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. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Neurobiol Aging. 2014 May 27;35(11):2486–2494. doi: 10.1016/j.neurobiolaging.2014.05.019

CSF sphingolipids, β-amyloid, and tau in adults at risk for Alzheimer's disease

Michelle M Mielke a,†,*, Norman J Haughey b,†, VVR Bandaru b, Henrik Zetterberg c, Kaj Blennow c, Ulf Andreasson c, Sterling C Johnson d,e,f, Carey E Gleason d,e,f, Hanna M Blazel d,f, Luigi Puglielli d,e,f, Mark A Sager d,f,g, Sanjay Asthana d,e,f, Cynthia M Carlsson d,e,f
PMCID: PMC4170854  NIHMSID: NIHMS601466  PMID: 24952994

Abstract

Cellular studies suggest sphingolipids may cause or accelerate amyloid-beta (Aβ) and tau pathology but in vivo human studies are lacking. We determined cerebrospinal fluid (CSF) levels of sphingolipids (ceramides, sphingomyelins), amyloid-beta (Aβ1–42, AβX-38, AβX-40, AβX-42) and tau (T-tau, p-tau181) in 91 cognitively normal individuals, aged 36–69 years, with a parental history of Alzheimer's disease (AD). The 18-carbon acyl chain length ceramide species was associated with AβX-38 (r = 0.312, p = 0.003), AβX-40 (r = 0.327, p = 0.002), and T-tau (r = 0.313, p = 0.003) but not with AβX-42 (r = 0.171, p = 0.106) or p-tau (r = 0.086, p = 0.418). All sphingomyelin species correlated (most p < 0.001) with all Aβ species and T-tau; many also correlated with p-tau. Results remained in regression models after controlling for age and APOE genotype. These results suggest in vivo relationships between CSF ceramides and sphingomyelins and Aβ and tau levels in cognitively normal individuals at increased risk for AD, indicating these sphingolipids may be associated with early pathogenesis.

Keywords: Sphingolipids, Ceramide, Sphingomyelin, Cerebrospinal fluid, Beta-amyloid, Tau, Alzheimer's disease

1. Introduction

As evidenced by the many failed treatment trials for Alzheimer's disease (AD), there appears to be minimal treatment benefit in the fully symptomatic stage of the disease. The pathogenesis of amyloid-beta (Aβ) plaques and neurofibrillary tangles begins decades before the emergence of clinical symptoms (Jack, et al., 2010; Reiman, et al., 2012) and impacts multiple cellular pathways. While much research has focused on Aβ as a biomarker and therapeutic target for AD, there is limited understanding of the cellular pathways that contribute to, or are caused by, Aβ aggregation and deposition or tau phosphorylation. Understanding the early mechanisms associated with AD pathology may be especially important for identifying disease-modifying therapeutic targets.

Lipidomic and targeted approaches have identified pathways and products of sphingolipid metabolism that are altered early in the course of AD and contribute to AD neuropathology (Haughey, et al., 2010; Mielke and Haughey, 2012). Sphingolipid metabolism is a dynamic process that modulates the formation of a number of bioactive metabolites. Sphingomyelins are among the most abundant lipids in many mammalian cells and tissues. They are central to the creation of lipid rafts and ordered membrane domains (Quinn and Wolf, 2009; Simons and Ikonen, 1997; Simons and Vaz, 2004), the functional regulation of membrane-spanning proteins (Contreras, et al., 2012), act as important regulators of plasma membrane and cell cholesterol homeostasis (Gatt and Bierman, 1980; Slotte and Bierman, 1988), and are precursors for other sphingolipids such as ceramides. Ceramides, the central molecular species of the sphingolipids pathway, have important structural roles in cell membranes and function as second messengers for critical intra- and inter-cellular signaling affecting cellular growth, differentiation, proliferation, and apoptosis. While ceramides are important for cell survival, injury-induced cytokine production, and activate stress-signaling protein phosphatases and kinases (Hannun, 1996), at high levels ceramides inhibit cell division and induce cellular dysfunction and apoptosis (Goodman and Mattson, 1996).

Several lines of evidence suggest both direct and indirect associations between ceramides and Aβ levels at the cellular level (Mattson, et al., 2005; Tamboli, et al., 2011). Recent studies have also suggested that ceramides, particularly the 18-carbon acyl chain length species, modulate tau phosphorylation (Chalfant, et al., 1999; Dobrowsky, et al., 1993; Goedert, et al., 1995; Gong, et al., 1994; Mukhopadhyay, et al., 2009). While one post-mortem study reported correlations between sphingomyelinase activity, Aβ and phosphorylated tau,(He, et al., 2010) CSF studies of sphingolipids in AD have not examined the in vivo relationship between sphingolipids and AD pathology (Kosicek, et al., 2012; Satoi, et al., 2005). Therefore, we examined the association between CSF ceramides and sphingomyelins, Aβ, and tau in cognitively normal individuals aged 36 to 69 years at increased risk for AD due to a parental family history.

2. Methods

2.1. Participants and recruitment

Participants included 91 cognitively normal middle-aged adults with parental history of AD enrolled in the ESPRIT (Evaluating Simvastatin's Potential Role in Therapy) trial who had available CSF Aβ, tau, and sphingolipid measures at baseline, prior to randomization. The ESPRIT study is a prospective, randomized, placebo-controlled clinical trial that aimed to determine the effects of nine months of simvastatin therapy on CSF biomarkers for AD including Aβ and phospho-tau. Participants were recruited from the Wisconsin Registry for Alzheimer's Prevention (WRAP) (Sager, et al., 2005) and also from the community. Prior to enrollment into the ESPRIT trial, the diagnosis of AD in one or both parents was confirmed for each participant by using the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria through clinical evaluation and/or chart review by Wisconsin Alzheimer's Disease Research Center physicians and neuropsychologists. Exclusion criteria included current use of cholesterol-lowering medications or medications known to interact with simvastatin, contraindication to lumbar puncture, active liver disease, diabetes mellitus, past adverse reaction to a statin, or elevated creatine kinase. The study was approved by the Institutional Review Board of the University of Wisconsin. Subjects provided written informed consent prior to participating. This clinical trial was registered with ClinicalTrials.gov (NCT00486044).

2.2. Study procedures

CSF collection was completed in the morning at approximately the same time at each visit. A Sprotte 22- or 25-gauge spinal needle was inserted into the L3-4 or L4-5 interspace and between 8 to 22 mL of CSF was removed. CSF samples were centrifuged (to remove red blood cells [RBCs]) at the University of Wisconsin Clinical Research Unit within 30 minutes of collection, aliquoted into 0.5 mL polypropylene storage tubes, and stored in a −80°C freezer until simultaneous analysis was conducted at the conclusion of the study. As ceramide and sphingomyelin levels can be elevated in RBCs, we also quantified RBCs in the CSF and found the concentrations to be low (mean 15.55, SD 82.5, median 1.00). This suggests that remaining RBCs would have little impact on our results.

2.3. Cognitive testing

All participants underwent cognitive testing by a trained technician according to protocols established for each test. The cognitive test battery targeted four domains: memory and learning (Hopkins Verbal Learning Test [HVLT] (Brandt, 1991); New York University [NYU] Paragraph Recall (Kluger, et al., 1999); Modified Taylor Complex Figure delayed recall (Hubley, 1996); Visual Spatial Learning Test [VSLT]) (Malec, et al., 1997); language (Controlled Oral Word Association Test) (Benton and Hamsher, 1989); visual-motor skills (Modified Taylor Complex Figure – Copy (Hubley, 1996); Grooved Pegboard) (Kløve, 1963); and executive functioning (Ruff 2 and 7 Selective Attention Test (Ruff, et al., 1986); Stroop Color-Word Test (Spreen and Strauss, 1998); Color Trails Test (D'Elia, et al., 1996); Wechsler Memory Scale – Third Edition [WMS-III] Mental Control, Letter-Number Sequencing, and Spatial Span subtests (Wechsler, 1997); Wechsler Adult Intelligence Scale [WAIS]-III Symbol Search and Digit Symbol Substitution Subtests) (Wechsler, 1991). Mini-Mental State Exam was administered to assess global cognitive function (Folstein, et al., 1975). To reduce multiple comparisons among cognitive measures, six cognitive variables were prospectively identified targeting memory, learning, and executive function: 1) HVLT total learning summary score (sum of trials 1–3); 2) HVLT delayed recall score; 3) WAIS-III Processing Speed Index; 4) WMS-III Mental Control total score; 5) WMS-III Working Memory score; and 6) Stroop Color-Word score.

2.4. Laboratory evaluation

Blood tests were collected following a 12-hour fast. Lipoproteins were analyzed by NMR spectroscopy (LipoScience, Inc., Raleigh, NC) (Otvos, 2002). CK, AST, and ALT levels were measured using enzymatic precipitation techniques. APOE genotype was measured using standard PCR and DNA sequencing techniques.

2.5. Sphingolipid assays

A crude lipid extraction of CSF was conducted using a modified Bligh and Dyer procedure as previously described (Haughey, et al., 2004). To control for slight differences in extraction efficiencies, and day to day variations in the efficiency of the mass spectrometer, purified standards of sphingomyelin C12:0 and ceramide C12:0 (10 nM each; Avanti Polar Lipids) were added directly to samples prior to extraction. Extractions were performed by the addition of 900 μl methanol containing ammonium formate (53 mM) to 300 μl of CSF. The mixture was vortexed, and 1200 μl of chloroform was added. The mixture was centrifuged at 1,000 g for 10 minutes. The chloroform (bottom) layer was carefully removed and dried in a nitrogen evaporator (OASYS model 11848. Organomation Associates, Inc.). Dried extracts were sealed and stored at −80°C. The dried extracts were re-suspended in 100% methanol prior to analysis.

Analyses of sphingolipids were performed on a high-performance liquid chromatography coupled electrospray ionization tandem mass spectrometer (LC/ESI/MS/MS) (API3000s, Sciex Inc., Thornhill, Ontario, Canada) using methods similar to those described in previous studies (Haughey, et al., 2004). Quantitation of sphingomyelins (d18:1/C16:0-C26:1) and simple and complex ceramides (d18:1/C16:0-C26:1) were conducted by multiple reaction monitoring (MRM). Samples were injected using a CTC PAL autosampler (LEAP technologies, Inc.) into a PerkElmer HPLC equipped with a 2.6 μm C18 100 Å LC Column 50 × 2.1 mm (Kinetex) and a guard column with identical packing material (Phenomenex). For a typical run, the LC column was first pre-equilibrated for 0.5 min with the first mobile phase consisting of 85% methanol, 15% H20, and 5 mM ammonium formate. The column was then eluted with the second mobile phase consisting of 99% methanol, 1% formic acid, and 5 mM ammonium formate at the flow rate of 400.0 μl/min. The eluted sample was injected into the ion source where the detection of each analyte was conducted by ESI/MS/MS in MRM mode monitoring the precursor, and products by ion scan.

Area under the curve was used to quantitate each sphingomyelin and simple and complex ceramide species using MultiQuant (AB Sciex). The resulting data were normalized to the corresponding internal standard for sphingomyelin (C18:1/C12:0) or ceramides (C18:1/C12:0). Each molecular species was analyzed separately to determine whether there were chain-specific associations within each sphingolipid species. A summary variable of all species within each class (sphingomyelins, ceramides) was also examined. Quantitation was reported as counts per second (cps). As concentrations were skewed to the right, similar to previous studies (Mielke, et al., 2010a; Mielke, et al., 2012), they were log-transformed prior to analyses.

2.6. CSF amyloid-beta and tau assays

Levels of β-amyloid1-42 (Aβ1-42), total-tau (T-tau) and phosphorylated tau 181 (ptau) in CSF were analyzed using the xMAP platform with the INNO-BIA AlzBio3 assay, as described previously in detail (Olsson, et al., 2005). Levels of CSF AβX-38, AβX-40 and AβX-42 were measured using Meso Scale Discovery (MSD®) electrochemiluminescence detection technology and the MSD Human/Rodent (4G8) Abeta Triplex Assay on a SECTOR™ Imager 2400 instrument as described by the manufacturer (Meso Scale Discovery, Gaithersburg, MD, USA). This assay employs Cterminally specific antibodies to capture AβX-38, AβX-40 and AβX-42, respectively, and a SULFO-TAG-labeled 4G8 antibody (directed against the Aβ mid-domain) to quantify them. All analyses were performed batch-wise by board-certified laboratory technicians who were blinded to all clinical data.

2.7. Statistical analysis

The relationship between the CSF sphingolipids and demographics, health characteristics, cognitive performance, and CSF Aβ and tau levels were examined using Spearman rank correlation for continuous variables and t-tests for dichotomous variables. Linear regression models were used to examine the relationship between the CSF sphingolipids and CSF Aβ and tau, controlling for age and APOE E4 genotype. Linear regression models were also used to examine the relationship between the CSF sphingolipids and performance on each cognitive test, controlling for age, education and APOE genotype. The a priori p-value was set at p < 0.05. All analyses were conducted using STATA Version 12.1 (StataCorp, College Station, TX).

3. Results

Characteristics of the 91 individuals included in this study are described in Table 1. On average, participants were 53.4 (SD = 7.9) years of age, had 16 years of education, were overweight (mean BMI 27.8, SD = 5.5) and had normal levels of cholesterol, triglycerides, and blood glucose. There were 63 women (69.2%); 33 participants (36.3%) had at least one APOE E4 allele. All individuals were considered cognitively normal and had levels of Aβ and tau that are thought to be in the `normal' range. There was a significant, positive correlation between Aβ1-42 using the xMAP assay and AβX-42 using the MSD assay (r = 0.766, p < 0.0001).

Table 1.

Characteristics of sample

Characteristics N Mean (SD)/n (%) Range
Age, years 91 53.4 (7.9) 36–69
Female 91 63 (69.2%)
Education, years 91 16.3 (2.9) 8–23
Any E4 allele 91 33 (36.3%)
Body mass index, kg/m2 91 27.8 (5.5) 19.2–47.8
Vitamin C supplements 91 22 (24.2%)
Vitamin E supplements 91 20 (22.0%)
Blood pressure medications 91 11 (12.1%)
Systolic blood pressure, mm Hg 91 121.6 (13.4) 94–161
Total cholesterol, mg/dL 91 190.7 (32.4) 114–299
HDL cholesterol, mg/dL 91 56.0 (16.5) 31–122
LDL cholesterol, mg/dL 91 118.8 (29.6) 41–215
Trigylcerides, mg/dL 91 99.7 (43.8) 46–246
Glucose, mg/dL 90 83.4 (7.2) 72–109
MMSE, points of 30 90 29.5 (0.7) 27–30
HVLT-total learning score 91 29.6 (3.4) 20–35
HVLT-delayed recall score 91 10.5 (1.8) 3–12
Processing speed index 91 90.8 (12.6) 57–119
Mental control total score 91 30.3 (4.9) 19–40
Working memory composite score 91 26.4 (4.2) 14–37
Stroop color-word score 91 43.1 (7.7) 26–69
CSF ABx-38 by MSD, ng/L 91 1575.7 (661.7) 405–4042
CSF ABx-40 by MSD, ng/L 91 6662.9 (2015.8) 2710–13,235
CSF ABx-42 by MSD, ng/L 91 524.6 (202.9) 155–1315
CSF AB1-42 by xMAP, ng/L 91 344.1 (69.0) 150–510
CSF tau by xMAP, ng/L 91 63.4 (25.0) 27–132
CSF p-tau by xMAP, ng/L 91 36.4 (18.6) 13–127
CSF nucleated cells, cells/uL 91 2.05 (2.42)
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Key: MMSE, Mini-Mental State Examination. HVLT, Hopkins Verbal Learning Test. CSF, Cerebrospinal Fluid. AB, amyloid-beta. MSD, Meso Scale Discovery electrochemiluminescence. xMAP. INNO-BIA AlzBio3.

Levels of CSF very long chain ceramides with chain lengths of C20–C26 were higher in APOE E4 carriers compared to non-carriers (p-values range from 0.016–0.045). However, levels of other ceramide species (C16:0 and C18:0) or sphingomyelins (C16:0-C26:1) did not vary by APOE genotype (all p > 0.10, data not shown). There were positive correlations between all sphingomyelins (total and each species) and age (e.g., total CSF sphingomyelin: r = 0.246, p = 0.019), but not between ceramides and age. Neither sphingomyelin nor ceramide levels differed by sex. There were also no associations between any sphingomyelins nor ceramides and use of vitamins or blood pressure medications, body mass index, and fasting serum cholesterol, triglycerides, or glucose (data not shown).

3.1. Relationship between CSF sphingolipids and CSF Aβand tau

Correlations between total ceramides, sphingomyelins and each individual species within these lipid classes and CSF Aβ and tau are shown in Table 2. Only ceramide C18:0 consistently correlated with Aβ and tau (Table 2 and Fig. 1). Notably, there were positive, significant, correlations with AβX-38 (r = 0.312, p = 0.003) and AβX-40 (r = 0.327, p = 0.002), but not with AβX-42 (r = 0.171, p = 0.106) or with Aβ1-42 (r = 0.117, p = 0.268). In contrast to the specific carbon-chain length findings for ceramides, virtually all CSF sphingomyelins were positively correlated with all CSF Aβ measures, and also with tau, p-tau, and the tau/Aβ1-42 ratio (Table 2 and Fig. 2).

Table 2.

Correlation between CSF sphingolipids and CSF amyloid-beta and tau measures

ABX38a ABX40a ABX42a AB1-42b taub p-tau181b tau/AB1-42b p-tau181/AB1-42b
Log CSF sphinqolipid r p value r p value r p value r p value r p value r p value r p value r p value
Ceramides
 d18:1–C16:0
 d18:1–C18:0 0.312 0.003 0.327 0.002 0.313 0.003 0.306 0.003
 d18:1–C20:0
 d18:1–C22:0
 d18:1–C24:0
 d18:1–C24:1 0.221 0.035
 d18:1–C26:0
Total ceramidesc
Sphingomyelins
 d18:1–C16:0 0.404 0.0001 0.412 <0.0001 0.301 0.004 0.259 0.013 0.410 <0.0001 0.311 0.003
 d18:1–C16:1 0.484 <0.0001 0.500 <0.0001 0.369 <0.001 0.253 0.016 0.441 <0.0001 0.277 0.008 0.331 0.001
 d18:1–C18:0 0.558 <0.0001 0.554 <0.0001 0.428 <0.0001 0.333 0.001 0.565 <0.0001 0.245 0.019 0.446 <0.0001
 d18:1–C18:1 0.552 <0.0001 0.559 <0.0001 0.419 <0.0001 0.281 0.007 0.535 <0.0001 0.306 0.003 0.426 <0.0001 0.214 0.042
 d18:1–C20:0 0.652 <0.0001 0.642 <0.0001 0.505 <0.0001 0.358 0.001 0.627 <0.0001 0.332 0.001 0.483 <0.0001 0.208 0.048
 d18:1–C20:1 0.421 <0.0001 0.456 <0.0001 0.334 0.001 0.266 0.011 0.359 <0.001 0.238 0.023
 d18:1–C22:0 0.613 <0.0001 0.611 <0.0001 0.488 <0.0001 0.354 <0.001 0.605 <0.0001 0.317 0.002 0.463 <0.0001
 d18:1–C22:1 0.386 <0.001 0.427 <0.0001 0.319 0.002 0.255 0.015 0.353 <0.001 0.239 0.023
 d18:1–C24:0 0.297 0.004 0.305 0.003 0.239 0.023 0.249 0.017 0.294 0.005 0.273 0.009 0.282 0.007
 d18:1–C24:1 0.387 <0.001 0.421 <0.001 0.326 0.002 0.257 0.014 0.384 <0.001
Total sphinogmyelinsc 0.595 <0.0001 0.598 <0.0001 0.462 <0.0001 0.34 0.001 0.580 <0.0001 0.29 0.005 0.443 <0.0001 0.271 0.010
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a

Assays conducted using Meso Scale Discovery (MSD) electrochemiluminescence.

b

Assays conducted using INNO-BIO AlzBio3 (xMAP).

c

Total refers to the sum of all carbon-chain lengths within the specific lipid class.

Fig. 1.

Fig. 1

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Correlations between CSF ceramide C18:0 and CSF levels of: A) AβX-38; B) AβX-40; C) AβX-42; D) Aβ1-42; E) tau; and F) p-tau181. In F (correlation between ceramide C18:0 and p-tau181), excluding the one outlier with p-tau181 level >121 had little effect on the results (excluding outlier: r = 0.068, p = 0.524).

Fig. 2.

Fig. 2

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Correlations between CSF total sphingomyelin and CSF levels of: A) AβX-38; B) AβX-40; C) AβX-42; D) Aβ1-42; E) tau; and F) p-tau181. In F (correlation between total sphingomyelins and p-tau181), excluding the one outlier with p-tau181 level >121 slightly reduced the significance of the results (excluding outlier: r = 0.270, p = 0.010).

3.2. Linear regression models and the effects of age and APOE genotype

Based on the above-described correlations, we focused on ceramide C18 in multivariate analyses because this compound was consistently associated with both CSF Aβ and tau. In contrast, since all sphingomyelins species were significantly associated with CSF Aβ and tau, linear regression models only examined total sphingomyelin levels. The associations between CSF ceramide C18:0 or sphingomyelins and CSF Aβ and tau remained in linear regression models controlling for age and APOE genotype (Table 3). Notably, the association between ceramide C18 and AβX-42 (b = 67.24, p = 0.031) and Aβ1-42 (b = 19.12, p = 0.071) were also stronger in the multivariate models.

Table 3.

Cross-sectional associations between CSF ceramides and sphingomyelins and CSF amyloid-beta and tau using multivariate linear regression

All individuals (n = 91)a Age <54 (n = 44) Age ≥54 (n = 47) No APOE E4 (n = 58) APOE E4 (n = 33)
Log sphingolipid b (SE) p value b (SE) p value b (SE) p value b (SE) p value b (SE) p value
Ceramide C 18:0 ABX-38b 302.75 (96.57) 0.002 163.11 (158.45) 0.309 418.91 (120.88) 0.001 286.68 (103.30) 0.008 358.88 (204.89) 0.090
ABX-40b 983.20 (291.75) 0.001 549.37 (474.95) 0.254 1349.24 (365.49) 0.001 868.16 (341.89) 0.014 1298.58 (549.95) 0.025
ABX-42b 67.24 (30.58) 0.031 21.21 (49.84) 0.522 90.07 (38.76) 0.025 66.01 (36.62) 0.077 69.02 (57.31) 0.238
AB1-42c 19.12 (10.44) 0.071 14.46 (14.37) 0.320 21.19 (15.10) 0.168 19.49 (11.88) 0.107 18.47 (21.07) 0.388
tauc 13.44 (3.49) <0.001 7.61 (5.41) 0.167 18.18 (4.63) <0.001 13.70 (4.03) 0.001 12.77 (6.91) 0.075
p-tau 181c 2.80 (2.84) 0.327 0.81 (4.77) 0.867 4.38 (3.55) 0.224 0.94 (2.92) 0.750 6.37 (6.24) 0.315
tau/AB1-42c 0.03 (0.01) 0.039 0.01 (0.01) 0.244 0.04 (0.02) 0.062 0.03 (0.01) 0.025 0.03 (0.03) 0.391
Total sphingomyelin ABX-38b 653.29 (128.14) <0.001 3.89 (202.17) 0.021 816.50 (158.79) <0.001 450.22 (149.83) 0.004 1152.83 (216.49) <0.001
ABX-40b 2037.57 (387.32) <0.001 1502.38 (605.20) 0.017 2553.43 (481.13) <0.001 1455.48 (492.0) 0.005 3490.53 (548.05) <0.001
ABX-42b 173.49 (41.01) <0.001 158.65 (62.64) 0.015 184.46 (53.72) 0.001 129.12 (52.42) 0.017 266.29 (66.48) <0.001
AB1-42c 47.00 (14.40) 0.002 45.39 (18.24) 0.017 45.43 (21.68) 0.042 35.79 (17.17) 0.042 71.80 (26.93) 0.012
tauc 24.56 (4.73) <0.001 18.69 (6.86) 0.009 30.68 (6.44) <0.001 18.22 (6.01) 0.004 37.74 (7.58) <0.001
p-tau181c 10.85 (3.92) 0.007 13.48 (6.06) 0.032 9.79 (5.11) 0.062 4.53 (4.24) 0.290 22.48 (7.91) 0.008
tau/AB1-42c 0.05 (0.02) 0.007 0.03 (0.01) 0.053 0.08 (0.03) 0.020 0.03 (0.02) 0.068 0.09 (0.04) 0.037
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Key: CSF, cerebrospinal fluid. SE, standard error.

a

Models control for age and APOE E4 allele; Models stratified by age control for APOE E4 genotype.

b

Assays conducted using Meso Scale Discovery (MSD) electrochemiluminescence.

c

Assays conducted using INNO-BIO AlzBio3 (xMAP).

In additional analyses, we separately stratified the above models by median age (≥54 vs <54 years) and the presence of an APOE E4 allele to further determine how these factors affected the above associations. Almost all relationships between CSF ceramide C18 or sphingomyelin and CSF Aβ and tau were stronger among individuals aged ≥54 compared to those less than 54 years (Table 3). While there were only 33 individuals with an APOE E4 allele, the associations between ceramide C18:0 and AβX38 and AβX40, and between total sphingomyelins and all measures were also stronger within this group compared to those without an E4 allele.

3.3. Relationship between CSF sphingolipids and cognitive performance

While all participants were cognitively normal, we did see some associations between CSF ceramides and cognitive performance, specifically in domains of memory performance. Using linear regression models and controlling for age, education, and APOE E4 genotype, higher ceramide C20:0 (b = −0.67, p = 0.034) and C22:0 (b = −0.69, p = 0.026) were associated with worse performance on the HVLT-delayed recall. Higher ceramide C26:0 (b = −1.32, p = 0.049) was also associated with worse performance on the Working Memory Composite Score. Examining Aβ and tau measures and controlling for the same variables, CSF tau was associated with better performance on Working Memory (b = 0.04, p = 0.042) while p-tau was associated with worse performance on the HVLT-immediate recall (b = −0.04, p = 0.047).

4. Discussion

In the present study, we found cross-sectional, positive associations between CSF ceramide C18:0 and total sphingomyelins and CSF Aβ and tau levels in a cohort of cognitively normal individuals aged 36–69 with a confirmed parental history of AD. These associations were stronger among individuals that were older than the median sample age (54 years) and among those with at least one APOE E4 allele. These results suggest in vivo associations between CSF sphingolipids and CSF Aβ and tau levels and, while cross-sectional, may suggest new pathways for the treatment or prevention of AD.

Cellular and animal studies have shown direct and indirect associations between sphingolipids and Aβ metabolism (Cutler, et al., 2004; Grimm, et al., 2005; Kalvodova, et al., 2005; Lee, et al., 2004; Mattson, et al., 2005; Puglielli, et al., 2003). Ceramides regulate Aβ(1-42) production by modulating the physical location of APP with beta-secretase, and stabilizing the activity of gamma-secretase (Kalvodova, et al., 2005; Puglielli, et al., 2003). Exposure of cultured neurons to Aβ(1-42) increases ceramide levels by activating sphingomyelinase (Grimm, et al., 2005; Lee, et al., 2004); blocking this ceramide increase protects neurons from Aβ-induced cell death (Cutler, et al., 2004). Lastly, Aβ(1-42) indirectly increases ceramides through an oxidative stress-mediated mechanism (Cutler, et al., 2004; Mattson, et al., 2005). Studies have also linked long-chain ceramides, specifically the 18-carbon acyl chain length species, to tau phosphorylation through modulation of PP2A activity (Chalfant, et al., 1999; Dobrowsky, et al., 1993; Goedert, et al., 1995; Gong, et al., 1994; Mukhopadhyay, et al., 2009). In this study, we extend these findings to humans to show in vivo correlations between CSF sphingolipids and CSF Aβ and tau measures. Notably, only ceramide C18:0 was associated with Aβ and tau levels, which is a direct in vivo translation of animal and cellular studies. However, further investigation is needed to determine why ceramide C18:0, the most abundant CSF ceramide species, was only associated with total tau and not phospho-tau.

An intriguing finding is that ceramide C18:0 was most strongly associated with Aβx-38 and Aβx-40. While the associations with Aβx-42 or Aβ1-42 were stronger among older individuals (>54 years) and those with an APOE E4 allele, most associations still did not reach statistical significance. These results suggest that the relationship between ceramides and Aβ may vary by Aβ isomer length. Alternatively, as these individuals are all cognitively normal and Aβ42 is the more toxic species of amyloid, the relationship between ceramide C18:0 and Aβ42 may strengthen with increasing pathology. Additional research is needed to replicate these findings and to longitudinally examine this association in persons with MCI.

Interestingly, while much basic science research, as described above, has focused on the associations between ceramides and Aβ and tau, we observed the strongest correlations between CSF sphingomyelins and CSF Aβ and tau. A previous study reported that sphingomyelin levels were elevated in prodromal AD and then decreased in mild to moderate AD (Kosicek, et al., 2012). However, the reason for the mechanism behind this sphingomyelin increase is not currently clear. As the authors of this study speculate (Kosicek, et al., 2012), it is possible that the increased sphingomyelin could be the result of a cellular response to initially increased ceramide. As ceramide can be catabolized to sphingomyelin, levels of this lipid would increase. An alternative explanation is that sphingomyelin is elevated as a result of brain atrophy and cellular degeneration since sphingomyelin is primarily a structural lipid located in membranes.

Previous clinical and epidemiological studies have reported associations of both CSF and plasma sphingolipids with AD severity. Two studies reported that CSF ceramide (Satoi, et al., 2005) and sphingomyelin levels varied by AD severity (Kosicek, et al., 2012), but they did not examine associations between these lipids and markers of AD pathology, including CSF Aβ or tau. High levels of plasma ceramides have also been associated with an increased risk of cognitive impairment and AD among cognitively normal individuals (Mielke, et al., 2010a; Mielke, et al., 2012), memory decline and hippocampal volume loss among patients with mild cognitive impairment (Mielke, et al., 2010b), and faster rates of cognitive decline among AD patients (Mielke, et al., 2011). A shotgun lipidomic approach also reported differences between plasma sphingolipids in AD patients and controls (Han, et al., 2011). This is one of the first studies to extend this research to examine associations between these sphingolipids and CSF biomarkers of AD. Examining the relationship between CSF sphingolipids and cognition, ceramides C20:0 and C22:0 were associated with worse performance on the HVLT-delayed recall and C26:0 with worse performance on the Working Memory Composite Score. There were also very few associations between Aβ and tau levels and cognitive performance. These limited associations might be expected in this group of relatively young (age 36–69) cognitively normal individuals. Notably, the associations between CSF sphingolipids and CSF Aβ and tau were stronger in individuals aged 54 years or older, at the time when AD pathology is thought to begin (Jack, et al., 2010), and also in APOE E4 carriers. This may suggest, in line with cellular studies, that sphingolipids are involved early in the etiopathogenesis of AD and may be viable therapeutic targets.

Serial measures of CSF and brain imaging are needed to better understand the temporality of these associations in humans. Similarly, back translational research in transgenic animals to understand the mechanism and timing by which CSF sphingomyelins and ceramide increase in relation amyloid is also critical. This research is particularly important to understand alterations in sphingolipids in the context of the current biomarker ordering of AD, with the hypothesis that amyloid is deposited first, followed by tau accumulation, neurodegeneration, and then cognitive symptoms (Jack, et al., 2013a). Although, recent research suggests that, among some individuals, neurodegeneration may appear prior to significant amyloid deposition (Jack, et al., 2013b).

Limitations of the study warrant consideration. First, the sample size was relatively small. However, despite this, we still detected significant associations between CSF sphingolipids and CSF Aβ and tau levels. Second, the sample consisted of cognitively normal individuals with `normal' levels of CSF Aβ and tau. While this is an advantage from the standpoint of understanding the earliest pathological changes, it is not known whether these correlations will be as robust in patients with MCI and AD. Such knowledge will help to determine whether CSF sphingolipids levels could be utilized as diagnostic or prognostic markers of disease severity. Third, the study was cross-sectional and cannot generate causal inferences. Longitudinal studies to assess the relationships between changes in CSF sphingolipids and changes in AD pathology are also needed to validate the pathological implications of these cross-sectional findings. Lastly, given the associations between plasma ceramides and cognitive decline across severities of AD (Mielke, et al., 2010a; Mielke, et al., 2012; Mielke, et al., 2010b), further research should be directed to elucidate the mechanistic relationship between plasma sphingolipids and CSF Aβ and tau.

Acknowledgements

This study was supported in part by the National Institutes of Health (grants AG026752, AG027161, 1UL1RR025011, AG033514, U01 AG037526, AA0017408, MH077542, MH075673, AG034849) and pilot funding from the University of Wisconsin, Department of Medicine.

Disclosure statement Dr. Mielke receives research support from the NIH/NIA and has served as a consultant for Eli Lilly. Dr. Haughey receives grant funding from NIH. Dr. Bandaru, Dr. Zetterberg, Dr. Andreasson, Dr. Gleason and Ms. Blazel have no disclosures to report. Dr. Johnson receives grant funding from NIH and the Department of Veterans Affairs. Dr. Puglielli receives grant funding from NIH and the Department of Veterans Affairs. Dr. Sager receives grant funding from NIH and the Helen Bader Foundation, Milwaukee, WI. Dr. Asthana receives grant funding from NIH and serves as a site PI for Alzheimer's disease treatment trials sponsored by Elan, Merck and Co., Inc., Pfizer, and Wyeth. Dr. Carlsson receives research support from the NIH/NIA, NIH/NINR, HRSA, and the University of Wisconsin, Department of Medicine.

Footnotes

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