Plasma Proteomics Biomarkers in Alzheimer's Disease: Latest Advances and Challenges.

The recent paradigm shift towards a more biologically oriented definition of Alzheimer's disease (AD) in clinical settings increases the need for sensitive biomarkers that can be applied in population-based settings. Blood plasma is easily accessible and contains a large number of proteins related to cerebral processes. It is therefore an ideal candidate for AD biomarker discovery. The present chapter provides an overview of the current research landscape in relation to blood-based AD biomarkers. Both clinical and methodological issues are covered. A brief summary is given on two relevant laboratory techniques to ascertain blood biomarker changes due to AD; methodological and clinical challenges in the field are also discussed.

LRP-1 polymorphism is associated with global and regional amyloid load in Alzheimer's Disease in humans in-vivo.

OBJECTIVE: Impaired amyloid clearance has been proposed to contribute to ß-amyloid deposition in sporadic late-onset Alzheimer's disease (AD). Low density lipoprotein receptor-related protein 1 (LRP-1) is involved in the active outward transport of ß-amyloid across the blood-brain barrier (BBB). The C667T polymorphism (rs1799986) of the LRP-1 gene has been inconsistently associated with AD in genetic studies. We aimed to elucidate the association of this polymorphism with in-vivo brain amyloid load of AD patients using amyloid PET with [(11)C]PiB. MATERIALS AND METHODS: 72 patients with very mild to moderate AD were examined with amyloid PET and C667T polymorphism was obtained using TaqMan PCR assays. The association of C667T polymorphism with global and regional amyloid load was calculated using linear regression and voxel based analysis, respectively. The effect of the previously identified modulator of amyloid uptake, the apolipoprotein E genotype, on this association was also determined. RESULTS: The regression analysis between amyloid load and C667T polymorphism was statistically significant (p = 0.046, ß = 0.236). In an additional analysis ApoE genotype and gender were identified to explain further variability of amyloid load. Voxel based analysis revealed a significant (p < 0.05) association between C667T polymorphism and amyloid uptake in the temporo-parietal cortex bilaterally. ApoE did not interact significantly with the LRP-1 polymorphism. DISCUSSION: In conclusion, C667T polymorphism of LRP-1 is moderately but significantly associated with global and regional amyloid deposition in AD. The relationship appears to be independent of the ApoE genotype. This finding is compatible with the hypothesis that impaired amyloid clearance contributes to amyloid deposition in late-onset sporadic AD.

Amyloid cascade and tau pathology cerebrospinal fluid markers in mild cognitive impairment with regards to Alzheimer's disease cerebral metabolic signature.

BACKGROUND: Biomarker relationships in early stages of Alzheimer's disease (AD) are elusive. Cerebrospinal fluid (CSF) levels of amyloid-ß 1-42 (Aß42) and total tau (tTau) as well as 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) contribute to help unravel AD pathology. Furthermore, peptides related to amyloid-ß protein precursor (AßPP) processing [e.g., soluble AßPPα and ß (sAßPPα and sAßPPß, respectively); sortilin-related receptor with A-type repeats (SORL1, also called LR11 or SORLA)] are factors crucially implicated in the formation of pathological hallmarks of AD. OBJECTIVE: To unveil differences in CSF concentrations of Aß42, sAßPPα, sAßPPß, tTau, and SORL1 between patients with mild cognitive impairment (MCI) who were categorized according to expert interpretation of FDG scans. METHODS: PET results were classified as suggesting high likelihood for AD (MCI-AD high), intermediate likelihood for AD (MCI-AD intermediate), or little likelihood for AD (MCI-AD unlikely). An AD dementia group was also included. Differences between the groups were tested by Kruskal- Wallis test, Mann-Whitney test, or χ2. Provided statistically significant differences were detected, multiple linear regression models were employed. RESULTS: Aß42 levels in patients with MCI-AD high (n = 15) were lower compared to MCI-AD intermediate (n = 18) and MCI-AD unlikely patients (n = 25) (p = 0.002), while they did not differ from patients with AD dementia (n = 17). The regression model revealed a significant impact of the metabolic pattern on Aß42 concentrations. SORL1, tTau, sAßPPα, and sAßPPß concentrations did not differ between the groups. CONCLUSION: These findings point to linkages between plaque pathology and glucose cerebral hypometabolism.

Heart-type fatty acid binding protein and vascular endothelial growth factor: cerebrospinal fluid biomarker candidates for Alzheimer's disease.

The main objective of the study was to validate the findings of previous cerebrospinal fluid (CSF) proteomic studies for the differentiation between Alzheimer's disease (AD) dementia and physiological ageing. The most consistently significant proteins in the separation between AD dementia versus normal controls using CSF proteomics were identified in the literature. The classification performance of the four pre-selected proteins was explored in 92 controls, 149 patients with mild cognitive impairment (MCI), and 69 patients with AD dementia. Heart-type fatty acid binding protein (hFABP) and vascular endothelial growth factor (VEGF) CSF concentrations distinguished between healthy controls and patients with AD dementia with a sensitivity and specificity of 57 and 35%, and 76 and 84%, respectively. The optimal classification was achieved by a combination of the two additional CSF biomarker candidates in conjunction with the three established markers Amyloid-ß (Aß)1-42, total-Tau (tTau), and phosphorylated-Tau (pTau)181, which resulted in a sensitivity of 83% and a specificity of 86%. hFABP also predicted the progression from MCI to AD dementia. The present study provides evidence in support of hFABP and VEGF in CSF as AD biomarker candidates to be used in combination with the established markers Aß1-42, tTau, and pTau181.

Plasma proteomics for the identification of Alzheimer disease.

Less-invasive biomarkers for early Alzheimer disease (AD) are urgently needed. The present study aimed to establish a panel of plasma proteins that accurately distinguishes early AD from physiological aging and to compare the findings with previous reports. Fifty-eight healthy controls (CON) and 109 patients with AD dementia were randomly split into a training (40%) and a test (60%) sample. Significant proteins to differentiate between the CON and AD dementia groups were identified in a comprehensive panel of 107 plasma analytes in the training sample; the accuracy in differentiating these 2 groups was explored in the test sample. A set of 5 plasma proteins was identified, which differentiated between the CON group and the AD dementia group with a sensitivity of 89.36% and a specificity of 79.17%. A biological pathway analysis showed that 4 of 5 proteins belonged to a common network with amyloid precursor protein and tau. Apolipoprotein E was the only protein that was both significant in the present report and in a previous proteomic study. The study provides a piece of evidence in support of the feasibility of a blood-based biomarker approach in AD diagnostics; however, further research is required because of issues with replicability.

The National Institute on Aging-Alzheimer's Association research criteria for mild cognitive impairment due to Alzheimer's disease: predicting the outcome.

The National Institute on Aging-Alzheimer's Association (NIA-AA) clinical research criteria for mild cognitive impairment (MCI) due to Alzheimer's disease (AD) incorporate the use of biomarkers to classify patients according to the likelihood of the presence of AD pathology. The aim of the study was to compare the risk of progression to AD dementia between the four NIA-AA MCI subgroups using data from the AD Neuroimaging Initiative. Patients with MCI were categorised according to the NIA-AA criteria into subgroups with high, intermediate, and low likelihood of the presence of AD pathology (MCI-high, MCI-intermediate, and MCI-unlikely, respectively) or into a group of patients that only met the MCI-core clinical criteria (MCI-core). Data of follow-up visits conducted 6-60 months after baseline were used to compare the relative risk of future AD dementia between the four subgroups employing a Cox regression model. The MCI-high subgroup (N = 22) had a 2.3 times higher risk of developing AD dementia compared with the MCI-core subgroup (N = 327; P = 0.002), while there was a trend for a higher risk in the MCI-high subgroup in contrast to the MCI-intermediate subgroup (N = 31, P = 0.08). No patients in the MCI-unlikely subgroup (N = 17) progressed to AD dementia. Patients with MCI-high have a higher risk for developing AD dementia. The new NIA-AA MCI criteria represent a valuable research instrument that could be incorporated into the diagnostic process of the MCI syndrome after optimisation and refinement.

ß-Site amyloid precursor protein-cleaving enzyme 1 activity is related to cerebrospinal fluid concentrations of sortilin-related receptor with A-type repeats, soluble amyloid precursor protein, and tau.

BACKGROUND: ß-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) activity determines the rate of APP cleavage and is therefore the main driver of amyloid ß production, which is a pathological hallmark of Alzheimer's disease (AD). METHODS: The present study explored the correlation between BACE1 activity and cerebrospinal fluid (CSF) markers of APP metabolism and axonal degeneration in 63 patients with mild AD and 12 healthy control subjects. RESULTS: In the AD group, positive correlations between BACE1 activity and soluble APP ß, the APP sorting receptor sortilin-related receptor with A-type repeats (also known as SorLA or LR11), and tau were detected. BACE1 activity was not associated with amyloid ß1-42 or soluble APP α concentrations in the AD group, and no associations between BACE1 activity and any of the protein concentrations were found in the control group. CONCLUSION: Our results confirm the relevance of BACE1 and sortilin-related receptor with A-type repeats within the amyloid cascade and also provide a further piece of evidence for the link between amyloid and tau pathology in AD.

Brain size and the compensation of Alzheimer's disease symptoms: a longitudinal cohort study.

BACKGROUND: Greater intracranial volume (ICV) has been associated with less severe Alzheimer's disease (AD) symptoms at a given level of cerebral pathology. In this study we examine whether ICV modulates the association between clinical disease progression on the one hand and brain atrophy or the apolipoprotein E genotype on the other. METHODS: Six hundred seventy-four subjects were studied from the AD Neuroimaging Initiative (ADNI). Subjects included 204 controls, 144 patients with AD dementia, and 326 with amnestic mild cognitive impairment (aMCI). Longitudinal analyses were conducted applying generalized estimating equations to examine the influence of ICV on clinical deterioration and atrophy progression. Follow-up data were available for up to 60 months after the baseline visit (mean 31.42 months, SD 13.12 months). RESULTS: ICV was not directly associated with clinical worsening or atrophy progression. However, ICV attenuated the impact of atrophy and the apolipoprotein E ε4 allele on clinical disease progression in aMCI. CONCLUSION: Greater ICV, that is, premorbid brain size, seems to protect against clinical deterioration in the face of AD-related brain atrophy in aMCI. The results support the theory of a compensatory role of brain reserve in contrast to a neuroprotective role. The protective effects of morphologic reserve seem to be limited to early clinical AD; once a certain threshold of neurodegenerative burden is passed, a larger premorbid brain no longer offers an advantage in this context.

SORL1 genetic variants and cerebrospinal fluid biomarkers of Alzheimer's disease.

The neuronal sortilin-related receptor with A-type repeats (SORL1, also called LR11 or sorLA) is involved in amyloidogenesis, and the SORL1 gene is a major risk factor for Alzheimer's disease (AD). We investigated AD-related CSF biomarkers for associations with SORL1 genetic variants in 105 German patients with mild cognitive impairment (MCI) and AD. The homozygous CC-allele of single nucleotide polymorphism (SNP) 4 was associated with increased Tau concentrations in AD, and the minor alleles of SNP8, SNP9, and SNP10 and the haplotype CGT of these SNPs were associated with increased SORL1 concentrations in MCI. SNP22 and SNP23, and the haplotypes TCT of SNP19-21-23, and TTC of SNP22-23-24 were correlated with decreased Ab42 levels in AD. These results strengthen the functional role of SORL1 in AD.

Cerebrospinal fluid BACE1 activity and brain amyloid load in Alzheimer's disease.

The secretase BACE1 is fundamentally involved in the development of cerebral amyloid pathology in Alzheimer's disease (AD). It has not been studied so far to what extent BACE1 activity in cerebrospinal fluid (CSF) mirrors in vivo amyloid load in AD. We explored associations between CSF BACE1 activity and fibrillar amyloid pathology as measured by carbon-11-labelled Pittsburgh Compound B positron emission tomography ([¹¹C]PIB PET). [¹¹C]PIB and CSF studies were performed in 31 patients with AD. Voxel-based linear regression analysis revealed significant associations between CSF BACE1 activity and [¹¹C]PIB tracer uptake in the bilateral parahippocampal region, the thalamus, and the pons. Our study provides evidence for a brain region-specific correlation between CSF BACE1 activity and in-vivo fibrillar amyloid pathology in AD. Associations were found in areas close to the brain ventricles, which may have important implications for the use of BACE1 in CSF as a marker for AD pathology and for antiamyloid treatment monitoring.

Interrelations between CSF soluble AßPPß, amyloid-ß 1-42, SORL1, and tau levels in Alzheimer's disease.

Recently, light has been shed on possible interrelations between the two most important pathological hallmarks of Alzheimer's disease (AD): the amyloid cascade and axonal degeneration. In this study, we investigated associations between sßAPPß, a product of the cleavage of the amyloid-ß protein precursor (AßPP) by ß-secretase, amyloid-ß 1-42 (Aß42), soluble SORL1 (also called LR11 or SORLA), a receptor that is involved in AßPP processing, and the marker of axonal degeneration tau in the cerebrospinal fluid (CSF) of 76 patients with mild cognitive impairment (MCI), 61 patients with AD, and 17 patients with frontotemporal dementia, which neuropathologically is not related to the amyloid pathology. In the AD group, significant associations between sAßPPß, tau (p < 0.001), and soluble SORL1 (p < 0.001) were detected according to linear regression models. In patients with MCI, sAßPPß correlated significantly with tau (p < 0.001) and soluble SORL1 (p = 0.003). In the FTD group, only SORL1 (p = 0.011) was associated with sAßPPß and not tau. Aß42 was found to be significantly related to tau levels in CSF in the MCI group (p < 0.001) and they tended to be associated in the AD group (p = 0.05). Our results provide further evidence for a link between the two facets of AD pathology, which is likely to be mediated by the binding of Aß oligomers to specifically targeted neurons, resulting in stimulating tau hyperphosphorylation and neurodegeneration.

Impact of SORL1 single nucleotide polymorphisms on Alzheimer's disease cerebrospinal fluid markers.

BACKGROUND: Recently, genetic variants of the neuronal sortilin-related receptor with A-type repeats (SORL1, also called LR11 or sorLA) have emerged as risk factors for the development of Alzheimer's disease (AD). METHODS: In this study, SORL1 gene polymorphisms, which have been shown to be related to AD, were analyzed for associations with cerebrospinal fluid (CSF) amyloid beta1-42 (Aß(1-42)), phosphorylated tau181, and total tau levels in a non-Hispanic Caucasian sample, which encompassed 100 cognitively healthy elderly individuals, 166 patients with mild cognitive impairment, and 87 patients with probable AD. The data were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (www.loni.ucla.edu/ADNI). Moreover, the impact of gene-gene interactions between SORL1 single nucleotide polymorphisms (SNPs) and the apolipoprotein E (APOE) ε4 allele, the major genetic risk factor for sporadic AD, on Aß(1-42) concentrations was investigated. RESULTS: Significant associations between CSF Aß(1-42) levels and the SORL1 SNPs 23 (rs3824968) and 24 (rs2282649) were detected in the AD group. The latter association became marginally statistically insignificant after Bonferroni correction for multiple comparisons. Carriers of the SORL1 SNP24 T allele and the SNP23 A allele both had lower CSF Aß(1-42) concentrations than non-carriers of these alleles. The analysis of the impact of interactions between APOE ε4 allele and SORL1 SNPs on CSF Aß(1-42) levels unraveled significant influences of APOE. CONCLUSIONS: Our findings provide further support for the notion that SORL1 genetic variants are related to AD pathology, probably by regulating the amyloid cascade.

A simplified method to assess affinity of insulin autoantibodies.

Insulin autoantibodies (IAA) precede type 1 diabetes, but not all IAA-positive children develop other islet autoantibodies and disease. Diabetes risk can be stratified by laborious IAA affinity measurement using competition with multiple ligand concentrations. Here, we identify a single competitor concentration that discriminates low- and high-affinity IAA. Discrimination was achieved among 122 IAA-positive sera using 7.0 nM competitor which is 54-fold that of the assay radioligand concentration. Relative-binding <60% at this competitor concentration identified all 85 sera with affinities ≥1.0×108 L/mol and none with lower affinities (P<0.0001), and 45 (96%) of 47 multiple islet autoantibody-positive sera (P<0.0001). IAA competition was further tested in a second set of 119 IAA-positive sera. Of these, 99 fulfilled high-affinity competition criteria of <60% relative-binding at 7.0 nM competitor including 89 (94%) of 95 sera with multiple islet autoantibodies (P<0.0001). Thus, increased IAA specificity can be achieved with simple modification to existing assays.

Tracing the Pathogenesis of Type 1 Diabetes: A Report on the 44th Annual Meeting of the European Association for the Study of Diabetes (EASD).

Clinical type 1 diabetes is preceded by autoimmune destruction of the pancreatic beta-cells. However, progression to disease is not uniform. One challenge facing current diabetes research is therefore to identify biomarker profiles that accurately reflect the individual stage of type 1 diabetes pathogenesis and develop new techniques to distinguish between these profiles and associated diabetes risks. This report highlights some of the recent studies on diabetes biomarkers, with a particular focus on zinc transporter ZnT8, presented at the EASD meeting in September 2008 in Rome, Italy.

Expression of interleukin-16 by tumor-associated macrophages/activated microglia in high-grade astrocytic brain tumors.

INTRODUCTION: Macrophages/microglial cells are considered as immune cells in the central nervous system. Interleukin (IL)-16 is a proinflammatory cytokine produced by activated monocytic cells. MATERIALS AND METHODS: Expression of IL-16 was analyzed by immunohistochemistry in human astrocytic brain tumors and the rat C6 glioblastoma tumor model. IL-16 was detected in both human astrocytic brain tumors and rat C6 glioma. RESULTS: Compared with human control brains, a significant increase in the percentages of parenchymal IL-16+ macrophages/microglia was observed already in grade II astrocytomas, indicating that IL-16+ immunostaining could be a descriptor of a macrophage/microglia subset in astrocytic brain tumors. A further increase was observed at the transition from grade II to III astrocytomas. This increase in IL-16 immunoreactivity correlated with WHO grades of human astrocytic brain tumors. CONCLUSIONS: Therefore, IL-16 might be a so far unknown factor in the regulation of the local inflammatory milieu of human and experimental astrocytomas.

Acute but not chronic stimulation of glial cells in rat spinal cord by systemic injection of lipopolysaccharide is associated with hyperalgesia.

We have analyzed development of mechanical hyperalgesia after repeated systemic lipopolysaccharide (LPS) injections and correlated these findings with stimulation of astrocytes and microglia in spinal cord. Male Lewis rats received a single or seven intraperitoneal injections of LPS. Mechanical hyperalgesia was measured as rat hindpaw withdrawal thresholds (PWTs). We observed that a single LPS injection elicited a specific change of PWTs while stimulated spinal glial activation was identified by immunoreactivities of specific markers, ED1, P2X4 receptor, endothelial monocyte activating polypeptide II (EMAP II) and glial fibrillary acidic protein (GFAP), respectively; multiple LPS treatments induced tolerance to mechanical hyperalgesia, whereas expression of ED1 and GFAP were further increased. In conclusion, we have demonstrated that the number of activated spinal glial cells was increased as an acute effect of LPS correlating with increased sensitivity to mechanical stimulation. However chronic exposure to LPS can develop a tolerance to mechanical hyperalgesia despite ongoing signs of CNS glial activation.

Combinations of TLR and NOD2 ligands stimulate rat microglial P2X4R expression.

As ATP-gated ion channels, P2X4 receptors (P2X4R) of microglial cells play a crucial role in central nervous system (CNS) inflammation. In this study, we used rat microglial cell cultures to examine P2X4R expression in response to stimulation by combination of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) receptors. Various TLR1-9 ligands and NOD2 agonist muramyldipeptide (MDP) were investigated. Our results showed that certain combination of ligands had additive effects on upregulating microglial P2X4R at both mRNA and protein levels, and induced nitric oxide increase and tumor necrosis factor-alpha production. Thus TLRs and NOD2 combinations are contributors to the signaling cascades resulting in purinergic microglial activation.

Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain.

P2X4 receptor (P2X4R) is an ion channel gated by adenosine 5'-triphosphate. Here we report the presence and the distribution of P2X4R in rat spinal cord by immunohistochemical analysis in an inflammatory pain model. Peripheral inflammation was induced by subcutaneous injection of 4% formalin into the rat hindpaw. Morphology, spatial localization, and activation state of P2X4R+ cells were described at 1, 5, 7, 14, and 28 days after injury. In normal and saline treated control rats, P2X4R was rarely seen. After formalin administration, an increase of P2X4R+ microglia were observed in the spinal cord dorsal horn on the side ipsilateral to the injection, reaching maximal levels by day 7, and then decreasing to normal levels by day 14. This implicates a role of P2X4R in the spinal inflammatory pain process. Furthermore, formalin-induced region-specific increase in activated microglia was confirmed by ED1 and endothelial monocytes activating polypeptide II (EMAP-II) expression. In conclusion, this is the first demonstration that P2X4R is expressed by microglia in the inflammatory pain.

Expression of P2X4 receptor in rat C6 glioma by tumor-associated macrophages and activated microglia.

Here we report an immunohistochemical analysis of P2X4 receptor (P2X4R), an ATP-gated ion channel, expression in rat C6 glioma model. Striking P2X4R expression was detected in compact and infiltrative tumor growth areas. Expression of P2X4R by perivascular cells was observed in normal control brain and in brain areas not affected by tumor growth. Double-immunolabeling revealed that P2X4R was co-expressed by ED1+, AIF-1+, and EMAP II+ tumor-infiltrating microglia/macrophages; whereas on GFAP+ and nestin+ astrocytes P2X4R was hardly seen. Our results indicate that P2X4R expression defines a distinct subset of tumor-associated macrophages and activated microglia in glioma.

Expression of interleukin-16 by microglial cells in inflammatory, autoimmune, and degenerative lesions of the rat brain.

Here we report a comparative analysis of interleukin-16 (IL-16) expression by microglial cells of the normal rat brain in trimethyltin (TMT) neurotoxicity, experimental autoimmune uveitis (EAU), encephalomyelitis (EAE), and viral infection (Borna disease, Borna disease virus) by immunohistochemistry. Striking differences were observed. In contrast to the human brain, IL-16 was not expressed constitutively in the rat brain. Remote activation of microglial cells of the optic tract in EAU did not result into IL-16 expression. TMT intoxication induced expression in microglial cells of the hippocampus. In EAE and BDV, massive IL-16(+) microglial cells could be seen. Thus, IL-16 is a descriptor of microglial cell activation that discriminates between different disease models, and might be a valuable marker for the detection of microglia activation in human and rat central nervous system (CNS) diseases.