Atrophic astrocytes in aged marmosets present tau hyperphosphorylation, RNA oxidation, and DNA fragmentation.

Astrocytes perform multiple essential functions in the brain showing morphological changes. Hypertrophic astrocytes are commonly observed in cognitively healthy aged animals, implying a functional defense mechanism without losing neuronal support. In neurodegenerative diseases, astrocytes show morphological alterations, such as decreased process length and reduced number of branch points, known as astroglial atrophy, with detrimental effects on neuronal cells. The common marmoset (Callithrix jacchus) is a non-human primate that, with age, develops several features that resemble neurodegeneration. In this study, we characterize the morphological alterations in astrocytes of adolescent (mean 1.75 y), adult (mean 5.33 y), old (mean 11.25 y), and aged (mean 16.83 y) male marmosets. We observed a significantly reduced arborization in astrocytes of aged marmosets compared to younger animals in the hippocampus and entorhinal cortex. These astrocytes also show oxidative damage to RNA and increased nuclear plaques in the cortex and tau hyperphosphorylation (AT100). Astrocytes lacking S100A10 protein show a more severe atrophy and DNA fragmentation. Our results demonstrate the presence of atrophic astrocytes in the brains of aged marmosets.

Activation of the NLRP3 inflammasome by CCl4 exacerbates hepatopathogenic diet-induced experimental NASH.

INTRODUCTION AND OBJECTIVES: Administration of carbon tetrachloride (CCl4), along with an hepatopathogenic diet, is widely employed as a chemical inducer to replicate human nonalcoholic steatohepatitis (NASH) in rodents; however, the role of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in this model remains unclear. We aimed to determine the relevance of NLRP3 inflammasome activation in the development of NASH induced by CCl4 along with an hepatopathogenic diet in male Wistar rats. MATERIALS AND METHODS: Animals were fed either a high fat, sucrose, and cholesterol diet (HFSCD) or a HFSCD plus intraperitoneal injections of low doses of CCl4 (400 mg/kg) once a week for 15 weeks. Liver steatosis, inflammation, fibrosis, and NLRP3 inflammasome activation were evaluated using biochemical, histological, ultrastructural, and immunofluorescence analyses, western blotting, and immunohistochemistry. RESULTS: Our experimental model reproduced several aspects of the human NASH pathophysiology. NLRP3 inflammasome activation was induced by the combined effect of HFSCD plus CCl4 and significantly increased levels of both proinflammatory and profibrogenic cytokines and collagen deposition in the liver; thus, NASH severity was higher in the HFSCD+CCl4 group than that in the HFSCD group, to which CCl4 was not administered. Hepatic stellate cells, the most profibrogenic cells, were activated by HFSCD plus CCl4, as indicated by elevated levels of α-smooth muscle actin. Thus, activation of the NLRP3 inflammasome, triggered by low doses of CCl4, exacerbates the severity of NASH. CONCLUSIONS: Our results indicate that NLRP3 inflammasome activation plays a key role and may be an important therapeutic target for NASH treatment.

Caffeine Inhibits NLRP3 Inflammasome Activation by Downregulating TLR4/MAPK/NF-κB Signaling Pathway in an Experimental NASH Model.

Caffeine elicits protective effects against liver diseases, such as NASH; however, its mechanism of action involving the pyrin domain-containing-3 (NLRP3) inflammasome signaling pathway remains to be elucidated. This study aimed to evaluate the effect of caffeine on the NLRP3 inflammasome signaling pathway in a rat model of NASH. NASH was induced by feeding rats a high-fat, -sucrose, and -cholesterol diet (HFSCD) for 15 weeks along with a weekly low dose (400 mg/kg, i.p.) of CCl4. Caffeine was administered at 50 mg/kg p.o. The effects of HFSCD+CCl4 and caffeine on the liver were evaluated using biochemical, ultrastructural, histological, and molecular biological approaches. The HFSCD+CCl4-treated rats showed fat accumulation in the liver, elevated levels of inflammatory mediators, NLRP3 inflammasome activation, antioxidant dysregulation, and liver fibrosis. Caffeine reduced necrosis, cholestasis, oxidative stress, and fibrosis. Caffeine exhibited anti-inflammatory effects by attenuating NLRP3 inflammasome activation. Moreover, caffeine prevented increases in toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) protein levels and mitigated the phosphorylation of mitogen-activated protein kinase (MAPK). Importantly, caffeine prevented the activation of hepatic stellate cells. This study is the first to report that caffeine ameliorates NASH by inhibiting NLRP3 inflammasome activation through the suppression of the TLR4/MAPK/NF-κB signaling pathway.

Increased oxidative stress, hyperphosphorylation of tau, and dystrophic microglia in the hippocampus of aged Tupaia belangeri.

Aging is a major risk factor for the development of neurodegenerative diseases. Alzheimer's disease and other neurodegenerative diseases are characterized by abnormal and prominent protein aggregation in the brain, partially due to deficiency in protein clearance. It has been proposed that alterations in microglia phagocytosis and debris clearance hasten the onset of neurodegeneration. Dystrophic microglia are abundant in aged humans, and it has been associated with the onset of disease. Furthermore, alterations in microglia containing ferritin are associated with neurodegenerative conditions. To further understand the process of microglia dysfunction during the aging process, we used hippocampal sections from Tupaia belangeri (tree shrews). Adult (mean age 3.8 years), old (mean age 6 years), and aged (mean age 7.5 years) tree shrews were used for histochemical and immunostaining techniques to determine ferritin and Iba1 positive microglia, iron tissue content, tau hyperphosphorylation and oxidized-RNA in dentate gyrus, subiculum, and CA1-CA3 hippocampal regions. Our results indicated that aged tree shrews presented an increased number of activated microglia containing ferritin, but microglia labeled with Iba1 with a dystrophic phenotype was more abundant in aged individuals. With aging, oxidative damage to RNA (8OHG) increased significantly in all hippocampal regions, while tau hyperphosphorylation (AT100) was enhanced in DG, CA3, and SUB in aged animals. Phagocytic inclusions of 8OHG- and AT100-damaged cells were observed in activated M2 microglia in old and aged animals. These data indicate that aged tree shrew may be a suitable model for translational research to study brain and microglia alterations during the aging process.

A Low Cost Antibody Signal Enhancer Improves Immunolabeling in Cell Culture, Primate Brain and Human Cancer Biopsy.

The use of antibodies to identify neuronal receptors, neurotransmitters, cytoskeletal elements or pathologic protein aggregates, ion channels, adhesion molecules or other cell-type specific markers, is common practice in neuroscience. Antibody detection systems are often based on confocal, epifluorescence or brightfield microscopy. Three types of technical issues can interfere with immunolabeling: low abundance of the target protein, low specific affinity of the antibody and/or signal background sometimes related to tissue fixation. Here, giving tribute to Professor Miledi's mentorship, we propose the application of an antibody signal enhancer (ASE) solution based on glycine, hydrogen peroxide and a detergent mix as a simple, low cost, protocol variation that significantly and specifically improves the signal to noise ratio during immunostaining experiments. We describe three new settings in which ASE improves the detection of a variety of antibodies applied on long-time stored non-human primate brain sections, cell culture monolayers and on squamous carcinomas retrieved from cervical cancer patients. The significant improvement of ASE over optimized immunohistochemical protocols used in clinical practice (i.e. cancer detection) combined with its simplicity and low cost makes it an attractive method for biomedical applications.

Corrigendum: Evidence of Tau Hyperphosphorylation and Dystrophic Microglia in the Common Marmoset.

[This corrects the article on p. 315 in vol. 8, PMID: 28066237.].

Evidence of Tau Hyperphosphorylation and Dystrophic Microglia in the Common Marmoset.

Common marmosets (Callithrix jacchus) have recently gained popularity in biomedical research as models of aging research. Basically, they confer advantages from other non-human primates due to their shorter lifespan with onset of appearance of aging at 8 years. Old marmosets present some markers linked to neurodegeneration in the brain such as amyloid beta (Aß)1-42 and Aß1-40. However, there are no studies exploring other cellular markers associated with neurodegenerative diseases in this non-human primate. Using immunohistochemistry, we analyzed brains of male adolescent, adult, old, and aged marmosets. We observed accumulation of Aß1-40 and Aß1-42 in the cortex of aged subjects. Tau hyperphosphorylation was already detected in the brain of adolescent animals and increased with aging in a more fibrillary form. Microglia activation was also observed in the aging process, while a dystrophic phenotype accumulates in aged subjects. Interestingly, dystrophic microglia contained hyperphosphorylated tau, but active microglia did not. These results support previous findings regarding microglia dysfunctionality in aging and neurodegenerative diseases as Alzheimer's disease. Further studies should explore the functional consequences of these findings to position this non-human primate as animal model of aging and neurodegeneration.