FTIR Spectroscopy and Blood-Derived Extracellular Vesicles Duo in Alzheimer's Disease.

Background: Alzheimer's disease (AD) diagnosis is difficult, and new accurate tools based on peripheral biofluids are urgently needed. Extracellular vesicles (EVs) emerged as a valuable source of biomarker profiles for AD, since their cargo is disease-specific and these can be easily isolated from easily accessible biofluids, as blood. Fourier Transform Infrared (FTIR) spectroscopy can be employed to analyze EVs and obtain the spectroscopic profiles from different regions of the spectra, simultaneously characterizing carbohydrates, nucleic acids, proteins, and lipids. Objective: The aim of this study was to identify blood-derived EVs (bdEVs) spectroscopic signatures with AD discriminatory potential. Methods: Herein, FTIR spectra of bdEVs from two biofluids (serum and plasma) and distinct sets of Controls and AD cases were acquired, and EVs' spectra analyzed. Results: Analysis of bdEVs second derivative peaks area revealed differences between Controls and AD cases in distinct spectra regions, assigned to carbohydrates and nucleic acids, amides, and lipids. Conclusions: EVs' spectroscopic profiles presented AD discriminatory value, supporting the use of bdEVs combined with FTIR as a screening or complementary tool for AD diagnosis.

Phosphoproteome Microarray Analysis of Extracellular Particles as a Tool to Explore Novel Biomarker Candidates for Alzheimer's Disease.

Phosphorylation plays a key role in Alzheimer's disease (AD) pathogenesis, impacting distinct processes such as amyloid-beta (Aß) peptide production and tau phosphorylation. Impaired phosphorylation events contribute to senile plaques and neurofibrillary tangles' formation, two major histopathological hallmarks of AD. Blood-derived extracellular particles (bdEP) can represent a disease-related source of phosphobiomarker candidates, and hence, in this pilot study, bdEP of Control and AD cases were analyzed by a targeted phosphoproteomics approach using a high-density microarray that featured at least 1145 pan-specific and 913 phosphosite-specific antibodies. This approach, innovatively applied to bdEP, allowed the identification of 150 proteins whose expression levels and/or phosphorylation patterns were significantly altered across AD cases. Gene Ontology enrichment and Reactome pathway analysis unraveled potentially relevant molecular targets and disease-associated pathways, and protein-protein interaction networks were constructed to highlight key targets. The discriminatory value of both the total proteome and the phosphoproteome was evaluated by univariate and multivariate approaches. This pilot experiment supports that bdEP are enriched in phosphotargets relevant in an AD context, holding value as peripheral biomarker candidates for disease diagnosis.

Effects of Exercise on Circulating Extracellular Vesicles in Cardiovascular Disease.

The evidence that physical exercise has multiple beneficial effects and is essential to a healthy lifestyle is widely accepted for a long-time. The functional and psychological changes promoted by exercise improve clinical outcomes and prognosis in several diseases, by decreasing mortality, disease severity, and hospital admissions. Nonetheless, the mechanisms that regulate the release, uptake, and communication of several factors in response to exercise are still not well defined. In the last years, extracellular vesicles have attracted significant interest in the scientific community due to their ability to carry and deliver proteins, lipids, and miRNA to distant organs in the body, promoting a very exciting crosstalk machinery. Moreover, increasing evidence suggests that exercise can modulate the release of those factors within EVs into the circulation, mediating its systemic adaptations.In this chapter, we summarize the effects of acute and chronic exercise on the extracellular vesicle dynamics in healthy subjects and patients with cardiovascular disease. The understanding of the changes in the cargo and kinetics of extracellular vesicles in response to exercise may open new possibilities of research and encourage the development of novel therapies that mimic the effects of exercise.

Monitoring clusterin and fibrillar structures in aging and dementia.

Objective: Clusterin is involved in a variety of physiological processes, including proteostasis. Several clusterin polymorphisms were associated with an increased risk of developing Alzheimer's disease, the world-leading cause of dementia. Herein, the effect of a clusterin polymorphism, aging and dementia in the levels of clusterin in human plasma were analysed in a primary care-based cohort, and the association of this chaperone with fibrillar structures discussed. Methods: 64 individuals with dementia (CDR≥1) and 64 age- and sex-matched Controls from a Portuguese cohort were genotyped for CLU rs1136000 polymorphism, and the plasma levels of clusterin and fibrils were assessed. Results: An increased prevalence of the CC genotype was observed for the dementia group, although no significant robustness was achieved. CLU rs11136000 SNP did not significantly change plasma clusterin levels in demented individuals. Instead, clusterin levels decreased with aging and even more in individuals with dementia. Importantly, plasma clusterin levels correlated with the presence of fibrillar structures in Control individuals, but not in those with dementia. Conclusion: This study reveals a significant decrease in plasma clusterin in demented individuals with aging, which related to altered clusterin-fibrils dynamics. Potentially, plasma clusterin and its association with fibrillar structures can be used to monitor dementia progression along aging.

Bioinformatic analysis of the SPs and NFTs proteomes unravel putative biomarker candidates for Alzheimer's disease.

Aging is the main risk factor for the appearance of age-related neurodegenerative diseases, including Alzheimer's disease (AD). AD is the most common form of dementia, characterized by the presence of senile plaques (SPs) and neurofibrillary tangles (NFTs), the main histopathological hallmarks in AD brains. The core of these deposits are predominantly amyloid fibrils in SPs and hyperphosphorylated Tau protein in NFTs, but other molecular components can be found associated with these pathological lesions. Herein, an extensive literature review was carried out to obtain the SPs and NFTs proteomes, followed by a bioinformatic analysis and further putative biomarker validation. For SPs, 857 proteins were recovered, and, for NFTs, 627 proteins of which 375 occur in both groups and represent the common proteome. Gene Ontology (GO) enrichment analysis permitted the identification of biological processes and the molecular functions most associated with these lesions. Analysis of the SPs and NFTs common proteins unraveled pathways and molecular targets linking both histopathological events. Further, validation of a putative phosphotarget arising from the in silico analysis was performed in serum-derived extracellular vesicles from AD patients. This bioinformatic approach contributed to the identification of putative molecular targets, valuable for AD diagnostic or therapeutic intervention.

Impaired Extracellular Proteostasis in Patients with Heart Failure.

BACKGROUND: Proteostasis impairment and the consequent increase of amyloid burden in the myocardium have been associated with heart failure (HF) development and poor prognosis. A better knowledge of the protein aggregation process in biofluids could assist the development and monitoring of tailored interventions. AIM: To compare the proteostasis status and protein's secondary structures in plasma samples of patients with HF with preserved ejection fraction (HFpEF), patients with HF with reduced ejection fraction (HFrEF), and age-matched individuals. METHODS: A total of 42 participants were enrolled in 3 groups: 14 patients with HFpEF, 14 patients with HFrEF, and 14 age-matched individuals. Proteostasis-related markers were analyzed by immunoblotting techniques. Fourier Transform Infrared (FTIR) Spectroscopy in Attenuated Total Reflectance (ATR) was applied to assess changes in the protein's conformational profile. RESULTS: Patients with HFrEF showed an elevated concentration of oligomeric proteic species and reduced clusterin levels. ATR-FTIR spectroscopy coupled with multivariate analysis allowed the discrimination of HF patients from age-matched individuals in the protein amide I absorption region (1700-1600 cm-1), reflecting changes in protein conformation, with a sensitivity of 73 and a specificity of 81%. Further analysis of FTIR spectra showed significantly reduced random coils levels in both HF phenotypes. Also, compared to the age-matched group, the levels of structures related to fibril formation were significantly increased in patients with HFrEF, whereas the ß-turns were significantly increased in patients with HFpEF. CONCLUSION: Both HF phenotypes showed a compromised extracellular proteostasis and different protein conformational changes, suggesting a less efficient protein quality control system.

A review on comparative studies addressing exosome isolation methods from body fluids.

Exosomes emerged as valuable sources of disease biomarkers and new therapeutic tools. However, extracellular vesicles isolation with exosome-like characteristics from certain biofluids is still challenging which can limit their potential use in clinical settings. While ultracentrifugation-based procedures are the gold standard for exosome isolation from cell cultures, no unique and standardized method for exosome isolation from distinct body fluids exists. The complexity, specific composition, and physical properties of each biofluid constitute a technical barrier to obtain reproducible and pure exosome preparations, demanding a detailed characterization of both exosome isolation and characterization methods. Moreover, some isolation procedures can affect downstream proteomic or RNA profiling analysis. This review compiles and discussed a set of comparative studies addressing distinct exosome isolation methods from human biofluids, including cerebrospinal fluid, plasma, serum, saliva, and urine, also focusing on body fluid specific challenges, physical properties, and other potential variation sources. This summarized information will facilitate the choice of exosome isolation methods, based on the type of biological samples available, and hopefully encourage the use of exosomes in translational and clinical research.

Extracellular vesicles in the study of Alzheimer's and Parkinson's diseases: Methodologies applied from cells to biofluids.

Extracellular vesicles (EVs) are gaining increased importance in fundamental research as key players in disease pathogenic mechanisms, but also in translational and clinical research due to their value in biomarker discovery, either for diagnostics and/or therapeutics. In the first research scenario, the study of EVs isolated from neuronal models mimicking neurodegenerative diseases can open new avenues to better understand the pathological mechanisms underlying these conditions or to identify novel molecular targets for diagnosis and/or therapeutics. In the second research scenario, the easy availability of EVs in body fluids and the specificity of their cargo, which can reflect the cell of origin or disease profiles, turn these into attractive diagnostic tools. EVs with exosome-like characteristics, circulating in the bloodstream and other peripheral biofluids, constitute a non-invasive and rapid alternative to study several conditions, including brain-related disorders. In both cases, several EVs isolation methods are already available, but each neuronal model or biofluid presents its own challenges. Herein, a literature overview on EVs isolation methodologies from distinct neuronal models (cellular culture and brain tissue) and body fluids (serum, plasma, cerebrospinal fluid, urine and saliva) was carried out. Focus was given to approaches employed in the context of Alzheimer's and Parkinson's diseases, and the main research findings discussed. The topics here revised will facilitate the choice of EVs isolation methodologies and potentially prompt new discoveries in EVs research and in the neurodegenerative diseases field.

Proteostasis Response to Protein Misfolding in Controlled Hypertension.

Hypertension is the most determinant risk factor for cardiovascular diseases. Early intervention and future therapies targeting hypertension mechanisms may improve the quality of life and clinical outcomes. Hypertension has a complex multifactorial aetiology and was recently associated with protein homeostasis (proteostasis). This work aimed to characterize proteostasis in easy-to-access plasma samples from 40 individuals, 20 with controlled hypertension and 20 age- and gender-matched normotensive individuals. Proteostasis was evaluated by quantifying the levels of protein aggregates through different techniques, including fluorescent probes, slot blot immunoassays and Fourier-transform infrared spectroscopy (FTIR). No significant between-group differences were observed in the absolute levels of various protein aggregates (Proteostat or Thioflavin T-stained aggregates; prefibrillar oligomers and fibrils) or total levels of proteostasis-related proteins (Ubiquitin and Clusterin). However, significant positive associations between Endothelin 1 and protein aggregation or proteostasis biomarkers (such as fibrils and ubiquitin) were only observed in the hypertension group. The same is true for the association between the proteins involved in quality control and protein aggregates. These results suggest that proteostasis mechanisms are actively engaged in hypertension as a coping mechanism to counteract its pathological effects in proteome stability, even when individuals are chronically medicated and presenting controlled blood pressure levels.

A Bioinformatics Approach Toward Unravelling the Synaptic Molecular Crosstalk Between Alzheimer's Disease and Diabetes.

BACKGROUND: Increasing evidence links impaired brain insulin signaling and insulin resistance to the development of Alzheimer's disease (AD). OBJECTIVE: This evidence prompted a search for molecular players common to AD and diabetes mellitus (DM). METHODS: The work incorporated studies based on a primary care-based cohort (pcb-Cohort) and a bioinformatics analysis to identify central nodes, that are key players in AD and insulin signaling (IS) pathways. The interactome for each of these key proteins was retrieved and network maps were developed for AD and IS. Synaptic enrichment was performed to reveal synaptic common hubs. RESULTS: Cohort analysis showed that individuals with DM exhibited a correlation with poor performance in the Mini-Mental State Examination (MMSE) cognitive test. Additionally, APOE ɛ2 allele carriers appear to potentially be relatively more protected against both DM and cognitive deficits. Ten clusters were identified in this network and 32 key synaptic proteins were common to AD and IS. Given the relevance of signaling pathways, another network was constructed focusing on protein kinases and protein phosphatases, and the top 6 kinase nodes (LRRK2, GSK3B, AKT1, EGFR, MAPK1, and FYN) were further analyzed. CONCLUSION: This allowed the elaboration of signaling cascades directly impacting AßPP and tau, whereby distinct signaling pathway play a major role and strengthen an AD-IS link at a molecular level.

Novel Exosome Biomarker Candidates for Alzheimer's Disease Unravelled Through Mass Spectrometry Analysis.

Exosomes are small extracellular vesicles (EVs) present in human biofluids that can transport specific disease-associated molecules. Consequently blood-derived exosomes have emerged as important peripheral biomarker sources for a wide range of diseases, among them Alzheimer's disease (AD). Although there is no effective cure for AD, an accurate diagnosis, relying on easily accessible peripheral biofluids, is still necessary to discriminate this disease from other dementias, test potential therapies and even monitor rate of disease progression. The ultimate goal is to produce a cost-effective and widely available alternative, which can also be employed as a first clinical screen. In this study, EVs with exosome-like characteristics were isolated from serum of Controls and AD cases through precipitation- and column-based methods, followed by mass spectrometry analysis. The resulting proteomes were characterized by Gene Ontology (GO) and multivariate analyses. Although GO terms were similar for exosomes' proteomes of Controls and ADs, using both methodologies, a clear segregation of disease cases was obtained when using the precipitation-based method. Nine significantly different abundant proteins were identified between Controls and AD cases, representing putative biomarker candidate targets. Among them are AACT and C4BPα, two Aß-binding proteins, whose exosome levels were further validated in individuals from independent cohorts using antibody-based approaches. The findings discussed represent an important contribution to the identification of novel exosomal biomarker candidates useful as potential blood-based tools for AD diagnosis.

Exosomal Aß-Binding Proteins Identified by "In Silico" Analysis Represent Putative Blood-Derived Biomarker Candidates for Alzheimer´s Disease.

The potential of exosomes as biomarker resources for diagnostics and even for therapeutics has intensified research in the field, including in the context of Alzheimer´s disease (AD). The search for disease biomarkers in peripheral biofluids is advancing mainly due to the easy access it offers. In the study presented here, emphasis was given to the bioinformatic identification of putative exosomal candidates for AD. The exosomal proteomes of cerebrospinal fluid (CSF), serum and plasma, were obtained from three databases (ExoCarta, EVpedia and Vesiclepedia), and complemented with additional exosomal proteins already associated with AD but not found in the databases. The final biofluids' proteomes were submitted to gene ontology (GO) enrichment analysis and the exosomal Aß-binding proteins that can constitute putative candidates were identified. Among these candidates, gelsolin, a protein known to be involved in inhibiting Abeta fibril formation, was identified, and it was tested in human samples. The levels of this Aß-binding protein, with anti-amyloidogenic properties, were assessed in serum-derived exosomes isolated from controls and individuals with dementia, including AD cases, and revealed altered expression patterns. Identification of potential peripheral biomarker candidates for AD may be useful, not only for early disease diagnosis but also in drug trials and to monitor disease progression, allowing for a timely therapeutic intervention, which will positively impact the patient's quality of life.

Diagnostic and therapeutic potential of exosomes in Alzheimer's disease.

Exosomes are small extracellular vesicles released by almost all cell types in physiological and pathological conditions. The exosomal potential to unravel disease mechanisms, or to be used as a source of biomarkers, is being explored, in particularly in the field of neurodegenerative diseases. Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the world and exosomes appear to have a relevant role in disease pathogenesis. This review summarizes the current knowledge on exosome contributions to AD as well as their use as disease biomarker resources or therapeutic targets. The most recent findings with respect to both protein and miRNA biomarker candidates for AD, herein described, highlight the state of the art in this field and encourage the use of exosomes derived from biofluids in clinical practice in the near future.

Potential of FTIR Spectroscopy Applied to Exosomes for Alzheimer's Disease Discrimination: A Pilot Study.

Alzheimer's disease (AD) diagnosis is based on psychological and imaging tests but can also include monitoring cerebrospinal fluid (CSF) biomarkers. However, CSF based-neurochemical approaches are expensive and invasive, limiting their use to well-equipped settings. In contrast, blood-based biomarkers are minimally invasive, cost-effective, and a widely accessible alternative. Blood-derived exosomes have recently emerged as a reliable AD biomarker source, carrying disease-specific cargo. Fourier-transformed infrared (FTIR) spectroscopy meets the criteria for an ideal diagnostic methodology since it is rapid, easy to implement, and has high reproducibility. This metabolome-based technique is useful for diagnosing a broad range of diseases, although to our knowledge, no reports for FTIR spectroscopy applied to exosomes in AD exist. In this ground-breaking pilot study, FTIR spectra of serum and serum-derived exosomes from two independent cohorts were acquired and analyzed using multivariate analysis. The regional UA-cohort includes 9 individuals, clinically diagnosed with AD, mean age of 78.7 years old; and the UMG-cohort comprises 12 individuals, clinically diagnosed with AD (based on molecular and/or imaging data), mean age of 73.2 years old. Unsupervised principal component analysis of FTIR spectra of serum-derived exosomes revealed higher discriminatory value for AD cases when compared to serum as a whole. Consistently, the partial least-squares analysis revealed that serum-derived exosomes present higher correlations than serum. In addition, the second derivative peak area calculation also revealed significant differences among Controls and AD cases. The results obtained suggest that this methodology can discriminate cases from Controls and thus be potential useful to assist in AD clinical diagnosis.

Monitoring plasma protein aggregation during aging using conformation-specific antibodies and FTIR spectroscopy.

The loss of proteostasis during aging has been well described using different models, however little is known with respect to protein aggregation levels in biofluids with aging. Therefore, the aim of this study was to assess the pattern of age-related protein aggregation in human plasma using two distinct approaches: analysis with conformation-specific antibodies and FTIR spectroscopy. The latter has been widely used in biomedical research to study protein conformational changes in health and disease. Samples from a primary care based-cohort from the Aveiro region, Portugal, were used for slot-blot analyses followed by immunodetection with conformation-specific antibodies and for the acquisition of FTIR spectra. Immunoblot analyses revealed an age-dependent evolution of the protein conformational profile in human plasma, towards a decrease in prefibrillar oligomers and an increase in fibrillar structures. This finding was also supported by PLS-R multivariate analysis of FTIR data, where a positive correlation between the age of the donors and secondary structure of plasma proteins could be observed. Samples from younger donors are characterized by antiparallel ß-sheet-containing structures while intermolecular ß-sheets characterized older samples. Exclusion of age-associated co-morbidities improved the correlation between protein conformational profiles and aging. The results reveal structural changes in human plasma proteins from middle to old age, confirming the age-associated changes in protein aggregation, and support the applicability of FTIR as a reliable approach to study proteostasis during aging.

Exosome isolation from distinct biofluids using precipitation and column-based approaches.

The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations' purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Toward Neuroproteomics in Biological Psychiatry: A Systems Approach Unravels Okadaic Acid-Induced Alterations in the Neuronal Phosphoproteome.

Neuroproteomics is an evolving field of postgenomic medicine, highlighting the convergence of psychiatry/neurology and proteomics, yet compared with neurogenetics, it has received little attention. This study in rat primary neuronal cultures provides an example of a neuroproteomic approach relevant to the study of psychiatric disease pathophysiology, focusing on Alzheimer's disease. In this context, okadaic acid (OA) is routinely used in experimental designs to investigate phosphorylation-mediated events. It is a potent protein phosphatase (PP) inhibitor, particularly of PP1 and PP2A. Typically, a single protein and its phosphorylation level are monitored upon OA exposure. Although useful, this can be misleading as protein phosphorylation-mediated events involve complex signaling cascades and an array of kinases, phosphatases, and substrates. Bearing in mind the involvement of multiple pathways and cascade cross talk, this study employed a systems approach to analyze OA-induced molecular responses through PP inhibition. We showed that upon OA exposure, the recovery rate of 245 phosphoproteins significantly increased, while that of 75 significantly decreased. The prominent biological processes affected included anatomical structural development, transport, cell differentiation, and signal transduction. The associated phosphointeraction networks identified nodes representing OA-responsive phosphoproteins. Many of these are key players of signaling cascades relevant to a range of pathologies. In summary, the data presented results from a neuroproteomic preclinical study offering an array of phosphoproteins as potential targets for future diagnostic and therapeutic strategies in biological psychiatry. We note, however, the nonspecificity of targeting PPs themselves and emphasize the need for future neuroproteomic approaches toward systems psychiatry.

Protein Phosphorylation is a Key Mechanism in Alzheimer's Disease.

Altered protein phosphorylation states of several proteins are closely associated with Alzheimer's disease (AD). Among these are the amyloid-ß protein precursor (AßPP) and the tau protein. In fact, altered protein phosphorylation states already provide strong biomarkers for AD diagnosis, as is the case with hyperphosphorylated tau. It follows that modulating signaling cascades provides an attractive avenue for exploring novel therapeutic strategies. This review focuses on some of the major protein kinases and protein phosphatases relevant to AD. Of particular relevance, posttranslational modifications dynamically regulate protein activity, subcellular localization, and stability. Protein phosphorylation states can mediate complex formation as well as regulate protein function, and this is important for cellular physiology but can likewise contribute to the development of neuropathological conditions. Furthermore, applying a system approach provides a more comprehensive understanding of the signaling events associated with AD and highlights possible convergence points that may contribute to the different AD pathological hallmarks.

Impact of Cytokines and Chemokines on Alzheimer's Disease Neuropathological Hallmarks.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder, neuropathologically characterized by aggregates of ß-amyloid peptides, which deposit as senile plaques, and of TAU protein, which forms neurofibrillary tangles. It is now widely accepted that neuroinflammation is implicated in AD pathogenesis. METHOD: Indeed, inflammatory mediators, such as cytokines and chemokines (chemotactic cytokines) can impact on the Alzheimer´s amyloid precursor protein by affecting its expression levels and amyloidogenic processing and/or ß -amyloid aggregation. Additionally, cytokines and chemokines can influence kinases' activities, leading to abnormal TAU phosphorylation. To date there is no cure for AD, but several therapeutic strategies have been directed to prevent neuroinflammation. Anti-inflammatory, but also anti-amyloidogenic compounds, such as flavonoids were shown to favourably modulate some pathological events associated with neurodegeneration. CONCLUSION: This review focuses on the role of cytokines and chemokines in AD-associated pathologies, and summarizes the potential anti-inflammatory therapeutic approaches aimed at preventing or slowing down disease progression.

Screening Younger Individuals in a Primary Care Setting Flags Putative Dementia Cases and Correlates Gastrointestinal Diseases with Poor Cognitive Performance.

BACKGROUND/AIMS: Diagnosing dementia is challenging in many primary care settings, given the limited human resources and the lack of current diagnostic tools. With this in mind, a primary care-based cohort was established in the Aveiro district of Portugal. METHODS: A total of 568 participants were evaluated using cognitive tests and APOE genotyping. RESULTS: The findings revealed a dementia prevalence of 12%. A strong correlation between increasing Clinical Dementia Rating (CDR) scores and education was clearly evident. Other highly relevant risk factors were activities of daily living (ADL), instrumental ADL, aging, depression, gender, the APOE ε4 allele, and comorbidities (depression as well as gastrointestinal, osteoarticular, and neurodegenerative diseases). A hitherto unreported, significant correlation between gastrointestinal disease and high CDR score was clearly observable. CONCLUSIONS: This study shows the merit of carrying out a dementia screening on younger subjects. Significantly, 71 subjects in the age group of 50-65 years were flagged for follow-up studies; furthermore, these cases with a potentially early onset of dementia were identified in a primary care setting.