The current study aimed to identify proteins which are significantly up and down regulated in the cerebrospinal fluid of AD patients with respect to controls. In the absence of similar work in the north Indian population, the study focused on both replicating previous findings from diverse experimental designs as also identifying tentative new biomarker proteins. LC-MS/MS analysis allowed us to identify 13 upregulated and 30 down regulated proteins. Out of these, 10 were found to interact in a closed network at high confidence level.
4.1. Up regulated Proteins:
LGALS3BP (Galectin-3-binding protein) is a protein involved in integrin-mediated cell adhesion. (UniProt) LGALS3BP contains a scavenger receptor cysteine-rich (SRCR) domain which is associated with the innate immunity response. It also harbors a BTB/POZ domain which is involved in a wide variety of biologic processes including transcription regulation, cytoskeletal rearrangement, and protein ubiquitination.21 LGALS3BP has been found upregulated in serum of various diseases such as cancer and HIV. It is also seen that LGALS3BP is a main ligand for sialic acid-binding immunoglobulin-like lectins (Siglecs) that transmit inhibitory signals via intracellular immune receptor and modulate the immune response.22 iTRAQ labeling and tandem mass spectrometry based analysis on whole protein extracts of Alzheimer’s brains identified LGALS3BP as one of the overexpressed polypeptides. 23 Similar findings emerged from our study with significantly high levels in CSF of AD subjects as compared to controls. Dysregulation in immune response is one of the mechanisms involved in AD pathogenesis, and LGALS3BP may play a role in the process.
APOE (Apolipoprotein E) mediates lipid transport between organs including the central nervous system via the plasma and interstitial fluids. Apolipoprotein E gene (APOE) is the major risk gene identified as yet for late onset AD (LOAD) and has three major isoforms: E2, E3, E4. The APOE4 allele has been found to be significantly associated with AD.24 APOE is involved in the production, conversion and clearance of plasma lipoproteins. APOE also plays an important role in regulation of neuronal survival and sprouting.25 APOE affects the clearance and aggregation of monomeric Aβ in the brain of mice by modulating the microglial response. APOE deficient mice have shown a decrease in plaque–associated micro-gliosis and also decreased expression of activated microglial gene.26 Neurons fulfill their need for cholesterol by APOE mediated endocytosis of lipoprotein. Interference in the delivery of cholesterol is associated with neurodegeneration.27 In addition, immuno-histochemical analysis of Alzheimer's disease brains suggested APOE interacts with β-amyloid and influences its aggregation.28
4.2 Down regulated Proteins:
F2 (Prothrombin) is the precursor of thrombin that is known to be expressed in the brain and possesses growth modulating properties. F2 has been hypothesized to be involved in the pathogenesis of Alzheimer's disease (AD).29 Plasma circulating prothrombin is deposited within the microvessel wall of the brain and surrounding the neuropil due to thinning and discontinuities within the vascular basement membrane of the blood-brain barrier. This leakage is more common in AD patients with at least one APOE4 allele, and is directly proportional to the severity of disease.30 Thrombin has been detected immuno-histochemically in brain tissue of Alzheimer's disease (AD) patients and age-matched controls. There have been suggestions that thrombin formation from prothrombin probably takes place in the AD brain.31 However, CSF F2 concentration has not been demonstrated to be altered in AD.29 In our study, we found significant down regulation of F2 in the CSF of AD patients. This could be a result of blood brain barrier leakage, prothrombin deposition in the microvasculature and conversion to thrombin.
PENK (Proenkephalin) is a precursor fragment of the neuropeptide hormone enkephalin which also has a role in blood brain barrier integrity.32 PENK is expressed in the nervous system33 and contributes to various physiologic functions including glial cell proliferation, responses to stress and aging.34 It is also a member of the opioid system that contributes to the hyper phosphorylation of tau. The dysfunction of the opioid system is linked to cholinergic neuronal degeneration and AD pathogenesis.35 In our study, we found significant down regulation of PENK which may be in line with opioid system dysfunction that happens in AD brains.
IGF2 (Insulin-like growth factor II) is a major fetal growth hormone in mammals which is also involved in tissue differentiation and glucose metabolism and acts as a ligand for integrin.36. Administration of recombinant IGF2 in the hippocampal region of aged wild-type mice enhances memory and promotes dendritic spine formation.37 Infusion of IGF2 in transgenic AD mice also replicated the same findings. IGF2 reduces the number of hippocampal Aβ40- and Aβ42-positive amyloid plaques in transgenic mice model. IGF2 also increases the expression of proteins involved in neurogenesis including NGF, BDNF, NT3 and IGF1 and of doublecortin in the hippocampal region. A significantly decreased expression of IGF2 was found in the current study in the CSF of AD patients. This may point towards a failure of the neuroprotective mechanism during disease pathogenesis. The finding is at variance to a previous report of significantly elevated level of immunoreactive IGF2 in both serum and CSF of AD patient group.38
APOH (Beta-2-glycoprotein 1) is a lipid-binding protein that prevents activation of the intrinsic pathway of blood coagulation. Studies have suggested that APOH has links to cognitive aging and Alzheimer’s disease. A study on the plasma of 664 subjects with mild cognitive impairment and normal cognition suggested the association of APOH with increased risk of cognitive decline in cognitively normal people and its potential as a biomarker.39 A multiplex immunoassay was utilized in CSF samples of moderate-to-severe AD subjects to analyze 90 proteins including APOH. Significant increase was found in APOH level only in those with moderate to severe AD.40 In our study we found a significant decrease in the expression of APOH in the CSF of AD subjects as compared to controls which differs from previous evidence. The reason for this is unclear.
SAA1 (Serum amyloid A-1 protein) and SAA2 (Serum amyloid A-2 protein) are major acute-phase reactant proteins. They play an important role in cell chemotaxis and cytokine-mediated signaling pathways.41,42 The exact mechanism how these affect neurodegeneration pathogenesis is less established but down regulation of SAA1 and SAA2 is seen in PD subjects.43 Potential involvement in tau hypo-phosphorylation is also seen in mice model. Lipopolysaccharides (LPS) induce the expression of SAA in the brain that leads to suppression of tau hyper phosphorylation by activating glial response.44 In the current study, we observed a down regulation of SAA1 and SAA2 proteins in the CSF of AD patients. Low SAA levels may result in tau hyperphosphorylation and the AD pathogenetic cascade.
AHSG (Alpha-2-HS-glycoprotein) also known as fetuin-A, is a highly glycosylated protein45 which is primarily secreted by the liver and promotes endocytosis and possesses opsonic properties.46 It is a physiological inhibitor of insulin receptor tyrosine kinase.47 Studies have also suggested that AHSG has an important role in neurodegenerative diseases. A significant decrease in the expression of AHSG is reported from the cerebral cortex, hippocampus, and thalamus in transgenic mice model of AD with chronic hypoperfusion.48 Our study found a decreased CSF level of AHSG in AD patients, a finding which concurred with previous observations.45
SPP1 (secreted phosphoprotein 1/ Osteopontin) is an extracellular matrix protein, involved in cellular migration, fusion, and motility which is biosynthesized by a variety of tissue types including the brain.49 A study by Comi et al revealed osteopontin (OPN) to be significantly increased in the CSF of AD patients and also higher in the early disease phases.50 A targeted proteomic multiple reaction monitoring (MRM)-based assay was used by another group to measure multiple candidate CSF biomarkers in AD patients and showed significant elevation of osteopontin.51 The downregulation of SPP1 observed in the current study is unexplained and needs validation in large populations.
CD44 (CD44 antigen) is a cell-surface receptor that plays an important role in cell-cell interaction, cell adhesion, and migration. CD44 also co-ordinates calcium mobilization signaling and actin-mediated cytoskeleton reorganization which are essential for cell migration and adhesion.52 The expression of CD44 splice variants is significantly higher in postmortem hippocampal samples of AD patients compared to non-AD controls. It has hence been suggested that CD44 splice variants contribute to AD pathology.53 Expression of CD44 is found predominantly in the white matter and also in both glial cells and neuronal cells of the human brain.54 In the grey matter, CD44 is expressed in astrocytes and it is found that expression of CD44 positive astrocytes increases dramatically in the AD brain. It serves as a receptor for various proteins like hyaluronic acid, collagen types I and VI.55 Increased expression of CD44 is also reported in the lymphocytes derived from AD subjects with respect to age-matched controls.56 In our study we found significant down regulation of CD44 in CSF. This finding needs further validation in larger sample sizes.
4.3 Tentative mechanisms in pathogenesis of AD- pointers from STRING analysis
The proteins found to be differentially expressed in the CSF of AD patients and involved in a closed PPI network were analyzed for functional enrichment in STRING. With respect to biological processes, the largest enrichment effect (strength > 2) was observed for regulation of behavioral fear response (APOE and PENK), followed by negative regulation of fibrinolysis (APOH and F2), locomotory exploration behavior (APOE and PENK), intrinsic pathway of blood coagulation (APOH and F2), negative regulation of platelet activation (APOE and F2), the steroid catabolic process (APOE and SPP1), acute phase response (F2, AHSG and SAA1), the response to vitamin D (PENK and SPP1), negative regulation of blood coagulation (APOE, APOH and F2), and negative regulation of endothelial cell proliferation (APOE and APOH) (Figure 5). We observed that APOE, APOH, F2 and PENK are involved in multiple of these biological processes. The interactions of each of PENK, F2, SPP1 and APOH with APOE, a known pathogenetic marker of AD may be of relevance, indicating putative involvement.
Among the local STRING network clusters, 6 clusters were significantly enriched. One that we found to be particularly relevant was regulation of insulin like growth factor transport and uptake which was a component of 3 out of the 6 clusters, and included AHSG, APOE, IGF2 and F2 (Figure 6). Insulin interacts with amyloid-β precursor protein (AβPP or APP) although the exact mechanisms remain unclear.57 Insulin resistance in brain has been demonstrated to correlate with AD pathogenesis.58 IGF-2 binds and activates insulin receptors and regulates neurotransmitter release, memory consolidation and neuroprotection.59 IGF-2 triggers its signaling processes through MAPK and PI3K/Akt signaling pathways.60 Extensive abnormalities in IGF-2 signaling mechanisms leads to reduced levels of insulin receptor substrate (IRS) mRNA and PI3K/Akt. This causes an increase in glycogen synthase kinase-3β activity that decreases glycogen synthesis and increases amyloid precursor protein mRNA expression.61 Decrease in IGF2 levels may have a decremental effect on its signaling pathways. Prothrombin proteolyzes the microtubule-associated protein tau.62 Lowering of prothrombin maybe an indirect marker of greater conversion to thrombin. The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1) which leads to insulin resistance by inactivating IRS/Akt signaling pathway.63 AHSG also acts on the insulin-signaling pathway. It may be an alternative marker for insulin resistance.64 It has been shown that the serum concentration of AHSG is significantly elevated in diet-induced obese mice and it inhibits the metabolic effects of insulin.65 AHSG inhibits insulin-stimulated insulin receptor autophosphorylation, insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS1).66 Evidence currently is lacking for the role of CSF AHSG in brain limited insulin resistance. Insulin binding to its receptor in the nucleus accumbens (NAc) of the brain has been shown to activate the opioid receptor-mediated release of presynaptic glutamate which influences cognition and the decision-making processes.67 The involvement of proenkephalin in similar interactions of the insulin and opioid pathways may be hypothesized. There is a already a large evidence pool for the link between APOE and cerebral insulin.68
As regards to molecular function, a process of interest for us was glycosaminoglycan (GAG) binding. Five proteins were annotated with GAG binding (APOE, APOH, F2, SAA1, and CD44) (Figure 7). GAGs are the major components of extracellular matrix which participate in tissue homeostasis and self-healing.69 In Alzheimer’s disease pathogenesis, Aβ plaques bind to different extracellular membrane components including GAGs. GAGs enhance the formation of Aβ fibrils that lead to its extracellular accumulation.70 The C-terminal domain of APOE binds with GAGs present on the cell surface. APOE4 has a greater ability to bind with GAGs which is physiologically significant.71 It is observed that GAGs along with acetaldehyde directly affects F2 to mediate the anticoagulant reaction.72 Acetaldehyde- a product of ethanol metabolism is involved in the cross linking of F2 and GAGs; how this anticoagulant property of GAGs is important in AD pathogenesis is not established yet. Surface plasmon resonance spectroscopy and molecular modeling based analysis have shown that cell surface HS (heparin sulphate) converts HDL-associated SAA into AA amyloid fibrils in cell culture.73 A similar interaction may be a supportive event in causing accumulation of amyloid proteins in the brain. To mediate cell adhesion and intracellular signaling, CD44 binds with the chondroitin sulfate (CS) proteoglycan of GAGs.74 CS proteoglycan is critical in spinal injury and diseases of the central nervous system. They play an inhibitory effect on glial scar formation and improve the regeneration and functional recovery of neurons.75 CD44 is also a major receptor for hyaluronan (HA) which is a remarkably versatile GAG involved in cell signaling and inflammatory pathologies.