Lee, A. Y.-F. | Search bioRxiv

bioRxiv2020

Generation of a DAT-Flp mouse line for intersectional genetic targeting of dopamine neuron subpopulations

Dopamine neurons project to diverse regions throughout the brain to modulate various brain processes and behaviors. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons, or mark a sub-set but concurrently label non-dopaminergic neurons. Here we establish a novel mouse line in which Flp recombinase is knocked-in to the endogenous Slc6a3 (dopamine active transporter, DAT) locus. DAT-Flp mice can be used with various Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by crossing DAT-Flp mice with NEX-Cre mice, to specifically label Neurod6-expressing dopamine neurons that project to the nucleus accumbens medial shell. DAT-Flp mice represent a novel tool, which will help parse the diverse functions mediated by dopaminergic circuits.

neuroscience↗

bioRxiv2019

IRG1 and iNOS act redundantly with other interferon gamma induced factors to restrict intracellular replication of Legionella pneumophila.

Interferon gamma (IFN{gamma}) restricts the intracellular replication of many pathogens, but how IFN{gamma} confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN{gamma}, but consistent with prior results, no individual genetic deficiency we tested compromised IFN{gamma}-mediated control. Intriguingly, however, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN{gamma}-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN{gamma}. Instead, we found that 2DG rescues bacterial replication predominantly by inhibiting the induction of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirm that loss of both iNOS and IRG1, but not individual deficiency in each gene, partially reduces IFN{gamma}-mediated restriction of L. pneumophila. Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we find that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3 and NOX2) results in a total loss of L. pneumophila restriction by IFN{gamma} in primary bone marrow macrophages. There are few, if any, other examples in which the complete set of cell-intrinsic factors required for IFN{gamma}-mediated restriction of an intracellular bacterial pathogen have been genetically identified. Our results highlight the combinatorial strategy used by hosts to block the exploitation of macrophages by pathogens.\n\nImportanceLegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN{gamma}) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has determined the exact molecular factors induced by IFN{gamma} that are required for its activity. We generated macrophages lacking different combinations of IFN{gamma}-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN{gamma}-mediated restriction of L. pneumophila. We successfully identified six genes that comprise the totality of the IFN{gamma}-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN{gamma} and highlight how redundancy downstream of IFN{gamma} is key to prevent exploitation of the macrophage niche by pathogens.

immunology↗