Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that 60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.[Keywords: translational profiling; hypocretin; orexin; bacTRAP; Lhx9; narcolepsy] Supplemental material is available for this article. Narcolepsy is a profound disorder of sleep regulation, characterized by excessive daytime sleepiness, sleep attacks, cataplexy, and sleep-onset REM (rapid eye movement) periods. Studies indicate that narcolepsy, especially when accompanied by cataplexy, is due to the absence of hypocretinergic neurons and signaling (Nishino et al. 2000;Thannickal et al. 2000;Bourgin et al. 2008). The hypocretins (Hcrts; also called orexins) are peptidergic neurotransmitters used by a discrete population of neurons in the lateral hypothalamus Sakurai et al. 1998). In model organisms, disruption of the gene encoding the Hcrt peptides or a Hcrt receptor (Chemelli et al. 1999;Lin et al. 1999) or, alternatively, the ablation of the neurons producing Hcrt (Gerashchenko et al. 2001;Hara et al. 2001;Zhang et al. 2007) recapitulates features of narcolepsy. In humans, there are two variations of the disorder: narcolepsy with cataplexy (NC) and narcolepsy without cataplexy (NwoC). NC is strongly linked and associated with the HLA DQB1*0602 allele, a T-cell receptor locus, and P2RY11, a receptor that can regulate immune cell survival (Hallmayer et al. 2009;Kornum et al. 2011). This strongly suggests involvement of the immune system in the etiology of NC, while the involvement in NwoC is less clear. Cold Spring Harbor Laboratory Press on May 13, 2018 -Published by genesdev.cshlp.org Downloaded from

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NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

Background Studies in psychiatric genetics have identified over 100 loci associated with disease risk, yet many of these loci are distant from protein coding genes. Recent characterization of the transcriptional landscape of cell lines and whole tissues has suggested widespread transcription in both coding and non-coding regions of the genome, including differential expression from loci that produce regulatory non-coding RNAs which function within the nucleus; however, the nuclear transcriptome of specific cell types in the brain has not been previously investigated. Methods Here we have defined the nuclear transcriptional landscape of the three major cellular divisions of the nervous system using flow sorting of genetically labeled nuclei from bacTRAP mouse lines. This was followed by characterization of the unique expression of coding, non-coding and intergenic RNAs in the mature mouse brain with RNAseq and validation with independent methods. Results Our findings reveal diverse expression across the cell-types of all classes of RNAs, including long non-coding RNAs – several of which were confirmed as highly enriched in the nuclei of specific cell-types using anatomical methods. Finally, we also discovered several examples of cell-type specific expression of tandem gene fusions, and report the first cell-type specific expression of circular RNAs, notably a neuron-specific and nuclear-enriched RNA arising from the gene Hnrnpu. Conclusion These data will provide an important resource for studies evaluating the function of a variety of ncRNAs in the brain, including those that may play a role in psychiatric disease.

SUMMARY Alternative translation initiation and stop codon readthrough in a few well-studied cases have been shown to allow the same transcript to generate multiple protein variants. Because the brain shows a particularly abundant use of alternative splicing, we sought to study alternative translation in CNS cells. We show that alternative translation is widespread and regulated across brain transcripts. In neural cultures, we identify alternative initiation on hundreds of transcripts, confirm several N-terminal protein variants, and show the modulation of the phenomenon by KCl stimulation. We also detect readthrough in cultures and show differential levels of normal and readthrough versions of AQP4 in gliotic diseases. Finally, we couple translating ribosome affinity purification to ribosome footprinting (TRAP-RF) for cell-type-specific analysis of neuronal and astrocytic translational readthrough in the mouse brain. We demonstrate that this unappreciated mechanism generates numerous and diverse protein isoforms in a cell-type-specific manner in the brain.

Much has been learned from studies of Limulus photoreceptors about the role of the circadian clock and light in the removal of photosensitive membrane. However, little is known in this animal about mechanisms regulating photosensitive membrane renewal, including the synthesis of proteins in, and associated with, the photosensitive membrane. To begin to understand renewal, this study examines diurnal changes in the levels of mRNAs encoding opsin, the integral membrane protein component of visual pigment, and the relative roles of light and the circadian clock in producing these changes. We show that at least two distinct opsin genes encoding very similar proteins are expressed in both the lateral and ventral eyes, and that during the day and night in the lateral eye, the average level of mRNA encoding opsinl is consistently higher than that encoding opsin2. Northern blot assays showed further that total opsin mRNA in the lateral eyes of animals maintained under natural illumination increases during the afternoon (9 & 12 h after sunrise) in the light and falls at night in the dark. This diurnal change occurs whether or not the eyes receive input from the circadian clock, but it is eliminated in eyes maintained in the dark. Thus, it is regulated by light and darkness, not by the circadian clock, with light stimulating an increase in opsin mRNA levels. The rise in opsin mRNA levels observed under natural illumination was seasonal; it occurred during the summer but not the spring and fall. However, a significant increase in opsin mRNA levels could be achieved in the fall by exposing lateral eyes to 3 h of natural illumination followed by 9 h of artificial light. The diurnal regulation of opsin mRNA levels contrasts sharply with the circadian regulation of visual arrestin mRNA levels (Battelle et al., 2000). Thus, in Limulus, distinctly different mechanisms regulate the levels of mRNA encoding two proteins critical for the photoresponse.

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Studies on regulation of gene expression have contributed substantially to understanding mechanisms for the long-term activity-dependent alterations in neural connectivity that are thought to mediate learning and memory. Most of these studies, however, have focused on the regulation of mRNA transcription. Here, we utilized high-throughput sequencing coupled with ribosome footprinting to globally characterize the regulation of translation in primary mixed neuronal-glial cultures in response to sustained depolarization. We identified substantial and complex regulation of translation, with many transcripts demonstrating changes in ribosomal occupancy independent of transcriptional changes. We also examined sequence-based mechanisms that might regulate changes in translation in response to depolarization. We found that these are partially mediated by features in the mRNA sequence—notably upstream open reading frames and secondary structure in the 5′ untranslated region—both of which predict downregulation in response to depolarization. Translationally regulated transcripts are also more likely to be targets of FMRP and include genes implicated in autism in humans. Our findings support the idea that control of mRNA translation plays an important role in response to neural activity across the genome.