Evolution of brain MRI lesions in paediatric myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD) and its relevance to disease course.

BACKGROUND: Lesion resolution is often observed in children with myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD), and asymptomatic lesions are less commonly reported in MOGAD than in multiple sclerosis (MS). OBJECTIVE: We aimed to evaluate brain MRI changes over time in paediatric MOGAD. METHODS: Retrospective study in eight UK paediatric neuroscience centres. Acute brain MRI and available follow-up MRIs were reviewed. Predictors for lesion dynamic were evaluated using multivariable regression and Kaplan-Meier survival analyses were used to predict risk of relapse, disability and MOG-Ab status. RESULTS: 200 children were included (MOGAD 97; MS 103). At first MRI post attack, new symptomatic and asymptomatic lesions were seen more often in MS versus MOGAD (52/103 vs 28/97; p=0.002 and 37/103 vs 11/97; p<0.001); 83% of patients with MOGAD showed at least one lesion's resolution at first follow-up scan, and 23% had normal MRI. Only 1 patient with MS had single lesion resolution; none had normal MRI. Disappearing lesions in MOGAD were seen in 40% after the second attack, 21% after third attack and none after the fourth attack.New lesions at first follow-up scan were associated with increased likelihood of relapse (p=0.02) and persistent MOG-Ab serostatus (p=0.0016) compared with those with no new lesions. Plasma exchange was associated with increased likelihood of lesion resolution (p=0.01). Longer time from symptom onset to steroids was associated with increased likelihood of new lesions; 50% increase at 20 days (p=0.01). CONCLUSIONS: These striking differences in lesion dynamics between MOGAD and MS suggest greater potential to repair. Early treatment with steroids and plasma exchange is associated with reduced likelihood of new lesions.

Measurement of phospholipase C by monitoring inositol phosphates using [³H]inositol labeling protocols in permeabilized cells.

Data on the production of inositol phosphates is a useful complement to measurements of intracellular Ca(2+). The basic principle is labeling of the inositol lipids by growing the appropriate cell line in culture in the presence of [3H]inositol for 2-3 days to reach labeling equilibrium. Lithium ions at 10 mM inhibits the degradation of inositol phosphates to free inositol and is used to trap the inositol in the inositol polyphosphate forms. Inositol phosphates can be separated with ease from free inositol by using anion exchange chromatography. A method capable of easily processing approximately 40-60 samples in a single day is presented.

Phosphatidylinositol- and phosphatidylcholine-transfer activity of PITPbeta is essential for COPI-mediated retrograde transport from the Golgi to the endoplasmic reticulum.

Vesicles formed by the COPI complex function in retrograde transport from the Golgi to the endoplasmic reticulum (ER). Phosphatidylinositol transfer protein beta (PITPbeta), an essential protein that possesses phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) lipid transfer activity is known to localise to the Golgi and ER but its role in these membrane systems is not clear. To examine the function of PITPbeta at the Golgi-ER interface, RNA interference (RNAi) was used to knockdown PITPbeta protein expression in HeLa cells. Depletion of PITPbeta leads to a decrease in PtdIns(4)P levels, compaction of the Golgi complex and protection from brefeldin-A-mediated dispersal to the ER. Using specific transport assays, we show that anterograde traffic is unaffected but that KDEL-receptor-dependent retrograde traffic is inhibited. This phenotype can be rescued by expression of wild-type PITPbeta but not by mutants defective in docking, PtdIns transfer and PtdCho transfer. These data demonstrate that the PtdIns and PtdCho exchange activity of PITPbeta is essential for COPI-mediated retrograde transport from the Golgi to the ER.

A unique phosphatidylinositol 4-phosphate 5-kinase is activated by ADP-ribosylation factor in Plasmodium falciparum.

In eukaryotes, calcium signalling has been linked to hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The final enzyme in the synthesis of this phosphoinositide, a Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), is activated by the small G protein ADP-ribosylation factor 1 (ARF1). In mammals, the ARF-PIP5K pathway is a key regulator of cell motility, secretion and cell signalling. We report the characterisation of a unique, putative bifunctional PIP5K in the human malaria parasite Plasmodium falciparum. The protein comprises a C-terminal, functional PIP5K domain with catalytic specificity for phosphatidylinositol 4-phosphate. The recombinant enzyme is activated by ARF1 but not phosphatidic acid. The protein also incorporates an unusual N-terminal domain with potential helix-loop-helix EF-hand-like motifs that is a member of the neuronal calcium sensor family (NCS). Intriguingly, NCS-1 has been shown to stimulate phosphatidylinositol 4-phosphate synthesis by activating mammalian and yeast phosphatidylinositol 4-kinase beta in vitro in a calcium-dependent manner. The unexpected physical attachment of an NCS-like domain to the plasmodial PIP5K might reflect a unique functional link between the calcium and PtdIns(4,5)P(2) pathways allowing modulation of PtdIns(4,5)P(2) production in response to changes in intracellular calcium concentrations within the parasite.

Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases.

The X-linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5-phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi-associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5-phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5-phosphatase activity of OCRL1 after membrane recruitment.

Differential expression of a C-terminal splice variant of phosphatidylinositol transfer protein beta lacking the constitutive-phosphorylated Ser262 that localizes to the Golgi compartment.

Mammalian PITPbeta (phosphatidylinositol transfer protein beta) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser262 present in the C-terminus of PITPbeta is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPbeta (PITPbeta-sp2) where the C-terminal 15 amino acids of PITPbeta-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPbeta-sp1 is the product of the first 11 exons, whereas PITPbeta-sp2 is a product of the first 10 exons followed by the twelfth exon--exon 11 being 'skipped'. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPbeta-sp2 being unable to transport SM. PITPbeta is ubiquitously expressed, with the highest amounts of PITPbeta found in HL60 cells and in rat liver; HL60 cells express only PITPbeta-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITPbeta-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITPbeta-sp1 are present. In contrast, PITPbeta-sp2 lacks the constitutively phosphorylated Ser262 (replaced with glutamine). Nonetheless, both PITPbeta variants localize to the Golgi and, moreover, dephosphorylation of Ser262 of PITPbeta-sp1 does not affect its Golgi localization. The presence of PITPbeta sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITPbeta gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

Measurement of phospholipase C by monitoring inositol phosphates using [³H]inositol-labeling protocols in permeabilized cells.

Hormones, neurotransmitters, chemoattractants, and growth factors all elicit intracellular responses on binding to cell surface receptors by activating inositol phospholipid-specific phospholipase C (PLC). Activated PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP(2)), a minor membrane phospholipid, to form two second messengers, diacylglycerol (DAG) and inositol (1,4,5)trisphosphate [Ins(1,4,5,)P(3)]. DAG is a direct activator of protein kinase C isozymes, and Ins(1,4,5)P(3) mobilizes intracellular Ca(2+). G protein-coupled receptors couple to the PLC-ß family via G proteins, and tyrosine kinase receptors activate PLC-γ isozymes. Regardless of the PLC isozyme activated, the product is invariantly Ins(1,4,5)P(3).

Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein alpha.

Mammalian phosphatidylinositol transfer protein alpha (PITP) is an intracellular lipid transporter with a binding site that can accommodate a single molecule of phosphatidylinositol (PI) or phosphatidylcholine (PC). Phospholipids are a heterogeneous population of molecular species that can be distinguished by their characteristic headgroups as well as their acyl chains at the sn-1 and sn-2 position. In this study, we have defined the acyl chain preference for PITPalpha when presented with a total population of cellular lipids. Recombinant PITPalpha loaded with bacterial lipid, phosphatidylglycerol (PG), was incubated with permeabilised HL60 cells, followed by recovery of PITPalpha by affinity chromatography. Lipids extracted from the PITPalpha were analysed by tandem electrospray ionisation mass spectrometry (ESI-MS) and showed total exchange of acquired bacterial lipids for HL60 cellular PI and PC. Detailed comparison of the molecular species composition of bound phospholipids with those in whole cells permitted the assessment of selectivity of acyl chain binding. For both phospholipid classes, progressive fractional enrichments in bound species possessing shorter acyl chains were apparent with a preference order: 16:1>16:0>18:1>18:0>20:4. A recapitulation of this specificity order was also seen from a dramatically altered range of molecular species present in HL60 cells enriched with arachidonate over many weeks of culture. We speculate that short-chain, saturate-binding preferences under both conditions may reflect properties in vivo. This is consistent with target cell membranes actively remodelling newly delivered phospholipids after transport rather than relying on the transport of the specific molecular species conventionally found in mammalian membranes.

Phosphorylation of a distinct structural form of phosphatidylinositol transfer protein alpha at Ser166 by protein kinase C disrupts receptor-mediated phospholipase C signaling by inhibiting delivery of phosphatidylinositol to membranes.

Phosphatidylinositol transfer protein alpha (PITPalpha) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) beta and gamma signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPalpha at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165-172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPalpha. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPalpha from rat brain or in vitro phosphorylated PITPalpha demonstrated that phosphorylated PITPalpha is structurally distinct from the non-phosphorylated form. Phosphorylated PITPalpha was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPalpha structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.

Structure-function analysis of human [corrected] phosphatidylinositol transfer protein alpha bound to phosphatidylinositol.

Phosphatidylinositol transfer protein alpha (PITPalpha) selectively transports and promotes exchange of phosphatidylinositol (PI) and phosphatidylcholine (PC) between lipid bilayers. In higher eukaryotes PITPalpha is required for cellular functions such as phospholipase C-mediated signaling, regulated exocytosis, and secretory vesicle formation. We have determined the crystal structure of human PITPalpha bound to its physiological ligand, PI, at 2.95 A resolution. The structure identifies the critical side chains within the lipid-headgroup binding pocket that define the exquisite specificity for PI. Mutational analysis of the PI binding pocket is in good agreement with the structural data and allows manipulation of functional properties of PITPalpha. Surprisingly, there are no major conformational differences between PI- and PC-loaded PITPalpha, despite previous predictions. In the crystal, PITPalpha-PI is dimeric, with two identical dimers in the asymmetric unit. The dimer interface masks precisely the sequence we identify as contributing to PITPalpha membrane interaction. Our structure represents a soluble, transport-competent form of PI-loaded PITPalpha.

Mechanism of ADP ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is required both as a substrate for the generation of lipid-derived second messengers as well as an intact lipid for many aspects of cell signaling, endo- and exocytosis, and reorganization of the cytoskeleton. ADP ribosylation factor (ARF) proteins regulate PI(4,5)P(2) synthesis, and here we have examined whether this is due to direct activation of Type I phosphatidylinositol 4-phosphate (PIP) 5-kinase or indirectly by phosphatidate (PA) derived from phospholipase D (PLD) in HL60 cells. ARF1 and ARF6 are both expressed in HL60 cells and can be depleted from the cells by permeabilization. Both ARFs increased the levels of PIP(2) (PI(4,5)P(2), PI(3,5)P(2), or PI(3,4)P(2) isomers) at the expense of PIP when added back to permeabilized cells. The PIP(2) could be hydrolyzed by phospholipase C, identifying it as PI(4,5)P(2). However, the ARF1-stimulated pool of PI(4,5)P(2) was accessible to the phospholipase C more efficiently in the presence of phosphatidylinositol transfer protein-alpha. To examine the role of PLD in the regulation of PI(4,5)P(2) synthesis, we used butanol to diminish the PLD-derived PA. PI(4,5)P(2) synthesis stimulated by ARF1 was not blocked by 0.5% butanol but could be blocked by 1.5% butanol. Although 0.5% butanol was optimal for maximal transphosphatidylation, PA production was still detectable. In contrast, 1.5% butanol was found to inhibit the activation of PLD by ARF1 and also decrease PIP levels by 50%. Thus the toxicity of 1.5% butanol prevented us from concluding whether PA was an important factor in raising PI(4,5)P(2) levels. To circumvent the use of alcohols, an ARF1 point mutant was identified (N52R-ARF1) that could selectively activate PIP 5-kinase alpha activity but not PLD activity. N52R-ARF1 was still able to increase PI(4,5)P(2) levels but at reduced efficiency. We therefore conclude that both PA derived from the PLD pathway and ARF proteins, by directly activating PIP 5-kinase, contribute to the regulation of PI(4,5)P(2) synthesis at the plasma membrane in HL60 cells.