Authors :
Presenting Author: Kelley Roark, BS, RMA – University of Maryland Baltimore School of Medicine
Sophie Bruckmeier, BS – PhD Student, Department of Neurology, University of Maryland Baltimore School of Medicine; Philip Iffland, Ph.D. – Assistant Professor, Director of Neurology Labs, Department of Neurology, University of Maryland Baltimore School of Medicine
Rationale: Malformations of Cortical Development (MCD) are a common cause of medically refractory epilepsy and neurodevelopmental disorders. Focal Cortical Dysplasia (FCD) is an MCD most commonly caused by genetic variants in the mTOR pathway genes
DEPDC5, NPRL2, and
NPRL3. Each of these genes code for proteins that form the GATOR1 complex-a negative regulator of the mTOR pathway. NPRL2 is a key component of this complex and forms its catalytic subunit. Loss of NPRL2 results in changes in neuronal morphology commonly associated with mTORopathies including increased phosphorylation of ribosomal S6 and increased soma size. However, there are conflicting data on the role NPRL2 loss plays in dendrite morphology. We hypothesize that
Nprl2 KO will result in mTOR-dependent changes in dendrite number and length.
Methods: An
Nprl2 knockout (KO) cell line was created using CRISPR/Cas9 in Neuro2a cells (N2aC). Successful Cas9 editing was confirmed using next-generation sequencing and qPCR. All experiments were performed with KO, scramble control (Scr), and wildtype (WT) N2aC with or without rapamycin or torin1 treatment. Cells were assayed via Western blotting for changes in mTOR pathway signaling as measured by PS6 (ser 240/244) in comparison to total S6 levels and loading control (
b-actin). Imaging cytometry was used to assess cell size in each experimental and treatment group. To induce dendrite growth in each cell line, cells were differentiated in 0.5% FBS EMEM media and 16 mM retinoic acid for 24 hours. Dendritic morphology was assayed using immunocytochemistry probing for the somatodendritic marker MAP2 and visualized using fluorescent secondary antibodies (Alexa 594). Imaging was performed using a spinning disc confocal microscope. Dendrite number and length were assessed in Fiji imaging software and quantified in Prism.Results: Nprl2
KO cells showed mismatched DNA pairing and reduced mRNA levels in comparison to Scr and WT. In Nprl2 KO cells we observed increases in PS6 (ser 240/244) and soma size (n > 3,000 per group) that were prevented with rapamycin or torin1 treatment (p< 0.05). Morphometric analysis of dendrites revealed mTOR-dependents increase in both the number and length of dendrites in Nprl2 KO cells compared to control (n=50 per group, p< 0.05).