Sphingolipids are important in many brain functions but their role in Alzheimer's disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired participants. In dementia, altered sphingolipid metabolism, decreased acid sphingomyelinase activity and its lost association with CSF amyloid β42 concentration, underscores the potential of sphingolipids as disease biomarkers, and acid sphingomyelinase as a target for AD diagnosis and/or treatment.
There is strong evidence that neuronal hyper-excitability underlies migraine, and may or may not be preceded by cortical spreading depression. However, the mechanisms for cortical spreading depression and/or migraine are not established. Previous studies reported that cerebrospinal fluid (CSF) [Na+] is higher during migraine, and that higher extracellular [Na+] leads to hyper-excitability. We raise the hypothesis that altered choroid plexus Na+, K+-ATPase activity can cause both migraine phenomena: inhibition raises CSF [K+] and initiates cortical spreading depression, while activation raises CSF [Na+] and causes migraine. In this study, we examined levels of specific Na+, K+-ATPase inhibitors, endogenous ouabain-like compounds (EOLC), in CSF from migraineurs and controls. CSF EOLC levels were significantly lower during ictal migraine (0.4 nM +/- 0.09) than from either controls (1.8 nM +/- 0.4) or interictal migraineurs (3.1 nM +/- 1.9). Blood plasma EOLC levels were higher in migraineurs than controls, but did not differ between ictal and interictal states. In a Sprague-Dawley rat model of nitroglycerin-triggered central sensitization, we changed the concentrations of EOLC and CSF sodium, and measured aversive mechanical threshold (von Frey hairs), trigeminal nucleus caudalis activation (cFos), and CSF [Na+] (ultra-high field 23Na MRI). Animals were sensitized by three independent treatments: intraperitoneal nitroglycerin, immunodepleting EOLC from cerebral ventricles, or cerebroventricular infusion of higher CSF [Na+]. Conversely, nitroglycerin-triggered sensitization was prevented by either vascular or cerebroventricular delivery of the specific Na+, K+-ATPase inhibitor, ouabain. These results affirm our hypothesis that higher CSF [Na+] is linked to human migraine and to a rodent migraine model, and demonstrate that EOLC regulates them both. Our data suggest that altered choroid plexus Na+, K+-ATPase activity is a common source of these changes, and may be the initiating mechanism in migraine.
Docosahexaenoic Acid (DHA) is one of the most abundant polyunsaturated fatty acid in the brain which plays an important role in neurogenesis. DHA brain metabolism is altered in individuals with Alzheimer's Disease (AD). Apolipoprotein E (ApoE) protein regulates lipid transport but its role in brain DHA metabolism is unknown. There are three major ApoE isoforms in human, ApoE2, ApoE3, and ApoE4, which have been shown to confer differential susceptibility to AD. ApoE4 is the major genetic risk variant for late onset AD, while ApoE2 is neuroprotective and reduces risk for AD when compared to ApoE3. We propose that ApoE4 is a poor transporter of brain DHA compared with other ApoE isoforms. The aim of this study is to examine brain DHA metabolism using cellular studies and in cerebrospinal fluid (CSF) HDL. ApoE particles from CSF were isolated by immunoprecipitation. The DHA content of ApoE particles was determined using gas chromatography/mass spectrometry. 14C-DHA was used to determine the DHA efflux out of cell. In ABCA1 expressing BHK cells, DHA efflux was significantly increased when ABCA1 was activated by ApoE or by an ApoE mimetic peptide that activates ABCA1 (CS-6253). Using immunoprecipitation experiments, Nascent HDL ApoE particles secreted from astrocytes were enriched with labeled DHA after inducing ABCA1 activity. This suggested a novel role for ABCA1 in DHA transport through the formation of native HDL. In ABCA1 expressing cells and mouse primary astrocytes, DHA transport was increased more by recombinant ApoE3 treatment than that of ApoE4. These findings were then confirmed in human CSF. There was a greater number of high-density lipoproteins (HDL) particles in ApoE2 carrier CSF than that of AopE3 and ApoE4. ApoE isolated from CSF of ApoE2 carriers (n=5) had significantly more DHA than that of ApoE3 and ApoE4 (n=10). These findings indicate the ApoE isotypes differentially regulate DHA release and transport through mechanisms that involve ABCA1 activity and ApoE HDL formation. Interventions that can enhance brain ABCA1 activity and HDL formation in the brain may enhance brain DHA transport.
Abstract Background We previously identified urine dicarboxylic acids (DCA) of carbon length 4‐9 as candidate biomarkers of probable Alzheimer’s disease (AD) and energy dysregulation, and found their levels correlated with hippocampal volume. Our aim was to evaluate these initial findings in a second cohort. Method Study participants (> 65 years) were classified clinically as cognitively healthy (CH, n=188) or with probable AD (n=66) based on Uniform Data System‐3 of the National Alzheimer’s Coordinating Center criteria. Cerebrospinal fluid (CSF) amyloid ß 42 (A) and phospho‐ tau (T) levels were measured by validated immunoassay. Urine was collected in a “spot” sample after a 12 h overnight fast; target DCAs were detected using gas chromatography coupled with negative ion chemical ionization mass spectrometry and each DCA was normalized to deuterated internal standards and to a percent of total fatty acids. Result The mean of short‐chain DCA’s (C4‐C5) were higher in CH (44.38% ±17.79) compared to AD individuals (34.79% ±12.39) (p=0.0002), while the long‐chain DCA’s (C7‐C9) were lower for CH (34.70% ±16.88) when compared to AD individuals (45.22% ±15.11) (p<0.0001). DCA levels in the subset of CH participants with A + /T + were in a transition to the levels of the AD group and C4‐C5 and C7‐C9 differed from those with A ‐ /T ‐ biomarkers (p=0.0001). Combining results into a ratio of short‐ to long‐chain DCAs distinguished the A ‐ /T ‐ (2.330 ±2.144) from the A + /T + group (1.178 ±0.946); p<0.0001. ROC curve of the DCA ratio between CH‐NAT and AD is 82%. Study participant clinical and DCA groups were not significantly correlated with the common confounders of age, sex, education or ApoE genotype. Conclusion Our data extends our initial finding that urine DCA measures have potential as biomarkers of AD pathology, including for cognitively healthy individuals who are positive for CSF AT biomarkers. We propose that measurement of urine DCAs provides a new approach to screen for metabolic fluctuations in early AD and to monitor the effect of novel therapies in clinical trials. While studies are needed, including with other populations, varied clinical classifications, simplified chemistry, this data supports DCA biomarkers for widespread screening since the necessary urine sample is available from everyone.
Chronic hepatitis increases the risk of hepatocellular carcinoma (HCC). To test whether circulating proteins reflect hepatic carcinogenesis, sera from patients and controls were albumin depleted, enriched for glycoproteins, digested with trypsin, and subjected to reverse phase chromatography and tandem mass spectrometry. Alpha-fetoprotein enhancer binding protein (AFPebp), a tumor suppressor, was repeatedly identified in sera from chronic HBV hepatitis patients. We independently identified and quantified AFPebp with a deuterated, phenylisocyanate-labeled synthetic peptide standard. Elevated AFPebp levels in sera from chronic HBV hepatitis patients decreased as cancer developed. These data suggest that rising AFPebp levels in chronic HBV hepatitis may be protective, while falling levels may contribute to HCC development.
Our previous studies demonstrated that inhibitors of arachidonate–phospholipid remodeling [i.e. the enzyme CoA-independent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate–phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.
The introduction of lumbar puncture into clinical medicine over 100 years ago marks the beginning of the study of central nervous system diseases using the human cerebrospinal fluid (CSF). Ever since, CSF has been analyzed extensively to elucidate the physiological and biochemical bases of neurological disease. The proximity of CSF to the brain makes it a good target for studying the pathophysiology of brain functions, but the barrier function of the CSF also impedes its diagnostic value. Today, measurements to determine alterations in the composition of CSF are central in the differential diagnosis of specific diseases of the central nervous system (CNS). In particular, the analysis of the CSF protein composition provides crucial information in the diagnosis of CNS diseases. This enables the assessment of the physiology of the blood‐CSF barrier and of the immunology of intrathecial responses. Besides those routine measurements, protein compositional studies of CSF have been extended recently to many other proteins in the expectation that comprehensive analysis of lower abundance CSF proteins will lead to the discovery of new disease markers. Disease marker discovery by molecular profiling of the CSF tissue has the enormous potential of providing many new disease relevant molecules. New developments in protein profiling techniques hold promise for the discovery and validation of relevant disease markers. In this review, we summarize the current efforts and progress in CSF protein profiling measurements using conventional and current protein analysis tools. We also discuss necessary development in methodology in order to have the highest impact on the study of the molecular composition of CSF proteins.
Abstract Background The health benefits of odd‐chain saturated fatty acids (ocSAFA) have recently been recognized. We previously showed decreased unesterified ocSAFA in cerebrospinal fluid (CSF) of AD compared to MCI and cognitively healthy (CH). However, changes in ocSAFA relative to even‐chain saturated fatty acids (ecSAFA) have not been determined in other biofluids. Therefore, we aim to determine if levels of ocSAFA and ecSAFA may reveal AD pathology in urine. Methods We used neuropsychology and CSF measures of Ab 42 and T‐tau to classify groups of elderly subjects as c ognitively h ealthy (CH) with negative A b 42 (CH‐Ab‐), cognitively healthy with positive Ab 42 (CH‐Ab + ), MCI, and AD. We collected urine after an overnight fast and quantified unesterified fatty acids (UFA) and esterified fatty acids (EFA) using gas chromatography coupled with negative‐ion chemical ionization mass spectrometry. For data normalization, we measured ocSAFA species (C15:0, C17:0, C19:0) and ecSAFA species (14:0, C16:0, C18:0, C20:0, C22:0, C24:0) as a proportion of the sum of all UFA or EFA > C14:0 in urine. We calculated group differences using ordinary ANOVA with Tukey’s multiple comparison tests. Results Urine unesterified C15:0 levels were significantly higher in AD than in MCI and CT (p = 0.0016). Similarly, esterified levels of ocSAFA were higher in AD than in MCI and CT (p<0.0001). Although Aβ 42 levels in CH‐Aβ + and AD are not significantly different, unesterified C15:0, esterified C17:0, C19:0, and total esterified ocSAFA were significantly higher in AD urine than in CH‐Aβ + . Urinary unesterified ecSAFA levels did not change in AD compared with CH and MCI. In contrast, esterified ecSAFA decreased in AD compared to CH. While esterified C16:0 decreased, esterified C18:0 increased in AD urine compared to CH and MCI. These changes result in a significant decrease in the C18:0/C16:0 ratio in AD (Fig. 1). The ratio of esterified ocSAFA to ocSAFA was higher in AD than CH and MCI (p<0.0001) (Fig. 2). Conclusions These studies suggest that urine saturated fatty acids can reveal AD‐associated pathology. Therefore, measurement of urine fatty acids can be used to monitor AD‐linked biochemical pathways or to monitor therapies targeting saturated fatty acid metabolism in AD.
