Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.

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.

Comparative Characterization of Phosphatidic Acid Sensors and Their Localization during Frustrated Phagocytosis.

Phosphatidic acid (PA) is the simplest phospholipid naturally existing in living organisms, but it constitutes only a minor fraction of total cell lipids. PA has attracted considerable attention because it is a phospholipid precursor, a lipid second messenger, and a modulator of membrane shape, and it has thus been proposed to play key cellular functions. The dynamics of PA in cells and in subcellular compartments, however, remains an open question. The recent generation of fluorescent probes for PA, by fusing GFP to PA-binding domains, has provided direct evidence for PA dynamics in different intracellular compartments. Here, three PA sensors were characterized in vitro, and their preferences for different PA species in particular lipidic environments were compared. In addition, the localization of PA in macrophages during frustrated phagocytosis was examined using these PA sensors and was combined with a lipidomic analysis of PA in intracellular compartments. The results indicate that the PA sensors display some preferences for specific PA species, depending on the lipid environment, and the localization study in macrophages revealed the complexity of intracellular PA dynamics.

Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages.

Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages.

Genetically encoded probes for phosphatidic acid.

In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267.4 macrophages. Interestingly, the two probes show a differential distribution in these cell types, indicating that they may have different affinities for PA or recognize different pools of PA. In conclusion, the development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.

Ral isoforms are implicated in Fc gamma R-mediated phagocytosis: activation of phospholipase D by RalA.

Phagocytosis is an essential element of the immune response permitting the elimination of pathogens, cellular debris, apoptotic cells, and tumor cells. Recently, both phospholipase D (PLD) isoforms, PLD1 and PLD2, were shown to be necessary for efficient FcgammaR-mediated phagocytosis. In this study, we investigated the role of a potential PLD regulator, the Ral GTPases RalA and RalB, in murine RAW 264.7 macrophages. Both Ral isoforms are expressed in macrophages and are transiently activated following FcgammaR stimulation. When Ral expression levels were varied using Ral mutants or interference RNA, phagocytosis assays revealed that Ral isoforms have antagonistic effects; RalA is a positive modulator, whereas RalB plays a negative role. We then focused on RalA and its possible relationship with PLD. The increase in PLD activity that occurs when phagocytosis is stimulated was inhibited in cells with reduced RalA protein, but it was unaffected by reduced levels of RalB. Furthermore, in macrophages transfected with dsRed-RalA and GFP-PLD1 or GFP-PLD2, RalA colocalized with PLD1 and PLD2 at the phagocytic cup during phagosome formation. Additional results obtained from immunoprecipitation of PLD from macrophages transfected with myc-RalA and hemagglutinin-tagged PLD1 or PLD2 indicated an enhanced interaction of RalA with both PLD isoforms during phagocytic stimulation. The increase in RalA and PLD1 interaction was transient and correlated with the time course of RalA activation. These findings reveal a novel pathway involving RalA and PLD in the regulation of FcgammaR-mediated phagocytosis.

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.

Doppel induces autophagic stress in prion protein-deficient Purkinje cells.

The ectopic expression of the prion protein homologue Doppel (Dpl) in brain neurons causes progressive cerebellar Purkinje cell death in prion protein-deficient Ngsk mice (NP0/0). The neurotoxicity caused by Dpl involves Bax-dependent apoptotic pathways as well as other yet to be characterized cell death mechanisms in the NP0/0 Purkinje cells. These neurons display increased amounts of several autophagy-related molecules such as the scrapie-responsive gene one (Scrg1), LC3B-II and p62 without showing any changes in mRNA expression; in addition, autolysosomes accumulate in all neuronal compartments including axon terminals. This suggests that Dpl toxicity provokes impairment in the autophagic flux, which may trigger apoptosis in these neurons, similar to the way neurodegeneration is thought to occur in Alzheimer and prion diseases. Purkinje cells feature early axonal autophagy in both NP0/0 and GluRdelta2Lc mutants, but no signs of autophagic flux impairment are evident in GluRdelta2Lc Lurcher, suggesting that different pathogenic stimuli (i.e., Dpl versus GluRdelta2Lc) trigger different cell death modalities involving autophagy and apoptosis in the same type of neuron. The interplay between these multiple pathways of programmed cell death needs to be further investigated in animal models of neurodegenerative diseases in order to develop new therapeutic approaches.

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis.

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.

Fibrillar prion peptide (106-126) and scrapie prion protein hamper phagocytosis in microglia.

The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106-126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106-126) is internalized and processed. However, both synthetic prion peptide PrP(106-126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106-126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106-126) did not prevent F-actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.

F-actin does not modulate the initial steps of the protein kinase C activation process in living nerve cells.

Actin is a major substrate for protein kinase C (PKC) and PKC is considered a modulator of the actin network. In addition in vitro studies (Biochemistry 39 (2000) 271) have suggested that all PKC isoforms bind to actin during the process of activation of the enzyme. To test the physiological significance of such a coupling we used living PC12 cells and primary cultures of cerebellar granule cells. When PC12 cells were treated with either latrunculin B, which impairs actin polymerization, or phalloidin, which stabilizes actin filaments, we observed a significant reduction of the [Ca2+]i response revealed by Fura-2 fluorescence, while the PKC conformational changes followed by Fim-1 fluorescence were unaffected. The responses induced either by cell depolarization or muscarinic receptor activation were similarly affected by the toxin treatment of PC12 cells. In cerebellar granule cells the [Ca2+]i response induced by KCl depolarization was increased by latrunculin treatment, whereas no effect was observed on the PKC response. Latrunculin had no effect on the NMDA-induced responses in these cells. Finally we also show that the response induced by a long-lasting depolarization, which mimics stimulation leading to neuronal plasticity, was not significantly altered by latrunculin or phalloidin treatment of the cells. These results suggest that the actin network is not involved in the initial steps of the PKC activation process in living nerve cells.

Noradrenergic, but not Adrenergic Chromaffin Cells in the Adrenal Gland Express Neuromodulin (GAP-43).

Neuroendocrine chromaffin cells of the adrenal gland express certain molecular markers either transiently during development or permanently. In the present study, the expression of neuromodulin (GAP-43), a neuronal protein often associated with neurite outgrowth, was examined in adult adrenals. Neuromodulin was detected by Western blot analysis in extracts of both rat adrenals and cultured bovine chromaffin cells, and was localized in situ in a subpopulation of chromaffin cells, as well as in nerve fibres and Schwann cells. The use of anti-tyrosine hydroxylase or anti-phenylethanolamine N-methyltransferase antibodies in combination with anti-neuromodulin antibodies in double immunofluorescent labelling of cryostat sections of rat glands demonstrated that neuromodulin is expressed by noradrenergic, and not by adrenergic chromaffin cells. The results provide further evidence that neuromodulin is not limited to neurons; it is also expressed in a subpopulation of neuroendocrine chromaffin cells. Neuromodulin may play a role in the development of the adrenal medulla or in the specific regulation of noradrenalin secretion from chromaffin cells.