Summary The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. We have now developed a new ‘BACarray’ methodology, based on affinity purification of polysomal mRNAs from genetically defined cell populations. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. Even two morphologically indistinguishable subclasses of MSNs display vastly different translational profiles. Striatopallidal neurons are characterized by a strong and cell-specific release of intracellular Ca2+ in response to sphingosine 1-phosphate, consistent with their selective expression of Gpr6. In contrast, striatonigral neurons demonstrate a selective cell-specific increase in GABAA receptor subunits in response to chronic cocaine treatment. BACarray translational profiling is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.

SignificanceIn aging, the brain becomes vulnerable to injury and cognitive function declines, but the mechanisms responsible are unknown. Astrocytes, the most abundant class of glial cells, are vital for the proper function of the central nervous system, and impairment of astrocyte function has been implicated in disease. Here we perform RNA sequencing of astrocytes from different brain regions across the lifespan of the mouse to identify age-related transcriptional changes that could contribute to cognitive decline. We find that aged astrocytes take on a reactive phenotype characteristic of neuroinflammatory reactive astrocytes, and that microglia play a role in inducing astrocyte activation. The aging astrocyte RNA sequencing profiles provide an important new resource for future studies exploring the role of astrocytes in cognitive decline.

Summary Comparative analysis of BACarray data can provide important insights into complex biological systems. As demonstrated in the accompanying paper, BACarray translational profiling permits comprehensive studies of translated mRNAs in genetically defined cell populations following physiological perturbations. To establish the generality of this approach, we present BACarray translational profiles for twenty four CNS cell populations, and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole tissue microarray studies, and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of sixteen transgenic mouse lines, their corresponding anatomic characterization, and BACarray translational profiles for cell types from a variety of CNS structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well known and previously uncharacterized neural cell populations.

SUMMARY When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this “Terminal UPR.” TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing micro-RNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing Caspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β-cell death during ER stress, and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death, and may be targeted to develop effective treatments for cell degenerative diseases.