these mini organs. Employing these technologies we will now proceed to study novel human mutations associated with epileptic encephalopathies.

Objectives: Studying novel mutations associated with epileptic encephalopathies.

Methods: In collaboration with Margot Ernst and geneticists at the Medical University we will identify interesting novel candidate genetic mutations to be studied in human neuronal cell culture and cerebral organoids. The Ernst lab will contribute for the case of GABA_A receptor subunit mutations the molecular/ functional phenotypes. Specifically we will:

• Generate induced pluripotent stem cells (iPSC) from patients with genetically defined epileptic disorders.
• Establish and study patient-derived neuronal cell culture (2D system) employing multielectrode arrays and calcium imaging.
• Establish and study patient-derived cerebral organoids (3D system) employing histological and transcriptomic techniques.