Identification of potential molecular markers that aid in elucidating the etiopathology associated with neurodegenerative diseases such as HIV induced neurodegeneration, are essential to facilitate disease diagnosis, monitor disease progression and assess response to existing and future treatments. Here, we hypothesized that a quantitative iTRAQ based proteomics approach would allow us to identify differential fingerprints in the plasma of monkeys, comparing immunodepleted samples before and after infection with SIV. Indeed, a number of significant differences were found. Of the differentially expressed proteins, we focused on afamin, a member of the albumin superfamily 21
, which was significantly down regulated after SIV infection.
Interestingly a recent study employing 2D-DIGE based mass spectrometry also found afamin to be down regulated (by 2.25 fold, comparable to our 2.77 fold) in immunodepleted sera that when comparing a group of HIV infected individuals with dementia to HIV infected individuals without CNS disease 22
. In our study, we also performed Western blot analysis using both immunodepleted and non-depleted plasmas from monkeys before and after SIV infection, which indeed revealed down-regulation of afamin, validating our proteomic data. Furthermore, analysis of plasma from monkeys that did not develop CNS disease revealed no alterations in the expression levels of afamin thus implying that the change not due to infection itself, and thus the decreased in afamin is linked to the presence of CNS disease
Several other proteomic studies have identified afamin as a potential biomarker in different disorders. Proteomic profiling of serum found afamin to be down regulated in patients with ovarian cancer 23
. 2-DE proteomic analysis on serum from patients with congenital disorders of glycosylation found down regulation of afamin when compared to the normal controls 24
. Another 2-DE based analysis afamin to be upregulated of maternal plasma from pregnant women with down syndrome fetuses in second trimester of pregnancy identified as compared to the controls with normal fetuses 25
. Proteome analysis of human follicular fluid revealed higher concentrations of afamin in the follicular fluid compared to serum thus proposing its significant role in folliculogenesis 26
. Thus alterations in afamin are not specific for HAND or these other conditions, but rather can be integrated with other measures in potential diagnostic, prognostic, and etiologic studies.
Afamin has been shown to be a specific binding protein for vitamin E 27
. The central role of vitamin E, comprising a set of eight related tocopherols and tocotrienols, is to maintain physiological cellular and tissue function via their antioxidant properties, and αTocH is the dominant biologically active form of vitamin E. In line with αTocH binding properties of afamin and neuroprotective properties of αTocH, it has been shown that αTocH-loaded afamin displays neuroprotection on primary neuronal cultures challenged by hydrogen peroxide or amyloid β25–35 28
To investigate the potential relationship of αTocH to our proteomic results on afamin, we measured the levels of αTocH via HPLC analysis in the plasma samples. The levels in five of the six monkeys that developed SIVE indeed dropped, but to varying degrees. However an identical result was found in animals that did not develop CNS disease. Thus the decrease in αTocH correlated with infection itself and not the development of CNS disease, in contrast to afamin that was decreased only in the animals that developed CNS disease. The independence of the levels of afamin and αTocH from each other in the plasma has indeed been reported by others 27
Whether the decrease in afamin as well as αTocH in the plasma of monkeys with CNS disease reflects similar changes in the brain was not assessed in this study, as appropriate samples were not available for analysis. However the cerebrospinal fluid (CSF) of HIV infected individuals was assessed by others for vitamin E levels in relationship to changes in cognitive status 29
. In this study, changes in CSF vitamin E were not associated directly with cognitive disorders, but increased CSF vitamin E preceded worsening of cognitive status. Whether this reflected an increased in the brain antioxidant defenses in the setting of incipient damage, or other potential causes, is unknown. In any case knowledge of the levels of αTocH and other forms of vitamin E in the brain itself during SIV/HIV induced CNS disease is a gap in our current knowledge.
Although advances in the search for biofluid-based biomarkers for neurodegenerative and other diseases have been made on several frontiers, limited success has been met. Amongst the emerging technologies, mass spectrometry based proteomics to assess CSF and plasma has greatly hastened the unbiased and high throughput for novel proteins as indicators of disease mechanisms and progression in a global manner. Further development of quantitative proteomics via differential tagging with isotopic reagents like ICAT and iTRAQ followed by multidimensional LC-MS/MS analysis has emerged as one of the most powerful methodologies in the quest for disease-linked proteins. Here we show the first successful application of the hybrid PQD/CID mode for iTRAQ analysis on an LTQ to identify disease-induced alterations in a disease model. PQD fragments peptides during MS/MS via a different mechanism than traditional CID, enabling the routine and reliable detection of ions. Peptide fragmentation occurs on a similar time scale compared to CID, thus producing high quality spectra peptide identification by sequence database searching.
Our cumulative data reveal that the antioxidant αTocH is decreased in the plasma following SIV infection, and that afamin is decreased in those infected monkeys that develop encephalitis. While afamin binds αTocH and other vitamin E molecules, the primary plasma transport of vitamin E is by lipoproteins 30
. However other molecules are crucial in the transport of vitamin E across barriers and into cells 31
. Interestingly a recent study has revealed that afamin is synthesized in the endothelial cells of the blood-brain barrier (BBB) and plays a role in αTocH transport across an in vitro
model of the BBB 19
. Given the neuroprotective role of αTocH-afamin during oxidative stress, further characterization of the role of afamin in SIV/HIV associated neurodegeneration, where the BBB can be compromised 32-35
, could reveal mechanistic clues for therapeutic intervention.