Cartography of teneurin and latrophilin expression reveals spatiotemporal axis heterogeneity in the mouse hippocampus during development.

Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.

Essential Role of Latrophilin-1 Adhesion GPCR Nanoclusters in Inhibitory Synapses.

Latrophilin-1 (Lphn1, a.k.a. CIRL1 and CL1; gene symbol Adgrl1) is an Adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knockin/knockout mice for Lphn1 that contain an extracellular myc-epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but contributes to inhibitory synaptic connections.Significance Statement Previously, the Adhesion-GPCRs Latrophilin-2/Latrophilin-3 have been shown to mediate excitatory, but not inhibitory, synapse assembly onto discreet dendritic compartments of hippocampal pyramidal neurons. Here we now show that Latrophilin-1 is targeted to both excitatory and inhibitory hippocampal synapses. Unexpectedly, we find that Latrophilin-1 is selectively essential for directing inhibitory synaptic connections to the neuronal soma. Our work supports a model by which Latrophilins are widely used as organizers of synaptic connectivity that act on a subcellular level. In the light of recent findings connecting haploinsufficiency of Latrophilin-1 to a plethora of neurodevelopmental and neuropsychiatric disorders, our study contributes to our understanding of the molecular mechanisms of Latrophilins that need to be targeted in order to address various pathologies.

Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1.

Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

Spatial transcriptomics reveal neuron-astrocyte synergy in long-term memory.

Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.

Correction: Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

[This corrects the article DOI: 10.1371/journal.pone.0158295.].

Alternative splicing of latrophilin-3 controls synapse formation.

The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.

Hippocampal place code plasticity in CA1 requires postsynaptic membrane fusion.

Rapid delivery of glutamate receptors to the postsynaptic membrane via vesicle fusion is a central component of synaptic plasticity. However, it is unknown how this process supports specific neural computations during behavior. To bridge this gap, we combined conditional genetic deletion of a component of the postsynaptic membrane fusion machinery, Syntaxin3 (Stx3), in hippocampal CA1 neurons of mice with population in vivo calcium imaging. This approach revealed that Stx3 is necessary for forming the neural dynamics that support novelty processing, spatial reward memory and offline memory consolidation. In contrast, CA1 Stx3 was dispensable for maintaining aspects of the neural code that exist presynaptic to CA1 such as representations of context and space. Thus, manipulating postsynaptic membrane fusion identified computations that specifically require synaptic restructuring via membrane trafficking in CA1 and distinguished them from neural representation that could be inherited from upstream brain regions or learned through other mechanisms.

Neutral lysophosphatidylcholine mediates α-synuclein-induced synaptic vesicle clustering.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

Efficient generation of functional neurons from mouse embryonic stem cells via neurogenin-2 expression.

The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.

Neuronal γ-secretase regulates lipid metabolism, linking cholesterol to synaptic dysfunction in Alzheimer's disease.

Presenilin mutations that alter γ-secretase activity cause familial Alzheimer's disease (AD), whereas ApoE4, an apolipoprotein for cholesterol transport, predisposes to sporadic AD. Both sporadic and familial AD feature synaptic dysfunction. Whether γ-secretase is involved in cholesterol metabolism and whether such involvement impacts synaptic function remains unknown. Here, we show that in human neurons, chronic pharmacological or genetic suppression of γ-secretase increases synapse numbers but decreases synaptic transmission by lowering the presynaptic release probability without altering dendritic or axonal arborizations. In search of a mechanism underlying these synaptic impairments, we discovered that chronic γ-secretase suppression robustly decreases cholesterol levels in neurons but not in glia, which in turn stimulates neuron-specific cholesterol-synthesis gene expression. Suppression of cholesterol levels by HMG-CoA reductase inhibitors (statins) impaired synaptic function similar to γ-secretase inhibition. Thus, γ-secretase enables synaptic function by maintaining cholesterol levels, whereas the chronic suppression of γ-secretase impairs synapses by lowering cholesterol levels.

Combinatorial expression of neurexins and LAR-type phosphotyrosine phosphatase receptors instructs assembly of a cerebellar circuit.

Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit.

Postsynaptic synucleins mediate endocannabinoid signaling.

Endocannabinoids are among the most powerful modulators of synaptic transmission throughout the nervous system, and yet little is understood about the release of endocannabinoids from postsynaptic compartments. Here we report an unexpected finding that endocannabinoid release requires synucleins, key contributors to Parkinson's disease. We show that endocannabinoids are released postsynaptically by a synuclein-dependent and SNARE-dependent mechanism. Specifically, we found that synuclein deletion blocks endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wild-type but not of mutant α-synuclein. Whole-cell recordings and direct optical monitoring of endocannabinoid signaling suggest that the synuclein deletion specifically blocks endocannabinoid release. Given the presynaptic role of synucleins in regulating vesicle lifecycle, we hypothesize that endocannabinoids are released via a membrane interaction mechanism. Consistent with this hypothesis, postsynaptic expression of tetanus toxin light chain, which cleaves synaptobrevin SNAREs, also blocks endocannabinoid-dependent signaling. The unexpected finding that endocannabinoids are released via a synuclein-dependent mechanism is consistent with a general function of synucleins in membrane trafficking and adds a piece to the longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

Cerebellin-neurexin complexes instructing synapse properties.

Cerebellins (Cbln1-4) are secreted adaptor proteins that connect presynaptic neurexins (Nrxn1-3) to postsynaptic ligands (GluD1/2 for Cbln1-3 vs. DCC and Neogenin-1 for Cbln4). Classical studies demonstrated that neurexin-Cbln1-GluD2 complexes organize cerebellar parallel-fiber synapses, but the role of cerebellins outside of the cerebellum has only recently been clarified. In synapses of the hippocampal subiculum and prefrontal cortex, Nrxn1-Cbln2-GluD1 complexes strikingly upregulate postsynaptic NMDA-receptors, whereas Nrxn3-Cbln2-GluD1 complexes conversely downregulate postsynaptic AMPA-receptors. At perforant-path synapses in the dentate gyrus, in contrast, neurexin/Cbln4/Neogenin-1 complexes are essential for LTP without affecting basal synaptic transmission or NMDA- or AMPA-receptors. None of these signaling pathways are required for synapse formation. Thus, outside of the cerebellum neurexin/cerebellin complexes regulate synapse properties by activating specific downstream receptors.

Astrocytic Neuroligins Are Not Required for Synapse Formation or a Normal Astrocyte Cytoarchitecture.

Astrocytes exert multifarious roles in the formation, regulation, and function of synapses in the brain, but the mechanisms involved remain unclear. Interestingly, astrocytes abundantly express neuroligins, postsynaptic adhesion molecules that bind to presynaptic neurexins. A pioneering recent study reported that loss-of-function of neuroligins in astrocytes impairs excitatory synapse formation and astrocyte morphogenesis. This study suggested a crucial synaptic function for astrocytic neuroligins but was puzzling given that constitutive neuroligin deletions do not decrease excitatory synapse numbers. Thus, we here examined the function of astrocytic neuroligins using a rigorous conditional genetic approach with deletion of all major neuroligins (Nlgn1-3) in astrocytes. Our results show that early postnatal deletion of neuroligins from astrocytes has no effect on cortical or hippocampal synapses and does not alter the cytoarchitecture of astrocytes. Thus, astrocytic neuroligins are unlikely to shape synapse formation or astrocyte development but may perform other important functions in astrocytes.

A combinatorial code of neurexin-3 alternative splicing controls inhibitory synapses via a trans-synaptic dystroglycan signaling loop.

Disrupted synaptic inhibition is implicated in neuropsychiatric disorders, yet the molecular mechanisms that shape and sustain inhibitory synapses are poorly understood. Here, we show through rescue experiments performed using Neurexin-3 conditional knockout mice that alternative splicing at SS2 and SS4 regulates the release probability, but not the number, of inhibitory synapses in the olfactory bulb and prefrontal cortex independent of sex. Neurexin-3 splice variants that mediate Neurexin-3 binding to dystroglycan enable inhibitory synapse function, whereas splice variants that don't allow dystroglycan binding do not. Furthermore, a minimal Neurexin-3 protein that binds to dystroglycan fully sustains inhibitory synaptic function, indicating that trans-synaptic dystroglycan binding is necessary and sufficient for Neurexin-3 function in inhibitory synaptic transmission. Thus, Neurexin-3 enables a normal release probability at inhibitory synapses via a trans-synaptic feedback signaling loop consisting of presynaptic Neurexin-3 and postsynaptic dystroglycan.