Osteopontin: A novel marker of pre-symptomatic sporadic Alzheimer's disease

Abstract

Introduction: We investigate the role of osteopontin (OPN) in participants with Pre-symptomatic Alzheimer's disease (AD), mild cognitive impairment (MCI), and in AD brains.

Methods: Cerebrospinal fluid (CSF) OPN, AD, and synaptic biomarker levels were measured in 109 cognitively unimpaired (CU), parental-history positive Pre-symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer's Disease (PREVENT-AD) participants, and in 167 CU and 399 participants with MCI from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. OPN levels were examined as a function of amyloid beta (Aβ) and tau positivity. Survival analyses investigated the link between OPN and rate of conversion to AD.

Results: In PREVENT-AD, CSF OPN was positively correlated with synaptic biomarkers. In PREVENT-AD and ADNI, OPN was elevated in CSF Aβ_42/40(+)/total tau(+) and CSF Aβ_42/40(+)/phosphorylated tau181(+) individuals. In ADNI, OPN was increased in Aβ(+) positron emission tomography (PET) and tau(+) PET individuals, and associated with an accelerated rate of conversion to AD. OPN was elevated in autopsy-confirmed AD brains.

Discussion: Strong associations between CSF OPN and key markers of AD pathophysiology suggest a significant role for OPN in tau neurobiology, particularly in the early stages of the disease.

Highlights: In the Pre-symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer's Disease cohort, we discovered that cerebrospinal fluid (CSF) osteopontin (OPN) levels can indicate early synaptic dysfunction, tau deposition, and neuronal loss in cognitively unimpaired elderly with a parental history. CSF OPN is elevated in amyloid beta(+) positron emission tomography (PET) and tau(+) PET individuals. Elevated CSF OPN is associated with an accelerated rate of conversion to Alzheimer's disease (AD). Elevated CSF OPN is associated with an accelerated rate of cognitive decline on the Alzheimer's Disease Assessment Scale-Cognitive subscale 13, Montreal Cognitive Assessment, Mini-Mental State Examination, and Clinical Dementia Rating Scale Sum of Boxes. OPN mRNA and protein levels are significantly upregulated in the frontal cortex of autopsy-confirmed AD brains.

Keywords: post mortem; Alzheimer's disease; Pre‐symptomatic; autopsied brains; biomarkers; cerebral ventricles; cerebrospinal fluid; mRNA; neuroinflammation; osteopontin; positron emission tomography; secreted phosphoprotein 1; synaptic markers.

FIGURE 1

CSF OPN is associated with CSF AD biomarkers and synaptic biomarkers in asymptomatic PREVENT‐AD participants. CSF OPN levels were measured using the Olink Proximity Extension Assay (n = 109); (A) Aβ₄₂, (B) p‐tau181, and (C) t‐tau were measured using validated Innotest ELISA kits, following the standardized protocols established by the BIOMARKAPD consortium (n = 101). The synaptic markers (D) SNAP‐25 (n = 106), (E) SYT1 (n = 106), (F) GAP43 (n = 46), and (G) NRGN (n = 46) were quantified using immunoprecipitation followed by mass spectrometry. Significant linear regressions are represented with a blue confidence region of the fitted line. R ², β, and p‐values are located in the top left corners of each panel. Analyses were adjusted for age, sex, and APOE ε4 carrier status. Aβ, amyloid beta; AD, Alzheimer's disease; APOE, apolipoprotein E; a.u., arbitrary units; BIOMARKAPD, Biomarkers for Alzheimer's Disease and Parkinson's Disease; CSF, cerebrospinal fluid; ELISA, enzyme‐linked immunosorbent assay; GAP43, growth‐associated protein 43; NPX, normalized protein expression; NRGN, neurogranin; OPN, osteopontin; p‐tau, phosphorylated tau; PREVENT‐AD, Pre‐symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer's Disease; SNAP‐25, synaptosomal‐associated protein 23; SYT1, synaptotagmin‐1; t‐tau, total tau.

FIGURE 2

CSF OPN is elevated in asymptomatic CSF Aβ_42/40(+)/t‐tau(+) and CSF Aβ_42/40(+)/p‐tau181(+) PREVENT‐AD participants. CSF OPN levels were measured using the Olink Proximity Extension Assay (n = 109). Aβ₄₂, p‐tau181, and t‐tau were measured using validated Innotest ELISA kits, following the standardized protocols established by the BIOMARKAPD consortium (n = 101). CSF Aβ₄₀ was measured using a previously described Meso Scale Discovery assay (n = 95). (A) 95 CU PREVENT‐AD participants were staged as CSF Aβ_42/40 and/or CSF t‐tau positive according to the thresholds of 0.157 and 336 pg/mL, respectively. Fourteen participants that were within ± 5% of either threshold were excluded a priori. Linear models, adjusted for age, sex, and APOE ε4 carrier status were used to examine mean differences in OPN protein levels across stages. (B) Mean differences in CSF OPN were contrasted across CSF Aβ_42/40 and t‐tau stages. (C) 95 CU PREVENT‐AD participants were staged as CSF Aβ_42/40 and/or CSF p‐tau181 positive according to the thresholds of 0.157 and 57 pg/mL, respectively. Thirteen participants that were within ± 5% of either threshold were excluded a priori. (D) Mean differences in CSF OPN were contrasted across CSF Aβ_42/40 and p‐tau181 stages. The data are represented as mean ± SEM. Aβ, amyloid beta; AD, Alzheimer's disease; APOE, apolipoprotein E; BIOMARKAPD, Biomarkers for Alzheimer's Disease and Parkinson's Disease; CSF, cerebrospinal fluid; CU, cognitively unimpaired; ELISA, enzyme‐linked immunosorbent assay; NPX, normalized protein expression; OPN, osteopontin; p‐tau, phosphorylated tau; PREVENT‐AD, Pre‐symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer's Disease; SEM, standard error of the mean; t‐tau, total tau.

FIGURE 3

CSF OPN is associated with tau PET burden in Braak stages II to III in asymptomatic PREVENT‐AD participants. CSF OPN levels were measured using the Olink Proximity Extension Assay (n = 109). (A) Global cortical Aβ deposition was measured using ¹⁸F‐NAV4694 (n = 46). Participants were staged as Aβ(+) PET (n = 9) based on a previously established SUVR threshold of 1.37. (B) Tau deposition was quantified using flortaucipir (n = 49). Participants were staged as tau(+) PET (n = 7) based on a previously established entorhinal cortex SUVR threshold of 1.23. Tau deposition was assessed in Braak stages II to III, most notably, (C) the entorhinal cortex, (D) fusiform gyrus, and (E) lingual gyrus. Significant linear regressions are represented with a blue confidence region of the fitted line. R ², β, and p‐values are located in the top left corners of each panel. Analyses were adjusted for age, sex, and APOE ε4 carrier status. Aβ, amyloid beta; APOE, apolipoprotein E; CSF, cerebrospinal fluid; NPX, normalized protein expression; OPN, osteopontin; PET, positron emission tomography; PREVENT‐AD, Pre‐symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer's Disease; SUVR, standardized uptake value ratio.

FIGURE 4

CSF OPN is associated with reductions in cerebral ventricle volume in asymptomatic PREVENT‐AD participants. CSF OPN levels were measured using the Olink Proximity Extension Assay (n = 109); (A) Lateral ventricle, (B) third ventricle, and (C) fourth ventricle volumes were computed from T1‐weighted MRI images, using a volumetric pipeline that has been previously described (n = 104). Significant linear regressions are represented with a blue confidence region of the fitted line. R ², β, and p‐values are located in the top right corners of each panel. Analyses were adjusted for total ICV, age, sex, and APOE ε4 carrier status. APOE, apolipoprotein E; CSF, cerebrospinal fluid; ICV, intracranial volume; MRI, magnetic resonance imaging; NPX, normalized protein expression; OPN, osteopontin; PREVENT‐AD, Pre‐symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer's Disease.

FIGURE 5

CSF OPN is elevated in CSF Aβ_42/40(+)/t‐tau(+), CSF Aβ_42/40(+)/p‐tau181(+), Aβ(+) PET, and tau(+) PET individuals from the ADNI cohort. CSF OPN levels were measured using the SomaScan proteomics assay. CSF Aβ_42/40 ratios were measured using the Lumipulse G β‐Amyloid assays from Fujirebio. CSF t‐tau and p‐tau181 were measured using the INNO‐BIA AlzBio3 immunoassay kits and the xMap Luminex platform. (A) Forty‐four CU participants and 118 participants with MCI from the ADNI cohort were staged as CSF Aβ_42/40 and/or CSF t‐tau positive according to the recommended thresholds of 0.058 and 93 pg/mL, respectively., However, as recommended by the FDA, CSF Aβ_42/40 ratios 0.059 to 0.072 were considered amyloid positive. Linear models, adjusted for age, sex, and APOE ε4 carrier status, were used to examine mean differences in OPN protein levels across stages. (B) Forty‐four CU participants and 118 participants with MCI from the ADNI cohort were staged as CSF Aβ_42/40 and/or CSF p‐tau181 positive according to the recommended thresholds of 0.058 and 23 pg/mL, respectively., However, as recommended by the FDA, CSF Aβ_42/40 ratios 0.059 to 0.072 were considered amyloid positive. (C) Aβ deposition in a cortical summary region of interest was quantified using florbetaben or florbetapir. One hundred twenty‐four CU participants and 273 participants with MCI from the ADNI cohort were staged as Aβ(+) PET based off of recommended SUVR thresholds of 1.08 and 1.11, respectively. (D) Tau deposition in a temporal meta‐region of interest (ROI) was determined using flortaucipir. Sixty CU participants and 59 participants with MCI from the ADNI cohort were staged as tau(+) PET (n = 21) if their meta‐ROI uptake surpassed two standard deviations from the mean of Aβ(–) PET participants that were CU at baseline (SUVR cut‐off = 1.37). (E) Aβ and tau PET staging combined was limited by the number of individuals that underwent tau PET scans (n = 119). The data are represented as mean ± SEM. Aβ, amyloid beta; ADNI, Alzheimer's Disease Neuroimaging Initiative; APOE, apolipoprotein E; CSF, cerebrospinal fluid; CU, cognitively unimpaired; FDA, US Food and Drug Administration; MCI, mild cognitive impairment; OPN, osteopontin; PET, positron emission tomography; p‐tau, phosphorylated tau; SEM, standard error of the mean; SUVR, standardized uptake value ratio; t‐tau, total tau.

FIGURE 6

Elevated CSF OPN is associated with greater rates of cognitive decline and conversion to AD in the ADNI cohort. CSF OPN levels were measured using the SomaScan proteomics assay in 399 participants with MCI. Cox proportional hazards models examined the association between baseline CSF OPN levels and rate of conversion to AD. Participants were classified into tertiles based on their CSF OPN measurements. Fifteen individuals with < 6 months of follow‐up were excluded, and 14 individuals with ambiguous conversion dates were excluded from analyses. Of the 370 individuals that were followed longitudinally, 154 individuals eventually received a clinical diagnosis of AD. (A) The initial Cox model was unadjusted for confounding variables, such as age, sex, and APOE ε4 carrier status. Individuals with CSF OPN values in the top tertile exhibited a significantly greater rate of conversion to AD, compared to the first tertile. Furthermore, participants underwent extensive neuropsychological assessments, including the (B) ADAS‐Cog13, (C) CDR‐SB, (D) MoCA, and (E) MMSE. Linear mixed models demonstrated that individuals in the top tertile exhibited greater rates of cognitive decline on all cognitive assessments compared to the middle and bottom tertiles. Linear mixed models were adjusted for age, sex, APOE ε4 carrier status, and years of education. AD, Alzheimer's disease; ADAS‐Cog, Alzheimer's Disease Assessment Scale‐Cognitive Subscale; ADNI, Alzheimer's Disease Neuroimaging Initiative; APOE, apolipoprotein E; CDR‐SB, Clinical Dementia Rating Scale Sum of Boxes; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; MMSE, Mini‐Mental State Examination; MoCA, Montreal Cognitive Assessment; OPN, osteopontin.

FIGURE 7

OPN mRNA and protein levels are elevated in the frontal cortex of autopsy‐confirmed AD brains. (A) Microarray technology was used to measure OPN mRNA levels in the frontal cortex of autopsy‐confirmed AD brains (n = 55) and age‐matched elderly controls (n = 31) from the QFP cohort. (B) OPN protein levels were measured in the frontal cortex of AD brains (n = 52) and control brains (n = 23) using a commercially available ELISA kit. p‐values are located in the top left corner of each panel. Analyses were adjusted for age, sex, APOE ε4 carrier status, and post mortem interval. The data are represented as mean ± SEM. AD, Alzheimer's disease; APOE, apolipoprotein E; CTL, control; ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA; OPN, osteopontin; QFP, Quebec Founder Population; SEM, standard error of the mean.