CSF Samples Research Articles

Proteomic comparison between CSF samples and CSF‐derived EVs from patients with Alzheimer’s disease, Parkinson’s disease and Lewy Body dementia

AbstractBackgroundExtracellular vesicles (EVs) have recently been considered as a potential biomarker source for a variety of diseases, including neurodegenerative disorders. EVs are important mediators of intercellular communication due to their capacity to transfer genetic material, lipids and proteins. By means of their communication role, interesting biomarkers are often enriched in EVs compared to total biofluid. In this study, this hypothesis is tested as untargeted proteomics is performed on both CSF samples and CSF‐derived EV samples from the same cohort with different dementia diseases.MethodCSF samples were collected from 297 patients (with Alzheimer’s disease (AD), Parkinson’s disease (PD), PD with cognitive deficit (PDCD), PD with dementia (PDD), Lewy Body dementia (LBD)) and healthy controls (HC). Furthermore, EVs were obtained from 500µL of the same CSF samples, isolated by SmartSEC HT. Total protein quantification was measured using absorbance at 280nm for CSF samples, and by fluorescence (NanoOrangeTM) for CSF‐derived EV samples. For both types of sample, LC‐MS/MS based proteomics analyses were performed.ResultTotal protein quantification showed multiple significant differences between all groups, both in CSF samples and in CSF‐derived EV samples. In both types of sample, PDCD group showed the highest protein concentration after normalization. In regard to LC‐MS/MS results, a first analysis resulted in a total of 3036 identified proteins (1564 protein groups) for the CSF samples, and 6627 proteins (3360 protein groups) for the CSF‐derived EV samples. The current analyses show four statistically differentially expressed proteins between CSF‐derived EV samples from HC and AD, AD and PD, LBD and PDCD. By the time of AAIC2023, also CSF samples will be analysed and a comparison will be drawn between both types of sample.ConclusionAs more in‐depth proteomic analyses will be performed by AAIC2023, a definite conclusion will be given at the conference. For now, already differences in total protein concentration were observed between the different disease groups and LC‐MS/MS of CSF‐derived EV samples resulted in four statistically differentially expressed proteins.

TREM2 AD risk variants impact pTau and microglia and astrocyte related biomarkers in cognitively unimpaired subjects

AbstractBackgroundIn the central nervous system, Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is expressed primarily in microglia. TREM2 receptors are important for microglial functions including phagocytosis, lipid metabolism and inflammatory responses. TREM2 coding variants (e.g. R47H rs75932628 and R62H rs143332484) increase the risk of late onset Alzheimer’s Diseases (AD). However, the impact of TREM2 risk variants on microglial cell functions in humans remains to be elucidated.MethodCSF and plasma samples were collected from AD patients (n = 120) and cognitively unimpaired control subjects (n = 53) with subjective complaints who do not meet biomarker‐based criteria for AD diagnosis. TREM2 AD risk variant carriers (n = 52) were screened from over 6000 samples of the Amsterdam dementia cohort using a combination of genotype array and exome sequencing. We performed extensive biomarker analysis of CSF and plasma samples with multidisciplinary approaches. We compared the effect of TREM2 mutations on AD disease and microglial and astrocytic biomarkers in AD patients and in cognitively unimpaired control subjects, respectively. A linear model was used with age and gender adjusted for the data analysis.ResultWe found higher levels of pTau181 and pTau231 in CSF and plasma in cognitively unimpaired TREM2 mutation carriers compared to non‐carriers, while no difference was observed between TREM2 genotype among the AD patients. Microglia and astrocyte biomarkers including sTREM2, FABP3, OLR1, YKL40 and GFAP were higher in the CSF from cognitively unimpaired TREM2 mutation carriers compared to non‐carriers, but no difference was detected between TREM2 genotype among the AD patients. We also found different levels of lipid metabolites in CSF and plasma samples from the TREM2 mutation carriers compared to non‐carriers in both AD patients and cognitively unimpaired control subjects.ConclusionTREM2 AD risk variants in cognitively unimpaired subjects increase pTau biomarkers and microglial and astrocytic related biomarkers. In AD patients, the biomarker differences between TREM2 genotype are diminished likely due to the extensive microglial responses to AD pathology. Moreover, our data indicates TREM2 AD risk variants may impact lipid‐metabolic processes in microglial cells. These data provide the biomarker insights on how TREM2 mutations may increase the risk of AD.

Imaging diagnosis and emotion recognition relationship in mild cognitive impairment

AbstractBackgroundRecent research has focused attention on social cognition impairment in Alzheimer Disease (AD). Specifically, emotion recognition can be evaluated with Reading the Mind in the Eyes Test (RMET). Some neuroanatomical changes could be related with social and cognitive deficits in patients. Therefore, future research combining cognitive indicators (RMET) with brain structure and function parameters (volumes, glucose metabolism, blood flow, amyloid deposits…) is required.MethodParticipants are patients from the Neurology Unit (Hospital Universitari i Politècnic La Fe). They were neuropsychologically evaluated, and CSF samples were obtained to determine some biomarkers (β‐amyloid‐42, β‐amyloid‐40, p‐Tau…). Also, brain imaging techniques (magnetic resonance (MR) and Positron Emission Tomography with [18F]Fluorodeoxyglucose (FDG‐PET/CT)) were applied to obtain brain structural and functional information, and to evaluate the relationship between them and emotion recognition evaluation (RMET). Imaging data were processed following SPM and CAT12 pipelines, a brain parcellation based on the Hammers atlas was used. Finally, 166 patients (with MR and/or FDG‐PET/CT) diagnosed with mild cognitive impairment (MCI) were included in the study.ResultThe correlation analysis between FDG‐PET/CT and RMET results showed significant correlations. Specifically, with prefrontal medial L and R (r = 0.36), posterior cingulate L and R (r = 0.34), parietal inferior L and R (r = 0.31), anterior cingulate L and R (r = 0.32), prefrontal lateral L and R (r = 0.32), insula R (r = 0.31) and basal ganglia R (r = 0.3). So, they are considered potential brain function biomarkers in MCI. Regarding the correlation analysis between MR (GM volumes (TIV‐corrected)) and RMET results, some significant correlations were observed. Specifically, with the straight gyrus L (r = 0.40), superior frontal gyrus L (r = 0.37), insula anterior inferior cortex R (r = 0.36), fusiform gyrus R (r = 0.36), anterior cingulate gyrus R (r = 0.36), insula anterior short gyrus L (r = 0.36), posterior and anterior cingulate gyrus L (r = 0.36, 0.35), and anterior orbital gyrus L (r = 0.35). So, they are considered potential brain structure biomarkers in MCI.ConclusionThe relationship between brain imaging parameters and emotion recognition impairment variables could provide important information for further biomarkers identification, as well as to identify potential physiopathological mechanisms involved in neurodegenerative development since early steps.

CSF sTREM2 and YKL‐40 modify the balance between soluble and fibrillary β‐amyloid in non‐demented individuals

AbstractBackgroundCerebrospinal fluid (CSF) Aβ concentrations and Positron Emission Tomography (PET) imaging provide information of two different Aβ pools. Identifying the factors affecting the balance between soluble and deposited Aβ may inform on the mechanism of cerebral Aβ aggregation. Microglia and astroglia respond to Aβ pathology, yet their role in promoting the aggregation of Aβ plaques is unclear. We addressed whether CSF sTREM2 and YKL‐40 modulate the association between soluble and deposited Aβ in a cohort of middle‐aged cognitively unimpaired (CU) individuals.MethodThe first consecutive 320 CU individuals of the ALFA+ study with a [18F]flutemetamol (FTM) PET scan and CSF samples were included. CSF Aβ40, Aβ42, sTREM2, and YKL‐40 were measured with the Roche NeuroToolKit robust prototype immunoassays (Roche Diagnostics International Ltd). Standardized uptake value ratio images were calculated and a voxel‐wise regression was performed to predict FTM uptake. The interaction terms between CSF Aβ42/40 and either CSF sTREM2 or YKL‐40 served as predictors, while adjusting for age, sex, years of education, and APOE‐ε4. Statistical threshold was set to p<0.001 with a cluster extent correction of 100 voxels.ResultTable 1 describes the sample’s characteristics. As expected, CSF Aβ42/40 concentrations were negatively associated with Aβ PET uptake in widespread areas of the brain, but this association was modified by CSF sTREM2 and YKL‐40. Higher levels of CSF sTREM2 were associated with a reduced Aβ PET uptake for any level of CSF Aβ42/40 in the hippocampus bilaterally. Conversely, higher CSF YKL‐40 related to a higher Aβ PET uptake as a function of CSF Aβ42/40 in the precuneus, prefrontal cortex, and inferior parietal areas.ConclusionIn CU individuals, microglia and astroglia reactivity modify the association between soluble (CSF) and deposited (amyloid PET) Aβ. While TREM2‐mediated microglial response (as measured by CSF sTREM2) may prevent Aβ deposition in individuals with decreased soluble Aβ, astroglial reactivity (as measured by CSF YKL‐40) may have the opposite effect.

A prospective study evaluating the clinical utility of tau‐PET in the diagnostic work‐up of patients with cognitive symptoms

AbstractBackgroundDetermining the added value of tau‐PET in the diagnostic work‐up of patients with cognitive symptoms is of great importance before implementation of the method in clinical practice. We therefore aimed to prospectively study the added clinical value of including tau‐PET in the clinical work‐up.Method878 patients referred for cognitive complaints to secondary Memory clinics in southern Sweden were consecutively recruited to the Swedish BioFINDER‐2 cohort study (May 2017 – Sept 2021). Participants underwent a baseline diagnostic work‐up including clinical examination, medical history, cognitive testing, blood and CSF sampling, and MRI of the brain. A tau‐PET ([18F]RO948) scan was added onto this clinical diagnostic work‐up. The tau‐PET scans were visually read as: normal, early AD pattern, late AD pattern or inconclusive scan. The primary end points were change in diagnosis and change in Alzheimer disease drug therapy or other drug treatment between the pre‐ and post‐PET visits. Secondary end point was the change in diagnostic certainty between the pre‐ and post‐PET visits.ResultThe tau‐PET visual read result led to a change in diagnoses in 66 participants (7.5%, p<0.001) and a change in medication in 48 participants (5.5%, p<0.001) (Figure 1). There was an overall increase in the diagnostic certainty after tau‐PET in the whole dataset [6.9±2.3 vs 7.4±2.4; p<0.0001]. The certainty was higher in participants with a pre‐PET diagnosis of AD [7.6±1.7 vs 8.2±2.0; p<0.0001] (Figure 2) and increased, as expected, even further in participants with a tau‐PET positive result supporting an AD diagnosis [8.0±1.4 vs 9.0±0.9; p<0.0001]. The impact of tau‐PET results was strongest in participants with pathological Ab‐status, whereas no significant change in diagnoses was seen in participants with normal Ab‐status.ConclusionWe report a significant change in diagnoses and medication of patients when tau‐PET is added to an already extensive diagnostic work‐up that included CSF AD biomarkers. Tau‐PET led to a significant increase in certainty of underlying etiology. The additive effect on certainty of etiology and to diagnosis was predominantly seen in Ab‐positives and we suggest that clinical use of tau‐PET is limited to populations with biomarkers indicating Ab‐positivity.

A novel fluid biomarker for TDP‐43 loss of function

AbstractBackgroundTDP‐43 nuclear clearance and cytoplasmic aggregation occur in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis‐frontotemporal dementia (ALS‐FTD), Alzheimer’s disease (AD), and limbic‐predominant age‐related TDP‐43 encephalopathy (LATE). Nuclear clearance of TDP‐43 leads to its failure to repress splicing of nonconserved cryptic exons. This loss of TDP‐43 splicing repression is well‐documented in postmortem tissues, but there is currently no method of detecting TDP‐43 dysfunction in living patients. As some cryptic exons are spliced in‐frame and translated into proteins carrying novel epitopes, we hypothesize that these “cryptic peptides” may be found in patient CSF as biomarkers of TDP‐43 dysfunction.MethodWe generated novel monoclonal antibodies against several TDP‐43 cryptic exon targets and then developed a sandwich ELISA using the Meso Scale Discovery (MSD) platform, which we validated in cells deficient in TDP‐43 for the cryptic exon target in HDGFL2. We then screened CSF samples of C9ORF72‐mutation carriers with or at risk of ALS‐FTD and control individuals for the presence of this cryptic peptide.ResultThe MSD signal for cryptic HDGFL2 was elevated in CSF of C9ORF72‐mutation carriers with ALS (n = 5, mean = 1601) compared to those of controls (n = 5, mean = ‐64.10; p<0.013), so we examined a larger cohort of C9ORF72‐mutation carriers. In symptomatic individuals (n = 27), cryptic peptide levels showed a correlation nearing significance with symptom duration (r = ‐0.368, p = 0.059), suggesting that levels of the cryptic peptide tend to be higher during the earlier symptomatic phase. We also found elevated cryptic peptide levels in CSF of several pre‐symptomatic individuals. To clarify cryptic peptide dynamics during disease progression, we began to assess longitudinal CSF samples and found that in 82.35% (14/17) of symptomatic individuals, levels of the cryptic peptide decreased with disease progression.ConclusionOur findings indicate that loss of TDP‐43 splicing repression occurs early in disease progression in ALS‐FTD, even pre‐symptomatically, and that detection of HDGFL2’s cryptic neoepitope may facilitate earlier diagnosis of TDP‐43‐related diseases. Further efforts linking the presence of cryptic HGDFL2 to TDP‐43 co‐pathology in AD may help expand biomarker development in AD. Additionally, evaluating dynamics of these biomarkers could provide a way of measuring target engagement for new therapeutics aimed at restoring TDP‐43 function.

Performance of clinical metagenomics in France: a prospective observational study

Metagenomic next-generation sequencing (mNGS) allows untargeted identification of a broad range of pathogens, including rare or novel microorganisms. Despite the recognition of mNGS as a valuable diagnostic tool for infections, the most relevant indications for this innovative strategy remain poorly defined. We aimed to assess the determinants of positivity and clinical utility of mNGS. In this observational study, we prospectively performed short-read shotgun metagenomics analysis as a second-line test (in cases of negative first-line test or when the symptoms were not fully explained by initial positive results) or as a first-line test in life-threatening situations requiring urgent non-targeted pathogen identification at the Necker-Enfants Malades Hospital (Paris, France). All sample types, clinical indications, and patient populations were included. Samples were accompanied by a mandatory form completed by the senior clinician or pathologist, on which the clinical level of suspected infection (defined as high or low) was indicated. We assessed the variables (gender, age, immune status, initial suspicion of infection, indication, and sample type) associated with mNGS pathogen detection using odds ratios (ORs) from multivariate logistic regression. Additional investigations were carried out using specific PCR or culture techniques, to confirm positive mNGS results, or when infectious suspicion was particularly high despite a negative mNGS result. Between Oct 29, 2019, and Nov 7, 2022, we analysed 742 samples collected from 523 patients. The initial suspicion of infection was either high (n=470, 63%) or low (n=272, 37%). Causative or possibly causative pathogens were detected in 117 (25%) samples from patients with high initial suspicion of infection, versus nine (3%) samples analysed to rule out infection (OR 9·1, 95% CI 4·6-20·4; p<0·0001). We showed that mNGS had higher odds of detecting a causative or possibly causative pathogenic virus on CNS biopsies than CSF samples (4·1, 1·7-10·7; p=0·0025) and in samples from immunodeficient compared with immunocompetent individuals (2·4, 1·4-4·1; p=0·0013). Concordance with conventional confirmatory tests results was 103 (97%) of 106, when mNGS detected causative or possibly causative pathogens. Altogether, among 231 samples investigated by both mNGS and subsequent specific tests, discordant results were found in 69 (30%) samples, of which 58 (84%) were mNGS positive and specific tests negative, and 11 (16%) mNGS negative and specific tests positive. Major determinants of pathogen detection by mNGS are immune status and initial level of suspicion of infection. These findings will contribute, along with future studies, to refining the positioning of mNGS in diagnostic and treatment decision-making algorithms. Necker-Enfants Malades Hospital and Institut Pasteur. For the French translation of the abstract see Supplementary Materials section.

Regional brain atrophy is associated with measures of impaired blood‐CSF barrier in patients with prodromal and overt dementia with Lewy bodies and Alzheimer’s disease dementia

AbstractBackgroundAlzheimer’s disease (ADdem) and Lewy body dementia (DLB) are characterized by cholinergic impairment and regional brain atrophy including the medial‐temporal and temporo‐parietal regions. In DLB, the overlapping AD pathology is associated with more prominent atrophy. However, other factors than AD pathology play role in brain atrophy and clinical impairmentl. The choroid plexus (CP), part of blood‐CSF barrier, is central in regulating inflammation and monitoring the CSF synthesis, composition, and circulation. However, association between measures of blood‐CSF barrier impairment and regional brain atrophy, and the effect of AD biomarkers in this association, remain unknown in prodromal and overt AD and DLB.MethodWe included 108 participants from the Czech Brain Aging Study (n = 30 prodromal AD, n = 24 ADdem, n = 10 prodromal DLB, n = 20 overt DLB, n = 24 cognitively unimpaired controls, CN) who underwent clinical/cognitive testing, brain MRI and CSF sampling. FreeSurfer automated algorithm and cholinergic basal forebrain ROIs were used to measure CP volumes and nucleus basalis of Meynert (NBM) and other regional cortical thickness and volumes. From CSF, the established AD biomarkers and an albumin quotient (QAlb), a proxy of blood‐CSF barrier impairment, were derived. Associations were measured using multivariate analysis of variance adjusted for age, sex; mediation analysis was used to assess effect of CSF AD biomarkers on associations between atrophy and blood‐CSF impairment.ResultCP volume was significantly larger and NBM volume was smaller in both DLB and AD compared to CN, and the CP volume increased from prodromal to overt dementia stages. Larger CP volumes were associated with smaller NBM volumes in AD and DLB prodromal and overt stages. Larger CP volumes were associated with abnormal QAlb, and with more abnormal levels of AD biomarkers. More abnormal AD biomarker levels mediated the association between larger CP volumes, abnormal AlbQ and regional atrophy in AD and DLB.ConclusionMeasures of impaired blood‐CSF barrier by enlarged CP and AlbQ are associated with more profound regional atrophy including cholinergic regions from prodromal to overt dementia stages. Whereas AD‐related pathology likely plays role in this, investigating the blood‐CSF barrier as a potential treatment target may be beneficial for AD and DLB patients.

CSF sTREM2 and YKL‐40 modify the balance between soluble and fibrillary β‐amyloid in non‐demented individuals

AbstractBackgroundCerebrospinal fluid (CSF) Aβ concentrations and Positron Emission Tomography (PET) imaging provide information of two different Aβ pools. Identifying the factors affecting the balance between soluble and deposited Aβ may inform on the mechanism of cerebral Aβ aggregation. Microglia and astroglia respond to Aβ pathology, yet their role in promoting the aggregation of Aβ plaques is unclear. We addressed whether CSF sTREM2 and YKL‐40 modulate the association between soluble and deposited Aβ in a cohort of middle‐aged cognitively unimpaired (CU) individuals.MethodThe first consecutive 320 CU individuals of the ALFA+ study with a [18F]flutemetamol (FTM) PET scan and CSF samples were included. CSF Aβ40, Aβ42, sTREM2, and YKL‐40 were measured with the Roche NeuroToolKit robust prototype immunoassays (Roche Diagnostics International Ltd). Standardized uptake value ratio images were calculated and a voxel‐wise regression was performed to predict FTM uptake. The interaction terms between CSF Aβ42/40 and either CSF sTREM2 or YKL‐40 served as predictors, while adjusting for age, sex, years of education, and APOE‐ε4. Statistical threshold was set to p&lt;0.001 with a cluster extent correction of 100 voxels.ResultTable 1 describes the sample’s characteristics. As expected, CSF Aβ42/40 concentrations were negatively associated with Aβ PET uptake in widespread areas of the brain, but this association was modified by CSF sTREM2 and YKL‐40. Higher levels of CSF sTREM2 were associated with a reduced Aβ PET uptake for any level of CSF Aβ42/40 in the hippocampus bilaterally. Conversely, higher CSF YKL‐40 related to a higher Aβ PET uptake as a function of CSF Aβ42/40 in the precuneus, prefrontal cortex, and inferior parietal areas.ConclusionIn CU individuals, microglia and astroglia reactivity modify the association between soluble (CSF) and deposited (amyloid PET) Aβ. While TREM2‐mediated microglial response (as measured by CSF sTREM2) may prevent Aβ deposition in individuals with decreased soluble Aβ, astroglial reactivity (as measured by CSF YKL‐40) may have the opposite effect.

The effect of BDNF Val66Met on memory, CSF total‐tau, CSF ptau181 and CSF Aβ42 in early sporadic Alzheimer’s disease

AbstractBackgroundAllelic variation in the brain‐derived neurotrophic factor (BDNF) Val66Met polymorphism has been shown to moderate rates of cognitive decline in preclinical sporadic Alzheimer’s disease (i.e., Aβ+ older adults), and pre‐symptomatic dominantly inherited Alzheimer’s disease (DIAD). In DIAD, Met66 was also associated with greater increases in CSF levels of total‐tau and phosphorylated tau (p‐tau181). This study sought to determine the extent to which BDNF Val66Met is also associated with changes in episodic memory and CSF total‐tau and p‐tau181 in Aβ+ older adults in early‐stage sporadic AD.MethodAβ+ Met66 carriers (n = 94) and Val66 homozygotes (n = 192) enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) who did not meet criteria for AD dementia were included. Only participants with at least one follow‐up neuropsychological and CSF assessment were included. Episodic memory was measured by the ADNI Memory Composite and CSF samples were collected via lumbar puncture to determine levels of total‐tau, p‐tau181 and Aβ42. A series of linear mixed models were conducted to investigate changes in each outcome variable over 10 years, covarying for CSF Aβ42 (except when examining changes in CSF Aβ42), APOE ε4 status, sex, age, baseline diagnosis and years of education.ResultWhen followed over 10 years, Aβ+ Met66 carriers demonstrated significantly faster memory decline (d = 0.33) and significantly greater increases in CSF total‐tau (d = 0.30) and p‐tau181 (d = 0.29) compared to Val66 homozygotes, despite showing equivalent rates of change in CSF Aβ42 (Table 1; Figure 1).ConclusionIn non‐demented Aβ+ older adults, Met66 carriers showed greater memory decline and greater increases in both CSF total‐tau and CSF p‐tau181 over 10 years compared to Val66 homozygotes, despite showing equivalent changes in CSF Aβ42. This suggests that Met66 carriage, which is associated with lower BDNF availability and lower neurotrophic support, may confer greater vulnerability to Aβ‐related increases in tau compared to Val66 homozygosity in the early stages of sporadic AD.

A novel analytic approach to examine the relationship between APOE4 allele status and CSF complement‐related proteins and signaling pathways

AbstractBackgroundThe mechanisms by which the APOE4 allele contributes to AD risk are unclear. Here, we tested the hypothesis that in cognitively normal adults, APOE4 allele is associated with a decrease in complement‐related proteins and downregulation of complement‐related pathways.MethodUntargeted LC‐MS/MS proteomics was conducted on CSF samples (n = 118) from APOE3/3, APOE3/4 and APOE4/4 individuals over age 60, with groups matched on age and sex. We developed a novel analytic approach (which we term “up/down analysis”) based on the directional consistency of the β‐coefficients from a linear model for the intensities of peptides mapping to a given protein with respect to APOE4 carrier status (APOE4‐/ APOE4+). The analysis involves two steps: 1) The direction of the beta‐coefficient (positive/up or negative/down) for the level of each peptide (the outcome variable) due to APOE4 carrier status was derived from linear regression models adjusting for age, sex, and study. 2) These β‐coefficients were tallied for all peptides from a given protein (i.e. number of positive/up versus negative/down β‐coefficients). We then tested the probability under a binomial distribution that the distribution of up versus down peptides from each protein would be as, or more extreme than what we found, with a = 0.05. Pathway enrichment analysis (using p‐values from binomial tests) was conducted using GAGE and functional annotations based on KEGG. We used Benjamini‐Hochberg for multiple comparison correction.ResultWe found 158 proteins for which the direction of peptide intensity was consistently up (+β‐coefficient) or down (‐β‐coefficient) with respect to APOE4 status beyond what would be expected by chance. Of these 158 proteins, 37 were from the complement pathway. Pathway enrichment analysis further supports the role of the complement system; as the top differentially expressed pathway (FDR &lt;0.05) was related to complement signaling.ConclusionOur results suggest that APOE4 carriers have lower CSF levels of key complement cascade proteins, which has previously been viewed as an indicator of complement activation. These results may provide insights into the mechanisms by which APOE4 and associated complement‐related proteins and signaling pathways ultimately contribute to increased AD risk.

Associations between metabolic syndrome and markers of brain pathologies: the role of sex‐differences

AbstractBackgroundIn old age, cardiovascular and metabolic disorders rarely occur in isolation, but rather cluster together, in the so‐called metabolic syndrome (MetS). Although individual MetS components (obesity, hypertension, diabetes, and dyslipidemia) have been linked to dementia, their combined effect on cognition remains poorly explored. We recently showed that MetS was associated with clinically relevant cognitive decrements in otherwise cognitively healthy septuagenarians. However, the mechanistic underpinnings are unknown. This study investigates the association between MetS and markers of neurodegenerative and cerebrovascular pathologies, considering sex‐differences.MethodWithin the Gothenburg H70‐Birth cohort 1944, we included 741 cognitively intact septuagenarians with available brain MRI (n = 286 with cerebrospinal fluid [CSF] samples). MetS was identified according to standard criteria as the presence of at least three conditions: central obesity, raised blood pressure, raised blood glucose, reduced HDL‐cholesterol, or elevated triglycerides. MRI markers of brain pathology included overall (mean cortical thickness) and Alzheimer’s disease (AD; average cortical thickness in signature areas) neurodegeneration as well as cerebrovascular pathology including small vessel disease (SVD) and DTI’s microstructural white‐matter changes. CSF biomarkers included t‐tau, neurofilament light, and neurogranin (overall neurodegeneration), Aβ42 and p‐tau (AD), and CSF/serum albumin ration (blood‐brain barrier integrity). Analyses included linear, quantile, and logistic regression models as well as interaction and stratification by sex.ResultOverall, 410 participants (53%) had MetS; 196 were women. In multi‐adjusted regressions (by sex, education, heart disease, and APOE‐ɛ4 allele), MetS was associated with increased likelihood of neurodegeneration (OR = 2.0, 95%CI 1.4–2.9) and AD (OR = 1.8, 95%CI 1.4–2.6), high SVD burden (presence of ≥3 SVD compared to none: OR = 2.1, 95%CI 1.1‐4.3), and greater microstructural white‐matter changes. In the CSF sample, MetS was associated with higher CSF/serum albumin ratio. In analyses stratified by sex, men with MetS (reference: no‐MetS) had increased odds of higher SVD burden, particularly of enlarged perivascular spaces (ePVS; OR = 2.9, 95%CI 1.7–5.0). Such differences were not observed between women with and without MetS. No associations were observed between MetS and amyloid and tau biomarkers.ConclusionMetS in late‐life increased the likelihood of neurodegenerative and cerebrovascular pathologies. SVD, especially ePVS, appears more prominent in men with MetS than women.

Sex differences of cerebrospinal fluid biomarkers in Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is more common in women than in men. Thus, to investigate the sex‐specific mechanism is important to understand the pathogenesis of AD. We examined the sex differences in levels of CSF biomarkers (tau, ptau‐181, Aβ42, HVA and 5HIAA) in AD.MethodThe subjects of this study consisted of clinically diagnosed 386 patients with AD (age 75.9±8.4 years, 237 women and 149 men). The clinical diagnosis was made based on the AD diagnostic criteria (NIA/AA, McKhann GM, et al., Alzheimers Dement 2011; 7: 263‐269). The CSF samples were obtained by lumbar puncture after informed consent was obtained. The levels of CSF tau, ptau‐181 and Aβ42 were measured by ELISA (Fujirebio, Belgium) according to the manufacturer’s protocol. The levels of CSF HVA and 5HIAA were measured by HPLC. We compared the levels of CSF biomarkers between women and men.ResultAge, the levels of CSF tau, ptau‐181 and Aβ42 showed no sex differences. The levels of CSF HVA (ng/ml) were 32.6±19.3 in women and 25.9±14.4 in men. The levels of 5HIAA were 19.1±11.7 in women and 14.8±7.7 in men. The levels of CSF HVA and 5HIAA were significantly higher in women than those in men (t‐test, HVA: p&lt;0.001, 5HIAA: p&lt;0.001).It has been reported that women have higher values of HVA and 5HIAA among normal subjects, due to higher monoamine oxidase (MAO) activity in women. The MAO‐A and MAO‐B genes are located on the X chromosome, and are controlled by XCI (X‐chromosome inactivation). However, the MAO genes may escape XCL MAO‐B has also been reported to be γ‐secretase associated protein and regulate neuronal amyloid β‐peptide levels. Thus, increased MAO‐B activity may be one of the reasons why AD is more common in women.ConclusionBoth CSF levels of HVA and 5HIAA were significantly higher in women in AD, presumably due to increased MAO activity.

Alzheimer’s Disease‐impacted Expression of tRNA‐derived Fragments in Body Fluid: Implication for New Disease Biomarkers

AbstractBackgroundAlzheimer’s disease (AD), the most common type of dementia, affects over six million Americans aged 65 years and older. Although numerous research efforts were made on AD, the knowledge of the mechanisms and therapeutic strategies is still very limited. At present, identifying the prodromal stages of AD remains a significant challenge. tRNA‐derived RNA fragments (tRFs), a family of recently discovered small non‐coding RNAs (sncRNAs), express high in both human and primate hippocampus. Our recent discovery also revealed that some hippocampus tRF expression is much higher in AD patients compared with age‐matched health individuals.MethodHerein, we further characterized the expression of tRFs in CSF and serum from healthy, mild cognitive impairment (MCI), and AD patients, using a combination of T4 polynucleotide kinase (T4 PNK)‐RNA‐seq and qRT‐PCR specific to tRF validation.ResultOur results indicated tRF5‐ProAGG altered by AD in both CSF and serum samples, and serum AD‐impacted tRF5‐ProAGG showed a correlation to the disease severity. Notably, serum tRF5‐ProAGG showed a change tendency when MCI progresses to AD, suggesting that it is a promising biomarker for AD. We also predicted putative targets of tRF5‐ProAGG using our in‐house computational program and confirmed that some targets, such as SV2B, CDH8, and NEFL, were decreased in AD’s hippocampus. Interestingly, SV2B and tRF5‐ProAGG showed a negative correlation.ConclusiontRF5‐ProAGG shows the potential as an AD biomarker and plays a vital role in disease progression.

Diagnostic performance and clinical applicability of plasma pTau181 to predict dementia risk in a large real‐world cohort of a memory clinic

AbstractBackgroundThe Alzheimer Association has recently remarked the diagnostic and prognostic revolution that plasma biomarkers will mean for AD diagnosis, and highlighting the need of further research to translate them as a realistic option in the clinical practice. However, many studies to date have assessed them in research cohorts. Here we evaluated the diagnostic performance and clinical applicability of plasma pTau181 as a predictive marker of dementia risk in a real‐world memory clinic cohort.MethodThe ACE cohort encompass 1649 patients with subjective cognitive decline (SCD), mild cognitive impairment (MCI), AD dementia and other dementias, with a complete battery of neurological tests, biochemistry data, and paired CSF and plasma samples of the Ace Alzheimer Center Barcelona memory clinic. CSF AB42, AB40, pTau181 and tTau, and plasma pTau181 were measured using the Lumipulse G1200 automatic platform (Fujirebio Inc.). Complete cohort were divided in the Testing cohort (n = 1000) and Validation cohort (n = 649).ResultPlasma pTau181 significantly correlated (P&lt;0.0001) with CSF pTau181 in MCI, AD dementia and other dementias, but not in SCD. Sex appeared not significant in plasma pTau181 levels, whereas age significantly influenced in the biomarker’s performance. ROC curve of A+T+ patients vs rest showed an AUC = 0.8250 and P&lt;0.0001. Specifically for MCI patients, ROC curve of MCI A+T+ vs MCI A‐T‐ showed and AUC = 0.8874 and P&lt;0.0001. A cut off value of 1.300 pg/ml of plasma pTau181 exhibited a 93.6% sensibility, 72.4% specificity, 77.8% precision and only 8.3% of false negatives in MCI patients. Hazard ratio of survival of conversion analysis showed that MCI patients above this cut off value exhibited a 4.570 higher risk to convert to dementia than MCI under the cut off value. Validation cohort confirmed these findings.ConclusionPlasma pTau181 could be useful as a pre‐screening biomarker of MCI patients with high risk to progress to dementia. However, positive patients will also require a CSF/PET confirmatory test. Plasma pTau181 by itself does not present a suitable performance in SCD patients. Another biomarker or a combination of biomarkers would be needed to this population. Further studies are need to obtain more clinical data and improve the biomarker performance to finally substitute CSF/PET biomarkers.

Effect of AZP2006, a new treatment for Alzheimer’s Disease and Progressive Supranuclear Palsy, in the Restoration of Lysosomal Homeostasis via the Progranulin‐Prosaposin Axis

AbstractBackgroundProgranulin (PGRN) is a secreted pleiotropic growth factor primarily expressed in neurons and microglia that regulates neuroinflammation, neurite branching and outgrowth. PGRN has been identified as a Cathepsin D chaperone, suggesting a role in lysosomal proteolysis. PGRN together with Prosaposin (PSAP) have been proved to be physiologically involved in lysosomal lipid catabolism. PSAP physically interacts with PGRN and facilitates glycosphingolipid degradation upon lysosomal processing, serving as activators of various lysosomal lipases. Thus, PGRN and PSAP play an essential role in the regulation of lysosomal homeostasis. Reduced lysosomal protein levels and activity, as well as accumulation of toxic proteins, have been observed in age‐dependent neurodegenerative diseases. The impact of the disturbance of the PGRN‐PSAP axis in these diseases is not yet fully understood.AZP2006 (INN: Ezeprogind) is a small molecule currently under development for the treatment of Progressive Supranuclear Palsy (PSP, Ph2a completed) and Alzheimer’s Disease (AD). AZP2006 was proved to protect neurons, to increase synaptogenesis and to reduce neuroinflammation in different models of proteinopathies. Importantly, its neuroprotective properties were fully abolished in absence of PGRN or PSAP. Although its exact mode of action (MOA) is still unclear, AZP2006 has been proved to target lysosomes and bind the complex PGRN‐PSAP. Considering this information, the AZP2006 lysosomal MOA was further investigated.MethodAged (18mo) C57Bl6 mice receiving Aβ1‐42 oligomers bilaterally injected into the CA1 area of the hippocampus, were orally treated (gavage) with AZP2006 (1 or 2mg/kg/day) for 22 days. At the end of the treatment, the brain, CSF and plasma of the animals were collected. Lysosomal markers were investigated (LAMP‐2, LC‐3b, PGRN, PSAP, α‐Syn and glycosphingolipids contents). Histological analysis of the CA1 hippocampal area was performed (LAMP‐2, LC‐3b and α‐Syn in neurons and microglial cells). In addition, plasma and CSF samples of PSP patients generated from PSP Ph2a were analyzed.ResultAZP2006 displayed beneficial effects in the Ab1‐42 oligomers injured aged mice. AZP2006 was able to correct the lysosomal dysfunction and to restore the lysosomal homeostasis.ConclusionAZP2006 represents an original therapeutic approach to protect the PGRN‐PSAP balance that is fundamental for the neuron health.

Longitudinal AD biomarkers in monozygotic twins: genetic contribution to AD biomarker concentrations over time

AbstractBackgroundUnderstanding the earliest pathophysiological changes in Alzheimer’s disease (AD) is important for development of therapeutic intervention strategies. Studying genetically identical twins provides unique information on genetic and environmental influences on development of AD. Here we studied change in AD biomarkers amyloid β1‐42 (aβ42), amyloid β1‐40 (aβ40), phosphorylated tau181 (p‐tau) and total tau (t‐tau) in CSF over time in a cohort of older cognitively normal monozygotic twins and tested the genetic contribution to biomarker concentrations at baseline and to change of biomarker concentrations over time.MethodWe included 103 twins (npairs = 42) from the EMIF‐AD preclinAD study with 2‐4 repeated CSF sampling available across a time period of 4.47±2 years (mean±SD age: 68.8±7 years, 54 (52%) female). We measured aβ42, aβ40, p‐tau and t‐tau using the Lumipulse platform (Fujirebio Diagnostics, Inc.). We used linear mixed models to investigate change in biomarker concentrations over time. We used twin‐pair intraclass correlation (ICC) analysis to estimate the genetic contribution to biomarker concentrations at baseline and at repeated measurements, and to annual change in biomarker concentrations.ResultAt baseline, 25 (24%) individuals had an abnormal aβ42/aβ40 ratio, 27 (26%) abnormal p‐tau and 35 (34%) abnormal t‐tau, and at last measurement respectively 34 (33%), 38 (37%) and 45 (44%). Across the group, aβ42/aβ40 decreased (β±SE ‐0.001±0, p‐value&lt;0.001), and p‐tau and t‐tau increased over time (respectively 1.37±0.3, p‐value&lt;0.001; 6.58±2.3, p‐value = 0.004). Increases in p‐tau levels were most pronounced in amyloid positive individuals (2.72±0.38, p‐value&lt;0.001) compared to amyloid negative individuals (0.65±0.24, p‐value = 0.007; p‐value interaction&lt;0.001). At baseline, the genetic contribution to biomarker concentrations was lowest for t‐tau (ICC = 0.5, p&lt;0.001) and highest for aβ40 (ICC = 0.79, p&lt;0.001). ICC’s remained similar at repeated measurements (table 1). Annual changes of aβ40 and p‐tau showed significant twin‐pair correlations (resp. ICC’s: 0.58 and 0.42; p‐values &lt;0.01; Figure 1), indicating a genetic contribution to change in these concentrations over time.ConclusionIn this sample of older cognitively normal monozygotic twins, we found that AD biomarkers became increasingly abnormal over time. Our unique twin design further indicates that these very early pathophysiological changes are under moderate to high genetic influence, which also indicates a role of environmental factors.

Longitudinal white matter integrity alterations as a function of Preclinical Alzheimer’s Pathology: findings from the EPAD non‐demented cohort

AbstractBackgroundCross‐sectional diffusion tensor imaging (DTI) studies report white matter (WM) microstructural alterations, mainly involving fornix and corpus callosum (CC), in symptomatic stages of Alzheimer’s Disease (AD). Exploring these changes longitudinally in relation to preclinical and prodromal AD pathology is essential to track disease progression. We investigated trajectories of WM alterations in non‐demented individuals from the European Prevention of Alzheimer’s Dementia (EPAD) cohort and their relationship with AD biomarkers.MethodEPAD inclusion criteria were age&gt;50 years and Clinical Dementia Rating = 0.5. We selected participants with baseline CSF samples, and longitudinal DTI scans. After defining AT groups(Ingala et al. 2021), A‐T+ participants were excluded (final N = 283). MRI acquisition and preprocessing was previously described(Lorenzini et al. 2022). We extracted fractional anisotropy (FA) in 14 regions(Mori et al. 2005) from DTI as a marker of WM integrity. Site harmonization of MRI‐derived data was performed with ComBat toolbox(Fortin et al. 2018). Age‐ and sex‐corrected linear mixed models with random intercepts on participants were used to estimate time‐effect on MRI‐derived phenotypes, and their interaction with AT status. Results were corrected for multiple testing.ResultAt baseline, A+T‐ participants showed lower FA in body/columns (ß = ‐0.04, p&lt;0.01) and crus of fornix (ß = ‐0.01, p = 0.04), and in splenium (ß = ‐0.007, p = 0.04) and body of CC (ß = ‐0.01, p = 0.03) compared to A‐T‐. In the whole group, FA decreased over time in splenium (ß = ‐0.003, p&lt;0.01), body (ß = ‐0.002, p&lt;0.01) and genu of CC (ß = ‐0.009, p&lt;0.01), body/columns (ß = ‐0.004, p&lt;0.01) and crus of fornix (ß = ‐0.005, p&lt;0.01), and inferior longitudinal fasciculus (ß = ‐0.007, p&lt;0.01). Compared to A‐T‐, A+T+ participants showed steeper FA reduction in superior (Interaction:ß = ‐0.004, p = 0.03) and anterior corona radiata (Interaction:ß = ‐0.006, p = 0.02).ConclusionOur results suggest that WM integrity in key AD regions may degenerate early in the AD continuum, showing greater impairment over short‐time intervals in individuals with higher AD‐specific pathological burden.

Tissue inhibitor of matrix metalloproteinases 4: a novel marker associated with CAA

AbstractBackgroundWe have previously shown that matrix metalloproteinase 9 (MMP9) and tissue inhibitor of proteinases 3 (TIMP3) are specifically expressed in the vasculature of patients with cerebral amyloid angiopathy (CAA). Moreover, we have demonstrated increased MMP9 and decreased TIMP3 levels, as well as an altered MMP9:TIMP3 ratio in the vasculature of CAA patients that suffered from intracerebral haemorrhages (CAA‐ICH) compared to CAA patients without ICH (CAA‐nonICH). We now extended our studies to investigate whether TIMP4 may have a similar role in CAA, as this has not been studied before.MethodUsing immunohistochemistry, we studied occipital lobe tissue of 44 controls (mean age 78.4 years; 50% female) and 58 CAA patients (77.5 years; 55% female; 40 CAA‐nonICH and 18 CAA‐ICH). Vascular TIMP4 staining was scored in a semiquantitative manner. Using ELISA, we measured TIMP4 CSF levels of 33 CAA patients (mean age 72.2 years; 47% female) and 35 controls (mean age 71.2 years; 51% female). For a subset of CSF samples (11 CAA samples and 14 controls), the levels of MMP2, MMP9, and MMP14 have been determined previously, and now their ratios to TIMP4 were calculated. Differences between CAA patients and controls were assessed using (ordinal) linear regression analysis.ResultIn brain tissue, CAA cases had increased vascular TIMP4 expression when compared to controls (p = 0.00002, figure 1). TIMP4 expression did not differ between CAA‐nonICH and CAA‐ICH cases. In the absence of vascular TIMP4 staining, we often observed perivascular staining. Decreased levels of perivascular TIMP4 staining were observed in CAA‐ICH cases when compared to CAA‐nonICH cases (p = 0.045) and to controls (p = 0.035). CSF levels of TIMP4 were decreased in CAA patients compared to controls (p = 0.03, figure 2). In addition, the ratios of MMP2 (p = 0.038), MMP9 (p = 0.005), and MMP14 (p = 0.025) to TIMP4 were increased in CAA patients compared to controls.ConclusionWe demonstrated increased TIMP4 expression in vessel walls of CAA patients compared to controls. In contrast, TIMP4 CSF levels were decreased in CAA patients compared to controls, whereas MMP:TIMP4 ratios were increased, indicating a disbalance of MMP/TIMP4 levels in CAA pathophysiology.Funding: BIONIC project (no. 733050822, ZonMW) and CAFÉ project (no. 5R01NS104147‐02, NIH).

Safety, Pharmacokinetics and Immunogenicity of Single and Multiple Ascending Doses of the Anti‐Tau Therapeutic Antibody E2814: A Phase 1, First‐In‐Human (FIH) Study in Healthy Subjects

AbstractBackgroundE2814 is an anti‐tau therapeutic monoclonal antibody (mAb) which is being developed as a potential disease‐modifying therapy for Alzheimer’s disease (AD). E2814 inhibits the propagation of pathological tau species by binding to the microtubule binding region (MTBR). This study evaluated safety, pharmacokinetics (PK), and immunogenicity of single and multiple doses of E2814.MethodsThis randomized, placebo‐controlled study evaluated 5 single ascending doses (SAD) and 4 multiple ascending doses (MAD) in healthy subjects. Subjects (n = 8/cohort) received single or multiple (every 4 weeks) 1‐hour IV infusions of E2814 (or placebo). Safety was evaluated through a review of adverse events (AE), clinical laboratory test results, physical examination, electrocardiogram results, and vital signs. Serial blood and CSF samples for measurement of serum and CSF E2814 concentrations by a validated electrochemiluminescence (ECL) assay were obtained at prespecified time‐points. The serum PK parameters were estimated using non‐compartmental analysis. Anti‐drug antibodies (ADA) in serum were measured by a validated ECL assay.ResultsE2814 has an adequate single and multiple dose safety profile. No treatment‐emergent serious AEs, severe AEs, infusion‐related reactions, or dose limiting events were observed. There was a dose‐related increase in serum and CSF E2814 exposures. The median time to maximum E2814 concentrations in serum was 1.5 to 2 h. The half‐life of E2814 was 23 days which is typical of mAb. In SAD, serum AUC and Cmax were approximately dose proportional at lower doses and greater than dose‐proportional at higher doses. In MAD, accumulation based on Cmax and AUC was ∼1.2 and 1.3‐fold, respectively by the third dose in all dose group. The serum‐to‐CSF concentration ratio ranged between 0.1 to 0.3%. The immunogenicity to E2814 was low. In SAD, 5 subjects had transient low level ADA titers by last study day. In MAD, 1 subject had a confirmatory positive ADA at baseline prior to dose on Day 1. PK in the ADA positive subjects was comparable to that of ADA negative subjects.ConclusionE2814 presented an adequate safety and immunogenicity profile in healthy adults. E2814 PK was comparable to that with other mAbs. These results support further development of E2814 as a potential disease‐modifying therapy for AD.