Expression profiling by high throughput sequencing
We report here the bacTRAP (bacterial artificial chromosome , translating ribosome affinity purification) profiling of 7 different types of neurons in the mouse, at three different ages: two neuron types very vulnerable to AD (principal cells of entorhinal cortex layer II - ECII), pyramidal cells of hippocampus CA1, and 5 types of neurons more resistant to AD (pyramidal cells of hippocampus CA2 and CA3, granule neurons of the dentate gyrus, pyramidal cells from layer IV of primary visual cortex V1, and pyramidal cells from layer II/III and V of primary somatosensory cortex S1). Using these profiles we generated molecular signatures for each of these cell types, proved that the molecular identity of these cell types is very well conserved across mouse and humans, and constructed functional networks for each cell type that allowed us to identify genes and pathways associated with selective neuronal vulnerability in AD. We also report the profiling of ECII neurons in APP/PS1 mice (a mouse model of Abeta accumulation) at 6 months of age.
We constructed bacTRAP mice overexpressing eGFP-L10a in a cell-type specific manner. Cell-type specific expression was achieved using bacterial artificial chromosomes (BAC) that contain the regulatory regions of cell-type specific genes. For ECII neurons, we used four different lines: two Rasgrp2-bacTRAP lines (using BACs RP23-199D5 and RP24-344N1), two different founder lines for Sh3bgrl2 (using BAC RP23-307B16); for CA1 neurons, two different lines: one SSTR4-bacTRAP (BAC RP23-126C5), and one Cck-bacTRAP line generated previously by the Heintz lab; for CA2 neurons: one Cacng5-bacTRAP line (BAC RP23- 329L1); for CA3 neurons: one Gprin3-bacTRAP line generated previously by the Heintz lab; for dentate gyrus granule neurons: one SSTR4-bacTRAP line (using BAC RP23-126C5, another founder from the one used for CA1, with ectopic expression in DG granule neurons); for V1: one Calca-bacTRAP line (BAC RP23-181A2); for S1: one Cartpt-bacTRAP line (BAC RP24-68J22). We used these mice to immunoprecipitate polysomes from these neurons at 4-5, 12 and 24 months of age, and profiled the isolated mRNA using RNAseq. Additionally, we profiled APP/PS1 mice crossed to ECII-bacTRAP mice (one of the Sh3bgrl2-bacTRAP lines) at 6 months of age to study the effects of Amyloid accumulation on ECII gene expression. In the end we had 3 to 12 replicates for each neuron type at each age. Each replicate consisted in the pooling of 2 different mice. Male mice were used for the profiling in wild-type mice. For the APP/PS1 profiling we used male and female mice.