APOE genotype dictates lipidomic signatures in primary human hepatocytes.

Apolipoprotein E (APOE) genetic variants are most notably known for their divergent impact on the risk of developing Alzheimer's disease. While APOE genotype has been consistently shown to modulate lipid metabolism in a variety of cellular contexts, the effect of APOE alleles on the lipidome in hepatocytes is unknown. In this study, we investigated the contribution of APOE alleles to lipidomic profiles of donor-derived primary human hepatocytes from 77 subjects. Lipidomic data obtained by liquid chromatography-mass spectrometry were analyzed across ε2/ε3, ε3/ε3, and ε3/ε4 genotypes to reveal how APOE modulates lipid relative levels over age and between groups. Hepatic APOE concentration, measured by ELISA, was assessed for correlation with lipid abundance in subjects grouped as per APOE genotype and sex. APOE genotype-specific differential lipidomic signatures associated with age for multiple lipid classes but did not differ between sexes. Compared to ε2/ε3, ε3/ε4 hepatocytes had higher abundance of acylcarnitines (AC) and acylphosphatidylglycerol (AcylPG) as a class, as well as higher medium and long-chain ACs, AcylPG, phosphatidylglycerol (PG), bis(monoacylglycerol)phosphate (BMP), monoacylglycerol (MG) and diacylglycerol (DG) species. The ε3/ε4 hepatocytes also exhibited a higher abundance of medium and long-chain ACs compared to the ε3/ε3 hepatocytes. Only in the ε3/ε4 hepatocytes, APOE concentration was lower and showed a negative correlation with BMP levels, specifically in females. APOE genotype dictates a differential lipidome in primary human hepatocytes. The lipids involved suggest mitochondrial dysfunction with accompanying alterations in neutral lipid storage, reflective of a general disturbance of free fatty acid metabolism in human hepatocytes with the ε4 allele.

Plasma apolipoprotein E levels, isoform composition, and dimer profile in relation to plasma lipids in racially diverse patients with Alzheimer's disease and mild cognitive impairment.

BACKGROUND: The APOEε4-promoted risk of Alzheimer's disease (AD) is lower in Black/African-Americans (B/AAs), compared to non-Hispanic whites (NHWs). Previous studies reported lower plasma apolipoprotein E (apoE) levels in NHW APOEε4-carriers compared to non-carriers, and low plasma apoE levels were directly associated with an increased risk of AD and all dementia. We further showed that APOEε3/ε3 AD patients exhibited reduced plasma apoE dimers compared to corresponding control subjects. Whether plasma apoE levels and apoE dimer formation differ between races/ethnicities and therefore may help explain AD risk racial disparity remains to be elucidated. METHODS: Using mass spectrometry, we determined total plasma apoE and apoE isoform levels in a cohort of B/AAs (n = 58) and NHWs (n = 67) including subjects with normal cognition (B/AA: n = 25, NHW: n = 28), mild cognitive impairment (MCI) (B/AA: n = 24, NHW: n = 24), or AD dementia (B/AA: n = 9, NHW: n = 15). Additionally, we used non-reducing western blot analysis to assess the distribution of plasma apoE into monomers/disulfide-linked dimers. Plasma total apoE, apoE isoform levels, and % apoE monomers/dimers were assessed for correlations with cognition, cerebrospinal fluid (CSF) AD biomarkers, sTREM2, neurofilament light protein (NfL), and plasma lipids. RESULTS: Plasma apoE was predominantly monomeric in both racial groups and the monomer/dimer distribution was not affected by disease status, or correlated with CSF AD biomarkers, but associated with plasma lipids. Plasma total apoE levels were not related to disease status and only in the NHW subjects we observed lower plasma apoE levels in the APOEε4/ε4-carriers. Total plasma apoE levels were 13% higher in B/AA compared to NHW APOEε4/ε4 subjects and associated with plasma high-density lipoprotein (HDL) in NHW subjects but with low-density lipoprotein levels (LDL) in the B/AA subjects. Higher plasma apoE4 levels, exclusively in APOEε3/ε4 B/AA subjects, were linked to higher plasma total cholesterol and LDL levels. In the controls, NHWs and B/AAs exhibited opposite associations between plasma apoE and CSF t-tau. CONCLUSIONS: The previously reported lower APOEε4-promoted risk of AD in B/AA subjects may be associated with differences in plasma apoE levels and lipoprotein association. Whether differences in plasma apoE levels between races/ethnicities result from altered APOEε4 expression or turnover, needs further elucidation.

APOE and immunity: Research highlights.

INTRODUCTION: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. METHODS: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. RESULTS: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). DISCUSSION: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development.

Behavioral and cognitive performance of humanized APOEε3/ε3 liver mice in relation to plasma apolipoprotein E levels.

Plasma apolipoprotein E levels were previously associated with the risk of developing Alzheimer's disease (AD), levels of cerebrospinal fluid AD biomarkers, cognition and imaging brain measures. Outside the brain, the liver is the primary source of apoE and liver transplantation studies have demonstrated that liver-derived apoE does not cross the blood-brain-barrier. How hepatic apoE may be implicated in behavioral and cognitive performance is not clear. In the current study, we behaviorally tested FRGN mice with humanized liver harboring the ε3/ε3 genotype (E3-human liver (HL)) and compared their behavioral and cognitive performance with that of age-matched ε3/ε3 targeted replacement (E3-TR) mice, the latter produces human apoE3 throughout the body whereas the E3-HL mice endogenously produce human apoE3 only in the liver. We also compared the liver weights and plasma apoE levels, and assessed whether plasma apoE levels were correlated with behavioral or cognitive measures in both models. E3-HL were more active but performed cognitively worse than E3-TR mice. E3-HL mice moved more in the open field containing objects, showed higher activity levels in the Y maze, showed higher activity levels during the baseline period in the fear conditioning test than E3-TR mice, and swam faster than E3-TR mice during training to locate the visible platform in the water maze. However, E3-HL mice showed reduced spatial memory retention in the water maze and reduced fear learning and contextual and cued fear memory than E3-TR mice. Liver weights were greater in E3-HL than E3-TR mice and sex-dependent only in the latter model. Plasma apoE3 levels were similar to those found in humans and comparable in female and male E3-TR mice but higher in female E3-HL mice. Finally, we found correlations between plasma apoE levels and behavioral and cognitive measures which were predominantly model-dependent. Our study demonstrates mouse-model dependent associations between plasma apoE levels, behavior and cognition in an 'AD-neutral' setting and suggests that a humanized liver might be sufficient to induce mouse behavioral and cognitive phenotypes.

APOE in the bullseye of neurodegenerative diseases: impact of the APOE genotype in Alzheimer's disease pathology and brain diseases.

ApoE is the major lipid and cholesterol carrier in the CNS. There are three major human polymorphisms, apoE2, apoE3, and apoE4, and the genetic expression of APOE4 is one of the most influential risk factors for the development of late-onset Alzheimer's disease (AD). Neuroinflammation has become the third hallmark of AD, together with Amyloid-ß plaques and neurofibrillary tangles of hyperphosphorylated aggregated tau protein. This review aims to broadly and extensively describe the differential aspects concerning apoE. Starting from the evolution of apoE to how APOE's single-nucleotide polymorphisms affect its structure, function, and involvement during health and disease. This review reflects on how APOE's polymorphisms impact critical aspects of AD pathology, such as the neuroinflammatory response, particularly the effect of APOE on astrocytic and microglial function and microglial dynamics, synaptic function, amyloid-ß load, tau pathology, autophagy, and cell-cell communication. We discuss influential factors affecting AD pathology combined with the APOE genotype, such as sex, age, diet, physical exercise, current therapies and clinical trials in the AD field. The impact of the APOE genotype in other neurodegenerative diseases characterized by overt inflammation, e.g., alpha- synucleinopathies and Parkinson's disease, traumatic brain injury, stroke, amyotrophic lateral sclerosis, and multiple sclerosis, is also addressed. Therefore, this review gathers the most relevant findings related to the APOE genotype up to date and its implications on AD and CNS pathologies to provide a deeper understanding of the knowledge in the APOE field.

Plasma apolipoprotein E levels in longitudinally followed patients with mild cognitive impairment and Alzheimer's disease.

BACKGROUND: Low levels of plasma apolipoprotein E (apoE) and presence of the APOE ε4 allele are associated with an increased risk of Alzheimer's disease (AD). Although the increased risk of AD in APOE ε4-carriers is well-established, the protein levels have received limited attention. METHODS: We here report the total plasma apoE and apoE isoform levels at baseline from a longitudinally (24 months) followed cohort including controls (n = 39), patients with stable amnestic mild cognitive impairment during 24 months follow up (MCI-MCI, n = 30), patients with amnestic MCI (aMCI) that during follow-up were clinically diagnosed with AD with dementia (ADD) (MCI-ADD, n = 28), and patients with AD with dementia (ADD) at baseline (ADD, n = 28). We furthermore assessed associations between plasma apoE levels with cerebrospinal fluid (CSF) AD biomarkers and α-synuclein, as well as both CSF and plasma neurofilament light chain (NfL), YKL-40 and kallikrein 6. RESULTS: Irrespective of clinical diagnosis, the highest versus the lowest apoE levels were found in APOE Îµ2/ε3 versus APOE Îµ4/ε4 subjects, with the most prominent differences exhibited in females. Total plasma apoE levels were 32% and 21% higher in the controls versus MCI-ADD and ADD patients, respectively. Interestingly, MCI-ADD patients exhibited a 30% reduction in plasma apoE compared to MCI-MCI patients. This decrease appeared to be associated with brain amyloid-ß (Aß42) pathology regardless of disease status as assessed using the Amyloid, Tau, and Neurodegeneration (A/T/N) classification. In addition to the association between low plasma apoE and low levels of CSF Aß42, lower apoE levels were also related to higher levels of CSF total tau (t-tau) and tau phosphorylated at Threonine 181 residue (p-tau) and NfL as well as a worse performance on the mini-mental-state-examination. In MCI-ADD patients, low levels of plasma apoE were associated with higher levels of CSF α-synuclein and kallikrein 6. No significant correlations between plasma apoE and the astrocytic inflammatory marker YKL40 were observed. CONCLUSIONS: Our results demonstrate important associations between low plasma apoE levels, Aß pathology, and progression from aMCI to a clinical ADD diagnosis.

Brain integrity is altered by hepatic APOE ε4 in humanized-liver mice.

Liver-generated plasma apolipoprotein E (apoE) does not enter the brain but nonetheless correlates with Alzheimer's disease (AD) risk and AD biomarker levels. Carriers of APOEε4, the strongest genetic AD risk factor, exhibit lower plasma apoE and altered brain integrity already at mid-life versus non-APOEε4 carriers. Whether altered plasma liver-derived apoE or specifically an APOEε4 liver phenotype promotes neurodegeneration is unknown. Here we investigated the brains of Fah-/-, Rag2-/-, Il2rg-/- mice on the Non-Obese Diabetic (NOD) background (FRGN) with humanized-livers of an AD risk-associated APOE ε4/ε4 versus an APOE ε2/ε3 genotype. Reduced endogenous mouse apoE levels in the brains of APOE ε4/ε4 liver mice were accompanied by various changes in markers of synaptic integrity, neuroinflammation and insulin signaling. Plasma apoE4 levels were associated with unfavorable changes in several of the assessed markers. These results propose a previously unexplored role of the liver in the APOEε4-associated risk of neurodegenerative disease.

Plasma Apolipoprotein E3 and Glucose Levels Are Associated in APOE ɛ3/ɛ4 Carriers.

BACKGROUND: Altered cerebral glucose metabolism, especially prominent in APOE ɛ4 carriers, occurs years prior to symptoms in Alzheimer's disease (AD). We recently found an association between a higher ratio of plasma apolipoprotein E4 (apoE4) over apoE3, and cerebral glucose hypometabolism in cognitively healthy APOE ɛ3/ɛ4 subjects. Plasma apoE does not cross the blood-brain barrier, hence we speculate that apoE is linked to peripheral glucose metabolism which is known to affect glucose metabolism in the brain. OBJECTIVE: Explore potential associations between levels of plasma insulin and glucose with previously acquired plasma apoE, cerebral metabolic rate of glucose (CMRgl), gray matter volume, and neuropsychological test scores. METHODS: Plasma insulin and glucose levels were determined by ELISA and a glucose oxidase assay whereas apoE levels were earlier quantified by mass-spectrometry in 128 cognitively healthy APOE ɛ3/ɛ4 subjects. Twenty-five study subjects had previously undergone FDG-PET and structural MRI. RESULTS: Lower plasma apoE3 associated with higher plasma glucose but not insulin in male subjects and subjects with a body mass index above 25. Negative correlations were found between plasma glucose and CMRgl in the left prefrontal and bilateral occipital regions. These associations may have functional implications since glucose levels in turn were negatively associated with neuropsychological test scores. CONCLUSION: Plasma apoE3 but not apoE4 may be involved in insulin-independent processes governing plasma glucose levels. Higher plasma glucose, which negatively affects brain glucose metabolism, was associated with lower plasma apoE levels in APOE ɛ3/ɛ4 subjects. High plasma glucose and low apoE levels may be a hazardous combination leading to an increased risk of AD.

Plasma Apolipoprotein E Monomer and Dimer Profile and Relevance to Alzheimer's Disease.

The APOEɛ4 gene variant is the strongest genetic risk factor for Alzheimer's disease (AD), whereas APOEɛ3 conventionally is considered as 'risk neutral' although APOEɛ3-carriers also develop AD. Previous studies have shown that the apolipoprotein E3 (apoE3) isoform occurs as monomers, homodimers and heterodimers with apolipoprotein A-II in human body fluids and brain tissue, but the relevance of a plasma apoE3 monomer/dimer profile to AD is unknown. Here we assessed the distribution of monomers, homodimers and heterodimers in plasma from control subjects and patients with mild cognitive impairment (MCI) and AD with either a homozygous APOEɛ3 (n = 31 control subjects, and n = 14 MCI versus n = 5 AD patients) or APOEɛ4 genotype (n = 1 control subject, n = 21 MCI and n = 7 AD patients). Total plasma apoE levels were lower in APOEɛ4-carriers and overall correlated significantly to CSF Aß42, p(Thr181)-tau and t-tau levels. Apolipoprotein E dimers were only observed in the APOEɛ3-carriers and associated with total plasma apoE levels, negatively correlated to apoE monomers, but were unrelated to plasma homocysteine levels. Importantly, the APOEɛ3-carrying AD patients versus controls exhibited a significant decrease in apoE homodimers (17.8±9.6% versus 26.7±6.3%, p = 0.025) paralleled by an increase in apoE monomers (67.8±18.3% versus 48.5±11.2%, p = 0.008). In the controls, apoE monomers and heterodimers were significantly associated with plasma triglycerides; the apoE heterodimers were also associated with levels of high-density lipoprotein cholesterol. The physiological relevance of apoE dimer formation needs to be further investigated, though the distribution of apoE in monomers and dimers appears to be of relevance to AD in APOEɛ3 subjects.

α-synuclein in the pathophysiology of Alzheimer's disease.

The Alzheimer's disease (AD) afflicted brain is neuropathologically defined by extracellular amyloid-ß (Aß) plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated tau protein. However, accumulating evidence suggests that the presynaptic protein α-synuclein (αSyn), mainly associated with synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), is involved in the pathophysiology of AD. Lewy-related pathology (LRP), primarily comprised of αSyn, is present in a majority of autopsied AD brains, and higher levels of αSyn in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) and AD have been linked to cognitive decline. Recent studies also suggest that the asymptomatic accumulation of Aß plaques is associated with higher CSF αSyn levels in subjects at risk of sporadic AD and in individuals carrying autosomal dominant AD mutations. Experimental evidence has further linked αSyn mainly to tau hyperphosphorylation, but also to the pathological actions of Aß and the APOEε4 allele, the latter being a major genetic risk factor for both AD and DLB. In this review, we provide a summary of the current evidence proposing an involvement of αSyn either as an active or passive player in the pathophysiological ensemble of AD, and furthermore describe in detail the current knowledge of αSyn structure and inferred function.

Associations Between APOE Variants, Tau and α-Synuclein.

Neurodegenerative diseases are characterized by the aggregation and deposition of misfolded proteins in the brain, most prominently amyloid-ß (Aß), tau and α-synuclein (α-syn), and are thus referred to as proteinopathies. While tau is a hallmark of Alzheimer's disease (AD) and other non-AD tauopathies, and α-synuclein is the pathological feature of the spectrum of synucleinopathies including Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB), the presence of co-pathologies is very frequent in all these diseases. Positive and synergistic associations between the different types of protein deposits have been reported, leading to worse prognosis and cognitive decline. A large variation in phenotypic clinical presentation of these diseases, largely due to the frequent presence of co-pathologies, makes differential diagnosis challenging. The observed clinico-pathological overlaps suggest common underlying mechanisms, in part due to shared genetic risk factors. The ε4 allele of the apolipoprotein (APOE) gene is one of the major genetic risk factors for the sporadic forms of proteinopathies, but the biological mechanisms linking APOE, tau and α-syn are not fully understood. This chapter describes current experimental evidence on the relationships between APOE variants, tau and α-syn, from clinical studies on fluid biomarkers and positron emission tomography (PET) imaging, and from basic experimental studies in cellular/molecular biology and animal models. The chapter focuses on recent advances and identifies knowledge gaps. In particular, no PET tracer for assessment of brain α-syn deposits is yet available, although it is subject of intense research and development, therefore experimental evidence on in vivo α-syn levels is based on measures in the cerebrospinal fluid (CSF) and plasma. Moreover, tau PET imaging studies comparing the patterns of tracer retention in synucleinopathies versus in other proteinopathies are scarce and much is still unknown regarding the relationships between APOE variants and fluid and/or imaging biomarkers of tau and α-syn. Further research incorporating multimodal imaging, fluid biomarkers and genetic factors will help elucidate the biological mechanisms underlying these proteinopathies, and contribute to differential diagnosis and patient stratification for clinical trials.

The relevance of cerebrospinal fluid α-synuclein levels to sporadic and familial Alzheimer's disease.

Accumulating evidence demonstrating higher cerebrospinal fluid (CSF) α-synuclein (αSyn) levels and αSyn pathology in the brains of Alzheimer's disease (AD) patients suggests that αSyn is involved in the pathophysiology of AD. To investigate whether αSyn could be related to specific aspects of the pathophysiology present in both sporadic and familial disease, we quantified CSF levels of αSyn and assessed links to various disease parameters in a longitudinally followed cohort (n = 136) including patients with sporadic mild cognitive impairment (MCI) and AD, and in a cross-sectional sample from the Dominantly Inherited Alzheimer's Network (n = 142) including participants carrying autosomal dominant AD (ADAD) gene mutations and their non-mutation carrying family members.Our results show that sporadic MCI patients that developed AD over a period of two years exhibited higher baseline αSyn levels (p = 0.03), which inversely correlated to their Mini-Mental State Examination scores, compared to cognitively normal controls (p = 0.02). In the same patients, there was a dose-dependent positive association between CSF αSyn and the APOEε4 allele. Further, CSF αSyn levels were higher in symptomatic ADAD mutation carriers versus non-mutation carriers (p = 0.03), and positively correlated to the estimated years from symptom onset (p = 0.05) across all mutation carriers. In asymptomatic (Clinical Dementia Rating < 0.5) PET amyloid-positive ADAD mutation carriers CSF αSyn was positively correlated to 11C-Pittsburgh Compound-B (PiB) retention in several brain regions including the posterior cingulate, superior temporal and frontal cortical areas. Importantly, APOEε4-positive ADAD mutation carriers exhibited an association between CSF αSyn levels and mean cortical PiB retention (p = 0.032). In both the sporadic AD and ADAD cohorts we found several associations predominantly between CSF levels of αSyn, tau and amyloid-ß1-40.Our results suggest that higher CSF αSyn levels are linked to AD pathophysiology at the early stages of disease development and to the onset of cognitive symptoms in both sporadic and autosomal dominant AD. We conclude that APOEε4 may promote the processes driven by αSyn, which in turn may reflect on molecular mechanisms linked to the asymptomatic build-up of amyloid plaque burden in brain regions involved in the early stages of AD development.

Multiple system atrophy and apolipoprotein E.

BACKGROUND: Dysregulation of the specialized lipid metabolism involved in myelin synthesis and maintenance by oligodendrocytes has been associated with the unique neuropathology of MSA. We hypothesized that apolipoprotein E, which is associated with neurodegeneration, may also play a role in the pathogenesis of MSA. OBJECTIVE: This study evaluated genetic associations of Apolipoprotein E alleles with risk of MSA and α-synuclein pathology, and also examined whether apolipoprotein E isoforms differentially affect α-synuclein uptake in a oligodendrocyte cell. METHODS: One hundred sixty-eight pathologically confirmed MSA patients, 89 clinically diagnosed MSA patients, and 1,277 control subjects were genotyped for Apolipoprotein E. Human oligodendrocyte cell lines were incubated with α-synuclein and recombinant human apolipoprotein E, with internalized α-synuclein imaged by confocal microscopy and cells analyzed by flow cytometry. RESULTS: No significant association with risk of MSA or was observed for either Apolipoprotein E ɛ2 or ɛ4. α-Synuclein burden was also not associated with Apolipoprotein E alleles in the pathologically confirmed patients. Interestingly, in our cell assays, apolipoprotein E ɛ4 significantly reduced α-synuclein uptake in the oligodendrocytic cell line. CONCLUSIONS: Despite differential effects of apolipoprotein E isoforms on α-synuclein uptake in a human oligodendrocytic cell, we did not observe a significant association at the Apolipoprotein E locus with risk of MSA or α-synuclein pathology. © 2018 International Parkinson and Movement Disorder Society.

Amyloid-beta 1-40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro.

The population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid-beta (Aß) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aß1-40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation-dependent impact of Aß1-40 on human NG2+ pericytes. Fibril-EP Aß1-40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aß1-40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer-EP Aß1-40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aß1-40 as potential key regulators of the brain pericyte population size.

Assessment of kallikrein 6 as a cross-sectional and longitudinal biomarker for Alzheimer's disease.

BACKGROUND: Kallikrein 6 (KLK6) is known to be an age-related protease expressed at high levels in the central nervous system. It was previously shown to be involved in proteolysis of extracellular proteins implicated in neurodegenerative diseases such as Alzheimer's disease (AD), prompting validation of KLK6 as a potential biomarker of disease. However, analyses of both plasma and cerebrospinal fluid (CSF) levels of KLK6 in patients with AD have been inconclusive. We present a detailed analysis of KLK6 in plasma and CSF in two separate cohorts in a cross-sectional and a longitudinal clinical setting. METHODS: The cross-sectional cohort included control subjects without dementia and patients with AD, and the longitudinal cohort included patients with MCI and patients with AD followed over a 2-year period. Plasma and CSF levels of KLK6 were quantified by use of a previously developed and validated enzyme-linked immunosorbent assay. Statistical analyses were performed to compare KLK6 levels between diagnostic groups and to identify potential associations between KLK6 level, age, apolipoprotein E (APOE) genotype, total apoE level and the classical CSF AD biomarkers. RESULTS: In the cross-sectional setting, KLK6 levels in plasma but not in CSF were significantly higher in the AD group than in control subjects. CSF but not plasma KLK6 levels were positively correlated with age in both the cross-sectional and longitudinal settings. In both cohorts, the CSF KLK6 levels were significantly and positively correlated with the CSF levels of core AD biomarkers. Total plasma and CSF apoE levels were positively associated with KLK6 in the cross-sectional study. Finally, during the 2-year monitoring period of the longitudinal cohort, CSF KLK6 levels increased with disease progression over time in the investigated patient groups. CONCLUSIONS: In two separate cohorts we have confirmed the previously reported correlation between age and CSF levels of KLK6. Increased plasma KLK6 levels in patients with AD with a more advanced disease stage suggest KLK6 as a potential biomarker in patients with AD with more severe dementia. Significant correlations between KLK6 levels and core CSF AD biomarkers suggest molecular links between KLK6 and AD-related pathological processes.

Peripheral apoE isoform levels in cognitively normal APOE ε3/ε4 individuals are associated with regional gray matter volume and cerebral glucose metabolism.

BACKGROUND: Carriers of the APOE ε4 allele are at increased risk of developing Alzheimer's disease (AD), and have been shown to have reduced cerebral metabolic rate of glucose (CMRgl) in the same brain areas frequently affected in AD. These individuals also exhibit reduced plasma levels of apolipoprotein E (apoE) attributed to a specific decrease in the apoE4 isoform as determined by quantification of individual apoE isoforms in APOE ε4 heterozygotes. Whether low plasma apoE levels are associated with structural and functional brain measurements and cognitive performance remains to be investigated. METHODS: Using quantitative mass spectrometry we quantified the plasma levels of total apoE and the individual apoE3 and apoE4 isoforms in 128 cognitively normal APOE ε3/ε4 individuals included in the Arizona APOE cohort. All included individuals had undergone extensive neuropsychological testing and 25 had in addition undergone FDG-PET and MRI to determine CMRgl and regional gray matter volume (GMV). RESULTS: Our results demonstrated higher apoE4 levels in females versus males and an age-dependent increase in the apoE3 isoform levels in females only. Importantly, a higher relative ratio of apoE4 over apoE3 was associated with GMV loss in the right posterior cingulate and with reduced CMRgl bilaterally in the anterior cingulate and in the right hippocampal area. Additional exploratory analysis revealed several negative associations between total plasma apoE, individual apoE isoform levels, GMV and CMRgl predominantly in the frontal, occipital and temporal areas. Finally, our results indicated only weak associations between apoE plasma levels and cognitive performance which further appear to be affected by sex. CONCLUSIONS: Our study proposes a sex-dependent and age-dependent variation in plasma apoE isoform levels and concludes that peripheral apoE levels are associated with GMV, CMRgl and possibly cognitive performance in cognitively healthy individuals with a genetic predisposition to AD.

Apolipoprotein E lipoprotein particles inhibit amyloid-ß uptake through cell surface heparan sulphate proteoglycan.

BACKGROUND: The accumulation, aggregation and deposition of amyloid-ß (Aß) peptides in the brain are central to the pathogenesis of Alzheimer's disease (AD). Alzheimer's disease risk increases significantly in individuals carrying one or two copies of APOE ε4 allele compared to individuals with an ε3/ε3 genotype. Growing evidence has demonstrated that apolipoprotein E (apoE) strongly influences AD pathogenesis by controlling Aß aggregation and metabolism. Heparan sulphate proteoglycans (HSPGs) are abundant cell surface molecules that bind to both apoE and Aß. HSPGs have been associated with Aß aggregation and deposition. Although several lines of research have shown that apoE influences Aß clearance in the brain, it is not clear how apoE influences HSPG-mediated cellular uptake of Aß. RESULTS: In this study, we show that apoE lipoprotein particles from conditioned media of immortalized astrocytes isolated from human APOE-targeted replacement (TR) mice significantly suppress cellular Aß42 and Aß40 uptake through cell surface HSPG. ApoE3 and apoE4 particles have similar binding affinity to heparin, while apoE4 particles are likely hypolipidated compared to apoE particles. We also found that the apoE particles antagonize Aß binding to cell surface, and inhibited Aß uptake in a concentration-dependent manner in Chinese hamster ovary (CHO) cells. While the effect was not apoE isoform-dependent, the suppressive effect of apoE particles on Aß uptake was not observed in HSPG-deficient CHO cells. We further demonstrated that apoE particles reduced the internalization of Aß in mouse primary neurons, an effect that is eliminated by the presence of heparin. CONCLUSIONS: Taken together, our findings indicate that apoE particles irrespective of isoform inhibit HSPG-dependent cellular Aß uptake. Modulating the ability of apoE particles to affect Aß cellular uptake may hold promises for developing new strategies for AD therapy.

The Inflammatory Marker YKL-40 Is Elevated in Cerebrospinal Fluid from Patients with Alzheimer's but Not Parkinson's Disease or Dementia with Lewy Bodies.

A major difference in the revised diagnostic criteria for Alzheimer's disease (AD) is the incorporation of biomarkers to support a clinical diagnosis and allow the identification of preclinical AD due to AD neuropathological processes. However, AD-specific fluid biomarkers which specifically distinguish clinical AD dementia from other dementia disorders are still missing. Here we aimed to evaluate the disease-specificity of increased YKL-40 levels in cerebrospinal fluid (CSF) from AD patients with mild to moderate dementia (n = 49) versus Parkinson's disease (PD) (n = 61) and dementia with Lewy bodies (DLB) patients (n = 36), and non-demented controls (n = 44). Second we aimed to investigate whether altered YKL-40 levels are associated with CSF levels of other inflammation-associated molecules. When correcting for age, AD patients exhibited 21.3%, 27.7% and 38.8% higher YKL-40 levels compared to non-demented controls (p = 0.0283), DLB (p = 0.0027) and PD patients (p<0.0001). The AD-associated increase in YKL-40 was not associated with CSF P-tau, T-tau or Aß42. No relationship between increased YKL-40 and levels of the astrocytic marker glial-fibrillary acidic protein (GFAP), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and interferon gamma-induced protein 10 (IP-10) could be identified. Our results confirm previous reports of an age-associated increased in CSF YKL-40 levels and further demonstrate increased CSF YKL-40 in AD patients versus non-demented controls and patients with DLB or PD. The increase in YKL-40 levels in the AD patients was unrelated to the established CSF AD biomarkers and the inflammatory markers GFAP, MCP-1, IP-10 and IL-8, proposing YKL-40 as a marker of yet to be identified AD-related pathological processes.

Involvement of matrix metalloproteinase-9 in amyloid-ß 1-42-induced shedding of the pericyte proteoglycan NG2.

Deposition of amyloid-ß (Aß) 1-42, the major component of senile plaques characteristic of Alzheimer disease, affects brain microvascular integrity and causes blood-brain barrier dysfunction, increased angiogenesis, and pericyte degeneration. To understand the cellular events underlying Aß1-42 effects on microvascular alterations, we investigated whether different aggregation forms of Aß1-42 affect shedding of the pericyte proteoglycan NG2 and whether they affect proteolytic cleavage mediated by matrix metalloproteinase (MMP)-9. We found decreased levels of soluble NG2, total MMP-9, and MMP-9 activity in pericyte culture supernatants in response to fibril-enriched preparations of Aß1-42. Conversely, oligomer-enriched preparations of Aß1-42 increased soluble NG2 levels in the supernatants. This increase was ablated by the MMP-9/MMP-2 inhibitor SB-3CT. There was also a trend toward increased MMP-9 activity observed after oligomeric Aß1-42 exposure. Our results, demonstrating an Aß1-42 aggregation-dependent effect on levels of NG2 and MMP-9, support previous studies showing an impact of Aß1-42 on vascular integrity and thereby add to our understanding of mechanisms behind the microvascular changes commonly found in patients with Alzheimer disease.