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BACKGROUND: Familial Alzheimer's disease (fAD) is driven by genetic predispositions affecting the expression and metabolism of the amyloid-ß protein precursor. Aluminum is a non-essential yet biologically-reactive metal implicated in the etiology of AD. Recent research has identified aluminum intricately and unequivocally associated with amyloid-ß in senile plaques and, more tentatively, co-deposited with neuropil-like threads in the brains of a Colombian cohort of donors with fAD. OBJECTIVE: Herein, we have assessed the co-localization of aluminum to immunolabelled phosphorylated tau to probe the potential preferential binding of aluminum to senile plaques or neurofibrillary tangles in the same Colombian kindred. METHODS: Herein, we have performed phosphorylated tau-specific immunolabelling followed by aluminum-specific fluorescence microscopy of the identical brain tissue sections via a sequential labelling method. RESULTS: Aluminum was co-localized with immunoreactive phosphorylated tau in the brains of donors with fAD. While aluminum was predominantly co-located to neurofibrillary tangles in the temporal cortex, aluminum was more frequently co-deposited with cortical senile plaques. CONCLUSION: These data suggest that the co-deposition of aluminum with amyloid-ß precedes that with neurofibrillary tangles. Extracellularly deposited amyloid-ß may also be more immediately available to bind aluminum versus intracellular aggregates of tau. Therapeutic approaches to reduce tau have demonstrated the amelioration of its synergistic interactions with amyloid-ß, ultimately reducing tau pathology and reducing neuronal loss. These data support the intricate associations of aluminum in the neuropathology of fAD, of which its subsequent reduction may further therapeutic benefits observed in ongoing clinical trials in vivo.
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BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-ß (Aß) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. METHODS: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-ß (Aß), Aß aggregation process in presence of CERTL, and the resulting changes in Aß toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. RESULTS: Here, we report that CERTL binds to APP, modifies Aß aggregation, and reduces Aß neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aß formation, and modulates microglia by decreasing their pro-inflammatory phenotype. CONCLUSION: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.
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Doença de Alzheimer , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Ceramidas , Modelos Animais de Doenças , Inflamação , Masculino , Camundongos , Camundongos Transgênicos , Placa AmiloideRESUMO
BACKGROUND: Protein misfolding disorders are frequently implicated in neurodegenerative conditions. Familial Alzheimer's disease (fAD) is an early-onset and aggressive form of Alzheimer's disease (AD), driven through autosomal dominant mutations in genes encoding the amyloid precursor protein and presenilins 1 and 2. The incidence of epilepsy is higher in AD patients with shared neuropathological hallmarks in both disease states, including the formation of neurofibrillary tangles. Similarly, in Parkinson's disease, dementia onset is known to follow neurofibrillary tangle deposition. OBJECTIVE: Human exposure to aluminum has been linked to the etiology of neurodegenerative conditions and recent studies have demonstrated a high level of co-localization between amyloid-ß and aluminum in fAD. In contrast, in a donor exposed to high levels of aluminum later developing late-onset epilepsy, aluminum and neurofibrillary tangles were found to deposit independently. Herein, we sought to identify aluminum and neurofibrillary tangles in fAD, Parkinson's disease, and epilepsy donors. METHODS: Aluminum-specific fluorescence microscopy was used to identify aluminum in neurofibrillary tangles in human brain tissue. RESULTS: We observed aluminum and neurofibrillary-like tangles in identical cells in all respective disease states. Co-deposition varied across brain regions, with aluminum and neurofibrillary tangles depositing in different cellular locations of the same cell. CONCLUSION: Neurofibrillary tangle deposition closely follows cognitive-decline, and in epilepsy, tau phosphorylation associates with increased mossy fiber sprouting and seizure onset. Therefore, the presence of aluminum in these cells may exacerbate the accumulation and misfolding of amyloidogenic proteins including hyperphosphorylated tau in fAD, epilepsy, and Parkinson's disease.
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Alumínio/metabolismo , Doença de Alzheimer/patologia , Emaranhados Neurofibrilares/patologia , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patologia , Epilepsia/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Doença de Parkinson/patologia , Fosforilação , Presenilina-1/metabolismo , Proteínas tau/metabolismoRESUMO
The use of vaccines containing aluminum (Al) adjuvants is widespread in ovine production. Al adjuvants induce an effective immune-response but lead to the formation of post-vaccination granulomas from which Al can disseminate. This work aims to study the accumulation of Al in the central nervous system of sheep subcutaneously inoculated with Al-hydroxide containing products. Lumbar spinal cord and parietal lobe from 21 animals inoculated with 19 doses of Vaccine (nâ¯=â¯7), Adjuvant-only (nâ¯=â¯7) or phosphate-buffered saline as Control (nâ¯=â¯7) were analyzed with transversely heated graphite furnace atomic absorption spectroscopy and lumogallion staining for Al analytical measurements and Al tisular localization respectively. In the lumbar spinal cord, Al median content was higher in both the Adjuvant-only and Vaccine group (pâ¯=â¯.001) compared with the Control group. Animals of the Adjuvant-only group showed the higher individual measurements in the lumbar spinal cord (14.36⯵g/g and 7.83⯵g/g). In the parietal lobe, Al median content tended to be higher in the Adjuvant-only group compared with Control group (pâ¯=â¯.074). Except for three replicates of the Adjuvant-only group, Al content was always below 1⯵g/g. In the lumbar spinal cord, lumogallion-reactive Al deposits were more abundant in the gray matter than in the white matter in both Vaccine (pâ¯=â¯.034) and Adjuvant-only groups (pâ¯=â¯.017) and Al deposits were mostly associated with glial-like cells (pâ¯=â¯.042). In the parietal lobe, few Al deposits, which were sometimes related to blood vessels, were found. In sheep, Al-hydroxide adjuvants inoculated in the subcutaneous tissue selectively accumulate in the lumbar spinal cord.
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Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/farmacocinética , Alumínio/farmacocinética , Lobo Parietal/metabolismo , Medula Espinal/metabolismo , Vacinas/administração & dosagem , Adjuvantes Imunológicos/farmacocinética , Hidróxido de Alumínio/administração & dosagem , Animais , Injeções Subcutâneas , Masculino , Lobo Parietal/efeitos dos fármacos , Lobo Parietal/imunologia , Ovinos , Medula Espinal/efeitos dos fármacos , Medula Espinal/imunologia , Distribuição TecidualRESUMO
Genetic predispositions associated with metabolism of the amyloid-ß protein precursor underlie familial Alzheimer's disease; a form of dementia characterized by early disease onset and elevated levels of cortical amyloid-ß. Human exposure to aluminum is linked to the etiology of Alzheimer's disease and recent research measured a high content of aluminum in brain tissue in familial Alzheimer's disease. To elaborate upon this finding, we have obtained brain tissues from a Colombian cohort of donors with familial Alzheimer's disease. We have used established methods to measure the aluminum content of these tissues and we have compared the data with a recently measured dataset for control brain tissues. We report significantly higher levels of aluminum in brain tissues in donors with familial Alzheimer's disease than in control tissues from donors without neurological impairment or neurodegeneration. We have used aluminum-specific fluorescence microscopy along with complementary imaging for amyloid-ß to demonstrate a very high degree of co-localization of these two risk factors in brain tissue in familial Alzheimer's disease. Aluminum and amyloid-ß were co-located in senile plaques as well as vasculature, the latter resembling cerebral amyloid angiopathy. Aluminum was also found separately from amyloid-ß in intracellular compartments including glia and neuronal axons. The research has identified an arguably unique association between high brain aluminum content and amyloid-ß and allows postulation that genetic predispositions defining familial Alzheimer's disease underlie this relationship.
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Alumínio/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Axônios/metabolismo , Química Encefálica , Angiopatia Amiloide Cerebral/metabolismo , Estudos de Coortes , Colômbia , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neuroglia/metabolismo , Placa Amiloide/metabolismo , Fatores de RiscoRESUMO
Within clinical vaccinations, recombinant antigens are routinely entrapped inside or adsorbed onto the surface of aluminium salts in order to increase their immunological potency in vivo. The efficacy of these immunisations is highly dependent upon the recognition and uptake of these complexes by professional phagocytes and their subsequent delivery to the draining lymph nodes for further immunological processing. While monocytes have been shown to internalise aluminium adjuvants and their adsorbates, the role of macrophages in this respect has not been fully established. Furthermore, this study explored the interaction of THP-1 macrophages with aluminium-based adjuvants (ABAs) and how this relationship influenced the survival of such cells in vitro. THP-1 macrophages were exposed to low concentrations of ABAs (1.7⯵g/mL Al) for a maximum of seven days. ABA uptake was determined using lumogallion staining and cell viability by both DAPI (4',6-diamidino-2-phenylindole) staining and LDH (lactate dehydrogenase) assay. Evidence of ABA particle loading was identified within cells at early junctures following treatment and appeared to be quite prolific (>90% cells positive for Al signal after 24â¯h). Total sample viability (% LDH release) in treated samples was predominantly similar to untreated cells and low levels of cellular death were consistently observed in populations positive for Al uptake. It can thus be concluded that aluminium salts can persist for some time within the intracellular environment of these cells without adversely affecting their viability. These results imply that macrophages may play a role in the systemic translocation of ABAs once administered in the form of an inoculation.
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Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Desdiferenciação Celular , Macrófagos/efeitos dos fármacos , Sobrevivência Celular , Humanos , Macrófagos/imunologia , Fagocitose , Células THP-1RESUMO
The use of vaccines including aluminum (Al)-based adjuvants is widespread among small ruminants and other animals. They are associated with the appearance of transient injection site nodules corresponding to granulomas. This study aims to characterize the morphology of these granulomas, to understand the role of the Al adjuvant in their genesis, and to establish the presence of the metal in regional lymph nodes. A total of 84 male neutered lambs were selected and divided into 3 treatment groups of 28 animals each: (1) vaccine (containing Al-based adjuvant), (2) adjuvant-only, and (3) control. A total of 19 subcutaneous injections were performed in a time frame of 15 months. Granulomas and regional lymph nodes were evaluated by clinicopathological means. All of the vaccine and 92.3% of the adjuvant-only lambs presented injection-site granulomas; the granulomas were more numerous in the group administered the vaccine. Bacterial culture in granulomas was always negative. Histologically, granulomas in the vaccine group presented a higher degree of severity. Al was specifically identified by lumogallion staining in granulomas and lymph nodes. Al median content was significantly higher ( P < .001) in the lymph nodes of the vaccine group (82.65 µg/g) compared with both adjuvant-only (2.53 µg/g) and control groups (0.96 µg/g). Scanning transmission electron microscopy demonstrated aggregates of Al within macrophages in vaccine and adjuvant-only groups. In these two groups, Al-based adjuvants induce persistent, sterile, subcutaneous granulomas with macrophage-driven translocation of Al to regional lymph nodes. Local translocation of Al may induce further accumulation in distant tissues and be related to the appearance of systemic signs.
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Adjuvantes Imunológicos/efeitos adversos , Alumínio/efeitos adversos , Granuloma/veterinária , Reação no Local da Injeção/veterinária , Doenças dos Ovinos/induzido quimicamente , Adjuvantes Imunológicos/administração & dosagem , Alumínio/administração & dosagem , Animais , Granuloma/induzido quimicamente , Granuloma/patologia , Reação no Local da Injeção/etiologia , Reação no Local da Injeção/patologia , Injeções Subcutâneas/efeitos adversos , Injeções Subcutâneas/veterinária , Linfonodos/patologia , Masculino , Ovinos , Doenças dos Ovinos/patologiaRESUMO
Alzheimer's disease is a debilitating neurodegenerative condition that progressively causes synaptic loss and major neuronal damage. Immunotherapy utilising Aß as an active immunogen or via passive treatment utilising antibodies raised to amyloid have shown therapeutic promise. The migratory properties of peripheral blood-borne monocytes and their ability to enter the central nervous system, suggests a beneficial role in mediating tissue damage and neuroinflammation. However, the intrinsic phagocytic properties of such cells have pre-disposed them to internalise misfolded amyloidogenic peptides that could act as seeds capable of nucleating amyloid formation in the brain. Mechanisms governing the cellular fate of amyloid therefore, may prove to be key in the development of future vaccination regimes. Herein, we have developed unequivocal and direct conformation-sensitive fluorescent molecular probes that reveal the intracytoplasmic and intranuclear persistence of amyloid in a monocytic T helper 1 (THP-1) cell line. Use of the pathogenic Aß42 species as a model antigen in simulated vaccine formulations suggested differing mechanisms of cellular internalisation, in which fibrillar amyloid evaded lysosomal capture, even when co-deposited on particulate adjuvant materials. Taken collectively, direct fluorescent labelling of antigen-adjuvant complexes may serve as critical tools in understanding subsequent immunopotentiation in vaccines directed against amyloidosis and wider dementia.
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Vacinas contra Alzheimer/farmacologia , Peptídeos beta-Amiloides/imunologia , Materiais Biomiméticos/farmacologia , Núcleo Celular/imunologia , Citoplasma/imunologia , Fragmentos de Peptídeos/imunologia , Coloração e Rotulagem/métodos , Adjuvantes Imunológicos/metabolismo , Alumínio/metabolismo , Vacinas contra Alzheimer/síntese química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Benzenossulfonatos/química , Benzotiazóis/química , Transporte Biológico , Materiais Biomiméticos/síntese química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Células THP-1RESUMO
The physicochemical properties of aluminum salts are key determinants of their resultant adjuvanticity in vivo when administered as part of a vaccine. While there are links between particle size and the efficacy of the immune response, the limited literature directly characterizing the PSD of aluminum adjuvants has stymied the elucidation of such a relationship for these materials. Hence, this comparative study was undertaken to monitor the PSD of aluminum adjuvants throughout the process of vaccine formulation using DLS. A significant proportion of the stock suspensions was highly agglomerated (>9 µm) and Alhydrogel® exhibited the smallest median size (2677 ± 120 nm) in comparison to Adju-Phos® or Imject alum® (7152 ± 308 and 7294 ± 146 nm respectively) despite its large polydispersity index (PDI). Dilution of these materials induced some degree of disaggregation within all samples with Adju-Phos® being the most significantly affected. The presence of BSA caused the median size of Alhydrogel® to increase but these trends were not evident when model vaccines were formulated with either Adju-Phos® or Imject alum®. Nevertheless, Alhydrogel® and Adju-Phos® exhibited comparable median sizes in the presence of this protein (4194 ± 466 and 4850 ± 501 nm respectively) with Imject alum® being considerably smaller (2155 ± 485 nm). These results suggest that the PSD of aluminum adjuvants is greatly influenced by dilution and the degree of protein adsorption experienced within the vaccine itself. The size of the resultant antigen-adjuvant complex may be important for its immunological recognition and subsequent clearance from the injection site.
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The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved.
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Adjuvantes Imunológicos/farmacocinética , Hidróxido de Alumínio/farmacocinética , Óxido de Alumínio/farmacocinética , Benzenossulfonatos/química , Flavonoides/química , Fosfatos/farmacocinética , Hidróxido de Alumínio/imunologia , Óxido de Alumínio/imunologia , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fagocitose/imunologia , Fosfatos/imunologia , Coloração e Rotulagem/métodosRESUMO
The observation of the co-deposition of metals and amyloid-beta(42) (Abeta(42)) in brain tissue in Alzheimer's disease prompted myriad investigations into the role played by metals in the precipitation of this peptide. Copper is bound by monomeric Abeta(12) and upon precipitation of the copper-peptide complex thereby prevents Abeta(42) from adopting a beta-sheet secondary structure. Copper is also bound by beta-sheet conformers of Abeta(42), and herein we have investigated how this interaction affects the conformation of the precipitated peptide. Copper significantly reduced the thioflavin T fluorescence of aged, fibrillar Abeta(42) with, for example, a 20-fold excess of the metal resulting in a ca 90% reduction in thioflavin T fluorescence. Transmission electron microscopy showed that copper significantly reduced the quantities of amyloid fibrils while Congo red staining and polarized light demonstrated a copper-induced abolition of apple-green birefringence. Microscopy under cross-polarized light also revealed the first observation of spherulites of Abeta(42). The size and appearance of these amyloid structures were found to be very similar to spherulites identified in Alzheimer's disease tissue. The combined results of these complementary methods strongly suggested that copper abolished the beta-sheet secondary structure of pre-formed, aged amyloid fibrils of Abeta(42). Copper may protect against the presence of beta-sheets of Abeta(42) in vivo, and its binding by fibrillar Abeta(42) could have implications for Alzheimer's disease therapy.