Neuropsychiatric SLE (NPSLE) is a potentially severe complication that varies in prevalence between 9.5% and 95% based on the literature and the diagnostic criteria [41
]. Findings suggest that NPSLE can be present in a mild form in as many as 95% of SLE patients [42
], making it more common but typically less severe than renal or cardiovascular complications. Mood disorders and cognitive disorders are common symptoms and can be particularly challenging to diagnose in pediatric and adolescent patients due to the rapid emotional and physical changes that occur at those ages. While acquisition of cerebral spinal fluid (CSF) is invasive, it has become routine in the diagnosis of many neurological diseases and, in addition to serum, is therefore a useful medium for proteomic study of NPSLE. NPSLE research has focused on both biomarker identification for its diagnostic value as well as autoantigen identification for improved mechanistic understanding of the disease state and potential therapeutic development.
Lu and colleagues [8
] found CSF and sera obtained from NPSLE patients to be reactive to rat, mouse and monkey brain homogenate when separated by two-dimensional electrophoresis. MALDI-TOF-TOF was used to identify immune-reactive proteins. A total of 275 serum samples and 177 CSF samples were included, giving a great deal of depth and statistical power to the study. Briefly, four antinuclear antibody (ANA)-positive serum samples, which did not react to Hep2 cells, were used as pilot samples. Initial analysis of the reactive spots revealed an interaction in three of four samples with α-internexin (INA), a highly conserved protein between rodents and humans (Table ). Recombinant human INA was then used in a western blot to confirm reactivity of sera as well as immunofluorescence with sera and anti-INA polyclonal antibody. Both the sera and the polyclonal antibody were found to react to cultured rat cortical neurons and the reactivity of sera was reduced by incubation with recombinant INA. These findings opened the door for analysis of the entire cohort of CSF and serum samples. Using two standard deviations as a cutoff for positivity, 41.7% of the NPSLE samples of CSF were found to be positive for anti-INA antibodies (compared to 0% of SLE with cerebral infarction, 7.1% of normal SLE controls, and 10% of other neurological diseases). In total, the data showed reactivity in more than 40% of NPSLE patients in a dose-dependent manner toward INA. Longitudinal studies found that antibody levels were inversely proportional to cognitive level and declined after initiation of therapy and clinical remission. Furthermore, mice immunized with INA plus lipopolysaccharide developed cognitive impairments similar to NPSLE and exhibited hippocampal and cortical neuron apoptosis, suggesting that the presence of anti-INA antibodies in the serum of patients was pathogenic.
Ndhlovu and colleagues [43
] identified α- and β-tubulin as target autoantigens for NPSLE using patient sera to probe isolated mitochondria from bovine brain followed by MALDI-TOF analysis. Autoantibodies to β-tubulin have been identified in other CNS targeting autoimmune diseases such as Guillain-Barre syndrome, multiple sclerosis, and Sydenham's chorea, but targeting of α-tubulin is potentially specific to NPSLE. α-Tubulin autoantibodies were present in 36% of NPSLE patient sera, only 4% of SLE patients without neurological symptoms and none of the multiple sclerosis, epileptic or healthy control samples (Table ). Possibly due to the small size of the NPSLE population in the study (n
= 37), no correlation was seen between specific symptoms of NPSLE and the presence of α-tubulin autoantibodies in sera. Larger patient population studies are needed to further explore the involvement of anti-α-tubulin antibodies in NPSLE.
LC-MS/MS incorporates a direct front-end separation of complex samples to greatly increase the sensitivity of the procedure for low abundance protein detection. LC-MS/MS may be superior to MALDI-based techniques when dealing with complex samples. Katsumata and colleagues found this to be true while attempting to uncover novel serum autoantibodies for SLE, in particular those involved in CNS syndromes [9
]. Whole protein extracts from human neuroblastoma and glioblastoma cell lines were separated using two-dimensional electrophoresis and reacted to patient sera. It was determined that MALDI-TOF sensitivity was too low for effective protein identification in this case. To address this concern, anti-sera from SLE patients with and without CNS syndromes was bound to protein G-sepharose beads and incubated with the same cell line extracts. Proteins pulled down with the sepharose beads were analyzed with a LC-MS/MS method using a Q-TOF instrument. Most of the 154 proteins identified by the LC-MS/MS approach were not identified in the two-dimensional electrophoresis/MALDI-TOF analysis and 50 were found in the CNS group but not the non-CNS group. After exclusion of previously described autoantigens, 11 recombinant candidates showed reactivity against pooled serum of 28 patients with active CNS SLE by western blot analysis (5 of which were only detected by LC-MS/MS). These 11 candidates were then probed with sera of 106 SLE patients (42 with active CNS syndromes) as well as 100 healthy controls, resulting in a list of 5 confirmed autoantigens, 3 of which were novel (crystalline αB, esterase D and APEX nuclease 1; Table ). When compared to other diseased sera (RA, systemic sclerosis, Sjögren's syndrome, and multiple sclerosis), levels of anti-crystalline αB were not found to be significantly different. Anti-APEX nuclease 1 and esterase D, on the other hand, had significantly higher levels in SLE than in these disease controls and between CNS and non-CNS SLE sera. APEX nuclease 1 is the major apurinic/apyrimidinic endonuclease in human cells and is found to be upregulated in tissues under oxidative stress and has a significant effect on apoptotic regulation in certain tissues [44
]. The effects of esterase D are less well known.
In a similar study, Kimura and colleagues [45
] used a combination two-dimensional electrophoresis and LC-MS/MS with an ESI-LCQ instrument to obtain higher sensitivity for low abundance proteins. Patient sera from a NPSLE patient with white matter hyperintensities on T2-weighted MRI scan was reacted to a two-dimensional electrophoresis rat cerebral lysate blot. In total, nine reactive spots were seen and five were analyzed by LC-MS/MS. The two spots of greatest intensity corresponded to 60 kDa heat shock protein (Hsp60) [45
], which functions to refold proteins that are misfolded and/or facilitate degradation of proteins that cannot be recovered [46
]. Enhanced expression of Hsp60 can induce endothelial cell apoptosis and Hsp60 expression on the surface of endothelial cells has been reported to be directly related to coronary artery disease [47
], opening the possibility of a similar relationship in the cerebral vasculature. In a follow-up study of 180 patients with neurological diseases (including 15 with SLE) and 23 healthy controls, Kimura and colleagues showed that the titer levels of anti-Hsp60 antibodies were directly correlated to severity of white matter hyperintensities in the brain, suggesting the presence of anti-Hsp60 antibodies as both a diagnostic tool as well as a mechanistic agent in the development of NPSLE [32
]. Although the authors chose to focus on Hsp60, the study also found SLE-specific antibodies to α-internexin, β-actin, and glial fibrillary acidic protein (Table ).
Another study using LC-MS/MS also suggested a relationship between NPSLE exhibiting psychosis with anti-Rab GDP dissociation inhibitor alpha (αGDI) [31
]. LC-MS/MS and western blot analysis of sera found that 4 of 5 patients with NPSLE with psychosis (80%) expressed anti-Rab αGDI (Table ), while 1 of 13 patients with NPSLE and none with psychosis (5.3%) expressed the autoantibody, along with no expression in patients with multiple sclerosis (n
= 12), infectious meningoencephalitis (n
= 13), polyneuropathy (n
= 10), psychotic syndromes (n
= 10), or healthy controls (n