Release of VAMP5-positive extracellular vesicles by retinal Müller glia in vivo.

Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo. We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro. Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neurons in vivo.

Niemann-Pick type C disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action in neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the CNS and intravitreal injections as mode of drug administration. Coupling CD to gold nanoparticles allowed us to trace its intracellular location. We report that CD enters the endosomal-lysosomal system of neurons in vivo and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Our data suggest that CD induces a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

Cytoplasmic FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and inhibitory synaptic defects.

Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These results indicate that FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, likely relevant also for other neurodegenerative diseases characterized by FUS mislocalization.

Protocol for electron microscopy ultrastructural localization of the fusogenic lipid phosphatidic acid on plasma membrane sheets from chromaffin cells.

The glycerophospholipid phosphatidic acid (PA) is a key player in regulated exocytosis, but little is known about its localization at the plasma membrane. Here, we provide a protocol for precisely determining the spatial distribution of PA at exocytotic sites by electron microscopy. Using primary bovine chromaffin cells expressing a PA sensor (Spo20p-GFP), we describe the process for cell stimulation and detergent-free preparation of plasma membrane sheets. The protocol can be applied to other cell models and to distinct membrane lipids. For complete details on the use and execution of this protocol, please refer to Tanguy et al. (2020).

Short-term plasticity at cerebellar granule cell to molecular layer interneuron synapses expands information processing.

Information processing by cerebellar molecular layer interneurons (MLIs) plays a crucial role in motor behavior. MLI recruitment is tightly controlled by the profile of short-term plasticity (STP) at granule cell (GC)-MLI synapses. While GCs are the most numerous neurons in the brain, STP diversity at GC-MLI synapses is poorly documented. Here, we studied how single MLIs are recruited by their distinct GC inputs during burst firing. Using slice recordings at individual GC-MLI synapses of mice, we revealed four classes of connections segregated by their STP profile. Each class differentially drives MLI recruitment. We show that GC synaptic diversity is underlain by heterogeneous expression of synapsin II, a key actor of STP and that GC terminals devoid of synapsin II are associated with slow MLI recruitment. Our study reveals that molecular, structural and functional diversity across GC terminals provides a mechanism to expand the coding range of MLIs.

Elimination of all redundant climbing fiber synapses requires granule cells in the postnatal cerebellum.

Different afferent synapse populations interact to control the specificity of connections during neuronal circuit maturation. The elimination of all but one climbing-fiber onto each Purkinje cell during the development of the cerebellar cortex is a particularly well studied example of synaptic refinement. The suppression of granule cell precursors by X irradiation during postnatal days 4 to 7 prevents this synaptic refinement, indicating a critical role for granule cells. Several studies of cerebellar development have suggested that synapse elimination has a first phase which is granule cell-independent and a second phase which is granule cell-dependent. In this study, we show that sufficiently-strong irradiation restricted to postnatal days 5 or 6 completely abolishes climbing fiber synaptic refinement, leaving the olivo-cerebellar circuit in its immature configuration in the adult, with up to 5 climbing fibers innervating the Purkinje cell in some cases. This implies that the putative early phase of climbing fiber synapse elimination can be blocked by irradiation-induced granule cell loss if this loss is sufficiently large, and thus indicates that the entire process of climbing fiber synapse elimination requires the presence of an adequate number of granule cells. The specific critical period for this effect appears to be directly related to the timing of Purkinje cell and granule cell development in different cerebellar lobules, indicating a close, spatiotemporal synchrony between granule-cell development and olivo-cerebellar synaptic maturation.

Cerebellar compartmentation of prion pathogenesis.

In prion diseases, the brain lesion profile is influenced by the prion "strain" properties, the invasion route to the brain, and still unknown host cell-specific parameters. To gain insight into those endogenous factors, we analyzed the histopathological alterations induced by distinct prion strains in the mouse cerebellum. We show that 22L and ME7 scrapie prion proteins (PrP22L , PrPME7 ), but not bovine spongiform encephalopathy PrP6PB1 , accumulate in a reproducible parasagittal banding pattern in the cerebellar cortex of infected mice. Such banding pattern of PrP22L aggregation did not depend on the neuroinvasion route, but coincided with the parasagittal compartmentation of the cerebellum mostly defined by the expression of zebrins, such as aldolase C and the excitatory amino acid transporter 4, in Purkinje cells. We provide evidence that Purkinje cells display a differential, subtype-specific vulnerability to 22L prions with zebrin-expressing Purkinje cells being more resistant to prion toxicity, while in stripes where PrP22L accumulated most zebrin-deficient Purkinje cells are lost and spongiosis accentuated. In addition, in PrP22L stripes, enhanced reactive astrocyte processes associated with microglia activation support interdependent events between the topographic pattern of Purkinje cell death, reactive gliosis and PrP22L accumulation. Finally, we find that in preclinically-ill mice prion infection promotes at the membrane of astrocytes enveloping Purkinje cell excitatory synapses, upregulation of tumor necrosis factor-α receptor type 1 (TNFR1), a key mediator of the neuroinflammation process. These overall data show that Purkinje cell sensitivity to prion insult is locally restricted by the parasagittal compartmentation of the cerebellum, and that perisynaptic astrocytes may contribute to prion pathogenesis through prion-induced TNFR1 upregulation.

Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions.

UNLABELLED: Aging and pathologic conditions cause intracellular aggregation of macromolecules and the dysfunction and degeneration of neurons, but the mechanisms are largely unknown. Prime examples are lysosomal storage disorders such as Niemann-Pick type C (NPC) disease, where defects in the endosomal-lysosomal protein NPC1 or NPC2 cause intracellular accumulation of unesterified cholesterol and other lipids leading to neurodegeneration and fatal neurovisceral symptoms. Here, we investigated the impact of NPC1 deficiency on rodent neurons using pharmacologic and genetic models of the disease. Improved ultrastructural detection of lipids and correlative light and electron microscopy identified lamellar inclusions as the subcellular site of cholesterol accumulation in neurons with impaired NPC1 activity. Immunogold labeling combined with transmission electron microscopy revealed the presence of CD63 on internal lamellae and of LAMP1 on the membrane surrounding the inclusions, indicating their origins from intraluminal vesicles of late endosomes and of a lysosomal compartment, respectively. Lamellar inclusions contained cell-intrinsic cholesterol and surface-labeled GM1, indicating the incorporation of plasma membrane components. Scanning electron microscopy revealed that the therapeutic drug candidate ß-cyclodextrin induces the subplasmalemmal location of lamellar inclusions and their subsequent release to the extracellular space. In parallel, ß-cyclodextrin mediated the NPC1-independent redistribution of cholesterol within neurons and thereby abolished a deleterious cycle of enhanced cholesterol synthesis and its intracellular accumulation, which was indicated by neuron-specific transcript analysis. Our study provides new mechanistic insight into the pathologic aggregation of macromolecules in neurons and suggests exocytosis as cellular target for its therapeutic reversal. SIGNIFICANCE STATEMENT: Many neurodegenerative diseases involve pathologic accumulation of molecules within neurons, but the subcellular location and the cellular impact are often unknown and therapeutic approaches lacking. We investigated these questions in the lysosomal storage disorder Niemann-Pick type C (NPC), where a defect in intracellular cholesterol transport causes loss of neurons and fatal neurovisceral symptoms. Here, we identify lamellar inclusions as the subcellular site of lipid accumulation in neurons, we uncover a vicious cycle of cholesterol synthesis and accretion, which may cause gradual neurodegeneration, and we reveal how ß-cyclodextrin, a potential therapeutic drug, reverts these changes. Our study provides new mechanistic insight in NPC disease and uncovers new targets for therapeutic approaches.

A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation.

Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, we investigated the function of the low-duplicated CYP715 cytochrome P450 gene family that appeared early in seed plants and evolved under strong negative selection. Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower-insect interaction.

Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis.

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament-bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2-induced actin bundling is apparently essential for generating active exocytotic sites.

Ataxia with cerebellar lesions in mice expressing chimeric PrP-Dpl protein.

Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.

Relevance of exocytotic glutamate release from retinal glia.

Glial cells release molecules that influence brain development, function, and disease. Calcium-dependent exocytosis has been proposed as potential release mechanism in astroglia, but the physiological relevance of "gliotransmission" in vivo remains controversial. We focused on the impact of glial exocytosis on sensory transduction in the retina. To this end, we generated transgenic mice to block exocytosis by Cre recombinase-dependent expression of the clostridial botulinum neurotoxin serotype B light chain, which cleaves vesicle-associated membrane protein 1-3. Ubiquitous and neuronal toxin expression caused perinatal lethality and a reduction of synaptic transmission thus validating transgene function. Toxin expression in Müller cells inhibited vesicular glutamate release and impaired glial volume regulation but left retinal histology and visual processing unaffected. Our model to study gliotransmission in vivo reveals specific functions of exocytotic glutamate release in retinal glia.

Selective recapture of secretory granule components after full collapse exocytosis in neuroendocrine chromaffin cells.

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-ß-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.

S100A10-mediated translocation of annexin-A2 to SNARE proteins in adrenergic chromaffin cells undergoing exocytosis.

In neuroendocrine cells, annexin-A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)-containing lipid microdomains that are required for calcium-regulated exocytosis. As soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin-A2-induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin-A2 to the plasma membrane, where it colocalizes with SNAP-25 and S100A10. Cross-linking experiments performed in stimulated chromaffin cells indicate that annexin-A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle-associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin-A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin-A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)-bisphosphate (PIP(2)) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin-A2-mediated lipid microdomains and SNAREs during exocytosis.

Autophagy and cell death of Purkinje cells overexpressing Doppel in Ngsk Prnp-deficient mice.

In Ngsk prion protein (PrP)-deficient mice (NP(0/0)), ectopic expression of PrP-like protein Doppel (Dpl) in central neurons induces significant Purkinje cell (PC) death resulting in late-onset ataxia. NP(0/0) PC death is partly prevented by either knocking-out the apoptotic factor BAX or overexpressing the anti-apoptotic factor BCL-2 suggesting that apoptosis is involved in Dpl-induced death. In this study, Western blotting and immunohistofluorescence show that both before and during significant PC loss, the scrapie-responsive gene 1 (Scrg1)--potentially associated with autophagy--and the autophagic markers LC3B and p62 increased in the NP(0/0) PCs whereas RT-PCR shows stable mRNA expression, suggesting that the degradation of autophagic products is impaired in NP(0/0) PCs. At the ultrastructural level, autophagic-like profiles accumulated in somatodendritic and axonal compartments of NP(0/0), but not wild-type PCs. The most robust autophagy was observed in NP(0/0) PC axon compartments in the deep cerebellar nuclei suggesting that it is initiated in these axons. Our previous and present data indicate that Dpl triggers autophagy and apoptosis in NP(0/0) PCs. As observed in amyloid neurodegenerative diseases, upregulation of autophagic markers as well as extensive accumulation of autophagosomes in NP(0/0) PCs are likely to reflect a progressive dysfunction of autophagy that could trigger apoptotic cascades.

Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage.

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.

Reinnervation of late postnatal Purkinje cells by climbing fibers: neosynaptogenesis without transient multi-innervation.

Synaptic partner selection and refinement of projections are important in the development of precise and functional neuronal connections. We investigated the formation of new synaptic connections in a relatively mature system to test whether developmental events can be recapitulated at later stages (i.e., after the mature synaptic organization has been established), using a model of postlesional reinnervation in the olivo-cerebellar pathway. During the development of this pathway, synaptic connections between climbing fibers (CFs) and Purkinje cells (PCs) are diffuse and redundant before synapse elimination refines the pattern. The regression of CFs during the first 2 postnatal weeks in the rat leads to mono-innervation of each PC. After unilateral transection of the rat olivo-cerebellar pathway and intracerebellar injection of BDNF 24 h after lesion, axons from the remaining inferior olive can sprout into the deafferented hemicerebellum and establish new contacts with denervated PCs at later developmental stages. We found that these contacts are first established on somatic thorns before the CFs translocate to the PC dendrites, recapitulating the morphological steps of normal CF-PC synaptogenesis, but on a relatively mature PC. However, electrophysiology of PC reinnervation by transcommissural CFs in these animals showed that each PC is reinnervated by only one CF. This mono-innervation contrasts with the reinnervation of grafted immature PCs in the same cerebellum. Our results provide evidence that relatively mature PCs do not receive several olivary afferents during late reinnervation, suggesting a critical role of the target cell state in the control of CF-PC synaptogenesis. Thus, synapse exuberance and subsequent elimination are not a prerequisite to reach a mature relationship between synaptic partners.