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Author(s): Esther Klingler [sup.1] , Ugo Tomasello [sup.1] , Julien Prados [sup.2] , Justus M. Kebschull [sup.3] [sup.6] , Alessandro Contestabile [sup.1] , Gregorio L. Galiñanes [sup.1] , Sabine Fièvre [...], Interconnectivity between neocortical areas is critical for sensory integration and sensorimotor transformations.sup.1-6. These functions are mediated by heterogeneous inter-areal cortical projection neurons (ICPN), which send axon branches across cortical areas as well as to subcortical targets.sup.7-9. Although ICPN are anatomically diverse.sup.10-14, they are molecularly homogeneous.sup.15, and how the diversity of their anatomical and functional features emerge during development remains largely unknown. Here we address this question by linking the connectome and transcriptome in developing single ICPN of the mouse neocortex using a combination of multiplexed analysis of projections by sequencing.sup.16,17 (MAPseq, to identify single-neuron axonal projections) and single-cell RNA sequencing (to identify corresponding gene expression). Focusing on neurons of the primary somatosensory cortex (S1), we reveal a protracted unfolding of the molecular and functional differentiation of motor cortex-projecting ([Formula omitted]) ICPN compared with secondary somatosensory cortex-projecting ([Formula omitted]) ICPN. We identify SOX11 as a temporally differentially expressed transcription factor in [Formula omitted] versus [Formula omitted] ICPN. Postnatal manipulation of SOX11 expression in S1 impaired sensorimotor connectivity and disrupted selective exploratory behaviours in mice. Together, our results reveal that within a single cortical area, different subtypes of ICPN have distinct postnatal paces of molecular differentiation, which are subsequently reflected in distinct circuit connectivities and functions. Dynamic differences in the expression levels of a largely generic set of genes, rather than fundamental differences in the identity of developmental genetic programs, may thus account for the emergence of intra-type diversity in cortical neurons. Combined analysis of the connectome and transcriptome in the mouse cortex indicates that dynamic differences in expression levels of largely generic sets of genes regulate differential targeting within neuronal subtypes.

Author(s): Yuan Pan [sup.1] , Jared D. Hysinger [sup.1] , Tara Barron [sup.1] , Nicki F. Schindler [sup.1] , Olivia Cobb [sup.2] , Xiaofan Guo [sup.2] , Belgin Yalçin [sup.1] [...], Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours.sup.1. Although the role of neurons in tumour progression has previously been demonstrated.sup.2, the importance of neuronal activity to tumour initiation is less clear--particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood.sup.3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1).sup.5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome. Mouse models of NF1-associated optic pathway glioma show that tumour initiation and growth are driven by aberrantly high levels of NLGN3 shedding in the optic nerve in response to retinal neuron activity.

Author(s): Wei Wu [sup.1] , Sarah E. Hill [sup.2] , William J. Nathan [sup.1] [sup.3] , Jacob Paiano [sup.1] , Elsa Callen [sup.1] , Dongpeng Wang [sup.1] , Kenta Shinoda [...], Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons.sup.1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair. DNA single-strand breaks in neurons accumulate at high levelsin functional enhancers.

Author(s): Vijendra Sharma [sup.1] [sup.2] , Rapita Sood [sup.1] [sup.2] , Abdessattar Khlaifia [sup.3] , Mohammad Javad Eslamizade [sup.1] [sup.2] [sup.3] , Tzu-Yu Hung [sup.1] [sup.2] , Danning Lou [sup.1] [...], An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory.sup.1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories.sup.5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF2.sup.11. Phosphorylation of the [alpha]-subunit of eIF2 (p-eIF2[alpha]), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds.sup.11-13. By contrast, inhibiting the ISR by mutating the eIF2[alpha] phosphorylation site, genetically.sup.11 and pharmacologically inhibiting the ISR kinases.sup.14-17, or mimicking reduced p-eIF2[alpha] with the ISR inhibitor ISRIB.sup.11, enhances long-term memory in health and disease.sup.18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2[alpha] phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2[alpha] in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2[alpha]-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons. Stimulation of de novo protein synthesis in both excitatory and inhibitory, somatostatin-expressing neurons in the mouse hippocampus enhances memory consolidation.

Author(s): Avni Baser [sup.1] [sup.2] , Maxim Skabkin [sup.1] , Susanne Kleber [sup.1] , Yonglong Dang [sup.1] [sup.2] , Gülce S. Gülcüler Balta [sup.1] [sup.2] , Georgios Kalamakis [sup.1] [sup.2] [...], Whether post-transcriptional regulation of gene expression controls differentiation of stem cells for tissue renewal remains unknown. Quiescent stem cells exhibit a low level of protein synthesis.sup.1, which is key to maintaining the pool of fully functional stem cells, not only in the brain but also in the bone marrow and hair follicles.sup.2-6. Neurons also maintain a subset of messenger RNAs in a translationally silent state, which react 'on demand' to intracellular and extracellular signals. This uncoupling of general availability of mRNA from translation into protein facilitates immediate responses to environmental changes and avoids excess production of proteins, which is the most energy-consuming process within the cell. However, when post-transcriptional regulation is acquired and how protein synthesis changes along the different steps of maturation are not known. Here we show that protein synthesis undergoes highly dynamic changes when stem cells differentiate to neurons in vivo. Examination of individual transcripts using RiboTag mouse models reveals that whereas stem cells translate abundant transcripts with little discrimination, translation becomes increasingly regulated with the onset of differentiation. The generation of neurogenic progeny involves translational repression of a subset of mRNAs, including mRNAs that encode the stem cell identity factors SOX2 and PAX6, and components of the translation machinery, which are enriched in a pyrimidine-rich motif. The decrease of mTORC1 activity as stem cells exit the cell cycle selectively blocks translation of these transcripts. Our results reveal a control mechanism by which the cell cycle is coupled to post-transcriptional repression of key stem cell identity factors, thereby promoting exit from stemness.Sequencing of total and ribosome-associated mRNAs enables identification of specific mRNAs that are differentially translationally regulated along the neuronal stem cell lineage, independently of their availability.

Author(s): Carlos A. Campos (corresponding author) [1]; Anna J. Bowen [1]; Carolyn W. Roman [1]; Richard D. Palmiter (corresponding author) [1] The parabrachial nucleus (PBN) receives ascending inputs from many [...], Animals must respond to various threats to survive. Neurons that express calcitonin gene-related peptide in the parabrachial nucleus (CGRP[sup.PBN] neurons) relay sensory signals that contribute to satiation and pain-induced fear behaviour, but it is unclear how they encode these distinct processes. Here, by recording calcium transients in vivo from individual neurons in mice, we show that most CGRP[sup.PBN] neurons are activated by noxious cutaneous (shock, heat, itch) and visceral stimuli (lipopolysaccharide). The same neurons are inhibited during feeding, but become activated during satiation, consistent with evidence that CGRP[sup.PBN] neurons prevent overeating. CGRP[sup.PBN] neurons are also activated during consumption of novel foods or by an auditory cue that has previously been paired with electrical footshocks. Correspondingly, silencing of CGRP[sup.PBN] neurons attenuates the expression of food neophobia and conditioned fear responses. Therefore, in addition to transducing primary sensory danger signals, CGRP[sup.PBN] neurons promote affective-behavioural states that limit harm in response to potential threats.

Administration of diphtheria toxin (DT) to [Agrp.sup.DTR] mice, which express the human DT receptor selectively in AgRP neurons, ablates nearly all AgRP neurons in the arcuate nucleus of the hypothalamus; [...], Hypothalamic neurons that co-express agouti-related protein (AgRP), neuropeptide Y and γ-aminobutyric acid (GABA) are known to promote feeding and weight gain by integration of various nutritional, hormonal, and neuronal signals (1,2). Ablation of these neurons in mice leads to cessation of feeding that is accompanied by activation of Fos in most regions where they project (3-6). Previous experiments have indicated that the ensuing starvation is due to aberrant activation of the parabrachial nucleus (PBN) and it could be prevented by facilitating [GABA.sub.A] receptor signalling in the PBN within a critical adaptation period (5). We speculated that loss of GABA signalling from AgRP-expressing neurons (AgRP neurons) within the PBN results in unopposed excitation of the PBN, which in turn inhibits feeding. However, the source of the excitatory inputs to the PBN was unknown. Here we show that glutamatergic neurons in the nucleus tractus solitarius (NTS) and caudal serotonergic neurons control the excitability of PBN neurons and inhibit feeding. Blockade of serotonin ([5-HT.sub.3]) receptor signalling in the NTS by either the chronic administration of ondansetron or the genetic inactivation of Tph2 in caudal serotonergic neurons that project to the NTS protects against starvation when AgRP neurons are ablated. Likewise, genetic inactivation of glutamatergic signalling by the NTS onto N-methyl D-aspartate-type glutamate receptors in the PBN prevents starvation. We also show that suppressing glutamatergic output of the PBN reinstates normal appetite after AgRP neuron ablation, whereas it promotes weight gain without AgRP neuron ablation. Thus we identify the PBN as a hub that integrates signals from several brain regions to bidirectionally modulate feeding and body weight.

During the course of exploring the roles of miR-9/9* and miR-124 (ref. 5), we noted that these miRNAs induced neuronal morphologies in cultured cells. To explore this effect in greater [...], Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons (1,2). However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function (3-6). Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions (4). Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function (5,7-13), we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/ 9*-124 can have an instructive role in neural fate determination.