To date, the development of disease-modifying therapies for Alzheimer’s disease (AD) has largely focused on the removal of amyloid beta Aβ fragments from the CNS. Proteomic profiling of patient fluids may help identify novel therapeutic targets and biomarkers associated with AD pathology. Here, we applied the Olink™ ProSeek immunoassay to measure 270 CSF and plasma proteins across 415 Aβ- negative cognitively normal individuals (Aβ- CN), 142 Aβ-positive CN (Aβ+ CN), 50 Aβ- mild cognitive impairment (MCI) patients, 75 Aβ+ MCI patients, and 161 Aβ+ AD patients from the Swedish BioFINDER study. A validation cohort included 59 Aβ- CN, 23 Aβ- + CN, 44 Aβ- MCI and 53 Aβ+ MCI. To compare protein concentrations in patients versus controls, we applied multiple linear regressions adjusting for age, gender, medications, smoking and mean subject-level protein concentration, and corrected findings for false discovery rate (FDR, q < 0.05). We identified, and replicated, altered levels of ten CSF proteins in Aβ+ individuals, including CHIT1, SMOC2, MMP-10, LDLR, CD200, EIF4EBP1, ALCAM, RGMB, tPA and STAMBP (− 0.14 < d < 1.16; q < 0.05). We also identified and replicated alterations of six plasma proteins in Aβ+ individuals OSM, MMP-9, HAGH, CD200, AXIN1, and uPA (− 0.77 < d < 1.28; q < 0.05). Multiple analytes associated with cognitive performance and cortical thickness (q < 0.05). Plasma biomarkers could distinguish AD dementia (AUC = 0.94, 95% CI = 0.87–0.98) and prodromal AD (AUC = 0.78, 95% CI = 0.68–0.87) from CN. These findings reemphasize the contributions of immune markers, phospholipids, angiogenic proteins and other biomarkers downstream of, and potentially orthogonal to, Aβ- and tau in AD, and identify candidate biomarkers for earlier detection of neurodegeneration.Electronic supplementary materialThe online version of this article (10.1186/s40478-019-0795-2) contains supplementary material, which is available to authorized users.

Apolipoprotein (apo) E4 is a risk factor for heart disease, Alzheimer's disease, and other forms of neurodegeneration, but the underlying mechanisms are unknown. Domain interaction, a structural property that distinguishes apoE4 from apoE2 and apoE3, results in more rapid turnover and lower plasma levels of apoE4. To determine whether domain interaction affects brain apoE levels, we analyzed brain homogenates from human apoE3 and apoE4 knock-in mice, wild-type mice, and Arg-61 apoE mice, in which domain interaction was introduced by gene targeting. As determined on Western blots, the hemibrain, cortex, hippocampus, and cerebellum of knock-in mice had 30 -40% lower levels of apoE4 than apoE3, and Arg-61 mice had 25-50% lower apoE levels than wild-type mice. In the CSF, Arg-61 apoE level was 40% lower than the wild-type level. Arg-61 apoE mRNA levels were similar to or slightly higher than wild-type apoE mRNA levels. Thus, the lower Arg-61 apoE levels were not attributable to decreased mRNA levels. In culture medium from heterozygous Arg-61/wild-type and apoE4/apoE3 primary astrocytes, Arg-61 apoE and apoE4 levels were lower than wild-type apoE and apoE3, respectively, suggesting that primary astrocytes secrete lower amounts of Arg-61 apoE and apoE4. These results demonstrate that domain interaction is responsible for the lower levels of both human apoE4 and mouse Arg-61 apoE in mouse brain. Cells may recognize apoE4 and Arg-61 apoE as misfolded proteins and target them for degradation or accumulation. Thus, degradation/accumulation or lower levels of apoE4 may contribute to the association of apoE4 with Alzheimer's disease.

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. AcetylCoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.Pantothenate kinase (PanK)

Domain interaction, a structural property of apolipoprotein E4 (apoE4), is predicted to contribute to the association of apoE4 with Alzheimer disease. Arg-61 apoE mice, a gene-targeted mouse model specific for domain interaction, have lower brain apoE levels and synaptic, functional, and cognitive deficits. We hypothesized that domain interaction elicits an endoplasmic reticulum (ER) stress in astrocytes and an unfolded protein response that targets Arg-61 apoE for degradation. Primary Arg-61 apoE astrocytes had less intracellular apoE than wild-type astrocytes, and unfolded protein response markers OASIS (old astrocyte specifically induced substance), ATF4, and XBP-1 and downstream effectors were up-regulated. ER stress appears to cause global astrocyte dysfunction as glucose uptake was decreased in Arg-61 apoE astrocytes, and astrocyte-conditioned medium promoted neurite outgrowth less efficiently than wild-type medium in Neuro-2a cell cultures. We showed age-dependent up-regulation of brain OASIS levels and processing in Arg-61 apoE mice. ER stress and astrocyte dysfunction represent a new paradigm underlying the association of apoE4 with neurodegeneration.

Pantothenate kinase (PanK) is thought to catalyze the first rate-limiting step in CoA biosynthesis. The fulllength cDNA encoding the human PanK1 ␣ protein was isolated, and the complete human PANK1 gene structure was determined. Bezafibrate (BF), a hypolipidemic drug and a peroxisome proliferator activator receptor-␣ (PPAR ␣ ) agonist, specifically increased hPANK1 ␣ mRNA expression in human hepatoblastoma (HepG2) cells as a function of time and dose of the drug, compared with hPANK1 ␤ , hPANK2 , and hPANK3 , which did not significantly increase. Four putative PPAR ␣ response elements were identified in the PANKI ␣ promoter, and BF stimulated hPANK1 ␣ promoter activity but did not alter the mRNA half-life. Pantothenate (Pan) is a B complex vitamin (B 5 ) and is a nutritional requirement in mammals. Pan is the precursor for the biosynthesis of CoA, the predominant acyl group carrier in cells. Acyl moieties are attached to the terminal sulfhydryl of CoA, and these compounds participate in over 100 different reactions in intermediary metabolism (1-4). Short-chain CoA thioesters, such as acetyl-CoA or succinyl-CoA, are the most abundant components of the CoA pool (5) and are important intermediates in the tricarboxylic acid cycle, which coordinates carbon utilization and oxidative energy production. CoA is also an essential cofactor in long-chain fatty acid metabolism. CoA transfers fatty acids to and from the mitochondrial carnitine transferases and carries all of the intermediates of fatty acid ␤ -oxidation in peroxisomes and mitochondria. CoA acyl-thioesters are also the substrates for the acyltransferases and desaturases involved in membrane phospholipid formation, triglyceride synthesis, and protein acylation. Thus, CoA participates in the major anabolic and catabolic pathways critical to cell growth and function.Pan is phosphorylated by pantothenate kinase (PanK) to yield 4 Ј -phosphopantothenate (P-Pan) in the first enzymatic reaction leading to CoA. PanK activity controls the cellular level of CoA in bacteria (6) and mammals (7,8), and there are multiple Pank genes that are differentially expressed and regulated in mammalian tissues (4, 9-11). The first mammalian gene identified was the mouse Pank1 gene, which encodes two proteins that differ at the N terminus due to alternative splicing of distinct initiating exons (9). PanK1 ␣ enzyme activity is feedback inhibited by both unacylated CoA and acetyl-CoA (9), whereas the PanK1 ␤ isoform is less sensitive to feedback regulation by acetyl-CoA and is stimulated by unacylated CoA (8). The human PANK2 gene also encodes two protein isoforms, Abbreviations: BF, bezafibrate; HSS, Hallervorden-Spatz syndrome; P-Pan, 4 Ј -phosphopantothenate; P-PanSH, 4 Ј -phosphopantetheine; Pan, pantothenate; PanK, pantothenate kinase; PKAN, pantothenate kinase-associated neurodegeneration; PPAR ␣ , peroxisome proliferator activator receptor-␣ ; RACE, rapid amplification of cDNA ends.

BackgroundApolipoprotein E4 (apoE4), the major genetic risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases, has three structural and biophysical properties that distinguish it from the other isoforms—domain interaction, reduced stability, and lack of cysteine. Assessing their relative contributions to effects of apoE4‐associated pathogenesis in AD is important from a mechanistic and therapeutic perspective, that is not possible using human apoE transgene or knock‐in models.MethodsWe analyzed Arg‐61 apoE mice, a gene‐targeted model that selectively displays domain interaction.ResultsThe mice displayed age‐dependent loss of the synaptic protein synaptophysin in neocortex and hippocampus and had lower levels of the postsynaptic neuroligin‐1. Activation of dentate gyrus granule neurons increased Arc expression 3.5‐fold in wildtype mice but only 2.3‐fold in Arg‐61 mice. The losses of synaptic proteins caused a mild memory deficit in Arg‐61 mice in the water‐maze test. Since synaptic integrity requires efficient glutamate uptake, we measured astrocyte glutamate transporter 1 in the hippocampus. The level was reduced in Arg‐61 mice, suggesting that inefficient glutamate uptake by astrocytes causes chronic excitotoxicity. Consistent with the reduced secretion of Arg‐61 apoE by astrocytes in this model, cholesterol secretion was also reduced 34%. This reduction could also contribute to the synaptic deficits by limiting the availability of cholesterol for neuronal repair.ConclusionsDomain interaction in the absence of other structural characteristics of apoE4 is sufficient to cause synaptic pathology and functional synaptic deficits, potentially associated with astrocyte dysfunction and impaired maintenance of neurons. Therapeutic targeting of domain interaction might blunt effects of apoE4 in neurodegenerative disease.

Apolipoprotein E (apoE), a key lipid transport protein in the brain, is predominantly produced by astrocytes. Astrocytes are the most numerous cell type in the brain and are the main support network for neurons. They play a critical role in the synthesis and delivery of cholesterol in the brain. Humans have three common apoE isoforms, apoE2, apoE3 and apoE4, that show a strong genotype effect on the risk and age of onset for sporadic and late onset forms of Alzheimer’s disease (AD). Carriers of an ε4 allele have an increased risk of developing AD, while those with an ε2 allele are protected. Investigations into the contribution of apoE to the development of AD has yielded conflicting results and there is still much speculation about the role of this protein in disease. Here, we review the opposing hypotheses currently described in the literature and the approaches that have been considered for targeting apoE as a novel therapeutic strategy for AD. Additionally, we provide our perspective on the rationale for targeting apoE and the challenges that arise with respect to “drug-ability” of this target.

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