A versatile tool for the analysis of neuronal survival.

To understand the principles that govern mechanisms of neuronal survival or death it is necessary to systematically model these processes. Methods involving overexpression or knockdown of a gene of interest using non-viral transfection of primary neurons can easily be adapted to study cell death pathways in primary neurons. However, common biochemical approaches to measure cell death are insufficient to measure neuronal viability in these systems. To investigate the functional role of genes in cultured neurons, we therefore established a cell-based assay using a cotransfection/cocultivation approach in primary cortical neurons from mouse or rat. Using this method, it is possible to use well-established cell culture models of neuronal damage, and to analyze cell survival in genetically different neurons on a single-cell basis following apoptotic stimuli under identical conditions. The duration of the entire protocol is 10 days. Finally, the method may be applicable to a wide range of damage models, primary cells, and cell lines as well as it can be used for high content screening (HCS) studies and downstream image cytometry.

Interferon-driven brain phenotype in a mouse model of RNaseT2 deficient leukoencephalopathy.

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2-/- mice using CRISPR/Cas9-mediated genome editing. Rnaset2-/- mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8+ effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2-/- mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies.

Investigating Gene Function for Neuronal Survival After Metabolic Stress Using Semi-Automated Fluorescence Microscopy and Automated Image Analysis.

Overexpression approaches and fluorescence microscopy techniques allow investigating important spatiotemporal aspects of gene regulation as well as quantifying gene function. Consequently, fluorescence microscopy techniques help answer important questions on gene regulation such as addressing the role of a specific gene product for neuronal survival under different treatments. Here, we describe a versatile tool to measure effects of a transfected gene of interest on neuronal survival upon metabolic stress. We focus on nutrient starvation of cultured rodent primary neurons as a model of metabolic stress but our approach can easily be generalized and adapted to other cell types or to investigate single gene function in regulating neuronal survival under various conditions.