An association of CSF apolipoprotein E glycosylation and amyloid-beta 42 in individuals who carry the APOE4 allele.

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer's disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aß42 levels (r = 0.53, p < 0.0001). These correlations were not observed for plasma apoE glycosylation. CSF total and secondary apoE glycosylation percentages also correlated with the concentration of CSF small high-density lipoprotein particles (s-HDL-P), which we have previously shown to be correlated with CSF Aß42 levels and measures of cognitive function. Desialylation of apoE purified from CSF showed reduced Aß42 degradation in microglia with E4 > E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aß metabolism and can be a potential target of treatment.

Eicosanoid lipidome activation in post-mortem brain tissues of individuals with APOE4 and Alzheimer's dementia.

BACKGROUND: Chronic neuroinflammation is one of the hallmarks of late-onset Alzheimer's disease (AD) dementia pathogenesis. Carrying the apolipoprotein ε4 (APOE4) allele has been associated with an accentuated response to brain inflammation and increases the risk of AD dementia progression. Among inflammation signaling pathways, aberrant eicosanoid activation plays a prominent role in neurodegeneration. METHODS: Using brains from the Religious Order Study (ROS), this study compared measures of brain eicosanoid lipidome in older persons with AD dementia to age-matched controls with no cognitive impairment (NCI), stratified by APOE genotype. RESULTS: Lipidomic analysis of the dorsolateral prefrontal cortex demonstrated lower levels of omega-3 fatty acids eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and DHA-derived neuroprotectin D1 (NPD-1) in persons with AD dementia, all of which associated with lower measures of cognitive function. A significant interaction was observed between carrying the APOE4 allele and higher levels of both pro-inflammatory lipids and pro-resolving eicosanoid lipids on measures of cognitive performance and on neuritic plaque burden. Furthermore, analysis of lipid metabolism pathways implicated activation of calcium-dependent phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and soluble epoxide hydrolase (sEH) enzymes. CONCLUSION: These findings implicate activation of the eicosanoid lipidome in the chronic unresolved state of inflammation in AD dementia, which is increased in carriers of the APOE4 allele, and identify potential therapeutic targets for resolving this chronic inflammatory state.

Effect of the ABCA1 agonist CS-6253 on amyloid-ß and lipoprotein metabolism in cynomolgus monkeys.

BACKGROUND: Inducing brain ATP-binding cassette 1 (ABCA1) activity in Alzheimer's disease (AD) mouse models is associated with improvement in AD pathology. The purpose of this study was to investigate the effects of the ABCA1 agonist peptide CS-6253 on amyloid-ß peptides (Aß) and lipoproteins in plasma and cerebrospinal fluid (CSF) of cynomolgus monkeys, a species with amyloid and lipoprotein metabolism similar to humans. METHODS: CS-6253 peptide was injected intravenously into cynomolgus monkeys at various doses in three different studies. Plasma and CSF samples were collected at several time points before and after treatment. Levels of cholesterol, triglyceride (TG), lipoprotein particles, apolipoproteins, and Aß were measured using ELISA, ion-mobility analysis, and asymmetric-flow field-flow fractionation (AF4). The relationship between the change in levels of these biomarkers was analyzed using multiple linear regression models and linear mixed-effects models. RESULTS: Following CS-6253 intravenous injection, within minutes, small plasma high-density lipoprotein (HDL) particles were increased. In two independent experiments, plasma TG, apolipoprotein E (apoE), and Aß42/40 ratio were transiently increased following CS-6253 intravenous injection. This change was associated with a non-significant decrease in CSF Aß42. Both plasma total cholesterol and HDL-cholesterol levels were reduced following treatment. AF4 fractionation revealed that CS-6253 treatment displaced apoE from HDL to intermediate-density- and low density-lipoprotein (IDL/LDL)-sized particles in plasma. In contrast to plasma, CS-6253 had no effect on the assessed CSF apolipoproteins or lipids. CONCLUSIONS: Treatment with the ABCA1 agonist CS-6253 appears to favor Aß clearance from the brain.

Low-Density Lipoprotein Particle Size Subfractions and Cerebral Amyloidosis.

Cerebral beta-amyloidosis (CA) is a condition in which amyloid-ß (Aß) proteins are deposited in the cerebral cortex and is a predictor of Alzheimer's disease (AD). The Aging Brain Study (ABS) investigated risk factors for CA in persons with diabetes and dyslipidemia. In the ABS, we identified that greater levels of LDL cholesterol and lower levels of HDL cholesterol were associated with increased CA. LDL particles comprise multiple species of varying size, density, and protein composition. For example, within a lipoprotein profile characteristic for persons with obesity and diabetic dyslipidemia, larger LDL particles have a greater ApoE to ApoB ratio, enhancing their binding affinity to LDL receptors. The goal of this study was to identify LDL particles that associate with CA in ABS. LDL particle size fractions were measured by ion mobility in plasma samples of 58 participants (40 women and 18 men). CA was assessed using Pittsburgh Compound B index-Positron Emission Tomography (PiB-PET) imaging. Among the LDL subfractions, greater plasma levels of large LDL particles were significantly associated with greater cerebral amyloidosis and lower hippocampal volumes independent of LDL cholesterol or triglyceride levels. Since Aß is cleared by the LDL receptor family, such as lipoprotein-like receptor 1 (LRP1), one potential mechanism for our findings is competition between ApoE enriched larger LDL particles and brain-derived Aß on hepatic Aß clearance and degradation. We conclude that assessing larger LDL particles in persons with atherogenic dyslipidemia may provide a mechanistic biomarker for the extent of CA.

Association of Serum Docosahexaenoic Acid With Cerebral Amyloidosis.

IMPORTANCE: Higher dietary intake of the essential fatty acid docosahexaenoic (DHA) has been associated with better cognitive performance in several epidemiological studies. Animal and in vitro studies also indicate that DHA prevents amyloid deposition in the brain. OBJECTIVE: To determine the association between serum DHA levels, cerebral amyloidosis, and the volumes of brain areas affected by Alzheimer disease. DESIGN, SETTINGS, AND PARTICIPANTS: Cross-sectional analysis of serum DHA levels together with measures of amyloid deposition (Pittsburgh Compound B index), brain volumes, and neuropsychological testing scores from 61 participants in the Aging Brain Study. The study was conducted between June 2008 and May 2013, and the data were analyzed between October 2015 and April 2016. Linear models were adjusted for age, sex, years of education, and apolipoprotein E status. MAIN OUTCOMES AND MEASURES: Serum DHA levels with cerebral amyloidosis measured using PIB PET. RESULTS: Samples were available from 61 Aging Brain Study participants (41 women and 20 men) who underwent amyloid PET imaging. The mean (SD) age of the participants was 77 (6) years and ranged from 67 to 88 years. Serum DHA levels (percentage of total fatty acids) were 23% lower in participants with cerebral amyloidosis than those without (0.97 vs 1.25, P = .007) and were inversely correlated with brain amyloid load (r = -0.32, P = .01) independent of age, sex, apolipoprotein E genotype, and years of education. Moreover, greater serum DHA levels were positively associated with brain volume in several subregions affected by AD, in particular the left subiculum (r = 0.38, P = .005) and the left entorhinal volumes (r = 0.51, P = .001). Serum DHA levels were also associated with nonverbal memory scores (r = 0.28, P = .03). CONCLUSIONS AND RELEVANCE: In this study, serum DHA levels were associated with pathogenesis of cerebral amyloidosis and with preservation of entorhinal and hippocampal volumes. These findings suggest an important role for DHA metabolism in brain amyloid deposition during the preclinical or early symptomatic stages of Alzheimer disease.

The association of plasma cystatin C proteoforms with diabetic chronic kidney disease.

BACKGROUND: Cystatin C (CysC) is an endogenous cysteine protease inhibitor that can be used to assess the progression of kidney function. Recent studies demonstrate that CysC is a more specific indicator of glomerular filtration rate (GFR) than creatinine. CysC in plasma exists in multiple proteoforms. The goal of this study was to clarify the association of native CysC, CysC missing N-terminal Serine (CysC des-S), and CysC without three N-terminal residues (CysC des-SSP) with diabetic chronic kidney disease (CKD). RESULTS: Using mass spectrometric immunoassay, the plasma concentrations of native CysC and the two CysC truncation proteoforms were examined in 111 individuals from three groups: 33 non-diabetic controls, 34 participants with type 2 diabetes (DM) and without CKD and 44 participants with diabetic CKD. Native CysC concentrations were 1.4 fold greater in CKD compared to DM group (p = 0.02) and 1.5 fold greater in CKD compared to the control group (p = 0.001). CysC des-S concentrations were 1.55 fold greater in CKD compared to the DM group (p = 0.002) and 1.9 fold greater in CKD compared to the control group (p = 0.0002). CysC des-SSP concentrations were 1.8 fold greater in CKD compared to the DM group (p = 0.008) and 1.52 fold greater in CKD compared to the control group (p = 0.002). In addition, the concentrations of CysC proteoforms were greater in the setting of albuminuria. The truncated CysC proteoform concentrations were associated with estimated GFR independent of native CysC concentrations. CONCLUSION: Our findings demonstrate a greater amount of CysC proteoforms in diabetic CKD. We therefore suggest assessing the role of cystatin C proteoforms in the progression of CKD.

The use of metformin is associated with decreased lumbar radiculopathy pain.

Lumbar radiculopathy pain represents a major public health problem, with few effective long-term treatments. Preclinical neuropathic and postsurgical pain studies implicate the kinase adenosine monophosphate activated kinase (AMPK) as a potential pharmacological target for the treatment of chronic pain conditions. Metformin, which acts via AMPK, is a safe and clinically available drug used in the treatment of diabetes. Despite the strong preclinical rationale, the utility of metformin as a potential pain therapeutic has not yet been studied in humans. Our objective was to assess whether metformin is associated with decreased lumbar radiculopathy pain, in a retrospective chart review. We completed a retrospective chart review of patients who sought care from a university pain specialist for lumbar radiculopathy between 2008 and 2011. Patients on metformin at the time of visit to a university pain specialist were compared with patients who were not on metformin. We compared the pain outcomes in 46 patients on metformin and 94 patients not taking metformin therapy. The major finding was that metformin use was associated with a decrease in the mean of "pain now," by -1.85 (confidence interval: -3.6 to -0.08) on a 0-10 visual analog scale, using a matched propensity scoring analysis and confirmed using a Bayesian analysis, with a significant mean decrease of -1.36 (credible interval: -2.6 to -0.03). Additionally, patients on metformin showed a non-statistically significant trend toward decreased pain on a variety of other pain descriptors. Our proof-of-concept findings suggest that metformin use is associated with a decrease in lumbar radiculopathy pain, providing a rational for larger retrospective trials in different pain populations and for prospective trials, to test the effectiveness of metformin in reducing neuropathic pain.

Proteomic and functional annotation analysis of injured peripheral nerves reveals ApoE as a protein upregulated by injury that is modulated by metformin treatment.

BACKGROUND: Peripheral nerve injury (PNI) results in a fundamental reorganization of the translational machinery in the injured peripheral nerve such that protein synthesis is increased in a manner linked to enhanced mTOR and ERK activity. We have shown that metformin treatment, which activates adenosine monophosphate-activated protein kinase (AMPK), reverses tactile allodynia and enhanced translation following PNI. To gain a better understanding of how PNI changes the proteome of the sciatic nerve and ascertain how metformin treatment may cause further change, we conducted a range of unbiased proteomic studies followed by biochemical experiments to confirm key results. RESULTS: We used multidimensional protein identification technology (MUDPIT) on sciatic nerve samples taken from rats with sham surgery, spinal nerve ligation (SNL) surgery or SNL + 200 mg/kg metformin treatment. MUDPIT analysis on these complex samples yielded a wide variety of proteins that were sorted according to their peptide counts in SNL and SNL + metformin compared to sham. These proteins were then submitted to functional annotation analysis to identify potential functional networks altered by SNL and SNL + metformin treatment. Additionally, we used click-chemistry-based labeling and purification of nascently synthesized proteins followed by MUDPIT to further identify peptides that were synthesized within the injured nerve. With these methods, we identified apolipoprotein E (ApoE) as a protein profoundly increased by PNI and further increased by PNI and metformin. This result was confirmed by Western Blot of samples from SNL rats and spared nerve injury (SNI) mice. Furthermore, we show that 7-day treatment with metformin in naïve mice leads to an increase in ApoE expression in the sciatic nerve. CONCLUSIONS: These proteomic findings support the hypothesis that PNI leads to a fundamental reorganization of gene expression within the injured nerve. Our data identify a key association of ApoE with PNI that is regulated by metformin treatment. We conclude from the known functions of ApoE in the nervous system that ApoE may be an intrinsic factor linked to nerve regeneration after PNI, an effect that is further enhanced by metformin treatment.