Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical Cterminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitopetagged ribosomal protein RPL22 HA , which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosomeassociated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuronspecific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.gene profiling ͉ immunopreciptation ͉ mouse genetics

SummaryLocal mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.

The 75 kDa neurotrophin receptor (p75NTR) and two neurotrophin receptor homologs (NRH1, NRH2) constitute a subfamily of the nerve growth factor/tumor necrosis factor receptor superfamily. NRH1 coexists with p75NTR in fish, amphibians, and birds but is absent in mammals, whereas NRH2 exists only in mammals. Unlike p75NTR and NRH1, NRH2 lacks a canonical extracellular ligand binding domain. The similarity of NRH2 to the product of metalloproteinase cleavage of p75NTR prompted us to examine the cleavage of p75NTR in greater detail. p75NTR, NRH1, and NRH2 undergo multiple proteolytic cleavages that ultimately release cytoplasmic fragments. For p75NTR, cleavage in the extracellular domain by a PMA-inducible membrane metalloproteinase is followed by cleavage within or near the transmembrane domain, releasing the intracellular domain into the cytoplasm. This processing resembles the alpha- and gamma-secretase-mediated processing of beta-amyloid precursor protein and the similar processing of Notch. Although neurotrophins did not regulate p75NTR processing, the alpha- and gamma-secretase-mediated cleavage of p75 is modulated by receptor tyrosine kinases (Trks) TrkA and TrkB but not TrkC. Surprisingly, although NRH1 and NRH2 also undergo proteolytic cytoplasmic release of intracellular domains, a different protease mediates the cleavage. Furthermore, whereas the p75NTR soluble intracellular domain accumulates only in the presence of proteasome inhibitors, the equivalent fragment of NRH2 is stable and localizes in the nucleus. Because soluble intracellular domains of p75NTR and NRH2 were found to activate NF-kappaB in concert with TNF receptor associated factor 6 (TRAF6), we propose that cleavage of these proteins may serve conserved cytoplasmic and nuclear signaling functions through distinct proteases.

The striatum regulates motor control, reward, and learning. Abnormal function of striatal GABAergic medium spiny neurons (MSNs) is believed to contribute to the deficits in these processes that are observed in many neuropsychiatric diseases. The orphan G-protein-coupled receptor (GPCR) GPR88 is robustly expressed in MSNs and regulated by neuropharmacological drugs, but its contribution to MSN physiology and behavior is unclear. Here we show that in the absence of GPR88, MSNs have increased glutamatergic excitation and reduced GABAergic inhibition that together promote enhanced firing rates in vivo, resulting in hyperactivity, poor motor-coordination, and impaired cue-based learning in mice. Targeted viral expression of GPR88 in MSNs rescues the molecular and electrophysiological properties and normalizes behavior, suggesting that aberrant MSN activation in the absence of GPR88 underlies behavioral deficits and its dysfunction may contribute to behaviors observed in neuropsychiatric disease.

The cAMP-dependent protein kinase holoenzyme is assembled from regulatory (R) and catalytic (C) subunits that are expressed in tissue-specific patterns. Despite the dispersion of the R and C subunit genes to different chromosomal loci, mechanisms exist that coordinately regulate the intracellular levels of R and C protein such that cAMP-dependent regulation is preserved. We have created null mutations in the RI␤ and RII␤ regulatory subunit genes in mice, and find that both result in an increase in the level of RI␣ protein in tissues that normally express the ␤ isoforms. Examination of RI␣ mRNA levels and the rates of RI␣ protein synthesis in wild type and RII␤ mutant mice reveals that the mechanism of this biochemical compensation by RI␣ does not involve transcriptional or translational control. These in vivo findings are consistent with observations made in cell culture, where we demonstrate that the overexpression of C␣ in NIH 3T3 cells results in increased RI␣ protein without increases in the rate of RI␣ synthesis or the level of RI␣ mRNA. Pulse-chase experiments reveal a 4 -5-fold increase in the half-life of RI␣ protein as it becomes incorporated into the holoenzyme. Compensation by RI␣ stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.The cAMP-dependent protein kinase (PKA) 1 is a key regulatory enzyme responsible for the intracellular transduction of a variety of extracellular signals and for the maintenance of numerous aspects of cellular homeostasis (1). The holoenzyme is composed of a regulatory (R) subunit dimer complexed with two catalytic (C) subunits. Two molecules of cAMP bind to each R subunit causing release of enzymatically active C subunits, which then modify the activity of target proteins by reversible phosphorylation of serine or threonine residues located within an appropriate consensus sequence (2).Four R subunit isoforms and two C subunit isoforms of PKA have been characterized in the mouse (3). They are highly conserved among mammals, encoded by unique genes located on separate chromosomes, and show unique patterns of gene expression. The ␣-isoforms are expressed ubiquitously while ␤ isoforms show more restricted patterns of expression. RI␤ is induced relatively late in development and is highly expressed in neural tissues (4 -6). RII␤ is expressed during embryogenesis in mouse brain, spinal cord, and liver (7). In adult mice RII␤ protein is most abundant in brain and brown and white adipose tissue, with lower expression in testis and ovary (8). C␤ is most abundant in the brain, but lower levels of C␤ mRNA are found in all tissues examined (9).PKA holoenzymes can be separated by ion-exchange chromatography and analysis of a variety of mammalian tissues has revealed significant differences in the ratio of type I (RI-containing) to type II (RII-containing) holoenzyme (10). In rats and mice, brain and adipose tissue contain principally the type II holoenzyme, while heart and liver contain mainly type I. The rat...