Control of neuronal excitation-inhibition balance by BMP-SMAD1 signalling.

Throughout life, neuronal networks in the mammalian neocortex maintain a balance of excitation and inhibition, which is essential for neuronal computation1,2. Deviations from a balanced state have been linked to neurodevelopmental disorders, and severe disruptions result in epilepsy3-5. To maintain balance, neuronal microcircuits composed of excitatory and inhibitory neurons sense alterations in neural activity and adjust neuronal connectivity and function. Here we identify a signalling pathway in the adult mouse neocortex that is activated in response to increased neuronal network activity. Overactivation of excitatory neurons is signalled to the network through an increase in the levels of BMP2, a growth factor that is well known for its role as a morphogen in embryonic development. BMP2 acts on parvalbumin-expressing (PV) interneurons through the transcription factor SMAD1, which controls an array of glutamatergic synapse proteins and components of perineuronal nets. PV-interneuron-specific disruption of BMP2-SMAD1 signalling is accompanied by a loss of glutamatergic innervation in PV cells, underdeveloped perineuronal nets and decreased excitability. Ultimately, this impairment of the functional recruitment of PV interneurons disrupts the cortical excitation-inhibition balance, with mice exhibiting spontaneous epileptic seizures. Our findings suggest that developmental morphogen signalling is repurposed to stabilize cortical networks in the adult mammalian brain.

A cell-type-specific alternative splicing regulator shapes synapse properties in a trans-synaptic manner.

The specification of synaptic properties is fundamental for the function of neuronal circuits. "Terminal selector" transcription factors coordinate terminal gene batteries that specify cell-type-specific properties. Moreover, pan-neuronal splicing regulators have been implicated in directing neuronal differentiation. However, the cellular logic of how splicing regulators instruct specific synaptic properties remains poorly understood. Here, we combine genome-wide mapping of mRNA targets and cell-type-specific loss-of-function studies to uncover the contribution of the RNA-binding protein SLM2 to hippocampal synapse specification. Focusing on pyramidal cells and somatostatin (SST)-positive GABAergic interneurons, we find that SLM2 preferentially binds and regulates alternative splicing of transcripts encoding synaptic proteins. In the absence of SLM2, neuronal populations exhibit normal intrinsic properties, but there are non-cell-autonomous synaptic phenotypes and associated defects in a hippocampus-dependent memory task. Thus, alternative splicing provides a critical layer of gene regulation that instructs specification of neuronal connectivity in a trans-synaptic manner.

Quantitative Detection of Protein Splice Variants by Selected Reaction Monitoring (SRM) Mass Spectrometry.

Molecular diversification of the cellular proteome through alternative splicing has emerged as an important biological principle. However, the lack of tools to specifically detect and quantify proteoforms (Smith et al., Nat Methods 10:186-187, 2013) is a major impediment to functional studies. Recently, biological mass spectrometry (MS) has undergone impressive advances (Mann, Nat Rev Mol Cell Biol 17:678, 2016), including the generation of a highly diverse set of biological applications (Aebersold and Mann, Nature 537:347-355, 2016), and has demonstrated to be an essential tool to address many biological questions (Savitski et al., Science 346:1255784, 2014; Rinner et al., Nat Methods 5:315-318, 2008). In particular, targeted LC-MS, with its high selectivity and specificity, is ideally suited for the precise and sensitive quantification of specific proteins and their proteoforms (Picotti and Aebersold, Nat Methods 9:555-566, 2012). We describe in detail the application of this workflow applied to dissect the molecular diversity of the synaptic adhesion proteins and their splicing-derived proteoforms (Schreiner et al., Elife 4:e07794, 2015).

Targeted proteoform mapping uncovers specific Neurexin-3 variants required for dendritic inhibition.

The diversification of cell adhesion molecules by alternative splicing is proposed to underlie molecular codes for neuronal wiring. Transcriptomic approaches mapped detailed cell-type-specific mRNA splicing programs. However, it has been hard to probe the synapse-specific localization and function of the resulting protein splice isoforms, or "proteoforms," in vivo. We here apply a proteoform-centric workflow in mice to test the synapse-specific functions of the splice isoforms of the synaptic adhesion molecule Neurexin-3 (NRXN3). We uncover a major proteoform, NRXN3 AS5, that is highly expressed in GABAergic interneurons and at dendrite-targeting GABAergic terminals. NRXN3 AS5 abundance significantly diverges from Nrxn3 mRNA distribution and is gated by translation-repressive elements. Nrxn3 AS5 isoform deletion results in a selective impairment of dendrite-targeting interneuron synapses in the dentate gyrus without affecting somatic inhibition or glutamatergic perforant-path synapses. This work establishes cell- and synapse-specific functions of a specific neurexin proteoform and highlights the importance of alternative splicing regulation for synapse specification.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

MyD88 signaling by neurons induces chemokines that recruit protective leukocytes to the virus-infected CNS.

Viral encephalitis initiates a series of immunological events in the brain that can lead to brain damage and death. Astrocytes express IFN-ß in response to neurotropic infection, whereas activated microglia produce proinflammatory cytokines and accumulate at sites of infection. Here, we observed that neurotropic vesicular stomatitis virus (VSV) infection causes recruitment of leukocytes into the central nervous system (CNS), which requires MyD88, an adaptor of Toll-like receptor and interleukin-1 receptor signaling. Infiltrating leukocytes, and in particular CD8+ T cells, protected against lethal VSV infection of the CNS. Reconstitution of MyD88, specifically in neurons, restored chemokine production in the olfactory bulb as well as leukocyte recruitment into the infected CNS and enhanced survival. Comparative analysis of the translatome of neurons and astrocytes verified neurons as the critical source of chemokines, which regulated leukocyte infiltration of the infected brain and affected survival.

Rescue of oxytocin response and social behaviour in a mouse model of autism.

A fundamental challenge in developing treatments for autism spectrum disorders is the heterogeneity of the condition. More than one hundred genetic mutations confer high risk for autism, with each individual mutation accounting for only a small fraction of cases1-3. Subsets of risk genes can be grouped into functionally related pathways, most prominently those involving synaptic proteins, translational regulation, and chromatin modifications. To attempt to minimize this genetic complexity, recent therapeutic strategies have focused on the neuropeptides oxytocin and vasopressin4-6, which regulate aspects of social behaviour in mammals7. However, it is unclear whether genetic risk factors predispose individuals to autism as a result of modifications to oxytocinergic signalling. Here we report that an autism-associated mutation in the synaptic adhesion molecule Nlgn3 results in impaired oxytocin signalling in dopaminergic neurons and in altered behavioural responses to social novelty tests in mice. Notably, loss of Nlgn3 is accompanied by a disruption of translation homeostasis in the ventral tegmental area. Treatment of Nlgn3-knockout mice with a new, highly specific, brain-penetrant inhibitor of MAP kinase-interacting kinases resets the translation of mRNA and restores oxytocin signalling and social novelty responses. Thus, this work identifies a convergence between the genetic autism risk factor Nlgn3, regulation of translation, and oxytocinergic signalling. Focusing on such common core plasticity elements might provide a pragmatic approach to overcoming the heterogeneity of autism. Ultimately, this would enable mechanism-based stratification of patient populations to increase the success of therapeutic interventions.

AAV2/DJ-mediated alpha-synuclein overexpression in the rat substantia nigra as early stage model of Parkinson's disease.

Parkinson's disease (PD) is pathologically characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and alpha-synucleinopathy. We mimic the disease pathology with overexpression of either the human α-syn wildtype (α-syn-WT) or E46K mutant form (α-syn-E46K) in DA neurons of the SNpc in adult rats using AAV2/DJ as a viral vector for the first time. Transduction efficiency was compared to an equal virus titer expressing the green fluorescent protein (GFP). Motor skills of all animals were evaluated in the cylinder and amphetamine-induced rotation test over a total time period of 12 weeks. Additionally, stereological quantification of DA cells and striatal fiber density measurements were performed every 4 weeks after injection. Rats overexpressing α-syn-WT showed a progressive loss of DA neurons with 40% reduction after 12 weeks accompanied by a greater loss of striatal DA fibers. In contrast, α-syn-E46K led to this reduction after 4 weeks without further progress. Insoluble α-syn positive cytoplasmic inclusions were observed in both groups within DA neurons of the SNpc and VTA. In addition, both α-syn groups developed a characteristic worsening of the rotational behavior over time. However, only the α-syn-WT group reached statistically significant different values in the cylinder test. Summarizing these effects, we established a motor symptom animal model of PD by using AAV2/DJ in the brain for the first time. Thereby, overexpressing of α-syn-E46K mimicked a rather pre-symptomatic stage of the disease, while the α-syn-WT overexpressing animals imitated an early symptomatic stage of PD.

A Sam68-dependent alternative splicing program shapes postsynaptic protein complexes.

Alternative splicing is one of the key mechanisms to increase the diversity of cellular transcriptomes, thereby expanding the coding capacity of the genome. This diversity is of particular importance in the nervous system with its elaborated cellular networks. Sam68, a member of the Signal Transduction Associated RNA-binding (STAR) family of RNA-binding proteins, is expressed in the developing and mature nervous system but its neuronal functions are poorly understood. Here, we perform genome-wide mapping of the Sam68-dependent alternative splicing program in mice. We find that Sam68 is required for the regulation of a set of alternative splicing events in pre-mRNAs encoding several postsynaptic scaffolding molecules that are central to the function of GABAergic and glutamatergic synapses. These components include Collybistin (Arhgef9), Gephyrin (Gphn), and Densin-180 (Lrrc7). Sam68-regulated Lrrc7 variants engage in differential protein interactions with signalling proteins, thus, highlighting a contribution of the Sam68 splicing program to shaping synaptic complexes. These findings suggest an important role for Sam68-dependent alternative splicing in the regulation of synapses in the central nervous system.

Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology.

In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.

γ-Protocadherins Interact with Neuroligin-1 and Negatively Regulate Dendritic Spine Morphogenesis.

The 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules are critical for the elaboration of complex dendritic arbors in the cerebral cortex. Here, we provide evidence that the γ-Pcdhs negatively regulate synapse development by inhibiting the postsynaptic cell adhesion molecule, neuroligin-1 (Nlg1). Mice lacking all γ-Pcdhs in the forebrain exhibit significantly increased dendritic spine density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 γ-Pcdh isoforms. Co-expression of γ-Pcdhs inhibits the ability of Nlg1 to increase spine density and to induce presynaptic differentiation in hippocampal neurons in vitro. The γ-Pcdhs physically interact in cis with Nlg1 both in vitro and in vivo, and we present evidence that this disrupts Nlg1 binding to its presynaptic partner neurexin1ß. Together with prior work, these data identify a mechanism through which γ-Pcdhs could coordinate dendrite arbor growth and complexity with spine maturation in the developing brain.

Synapse biology in the 'circuit-age'-paths toward molecular connectomics.

The neural connectome is a critical determinant of brain function. Circuits of precisely wired neurons, and the features of transmission at the synapses connecting them, are thought to dictate information processing in the brain. While recent technological advances now allow to define the anatomical and functional neural connectome at unprecedented resolution, the elucidation of the molecular mechanisms that establish the precise patterns of connectivity and the functional characteristics of synapses has remained challenging. Here, we describe the power and limitations of genetic approaches in the analysis of mechanisms that control synaptic connectivity and function, and discuss how recent methodological developments in proteomics might be used to elucidate the molecular synaptic connectome that is at the basis of the neural connectome.

An alternative splicing switch shapes neurexin repertoires in principal neurons versus interneurons in the mouse hippocampus.

The unique anatomical and functional features of principal and interneuron populations are critical for the appropriate function of neuronal circuits. Cell type-specific properties are encoded by selective gene expression programs that shape molecular repertoires and synaptic protein complexes. However, the nature of such programs, particularly for post-transcriptional regulation at the level of alternative splicing is only beginning to emerge. We here demonstrate that transcripts encoding the synaptic adhesion molecules neurexin-1,2,3 are commonly expressed in principal cells and interneurons of the mouse hippocampus but undergo highly differential, cell type-specific alternative splicing. Principal cell-specific neurexin splice isoforms depend on the RNA-binding protein Slm2. By contrast, most parvalbumin-positive (PV+) interneurons lack Slm2, express a different neurexin splice isoform and co-express the corresponding splice isoform-specific neurexin ligand Cbln4. Conditional ablation of Nrxn alternative splice insertions selectively in PV+ cells results in elevated hippocampal network activity and impairment in a learning task. Thus, PV-cell-specific alternative splicing of neurexins is critical for neuronal circuit function.

Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function.

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis.

Protein Kinase C Phosphorylation of a γ-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization.

The γ-protocadherins (γ-Pcdhs) are a family of 22 adhesion molecules with multiple critical developmental functions, including the proper formation of dendritic arbors by forebrain neurons. The γ-Pcdhs bind to and inhibit focal adhesion kinase (FAK) via a constant C-terminal cytoplasmic domain shared by all 22 proteins. In cortical neurons lacking the γ-Pcdhs, aberrantly high activity of FAK and of PKC disrupts dendrite arborization. Little is known, however, about how γ-Pcdh function is regulated by other factors. Here we show that PKC phosphorylates a serine residue situated within a phospholipid binding motif at the shared γ-Pcdh C terminus. Western blots using a novel phospho-specific antibody against this site suggest that a portion of γ-Pcdh proteins is phosphorylated in the cortex in vivo. We find that PKC phosphorylation disrupts both phospholipid binding and the γ-Pcdh inhibition of (but not binding to) FAK. Introduction of a non-phosphorylatable (S922A) γ-Pcdh construct into wild-type cortical neurons significantly increases dendrite arborization. This same S922A construct can also rescue dendrite arborization defects in γ-Pcdh null neurons cell autonomously. Consistent with these data, introduction of a phosphomimetic (S/D) γ-Pcdh construct or treatment with a PKC activator reduces dendrite arborization in wild-type cortical neurons. Together, these data identify a novel mechanism through which γ-Pcdh control of a signaling pathway important for dendrite arborization is regulated.

Quantitative isoform-profiling of highly diversified recognition molecules.

Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands.

Targeted combinatorial alternative splicing generates brain region-specific repertoires of neurexins.

Molecular diversity of surface receptors has been hypothesized to provide a mechanism for selective synaptic connectivity. Neurexins are highly diversified receptors that drive the morphological and functional differentiation of synapses. Using a single cDNA sequencing approach, we detected 1,364 unique neurexin-α and 37 neurexin-ß mRNAs produced by alternative splicing of neurexin pre-mRNAs. This molecular diversity results from near-exhaustive combinatorial use of alternative splice insertions in Nrxn1α and Nrxn2α. By contrast, Nrxn3α exhibits several highly stereotyped exon selections that incorporate novel elements for posttranscriptional regulation of a subset of transcripts. Complexity of Nrxn1α repertoires correlates with the cellular complexity of neuronal tissues, and a specific subset of isoforms is enriched in a purified cell type. Our analysis defines the molecular diversity of a critical synaptic receptor and provides evidence that neurexin diversity is linked to cellular diversity in the nervous system.

Polymorphic receptors: neuronal functions and molecular mechanisms of diversification.

The formation of neuronal circuits is driven by complex developmental programs. A key feature of such programs is the precise spatiotemporal control of cell surface recognition molecules in genetically defined cells. Moreover, epigenetic modifications and alternative splicing processes have emerged that are not genetically predetermined but stochastic in nature. Recent studies have highlighted critical functions of such stochastic processes in neuronal wiring and neuronal self-recognition. In this review, we will illustrate recently identified principles that control the molecular diversity of neuronal cell surface receptors, and the function of such receptors in encoding a dynamic or stable neuronal identity.