A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Astrocytes locally translate transcripts in their peripheral processes.

Local translation in neuronal processes is key to the alteration of synaptic strength necessary for long-term potentiation, learning, and memory. Here, we present evidence that regulated de novo protein synthesis occurs within distal, perisynaptic astrocyte processes. Astrocyte ribosomal proteins are found adjacent to synapses in vivo, and immunofluorescent detection of peptide elongation in acute slices demonstrates robust translation in distal processes. We have also developed a biochemical approach to define candidate transcripts that are locally translated in astrocyte processes. Computational analyses indicate that astrocyte-localized translation is both sequence-dependent and enriched for particular biological functions, such as fatty acid synthesis, and for pathways consistent with known roles for astrocyte processes, such as GABA and glutamate metabolism. These transcripts also include glial regulators of synaptic refinement, such as Sparc Finally, the transcripts contain a disproportionate amount of a binding motif for the quaking RNA binding protein, a sequence we show can significantly regulate mRNA localization and translation in the astrocytes. Overall, our observations raise the possibility that local production of astrocyte proteins may support microscale alterations of adjacent synapses.

Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.

Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

Astrocyte-secreted neurocan controls inhibitory synapse formation and function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. Several astrocyte-secreted synaptogenic proteins controlling excitatory synapse development were identified; however, those that induce inhibitory synaptogenesis remain elusive. Here, we identify neurocan as an astrocyte-secreted inhibitory synaptogenic protein. After secretion from astrocytes, neurocan is cleaved into N- and C-terminal fragments. We found that these fragments have distinct localizations in the extracellular matrix. The neurocan C-terminal fragment localizes to synapses and controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic domain have reduced inhibitory synapse numbers and function. Through super-resolution microscopy, in vivo proximity labeling by secreted TurboID, and astrocyte-specific rescue approaches, we discovered that the synaptogenic domain of neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of ERM Phosphorylation.

Astrocytes, a major glial cell type of the brain, regulate synapse numbers and function. However, whether astrocyte dysfunction can cause synaptic pathologies in neurological disorders such as Parkinson's Disease (PD) is unknown. Here, we investigated the impact of the most common PD-linked mutation in the leucine-rich repeat kinase 2 (LRRK2) gene (G2019S) on the synaptic functions of astrocytes. We found that both in human and mouse cortex, the LRRK2 G2019S mutation causes astrocyte morphology deficits and enhances the phosphorylation of the ERM proteins (Ezrin, Radixin, and Moesin), which are important components of perisynaptic astrocyte processes. Reducing ERM phosphorylation in LRRK2 G2019S mouse astrocytes restored astrocyte morphology and corrected excitatory synaptic deficits. Using an in vivo BioID proteomic approach, we found Ezrin, the most abundant astrocytic ERM protein, interacts with the Autophagy-Related 7 (Atg7), a master regulator of catabolic processes. The Ezrin/Atg7 interaction is inhibited by Ezrin phosphorylation, thus diminished in the LRRK2 G2019S astrocytes. Importantly, Atg7 function is required to maintain proper astrocyte morphology. These studies reveal an astrocytic molecular mechanism that could serve as a therapeutic target in PD.

Astrocyte-Secreted Neurocan Controls Inhibitory Synapse Formation and Function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. To date, several astrocyte-secreted synaptogenic proteins controlling different stages of excitatory synapse development have been identified. However, the identities of astrocytic signals that induce inhibitory synapse formation remain elusive. Here, through a combination of in vitro and in vivo experiments, we identified Neurocan as an astrocyte-secreted inhibitory synaptogenic protein. Neurocan is a chondroitin sulfate proteoglycan that is best known as a protein localized to the perineuronal nets. However, Neurocan is cleaved into two after secretion from astrocytes. We found that the resulting N- and C-terminal fragments have distinct localizations in the extracellular matrix. While the N-terminal fragment remains associated with perineuronal nets, the Neurocan C-terminal fragment localizes to synapses and specifically controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic region have reduced inhibitory synapse numbers and function. Through super-resolution microscopy and in vivo proximity labeling by secreted TurboID, we discovered that the synaptogenic domain of Neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte-neuron cadherin interactions.

Astrocytes control the formation of specific synaptic circuits via cell adhesion and secreted molecules. Astrocyte synaptogenic functions are dependent on the establishment of their complex morphology. However, it is unknown if distinct neuronal cues differentially regulate astrocyte morphogenesis. δ-Catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. We found δ-catenin is also highly expressed by astrocytes and required both in astrocytes and neurons for astrocyte morphogenesis. δ-Catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and biochemical analyses to reveal that δ-catenin interacts with the N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

Training-induced circuit-specific excitatory synaptogenesis in mice is required for effort control.

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC→DMS). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ-1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ-1 in the adult ACC→DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC→DMS neurons rescues or phenocopies the behaviors of the α2δ-1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion.

Activity-dependent translation dynamically alters the proteome of the perisynaptic astrocyte process.

Within eukaryotic cells, translation is regulated independent of transcription, enabling nuanced, localized, and rapid responses to stimuli. Neurons respond transcriptionally and translationally to synaptic activity. Although transcriptional responses are documented in astrocytes, here we test whether astrocytes have programmed translational responses. We show that seizure activity rapidly changes the transcripts on astrocyte ribosomes, some predicted to be downstream of BDNF signaling. In acute slices, we quantify the extent to which cues of neuronal activity activate translation in astrocytes and show that this translational response requires the presence of neurons, indicating that the response is non-cell autonomous. We also show that this induction of new translation extends into the periphery of astrocytes. Finally, synaptic proteomics show that new translation is required for changes that occur in perisynaptic astrocyte protein composition after fear conditioning. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.

Loss of Quaking RNA binding protein disrupts the expression of genes associated with astrocyte maturation in mouse brain.

Quaking RNA binding protein (QKI) is essential for oligodendrocyte development as myelination requires myelin basic protein mRNA regulation and localization by the cytoplasmic isoforms (e.g., QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we define the targets of QKI in the mouse brain via CLIPseq and we show that QKI-6 binds 3'UTRs of a subset of astrocytic mRNAs. Binding is also enriched near stop codons, mediated partially by QKI-binding motifs (QBMs), yet spreads to adjacent sequences. Using a viral approach for mosaic, astrocyte-specific gene mutation with simultaneous translating RNA sequencing (CRISPR-TRAPseq), we profile ribosome associated mRNA from QKI-null astrocytes in the mouse brain. This demonstrates a role for QKI in stabilizing CLIP-defined direct targets in astrocytes in vivo and further shows that QKI mutation disrupts the transcriptional changes for a discrete subset of genes associated with astrocyte maturation.

Control of neural development and function by glial neuroligins.

Neuroligins are a family of cell adhesion molecules, which are best known for their functions as postsynaptic components of the trans-synaptic neurexin-neuroligin complexes. Neuroligins are highly conserved across evolution with important roles in the formation, maturation and function of synaptic structures. Mutations in the genes that encode for neuroligins have been linked to a number of neurodevelopmental disorders such as autism and schizophrenia, which stem from synaptic pathologies. Owing to their essential functions in regulating synaptic connectivity and their link to synaptic dysfunction in disease, previous studies on neuroligins have focused on neurons. Yet a recent work reveals that neuroligins are also expressed in the central nervous system by glial cells, such as astrocytes and oligodendrocytes, and perform important roles in controlling synaptic connectivity in a non-cell autonomous manner. In this review, we will highlight these recent findings demonstrating the important roles of glial neuroligins in regulating the development and connectivity of healthy and diseased brains.

Different Mixed Astrocyte Populations Derived from Embryonic Stem Cells Have Variable Neuronal Growth Support Capacities.

Central nervous system injury often leads to functional impairment due, in part, to the formation of an inhibitory glial scar following injury that contributes to poor regeneration. Astrocytes are the major cellular components of the glial scar, which has led to the belief that they are primarily inhibitory following injury. Recent work has challenged this by demonstrating that some astrocytes are required for spinal cord regeneration and astrocytic roles in recovery depend on their phenotype. In this work, two mixed populations containing primarily either fibrous or protoplasmic astrocytes were derived from mouse embryonic stem cells (mESCs). Motoneuron and V2a interneuron growth on live cultures, freeze-lysed cultures, or decellularized extracellular matrix (ECM) from astrocytes were assessed. Both neuronal populations were found to extend significantly longer neurites on protoplasmic-derived substrates than fibrous-derived substrates. Interestingly, neurons extended longer neurites on protoplasmic-derived ECM than fibrous-derived ECM. ECM proteins were compared with in vivo astrocyte expression profiles, and it was found that the ESC-derived ECMs were enriched for astrocyte-specific proteins. Further characterization revealed that protoplasmic ECM had significantly higher levels of axon growth promoting proteins, while fibrous ECM had significantly higher levels of proteins that inhibit axon growth. Supporting this observation, knockdown of spondin-1 improved neurite growth on fibrous ECM, while laminin α5 and γ1 knockdown decreased neurite growth on protoplasmic ECM. These methods allow for scalable production of specific astrocyte subtype-containing populations with different neuronal growth support capacities, and can be used for further studies of the functional importance of astrocyte heterogeneity.

A Comprehensive Analysis of Cell Type-Specific Nuclear RNA From Neurons and Glia of the Brain.

BACKGROUND: Studies in psychiatric genetics have identified >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 noncoding regions of the genome, including differential expression from loci that produce regulatory noncoding RNAs that function within the nucleus; however, the nuclear transcriptome of specific cell types in the brain has not been previously investigated. METHODS: We 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. Next, we characterized the unique expression of coding, noncoding, and intergenic RNAs in the mature mouse brain with RNA-Seq and validation with independent methods. RESULTS: We found diverse expression across the cell types of all classes of RNAs, including long noncoding RNAs, several of which were confirmed as highly enriched in the nuclei of specific cell types using anatomic methods. We also discovered several examples of cell type-specific expression of tandem gene fusions, and we report the first cell type-specific expression of circular RNAs-a neuron-specific and nuclear-enriched RNA arising from the gene Hnrnpu. CONCLUSIONS: These data provide an important resource for studies evaluating the function of various noncoding RNAs in the brain, including noncoding RNAs that may play a role in psychiatric disease.