Mapping and targeting of C1ql1-expressing cells in the mouse.

The C1Q complement protein C1QL1 is highly conserved in mammals where it is expressed in various tissues including the brain. This secreted protein interacts with Brain-specific Angiogenesis Inhibitor 3, BAI3/ADGRB3, and controls synapse formation and maintenance. C1ql1 is expressed in the inferior olivary neurons that send projections to cerebellar Purkinje cells, but its expression in the rest of the brain is less documented. To map C1ql1 expression and enable the specific targeting of C1ql1-expressing cells, we generated a knockin mouse model expressing the Cre recombinase under the control of C1ql1 regulatory sequences. We characterized the capacity for Cre-driven recombination in the brain and mapped Cre expression in various neuron types using reporter mouse lines. Using an intersectional strategy with viral particle injections, we show that this mouse line can be used to target specific afferents of Purkinje cells. As C1ql1 is also expressed in other regions of the brain, as well as in other tissues such as adrenal glands and colon, our mouse model is a useful tool to target C1ql1-expressing cells in a broad variety of tissues.

Determination of sex in cDNA samples from mice.

The need to take sex into account in biomedical research is now recognized and mandated by funding institutions. In laboratory rodents, such as mice, sexing is usually performed anatomically or by genotyping using multiplex or simplex PCR techniques on genomic DNA. Here we present a simple RT-PCR-based method targeting Kdm5c and Kdm5d to determine genetic sex in mouse cDNA samples, allowing for retrospective sex determination.

Synapse Formation and Function Across Species: Ancient Roles for CCP, CUB, and TSP-1 Structural Domains.

The appearance of synapses was a crucial step in the creation of the variety of nervous systems that are found in the animal kingdom. With increased complexity of the organisms came a greater number of synaptic proteins. In this review we describe synaptic proteins that contain the structural domains CUB, CCP, or TSP-1. These domains are found in invertebrates and vertebrates, and CUB and CCP domains were initially described in proteins belonging to the complement system of innate immunity. Interestingly, they are found in synapses of the nematode C. elegans, which does not have a complement system, suggesting an ancient function. Comparison of the roles of CUB-, CCP-, and TSP-1 containing synaptic proteins in various species shows that in more complex nervous systems, these structural domains are combined with other domains and that there is partial conservation of their function. These three domains are thus basic building blocks of the synaptic architecture. Further studies of structural domains characteristic of synaptic proteins in invertebrates such as C. elegans and comparison of their role in mammals will help identify other conserved synaptic molecular building blocks. Furthermore, this type of functional comparison across species will also identify structural domains added during evolution in correlation with increased complexity, shedding light on mechanisms underlying cognition and brain diseases.

Modified climbing fiber/Purkinje cell synaptic connectivity in the cerebellum of the neonatal phencyclidine model of schizophrenia.

Environmental perturbations during the first years of life are a major factor in psychiatric diseases. Phencyclidine (PCP), a drug of abuse, has psychomimetic effects, and neonatal subchronic administration of PCP in rodents leads to long-term behavioral changes relevant for schizophrenia. The cerebellum is increasingly recognized for its role in diverse cognitive functions. However, little is known about potential cerebellar changes in models of schizophrenia. Here, we analyzed the characteristics of the cerebellum in the neonatal subchronic PCP model. We found that, while the global cerebellar cytoarchitecture and Purkinje cell spontaneous spiking properties are unchanged, climbing fiber/Purkinje cell synaptic connectivity is increased in juvenile mice. Neonatal subchronic administration of PCP is accompanied by increased cFos expression, a marker of neuronal activity, and transient modification of the neuronal surfaceome in the cerebellum. The largest change observed is the overexpression of Ctgf, a gene previously suggested as a biomarker for schizophrenia. This neonatal increase in Ctgf can be reproduced by increasing neuronal activity in the cerebellum during the second postnatal week using chemogenetics. However, it does not lead to increased climbing fiber/Purkinje cell connectivity in juvenile mice, showing the complexity of PCP action. Overall, our study shows that administration of the drug of abuse PCP during the developmental period of intense cerebellar synaptogenesis and circuit remodeling has long-term and specific effects on Purkinje cell connectivity and warrants the search for this type of synaptic changes in psychiatric diseases.

Sushi domain-containing protein 4 controls synaptic plasticity and motor learning.

Fine control of protein stoichiometry at synapses underlies brain function and plasticity. How proteostasis is controlled independently for each type of synaptic protein in a synapse-specific and activity-dependent manner remains unclear. Here, we show that Susd4, a gene coding for a complement-related transmembrane protein, is expressed by many neuronal populations starting at the time of synapse formation. Constitutive loss-of-function of Susd4 in the mouse impairs motor coordination adaptation and learning, prevents long-term depression at cerebellar synapses, and leads to misregulation of activity-dependent AMPA receptor subunit GluA2 degradation. We identified several proteins with known roles in the regulation of AMPA receptor turnover, in particular ubiquitin ligases of the NEDD4 subfamily, as SUSD4 binding partners. Our findings shed light on the potential role of SUSD4 mutations in neurodevelopmental diseases.

Longitudinal Analyses of Blood Transcriptome During Conversion to Psychosis.

The biological processes associated with the onset of schizophrenia remain largely unknown. Current hypotheses favor gene × environment interactions as supported by our recent report about DNA methylation changes during the onset of psychosis. Here, we conducted the first longitudinal transcriptomic analysis of blood samples from 31 at-risk individuals who later converted to psychosis and 63 at-risk individuals who did not. Individuals were followed for a maximum of 1 year. Blood samples were collected at baseline and at the end of follow-up and individuals served as their own controls. Differentially expressed genes between the 2 groups were identified using the RNA sequencing of an initial discovery subgroup (n = 15 individuals). The most promising results were replicated using high-throughput real-time qPCR in the whole cohort (n = 94 individuals). We identified longitudinal changes in 4 brain-expressed genes based on RNAseq analysis. One of these genes (CPT1A) was replicated in the whole cohort. The previously observed hypermethylation in NRP1 and GSTM5 during the onset of psychosis correlated with a decrease in corresponding gene expression. RNA sequencing also identified 2 co-expression networks that were impaired after conversion compared with baseline-the Wnt pathway including AKT1, CPT1A and semaphorins, and the Toll-like receptor pathway, related to innate immunity. This longitudinal study of transcriptomic changes in individuals with at-risk mental state revealed alterations during conversion to psychosis in pathways and genes relevant to schizophrenia. These results may be a first step toward better understanding psychosis onset. They may also help to identify new biomarkers and targets for disease-modifying therapeutic strategies.

Smooth 2D manifold extraction from 3D image stack.

Three-dimensional fluorescence microscopy followed by image processing is routinely used to study biological objects at various scales such as cells and tissue. However, maximum intensity projection, the most broadly used rendering tool, extracts a discontinuous layer of voxels, obliviously creating important artifacts and possibly misleading interpretation. Here we propose smooth manifold extraction, an algorithm that produces a continuous focused 2D extraction from a 3D volume, hence preserving local spatial relationships. We demonstrate the usefulness of our approach by applying it to various biological applications using confocal and wide-field microscopy 3D image stacks. We provide a parameter-free ImageJ/Fiji plugin that allows 2D visualization and interpretation of 3D image stacks with maximum accuracy.

The immunoglobulin-like superfamily member IGSF3 is a developmentally regulated protein that controls neuronal morphogenesis.

The establishment of a functional brain depends on the fine regulation and coordination of many processes, including neurogenesis, differentiation, dendritogenesis, axonogenesis, and synaptogenesis. Proteins of the immunoglobulin-like superfamily (IGSF) are major regulators during this sequence of events. Different members of this class of proteins play nonoverlapping functions at specific developmental time-points, as shown in particular by studies of the cerebellum. We have identified a member of the little studied EWI subfamily of IGSF, the protein IGSF3, as a membrane protein expressed in a neuron specific- and time-dependent manner during brain development. In the cerebellum, it is transiently found in membranes of differentiating granule cells, and is particularly concentrated at axon terminals. There it co-localizes with other IGSF proteins with well-known functions in cerebellar development: TAG-1 and L1. Functional analysis shows that IGSF3 controls the differentiation of granule cells, more precisely axonal growth and branching. Biochemical experiments demonstrate that, in the developing brain, IGSF3 is in a complex with the tetraspanin TSPAN7, a membrane protein mutated in several forms of X-linked intellectual disabilities. In cerebellar granule cells, TSPAN7 promotes axonal branching and the size of TSPAN7 clusters is increased by downregulation of IGSF3. Thus IGSF3 is a novel regulator of neuronal morphogenesis that might function through interactions with multiple partners including the tetraspanin TSPAN7. This developmentally regulated protein might thus be at the center of a new signaling pathway controlling brain development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 75-92, 2017.

Adhesion GPCRs as Novel Actors in Neural and Glial Cell Functions: From Synaptogenesis to Myelination.

Adhesion G-protein-coupled receptors (aGPCRs) are emerging as key regulators of nervous system development and health. aGPCRs can regulate many aspects of neural development, including cell signaling, cell-cell and cell-matrix interactions, and, potentially, mechanosensation. Here, we specifically focus on the roles of several aGPCRs in synapse biology, dendritogenesis, and myelinating glial cell development. The lessons learned from these examples may be extrapolated to other contexts in the nervous system and beyond.

The Secreted Protein C1QL1 and Its Receptor BAI3 Control the Synaptic Connectivity of Excitatory Inputs Converging on Cerebellar Purkinje Cells.

Precise patterns of connectivity are established by different types of afferents on a given target neuron, leading to well-defined and non-overlapping synaptic territories. What regulates the specific characteristics of each type of synapse, in terms of number, morphology, and subcellular localization, remains to be understood. Here, we show that the signaling pathway formed by the secreted complement C1Q-related protein C1QL1 and its receptor, the adhesion-GPCR brain angiogenesis inhibitor 3 (BAI3), controls the stereotyped pattern of connectivity established by excitatory afferents on cerebellar Purkinje cells. The BAI3 receptor modulates synaptogenesis of both parallel fiber and climbing fiber afferents. The restricted and timely expression of its ligand C1QL1 in inferior olivary neurons ensures the establishment of the proper synaptic territory for climbing fibers. Given the broad expression of C1QL and BAI proteins in the developing mouse brain, our study reveals a general mechanism contributing to the formation of a functional brain.

Cerebellum involvement in cortical sensorimotor circuits for the control of voluntary movements.

Sensorimotor integration is crucial to perception and motor control. How and where this process takes place in the brain is still largely unknown. Here we analyze the cerebellar contribution to sensorimotor integration in the whisker system of mice. We identify an area in the cerebellum where cortical sensory and motor inputs converge at the cellular level. Optogenetic stimulation of this area affects thalamic and motor cortex activity, alters parameters of ongoing movements and thereby modifies qualitatively and quantitatively touch events against surrounding objects. These results shed light on the cerebellum as an active component of sensorimotor circuits and show the importance of sensorimotor cortico-cerebellar loops in the fine control of voluntary movements.

Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge.

Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.

The biochemical anatomy of cortical inhibitory synapses.

Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.

Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse.

Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKgamma, are major unrecognized components of this synapse type. We demonstrate that MRCKgamma can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.

Survival of interneurons and parallel fiber synapses in a cerebellar cortex deprived of Purkinje cells: studies in the double mutant mouse Grid2Lc/+;Bax(-/-).

The Lurcher mutation in the Grid2 gene causes the cell autonomous death of virtually all cerebellar Purkinje cells and the target-related death of 90% of the granule cells and 60-75% of the olivary neurons. Inactivation of Bax, a pro-apoptotic gene of the Bcl-2 family, in heterozygous Lurcher mutants (Grid2Lc/+) rescues approximately 60% of the granule cells, but does not rescue Purkinje or olivary neurons. Given the larger size of the cerebellar molecular layer in Grid2Lc/+;Bax(-/-) double mutants compared to Grid2Lc/+ mutants, we analyzed the survival of the stellate and basket interneurons as well as the synaptic connectivity of parallel fibers originating from the surviving granule cells in the absence of their Purkinje cell targets in the Grid2Lc/+;Bax(-/-) cerebellum. Quantification showed a significantly higher density of interneurons ( approximately 60%) in the molecular layer of the Grid2Lc/+;Bax(-/-) mice compared to Grid2Lc/+, suggesting that interneurons are subject to a BAX-dependent target-related death in the Lurcher mutants. Furthermore, electron microscopy showed the normal ultrastructural aspect of a number of parallel fibers in the molecular layer of the Grid2Lc/+; Bax(-/-) double mutant mice and preserved their numerous synaptic contacts on interneurons, suggesting that interneurons could play a trophic role for axon terminals of surviving granule cells. Finally, parallel fibers varicosities in the double mutant established "pseudo-synapses" on glia as well as displayed autophagic profiles, suggesting that the connections established by the parallel fibers in the absence of their Purkinje cell targets were subject to a high turnover involving autophagy.

Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells.

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.

A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice.

Autophagy is a pathway for bulk degradation of subcellular constituents that is hyperactivated in many neurodegenerative conditions. It has been considered a second form of programmed cell death. Death of cerebellar Purkinje cells in lurcher animals is due to a mutation in GluRdelta2 that results in its constitutive activation. Here we have identified protein interactions between GluRdelta2, a novel isoform of a PDZ domain-containing protein (nPIST) that binds to this receptor, and Beclin1. nPIST and Beclin1 can synergize to induce autophagy. GluRdelta2(Lc), but not GluRdelta2(wt), can also induce autophagy. Furthermore, dying lurcher Purkinje cells contain morphological hallmarks of autophagic death in vivo. These results provide strong evidence that a direct link exists between GluRdelta2(Lc) receptor and stimulation of the autophagic pathway in dying lurcher Purkinje cells.