Three-dimensional reengineering of neuronal microcircuits : The cortical column in silico

The presented thesis will describe a pipeline to reengineer three-dimensional, anatomically realistic, functional neuronal networks with subcellular resolution. The pipeline consists of five methods: 1. "NeuroCount" provides the number and three-dimensional distribution of all neuron somata in large brain regions. 2. "NeuroMorph" provides authentic neuron tracings, comprising dendrite and axon morphology. 3. "daVinci" registers the neuron morphologies to a standardized reference framework. 4. "NeuroCluster" objectively groups the standardized tracings into anatomical neuron types. 5. "NeuroNet" combines the number and distribution of neurons and neuron-types with the standardized tracings and determines the neuron-type- and position-specific number of synaptic connections for any two types of neuron. The developed methods are demonstrated by reengineering the thalamocortical lemniscal microcircuit in the somatosensory system of rats. There exists an one-to-one correspondence between the sensory information obtained by a single facial whisker and segregated areas in the thalamus and cortex. The reengineering of this pathway results in a column-shaped network model of ~15200 excitatory full-compartmental cortical neurons. This network is synaptically connected to ~285 pre-synaptic thalamic neurons. Animation of this "cortical column in silico" with measured physiological input will help to gain a mechanistic understanding of neuronal sensory information processing in the mammalian brain

Exploring the link between CHD2 mutations and double strand break repair in developing neurons

Introduction: Heterozygous mutations CHD2 cause intellectual disability and refractory epilepsy. Functional studies demonstrate a deficit in DNA double strand break (DSB) repair via nonhomologous end joining (NHEJ) in CHD2 deficient cells. This project aims to investigate the impact of CHD2 mutations on the outcomes of DSB repair in developing neurons. Methods: A doxycycline inducible CRISPR-Cas9 (iCas9) construct was integrated into the AAVS1 safe harbour. A pipeline for high-throughput analysis of CRISPR experiments was designed and implemented based on nanopore sequencing. This pipeline was used to create a CHD2- deficient human induced pluripotent stem cell (hIPSC) line, which was used to investigate the effects of CHD2 on neurodifferentiation and DSB repair. The pipeline was then adapted to monitor repair outcomes of targeted DSBs in differentiating cells. Spontaneously occurring DSBs were examined using a novel next-generation sequencing technique, INDUCE-Seq, in which unrepaired DSBs are captured from permeabilised cells in-situ and sequenced in order to provide a snapshot of DSBs existing at the time of extraction in differentiating cells. Results: Cas9 induction of DSBs demonstrated changes in the repair, with an increased rate of large deletions in the CHD2-deficient cell line. No reproducible change in the pattern of smaller indels was identified. There was a significant increase of the number of DSBs captured by INDUCE-seq at D19 of differentiation in WT cells, which was not present until D40 in CHD2-deficient cells. Differences in the enrichment of DSBs at various histone markers, gene bodies and transcription start sites (TSS) were identified between CHD2-deficient cells and WT cells. Conclusions: This study demonstrates an impact on the occurrence and repair of DSBs in CHD2- deficient cell lines. Integration of RNA-Seq data and analysis of the pattern of spontaneous breakage suggests that altered DSB repair physiology could contribute towards the phenotype exhibited by patients with CHD2 mutations