CERT\u3csub\u3eL\u3c/sub\u3e Reduces C16 Ceramide, Amyloid-β Levels, and Inflammation in a Model of Alzheimer’s Disease

BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer\u27s 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-β (Aβ) 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-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ 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 Aβ aggregation, and reduces Aβ 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 Aβ 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

CERTL reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

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-β (Aβ) 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-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ 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 Aβ aggregation, and reduces Aβ 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 Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion: Our results demonstrate a crucial role of CERTL in r

Aluminum translocation to the central nervous system of sheep repetitively inoculated with aluminum-adjuvants

Trabajo presentado en el Joint Congress of the Veterinary Pathology and Veterinary Clinical Pathology, celebrado en Arnhem (Países Bajos), del 25 al 28 de septiembre de 2019Ovine management systems usually involve prophylactic vaccination. Most ovine vaccines use aluminium hydroxide as adjuvant. In sheep, aluminium hydroxide promotes an effective immune response, but also induces local granulomatous reactions at the injection site. Aluminium can reach the regional lymph node. The aim of this work was to describe the translocation of subcutaneously inoculated aluminium-based adjuvants to the central nervous system (CNS)

Detection of aluminum in lumbar spinal cord of sheep subcutaneously inoculated with aluminum-hydroxide containing products

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 (n = 7), Adjuvant-only (n = 7) or phosphate-buffered saline as Control (n = 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 (p = .001) compared with the Control group. Animals of the Adjuvant-only group showed the higher individual measurements in the lumbar spinal cord (14.36 μg/g and 7.83 μg/g). In the parietal lobe, Al median content tended to be higher in the Adjuvant-only group compared with Control group (p = .074). Except for three replicates of the Adjuvant-only group, Al content was always below 1 μg/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 (p = .034) and Adjuvant-only groups (p = .017) and Al deposits were mostly associated with glial-like cells (p = .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.RM is a PhD student funded by the Department of Innovation, Research and University of Aragon, Spain. JA and ARL are PhD students funded by the Spanish Ministry of Science, Innovation and Universities (formerly Spanish Ministry of Education). This work was funded by grants from the Spanish Ministry of Economy, Industry and Competitiveness (AGL2013-49137-C3-1-R and RTI2018-096172-B-C33), the Ministry of Science, Innovation and Universities (RTI2018-096172-B-C31 and RTI2018-096172-B-C33) and the Government of Aragón (A17_17R, Animal Health and Reproduction)

Granulomas Following Subcutaneous Injection With Aluminum Adjuvant-Containing Products in Sheep

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.Peer reviewe

Studies on injection site reactions caused by aluminium-containing products in sheep

Trabajo presentado en el 3rd joint European Congress of the ESVP, ESTP and ECVP, celebrado en Lyon (Francia), del 30 de agosto al 2 de septiembre de 2017Introduction: Aluminium (Al) containing adjuvants are widely used in sheep vaccines to promote an effective immune reaction against antigens but induce local injection-site reactions. This work aims to characterize these reactions and to determine the role of the Al in its genesis. Materials and Methods: 84 lambs divided into 3 groups (n=28 each) were inoculated with a different substance: A) Vaccines containing aluminium hydroxide; B) Aluminium hydroxide only; C) PBS. Animals received 19 subcutaneous inoculations along 15 months. Injection-site reactions and the regional lymph node (LN) were studied by gross and microscopic pathology, microbiology, fluorescence microscopy with lumogallion, Transmission Electron Microscopy (TEM), Energy Dispersive X-Ray Spectroscopy (EDS) and Graphite Furnace Atomic Absorption Spectroscopy (GFAAS). Results: Injection-site reactions consisted ofgranulomas that were more numerous (p<0.001) and with more severe central necrosis (p=0.021) in group A than in group B. Most of the vaccinated lambs (76.9%) showed more than 7 granulomas and all granulomas could be recovered in certain cases. Macrophages in the granulomas showed an orange fluorescence emission (590 nm), typical of Al. Similar groups of macrophages were observed in the regional LN. By TEM, macrophages in the granulomas contained aggregates of a spiculated electrondense material identified as Al by EDS. Group A showed longer Al particles than group B (p<0.001). By GFAAS, group A showed higher Al concentration in the regional LN than group B (p<0.001). Conclusions: Al induces persistent, immunomediated subcutaneous granulomas and the reactions are more severe in vaccinated animals. Al can reach the regional lymph nodPeer reviewe

Clinicopathological studies in lambs repetitively inoculated with products containing alimunium adjuvants

Trabajo presentado en el 11th International Congress on Autoimmunity, celebrado en Lisboa (Portugal), del 16 al 20 de mayo de 2018The use of aluminum-containing vaccine adjuvants is widespread in the Spanish small ruminant industry. These compounds were related to an episode of vaccine adverse reactions which gave rise to a process known today as the ovine ASIA syndrome. An in vivo model of this syndrome was established. Eighty-four lambs were selected, divided into three groups (n=28 each) and submitted to an intensive inoculation program with: i) Vaccines containing aluminum hydroxide as adjuvant; ii) The adjuvant only; iii) PBS. Nineteen inoculations were performed during 15 months. A comprehensive in vivo follow-up was performed, including clinical examinations and behavioral and cognitive tests. After euthanasia, the pathology of different tissues was studied grossly, microscopically and by electron microscopy. The presence of aluminum in tissues was studied by energy dispersive X-ray spectroscopy, graphite furnace atomic absorption spectroscopy and lumogallion fluorescent staining. Animals in the vaccinated and adjuvant-inoculated groups presented persistent injection-site granulomas with intramacrophagic aluminum. Persistency was higher in the vaccinated group (p<0.001), reaching 15 months in some cases. There was translocation of aluminum to the regional lymph nodes (p<0.001) and lumbar spinal cord (p<0.001). Vaccinated and adjuvant-inoculated animals showed an increase in aggressive interactions (p<0.001) and stereotypies (p<0.001) and a decrease in affiliative interactions (p<0.001) when compared with the control group. Differences were more marked with higher number of doses applied. Repetitive inoculation of aluminum-hydroxide only or combined into commercial vaccines to experimental lambs induces highly persistent injection site granulomas, accumulation of aluminum in distant tissues and changes in the inter-individual interaction patterns.Peer reviewe