搜索结果：Methods in cell biology

Yes-associated protein 1 (YAP1) plays essential roles in somatic cell proliferation and differentiation and is regulated by cell cycle proteins and mechanical cues transduced by the actin cytoskeleton. YAP1 has been localized to the mitotic spindle. Whether YAP1 may participate in oocyte maturation is not known. Determine effect of YAP inhibitor on oocyte maturation and the potential involvement of cell cycle regulators and actin polymerization modulators on YAP localization and phosphorylation status during oocyte maturation. Mouse oocytes were cultured in vitro alone or in the presence of various small molecule inhibitors blocking YAP1, cyclin dependent kinase 1 (CDK1), phosphatase protein 2 A (PP2A), actin polymerization and the resulting effects on maturation, YAP localization by immunocytochemistry and/or YAP phosphorylation by western blot were determined. Blocking YAP1 activity with verteporfin hampers oocyte maturation, arresting the majority of oocytes at metaphase I (MI), with a distorted spindle structure. Moreover, YAP1 localized to the meiotic spindle and is phosphorylated on T119 beginning at metaphase I, which is regulated in part by PP2A phosphatase but is not strongly affected by CDK1 inhibitors (RO-3306). The increase in YAP1 phosphorylated on T119 (pYAP1) at MI mirrors the rise in CDK1 activity, suggesting an association between CDK1, pYAP1-T119 and PP2A. The results link pYAP1-T119 with cell cycle regulators CDK1, PP2A, and the meiotic spindle, but not with changes in actin polymerization. These findings suggest a previously unknown role of YAP1 in regulating meiotic spindle shape during oocyte maturation. Modulation of YAP1 activity during oocyte maturation may allow for better in vitro maturation procedures to be developed.

Irisin, a myokine released during physical activity, has been proposed as a mediator of exercise's protective effects against breast cancer (BC). This review underscores the critical role of irisin in mediating the anticancer effects of exercise and its potential application in BC prevention and prognosis. Studies published up to 2025 were identified in PubMed, Scopus, and Web of Science databases. Data from experimental models, clinical trials, and observational studies were analyzed with emphasis on exercise-induced irisin secretion and its effects on cancer-related pathways. Irisin, derived from the precursor FNDC5 upon PGC-1α activation in skeletal muscle, regulates cancer-associated pathways by activating AMP-activated protein kinase (AMPK), inhibiting mammalian target-of-rapamycin (mTOR), modulating phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor kappa B (NF-κB) signaling, and influencing transforming growth factor beta (TGF-β) activity. These actions reduce chronic inflammation, tumor proliferation, angiogenesis, and epithelial-mesenchymal transition, while enhancing apoptosis and metabolic balance. Preclinical studies demonstrate irisin's capacity to limit BC cell viability, migration, and metastasis. Clinically, higher circulating irisin levels correlate with reduced tumor aggressiveness, fewer metastases, and better survival, though tumor may overexpress irisin as a local adaptive response. Regular moderate physical activity appears most effective in stimulating irisin secretion, although optimal exercise parameters remain to be determined. Irisin exerts multifaceted anticancer effects and holds promise as a biomarker and therapeutic target in BC. Its role as a mediator of exercise benefits supports the inclusion of regular moderate physical activity in BC prevention and prognosis strategies. Further research is needed to define clinical applications and optimal exercise regimens for maximizing irisin potential.

Albumin is abundant in human plasma and has been widely studied in cancer mainly in the context of systemic nutrition or the tumor microenvironment; however, the clinicopathologic significance and intracellular role of tumor-cell albumin in gastric adenocarcinoma remain unclear. We analyzed 187 patients who underwent gastrectomy for gastric adenocarcinoma between 2000 and 2010. Albumin expression was evaluated by immunohistochemistry on tissue microarrays and classified as high versus low based on intensity relative to intra-tumoral stromal cells. Associations with clinicopathologic variables were examined, and disease-free survival (DFS) and disease-specific survival (DSS) were assessed using Kaplan-Meier and Cox regression. Albumin mRNA/protein expression was examined in three metastatic gastric cancer cell lines, and functional assays (wound healing and proliferation) were performed after siRNA-mediated albumin knockdown in Hs746T cells. High albumin expression was significantly associated with larger tumor size and advanced T and N stages. Albumin expression was not significantly associated with DFS or DSS in univariate or multivariate analyses, whereas T stage and N stage remained independent prognostic factors. In vitro, albumin knockdown significantly impaired migration and reduced proliferative capacity, despite limited detectable reduction in protein levels. Tumor-cell albumin correlates with gastric cancer progression and functionally contributes to motility and growth at the intracellular level, supporting its role as a marker of aggressive tumor biology rather than an independent prognostic biomarker.

Methylene blue is a versatile dye used in cell biology to quantify cell adhesion and indirectly assess cell death. Unlike viability assays that rely on metabolic activity or membrane integrity, methylene blue binds to cellular proteins, making it particularly effective for staining adherent cells. This property is the basis of the assay we describe here, which allows direct measurement of cell detachment, a hallmark of various cell death processes, including apoptosis and anoikis. Using a simple staining and washing procedure, methylene blue selectively labels cells that remain attached to the culture surface after detached or dying cells have been removed. After washing, the retained dye can then be eluted and quantified spectrophotometrically, providing a reliable and cost-effective method for evaluating cytotoxicity, drug responses, and adhesion dynamics. This chapter outlines the principles, advantages, and practical applications of methylene blue staining in cell-based assays, highlighting its role as a powerful tool for studying cell survival, or detachment in both basic and applied research.

Various mouse models are used to study pollen allergies, but a systematic experimental comparisvon is lacking. We aimed to establish a physiologically relevant adjuvant-free birch pollen allergy model to understand the dynamics of the allergic immune response and to compare the sensitising potential of different self-collected birch pollen extracts in different distinct in vivo and in vitro routes and models. Different intranasal (i. n.) models in BALB/c mice and IL-4 reporter mice (BALB/c:4Get), an intradermal (i. d.) model (BALB/c:4Get), and human dendritic cells (moDCs) were used to investigate the sensitising and inflammatory effects of birch pollen extracts (BPE). A timeseries experiment was performed in the i. n. model to determine the onset of Th2 responses. Bronchioalveolar lavage fluid (BALF), lungs, draining lymph nodes, and serum were analysed for cell infiltrate, cytokines, and antibodies. Repeated i. n. instillations of adjuvant-free BPE prepared from commercial pollen resulted in BALF Th2 cells, eosinophilic lung inflammation, and specific serum immunoglobulins in BALB/c mice. After 6 i. n. instillations, eosinophils and CD4+ T cells peaked in BALF, and CD4+IL-4+ and CD4+IL4Rα+ cells peaked in mediastinal lymph nodes. Both, i. n. and i. d. models detected subtle differences in the sensitising potential of BPEs from two self-collected pollen samples. MoDCs showed higher IL-10 release towards the less inflammatory extract. Adjuvant-free murine sensitisation models, including intranasal and intradermal routes, as well as a human DC model, covered different aspects of the sensitisation route and temporal resolution. The models may be broadly helpful in screening approaches in studying mechanisms of pollen allergy.

Here we detail a method for the exploration of patient-derived xenografts (PDX) based on organoids (PDX-O), using antibodies validated for immunohistochemistry. Importantly this approach can be used to characterize the response of malignant cells to antineoplastic treatments. Cancer progression and metastasis reflect tumor heterogeneity and plasticity. Here we analyzed cells in PDX grown as spheroids or tumoroids embedded in collagen/basement membrane extract (BME) matrix. The use of 3D cellular models instead of 2D allows the inclusion in the study of non-adherent tumors. Even if the cells are adherent the spheroid and tumoroids conformation or organization in space and cellular polarity differ for 2D. This difference can change the response to treatment. Adding a matrix component is an important addition to the tumoral microenvironment that can change also the cellular response to treatment. We combined this tridimensional model with a multiplexed staining protocol to analyze tumor cell differentiation and responses to drugs. We analyzed paraffin embedded organoid sections, allowing to analyze the core of cell aggregates, and analyzed cell plasticity, differentiation and proliferation with and without treatment with palbociclib. Spheroids included an outer layer of luminal cytokeratin (CK) 8-positive cells surrounding a mixed population of cells expressing both CK5 and CK8. Palbociclib treatment resulted in a proliferation arrest, linked to dedifferentiation of the cancer cells that switched to a mostly basal phenotype, supporting a hybrid CK5/CK8 phenotype. In summary, this method offers a simple and versatile strategy to analyze tumor cell responses to drugs, using routine antibodies validated for immunohistochemistry and multiplex analysis.

Apoptosis and other regulated cell death pathways display complex, dynamic, and heterogeneous behaviors that are frequently masked by conventional endpoint assays relying on bulk measurements. Here, we describe a live-cell imaging workflow for temporally resolved, single-cell quantification of both apoptotic and non-apoptotic cell death in cell cultures. In colorectal carcinoma cell lines, cell death was triggered by agents including Staurosporine, RSL3, and Cisplatin, and continuously tracked using a live-cell imaging platform with fluorescent markers such as Annexin V-FITC, Caspase-3/7 activity dye, and propidium iodide. Parameters of cell death were monitored for 24-48 h, allowing precise discrimination between early and late apoptotic events as well as necrosis. This methodology enables reproducible, quantitative profiling of cell death kinetics at single-cell resolution, reduces artifacts from manual handling, and offers valuable insights into the temporal dynamics and interplay of different cell death modalities.

Rare diseases, characterized by their low prevalence, cumulatively affect millions of people around the world and place significant burden on the healthcare system. With limited clinical expertise and infrastructure in this field, patients encounter barriers in obtaining an accurate diagnosis and accessing treatment. Rare diseases are commonly attributable to genetic alterations; thus, we can optimize modern genetic technologies to pinpoint pertinent genes and molecular pathways involved in disease phenotypes. In this article, we discuss rare diseases in context of multi-omics, an integrative approach combining data from various sources, including genomics, transcriptomics, and epigenomics. Advancements in multi-omics have facilitated the collection of more high-dimensional data, particularly useful for rare diseases comprising limited sample sizes. Artificial intelligence (AI) and machine learning (ML) are powerful tools for extracting disease-relevant patterns from complex datasets and unraveling causative markers underlying disease. Together, these tools are invaluable for incorporating precision medicine in rare diseases through guiding therapeutic strategies aimed at modifying the structure and functionality of specific genes to address the root cause of disease. Specifically, we curate a list of twenty-three rare diseases, prioritized by the medical community based on unmet medical needs and prevalence. To illustrate the current landscape of precision medicine for these diseases, we summarize advancements in genomic sequencing and computational methods for their diagnosis, and utilization of gene-editing technologies for personalized treatment. Overall, the various bioinformatic strategies discussed in this paper help formulate an end-to-end workflow of the integration of gene testing, multi-omics, and AI/ML to guide effective rare disease management.

We examined gene expression profiles in abdominal aortic aneurysm (AAA) lesions vs. normal aortas by cDNA microarray and real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Phosphorus (32P)-labeled cDNA from AAA specimens (mean AAA size 6.65 cm) and normal aortas were hybridized with a 588-gene microarray primarily of the cardiovascular system. The results were validated by qRT-PCR. A total of 35 out of the 588 genes were differentially expressed, with either log2 ratio of AAAs/controls ≥1 (upregulated; 20 genes) or ≤-1 (downregulated; 15 genes) in AAA lesions vs. normal aorta, and 25 of these were significantly different (71%). Expression of matrix metalloproteinase 9, TIMP metallopeptidase inhibitor 3, collagen type I α 1 chain (COL1A1), COL6A3, COL15A1, intercellular adhesion molecule 1 (ICAM1), ICAM2, decorin, endoglin, apolipoprotein D (APOD), APOE, phospholipid transfer protein, calcium and integrin binding 1 (CIB1), phospholipase A2 group IIA, von Willebrand factor, serpin family B member 6 (SERPINB6), urokinase-type plasminogen activator, H19, C-C motif chemokine ligand 2, and platelet-derived growth factor receptor beta was upregulated in AAA vs. normal aorta. Expression of collagen type IV α 4 chain (COL4A4), COL11A2, gap junction protein α 1 (GJA1), biglycan, integrin subunit α 8, galectin-1, low-density lipoprotein receptor-related protein 1, acetyl-CoA acyltransferase, serpin family E member 1, melanoma cellular adhesion molecule, sodium channel epithelial 1 subunit beta (SCNN1B), natriuretic peptide receptor 1 (NPR1), superoxide dismutase 3, actinin α 1 (ACTN1) and cardiac phospholamban (PLN) was downregulated. Eleven genes differentially expressed (p≤0.05) in AAA lesions vs. normal aortas were not reported previously: upregulated: COL6A3, COL15A1, ICAM2, APOD, CIB and SERPINB6; downregulated: GJA1, SCNN1B, NPR1, ACTN1 and PLN. Remaining results confirmed previous reports regarding 21 genes differentially expressed in AAA. qRT-PCR results were in general in agreement with microarray results.

To maintain homeostasis, cells undergo a tightly regulated process called programmed cell death. Some forms of programmed cell death, such as pyroptosis, can elicit a strong inflammatory response by releasing cytokines through small protein pores. The terminal event of pyroptosis in most cells is plasma membrane rupture (PMR), which breaks down large sections of the plasma membrane and emits intracellular contents that can further amplify the inflammatory signal. In opposition to the previous dogma that PMR is a passive event, it was recently discovered that the transmembrane protein, Ninjurin-1 (NINJ1), is the key executor of PMR. Two models of NINJ1-mediated PMR predict that NINJ1 oligomerizes into filaments or ring-like structures to either open large pores or to excise sections of the membrane. Both models underpin how NINJ1 must oligomerize to execute PMR. When at rest, NINJ1 will autoinhibit oligomerization and activation by forming face-to-face dimer-dimer structures on the plasma membrane. Follow-up studies have shown that NINJ1 executes PMR for other forms of programmed cell death, including apoptosis, ferroptosis, and PANoptosis, as well as mechanical cell death. Thus, assessing NINJ1 function directly through quantification of NINJ1 oligomerization is important to expanding our understanding of both programmed and mechanistic cell death. Here, we describe methods to visualize and quantify NINJ1 oligomerization via immunofluorescence imaging of NINJ1 puncta. This protocol enables more precise and accurate measurement of NINJ1 function during PMR, surpassing conventional methods that just quantify PMR by-products.

Apoptosis, a tightly regulated form of programmed cell death, eliminates damaged or malignant cells and is triggered by internal or external stress signals. A critical decision point is mitochondrial outer membrane permeabilization (MOMP), governed by BCL-2 family proteins. Pro-apoptotic members such as BAX and BAK form pores in the mitochondrial outer membrane, releasing intermembrane space proteins like cytochrome c into the cytoplasm. Once cytosolic, cytochrome c binds APAF-1 to form the apoptosome, which activates caspase-9 and subsequently caspase-3, driving apoptosis through cleavage of key cellular substrates. Cytochrome c release serves as a hallmark and point of no return in the apoptotic cascade. However, cytochrome c release can be variable, occurring at submaximal levels or from only a subset of mitochondria, which complicates detection in heterogeneous cell populations. To address this, we developed a semi-automated imaging-based method to quantify cytochrome c release at the single-cell level using immunofluorescence microscopy. Our approach uses CellProfiler, an open-source image analysis platform, to implement a pipeline that segments adherent cells into nuclear, mitochondrial, and cytoplasmic compartments based on compartment-specific reference stains. The pipeline quantifies cytochrome c distribution across these compartments, calculating the ratio of mitochondrially retained to cytoplasmic cytochrome c for each cell. Automation of segmentation and measurement ensures rapid, robust, and reproducible analysis, with only image acquisition and data interpretation performed manually. This method provides a quantitative readout of MOMP and can be readily adapted to any immunofluorescence-detectable protein given an appropriate compartmental marker, expanding its utility for broader cellular studies.

Intentional destruction of ultrasound microbubble (MB) contrast agents by induced cavitation is a well-established technique in contrast imaging applications and may translate to pulmonary imaging. However, the safety of exposing human bronchial epithelial (hBE) cells to an inhalable contrast agent with intentional MB destruction is not known. We conducted an in vitro evaluation by exposing hBE cell cultures to a mucus-targeting, cationic MB contrast agent. A total of 12 hBE cell cultures, from 3 healthy donors (4 cultures each), were exposed to 4 experimental conditions: (i) No-contrast imaging with no MBs, using cadence contrast pulse sequencing followed by a MB destruction pulse exposure (mechanical index [MI] ramped from 0.14 to 1.90); (ii) contrast imaging using MBs with contrast pulse sequencing at low MI (MI maintained at 0.14); (iii) contrast imaging with intentional MB destruction by MB destruction pulse exposure; and (iv) Triton-X (positive control, inducing cellular death). Cell culture viability was evaluated pre- and 24 hours post-exposure by quantifying percent ciliated area and ciliary beat frequency. The contrast signal was visible for all cultures administered MBs, with signal loss only apparent after the MB destruction pulse. Culture viability (percent ciliated area) was comparable at pre- and post-exposure, for all imaging conditions (coefficient of variation range, 3%-8%), with no decline in ciliary beat frequency. No ciliated area persisted after exposure to Triton-X. Intentional MB destruction induced by a high MI does not induce cellular death to the respiratory epithelium. These findings support further development of an inhaled MB contrast agent for pulmonary imaging applications.

Coronavirus Disease 19 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), stimulated intensive drug-development efforts throughout the world. As COVID-19 has become endemic in many regions of the world, phytochemicals used in ethnomedicine may have utility in the prevention or treatment of COVID-19. Here, we employed machine learning/deep learning drug discovery tools to evaluate compounds previously identified in roots of plants of the Paeonia genus for their drug-like properties and potential to inhibit 3CLpro, the main protease of SARS-CoV2. Our results identify Paeonia-derived compounds that have favorable drug-like properties and are predicted to have a high binding affinity for 3CLpro. Molecular dynamics simulations supported the binding affinity of Paeonia-derived compounds for 3CLpro. We validated these in silico computational results by experimentally determining the 50% inhibitory concentration (IC50) of two Paeonia-derived compounds, paeoniflorigenone and 3-O-methylquercetin, using purified 3CLpro with baicalein as a control. The IC50 values for paeoniflorigenone, 3-O-methylquercetin and baicalein were 9.33 µM, 43.94 and 43.85 µM, respectively. Root extracts from five Paeonia species were found to have minimal cytotoxicity against three different human cell types. Our results represent a proof-of-concept study demonstrating that in silico techniques, including machine and deep learning methods, can be used to identify phytochemicals as starting points for the discovery of antiviral compounds against SARS-CoV-2. The online version contains supplementary material available at10.1007/s40203-026-00630-7.

Lipid droplets (LDs) are dynamic organelles that not only store energy and regulate metabolism but also serve as key modulators of cell death signaling and disease progression. LDs influence cell fate by buffering lipid peroxidation or releasing fatty acids during ferroptosis, modulating apoptotic protein expression, and facilitating autophagic degradation. Their multifunctional roles are context-dependent and span multiple cell death pathways. In disease, aberrant LD accumulation is closely linked to metabolic disorders, neurodegeneration, cancer therapy resistance, and pathogen infection. Alterations in LD morphology and abundance have emerged as diagnostic indicators. Thus, precise detection and efficient isolation of LDs are critical for elucidating disease mechanisms, advancing targeted therapies, and translating LD biology into clinical applications. This chapter outlines key methodologies-including lipid staining, fluorescent probes, high-content microscopy, density gradient centrifugation, and immunoaffinity purification-for evaluating lipid droplet function and achieving their isolation in the context of cell death.

Three-dimensional (3D) models more closely represent the in vivo situation and are therefore more relevant models for drug screening. Among the more than twelve regulated cell death (RCD) modalities, immunogenic cell death (ICD) stands out for its ability to initiate efficient anti-tumor immune response. For example, ferroptosis, which can be induced via the inhibition of GPX4 leading to uncontrolled lipid peroxidation, might be immunogenic under certain conditions. It is crucial to identify which cell death modality is induced, as certain cancer types exhibit resistance to specific forms of cell death. However, a major limitation of 3D models is the lack of high-throughput assays, which often require dissociation of the 3D models, potentially leading to misinterpretation of results. Here, we describe a protocol for identifying and quantifying the induction of RCD modalities in 3D models, such as spheroids. This method eliminates the need for tumor spheroid dissociation and is compatible with other screening techniques, including confocal microscopy. This protocol enables high-throughput screening of various cell death inducers in intact 3D models, serving as a crucial first step in the identification of novel inducers and their specificity for particular ICD and RCD types.

Knowledge of the copy numbers of transcripts and genomic loci in their natural spatial contexts plays an important role in our understanding of biology and medicine. Fluorescent hybridization probes have emerged as a powerful tool to profile transcripts and genomic loci in single cells in situ. However, the number of different nucleic acid species that can be quantified by fluorescence imaging-based methods is limited. Here, we report a highly multiplexed in situ hybridization approach for spatial transcriptomics and genomics analysis. In this approach, each nucleic acid molecule is visualized as a fluorescent spot at its natural cellular context throughout the consecutive cycles of fluorescence in situ hybridization. In each analysis cycle, fluorescent oligonucleotide probes stain the nucleic acid targets by hybridizing to the probes applied in the previous cycle. And these probes also introduce the binding sites for the next cycle probes. Through reiterative cycles of hybridization, imaging, and photobleaching, unique color sequences are generated as barcodes for varied nucleic acids. With multi-color staining and reiterative cycles, tens of thousands of different transcripts or genomic loci could be precisely profiled in individual cells in situ.

The inflammasome is a multiprotein cytosolic signalling platform that initiates inflammatory responses upon detection of microbial and danger-associated stimuli. Inflammasome assembly occurs due to the involvement of specific cytosolic pattern recognition receptors (PRRs) in response to pathogens and danger signals in host cells. This causes activation of inflammatory caspases that results in cytokine release and pyroptosis. A central player in inflammasome formation is the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD), which bridges activated PRRs to pro-Caspase-1, leading to the formation of the inflammasome complex. This chapter provides detailed protocols for analysing inflammasome assembly by monitoring ASC speck formation using confocal microscopy and biochemically by detecting ASC oligomerisation using DSS-mediated crosslinking. Together, these methods offer complementary insights into spatial organisation and activity of inflammasomes in response to pathogens or cellular damage.

Alkaliptosis is a pH-dependent form of regulated cell death induced by the small molecule JTC801, characterized by disruption of lysosomal and cytoplasmic pH homeostasis. Unlike classical forms of cell death such as apoptosis, necroptosis, or ferroptosis, alkaliptosis is driven by dysregulation of intracellular pH-regulatory proteins, leading to aberrant alkalinization and metabolic dysfunction. This mechanism is particularly effective against apoptosis-resistant cancers, including pancreatic ductal adenocarcinoma, and represents a promising strategy for overcoming therapeutic resistance. Monitoring alkaliptosis remains challenging, as it is not detected by conventional cell death markers such as caspases or pore-forming proteins. This chapter introduces methodologies for assessing alkaliptosis, including dynamic pH measurement, analysis of alkaliptosis-related protein expression, and evaluation of lysosomal function. Improved detection strategies will advance the understanding of non-canonical cell death pathways and support the development of novel therapeutic interventions targeting alkaliptosis.

Induced pluripotent stem cells (iPSCs) represent an innovative tool to model neurodegenerative disorders, providing access to disease-relevant cell types such as neurons that are otherwise inaccessible. In the context of Alzheimer's disease (AD), iPSC-derived neural cultures offer a unique opportunity to investigate pathological mechanisms in a controlled environment, overcoming limitations of animal models. Central to AD pathogenesis is the amyloid cascade hypothesis, which is based on the concept that accumulation and aggregation of the toxic oligomeric species, initiates a cascade of events leading to synaptic dysfunction, neuronal loss, and cognitive decline. It is known that application of the Aβ oligomers to neurons reproduces key features of synaptic impairment, preceding overt neuronal death. In this study, we proposed an optimized protocol employing iPSC-derived neurons exposed to Aβ1-42 peptide. This approach provides a clear and reliable method to evaluate neurotoxic effects of Aβ peptide on neuronal morphology and viability. Indeed, on the one hand neuronal morphology, assessed through immunofluorescence using specific neuronal markers, allows precise monitoring of neurite length and synaptic connectivity, crucial parameters to evaluate neuronal health. On the other hand, cytotoxicity is directly quantified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, which confirm Aβ-induced neuronal injury. Notably, this combined approach provides novel insights into early Aβ-driven neurodegenerative processes and offers a platform to identify therapeutic strategies targeting the initial phases of AD pathology.