Immune synaptopathies: how maternal immune activation impacts synaptic function during development.

In the last two decades, the term synaptopathy has been largely used to underline the concept that impairments of synaptic structure and function are the major determinant of brain disorders, including neurodevelopmental disorders. This notion emerged from the progress made in understanding the genetic architecture of neurodevelopmental disorders, which highlighted the convergence of genetic risk factors onto molecular pathways specifically localized at the synapse. However, the multifactorial origin of these disorders also indicated the key contribution of environmental factors. It is well recognized that inflammation is a risk factor for neurodevelopmental disorders, and several immune molecules critically contribute to synaptic dysfunction. In the present review, we highlight this concept, which we define by the term "immune-synaptopathy," and we discuss recent evidence suggesting a bi-directional link between the genetic architecture of individuals and maternal activation of the immune system in modulating brain developmental trajectories in health and disease.

Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications.

Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson's disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation.

Unique properties of dually innervated dendritic spines in pyramidal neurons of the somatosensory cortex uncovered by 3D correlative light and electron microscopy.

Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.

Trans-synaptic interactions of ionotropic glutamate receptors.

Trans-synaptic interactions organize the multiple steps of synaptic development and are critical to generate fully functional neuronal circuits. While trans-synaptic interactions are primarily mediated by cell adhesion molecules (CAMs), some directly involve ionotropic glutamate receptors (iGluRs). Here, we review the expanding extracellular and trans-synaptic proteome of iGluRs. We discuss the role of these molecular networks in specifying the formation of excitatory and inhibitory circuits and in the input-specific recruitment of iGluRs at synapses in various cell types and brain regions. We also shed light on human-specific mutations affecting iGluR-mediated trans-synaptic interactions that may provide unique features to the human brain and contribute to its susceptibility to neurodevelopmental disorders. Together, these data support a view in which iGluR function goes far beyond fast excitatory synaptic transmission by shaping the wiring and the functional properties of neural circuits.

Ubiquitin and Ubiquitin-Like Proteins in the Critical Equilibrium between Synapse Physiology and Intellectual Disability.

Posttranslational modifications (PTMs) represent a dynamic regulatory system that precisely modulates the functional organization of synapses. PTMs consist in target modifications by small chemical moieties or conjugation of lipids, sugars or polypeptides. Among them, ubiquitin and a large family of ubiquitin-like proteins (UBLs) share several features such as the structure of the small protein modifiers, the enzymatic cascades mediating the conjugation process, and the targeted aminoacidic residue. In the brain, ubiquitination and two UBLs, namely sumoylation and the recently discovered neddylation orchestrate fundamental processes including synapse formation, maturation and plasticity, and their alteration is thought to contribute to the development of neurological disorders. Remarkably, emerging evidence suggests that these pathways tightly interplay to modulate the function of several proteins that possess pivotal roles for brain homeostasis as well as failure of this crosstalk seems to be implicated in the development of brain pathologies. In this review, we outline the role of ubiquitination, sumoylation, neddylation, and their functional interplay in synapse physiology and discuss their implication in the molecular pathogenesis of intellectual disability (ID), a neurodevelopmental disorder that is frequently comorbid with a wide spectrum of brain pathologies. Finally, we propose a few outlooks that might contribute to better understand the complexity of these regulatory systems in regard to neuronal circuit pathophysiology.

Trans-Synaptic Signaling through the Glutamate Receptor Delta-1 Mediates Inhibitory Synapse Formation in Cortical Pyramidal Neurons.

Fine orchestration of excitatory and inhibitory synaptic development is required for normal brain function, and alterations may cause neurodevelopmental disorders. Using sparse molecular manipulations in intact brain circuits, we show that the glutamate receptor delta-1 (GluD1), a member of ionotropic glutamate receptors (iGluRs), is a postsynaptic organizer of inhibitory synapses in cortical pyramidal neurons. GluD1 is selectively required for the formation of inhibitory synapses and regulates GABAergic synaptic transmission accordingly. At inhibitory synapses, GluD1 interacts with cerebellin-4, an extracellular scaffolding protein secreted by somatostatin-expressing interneurons, which bridges postsynaptic GluD1 and presynaptic neurexins. When binding to its agonist glycine or D-serine, GluD1 elicits non-ionotropic postsynaptic signaling involving the guanine nucleotide exchange factor ARHGEF12 and the regulatory subunit of protein phosphatase 1 PPP1R12A. Thus, GluD1 defines a trans-synaptic interaction regulating postsynaptic signaling pathways for the proper establishment of cortical inhibitory connectivity and challenges the dichotomy between iGluRs and inhibitory synaptic molecules.

Epilepsy and intellectual disability linked protein Shrm4 interaction with GABABRs shapes inhibitory neurotransmission.

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.

SRGAP2 and Its Human-Specific Paralog Co-Regulate the Development of Excitatory and Inhibitory Synapses.

The proper function of neural circuits requires spatially and temporally balanced development of excitatory and inhibitory synapses. However, the molecular mechanisms coordinating excitatory and inhibitory synaptogenesis remain unknown. Here we demonstrate that SRGAP2A and its human-specific paralog SRGAP2C co-regulate the development of excitatory and inhibitory synapses in cortical pyramidal neurons in vivo. SRGAP2A promotes synaptic maturation, and ultimately the synaptic accumulation of AMPA and GABAA receptors, by interacting with key components of both excitatory and inhibitory postsynaptic scaffolds, Homer and Gephyrin. Furthermore, SRGAP2A limits the density of both types of synapses via its Rac1-GAP activity. SRGAP2C inhibits all identified functions of SRGAP2A, protracting the maturation and increasing the density of excitatory and inhibitory synapses. Our results uncover a molecular mechanism coordinating critical features of synaptic development and suggest that human-specific duplication of SRGAP2 might have contributed to the emergence of unique traits of human neurons while preserving the excitation/inhibition balance.

An investigation of the effect of membrane curvature on transmembrane-domain dependent protein sorting in lipid bilayers.

Sorting of membrane proteins within the secretory pathway of eukaryotic cells is a complex process involving discrete sorting signals as well as physico-chemical properties of the transmembrane domain (TMD). Previous work demonstrated that tail-anchored (TA) protein sorting at the interface between the Endoplasmic Reticulum (ER) and the Golgi complex is exquisitely dependent on the length and hydrophobicity of the transmembrane domain, and suggested that an imbalance between TMD length and bilayer thickness (hydrophobic mismatch) could drive long TMD-containing proteins into curved membrane domains, including ER exit sites, with consequent export of the mismatched protein out of the ER. Here, we tested a possible role of curvature in TMD-dependent sorting in a model system consisting of Giant Unilamellar Vesicles (GUVs) from which narrow membrane tubes were pulled by micromanipulation. Fluorescent TA proteins differing in TMD length were incorporated into GUVs of uniform lipid composition or made of total ER lipids, and TMD-dependent sorting and diffusion, as well as the bending rigidity of bilayers made of microsomal lipids, were investigated. Long and short TMD-containing constructs were inserted with similar orientation, diffused equally rapidly in GUVs and in tubes pulled from GUVs, and no difference in their final distribution between planar and curved regions was detected. These results indicate that curvature alone is not sufficient to drive TMD-dependent sorting at the ER-Golgi interface, and set the basis for the investigation of the additional factors that must be required.

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER.

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward-bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi-to-plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal-deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail-anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy- and Rab6-dependent, and Rab6 inhibition accelerated signal-deleted VSVG's transport to the cell surface. Our results extend the dynamic bi-directional relationship between the Golgi and ER to include surface-directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.

Visualization of endoplasmic reticulum subdomains in cultured cells.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

Analysis of neuromuscular junctions and effects of anabolic steroid administration in the SOD1G93A mouse model of ALS.

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.

Restructured endoplasmic reticulum generated by mutant amyotrophic lateral sclerosis-linked VAPB is cleared by the proteasome.

VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56S-VAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein.

Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.

A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum.

VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-resident tail-anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell-free systems and in cultured mammalian cells were used to investigate P56S-VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post-translationally into ER membranes in a manner indistinguishable from the wild-type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild-type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a approximately 30 nm-thick electron-dense cytosolic layer. Our results demonstrate that the ALS-linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons.