Unlocking Preclinical Alzheimer's: A Multi-Year Label-Free In Vitro Raman Spectroscopy Study Empowered by Chemometrics.

Alzheimer's disease is a progressive neurodegenerative disorder, the early detection of which is crucial for timely intervention and enrollment in clinical trials. However, the preclinical diagnosis of Alzheimer's encounters difficulties with gold-standard methods. The current definitive diagnosis of Alzheimer's still relies on expensive instrumentation and post-mortem histological examinations. Here, we explore label-free Raman spectroscopy with machine learning as an alternative to preclinical Alzheimer's diagnosis. A special feature of this study is the inclusion of patient samples from different cohorts, sampled and measured in different years. To develop reliable classification models, partial least squares discriminant analysis in combination with variable selection methods identified discriminative molecules, including nucleic acids, amino acids, proteins, and carbohydrates such as taurine/hypotaurine and guanine, when applied to Raman spectra taken from dried samples of cerebrospinal fluid. The robustness of the model is remarkable, as the discriminative molecules could be identified in different cohorts and years. A unified model notably classifies preclinical Alzheimer's, which is particularly surprising because of Raman spectroscopy's high sensitivity regarding different measurement conditions. The presented results demonstrate the capability of Raman spectroscopy to detect preclinical Alzheimer's disease for the first time and offer invaluable opportunities for future clinical applications and diagnostic methods.

Accelerated long-term forgetting in individuals with subjective cognitive decline and amyloid-ß positivity.

OBJECTIVES: We studied a sample of cognitively unimpaired individuals, with and without subjective cognitive decline (SCD), in order to investigate accelerated long-term forgetting (ALF) and to explore the relationships between objective and subjective cognitive performance and cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers. METHODS: Fifty-two individuals were included and SCD was quantified through the Subjective Cognitive Decline Questionnaire (SCD-Q), using its validated cutoff to classify participants as Low SCD-Q (n = 21) or High SCD-Q (n = 31). These groups were further subdivided according to the presence or absence of abnormal levels of CSF Aß42 . Objective cognitive performance was assessed with the Ancient Farming Equipment Test (AFE-T), a new highly-demanding test that calls for acquisition and retention of novel object/name pairs and allows measuring ALF over a 6-month period. RESULTS: The High SCD-Q group showed a significantly higher free forgetting rate at 3 months compared to the Low SCD-Q (F [1,44] = 4.72; p < 0.05). When stratifying by amyloid status, High SCD-Q/Aß+ showed a significantly lower performance than High SCD-Q/Aß-on the final free and cued learning scores (F [1,27] = 6.44, p < 0.05 and F [1,27] = 7.51, p < 0.05, respectively), the 1-week free and cued recall (F [1,24] = 4.49; p < 0.05 and F [1,24] = 7.10; p < 0.01, respectively), the 1-week cued forgetting rate (F [1,24] = 5.13; p < 0.05), and the 3-month cued recall (F [1,24] = 4.27; p < 0.05). Linear regression analyses showed that higher SCD-Q scores were associated with higher forgetting rates on the AFE-T (ß = -0.212; p < 0.05). CONCLUSIONS: It is possible to detect ALF in individuals with high SCD ratings, appearing especially in those with abnormal CSF Aß42 levels. Both in research and the clinical field, there is an increasing need of using more demanding cognitive measures, such as the AFE-T, for identifying and tracking the earliest cognitive changes in these populations.

Cortical microstructural changes along the Alzheimer's disease continuum.

INTRODUCTION: Cortical mean diffusivity (MD) and free water fraction (FW) changes are proposed biomarkers for Alzheimer's disease (AD). METHODS: We included healthy control subjects (N = 254), mild cognitive impairment (N = 41), and AD dementia (N = 31) patients. Participants underwent a lumbar puncture and a 3 T magnetic resonance imaging. Healthy control subjects were classified following National Institute on Aging-Alzheimer's Association stages (stage 0, N = 220; stage 1, N = 25; and stage 2/3, N = 9). We assessed the cortical MD, cortical FW, and cortical thickness (CTh) changes along the AD continuum. RESULTS: Microstructural and macrostructural changes show a biphasic trajectory. Stage 1 subjects showed increased CTh and decreased MD and FW with respect the stage 0 subjects. Stage 2/3 subjects showed decreased CTh and increased cortical MD and FW, changes that were more widespread in symptomatic stages. DISCUSSION: These results support a biphasic model of changes in AD, which could affect the selection of patients for clinical trials and the use of magnetic resonance imaging as a surrogate marker of disease modification.

GOIZ ZAINDU study: a FINGER-like multidomain lifestyle intervention feasibility randomized trial to prevent dementia in Southern Europe.

BACKGROUND: GOIZ ZAINDU ("caring early" in Basque) is a pilot study to adapt the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) methodology to the Basque population and evaluate the feasibility and adherence to a FINGER-like multidomain intervention program. Additional aims included the assessment of efficacy on cognition and data collection to design a large efficacy trial. METHOD: GOIZ ZAINDU is a 1-year, randomized, controlled trial of a multidomain intervention in persons aged 60+ years, with Cardiovascular Risk Factors, Aging and Dementia (CAIDE) risk score ≥ 6, no diagnosis of dementia, and below-than-expected performance in at least one of three cognitive screening tests. Randomization to a multidomain intervention (MD-Int) or regular health advice (RHA) was stratified by sex, age (>/≤ 75), and cognitive status (mild cognitive impairment (MCI)/normal cognition). MD-Int included cardiovascular risk factor control, nutritional counseling, physical activity, and cognitive training. The primary outcomes were retention rate and adherence to the intervention program. Exploratory cognitive outcomes included changes in the Neuropsychological Test Battery z-scores. Analyses were performed according to the intention to treat. RESULTS: One hundred twenty-five participants were recruited (mean age: 75.64 (± 6.46); 58% women). The MD-Int (n = 61) and RHA (n = 64) groups were balanced in terms of their demographics and cognition. Fifty-two (85%) participants from the RHA group and 56 (88%) from the MD-Int group completed the study. More than 70% of the participants had high overall adherence to the intervention activities. The risk of cognitive decline was higher in the RHA group than in the MD-Int group in terms of executive function (p =.019) and processing speed scores (p =.026). CONCLUSIONS: The GOIZ-ZAINDU study proved that the FINGER methodology is adaptable and feasible in a different socio-cultural environment. The exploratory efficacy results showed a lower risk of decline in executive function and processing speed in the intervention group. These results support the design of a large-scale efficacy trial. TRIAL REGISTRATION: GOIZ ZAINDU feasibility trial was approved and registered by the Euskadi Drug Research Ethics Committee (ID: PI2017134) on 23 January 2018. Retrospectively registered in ClinicalTrials.gov (NCT06163716) on 8 December 2023.

Accelerated long-term forgetting over three months in asymptomatic APOE ɛ4 carriers.

Accelerated long-term forgetting (ALF) refers to a rapid loss of information over days or weeks despite normal acquisition/encoding. Notwithstanding its potential relevance as a presymptomatic marker of cognitive dysfunction, no study has addressed the relationship between ALF and Alzheimer's disease (AD) biomarkers. We examined ALF in APOE ɛ4 carriers versus noncarriers, and its relationships with AD cerebrospinal fluid (CSF) biomarkers. We found ALF over three months in APOE ɛ4 carriers (F(1,19) = 5.60; P < 0.05; Cohen's d = 1.08), and this performance was associated with abnormal levels of the CSF Aß42 /ptau ratio (r = -.614; P < 0.01). Our findings indicate that ALF is detectable in at-risk individuals, and that there is a relationship between ALF and the pathophysiological processes underlying AD.

Plasma lipids are associated with white matter microstructural changes and axonal degeneration.

Dislipidemia is a risk factor for cognitive impairment. We studied the association between interindividual variability of plasma lipids and white matter (WM) microstructure, using diffusion tensor imaging (DTI) in 273 healthy adults. Special focus was placed on 7 regions of interest (ROI) which are structural components of cognitive neurocircuitry. We also investigated the effect of plasma lipids on cerebrospinal fluid (CSF) neurofilament light chain (NfL), an axonal degeneration marker. Low density lipoprotein (LDL) and triglyceride (TG) levels showed a negative association with axial diffusivity (AxD) in multiple regions. High density lipoproteins (HDL) showed a positive correlation. The association was independent of Apolipoprotein E (APOE) genotype, blood pressure or use of statins. LDL moderated the relation between NfL and AxD in the body of the corpus callosum (p = 0.041), right cingulum gyrus (p = 0.041), right fornix/stria terminalis (p = 0.025) and right superior longitudinal fasciculus (p = 0.020) and TG in the right inferior longitudinal fasciculus (p = 0.004) and left fornix/stria terminalis (p = 0.001). We conclude that plasma lipids are associated to WM microstructural changes and axonal degeneration and might represent a risk factor in the transition from healthy aging to disease.

Cerebrospinal Fluid 7-Ketocholesterol Level is Associated with Amyloid-ß42 and White Matter Microstructure in Cognitively Healthy Adults.

BACKGROUND: Abnormal cholesterol metabolism changes the neuronal membrane and may promote amyloidogenesis. Oxysterols in cerebrospinal fluid (CSF) are related to Alzheimer's disease (AD) biomarkers in mild cognitive impairment and dementia. Cholesterol turnover is important for axonal and white matter (WM) microstructure maintenance. OBJECTIVE: We aim to demonstrate that the association of oxysterols, AD biomarkers, and WM microstructure occurs early in asymptomatic individuals. METHODS: We studied the association of inter-individual variability of CSF 24-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), 7-ketocholesterol (7-KC), 7ß-hydroxycholesterol (7ß-OHC), amyloid-ß42 (Aß42), total-tau (t-tau), phosphorylated-tau (p-tau), neurofilament (NfL), and WM microstructure using diffusion tensor imaging, generalized linear models and moderation/mediation analyses in 153 healthy adults. RESULTS: Higher 7-KC levels were related to lower Aß42, indicative of greater AD pathology (p = 0.041) .  Higher 7-KC levels were related to lower fractional anisotropy (FA) and higher mean (MD), axial (AxD), and radial (RD) diffusivity. 7-KC modulated the association between AxD and NfL in the corpus callosum splenium (B = 39.39, p = 0.017), genu (B = 68.64, p = 0.000), and fornix (B = 10.97, p = 0.000). Lower Aß42 levels were associated to lower FA and higher MD, AxD, and RD in the fornix, corpus callosum, inferior longitudinal fasciculus, and hippocampus. The association between AxD and Aß42 was moderated by 7K-C (p = 0.048). CONCLUSION: This study adds clinical evidence to support the role of 7K-C on axonal integrity and the involvement of cholesterol metabolism in the Aß42 generation process.

Advances on Automatic Speech Analysis for Early Detection of Alzheimer Disease: A Non-linear Multi-task Approach.

OBJECTIVE: Nowadays proper detection of cognitive impairment has become a challenge for the scientific community. Alzheimer's Disease (AD), the most common cause of dementia, has a high prevalence that is increasing at a fast pace towards epidemic level. In the not-so-distant future this fact could have a dramatic social and economic impact. In this scenario, an early and accurate diagnosis of AD could help to decrease its effects on patients, relatives and society. Over the last decades there have been useful advances not only in classic assessment techniques, but also in novel non-invasive screening methodologies. METHODS: Among these methods, automatic analysis of speech -one of the first damaged skills in AD patients- is a natural and useful low cost tool for diagnosis. RESULTS: In this paper a non-linear multi-task approach based on automatic speech analysis is presented. Three tasks with different language complexity levels are analyzed, and promising results that encourage a deeper assessment are obtained. Automatic classification was carried out by using classic Multilayer Perceptron (MLP) and Deep Learning by means of Convolutional Neural Networks (CNN) (biologically- inspired variants of MLPs) over the tasks with classic linear features, perceptual features, Castiglioni fractal dimension and Multiscale Permutation Entropy. CONCLUSION: Finally, the most relevant features are selected by means of the non-parametric Mann- Whitney U-test.

Increased CAIDE dementia risk, cognition, CSF biomarkers, and vascular burden in healthy adults.

OBJECTIVE: To investigate the cognitive profile of healthy individuals with increased Cardiovascular Risk Factors, Aging and Dementia (CAIDE) dementia risk score and to explore whether this association is related to vascular burden and CSF biomarkers of amyloidosis and neurodegeneration. METHOD: Cognitively normal participants (mean age 57.6 years) from the Gipuzkoa Alzheimer Project study were classified as having high risk (HR; n = 82) or low risk (LR; n = 293) for dementia according to a CAIDE score cutoff of 9. Cognitive composites were compared between groups. We explored using generalized linear models the role of APOE genotype, MRI white matter hyperintensities (WMH), and CSF (n = 218) levels of ß-amyloid1-42 (Aß1-42), total tau (t-tau), and phosphorylated tau (p-tau) in the association between CAIDE score and cognition. RESULTS: HR participants obtained lower scores on executive function (EF) (p = 0.001) and visual perception and construction (VPC) (p < 0.001) composites. EF composite was associated with CAIDE score × p-tau (p = 0.001), CAIDE score × t-tau (p = 0.001), and WMH (p = 0.003). VPC composite was associated with APOE (p = 0.001), Aß1-42 (p = 0.004), the interaction APOE × Aß1-42 (p = 0.003), and WMH (p = 0.004). Performance on global memory was associated with Aß1-42 (p = 0.006), APOE (p = 0.008), and their interaction (p = 0.006). Analyses were adjusted for age, education, sex, premorbid intelligence, and stress. CONCLUSION: Healthy participants at increased dementia risk based on CAIDE scores show lower performance in EF and VPC. This difference is related to APOE, WMH, and Alzheimer biomarkers.

Functional brain network centrality is related to APOE genotype in cognitively normal elderly.

INTRODUCTION: Amyloid plaque deposition in the brain is an early pathological change in Alzheimer's disease (AD), causing disrupted synaptic connections. Brain network disruptions in AD have been demonstrated with eigenvector centrality (EC), a measure that identifies central regions within networks. Carrying an apolipoprotein (APOE)-ε4 allele is a genetic risk for AD, associated with increased amyloid deposition. We studied whether APOE-ε4 carriership is associated with EC disruptions in cognitively normal individuals. METHODS: A total of 261 healthy middle-aged to older adults (mean age 56.6 years) were divided into high-risk (APOE-ε4 carriers) and low-risk (noncarriers) groups. EC was computed from resting-state functional MRI data. Clusters of between-group differences were assessed with a permutation-based method. Correlations between cluster mean EC with brain volume, CSF biomarkers, and psychological test scores were assessed. RESULTS: Decreased EC in the visual cortex was associated with APOE-ε4 carriership, a genetic risk factor for AD. EC differences were correlated with age, CSF amyloid levels, and scores on the trail-making and 15-object recognition tests. CONCLUSION: Our findings suggest that the APOE-ε4 genotype affects brain connectivity in regions previously found to be abnormal in AD as a sign of very early disease-related pathology. These differences were too subtle in healthy elderly to use EC for single-subject prediction of APOE genotype.

Amyloid-ß, Tau, and Cognition in Cognitively Normal Older Individuals: Examining the Necessity to Adjust for Biomarker Status in Normative Data.

We investigated whether amyloid-ß (Aß) and tau affected cognition in cognitively normal (CN) individuals, and whether norms for neuropsychological tests based on biomarker-negative individuals would improve early detection of dementia. We included 907 CN individuals from 8 European cohorts and from the Alzheimer's disease Neuroimaging Initiative. All individuals were aged above 40, had Aß status and neuropsychological data available. Linear mixed models were used to assess the associations of Aß and tau with five neuropsychological tests assessing memory (immediate and delayed recall of Auditory Verbal Learning Test, AVLT), verbal fluency (Verbal Fluency Test, VFT), attention and executive functioning (Trail Making Test, TMT, part A and B). All test except the VFT were associated with Aß status and this influence was augmented by age. We found no influence of tau on any of the cognitive tests. For the AVLT Immediate and Delayed recall and the TMT part A and B, we calculated norms in individuals without Aß pathology (Aß- norms), which we validated in an independent memory-clinic cohort by comparing their predictive accuracy to published norms. For memory tests, the Aß- norms rightfully identified an additional group of individuals at risk of dementia. For non-memory test we found no difference. We confirmed the relationship between Aß and cognition in cognitively normal individuals. The Aß- norms for memory tests in combination with published norms improve prognostic accuracy of dementia.

Beneficial effect of bilingualism on Alzheimer's disease CSF biomarkers and cognition.

Bilingualism as a component of cognitive reserve has been claimed to delay the onset of Alzheimer's disease (AD). However, its effect on cerebrospinal fluid (CSF) AD-biomarkers has not been investigated. We assessed cognitive performance and CSF AD-biomarkers, and potential moderation effect of bilingualism on the association between age, CSF AD-biomarkers, and cognition. Cognitively healthy middle-aged participants classified as monolinguals (n = 100, nCSF = 59), early (n = 81, nCSF = 55) and late bilinguals (n = 97, nCSF = 52) were evaluated. Models adjusted for confounders showed that bilinguals performed better than monolinguals on digits backwards (early-bilinguals p = 0.003), Judgment of Line Orientation (JLO) (early-bilinguals p = 0.018; late-bilinguals p = 0.004), and Trail Making Test-B (late-bilinguals p = 0.047). Early bilingualism was associated with lower CSF total-tau (p = 0.019) and lower prevalence of preclinical AD (NIA-AA classification) (p = 0.02). Bilingualism showed a moderation effect on the relationship between age and CSF AD-biomarkers and the relationship between age and executive function. We conclude that bilingualism contributes to cognitive reserve enhancing executive and visual-spatial functions. For the first time, this study reveals that early bilingualism is associated with more favorable CSF AD-biomarker profile.

Early Detection of Learning Difficulties when Confronted with Novel Information in Preclinical Alzheimer's Disease Stage 1.

We employed a highly demanding experimental associative learning test (the AFE-T) to explore memory functioning in Preclinical Alzheimer's Disease stage 1 (PreAD-1) and stage 2 (PreAD-2). The task consisted in the learning of unknown object/name pairs and our comprehensive setup allowed the analysis of learning curves, immediate recall, long-term forgetting rates at one week, three months, and six months, and relearning curves. Forty-nine cognitively healthy subjects were included and classified according to the presence or absence of abnormal CSF biomarkers (Control, n = 31; PreAD-1, n = 14; PreAD-2, n = 4). Control and PreAD-1 performances on the experimental test were compared by controlling for age and education. These analyses showed clear learning difficulties in PreAD-1 subjects (F = 6.98; p = 0.01). Between-group differences in long-term forgetting rates were less notable, reaching statistical significance only for the three-month cued forgetting rate (F = 4.83; p = 0.03). Similarly, relearning sessions showed only statistical trends between the groups (F = 3.22; p = 0.08). In the whole sample, significant correlations between CSF Aß42/tau ratio and the AFE-T were found, both in the total learning score (r = 0.52; p < 0.001) and in the three-month cued forgetting rate (r = -0.38; p < 0.01). Descriptive subanalyses involving PreAD-2 suggested greater learning and recall difficulties in these subjects when compared with the PreAD-1 group. The present results suggest that explicit learning difficulties when binding information could be one of the earliest signs of the future emergence of episodic memory difficulties on the Alzheimer's disease continuum. Our findings indicate that the AFE-T is a sensitive test, capable of detecting subtle memory difficulties in PreAD-1.

Cerebrospinal fluid mitochondrial DNA in the Alzheimer's disease continuum.

Low levels of cell-free mitochondrial DNA (mtDNA) in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients have been identified and proposed as a novel biomarker for the disease. The lack of validation studies of previous results prompted us to replicate this finding in a comprehensive series of patients and controls. We applied droplet digital polymerase chain reaction in CSF specimens from 124 patients representing the AD spectrum and 140 neurologically healthy controls. The following preanalytical and analytical parameters were evaluated: the effect of freeze-thaw cycles on mtDNA, the linearity of mtDNA load across serial dilutions, and the mtDNA levels in the diagnostic groups. We found a wide range of mtDNA copies, which resulted in a high degree of overlap between groups. Although the AD group presented significantly higher mtDNA counts, the receiver-operating characteristic analysis disclosed an area under the curve of 0.715 to distinguish AD patients from controls. MtDNA was highly stable with low analytical variability. In conclusion, mtDNA levels in CSF show a high interindividual variability, with great overlap within phenotypes and presents low sensitivity for AD.

Amyloid precursor protein metabolism and inflammation markers in preclinical Alzheimer disease.

OBJECTIVE: To investigate CSF markers involved in amyloid precursor protein processing, neuronal damage, and neuroinflammation in the preclinical stages of Alzheimer disease (AD) and participants with suspected non-Alzheimer pathology (SNAP). METHODS: We collected CSF from 266 cognitively normal volunteers participating in a cross-sectional multicenter study (the SIGNAL study) to investigate markers involved in amyloid precursor protein processing (Aß42, sAPPß, ß-secretase activity), neuronal damage (total-tau [t-tau], phospho-tau [p-tau]), and neuroinflammation (YKL-40). We analyzed the relationship among biomarkers, clinical variables, and the APOE genotype, and compared biomarker levels across the preclinical stages of the National Institute on Aging-Alzheimer's Association classification: stage 0, 1, 2, 3, and SNAP. RESULTS: The median age in the whole cohort was 58.8 years (range 39.8-81.6). Participants in stages 2-3 and SNAP had higher levels of YKL-40 than those in stages 0 and 1. Participants with SNAP had higher levels of sAPPß than participants in stage 0 and 1. No differences were found between stages 0, 1, and 2-3 in sAPPß and ß-secretase activity in CSF. Age correlated with t-tau, p-tau, and YKL-40. It also correlated with Aß42, but only in APOE ε4 carriers. Aß42 correlated positively with t-tau, sAPPß, and YKL-40 in participants with normal Aß42. CONCLUSIONS: Our findings suggest that inflammation in the CNS increases in normal aging and is intimately related to markers of neurodegeneration in the preclinical stages of AD and SNAP. sAPPß and ß-secretase activity are not useful diagnostic or staging markers in preclinical AD.