extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine Psychology

text:
Antiribosomal-P autoantibodies from psychiatric lupus target a novel neuronal surface protein causing calcium influx and apoptosis

The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P–mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by a wide range of clinical manifestations and the production of a variety of autoantibodies frequently directed against intracellular components ( 1 ). In most cases, the relationship between particular autoantibodies and disease manifestations is obscure. This is especially true for the abnormalities of the central nervous system (CNS). Neuropsychiatric (NP) symptoms or cognitive decline can be found in diff erent series aff ecting a range from 6 to 95% of the patients ( 2 -5 ). The CNS compromise varies from mild to severe but can be the most devastating manifestation of the disease, often being diffi cult to diagnose and treat ( 3, 6 -9 ). In contrast with focal organic brain syndromes, such as strokes and seizures resulting from thrombotic and vasculitic alterations, the pathogenic mechanisms of diff use brain manifestations such as mood and anxiety disorders or psychosis, as well as cognitive dysfunction, remain largely unknown ( 3,8 ). Even without overt CNS symptoms, the brain of SLE patients presents nonfocal atrophy and cortical and subcortical functional alterations of unknown origin ( 10 ).
Several autoantibodies have been associated with NP-SLE. These include autoantibodies against antiribosomal-P proteins (anti-Ps) ( 11 ), The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confi rmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P -mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium infl ux in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.
lupus, occasionally extending it to severe depression and other CNS-diff use manifestations ( 8, 25, 31 -33 ). The serum titer of anti-P has been reported to follow the psychiatric manifestations ( 29 ). Anti-P antibodies have also been detected in the cerebro spinal fl uid of NP-SLE patients ( 34,35 ). Furthermore, a recent study reported that injection of anti-P antibodies into the mice brain provokes a depression-like behavior ( 36 ). All of these data constitute strong evidence for a pathogenic role of anti-P antibodies ( 30,37 ). However, several contradicting reports failed to fi nd any association with NP-SLE ( 5,26,38 ), thus creating a controversy. Methodological and clinical considerations could explain certain discrepancies but are in fact insuffi cient to dissipate the doubts raised on the actual role of anti-P antibodies in NP-SLE ( 2,5,25,37 ). Contributing to this uncertainty is the lack of explanatory mechanisms able to causally relate anti-P antibodies with CNS dysfunction ( 25 ). Actually, molecular, cellular, and physiological bases for the postulated anti-P neuropathogenicity remain unknown, and the direct eff ects of these antibodies on neurons have not been reported.
Anti-P autoantibodies recognize a C-terminal epitope of 22 amino acids shared by three highly conserved phosphoproteins called P proteins Ϫ P0 (38 kD), P1 (19 kD), and P2 (17 kD)of the large ribosomal subunit ( 23,24 ). Further refi nement has restricted the P epitope location to the last 11 C-terminal residues ( 39 ). Being intracellular, these ribosomal antigens are normally inaccessible to circulating antibodies. However, a crucial advance toward understanding the pathogenic role of anti-P antibodies has been the observation that they bind to the surface of several kinds of cells, including human neuroblastoma and hepatoma cells ( 40 ). The cell-surface target for anti-P antibodies is currently believed to be a 38-kD protein, which is assumed to correspond to a cell-surface form of the P0 ribosomal protein ( 37 ). Subsequent studies led to the notion that anti-P antibodies neurofi laments ( 12 ), microtubule-associated protein 2 ( 13 ), N -methyl-D-aspartate receptor (NMDAR) ( 14,15 ), endothelial cells ( 16 ), and neuronal cells ( 17 ) (for reviews see references 3,8 ). However, excluding anti-NMDAR antibodies, all of the other associations lack mechanistic explanations. Immunocomplex-mediated small vessel infl ammation, which typically leads to tissue injury in the kidney and other organs, seems not to be important in the psychiatric manifestations ( 8 ). Evidence from other autoimmune diseases indicates that certain autoantibodies can directly cause damage to neuronal physiology by interfering with the function of cell-surface elements such as receptors and ion channels ( 18 ). In SLE, this possibility has been suggested by the fi nding of a subset of anti -double-stranded DNA autoantibodies that cross react with NMDAR and induce neurotoxicity and cognitive alterations ( 19 -22 ). It thus seems likely that other autoantibodies, as yet unknown, could also display neuropathogenic properties by interacting with a suitable neuronal surface target to contribute to NP lupus.
Anti-P autoantibodies ( 23,24 ) have been viewed for a long time as the most reiterative but also controversial candidates for a neuropathogenic role in SLE ( 5,8,25,26 ). These antibodies are quite specifi c for SLE, but their reported prevalence varies widely from 6 -45%, seemingly depending on diff erences in methodology and ethnic backgrounds ( 5,26 ). There is also evidence that healthy individuals express anti-P antibodies masked by antiidiotypic antibodies, suggesting that anti-P antibodies could become detectable in SLE patients because of a loss of this regulatory network ( 27,28 ). In 1987, Bonfa et al. made the seminal observation that anti-P autoantibodies associate with lupus psychosis ( 29 ). Afterward, anti-P has also been associated with lupus hepatitis and nephritis ( 30 ), but its pathogenic role remains controversial. Several studies have confi rmed the clinical association between anti-P antibodies and psychiatric with affi nity-purifi ed anti-P antibodies ( ␣ -hP 11 ) shows reaction with the P ribosomal proteins P0 (38 kD), P1 (19 kD), and P2 (17 kD; lane 1), which is blocked by the P peptide (lane 2). (B) Anti-P staining of the neuronal surface. Brain cortical primary culture cells were incubated with ␣ -hP 11 antibody at 4 ° C, and fi xed and incubated with secondary FITC -anti -human antibody. Some neurons show intense surface staining. Bar, 10 m. (C) Detection of anti-P target at the surface of brain cortical neurons. Neurons in primary culture were metabolically labeled for 16 h with 100 Ci [ 35 S]methionine-cysteine, biotinylated at 4 ° C, and subjected to immunoprecipitation with either control P ( Ϫ ) serum from an SLE patient (lane 1) or ␣ -hP 11 antibodies (lane 2). The immunoprecipitated proteins were separated from the sepharose beads by heating in SDS buffer, and the biotinylated proteins were precipitated with immobilized neutravidin protein. A single high molecular mass protein (p331) is detected. precipitated with anti-P antibodies and then assessed the biotinylated proteins in the immunocomplexes by blotting with streptavidin conjugates ( 42,43,45 ). This stepwise order of the experiment ensured that the detected proteins are indeed biotinylated and, therefore, exposed at the cell surface. Only the p331 showed up in the cell surface of N2a cells ( Fig. 2 B ). might somehow penetrate into living cells and provoke deleterious eff ects ( 41 ). In this scenario, anti-P antibodies would have a generalized deleterious potential to cause dysfunctions in a variety of diff erent cells ( 30,37 ).
In this paper, we provide a novel mechanistic framework that is more specifi cally applicable to a neuropathogenic role for the anti-P antibodies. We demonstrate that a new nonribosomal protein present at the cell surface of certain neuronal populations is recognized by anti-P autoantibodies, with consequences for neuronal calcium homeostasis and survival.

RESULTS
A high molecular mass cell-surface protein (p331) exposes an extracellular P epitope In a previous paper, we found anti-P antibodies in 15% of our cohort of 141 SLE patients, including 2 with psychosis ( 33 ). From these two patients with psychosis, we isolated specifi c anti-P antibodies by affi nity chromatography using a synthetic P peptide of 11 residues (P 11 ) ( 39 ). Unless otherwise specifi ed, all of the experiments were performed indistinctly with either one of these sera, as both produced similar results. In the immunoblot, these affi nity-purifi ed human anti-P against P 11 ( ␣ -hP 11 ) antibodies showed reactivity with the three ribosomal P proteins: P0 (38 kD), P1 (19 kD), and P2 (17 kD; Fig. 1 A ), which was blocked by competition with the synthetic P 11 peptide, thus indicating specifi city. With these affi nity-purifi ed antibodies, we analyzed neurons in primary culture and neuroblastoma N2a cells, searching for cell-surface anti-P immunoreactivity and molecular targets. Two complementary approaches, biotinylation assays and direct cell-surface protein immunocapture and precipitation, which we had used in previous studies to detect proteins at the cell surface ( 42 -44 ), unexpectedly revealed not the described 38-kD protein ( 37 ) but a very high molecular mass protein ( > 200 kD) that exposes a cell-surface P epitope ( Figs. 1 and 2 ). We provisionally named this protein p331 based on its sequence data (see next section).
Indirect immunofl uorescence showed anti-P reactivity in rat brain cortex cells in primary culture. To avoid permeabilization during fi xation, we initially incubated intact cells with the antibodies at 4 ° C and, after washing and fi xing them, added the secondary antibody. Only certain neurons displayed strong fl uorescence staining ( Fig. 1 B ), suggesting that a subset of cortex neurons express a cell-surface P antigen, at least at immunofl uorescence-detectable levels. To identify the anti-P molecular target, we had to increase the sensitivity of the biotinylation assays by using metabolically labeled cells and phosphoroimaging detection ( 45 ). This strategy disclosed a single protein of high molecular mass (p331) in the neuronal surface ( Fig. 1 C ).
Rat neuroblastoma N2a cells gave coincident results, providing a more homogeneous cellular system that facilitated further analysis. These cells displayed strong and asymmetrically localized anti-P cell-surface staining ( Fig. 2 A ). Regular biotinylation assays indicated that most of this cell-surface immunoreactivity is attributable to the high molecular mass p331 antigen. After biotinylation of the cell surface, we initially immuno- molecular mass of 331 kD), which contains a calcium-binding domain, an anaphase-promoting complex 10 domain, a CUB domain, and two zinc-binding domains. It possesses 8 -11 hydrophobic regions, each encompassing 19 -23 residues, but its large size did not allow us to predict which of them correspond to membrane-spanning domains. Because it lacks an N-terminal signal peptide, its membrane insertion is most likely directed by an internal signal peptide that is also a transmembrane domain, similar to type II and to several polytopic proteins, but the actual signal peptide remains unpredictable by the available algorithms. Predicting transmembrane domains and discriminating them from signal peptides that currently look the same has inherent diffi culties, which can only be circumvented by experimental data ( 49,50 ). The sequence of NP055928 is available from The blocking eff ect of the P 11 peptide indicated that p331 contains a P epitope ( Fig. 2

, A and B ).
This unexpected p331 anti-P cell-surface target was further confi rmed by direct immunocapture from metabolically labeled cells. N2a cells were initially metabolically labeled and then incubated intact with ␣ -hP 11 antibodies at 4 ° C. The immunocomplexes formed at the cell surface were isolated by protein A -sepharose precipitation, as previously described ( 42,43 ). This approach also revealed p331 as the main, if not the only, protein exposing a P epitope at the cell surface ( Fig. 2 C ). Similar experiments made with whole serum instead of affi nity-purifi ed ␣ -hP 11 antibodies also demonstrated p331 as the cell-surface anti-P target ( Fig. 2 D ). Anti-P ( Ϫ ) sera showed no reaction with this p331 protein, whereas anti-P (+) sera from fi ve diff erent lupus patients, including one patient with lupus psychosis (lane 5) and four patients without NP symptoms (lanes 6 -9), immunoprecipitated only the p331 from the cell surface ( Fig. 2 D ). All of these results disclosed a new P antigen -bearing protein accessible to anti-P autoantibodies from the extracellular medium, expressed by at least certain neurons in the brain (as shown in Fig. 1 ). p331 is a novel integral membrane protein of unknown function expressed in brain neurons termed neuronal surface P antigens (NSPAs) Rat brain synaptosomal fractions highly enriched in synaptic regions ( 46 ) provided additional evidence of the presence of p331 in brain neurons. Only P (+) sera, either from a patient with lupus psychosis or from patients without NP-SLE, recognized the p331 protein in synaptosomes ( Fig. 3 A ). Given the variety of autoantibodies produced by lupus patients, some sera also showed other immunoblot bands. However, when using affi nity-purifi ed ␣ -hP 11 antibodies instead of serum, p331 appeared as the main detectable anti-P target at the synaptic region (synaptosomes; Fig. 2 B , lane 1).
Synaptosomes provided abundant material for distinguishing between extrinsic and intrinsic membrane proteins by using the standard method of sodium carbonate extraction ( 47 ), as well as for preparative immunoprecipitations. Resistance to alkaline extraction (e.g., recovery of the protein in the pellet of extracted membranes) indicated that p331 is an integral membrane protein ( Fig. 3 B , lanes 2 and 3). Preparative immunoprecipitations from synaptosomal fractions (P2 fraction) ( 46 ) yielded enough p331 to perform mass spectrometric analysis and microsequencing ( Fig. 4 A ). The major accompanying protein of 180 kD corresponded to the clathrin heavy chain. It might be that p331 interacts with clathrin, which is a cytosolic protein and certainly not an anti-P target. In contrast, p331 turned out to be a novel protein. Six internal peptides (P1 -6) were microsequenced and found to align with the sequence of the human protein NP055928 ( Fig. 4 B ), which is of unknown function. This protein is encoded by the complementary DNA NM015113 originally reported in a high molecular weight complementary DNA library of the human brain ( 48 ). Its unique gene, located in chromosome 17, lacks any homologues in the entire human genome and encodes a protein of 2,961 residues (with an estimated Figure 3. The anti-P target p331 is an integral membrane protein present in rat brain synaptosomes. (A) Detection of p331 in rat brain synaptosomes. Membrane fractions prepared from rat brain synaptosomes were analyzed by immunoblot (IB) with control sera from healthy individuals (lanes 1 and 2), anti-P ( Ϫ ) from two SLE patients (lanes 3 and 4), and anti-P (+) from six SLE patients (lanes 5 -10), including one with psychosis (lane 10). All P (+) serum detected p331, providing further evidence of its expression in brain neurons. (B) p331 is an integral membrane protein and the main ␣ -hP 11 target in synaptosomes. Immunoblot of synaptosomes (Syn) with affi nity-purifi ed ␣ -hP 11 antibodies shows p331 as the main target (lane 1). The lower bands are degradation products that vary in different experiments. Sodium carbonate extraction of synaptosomal membranes shows that p331 distributes exclusively to the pellet (P; lane 2), similar to the integral membrane protein dopamine receptor (D2DR), whereas peripherally associated clathrin distributes to the supernatant (SN; lane 3).
antibodies. Affi nity chromatography with this peptide isolated a subset of anti-P antibodies ( ␣ -hP 4 ) from P (+) sera. These ␣ -hP 4 antibodies mimicked the previously characterized ␣ -hP 11 antibodies, recognizing the P ribosomal proteins from rat liver ( Fig. 5 B ) and the p331 in carbonate-extracted synaptosomal membranes ( Fig. 5 C ), and displaying the regionalized immunofl uorescent pattern at the surface of N2a cells, which was completely abrogated by the 642 SS DDLG ED 649 peptide ( Fig. 5 D ). Therefore, our results indicate that NSPA exposes two regions to the cell surface encompassing the residues 644 DDLG 647 and 2881 GLFE 2884 , which are recognized by anti-P antibodies, most likely within the context of a conformational epitope given their mutually distant locations.
In the brain, NSPA is exclusively expressed by neurons at specifi c zones The fi nding of NSPA prompted us to produce rabbit antibodies suitable for immunohistochemistry and determine which brain cells and regions might be targets for anti-P autoantibodies. Anti-P antibodies are not convenient tools for this purpose because of their interaction with ubiquitous ribosomal proteins. We tried diff erent peptides of NSPA as immunogens. Interestingly, a polyclonal antibody raised against an NSPA peptide containing the 2881 GLFE 2884 motif (THMEY GLFE DV; Fig. 4 B ) did not recognize any of the P ribosomal proteins (not depicted), whereas it did interact with NSPA ( Fig. 6 ). Thus, anti-P autoantibodies seem to diff er from the antibodies produced by immunization, at least in the recognition of the GLFE sequence. However, these ␣ -NSPA antibodies recognized the integral membrane protein p331 in alkaline-extracted synaptosomal membranes ( Fig. 6 A ), and showed cross reaction ( Fig. 6 B ) and cell-surface colocalization with ␣ -hP 11 antibodies ( Fig. 6 C ), indicating that they interact with the same protein recognized by ␣ -hP 11 antibodies.
Strikingly, immunohistochemistry of the rat brain showed that ␣ -NSPA antibody stained only neurons and only at specifi c GenBank/EMBL/DDBJ under accession no. NP_055928 . Because our results indicated that p331 (NP055928) exposes a P epitope to the cell surface, we termed this protein NSPA.

Possible P epitopes displayed by NSPA at the cell surface
Optimal binding of anti-P antibodies to the last 11 C-terminal residues of P ribosomal proteins (SE/DDDMGFGLFD) depends on clusters of acidic (E/DDD) and hydrophobic (GF-GLFD) residues, which very likely correspond to two overlapping epitopes ( 51,52 ). The phosphorylated amino acids of the P proteins are not critical epitope determinants ( 51 ). Analysis of peptide arrays has suggested that the GFGLFD motif is the key determinant recognized by anti-P antibodies ( 52 ). In NSPA, a region encompassing residues 2881 GLFE 2884 partially coincides with this P ribosomal epitope ( Fig. 4 B ), which accepts the conservative D/E substitution ( 53 ). Other studies modeled the paratope -epitope interaction on a monoclonal antibody and suggested that the DDxGF sequence, present in all members of the P protein family (i.e., P0, P1, and P2), conforms to a P epitope in which any residue can occupy the x position ( 54 ). In NSPA, a motif like this is represented by the sequence 644 DDLG 647 ( Fig. 4 B ). To assess whether these two motifs, in the context of NSPA, could mediate interactions with anti-P antibodies, we tested the blocking capability of synthetic peptides encompassing these regions ( Fig. 5 A ). NSPA synthetic peptides 642 SS DDLG ED 649 and 2876 THMEY GLFED V 2886 (underlined residues could conform a P epitope) dramatically decreased the characteristic asymmetrical cell-surface staining of ␣ -hP 11 antibodies, whereas the scrambled peptides containing the same residues in random positions had minor eff ects ( Fig. 5 A ). Because the peptide-array analysis of Mahler et al. ( 52 ) did not attribute a major role to the acidic cluster in the anti-P antibody recognition, contrasting with the fi ndings of Hasler et al. ( 51 ), we tested more directly whether the NSPA peptide 642 SS DDLG ED 649 could eff ectively interact with anti-P Presynaptosomal fractions (P2) obtained from 50 rat brains were subjected to immunoprecipitation with affi nity-purifi ed anti-P antibodies. SDS-PAGE and Coomassie blue staining show two main bands (asterisks; IgG-HC, IgG -heavy chain) that were analyzed by mass spectrometry and microsequencing. The 180-kD protein corresponds to clathrin, whereas p331 corresponds to the human protein NP055928 of unknown function. (B) Sequence of p331-derived peptides, possible P epitopes, and the immunogenic NSPA peptide. Six out of six peptides derived from p331 (P1 -6) matched perfectly with the putative human protein NP055928 at the indicated regions. We named this protein NSPA. The NSPA sequences 644 DDLG 647 and 2884 GLFD 2887 are homologous to the ribosomal DDMG and GLFD P epitopes within the 11 C-terminal region. The peptide sequence 2876 -2886 used to immunize rabbits to obtain anti-NSPA antibodies is also shown.
in the stratium and the lack of immunoblot detection of NSPA in synaptosomes isolated from this region; Fig. 7 C ). In synaptosomes isolated from brain cortex, we detected NSPA by using not only ␣ -NSPA or ␣ -hP 11 antibodies ( Fig. 7 C ) but also rabbit antibodies raised against a peptide of 11 residues of the C-terminal region of the ribosomal p38 (not depicted). The results summarized in Fig. 7 C indicate that NSPA is expressed by neurons of certain regions of the brain, which might well be involved in NP-SLE.

Both anti-P and anti-NSPA antibodies elicit calcium entry into cortical neurons in primary culture
Having shown that anti-P antibodies interact with a protein located at the neuronal surface, we went on to study their possible impact on neuronal function. Initially, we monitored cytosolic calcium in rat cortical cultures loaded with fl uorescent brain zones ( Fig. 7 ). In the neocortex, ␣ -NSPA stained neurons of layers I, II, III, and V with somatodendritic distribution. Layer IV lacks staining. In the hippocampus, ␣ -NSPA staining predominates in somatodendritic regions of interneurons ( Fig. 7 A , arrows). Neurons of the bed nucleus of stria terminalis show NSPA in a pattern characteristic of synaptic buttons. In the central nucleus of the amygdala, NSPA also appeared concentrated in synaptic regions, including calyx-like terminals ( Fig. 7 A , arrows), very much like the CGRP-immunoreactive calices that originate from the lateral parabrachial nucleus ( 55 ). The brain regions displaying ␣ -NSPA immunostaining ( Fig.  7 C ) included regions preferentially involved in higher brain functions such as cognition, emotion, and memory. The specifi city of the immunostaining was shown by its complete abrogation with the immunogenic peptide ( Fig. 7 B ) and by its correlation with NSPA expression (e.g., the lack of staining  11 in nonpermeabilized N2a cells either in the absence or presence of NSPA peptides P DDLG ( 642 SSDDLGED 649 ) and P GLFE ( 2876 THMEYGLFEDV 2886 ) or their corresponding scrambled peptides, randomized P DDLG (LGDSSEDLD) and randomized P GLFE (DEYTEHFGLVM). Both P DDLG and P GLFE peptides, but not their randomized versions, decreased the cell-surface ␣ -hP 11 immunostaining. An IgG fraction isolated from a P ( Ϫ ) serum gives no staining. In all of the images, some staining background is depicted on purpose to visualize the cells. Bar, 10 m. (B -D) A subset of anti-P antibodies ( ␣ -hP 4 ) recognize P ribosomal proteins and NSPA. A P (+) serum was subjected to affi nity chromatography with the NSPA peptide DDLGEDD containing a putative P epitope (DDLG). The isolated ␣ -hP 4 antibodies, similar to the ␣ -hP 11 antibodies, recognize in immunoblot (IB) the P ribosomal proteins in ribosomes isolated from rat liver (B), and the high molecular mass protein (p331) the in synaptosomes (Syn; lane 1) and pellet (P; lane 2) but not the supernatant (SN; lane 3) of carbonate-extracted membranes (C). (D) Indirect immunofl uorescence with ␣ -hP 4 antibodies shows the characteristic asymmetrical cell-surface staining, which was abrogated by the P DDLG ( 642 SSDDLGED 649 ) peptide. Bar, 10 m.
Anti-P acted selectively on neurons. Astrocytes, in which we could not detect NSPA by immunohistochemistry, responded very weakly or not at all, neither to human anti-P nor to rabbit anti-NSPA antibodies ( Fig. 8 F ). These results demonstrate that anti-P antibodies can eff ectively interfere with neuronal function through specifi c interaction with an extracellularly exposed P epitope. Because NSPA was the only anti-P target that we could detect in neurons and the antibodies against it also triggered calcium infl ux, it seems very likely that NSPA mediates the eff ects of anti-P antibodies.

Anti-P antibodies induce apoptosis and loss of neurons both in vitro and in vivo
In neurons, moderate but sustained Ca 2 + overloads are known to cause cell death ( 56 ). Consistently, brain cultures exposed to anti-P antibodies developed several signs of neuronal stress, including morphological changes, dramatic decrease of neuronal processes, nuclear alterations, and activation of the apoptotic marker caspase-3 ( Fig. 9 A ). Cell viability decreased, resulting in a loss of cells from the culture ( Fig. 9 B ).
Defi nitive proof of a neuropathogenic role of anti-P antibodies requires demonstration of their neurotoxicity in the brain. To this end, we performed in vivo experiments. Injection of anti-P antibodies into the primary motor cortex of living rats resulted in neuronal apoptosis, as revealed by caspase-3 activation, and loss of neurons ( Fig. 9 C ). Similar results were observed in four rats and with ␣ -hP 11 antibodies from two diff erent SLE patients, including one with psychosis. Therefore, anti-P antibodies do have a strong neurotoxic potential to lead to CNS compromise.

Anti-P antibodies isolated from patients without NP-SLE also induce calcium infl ux and neuronal apoptosis
In all previous functional experiments, we used anti-P antibodies affi nity purifi ed from two SLE patients that presented psychosis at the moment of obtaining the serum samples. However, our results showed that anti-P (+) serum from patients that manifested no NP symptoms also recognized NSPA ( Figs. 2 and 3 ). Thus, it seems crucial to determine whether the anti-P antibodies produced by these two groups of patients display relevant functional diff erences to account for developing or not developing NP-SLE. Strikingly, anti-P antibodies affi nity purifi ed from two SLE patients that had never presented NP symptoms elicited sustained calcium infl ux and apoptosis (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20071285/DC1), thus revealing a pathogenic equivalence to the anti-P antibodies produced by SLE patients with psychosis.

DISCUSSION
We found a new target for anti-P autoantibody, termed NSPA, that is expressed at the surface of brain neurons and can potentially mediate anti-P -dependent NP symptoms. Sequence data and biochemical characterization showed that NSPA corresponds to a new integral membrane protein of high molecular mass (331 kD). A polyclonal antibody suitable for immunohistochemistry demonstrated that NSPA is expressed by neurons from specific regions of the brain, including calcium indicators. Calibration experiments showed that resting calcium levels in these cultures fl uctuate at ف 50 nM for astrocytes and 80 nM for neurons. Exposure to anti-P antibodies caused a rapid and sustained increase in cytosolic calcium in neurons ( Fig. 8 A ). The eff ect, which was readily reversible upon removal of the antibody, was blocked by the synthetic P peptide ( Fig. 8 B ), showed dependency on antibody concentration ( Fig. 8 C ), and refl ected a calcium infl ux from the extracellular medium, as judged by inhibition with EGTA ( Fig. 8 D ). Affi nity-purifi ed anti-P antibodies from three different lupus patients that developed psychosis gave similar results, whereas the IgG fraction from the serum of normal individuals showed no effect (unpublished data). Strikingly, our anti-NSPA antibodies mimicked the eff ects of the anti-P antibodies, though in a lesser magnitude at a similar concentration ( Fig. 8 E ). Rabbit affi nity-purifi ed control antibodies against proteins unrelated to ribosomal P proteins caused no eff ect (unpublished data).   11 show p331 only in synaptosomes from the cortex. In contrast, the dopamine transporter (DAT), which is 90% expressed in the striatum (reference 73 ), was exclusively detected in this region, thus showing that these two brain areas were effectively isolated. on neurons in culture and in situ. Thus, our results contribute to mechanistic support for the role of anti-P autoantibodies in NP-SLE that has been studied for more than 20 years. regions involved in emotional responses, memory, and other higher brain functions that become affected in neuropshychiatric lupus. In addition, we disclosed for the fi rst time that anti-P autoantibodies have neurotoxic potential, as shown  11 are specifi c and reversible. The increased calcium levels elicited by ␣ -hP 11 returned to basal levels after washing the antibody (white bar) and were abrogated by preincubating the antibodies with the P peptide (black bar). (C) Dose-dependent responses to ␣ -hP 11 . The same cell showed increased responses to increasing amounts of antibody added after a wash out. Horizontal bars represent incubation time periods. (D) Chelation of extracellular Ca 2 + with 10 mM EGTA abolished the effects of ␣ -hP 11 . (E) 1 g/ml of ␣ -NSPA antibodies also provoke an increase in neuronal calcium levels of lower magnitude, which is reversible, specifi cally inhibited by the corresponding immunogenic peptide, and abolished by EGTA. (F) Neurons predominantly responded to both ␣ -hP 11 and ␣ -NSPA. Although 67 and 51% of neurons increased their calcium levels in response to either ␣ -hP 11 or ␣ -NSPA, respectively, only 5 and 3% of the astrocytes showed detectable responses. Control experiments showed that astrocytes responded to glutamate (not depicted). Error bars represent the mean ± SEM.
The following evidence indicates that NSPA is a neuronal target of anti-P antibodies able to mediate neurotoxic eff ects. First, affi nity-purifi ed anti-P antibodies used in biotinylation and immunocapture assays detected NSPA as the only cell-surface target, both in N2a cells and in cortex neurons in primary culture. Experiments of alkaline extraction of synaptosomal membranes also showed NSPA as the only protein recognized by anti-P antibodies. Second, peptide competition blocked the interaction of anti-P antibodies with the cell surface, indicating that NSPA exposes a P epitope at the cell surface, very likely conformed by two separate regions encompassing residues 644 DDLG 647 and 2881 GLFE 2884 . Third, affi nity-purifi ed anti-P antibodies from two SLE patients with psychosis and rabbit anti-NSPA antibodies both triggered calcium infl ux into neurons in primary culture showing specifi c target dependency, as demonstrated by the corresponding blocking peptide. Neither anti-P nor anti-NSPA antibodies elicited calcium infl ux in astrocytes that do not express NSPA. Fourth, anti-P antibodies elicited caspase-3 activation and neuronal death both in vitro and in the rat brain in vivo. Finally, experiments with rabbit anti-P More than 100 autoantibodies have been described in SLE, but very few can be associated with clinical outcomes and even fewer revealed their pathogenic mechanism ( 1,8 ). Particularly elusive has been the role of autoantibodies in the pathogenesis of NP-SLE ( 2, 6 ). The subset of anti -double-stranded DNA reported to cross react with NMDAR, resulting in apoptotic neuronal loss and cognitive impairment in mice, so far constitute the best documented factor with neuropathogenic potential to mediate CNS dysfunctions ( 19 -22 ). However, the clinical evidence still seems limited ( 57 ). Two series coincidently reported their association with depression but diff ered in their association with cognitive decline in lupus patients ( 14,15 ). In contrast, anti-P antibodies had remained for a long time as the most promising candidates for a neuropathogenic role in SLE, supported by numerous clinical data that reported their association with lupus psychosis and/or depression ( 25,34,35 ). However, contradictory studies ( 5,26,38 ), combined with the lack of any suitable neuronal target or evidence of deleterious actions on neuronal function, have consistently raised doubts about the pathogenic role of anti-P antibodies ( 25, 37 ).  11 antibodies induce apoptosis in neurons in situ. The rat brain cortex was injected with 0.7 g of either control IgG or ␣ -hP 11 in contralateral sides, fi xed after 24 h, and either stained with Cresyl violet or treated for immunohistochemistry of activated caspase-3. The region injected with ␣ -hP 11 shows caspase-3 activation (bottom right and inset) and lower cell density (top right) and. Bars, 100 m. ment of disrupting the blood -brain barrier (BBB) for autoantibody-mediated brain symptoms, which varied according to the region of the BBB breach ( 21,22 ). Evidence of antibody production in situ in the brain and passage across the BBB have been described in SLE patients ( 22,61 ). There are also several conditions that promote BBB breach, including infection, stress, hypertension, and nicotine exposure ( 21,22,62 ), which can vary among patients. In addition, the variety of NP symptoms in SLE patients very likely involves coexpression of autoantibodies with direct or indirect neuropathogenic potential, such as anti-NMDAR antibodies ( 19 ). In this paper, we show that anti-NSPA antibodies share with ␣ -hp 11 antibodies the capacity to trigger neuronal calcium infl ux. Future studies should determine whether SLE patients produce autoantibodies against NSPA, which might be distinct from anti-P antibodies. All of these factors could contribute to the NP outcome of SLE.
The identifi cation of new neuronal surface molecules as autoimmune targets in the CNS also entails general interest. Disorders aff ecting the neuromuscular junction involve autoantibodies against nicotinic acetylcholine receptors in myasthenia gravis, calcium channels in the Lambert-Eaton syndrome, and potassium channels in Isaac ' s syndrome ( 18,63 ). Whether autoantibodies underlie certain CNS disorders remains less apparent, as they would require a mechanism to access CNS targets ( 22 ). Autoantibody targets with pathogenic potential include the glutamate receptor 3 in Rasmussen ' s encephalitis ( 64 ), metabotropic glutamate receptor 1 in paraneoplastic cerebellar ataxia ( 65 ), the NMDAR in SLE ( 19 ), and neuropeptides in eating disorders ( 66 ). The protein that we described in this study, NSPA, is a candidate for alterations caused either by autoantibodies or mutations that may eventually promote psychiatric disease, not only in SLE. Although the function of NSPA is unknown, we can speculate, by analogy with the mentioned disorders, that it may be related to cell-surface receptors and/or ion channels.
In summary, we have contributed a mechanistic link to the vast body of clinical evidence supporting a role of anti-P antibodies in psychiatric lupus that can explain why they might be pathogenic. Deleterious calcium infl uxes can be triggered by anti-P antibodies in brain neuronal cells through interaction with NSPA at specifi c brain zones.

Cell culture and viability assay
Neuroblastoma N2a cells were grown in DMEM with 7.5% FBS and antibiotics (200 U/ml penicillin, 0.1 mg/ml streptomycin). Primary cultures of cortical neurons were prepared from rat embryos (E18), as previously described ( 67 ), and were maintained for at least 7 d before the experiments, either without or with 4 μ m of cytosine arabinoside (Sigma-Aldrich) added 48 h after plating to prevent nonneuronal cell proliferation ( 68 ). Cell viability assays were performed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT; Sigma-Aldrich) reduction, as previously described ( 69 ), except that the lysis buff er lacks formamide. All of the experiments were performed in triplicate.

Antibodies and affi nity chromatography
Anti-P -positive sera from patients with SLE controlled at our Rheumatology Department of the Pontifi cia Universidad Católica de Chile were screened antibodies raised against the 11 ribosomal C-terminal residues containing the P epitopes mimicked the reactivity and the eff ects of both ␣ -hP11 and ␣ -NSPA antibodies, including the recognition of cell surface and synaptosomal NSPA, and the induction of calcium infl ux and apoptosis in neurons (unpublished data). Collectively, these results indicate that anti-P antibodies interact with NSPA through a P epitope exposed at the cell surface, and as a consequence of such interaction, neurons experience enhanced calcium entry and undergo apoptosis.
Our anti-NSPA antibodies allowed us to defi ne the distribution of a cell-surface protein bearing a P epitope in the brain. In immunohistochemistry, we found NSPA being expressed by neurons at specifi c brain regions, including neocortical layers II, V, and VI, and other zones of relevance for the pathogenesis of NP-SLE, such as the amygdala, which is involved in arousal and emotional responses ( 58 ), the cortex and hippocampus, which are involved in memory and higher brain functions ( 59 ), and the ventral tegmental area, which is associated with reward processing and drug addiction ( 60 ). A recent study reported that anti-P antibodies selectively stained certain limbic regions of the mice brain ( 36 ), but it did not clarify whether the immunostaining involved neuronal or glial cell bodies, or axons, and did not identify the antigen recognized by anti-P antibodies. Anti-P antibodies might decorate ribosomes in cells that could become inadvertently permeabilized during the immunohistochemistry. Instead, our ␣ -NSPA antibody only showed immunohistochemical staining in neurons (neural bodies), and in some places we could distinguish immunostaining in axon terminals (synaptic buttons), including the calyx-like axon terminal in the central nucleus of the amygdala. Controls indicating that the immunohistochemical staining corresponds specifi cally to NSPA included competition with the immunogenic NSPA peptide and correlation with immunoblot detection in synaptosomes obtained from either the cortex or striatum, which displayed or lacked ␣ -NSPA staining, respectively. Thus, our present work not only identifi es a new P antigen that is expressed at the neuronal cell surface but also discloses in great detail its distribution in the brain. At least through interaction with NSPA, anti-P antibodies could provoke serious dysfunctions in the CNS and contribute to developing NP disorders.
Because not all SLE patients that produce anti-P antibodies develop an NP disease, an important yet unsolved question is whether anti-P antibodies from SLE patients that do not manifest psychiatric or other diff use CNS symptoms are functionally equivalent to those produced by SLE patients with psychiatric compromise. We showed that anti-P antibodies from SLE patients without NP disease not only recognize NSPA but also induce calcium infl ux and apoptosis in neurons, indicating that they possess an equivalent pathogenic potential as psychiatrically associated anti-P antibodies. Therefore, besides the production of anti-P antibodies, additional risk factors are certainly required for eliciting psychiatric manifestations.
Both accessibility into the brain and the presence of other autoantibodies very likely cooperate in developing NP-SLE. Experiments in mice that produce or have been injected with antibodies against NMDAR recently highlighted the require-bound to 100 mg of protein A -sepharose for 2 h at 4 ° C. The immunocomplexes were exhaustively washed for 24 h at 4 ° C, resolved in preparative 7.5% SDS-PAGE, and stained with Coomassie blue. Relevant bands were isolated and treated for matrix-assisted laser desorption/ionization -time of fl ight mass spectrometry (TofSpec 2E; Micromass) and analysis (Protein Prospector, available at http://www.abcc.ncifcrf.gov/prospect.htm). The most abundant mass peaks were subjected to electrospray ionization for mass spectrometric sequencing (ESI-MS/MS; QT [Micromass]). In addition, peptide sequences were obtained by Edman ' s degradation at the Max-Delbr ü ck-Center.

Rat brain immunohistochemistry
Male Sprague-Dawley rats were deeply anaesthetized, and the brains were fi xed and processed for immunohistochemistry exactly as previously described ( 71 ), using 12 g/ml of the affi nity-purifi ed anti-NSPA antibody or 1:500 dilution of anticleaved caspase-3 antibody (Cell Signaling Technology). The sec tions were incubated with the secondary antibody (1:1,000 dilution) of Biotin-SP -conjugated Affi niPure goat anti -rabbit IgG (H+L; Jackson Immuno-Research Laboratories). Preadsorption of the anti-P antisera with the peptide K-2876 THMEYGLFEDV 2886 was used to control specifi city.

Surgery and stereotaxis
Animal handling and care were performed according to the recommendations of the National Institutes of Health Guide for Animal Experimentation Care. The Commission on Bioethics and Biosafety of the Facultad de Ciencias Biol ó gicas at the Pontifi cia Universidad Cat ó lica de Chile approved the study protocols. Adult Male Sprague-Dawley rats were anesthetized with 0.01%/100 g ketamine, placed in a stereotaxic frame, and bilaterally injected in the primary motor cortex with 0.7 g of affi nity-purifi ed human anti-P antibody in 1 l of sterile PBS, according to coordinates measured from bregma ( 72 ): anteropos terior, 1 mm; lateral, 2 mm; and horizontal, 1 mm from the skull. The rats ( n = 4) were killed 24 h after lesion. As a control, rats were bilaterally injected with 0.7 g of human IgG antibody purifi ed by adsorption to protein A -sepharose.

Intracellular calcium levels
Confocal microscopy was used to assess intracellular calcium in cortical neurons (400,000 cells plated on coverslips) loaded for 30 min with 5 M Fluo-3 AM and Fura Red AM (Invitrogen), as previously described ( 67 ). Real-time fl uorophore emissions were registered at room temperature (23 -26 ° C) in an inverted laser-scanning confocal microscope (LSM5 PASCAL; Carl Zeiss, Inc.) with a 40 × objective (numerical aperture, 1.3). Cultures were excited at 488 nm every 15 -30s, or every 2 min in Fig. 8 A , and Fluo-3 AM and Fura Red AM were imaged simultaneously. A ratio (R) minus background ( ⌬ R) was calculated between the green (Fluo-3 AM) and red (Fura Red AM) emissions, which is proportional to the concentration of cytosolic calcium. Data are presented as ⌬ R/Ro, where Ro is the ratio at the beginning of each experiment.

Online supplemental material
Fig. S1 depicts the results obtained with anti-P antibodies affi nity purifi ed from SLE patients without NP symptoms. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20071285/DC1.

Detection of P epitope -bearing proteins at the cell surface
Cell-surface biotinylation assays. Cell-surface biotinylation was performed with Sulfo-NHS-biotin (Thermo Fisher Scientifi c) according to a previously established procedure ( 42,43,45 ). The cells were lysed in buff er A (50 mM Hepes, 150 mM NaCl, 1 mM EGTA, 2 mM MgCl 2 , 10% glycerol, and 1% Triton X-100, supplemented with a cocktail of protease inhibitors including 50 μ g/ml pepstatin A and 1 mM PMSF) for 30 min at 4 ° C, and immunoprecipitation was performed with 10 μ g of affi nity-purifi ed anti-P antibody prebound to protein A -sepharose, either with or without 50 g of competing P peptide. After resolving the immunocomplexes by 7.5% SDS-PAGE, biotinylated proteins were detected with horseradish peroxidase -streptavidin ( 42 ). For brain cortical primary cultures, the biotinylation assay was performed in metabolically labeled cells for 16 h with 100 Ci [ 35 S]methionine-cysteine. The immunocomplexes were resuspended in 10% SDS and heated to 100 ° C for 5 min, and after pelleting the beads, the supernatant was collected and incubated with immobilized neutravidin protein for 18 h at 4 ° C, thus separating by precipitation the cell-surface biotinylated proteins. Samples were analyzed by SDS-PAGE and revealed by phosphorimager scanning (Cyclone Storage Phosphor Scanner; PerkinElmer).
Immunocapture of cell-surface P epitope -bearing proteins. N2a cells grown to confl uence in 3.5-cm well dishes were metabolically labeled with 100 Ci [ 35 S]methionine-cysteine for 18 h and incubated intact for 1 h at 4 ° C with 30 l of either P-positive serum, to capture P epitope -bearing cellsurface proteins. After exhaustively washing out the unbound antibodies, the cells were lysed in buff er A, protein A -sepharose was added, and the immunocomplexes were sedimented by centrifugation, resolved by 7.5% SDS-PAGE, and revealed by fl uorography ( 42,43 ). To detect the high molecular mass anti-P target (p331), it is crucial to use fresh antiproteases, run the dye front out of the gel, and continue the electrophoretic migration for 90 min extra.

Detection of P epitope -bearing proteins in synaptosomes
Synaptosomes were isolated from the rat brain with the Percoll gradient method ( 46 ) and lysed in buff er A. Immunoblots were performed with a 1:1,000 dilution of anti-P (+) sera and revealed with electrochemiluminescence (ECL). Immune cross reaction of NSPA was assessed by immunoprecipitation with 10 g of affi nity-purifi ed anti-P antibody prebound to 30 l of protein A -sepharose (Sigma-Aldrich) and immunoblotting with either preimmune serum or affi nitypurifi ed anti-NSPA antibody, followed by peroxidase-labeled anti -rabbit (Rockland) and ECL. Sodium carbonate extraction was performed exactly as previously described ( 47 ).

Immunofl uorescence microscopy
N2a cells grown in 10-mm coverglasses (VWR Scientifi c) were fi xed and treated for indirect immunofl uorescence either in intact cells or in cells permeabilized by 0.2% Triton X-100, as previously described ( 42,70 ). Digital images of fl uorescence and Nomarski interference were acquired on a microscope (Axiophot; Carl Zeiss, Inc.) with a 63 × immersion objective and a 14-bit camera (Axiocam), and were transferred to a computer workstation running imaging software (Axiovision) ( 70 ).

Preparative immunoisolation of P epitope -bearing proteins and internal peptide sequence analysis
A presynaptosomal membrane fraction (P2) prepared from the brains of 50 rats was solubilized with lysis buff er A and used for immunoprecipitation of P-bearing proteins by adding 400 g of affi nity-purifi ed anti-P antibody previously


s2fieldsofstudy: Biology Medicine

text:
Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study

Introduction Subclinical atherosclerosis (SCA) measures in multiple arterial beds are heritable phenotypes that are associated with increased incidence of cardiovascular disease. We conducted a genome-wide association study (GWAS) for SCA measurements in the community-based Framingham Heart Study. Methods Over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1345 subjects from 310 families. We calculated sex-specific age-adjusted and multivariable-adjusted residuals in subjects tested for quantitative SCA phenotypes, including ankle-brachial index, coronary artery calcification and abdominal aortic calcification using multi-detector computed tomography, and carotid intimal medial thickness (IMT) using carotid ultrasonography. We evaluated associations of these phenotypes with 70,987 autosomal SNPs with minor allele frequency ≥ 0.10, call rate ≥ 80%, and Hardy-Weinberg p-value ≥ 0.001 in samples ranging from 673 to 984 subjects, using linear regression with generalized estimating equations (GEE) methodology and family-based association testing (FBAT). Variance components LOD scores were also calculated. Results There was no association result meeting criteria for genome-wide significance, but our methods identified 11 SNPs with p < 10-5 by GEE and five SNPs with p < 10-5 by FBAT for multivariable-adjusted phenotypes. Among the associated variants were SNPs in or near genes that may be considered candidates for further study, such as rs1376877 (GEE p < 0.000001, located in ABI2) for maximum internal carotid artery IMT and rs4814615 (FBAT p = 0.000003, located in PCSK2) for maximum common carotid artery IMT. Modest significant associations were noted with various SCA phenotypes for variants in previously reported atherosclerosis candidate genes, including NOS3 and ESR1. Associations were also noted of a region on chromosome 9p21 with CAC phenotypes that confirm associations with coronary heart disease and CAC in two recently reported genome-wide association studies. In linkage analyses, several regions of genome-wide linkage were noted, confirming previously reported linkage of internal carotid artery IMT on chromosome 12. All GEE, FBAT and linkage results are provided as an open-access results resource at . Conclusion The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis.

Conclusion:
The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis.

Background
Myocardial infarction, stroke and other atherosclerotic cardiovascular diseases comprise the leading cause of death for men and women in the U.S. [1], and will soon become the leading cause of death worldwide [2]. Atherosclerosis in the arterial wall precedes the onset of most cases of clinically apparent cardiovascular disease by decades, and subclinical atherosclerosis (SCA) is quite common in young and middle-aged persons [3][4][5]. SCA can be detected and quantified in major arteries, such as the carotid and coronary arteries, which provide essential blood flow to major organs, using noninvasive, high resolution imaging modalities. Such measures include peripheral arterial atherosclerosis detected by the anklebrachial index (ABI); internal and common carotid intimal medial thickness (IMT) detected by B-mode ultrasound; and coronary artery calcium (CAC) and abdominal aortic calcium (AAC) deposits by multidetector computed tomography (MDCT). We have previously reported evidence for incomplete correlations between carotid IMT, CAC and aortic calcium [6]. Both genetic and environmental factors underlie interindividual variability in SCA, and significant heritability has been found for measures of SCA in the peripheral vasculature [7], carotid arteries [8], aorta [9], and coronary arteries [10]. Further, while there is evidence for partial correlation of major cardiovascular risk factors with atherosclerosis, multiple SCA measures, including ABI, carotid IMT, CAC, and AAC, have been shown to predict future risks for cardiovascular disease independent of risk factors [11][12][13][14].
While SCA measures are heritable, relatively little is known regarding the role of genetic variants in the interindividual variability in these quantitative measures of atherosclerosis. To date, results from candidate gene association studies for clinically apparent cardiovascular disease or subclinical disease have been inconsistent, though overviews of multiple studies seem to provide evidence for modest associations for variants in a number of candidate genes, such as APOE and ACE [8,15]. Genome-wide association studies (GWAS) using densely spaced single nucleotide polymorphisms (SNPs) provide a more comprehensive approach unconstrained by existing knowledge to test common genetic variation across the genome using high-throughput genotyping arrays. From GWAS, several previously unrecognized genes have been identified that may contribute to disease, including CFH and age-related macular degeneration [16], INSIG2 and obesity [17], NOS1AP with QT interval variation [18], and several genes including TCF7L2 [19] as well as IGF2BP2 and CDKA1 with diabetes mellitus [20][21][22]. Additionally, two other GWAS have identified an association with coronary heart disease of SNPs in a region of chromosome 9p21 [23,24] that was also associated with diabetes mellitus in three previous GWAS [20][21][22]. Of note, there was an association of this chromosome 9 region with the SCA measure of coronary artery calcium in one replication study [19,24].
With the availability of high resolution imaging of atherosclerosis phenotypes in specific arterial beds in community-based cohorts such as the Framingham Heart Study, GWAS analysis is now possible for quantitative SCA phenotypes in major arterial beds. Recently, over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1,345 subjects from 310 pedigrees in the Framingham Heart Study. The primary objective of this report is to present a brief summary of the results of this GWAS for SCA detected in the lower extremity arteries, carotid arteries, aorta and coronary arteries, by conduct of genetic association analyses and genetic linkage mapping. This report is one of a series of manuscripts from a collaborative project conducted by Framingham Heart Study investigators; the overall approach to this project's statistical genetic methods is summarized in an Overview manuscript that summarizes the 100K genome-wide association study [25].

Study sample
Participants from the Offspring Cohort of the Framingham Heart Study who underwent one or more SCA measurements and genotyping with the Affymetrix 100K GeneChip are included in the study sample; genotyping was completed in a total of 1,345 subjects (1,084 Offspring cohort subjects). Original cohort subjects did not undergo the recently conducted MDCT or carotid IMT testing and as such were not included in this study. Of the 1,084 Offspring cohort subjects with genotyping, up to 984 participants with SCA phenotype information were analyzed. Details regarding selection of participants and genotyping are provided in the Overview [25].

Phenotype definitions & methods
Determination of the clinical characteristics in Offspring participants reported here was obtained at study entry (baseline) and at each follow-up examination, including Offspring examinations 6 and 7. The methods of measurement of the covariates (blood pressure, body height and weight, lipids, diabetes mellitus, smoking and other clinical characteristics) used in these analyses have been previously described [26]. For imaging tests conducted between two examination cycles, covariates used in the analysis were obtained from the earlier examination cycle.

Carotid ultrasonography for carotid IMT
Participants underwent carotid ultrasonography according to a previously reported standardized protocol at Offspring examination 6 (1995 to 1998) [27]. Imaging was conducted with a Toshiba SSH-140A imaging unit using a high resolution 7.5 MHz transducer for the common carotid artery, and a 5.0 MHz transducer for the internal carotid artery. Images were gated to an electrocardiogram; end-diastolic images were acquired. As previously described, correlation coefficients for the mean and maximum internal carotid artery were 0.83 and 0.84, respectively, based upon 25 readings by two separate readers [28].
All studies were recorded on optical disk and read according to a standardized protocol [29]. To quantify the degree of thickening of the carotid artery walls, the measures of IMT were summarized into two variables: one for common carotid artery and one for internal carotid artery. Mean and maximum wall thicknesses of the common and internal carotid artery were defined as the mean of the wall thickness or the mean of the maximum wall thickness for the near and far wall on the left and right sides. The number of available measurements for averaging ranged from 1-4 for the common carotid artery, and 1-8 for the internal carotid artery.

MDCT for CAC and AAC
Measures of CAC and AAC were obtained between 2002 and 2005 in Offspring participants, between examinations 7 and 8. All eligible participants were imaged with an eight-slice multidetector computed tomography (Lightspeed Ultra, GE, Milwaukee, WI, USA) of the chest, as previously described [30], as well as the abdomen. Each subject underwent two chest CT scans and one abdominal scan that were performed using a sequential scan protocol with a slice collimation of 8 mm × 2.5 mm (120 KVp, 320/400 mA for .220 lbs body weight, respectively) during a single end-inspiratory breath hold (typical duration 18 s). Image acquisition (330 ms) was prospectively initiated at 50% of the cardiac cycle. For the abdominal scan, thirty contiguous 5 mm thick slices of the abdomen were acquired covering 150 mm above the level of S1. A cali-bration phantom (Image Analysis, Lexington, KY, USA) that contained rods of water and 75 and 150 mg/cm3 calcium hydroxyapatite, was placed underneath each subject.
Calcium measurements were performed on an offline workstation (Acquarius, Terarecon, San Matteo, CA, USA) by a trained technician. Scoring of coronary calcification has been previously described, and we reported excellent intra and inter reader reproducibility for the CAC measurements [30]. A calcified lesion in either the coronary arteries or in the aorta was defined as an area of at least three connected pixels with CT attenuation >130 Hounsfield Units using 3D connectivity criteria. A score for AAC (for the abdominal scan) and CAC (for each of the two chest scans) was calculated by multiplying the area of a calcified lesion with a weighted CT attenuation score dependent on the maximal CT attenuation (Hounsfield Units) within a lesion. In modification to the original Agatston Score that was originally developed for electron beam CT, we applied this algorithm to our MDCT scan protocol to score for CAC, as used in numerous previous studies [30], as well as for AAC.

Ankle brachial index (ABI) for peripheral arterial disease
Ankle-brachial systolic blood pressure measurements were obtained at Offspring examinations 6 and 7 according to a standard protocol by trained technicians, as previously described [7]. Participants rested for a minimum of five minutes in the supine position on the examining table prior to blood pressure measurement. Blood pressure cuffs were applied to bare ankles with the midpoint of the bladder over the posterior tibial artery approximately three centimeters above the medial malleolus. Systolic blood pressure was measured using an 8 megahertz Doppler pen probe and an ultrasonic Doppler flow detector (Parks Medical Electronics, Inc.). For each limb, (right and left arms, right and left ankles) the cuff was inflated quickly to the maximal inflation level and deflated at a rate of 2 mmHg per second until the systolic blood pressure became audible. All limb blood pressures were repeated in reverse order. If the initial and repeat blood pressures differed by more than 10 mmHg at any one site, a third measurement was taken. Measurement was taken from the dorsalis pedis artery only if the posterior tibial pulse could not be located by palpation or with the Doppler probe [7].
The ABI is defined as the ratio of the average systolic blood pressure in the ankle divided by the average systolic blood pressure in the arm. The higher arm mean was used to calculate the ankle-brachial index for each leg. The lower of the two ankle-brachial index measurements was used for analysis.

Genotyping methods
Details of the genotyping methods are available in the Overview [25]. Briefly, 112,990 SNPs on the Affymetrix 100K chip were genotyped using DNA from family members of the Framingham Heart Study. For this report, SNPs were excluded for the following reasons: minor allele frequency ≤10% (n = 38,062); genotypic call rate ≤80% (n = 2346); Hardy Weinberg Equilibrium p-value ≤ 0.001(n = 1,595), leaving 70,987 SNPs available for analysis [25]. Results for all SNPs are reported on the open-access results website.

Statistical analysis methods
In total, of the 1,084 Offspring cohort subjects with genotyping, 673 to 984 participants with analyzable phenotype information were available for analysis. Residuals were created from multiple linear regression models in all subjects with the SCA measures, regardless of whether they were genotyped or not, to adjust phenotypes for covariates; these residuals were created in women and men separately. Covariate adjustment for blood pressure and/ or hypertension was performed is several different ways. For ABI, a dichotomous measure for hypertension (systolic blood pressure > 140 or diastolic blood pressure > 90 or on treatment) was used. For CAC and AAC, systolic blood pressure and use of anti-hypertensive treatment were used as covariates. For carotid IMT, treatmentadjusted systolic blood pressure was used. As previously described, systolic blood pressures for those on treatment are imputed to estimate what these values would be if the subject were not on treatment [31]. Ranked normalized deviates, created from the standardized residuals from the regression models, were used in the genetic analyses. For each sex, SCA phenotypes were age-adjusted and multivariable-adjusted; details of the covariates included in the multivariable adjustment for each phenotype are presented in Table 1.
All association analyses were performed using either generalized estimating equations (GEE) or family-based association testing (FBAT). We evaluated associations of the SCA phenotypes using an additive genetic model with 70,987 SNPs meeting criteria above. Details regarding these analytic methods are provided in the Overview [25]. Only results of multivariable-adjusted SCA phenotypes are displayed in Table 2. Linkage analysis was performed using variance components methods on a subset of the 100K chip SNPs in linkage equilibrium and Marshfield STR markers that were previously obtained [25].
We further sought to identify SNPs with consistent associations with phenotypes within measurement groups using GEE and FBAT analyses. SNPs were selected based on associations with multiple correlated traits in both population-and family-based tests. We evaluated the fol-lowing five phenotypic subgroups using sex-specific ageadjusted and multivariable-adjusted residuals (Table 1): ABI, common carotid artery IMT, internal carotid artery IMT, AAC, and CAC. For each SNP we calculated the proportion of phenotypes significantly associated with the SNP with P < 0.01 in both GEE and FBAT. For instance, for ABI, we evaluated GEE and FBAT results for both age-sexadjusted and multivariable-adjusted measures of anklebrachial index from both examination cycle 6 and 7 (in Table 1, these variables are named RANKLEBI6, RANKLEBI6MV, RANKLEBI7, and RANKLEBI7MV). Results are displayed in Table 3 of the top 5 SNPs with the highest proportions of significantly (P < 0.01) associated phenotypes. For identical proportions of significant phenotypes, SNPs were additionally ranked by the logarithm of the mean of GEE p-values.

Results
Ten SCA atherosclerosis measures were studied (Table 1). For each SCA measurement, sex-specific, age-adjusted phenotypes as well as age-and multivariable-adjusted phenotypes were evaluated. Measures for ABI and carotid IMT phenotypes were available in 880-984 participants, whereas measures for MDCT phenotypes were available in 673-680 participants.
Tables 2a, 2b and 2c provides a summary of the most significant findings in GEE, FBAT and linkage analyses, respectively, across a number of selected multivariableadjusted phenotypes described in Table 1. Full-disclosure of all results for all associations can be found at http:// www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/ study.cgi?id=phs000007. The top 25 most statistically significant associated SNPs in GEE analyses are presented in Table 2a. There were 11 SNPs with p < 10 -5 in GEE analyses. The top three associated SNPs are rs1376877 (p < 1*10 -6 , located in ABI2) associated with maximum internal carotid IMT, rs2390582 (p = 1*10 -6 , not located near a known gene) associated with maximum CAC score, and rs3849150 (p = 2*10 -6 , located near LRRC18) associated with AAC score. The top 25 most significantly associated SNPs in FBAT analyses are shown in Table 2b. There were 5 SNPs with p < 10 -5 by FBAT. The top three associated SNPs by FBAT are rs4814615 (p = 3*10 -6 , located in PCSK2) associated with maximum common carotid IMT, rs6053733 (p = 4*10 -6 , located near FLJ25067) associated with mean common carotid IMT, and rs10499903 (p = 4*10 -6 , located near PFTK1) associated with ABI.
When we examined below the top 25 associations on our list of nominally significant GEE results for various SCA phenotypes, there were significant associations with multivariable-adjusted CAC for several SNPs on chromosome 9, including three SNPs (rs10511701, rs1556516 and rs1537371; p-values for association 1.1 × 10 -4 , 8.8 × 10 -5 , and 1.7 × 10 -4 , respectively) lying within a 15 kB region implicated in recent GWAS's for coronary heart disease. Similar associations were noted for age-and sex-adjusted residuals for CAC.
Results for all multipoint LOD scores > 2.0 are displayed in Table 2c. There were four LOD score results exceeding 3.0 and ten additional LOD score results exceeding 2.0. Results for linkage of internal carotid artery IMT phenotypes to chromosome 12 and 1 (the top three LOD score results) and for chromosome 11 are consistent with findings from our previous report [32]. Upon further inspection of the list of associations with p < 0.01 for each of the phenotypes, SNPs in or near biologically plausible genes were noted, including: fibroblast growth factor (FGF1) for AAC, adrenergic, beta-2-, receptor (ADRB2) for CAC, myocyte enhancer factor 2C (MEF2C) and thrombospondin 2 (THBS2) for common carotid artery IMT, and cAMP-specific phosphodiesterase 4D (PDE4D) for ankle brachial index.
In Table 3, we provide a summary of the top five associated SNPs for each of four phenotypic categories -ABI, common carotid artery IMT, internal carotid artery IMT, and CAC. SNPs were rank ordered first according to percent of phenotypes with p < 0.01 for GEE and FBAT associations and second by geometric mean of GEE p-values. A number of SNPs were common to the top 25 associated SNPs by GEE or FBAT reported in Tables 2a and 2b, respectively, including rs28207 and rs4814615 for common carotid IMT and rs10483853 and rs10507130 for CAC.
We further examined associations with SNPs in or near regions of 37 candidate genes previously reported to have been associated with SCA or overt coronary heart disease.  Abbreviations: SNP = single nucleotide polymorphism; Chr = chromosome; GEE = generalized estimating equations; FBAT = family based association testing; LOD = logarithm of the odds; IMT = intimal medial thickness;. Gaps appear in the rank order for markers that appear more than once. In the column entitled "Phenotype", 'internal carotid artery IMT' denotes either mean or maximum internal carotid IMT, 'common carotid artery IMT' denotes either mean or maximum common carotid IMT, 'coronary artery calcification' denotes either mean CAC or max CAC, and 'ankle brachial index' denotes and ankle brachial index from either examination cycle 6 or cycle 7. For proximity to known genes (Table columns nine and ten), "IN" refers to a SNP within a protein-coding gene intron or exon, "OUT" refers to a SNP greater than 60 kb away from a protein-coding gene gene, and "NEAR" refer to a SNP within 60 kb of a protein-coding gene.

Table 2: 25 Most significant results for multivariable-adjusted subclinical atherosclerosis measures in multiple arterial territories by GEE (2a), FBAT (2b) and linkage (2c) analyses (Continued)
Of these genes, there were 28 genes with SNPs on the 100K array that were in or nearby (within 60 kb of) the gene. In Table 4, we report significant associations for SNPs in or near nine of these 28 candidate genes with p < 0.01 in GEE or FBAT analyses or p < 0.05 in both GEE and FBAT analyses. Associations were noted with one or more SCA measure for: CCR5, FGB, ESR1, IL6, NOS3, TNFRSF11, ADM, CD44, and CCL2 (Table 4).

Discussion
In this manuscript, we report the principle findings for a GWAS of SNPs from a 100K Affymetrix scan with SCA phenotypes in multiple major arterial beds. A conservative Bonferroni correction using this number of tests (0.05/1,000,000) would yield an approximate threshold of genome-wide significance to be 5*10 -8 , and none of our association findings meet these criteria for genomewide association., However, we did note a number of associations with p < 10 -5 after rank-ordering SNP associations using GEE and/or FBAT analysis. While we expect that many of these genotype-phenotype associations may be false positives, the SNPs identified in this manner are nonetheless hypothesized to include SNPs that merit further follow-up. To our knowledge, this is the first report of a GWAS using SCA phenotypes.
The first replicated GWAS findings for clinically apparent coronary heart disease were recently reported by two independent groups, identifying an association with SNPs in a region of chromosome 9p21 [23,24]. Interestingly, there was an association of this chromosome 9 region with CAC in one replication study [24], and this same chromosome 9 region was associated with diabetes mellitus in three other recent GWAS [20][21][22]. When we examined our GEE results for various SCA phenotypes, there were nominally significant associations with multivariable-adjusted CAC for several SNPs on chromosome 9 lying within a 15 kB region implicated in the recent GWAS's for coronary heart disease. While these SNPs were not our top-ranked SNPs, their specific association with coronary artery atherosclerosis (CAC) phenotypes likely represent strong evidence of replication in light of the recent GWAS. Abbreviations: SNP = single nucleotide polymorphism; Chr = chromosome; GEE = generalized estimating equations; FBAT = family based association testing; IMT = intimal medial thickness; MAF = minor allele frequency. Column Definitions: For each Phenotype Group (Table column one), a cluster analysis is conducted for several phenotype variables defined in Table 1. For "Ankle brachial index," GEE and FBAT results were evaluated for RANKLEBI6, RANKLEBI6MV, RANKLEBI7, and RANKLEBI7MV; for "Common carotid artery IMT," RNKCAROTCCAMAXAS6, RNKCAROTCCAMAXMV6, RNKCAROTCCAMEANAS6, and RNKCAROTCCAMEANMV6; for "Internal carotid artery IMT," RNKCAROTICAMAXAS6, RNKCAROTICAMAXMV6, RNKCAROTICAMEANAS6, and RNKCAROTICAMEANMV6; and for "Coronary artery calcification," RESMDCTCACMAXMV7 and RESMDCTCACMV7. The percent of phenotypes in the cluster analysis for which there was a p < 0.01 by both GEE and FBAT analyses is shown in Table column five. The geometric mean p-value is shown in Table column six for GEE associations for all phenotypes in the cluster analyses. For proximity to known genes (Gene position, Table column eight), "IN" refers to a SNP within a protein-coding gene intron or exon, "OUT" refers to a SNP greater than 60 kb away from a proteincoding gene gene, and "NEAR" refer to a SNP within 60 kb of a protein-coding gene.
Because there may be distinct genetic determination for atherosclerosis occurring in individual vascular beds (e.g., carotid arteries versus coronary arteries versus aorta versus peripheral arteries), we further examined for consistency of association of specific SNPs within five trait groups -ABI, common carotid artery IMT, internal carotid artery IMT, AAC and CAC. By this method of ranking associations, we identified distinct sets of SNPs, for which there was consistency of overlap with top SNP results by both GEE and FBAT (Table 3). There is evidence for partial correlation between SCA measures [6], and it is interesting to note that there were some SNPs for which there were nominally significant associations with more than one SCA measure. For example, rs10263213 is associated with both multivariable adjusted CAC (p = 0.001) and multivariable adjusted wall thickness of the carotid bulb (p = 0.013). Further research is warranted to determine whether SNPs showing association with two or more SCA measures are more likely to replicate in follow-up studies. Again, because no results from these approaches met criteria for genome-wide association, further validation studies will be required to confirm such associations. An extensive literature exists for prior studies of candidate gene variation associated with clinical atherosclerotic cardiovascular disease, in particular coronary heart disease [15], as well as SCA, particularly carotid IMT [8]. While many associations are not consistently noted from study to study, a review of the available studies reveals a modest overall association of clinical cardiovascular disease or SCA with variants in candidate genes, such as APOE, ACE, and NOS3 [15]. More recently, a number of screens of larger numbers of variants have revealed new candidate gene hypotheses, such as ALOX5AP [33,34] and LGALS2 [35]. When we examined for associations between SCA and SNPs on the 100K chip that reside in or near some previously studied candidate genes, we confirmed a number of modestly significant SNP associations with SCA phenotypes (Table 4). These and other associations between SNPs and various SCA measures may be viewed as supportive though not strongly confirmatory of prior hypotheses. However, it should be noted that 100K SNP coverage of any given gene region may be insufficient to exclude real associations. Better coverage may be afforded by newer, more dense SNP arrays. Nevertheless, these data suggest that GWAS may be used to contribute substantial information to the literature on the presence and strength of previously reported "candidate gene" associations, including those identified from other GWAS, as well as for unbiased searches for novel variants.
Our study was conducted in a moderate-sized, well-characterized community-based sample, and the conduct of SCA imaging was conducted without ascertainment for prior cardiovascular disease. Strengths and limitations of the GWAS in this sample using a 100K screen are discussed in the Overview [25]. A strength of this particular study is the substantial heritability of SCA phenotypes determined by high resolution imaging conducted in a reproducible manner. While these SCA phenotypes represent state of the art, non-invasive imaging in populations, it must be acknowledged that the available modalities allow a focus on only fixed anatomic components (e.g., calcific plaque or IMT) rather than dynamic or metabolically active components. Moreover, the occurrence and distribution of these modalities may differ by race or environmental background. Thus, our results may not extrapolate to non-white populations.
In conclusion, using a GWAS unconstrained by existing knowledge, we have identified new candidate SNP association hypotheses and further confirm some existing candidate gene and candidate SNP hypotheses. In particular, the evidence for association of several SNPs with CAC in the region of chromosome 9 that was recently reported to be associated with CHD or CAC in tens of thousands of subjects [23,24] provides evidence that we were able to identify true associations. These findings provide evidence for the exciting promise of GWAS of SCA to identify novel genetic variants underlying atherosclerosis within specific arterial territories or across multiple arterial territories. In this manuscript and in the accompanying web-posted results http://www.ncbi.nlm.nih.gov/projects/gap/cgibin/study.cgi?id=phs000007., we provide a full disclosure of the totality of our results, including all "non-significant" associations. In light of the multiple association tests that result from a GWAS, a powerful and efficient follow-up approach to increase the statistical confidence for an association of common variants with complex traits is a staged design, in which a modest number of results from the first stage are then tested in a second independent sample and the combined statistical evidence considered [36]. A further approach may be to seek evidence of in silico replication of our initial findings in other GWAS studies. The latter approach will be feasible with completion of the planned dense GWAS (the NHLBI's SNP Health Association [SHARe] Study) in over 9,000 men and women from the Framingham Heart Study [37].