Yannick Bailly

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.

Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

NMDA RECEPTOR CONTRIBUTION TO THE CLIMBING FIBER RESPONSE IN THE ADULT MOUSE PURKINJE CELL

Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at ∼21 d after birth and control the complex spike waveform.

The Cerebellum Harbors a Circadian Oscillator Involved in Food Anticipation

The cerebellum participates in motor coordination as well as in numerous cerebral processes, including temporal discrimination. Animals can predict daily timing of food availability, as manifested by food-anticipatory activity under restricted feeding. By studying ex vivo clock gene expression by in situ hybridization and recording in vitro Per1-luciferase bioluminescence, we report that the cerebellum contains a circadian oscillator sensitive to feeding cues (i.e., whose clock gene oscillations are shifted in response to restricted feeding). Food-anticipatory activity was markedly reduced in mice injected intracerebroventricularly with an immunotoxin that depletes Purkinje cells (i.e., OX7-saporin). Mice bearing the hotfoot mutation (i.e., Grid2ho/ho) have impaired cerebellar circuitry and mild ataxic phenotype. Grid2ho/ho mice fed ad libitum showed regular behavioral rhythms and day–night variations of clock gene expression in the hypothalamus and cerebellum. When challenged with restricted feeding, however, Grid2ho/ho mice did not show any food-anticipatory rhythms, nor timed feeding-induced changes in cerebellar clock gene expression. In hypothalamic arcuate and dorsomedial nuclei, however, shifts in Per1 expression in response to restricted feeding were similar in cerebellar mutant and wild-type mice. Furthermore, plasma corticosterone and metabolites before mealtime did not differ between cerebellar mutant and wild-type mice. Together, these data define a role for the cerebellum in the circadian timing network and indicate that the cerebellar oscillator is required for anticipation of mealtime.

Synaptic β-amyloid precursor proteins increase with learning capacity in rats

Abstract The precursor proteins of Alzheimers disease β -amyloid peptide, the β -amyloid precursor protein isoforms, comprise a family of neuronal proteins with synaptic localization whose physiological roles in brain are poorly understood. One possible role for synaptic proteins is involvement in neuronal plasticity. After exposure to an enriched environment compared to impoverished conditions, rats exhibited superior cognitive capacity. Up to ∼four-fold increased overall levels of β -amyloid precursor proteins were found in cortical/subcortical tissue of the enriched animals displaying significantly more synapses immunoreactive for the different β -amyloid precursor protein isoforms ( β -amyloid precursor protein 695 - and β -amyloid precursor protein 751/770 ) in hippocampus and adjacent occipital cortex. This correlation thus provides in vivo evidence for an association of β -amyloid precursor proteins with plastic changes induced by complex environment with consequences for cognitive functions and suggests that impaired β -amyloid precursor protein metabolism at synapses might contribute to brain dysfunction in Alzheimers disease.

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.

PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases

α-secretase–mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-β (Aβ) peptides, and α-cleavage of cellular prion protein (PrPC) prevents its conversion into misfolded, pathogenic prions (PrPSc). The mechanisms leading to decreased α-secretase activity in Alzheimers and prion disease remain unclear. Here, we find that tumor necrosis factor-α–converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrPSc or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide–dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aβ deposition and in the brains of individuals with Alzheimers disease. PDK1 induces phosphorylation and caveolin-1–mediated internalization of TACE. This dysregulation of TACE increases PrPSc and Aβ accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrPC and TNFR1, and attenuates PrPSc- and Aβ-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrPSc infection and in APP-transgenic mice reduces Alzheimers disease-like pathology and memory impairment.

The vascular and epithelial serotonergic innervation of the actinopterygian gill filament with special reference to the trout, Salmo gairdneri

SummaryAntibodies against serotonin and 5-methoxytryptamine reveal indolaminergic neurons innervating the proximal part of the efferent arterial vasculature, the filament epithelia, the central venous sinus, and certain other serotonergic cells of the teleost gill filament. In the same area, acetylcholinesterase-positive and indoleaminergic neurons have already been described. We propose that these populations of neurons belong to a single neuronal type but express different agents. Our current results support this idea; in particular, they point to the presence of a single type of serotonin-containing nerve terminal, impinging on vascular smooth muscle. These results are in agreement with physiological data showing (i) the existence of non-cholinergic (atropine-resistant) vasoconstriction of the gill vasculature after nerve stimulation, and (ii) a potent vasoconstrictory action of infused serotonin. In addition, the above-mentioned serotonergic neurons have synaptic contacts with catecholaminergic nerve fibers, suggesting the existence of a modulatory relationship between the sympathetic and the cranial autonomic nerves supplying the teleost gill. Finally, these neurons show morphological relationships with a previously undescribed type of branchialserotonergic cell. The role of the parasympathetic nerve plexus of the teleost gill filament in the control of respiration and ionoregulation is discussed.

Synaptic prion protein immuno‐reactivity in the rodent cerebellum

The cellular prion protein PrPc is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrPc is converted to infectious protease‐resistant isoforms PrPres or prions. Prions provoque predictable and distinctive patterns of PrPres accumulation and neurodegeneration depending on the prion strain and on regional cell‐specific properties modulating PrPc affinity for infectious PrPres in the host brain. Synaptolysis and synaptic accumulation of PrPres during PrP‐related diseases suggests that the synapses could be primary sites able to propagate PrPres and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno‐cytochemical analysis shows that distinct types of synapses display differential expression of PrPc, suggesting that synapse‐specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrPc remain unknown, the concentration of PrPc almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies. Microsc. Res. Tech. 50:66–75, 2000.

Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain.

Expression of the cellular prion protein (PrPc) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrPc is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrPc in the mouse brain, the immunocytochemical localization of PrPc was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrPc was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrPc antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrPc immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrPc. Transgenic mice expressing PrP‐GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases. J. Comp. Neurol. 473:244–269, 2004.