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.

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

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.

Novel therapeutic strategies targeting mitochondria as a gateway in neurodegeneration.

In recent years, multiple disciplines have focused on mitochondrial biology and contributed to understanding its relevance towards adult-onset neurodegenerative disorders. These are complex dynamic organelles that have a variety of functions in ensuring cellular health and homeostasis. The plethora of mitochondrial functionalities confers them an intrinsic susceptibility to internal and external stressors (such as mutation accumulation or environmental toxins), particularly so in long-lived postmitotic cells such as neurons. Thus, it is reasonable to postulate an involvement of mitochondria in aging-associated neurological disorders, notably neurodegenerative pathologies including Alzheimer's disease and Parkinson's disease. On the other hand, biological effects resulting from neurodegeneration can in turn affect mitochondrial health and function, promoting a feedback loop further contributing to the progression of neuronal dysfunction and cellular death. This review examines state-of-the-art knowledge, focus on current research exploring mitochondrial health as a contributing factor to neuroregeneration, and the development of therapeutic approaches aimed at restoring mitochondrial homeostasis in a pathological setting.

Mitochondria dysfunction and impaired response to oxidative stress promotes proteostasis disruption in aged human cells.

The plastic architecture of the mitochondrial network and its dynamic structure play crucial roles ensuring that varying energetic demands are rapidly met. Given the brain's high energy demand, mitochondria play a particularly critical role in neuronal and axonal energy homeostasis. With ageing physiological properties of the organism deteriorate, and are associated with loss of cellular homeostasis, accumulation of dysfunctional organelles and damaged macromolecules. Thus, mitochondrial loss of efficiency is likely to be both a cause and a consequence of ageing. Additionally distinct cellular events can contribute to oxidative stress, disruption of metabolism and mitochondria homeostasis, resulting in neuropathology. However, although the correlation between ageing and mitochondria disfunction is well established, the response to oxidative stress, particularly proteostasis, remains to be fully elucidated. The work here described explores the degradation of mitochondria oxidative stress-response mechanisms with ageing in human cells, addressing the physiological effects on proteostasis, focused on its role in differentiating between healthy and pathological ageing. Increased protein aggregation appears to be tightly related to impairment of ageing mitochondria response to oxidative stress, and antioxidative agents are shown to have a progressive protective effect with age; cells from old individuals show higher susceptibility to oxidative stress, in terms of protein aggregation, cell viability, or mitochondria homeostasis. These results support the antioxidant properties of flavonoids as a good therapeutic strategy for age-related diseases. Given their protective effect, this family of compounds can be of strategic therapeutic value for protein-aggregation related diseases.

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.

Nuclear Envelope Alterations in Myotonic Dystrophy Type 1 Patient-Derived Fibroblasts.

Myotonic dystrophy type 1 (DM1) is a hereditary and multisystemic disease characterized by myotonia, progressive distal muscle weakness and atrophy. The molecular mechanisms underlying this disease are still poorly characterized, although there are some hypotheses that envisage to explain the multisystemic features observed in DM1. An emergent hypothesis is that nuclear envelope (NE) dysfunction may contribute to muscular dystrophies, particularly to DM1. Therefore, the main objective of the present study was to evaluate the nuclear profile of DM1 patient-derived and control fibroblasts and to determine the protein levels and subcellular distribution of relevant NE proteins in these cell lines. Our results demonstrated that DM1 patient-derived fibroblasts exhibited altered intracellular protein levels of lamin A/C, LAP1, SUN1, nesprin-1 and nesprin-2 when compared with the control fibroblasts. In addition, the results showed an altered location of these NE proteins accompanied by the presence of nuclear deformations (blebs, lobes and/or invaginations) and an increased number of nuclear inclusions. Regarding the nuclear profile, DM1 patient-derived fibroblasts had a larger nuclear area and a higher number of deformed nuclei and micronuclei than control-derived fibroblasts. These results reinforce the evidence that NE dysfunction is a highly relevant pathological characteristic observed in DM1.

Revisiting APP secretases: an overview on the holistic effects of retinoic acid receptor stimulation in APP processing.

Alzheimer's disease (AD) is the leading cause of dementia worldwide and is characterized by the accumulation of the ß-amyloid peptide (Aß) in the brain, along with profound alterations in phosphorylation-related events and regulatory pathways. The production of the neurotoxic Aß peptide via amyloid precursor protein (APP) proteolysis is a crucial step in AD development. APP is highly expressed in the brain and is complexly metabolized by a series of sequential secretases, commonly denoted the α-, ß-, and γ-cleavages. The toxicity of resulting fragments is a direct consequence of the first cleaving event. ß-secretase (BACE1) induces amyloidogenic cleavages, while α-secretases (ADAM10 and ADAM17) result in less pathological peptides. Hence this first cleavage event is a prime therapeutic target for preventing or reverting initial biochemical events involved in AD. The subsequent cleavage by γ-secretase has a reduced impact on Aß formation but affects the peptides' aggregating capacity. An array of therapeutic strategies are being explored, among them targeting Retinoic Acid (RA) signalling, which has long been associated with neuronal health. Additionally, several studies have described altered RA levels in AD patients, reinforcing RA Receptor (RAR) signalling as a promising therapeutic strategy. In this review we provide a holistic approach focussing on the effects of isoform-specific RAR modulation with respect to APP secretases and discuss its advantages and drawbacks in subcellular AD related events.

The role of the integral type II transmembrane protein BRI2 in health and disease.

BRI2 is a type II transmembrane protein ubiquitously expressed whose physiological function remains poorly understood. Although several recent important advances have substantially impacted on our understanding of BRI2 biology and function, providing valuable information for further studies on BRI2. These findings have contributed to a better understanding of BRI2 biology and the underlying signaling pathways involved. In turn, these might provide novel insights with respect to neurodegeneration processes inherent to BRI2-related pathologies, namely Familial British and Danish dementias, Alzheimer's disease, ITM2B-related retinal dystrophy, and multiple sclerosis. In this review, we provided a state-of-the-art outline of BRI2 biology, both in physiological and pathological conditions, and discuss the proposed molecular underlying mechanisms. Overall, the BRI2 knowledge here reviewed is of extreme importance and may contribute to propose BRI2 and/or BRI2 proteolytic fragments as novel therapeutic targets for neurodegenerative diseases, such as Alzheimer's disease.

Fourier-Transform Infrared Spectroscopy as a Discriminatory Tool for Myotonic Dystrophy Type 1 Metabolism: A Pilot Study.

Myotonic dystrophy type 1 (DM1) is a hereditary disease characterized by progressive distal muscle weakness and myotonia. Patients with DM1 have abnormal lipid metabolism and a high propensity to develop a metabolic syndrome in comparison to the general population. It follows that metabolome evaluation in these patients is crucial and may contribute to a better characterization and discrimination between DM1 disease phenotypes and severities. Several experimental approaches are possible to carry out such an analysis; among them is Fourier-transform infrared spectroscopy (FTIR) which evaluates metabolic profiles by categorizing samples through their biochemical composition. In this study, FTIR spectra were acquired and analyzed using multivariate analysis (Principal Component Analysis) using skin DM1 patient-derived fibroblasts and controls. The results obtained showed a clear discrimination between both DM1-derived fibroblasts with different CTG repeat length and with the age of disease onset; this was evident given the distinct metabolic profiles obtained for the two groups. Discrimination could be attributed mainly to the altered lipid metabolism and proteins in the 1800-1500 cm-1 region. These results suggest that FTIR spectroscopy is a valuable tool to discriminate both DM1-derived fibroblasts with different CTG length and age of onset and to study the metabolomic profile of patients with DM1.

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.

APP Binds to the EGFR Ligands HB-EGF and EGF, Acting Synergistically with EGF to Promote ERK Signaling and Neuritogenesis.

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well-described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders, including AD. Pro-HB-EGF arose as a putative novel APP interactor in a human brain cDNA library yeast two-hybrid screen. Based on their structural and functional similarities, we first aimed to verify if APP could bind to (HB-)EGF proforms. Here, we show that APP interacts with these two EGFR ligands, and further characterized the effects of APP-EGF interaction in ERK activation and neuritogenesis. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were observed to synergistically activate the ERK pathway, crucial for neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in APP overexpression and EGF exposure conditions confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying APP/EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP/EGF-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration.

The impact of the early phase of the COVID-19 pandemic on mental-health services in Europe.

PURPOSE: The current COVID-19 pandemic confronts psychiatric patients and mental health services with unique and severe challenges. METHODS: In order to identify these trans-national challenges across Europe, an ad-hoc survey was conducted among 23 experts, each answering for one European or aligned country. RESULTS: A number of important themes and issues were raised for the impact of COVID-19 on mental health and mental health services, barriers to service provision and future consequences. A number of key issues were reported by colleagues across several jurisdictions, even though these were at different stages of their national epidemics. CONCLUSIONS: Based on these findings, we articulate some important learnings from the early stages of the COVID-19 European pandemic, and highlight key considerations for all countries' mental health services as the current pandemic develops and for future pandemics.

IL-8 and MCP-1 Impact on Tau Phosphorylation and Phosphatase Activity.

BACKGROUND: Chronic inflammation is a feature of Alzheimer´s disease (AD), resulting in excessive production of inflammatory mediators that can lead to neuroinflammation, contributing to alterations in Aß production and deposition as Senile Plaques (SPs), and to neurofibrillary tangles (NFTs) formation, due to hyperphosphorylated Tau protein. OBJECTIVE: This work addressed the impact of the interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), two chemokines, on Tau phosphorylation; and also evaluated the chemokines' levels in plasma using samples from a regional cohort. METHODS: Human neuronal SH-SY5Y cells exposed to IL-8 and MCP-1 chemokines were monitored for their protein and phosphorylated protein levels by western blotting analysis. A serine/threonine protein phosphatase (PPs) activity assay was employed to monitor PPs activity. Subsequently, flow cytometry was used to monitor chemokines levels in plasma samples from individuals with cognitive deficits. RESULTS: Chemokines' exposure resulted only in minor cytotoxicity effects on SH-SY5Y, and in increased Tau phosphorylation, particularly at the S396 residue. Tau phosphorylation correlated with PPs inhibition and was consistent with GSK3ß phosphorylation-mediated inhibition. Subsequent analysis of plasma from individuals with cognitive deficits showed that IL-8 levels were decreased. CONCLUSION: Data shows that both chemokines tested can exert an effect on GSK3ß phosphorylation and modulate PPs activity, potentially resulting in increased Tau phosphorylation and subsequent NFTs formation. One can deduce that increased chemokines stimulation during chronic inflammation can exacerbate this event. The work contributes to a better understanding of the mode of action of these chemokines on AD pathogenesis and opens novel research avenues.

Nuclear Accumulation of LAP1:TRF2 Complex during DNA Damage Response Uncovers a Novel Role for LAP1.

Lamina-associated polypeptide 1 (LAP1) is a nuclear envelope (NE) protein whose function remains poorly characterized. In a recent LAP1 protein interactome study, a putative regulatory role in the DNA damage response (DDR) has emerged and telomeric repeat-binding factor 2 (TRF2), a protein intimately associated with this signaling pathway, was among the list of LAP1 interactors. To gain insights into LAP1's physiological properties, the interaction with TRF2 in human cells exposed to DNA-damaging agents was investigated. The direct LAP1:TRF2 binding was validated in vitro by blot overlay and in vivo by co-immunoprecipitation after hydrogen peroxide and bleomycin treatments. The regulation of this protein interaction by LAP1 phosphorylation was demonstrated by co-immunoprecipitation and mass spectrometry following okadaic acid exposure. The involvement of LAP1 and TRF2 in the DDR was confirmed by their increased nuclear protein levels after bleomycin treatment, evaluated by immunoblotting, as well as by their co-localization with DDR factors at the NE and within the nucleoplasm, assessed by immunocytochemistry. Effectively, we showed that the LAP1:TRF2 complex is established during a cellular response against DNA damage. This work proposes a novel functional role for LAP1 in the DDR, revealing a potential biological mechanism that may be disrupted in LAP1-associated pathologies.