ABSTRACT
Alzheimer's disease (AD) is a progressive neurological condition characterized by impaired cognitive function and behavioral alterations. While AD research historically centered around mis-folded proteins, advances in mass spectrometry techniques have triggered increased exploration of the AD lipidome with lipid dysregulation emerging as a critical player in AD pathogenesis. Gangliosides are a class of glycosphingolipids enriched within the central nervous system. Previous work has suggested a shift in a-series gangliosides from complex (GM1) to simple (GM2 and GM3) species may be related to the development of neurodegenerative disease. In addition, complex gangliosides with 20 carbon sphingosine chains have been shown to increase in the aging brain. In this study, we utilized matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to interrogate the in situ relationship of a-series gangliosides with either 18 or 20 carbon sphingosine chains (d18:1 or d20:1, respectively) in the post-mortem human AD brain. Here, we expanded upon previous literature and demonstrated a significant decrease in the GM1 d20:1 to GM1 d18:1 ratio in regions of the dentate gyrus and entorhinal cortex in AD relative to control brain tissue. Then, we demonstrated that the MALDI-MSI profile of GM3 co-localizes with histologically confirmed amyloid beta (Aß) plaques and found a significant increase in both GM1 and GM3 in proximity to Aß plaques. Collectively, this study demonstrates a perturbation of the ganglioside profile in AD, and validates a pipeline for MALDI-MSI and classic histological staining in the same tissue sections. This demonstrates feasibility for integrating untargeted mass spectrometry imaging approaches into a digital pathology framework.
Subject(s)
Alzheimer Disease , Gangliosides , Plaque, Amyloid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Humans , Alzheimer Disease/pathology , Alzheimer Disease/metabolism , Gangliosides/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Plaque, Amyloid/pathology , Plaque, Amyloid/metabolism , Aged , Aged, 80 and over , Brain/pathology , Brain/metabolism , Male , FemaleABSTRACT
OBJECTIVES: Neuropsychiatric symptoms (NPS) increase risk of developing dementia and are linked to various neurodegenerative conditions, including mild cognitive impairment (MCI due to Alzheimer's disease [AD]), cerebrovascular disease (CVD), and Parkinson's disease (PD). We explored the structural neural correlates of NPS cross-sectionally and longitudinally across various neurodegenerative diagnoses. METHODS: The study included individuals with MCI due to AD, (n = 74), CVD (n = 143), and PD (n = 137) at baseline, and at 2-years follow-up (MCI due to AD, n = 37, CVD n = 103, and PD n = 84). We assessed the severity of NPS using the Neuropsychiatric Inventory Questionnaire. For brain structure we included cortical thickness and subcortical volume of predefined regions of interest associated with corticolimbic and frontal-executive circuits. RESULTS: Cross-sectional analysis revealed significant negative correlations between appetite with both circuits in the MCI and CVD groups, while apathy was associated with these circuits in both the MCI and PD groups. Longitudinally, changes in apathy scores in the MCI group were negatively linked to the changes of the frontal-executive circuit. In the CVD group, changes in agitation and nighttime behavior were negatively associated with the corticolimbic and frontal-executive circuits, respectively. In the PD group, changes in disinhibition and apathy were positively associated with the corticolimbic and frontal-executive circuits, respectively. CONCLUSIONS: The observed correlations suggest that underlying pathological changes in the brain may contribute to alterations in neural activity associated with MBI. Notably, the difference between cross-sectional and longitudinal results indicates the necessity of conducting longitudinal studies for reproducible findings and drawing robust inferences.
Subject(s)
Alzheimer Disease , Cerebrovascular Disorders , Cognitive Dysfunction , Parkinson Disease , Humans , Cross-Sectional Studies , Parkinson Disease/psychology , Longitudinal Studies , Cognitive Dysfunction/psychology , Alzheimer Disease/psychology , Brain/diagnostic imaging , Brain/pathology , Cerebrovascular Disorders/complications , Neuropsychological TestsABSTRACT
INTRODUCTION: Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure. METHODS: We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls. RESULTS: Circulating amyloid beta (Aß), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235. DISCUSSION: This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools. HIGHLIGHTS: Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches.
ABSTRACT
INTRODUCTION: We investigated whether novel plasma biomarkers are associated with cognition, cognitive decline, and functional independence in activities of daily living across and within neurodegenerative diseases. METHODS: Glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), phosphorylated tau (p-tau)181 and amyloid beta (Aß)42/40 were measured using ultra-sensitive Simoa immunoassays in 44 healthy controls and 480 participants diagnosed with Alzheimer's disease/mild cognitive impairment (AD/MCI), Parkinson's disease (PD), frontotemporal dementia (FTD) spectrum disorders, or cerebrovascular disease (CVD). RESULTS: GFAP, NfL, and/or p-tau181 were elevated among all diseases compared to controls, and were broadly associated with worse baseline cognitive performance, greater cognitive decline, and/or lower functional independence. While GFAP, NfL, and p-tau181 were highly predictive across diseases, p-tau181 was more specific to the AD/MCI cohort. Sparse associations were found in the FTD and CVD cohorts and for Aß42/40 . DISCUSSION: GFAP, NfL, and p-tau181 are valuable predictors of cognition and function across common neurodegenerative diseases, and may be useful in specialized clinics and clinical trials.
Subject(s)
Alzheimer Disease , Cardiovascular Diseases , Cognitive Dysfunction , Frontotemporal Dementia , Neurodegenerative Diseases , Humans , Activities of Daily Living , Amyloid beta-Peptides , Ontario , Cognition , Biomarkers , tau ProteinsABSTRACT
Guilt is a negative emotion, elicited by realizing one has caused actual or perceived harm to another person. Anecdotally, guilt often is described as a visceral and physical experience. However, while the way that the body responds to and contributes to emotions is well known in basic emotions, little is known about the characteristics of guilt as generated by the autonomic nervous system. This study investigated the physiologic signature associated with guilt in adults with no history of psychological or autonomic disorder. Healthy adults completed a novel task, including an initial questionnaire about their habits and attitudes, followed by videos designed to elicit guilt, as well as the comparison emotions of amusement, disgust, sadness, pride, and neutral. During the video task, participants' swallowing rate, electrodermal activity, heart rate, respiration rate, and gastric activity rate were continuously recorded. Guilt was associated with alterations in gastric rhythms, electrodermal activity, and swallowing rate relative to some or all the comparison emotions. These findings suggest that there is a mixed pattern of sympathetic and parasympathetic activation during the experience of guilt. These results highlight potential therapeutic targets for modulation of guilt in neurologic and psychiatric disorders with deficient or elevated levels of guilt, such as frontotemporal dementia, posttraumatic stress disorder, and Obsessive-compulsive disorder.
Subject(s)
Obsessive-Compulsive Disorder , Stress Disorders, Post-Traumatic , Adult , Humans , Guilt , Emotions/physiology , PsychophysiologyABSTRACT
Pick's disease (PiD) is a rare form of frontal temporal lobar degeneration. The pathognomonic feature is atrophy of the frontotemporal lobes and intraneuronal deposits of 3R-τ inclusions, the Pick body. Corticobasal syndrome (CBS) is an atypical parkinsonian syndrome with a heterogeneous spectrum of underlying pathologies. We report a case of clinically diagnosed CBS with a post-mortem diagnosis of PiD and conduct a clinicopathological review of the literature on this unusual presentation.
Subject(s)
Corticobasal Degeneration , Pick Disease of the Brain , Humans , Pick Disease of the Brain/pathology , Atrophy , tau ProteinsABSTRACT
INTRODUCTION: Understanding synergies between neurodegenerative and cerebrovascular pathologies that modify dementia presentation represents an important knowledge gap. METHODS: This multi-site, longitudinal, observational cohort study recruited participants across prevalent neurodegenerative diseases and cerebrovascular disease and assessed participants comprehensively across modalities. We describe univariate and multivariate baseline features of the cohort and summarize recruitment, data collection, and curation processes. RESULTS: We enrolled 520 participants across five neurodegenerative and cerebrovascular diseases. Median age was 69 years, median Montreal Cognitive Assessment score was 25, median independence in activities of daily living was 100% for basic and 93% for instrumental activities. Spousal study partners predominated; participants were often male, White, and more educated. Milder disease stages predominated, yet cohorts reflect clinical presentation. DISCUSSION: Data will be shared with the global scientific community. Within-disease and disease-agnostic approaches are expected to identify markers of severity, progression, and therapy targets. Sampling characteristics also provide guidance for future study design.
Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Neurodegenerative Diseases , Humans , Male , Aged , Neurodegenerative Diseases/epidemiology , Activities of Daily Living , Ontario , Cohort Studies , Longitudinal StudiesABSTRACT
BACKGROUND: Individuals with frontotemporal dementia (FTD) often present with poor decision-making, which can affect both their financial and social situations. Delineation of the specific cognitive impairments giving rise to impaired decision-making in individuals with FTD may inform treatment strategies, as different neurotransmitter systems have been associated with distinct patterns of altered decision-making. OBJECTIVE: To use a reversal-learning paradigm to identify the specific cognitive components of reversal learning that are most impaired in individuals with FTD and those with Alzheimer disease (AD) in order to inform future approaches to treatment for symptoms related to poor decision-making and behavioral inflexibility. METHOD: We gave 30 individuals with either the behavioral variant of FTD or AD and 18 healthy controls a stimulus-discrimination reversal-learning task to complete. We then compared performance in each phase between the groups. RESULTS: The FTD group demonstrated impairments in initial stimulus-association learning, though to a lesser degree than the AD group. The FTD group also performed poorly in classic reversal learning, with the greatest impairments being observed in individuals with frontal-predominant atrophy during trials requiring inhibition of a previously advantageous response. CONCLUSION: Taken together, these results and the reversal-learning paradigm used in this study may inform the development and screening of behavioral, neurostimulatory, or pharmacologic interventions aiming to address behavioral symptoms related to stimulus-reinforcement learning and response inhibition impairments in individuals with FTD.
Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Frontotemporal Dementia , Alzheimer Disease/psychology , Frontotemporal Dementia/psychology , Humans , Reversal LearningABSTRACT
Periventricular white matter hyperintensities (pvWMHs) are a neurological feature detected with magnetic resonance imaging that are clinically associated with an increased risk of stroke and dementia. pvWMHs represent white matter lesions characterized by regions of myelin and axon rarefaction and as such likely involve changes in lipid composition; however, these alterations remain unknown. Lipids are critical in determining cell function and survival. Perturbations in lipid expression have previously been associated with neurological disorders. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an emerging technique for untargeted, high-throughput investigation of lipid expression and spatial distribution in situ; however, the use of MALDI IMS has been previously been limited by the need for non-embedded, non-fixed, fresh-frozen samples. In the current study, we demonstrate the novel use of MALDI IMS to distinguish regional lipid abnormalities that correlate with magnetic resonance imaging (MRI) defined pvWMHs within ammonium formate washed, formalin-fixed human archival samples. MALDI IMS scans were conducted in positive or negative ion detection mode on tissues sublimated with 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene matrices, respectively. Using a broad, untargeted approach to lipid analysis, we consistently detected 116 lipid ion species in 21 tissue blocks from 11 different post-mortem formalin-fixed human brains. Comparing the monoisotopic mass peaks of these lipid ions elucidated significant differences in lipid expression between pvWMHs and NAWM for 31 lipid ion species. Expanding our understanding of alterations in lipid composition will provide greater knowledge of molecular mechanisms underpinning ischemic white matter lesions and provides the potential for novel therapeutic interventions targeting lipid composition abnormalities.
Subject(s)
Brain/pathology , Lipids/chemistry , Magnetic Resonance Imaging , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , White Matter/pathology , Diagnosis , Humans , White Matter/metabolismABSTRACT
Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop methods for the early diagnosis of ovarian cancer. A step towards early and non-invasive cancer diagnosis is through the utilization of extracellular vesicles (EVs), which are nanoscale, membrane-bound vesicles that contain proteins and genetic material reflective of their parent cell. Thus, EVs secreted by cancer cells can be thought of as cancer biomarkers. In this paper, we present gold nanohole arrays for the capture of ovarian cancer (OvCa)-derived EVs and their characterization by surface-enhanced Raman spectroscopy (SERS). For the first time, we have characterized EVs isolated from two established OvCa cell lines (OV-90, OVCAR3), two primary OvCa cell lines (EOC6, EOC18), and one human immortalized ovarian surface epithelial cell line (hIOSE) by SERS. We subsequently determined their main compositional differences by principal component analysis and were able to discriminate the groups by a logistic regression-based machine learning method with â¼99% accuracy, sensitivity, and specificity. The results presented here are a great step towards quick, facile, and non-invasive cancer diagnosis.
Subject(s)
Extracellular Vesicles , Ovarian Neoplasms , Apoptosis , Cell Line, Tumor , Female , Humans , Ovarian Neoplasms/diagnosis , Spectrum Analysis, RamanABSTRACT
Creutzfeldt-Jakob disease (CJD) is a fatal neurological illness for which accurate diagnosis is paramount. Real-time quaking-induced conversion (RT-QuIC) is a prion-specific assay with high sensitivity and specificity for CJD. The Canadian endpoint quaking-induced conversion (EP-QuIC) test is similar, but unlike RT-QuIC there is little data regarding its diagnostic utility in clinical practice. In this exploratory predictive value analysis of EP-QuIC in CJD, the negative predictive value (NPV) and positive predictive value (PPV) was 100% and 83%, respectively, with one false-positive result identified. Re-testing this sample with an optimized EP-QuIC protocol eliminated this false-positive result, leading to a PPV of 100%.
La valeur prédictive de la méthode diagnostique dite de conversion provoquée par tremblement au point final dans le cas de la maladie de Creutzfeldt-Jakob. La maladie de Creutzfeldt-Jakob (MCJ) est une maladie neurologique qui entraîne à terme un décès et pour laquelle l'établissement d'un diagnostic précis est primordial. La conversion provoquée par tremblement en temps réel (RT-QuIC en anglais) est une méthode diagnostique qui repose sur la détection de la protéine prion. Dans le cas de la MCJ, cette méthode est réputée posséder une sensibilité et une spécificité élevées. La méthode canadienne dite de conversion provoquée par tremblement au point final (EP-QuIC en anglais) se veut similaire mais, à la différence de la RT-QuIC, il existe peu de données en ce qui concerne son utilité diagnostique dans le cadre d'une pratique clinique. Dans cette analyse prédictive exploratoire de la EP-QuIC en lien avec la MCJ, la valeur prédictive négative (VPN) et la valeur prédictive positive (VPP) ont été respectivement de 100 % et de 83 %, un seul résultat faux-positif ayant été identifié. Le fait de soumettre notre échantillon à un nouveau test effectué à l'aide d'un protocole d'EP-QuIC optimisé a permis d'éliminer ce résultat faux-positif, ce qui a débouché sur une VPP de 100 %.
Subject(s)
Creutzfeldt-Jakob Syndrome/diagnosis , PrPSc Proteins/analysis , Prion Proteins/analysis , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Predictive Value of TestsABSTRACT
The Montreal Cognitive Assessment (MoCA), a brief screening test developed to detect patients with mild cognitive impairment, is used in clinical settings across North America [Nasreddine et al.: J Am Geriatr Soc 2005;53:695-699]. The MoCA has been demonstrated to be sensitive to cognitive deficits in frontotemporal dementias (FTD) and related disorders [Coleman et al.: Alzheimer Dis Assoc Disord 2016;30:258-263]. Given attentional impairments in patients with FTD, whether and to what extent the abbreviated items on the MoCA may predict performance on corresponding assessments is not known. Testing and demographic data were extracted from a clinical database using a sample of 91 patients with FTD and related disorders. The relationship between MoCA items and corresponding neuropsychological tasks was assessed through McNemar tests and Spearman correlations. While some MoCA items such as letter fluency, orientation, and clock drawing were strongly correlated with the corresponding standard cognitive test, the MoCA trails were insensitive to impairment compared to the full Trail Making B Test (p = 0.01). In contrast, MoCA naming and delayed recall sub-items detected cognitive impairment more frequently than available comparison tests. The MoCA is a sensitive screening measure to detect impairment in patients with FTD and related disorders, but cognitive deficits specific to FTD result in differential performance on MoCA items compared to longer standard cognitive tests.
Subject(s)
Cognitive Dysfunction , Frontotemporal Dementia , Aged , Aged, 80 and over , Cognition , Cognitive Dysfunction/diagnosis , Cognitive Dysfunction/psychology , Female , Frontotemporal Dementia/diagnosis , Frontotemporal Dementia/psychology , Geriatric Assessment/methods , Humans , Male , Mental Recall , Mental Status and Dementia Tests , Middle Aged , Neuropsychological TestsABSTRACT
Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.
Subject(s)
Image Interpretation, Computer-Assisted/methods , Models, Biological , Molecular Imaging/methods , Multiprotein Complexes/metabolism , Protein Interaction Mapping/methods , Software , Algorithms , Computer Graphics , Computer Simulation , Models, Chemical , Multiprotein Complexes/ultrastructure , Reproducibility of Results , Sensitivity and Specificity , User-Computer InterfaceABSTRACT
The Montreal Cognitive Assessment (MoCA) is a cognitive screening tool used by practitioners worldwide. The efficacy of the MoCA for screening frontotemporal dementia (FTD) and related disorders is unknown. The objectives were: (1) to determine whether the MoCA detects cognitive impairment (CI) in FTD subjects; (2) to determine whether Alzheimer disease (AD) and FTD subtypes and related disorders can be parsed using the MoCA; and (3) describe longitudinal MoCA performance by subtype. We extracted demographic and testing data from a database of patients referred to a cognitive neurology clinic who met criteria for probable AD or FTD (N=192). Logistic regression was used to determine whether dementia subtypes were associated with overall scores, subscores, or combinations of subscores on the MoCA. Initial MoCA results demonstrated CI in the majority of FTD subjects (87%). FTD subjects (N=94) performed better than AD subjects (N=98) on the MoCA (mean scores: 18.1 vs. 16.3; P=0.02). Subscores parsed many, but not all subtypes. FTD subjects had a larger decline on the MoCA within 13 to 36 months than AD subjects (P=0.02). The results indicate that the MoCA is a useful tool to identify and track progression of CI in FTD. Further, the data informs future research on scoring models for the MoCA to enhance cognitive screening and detection of FTD patients.
Subject(s)
Alzheimer Disease/diagnosis , Cognitive Dysfunction , Frontotemporal Dementia/diagnosis , Neuropsychological Tests/statistics & numerical data , Aged , Cross-Sectional Studies , Disease Progression , Female , Frontotemporal Dementia/classification , Humans , MaleABSTRACT
OBJECTIVE: The Montreal Cognitive Assessment (MoCA) is a general cognitive screening tool that has shown sensitivity in detecting mild levels of cognitive impairment in various clinical populations. Although mood dysfunction is common in referrals to memory clinics, the influence of mood on the MoCA has to date been largely unexplored. METHOD: In this study, we examined the impact of mood dysfunction on the MoCA using a memory clinic sample of individuals with depressive symptoms who did not meet criteria for a neurodegenerative disease. RESULTS: Half of the group with depressive symptoms scored below the MoCA-suggested cutoff for cognitive impairment. As a group, they scored below healthy controls, but above individuals with Alzheimer's disease and frontotemporal dementia. A MoCA subtask analysis revealed a pattern of executive/attentional dysfunction in those with depressive symptoms. CONCLUSIONS: This observed negative impact of depressive symptomatology on the MoCA has interpretative implications for its utility as a cognitive screening tool in a memory clinic setting.
Subject(s)
Cognition Disorders/diagnosis , Cognition Disorders/etiology , Depression/complications , Neuropsychological Tests , Adult , Aged , Aged, 80 and over , Alzheimer Disease/complications , Alzheimer Disease/diagnosis , Female , Frontotemporal Dementia/complications , Frontotemporal Dementia/diagnosis , Humans , Longitudinal Studies , Male , Middle Aged , Psychiatric Status Rating Scales , Reproducibility of ResultsABSTRACT
The high-affinity choline transporter (CHT) is responsible for choline uptake into cholinergic neurons, with this being the rate-limiting step for acetylcholine production. Altering CHT protein disposition directly impacts choline uptake activity and cholinergic neurotransmission. Amyloid precursor protein (APP) interacts with CHT proteins and increases their endocytosis from the cell surface. The goal of this study was to examine regulation of CHT trafficking and activity by wild-type APP (APPwt) and determine if this differs with Swedish mutant APP (APPSwe) in SH-SY5Y human neuroblastoma cells. APPSwe differs from APPwt in its trafficking from the cell surface through endosomes. We report for the first time that CHT interacts significantly less with APPSwe than with APPwt. Surprisingly, however, CHT cell surface levels and choline uptake activity are decreased to the same extent and CHT co-localization to early endosomes increased similarly in cells expressing either APPwt or APPSwe. A critical observation is that CHT co-immunoprecipitates with ßCTF from APPSwe-expressing cells. We propose that decreased CHT function is mediated differently by APPwt and APPSwe; APPwt interaction with CHT facilitates its endocytosis from the cell surface, whereas the effect of APPSwe on CHT is mediated indirectly potentially by binding to the ßCTF fragment or by Aß released from cells. High-affinity choline transporter (CHT) takes choline into cholinergic neurons for acetylcholine synthesis. Amyloid precursor protein (APP) interacts with CHT proteins, but this is decreased for Swedish mutant APP (APPSwe). CHT cell surface levels and localization to early endosomes, and choline uptake activity are changed similarly by APPwt or APPSwe. APPSwe mediates effects indirectly potentially by ßCTF or Aß.
Subject(s)
Amyloid beta-Protein Precursor/genetics , Membrane Transport Proteins/physiology , Mutation/genetics , Animals , Cell Line, Tumor , Humans , Protein Transport/physiology , RatsSubject(s)
Alzheimer Disease/genetics , Mutation/genetics , Presenilin-2/genetics , Female , Humans , Middle AgedABSTRACT
The overproduction of the toxic peptide amyloid-beta (Aß) generated from the cleavage of amyloid precursor protein (APP) is proposed to be a critical event in the development of Alzheimer's disease. Evidence suggests that the cleavage of APP occurs after its internalization from the cell surface. Previously, we identified a novel pathway for APP internalization, which trafficks cell surface APP directly to lysosomes by macropinocytosis, leading to its processing into Aß. We also demonstrated that ADP-ribosylation factor 6 (Arf6) is required for the macropinocytosis of APP. Here, we characterized the roles of Arf6's downstream effectors Rac1, Cdc42 and RhoA. Both pharmacological inhibition and siRNA knockdown of these proteins reduced the amount of APP colocalized with LAMP1-labeled lysosomes without affecting APP transport to early endosomes. Decreases in the production of both Aß40 and Aß42 were also observed by ELISA in response to inhibitor treatment. These findings together demonstrate that Rac1, Cdc42 and RhoA are components of the mechanism regulating the macropinocytosis of APP and targeting these components can reduce the production of Aß.
ABSTRACT
Alzheimer's disease (AD) is a progressive neurological condition characterized by impaired cognitive function and behavioural alterations. While AD research historically centered around mis-folded proteins, advances in mass spectrometry techniques have triggered increased exploration of the AD lipidome with lipid dysregulation emerging as a critical player in AD pathogenesis. Gangliosides are a class of glycosphingolipids enriched within the central nervous system. Previous work has suggested a shift in a-series gangliosides from complex (GM1) to simple (GM2 and GM3) species may be related to the development of neurodegenerative disease. Additionally, complex gangliosides with 20 carbon sphingosine chains have been shown to increase in the aging brain. In this study, we utilized matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to interrogate the in situ relationship of a-series gangliosides with either 18 or 20 carbon sphingosine chains (d18:1 or d20:1 respectively) in the post-mortem human AD brain. Here, we expanded upon previous literature and demonstrated a significant decrease in the GM1 d20:1:GM1 d18:1 ratio in regions of the dentate gyrus and entorhinal cortex in AD relative to control brain tissue. Then we demonstrated that the MALDI-MSI profile of GM3 co-localizes with histologically confirmed amyloid beta (Aß) plaques and found a significant increase in both GM1 and GM3 in proximity to Aß plaques. Collectively these results support past literature and demonstrate a perturbation of the ganglioside profile in AD. Moreover, this work validates a pipeline for MALDI-MSI and classic histological staining in the same tissue sections. This demonstrates feasibility for integrating untargeted mass spectrometry imaging approaches into a digital pathology framework.
ABSTRACT
We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions.