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ImportanceIdentification of proteins and genetic factors that reduce Alzheimer disease (AD) pathology is of importance when searching for novel AD treatments. Heterozygosity of the KL-VS haplotype has been associated with reduced amyloid and tau burden. Whether this association is mediated by the Klotho protein remains unclear.ObjectivesTo assess concentrations of Klotho in cerebrospinal fluid (CSF) and plasma among cognitively healthy controls and patients with AD and to correlate these findings with KL-VS heterozygosity status and amyloid and tau burden.Design, Setting, and ParticipantsThis case-control study combined 2 independent case-control AD cohorts consisting of 243 referred patients with AD and volunteer controls recruited from January 1, 2009, to December 31, 2018. Klotho levels were measured in CSF and plasma and correlated with KL-VS heterozygosity status and levels of CSF amyloid-β 42 (Aβ42), total tau, and phosphorylated tau. Statistical analysis was performed from January 1, 2021, to March 1, 2022.Main Outcomes and MeasuresAssociations of Klotho levels in CSF and plasma with levels of CSF biomarkers were analyzed using linear regression. Association analyses were stratified separately by clinical groups, APOE4 status, and KL-VS heterozygosity. Pearson correlation was used to assess the correlation between CSF and plasma Klotho levels.ResultsA total of 243 participants were included: 117 controls (45 men [38.5%]; median age, 65 years [range, 41-84 years]), 102 patients with mild cognitive impairment due to AD (AD-MCI; 59 men [57.8%]; median age, 66 years [range, 46-80 years]), and 24 patients with dementia due to AD (AD-dementia; 12 men [50.0%]; median age, 64.5 years [range, 54-75 years]). Median CSF Klotho levels were higher in controls (1236.4 pg/mL [range, 20.4-1726.3 pg/mL]; β = 0.103; 95% CI, 0.023-0.183; P = .01) and patients with AD-MCI (1188.1 pg/mL [range, 756.3-1810.3 pg/mL]; β = 0.095; 95% CI, 0.018-0.172; P = .02) compared with patients with AD-dementia (1073.3 pg/mL [range, 698.2-1661.4 pg/mL]). Higher levels of CSF Klotho were associated with lower CSF Aβ42 burden (β = 0.519; 95% CI, 0.201-0.836; P P P KL-VS heterozygosity, or APOE4 status. There was a weak correlation between Klotho CSF and plasma levels among the entire cohort (Pearson correlation r = 0.377; P Conclusions and RelevanceThe findings of this case-control study suggest that Klotho protein levels were associated with clinical stages of AD, cognitive decline, and amyloid and tau burden and that these outcomes were more clearly mediated by the protein directly rather than the KL-VS heterozygosity variant. When selecting individuals at risk for clinical trials, the Klotho protein level and not only the genetic profile should be considered.

Background: The unbiased amyloid, tau, and neurodegeneration (A/T/N) classification is designed to characterize individuals in the Alzheimer continuum and is currently little explored in clinical cohorts. Objective: A retrospective comparison of the A/T/N classification system with the results of a two-year clinical study, with extended follow-up up to 10 years after inclusion. Methods: Patients (n = 102) clinically diagnosed as Alzheimer’s disease (AD) with dementia or amnestic mild cognitive impairment (MCI), and 61 cognitively healthy control individuals were included. Baseline cerebrospinal fluid core biomarkers for AD (A42, phosphorylated tau, and total tau) were applied to the A/T/N classification using the final clinical diagnosis at extended follow-up as the gold standard. Results: A + T + N+ was a strong predictor for AD dementia, even among cognitively healthy individuals. Amnestic MCI was heterogenous, considering both clinical outcome and distribution within A/T/N. Some individuals with amnestic MCI progressed to clinical AD dementia within all four major A/T/N groups. The highest proportion of progression was among triple positive cases, but progression was also common in individuals with suspected non-Alzheimer pathophysiology (AT + N+), and those with triple negative status. A-T-N- individuals who were cognitively healthy overwhelmingly remained cognitively intact over time, but in amnestic MCI the clinical outcome was heterogenous, including AD dementia, other dementias, and recovery. Conclusion: The A/T/N framework accentuates biomarkers over clinical status. However, when selecting individuals for research, a combination of the two may be necessary since the prognostic value of the A/T/N framework depends on clinical status. This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0).

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 Aβ42, 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.

Background: There is substantial variation caused by preanalytical procedures in the measurement of cerebrospinal fluid (CSF) biomarkers for Alzheimer’s disease (AD) reported in the literature. Objective: Determine whether the detergent Tween-20 improves diagnostic accuracy. Methods: CSF proteins (Aβ42, Aβ40, total tau, and phosphorylated tau) were measured by standard ELISA, in uncentrifuged CSF with or without 0.05% Tween-20 from patients with AD or amnestic mild cognitive impairment, and healthy elderly controls. In the main study, collection tubes containing Tween-20 (Sarstedt 15 mL) were filled with 5 mL CSF to ensure consistent detergent concentration across subsequent aliquots into Corning 2 mL tubes. These latter were also the primary collection vessel for samples without Tween-20. The effect of centrifugation, and extra tube transfer of samples with Tween-20 were also examined. Results: 0.05% Tween-20 significantly increased mean measured CSF concentration of Aβ42 (30% ), Aβ40 (23% ), and total tau (4% ), but not phosphorylated tau. Generally, these increases were similar in all groups, although for Aβ42, the mean percentage increase with Tween-20 was slightly larger for AD. Areas under receiver-operator characteristic curves were similar whether Tween-20 was present or not. Centrifuged CSF without Tween-20 significantly reduced the measured concentration of Aβ42 versus non-centrifuged samples, a difference not seen when detergent was added. Similar CSF Aβ42 levels were found whether Tween-20 was added at collection in an extra tube or directly to the main collection tube. Conclusion: Addition of Tween-20 to CSF did not improve differentiation of patients from controls.

Introduction: Amyloid beta 1-43 (Aβ43), with its additional C-terminal threonine residue, is hypothesized to play a role in early Alzheimer’s disease pathology possibly different from that of amyloid beta 1-42 (Aβ42). Cerebrospinal fluid (CSF) Aβ43 has been suggested as a potential novel biomarker for predicting conversion from mild cognitive impairment (MCI) to dementia in Alzheimer’s disease. However, the relationship between CSF Aβ43 and established imaging biomarkers of Alzheimer’s disease has never been assessed. Materials and Methods: In this observational study, CSF Aβ43 was measured with ELISA in 89 subjects; 34 with subjective cognitive decline (SCD), 51 with MCI, and four with resolution of previous cognitive complaints. All subjects underwent structural MRI; 40 subjects on a 3T and 50 on a 1.5T scanner. Forty subjects, including 24 with SCD and 12 with MCI, underwent 18F-Flutemetamol PET. Seventy-eight subjects were assessed with 18F-fluorodeoxyglucose PET (21 SCD/7 MCI and 11 SCD/39 MCI on two different scanners). Ten subjects with SCD and 39 with MCI also underwent diffusion tensor imaging. Results: Cerebrospinal fluid Aβ43 was both alone and together with p-tau a significant predictor of the distinction between SCD and MCI. There was a marked difference in CSF Aβ43 between subjects with 18F-Flutemetamol PET scans visually interpreted as negative (37 pg/ml, n = 27) and positive (15 pg/ml, n = 9), p < 0.001. Both CSF Aβ43 and Aβ42 were negatively correlated with standardized uptake value ratios for all analyzed regions; CSF Aβ43 average rho -0.73, Aβ42 -0.74. Both CSF Aβ peptides correlated significantly with hippocampal volume, inferior parietal and frontal cortical thickness and axial diffusivity in the corticospinal tract. There was a trend toward CSF Aβ42 being better correlated with cortical glucose metabolism. None of the studied correlations between CSF Aβ43/42 and imaging biomarkers were significantly different for the two Aβ peptides when controlling for multiple testing. Conclusion: Cerebrospinal fluid Aβ43 appears to be strongly correlated with cerebral amyloid deposits in the same way as Aβ42, even in non-demented patients with only subjective cognitive complaints. Regarding imaging biomarkers, there is no evidence from the present study that CSF Aβ43 performs better than the classical CSF biomarker Aβ42 for distinguishing SCD and MCI.

Background α-Synuclein has been proposed as a potential biomarker for Alzheimer’s disease (AD) and amnestic mild cognitive impairment (aMCI). However, results from α-synuclein measurements in cerebrospinal fluid (CSF) have been inconclusive, and to our knowledge, longitudinal studies of changes prior to the AD diagnosis have not been investigated. Methods Levels of α-synuclein at baseline and after one and two years were measured in CSF, by enzyme-linked immunosorbent assay. Twenty-six patients with early AD (AD-AD), 48 patients with aMCI, subdivided as 23 that developed AD during follow-up (MCI-AD), and 25 that did not (MCI-MCI), and 25 healthy control individuals, were included. One-way ANOVA was applied to compare mean α-synuclein baseline values between all four study groups, and a linear mixed model was used to compare mean change over time between the three patient groups. Linear associations between α-synuclein and amyloid-β 1–42 (Aβ42), amyloid-β 1–40 (Aβ40), total tau and phosphorylated tau were also examined. Results A large variation in individual α-synuclein CSF levels was observed, particularly in the MCI-AD group. No significant differences were found in mean α-synuclein levels between all the study groups at baseline. When using a linear mixed model, no significant differences were found at follow-up for estimated mean changes between the patient groups. MCI-AD patients with short duration of symptoms prior to inclusion in the study (≤2 years) had considerably higher mean CSF α-synuclein levels compared to patients with a longer symptom duration (802.2 vs. 442.8 pg/mL, p = 0.01). No such difference was seen in the MCI-MCI or AD-AD groups. Significant linear associations (p

CSF was collected from patients or healthy elderly volunteers by lumbar puncture. Patients were punctured every 6 months over a 2-year period (5 collections), whereas controls were only punctured at baseline. CSF was collected directly into small cryovials in ice-water, and frozen within 30 minutes. No CSF samples were centrifuged. Erythrocyte counts in the CSF were on average 1.7/mL (range 1 5). Polypropylene tubes (Corning) were used throughout, and all samples were stored at -80 oC until analysis.