Brains Of Young Mice Research Articles

Negligible senescence in naked mole rats may be a consequence of well-maintained splicing regulation

Naked mole-rats (NMRs) have amongst the longest lifespans relative to body size of any known, non-volant mammalian species. They also display an enhanced stress resistance phenotype, negligible senescence and very rarely are they burdened with chronic age-related diseases. Alternative splicing (AS) dysregulation is emerging as a potential driver of senescence and ageing. We hypothesised that the expression of splicing factors, important regulators of patterns of AS, may differ in NMRs when compared to other species with relatively shorter lifespans. We designed assays specific to NMR splicing regulatory factors and also to a panel of pre-selected brain-expressed genes known to demonstrate senescence-related alterations in AS in other species, and measured age-related changes in the transcript expression levels of these using embryonic and neonatal developmental stages through to extreme old age in NMR brain samples. We also compared splicing factor expression in both young mouse and NMR spleen and brain samples. Both NMR tissues showed approximately double the expression levels observed in tissues from similarly sized mice. Furthermore, contrary to observations in other species, following a brief period of labile expression in early life stages, adult NMR splicing factors and patterns of AS for functionally relevant brain genes remained remarkably stable for at least two decades. These findings are consistent with a model whereby the conservation of splicing regulation and stable patterns of AS may contribute to better molecular stress responses and the avoidance of senescence in NMRs, contributing to their exceptional lifespan and prolonged healthspan.

Melatonin Effects in Young and Aging Mice with the Toxic Cuprizone-Induced Demyelination

The following indices were assessed in 3–5-month old and 15–17-month old 129/Sv mice treated with cuprizone and melatonin: the number of activated T-lymphocytes, macrophages, neural stem cells (determined by CD3, Mac1, and nestin markers), structurally unchanged neurons, malondialdehyde (MDA) content, antioxidant enzyme activities in the brain, the blood level of the thymic hormone thymulin, and the behavioral indices. The animals were fed with cuprizone for 3 weeks. From the eighth day of cuprizone treatment, mice were injected with melatonin (1 mg/kg, at 6 p.m. daily). The results demonstrated an increase in the number of CD3, Mac1, and nestin cells and the MDA level and a decrease in the activity of glutathione peroxidase (GP) in the brains of young and aging mice fed with cuprizone. Mice of both age groups demonstrated a decrease in the proportion of unchanged neurons in the central nervous system, the motor and emotional activity, and the muscle tone. Regardless of animal age, melatonin injections led to a decrease in the number of CD3 and Mac1 cells and the MDA level and an increase in GP activity and the thymulin level. The decrease in the number of nestin cells was accompanied by an increase in the number of unchanged neurons. The effects of both neurotoxin and melatonin on the immune factors and the structure and functional state of the neurons were more pronounced in young mice. Thus, the positive effect of melatonin in young and aging mice upon cuprizone-induced demyelination was mainly mediated by pathogenic factors.

Increased Expression of Immature Mannose-Containing Glycoproteins and Sialic Acid in Aged Mouse Brains.

Aging represents the accumulation of changes in an individual over time, encompassing physical, psychological, and social changes. Posttranslational modifications of proteins such as glycosylation, including sialylation or glycation, are proposed to be involved in this process, since they modulate a variety of molecular and cellular functions. In this study, we analyzed selected posttranslational modifications and the respective proteins on which they occur in young and old mouse brains. The expression of neural cell adhesion molecule (NCAM), receptor for advanced glycation endproducts (RAGE), as well as the carbohydrate-epitopes paucimannose and high-mannose, polysialic acid, and O-GlcNAc were examined. We demonstrated that mannose-containing glycans increased on glycoproteins in aged mouse brains and identified synapsin-1 as one major carrier of paucimannose in aged brains. In addition, we found an accumulation of so-called advanced glycation endproducts, which are generated by non-enzymatic reactions and interfere with protein function. Furthermore, we analyzed the expression of sialic acid and found also an increase during aging.

Every-Other-Day Feeding Decreases Glycolytic and Mitochondrial Energy-Producing Potentials in the Brain and Liver of Young Mice.

Intermittent fasting is used to reduce body mass in obese adult humans and animals. However, information on the impact of one type of intermittent fasting (IF) called every-other-day feeding (EODF) on young animals is scarce. In this study, 1-month-old mice of both sexes were subjected to a 4-week regimen of EODF using age-matched counterparts fed ad libitum as controls. At the end of EODF exposure, experimental male and female mice weighed 14 and 13% less than the control counterparts. The EODF regimen resulted in lower liver levels of glycogen, glucose, and lactate, but did not affect lactate level in mouse cerebral cortex of both sexes. Activities of key glycolytic enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) in liver of experimental mice were lower than those in controls. In the cerebral cortex, only hexokinase and pyruvate kinase activities were lower than in controls, but phosphofructokinase activity was not affected in IF females and was higher in IF males as compared with ad libitum fed males. Mitochondria isolated from liver of IF mice had lower respiratory control ratios, but those from the cortex had the same values as control animals. The concentration of β-hydroxybutyrate and the activity of β-hydroxybutyrate dehydrogenase were lower in the IF mouse liver, but not changed or enhanced in the IF cerebral cortex. Thus, animal responses to IF do not depend significantly on sex and are directed to decrease energy metabolism to save resources, and the effects are more pronounced in the liver than in the brain.

Single-cell transcriptomic profiling of the aging mouse brain.

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.

BSCI-17. YOUNG AGE PROMOTES BREAST CANCER METASTASIS TO THE BRAIN

Younger women (< 40 years old) diagnosed with breast cancer often have a poorer outcome and a higher risk of developing brain metastases compared to women diagnosed at an older age. Multi-variate analyses have shown that even after accounting for differences in primary tumor characteristics, young age is still an independent predictor of poorer outcome. We therefore hypothesize that rather than intrinsic tumor properties, age-related changes to microenvironmental factors can affect breast cancer metastasis. To test this hypothesis, human and mouse breast cancer cells were injected into young (< 6 month) and old (> 13 month) mice and metastatic tumor burden was quantified. Young mice injected with brain-seeking breast cancer cells (MDA-MB-231BR, 4T1-BR, and 99LN-BrM) developed significantly more brain metastases compared to their older counterparts. In contrast, age had no effect on lung metastatic tumor burden in five breast cancer models. The effect of age is organ-specific, and the young brain is more permissive for breast cancer metastasis. To gain mechanistic insight, the transcriptome of young and old mouse brains were analyzed by RNAseq, the metastatic microenvironment was analyzed by laser capture microdissection and mass spectrometry, immune populations have been identified by flow cytometry, and functional immune contributions analyzed by immunodepleting antibodies. Multiple brain immune subsets were altered with age. In vivo depletion experiments showed no significant contribution of CD4+ T-cells and GR1+ myeloid cells to baseline brain metastatic colonization. A subpopulation of microglia in aged metastatic brains had a high side-scatter profile, which is consistent with published reports that aged microglia are in a “pro-inflammatory” state. Depletion of microglia reduced baseline brain metastatic colonization by 50% and experiments are underway to determine their contribution to an age effect.

Altered Levels of Proteins and Phosphoproteins, in the Absence of Early Causative Transcriptional Changes, Shape the Molecular Pathogenesis in the Brain of Young Presymptomatic Ki91 SCA3/MJD Mouse

Spinocerebellar ataxia type 3 (SCA3/MJD) is a polyQ neurodegenerative disease where the presymptomatic phase of pathogenesis is unknown. Therefore, we investigated the molecular network of transcriptomic and proteomic triggers in young presymptomatic SCA3/MJD brain from Ki91 knock-in mouse. We found that transcriptional dysregulations resulting from mutant ataxin-3 are not occurring in young Ki91 mice, while old Ki91 mice and also postmitotic patient SCA3 neurons demonstrate the late transcriptomic changes. Unlike the lack of early mRNA changes, we have identified numerous early changes of total proteins and phosphoproteins in 2-month-old Ki91 mouse cortex and cerebellum. We discovered the network of processes in presymptomatic SCA3 with three main groups of disturbed processes comprising altered proteins: (I) modulation of protein levels and DNA damage (Pabpc1, Ddb1, Nedd8), (II) formation of neuronal cellular structures (Tubb3, Nefh, p-Tau), and (III) neuronal function affected by processes following perturbed cytoskeletal formation (Mt-Co3, Stx1b, p-Syn1). Phosphoproteins downregulate in the young Ki91 mouse brain and their phosphosites are associated with kinases that interact with ATXN3 such as casein kinase, Camk2, and kinases controlled by another Atxn3 interactor p21 such as Gsk3, Pka, and Cdk kinases. We conclude that the onset of SCA3 pathology occurs without altered transcript level and is characterized by changed levels of proteins responsible for termination of translation, DNA damage, spliceosome, and protein phosphorylation. This disturbs global cellular processes such as cytoskeleton and transport of vesicles and mitochondria along axons causing energy deficit and neurodegeneration also manifesting in an altered level of transcripts at later ages.

Absence of BBSome function leads to astrocyte reactivity in the brain

In humans, dysfunctional primary cilia result in Bardet-Biedl syndrome (BBS), which presents with clinical features including intellectual disabilities, obesity, and retinal degeneration, and, in mouse models, the added feature of hydrocephalus. We observed increased Glial Fibrillary Acidic Protein (GFAP) immunoreactivity in BBS mouse brains. Increased GFAP expression is a hallmark of astrocyte reactivity that is associated with microglia activation and neuro-inflammation. To gain a better understanding of reactive astrocytes observed in BBS mice, we used two mouse models of BBS8, a BBSome protein, to characterize the reactive astrocyte phenotype. The finding of reactive astrocytes in young BBS mouse brains led us to hypothesize that loss of BBSome function leads to reactive astrocytes prior to hydrocephalus and obesity. By using two mouse models of BBS8, a congenital BBS8 knockout with hydrocephalus, and a tamoxifen-inducible BBS8 knockout without hydrocephalus, we were able to molecularly phenotype the reactive astrocytes. Molecular phenotype of reactive astrocytes shows differential regulation of inducers of Pan, A1 neurotoxic, and A2 neuroprotective astrocytes that are significantly altered in brains of both congenital and induced knockouts of BBS8, but without microglia activation. We find evidence for neuroinflammation in the brains of congenital knockout mice, but not in induced knockout mice. Protein levels of GFAP, SERPINA3N and post-synaptic density 95 (PSD95) are significantly increased in congenital knockout mice, but remain unchanged in induced knockout mice. Thus, despite the reactive astrocyte phenotype being present in both models, the molecular signature of reactive astrocytes in BBS8 mice models are distinct. Together, these findings suggest that BBS8, and by extension the BBSome, plays a role in neuro-astrocyte functions independent of hydrocephalus, and its dysregulation is associated with astrocyte reactivity without microglia activation. (Total word count 278).

Lipidomic Alterations in the Mitochondria of Aged Parkin Null Mice Relevant to Autophagy.

Mitochondrial quality control is important in neurological diseases, but in genetic Parkinson’s disease caused by mutations in PINK and parkin mitochondrial degradation through autophagy is crucial. Reductions in autophagy and mitophagy are implicated in aging, age related diseases and Parkinson. The parkin null mice (PK-KO) show only a subtle phenotype, apparent with age or with stressors. We have studied the changes in the lipidomic composition of the mitochondrial membranes isolated from the brains of young and old PK-KO mice and compared them to wild type in order to determine possible implications for Parkinson’s disease pathology. We observed an increase in the levels of phosphatidylethanolamine in the young PK-KO mice that is lost in the old and correlate to changes in the phosphatidylserine decarboxylase. PK-KO old mice mitochondria showed lower phosphatidylglicerol and phosphatidylinositol levels and higher levels of some forms of hydroxylated ceramides. Regarding cardiolipins there were changes in the degree of saturation mainly with age. The lipidomic composition discriminates between the study groups using partial least square discriminant analysis. We discuss the relevance of the lipid changes for the autophagic activity, the mitophagy, the mitochondrial activity and the Parkinson’s disease pathology in absence of parkin.

Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system.

The protein doublecortin is mainly expressed in migrating neuroblasts and immature neurons. The X-linked gene MECP2, associated to several neurodevelopmental disorders such as Rett syndrome, encodes the protein methyl-CpG-binding protein 2 (MeCP2), a regulatory protein that has been implicated in neuronal maturation and refinement of olfactory circuits. Here, we explored doublecortin immunoreactivity in the brain of young adult female Mecp2-heterozygous and male Mecp2-null mice and their wild-type littermates. The distribution of doublecortin-immunoreactive somata in neurogenic brain regions was consistent with previous reports in rodents, and no qualitative differences were found between genotypes or sexes. Quantitatively, we found a significant increase in doublecortin cell density in the piriform cortex of Mecp2-null males as compared to WT littermates. A similar increase was seen in a newly identified population of doublecortin cells in the olfactory tubercle. In these olfactory structures, however, the percentage of doublecortin immature neurons that also expressed NeuN was not different between genotypes. By contrast, we found no significant differences between genotypes in doublecortin immunoreactivity in the olfactory bulbs. Nonetheless, in the periglomerular layer of Mecp2-null males, we observed a specific decrease of immature neurons co-expressing doublecortin and NeuN. Overall, no differences were evident between Mecp2-heterozygous and WT females. In addition, no differences could be detected between genotypes in the density of doublecortin-immunoreactive cells in the hippocampus or striatum of either males or females. Our results suggest that MeCP2 is involved in neuronal maturation in a region-dependent manner.

Click histochemistry for whole-mount staining of brain structures

Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs. Here we describe a method for labeling dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and their further detection in whole brain structures (for example, hippocampus) using the Cu (I) -catalyzed [3 + 2] cycloaddition reaction (so-called click-reaction). The presented method can be used for brain neurogenesis studies as well as for whole-mount staining of any preparations in which the terminal ethynyl group has been introduced.•New click histochemistry method based on Cu (I) -catalyzed [3 + 2] cycloaddition reaction allows whole-mount staining of brain structures and other tissues.•Our whole-mount click histochemistry method allows to visualize dividing cells in 3D and can be used in neurogenesis studies, i.e. for birthdating dividing early progenitors and further tracking of proliferation, survival, migration, differentiation, and fate of their progeny.•Our whole-mount click histochemistry staining demonstrates high staining specificity, high signal intensity, and low background levels in young and adult mouse brain tissue.

Age-related alteration in the distribution of methylglyoxal and its metabolic enzymes in the mouse brain

Methylglyoxal (MG) is an α-dicarbonyl compound that is naturally produced in vivo through glucose metabolism. In general, MG is metabolized by the glyoxalase 1(GLO1)/GLO2 system and aldose reductase (AR); however, excessive MG can react with proteins and nucleic acids to induce the accumulation of advanced glycation end products (AGEs). Recently, the accumulation of AGEs in the brain has been presumed to be related to neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, respectively. Research investigating the role of AGEs in such diseases is ongoing. However, the changes in MG concentration that occur in the brain during healthy ageing remain unclear. Therefore, we performed fractionation of the brains of aged and young mice, measured the MG concentration in each part of the brain, and then examined the distribution. We also investigated the expression levels of GLO1 and AR, the main metabolizing enzymes of MG, in various brain regions, across age groups. We show that MG concentration varies among different regions of the brain, and that MG concentration in aged mice is significantly lower than that in young mice across all regions of the brain, except the brain stem. In addition, although the expression level of the GLO1 protein in the brain did not change with ageing, the expression level of AR was higher in aged than in young mice. Moreover, although a significant positive correlation was observed between GLO1 expression and MG concentration in the brains of young mice, no significant correlations were observed in the brains of aged mice. Meanwhile, the production of protein carbonyls and the accumulation of AGEs were not observed in the brains of aged mice. These results suggest that the accumulation of MG in the brain, along with the carbonyl stress are suppressed and regionally controlled during healthy ageing. This finding is useful as the foundation for further studies to investigate the role and toxicity of MG in various age-related disease conditions.

Contribution of the inflammasome to inflammaging

BackgroundInflammation is a natural part of the aging process. This process is referred to as inflammaging. Inflammaging has been associated with deleterious outcomes in the aging brain in diseases such as Alzheimer’s disease and Parkinson’s disease. The inflammasome is a multi-protein complex of the innate immune response involved in the activation of caspase-1 and the processing of the inflammatory cytokines interleukin (IL)-1β and IL-18. We have previously shown that the inflammasome plays a role in the aging process in the brain. In this study, we analyzed the brain of young (3 months old) and aged (18 months old) mice for the expression of inflammasome proteins.ResultsOur findings indicate that the inflammasome proteins NLRC4, caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and IL-18 are elevated in the cytosol of cortical lysates in aged mice when compared to young. In addition, in the cytosolic fraction of hippocampal lysates in aged mice, we found an increase in NLRC4, caspase-1, caspase-11, ASC and IL-1β. Moreover, we found higher levels of ASC in the mitochondrial fraction of aged mice when compared to young, consistent with higher levels of the substrate of pyroptosis gasdermin-D (GSDM-D) and increased pyroptosome formation (ASC oligomerization). Importantly, in this study we obtained fibroblasts from a subject that donated his cells at three different ages (49, 52 and 64 years old (y/o)) and found that the protein levels of caspase-1 and ASC were higher at 64 than at 52 y/o. In addition, the 52 y/o cells were more susceptible to oxidative stress as determined by lactose dehydrogenase (LDH) release levels. However, this response was ameliorated by inhibition of the inflammasome with Ac-Tyr-Val-Ala-Asp-Chloromethylketone (Ac-YVAD-CMK). In addition, we found that the protein levels of ASC and IL-18 are elevated in the serum of subjects over the age of 45 y/o when compared to younger subjects, and that ASC was higher in Caucasians than Blacks and Hispanics, whereas IL-18 was higher in Caucasians than in blacks, regardless of age.ConclusionsTaken together, our data indicate that the inflammasome contributes to inflammaging and that the inflammasome-mediated cell death mechanism of pyroptosis contributes to cell demise in the aging brain.

Age-specific changes in genome-wide methylation enrich for Foxa2 and estrogen receptor alpha binding sites.

The role of DNA methylation patterns in complex phenotypes remains unclear. To explore this question, we adapted our methods for rare variant analysis to characterize genome-wide murine DNA hybridization array to investigate methylation at CpG islands, shores, and regulatory elements. We have applied this platform to compare age and tissue- specific methylation differences in the brain and spleen of young and aged mice. As expected from prior studies, there are clear global differences in organ-specific, but not age-specific, methylation due mostly to changes at repetitive elements. Surprisingly, out of 200,000 loci there were only 946 differentially methylated cytosines (DMCs) between young and old samples (529 hypermethylated, 417 hypomethylated in aged mice) compared to thousands of tissue-specific DMCs. Hypermethylated loci were clustered around the promoter region of Sfi1, exon 2 of Slc11a2, Drg1, Esr1 and Foxa2 transcription factor binding sites. In particular, there were 75 hypermethylated Foxa2 binding sites across a 2.7 Mb region of chromosome 11. Hypomethylated loci were clustered around Mid1, Isoc2b and genome-wide loci with binding sites for Foxa2 and Esr1, which are known to play important roles in development and aging. These data suggest discreet tissue-independent methylation changes associated with aging processes such as cell division (Sfi1, Mid1), energy production (Drg1, Isoc2b) and cell death (Foxa2, Esr1).

P53 Loss Mitigates Early Volume Deficits in the Brains of Irradiated Young Mice

Pediatric cranial radiation therapy results in lasting changes in brain structure. Though different facets of radiation response have been characterized, the relative contributions of each to altered development is unclear. We sought to determine the role of radiation-induced programmed cell death, as mediated by the Trp53 (p53) gene, on neuroanatomic development. Mice having a conditional knockout of p53 (p53KO) or wildtype p53 (WT) were irradiated with a whole-brain dose of 7Gy (IR; n=30) or 0Gy (sham; n=28) at 16days of age. Invivo magnetic resonance imaging was performed before irradiation and at 4 time points after irradiation, until 3months posttreatment, followed by exvivo magnetic resonance imaging and immunohistochemistry. The role of p53 in development was assessed at 6weeks of age in another group of untreated mice (n=37). Neuroanatomic development in p53KO mice was normal. After cranial irradiation, alterations in neuroanatomy were detectable in WT mice and emerged through 2 stages: an early volume loss within 1week and decreased growth through development. In many structures, the early volume loss was partially mitigated by p53KO. However, p53KO had a neutral or negative impact on growth; thus, p53KO did not widely improve volume at endpoint. Partial volume recovery was observed in the dentate gyrus and olfactory bulbs of p53KO-IR mice, with corresponding increases in neurogenesis compared with WT-IR mice. Although p53 is known to play an important role in mediating radiation-induced apoptosis, this is the first study to look at the cumulative effect of p53KO through development after cranial irradiation across the entire brain. It is clear that apoptosis plays an important role in volume loss early after radiation therapy. This early preservation alone was insufficient to normalize brain development on the whole, but regions reliant on neurogenesis exhibited a significant benefit.

Assessment of pathological features in Alzheimer's disease brain tissue with a large field-of-view visible-light optical coherence microscope.

.We implemented a wide field-of-view visible-light optical coherence microscope (OCM) for investigating ex-vivo brain tissue of patients diagnosed with Alzheimer’s disease (AD) and of a mouse model of AD. A submicrometer axial resolution in tissue was achieved using a broad visible light spectrum. The use of various objective lenses enabled reaching micrometer transversal resolution and the acquisition of images of microscopic brain features, such as cell structures, vessels, and white matter tracts. Amyloid-beta plaques in the range of 10 to were visualized. Large field-of-view images of young and old mouse brain sections were imaged using an automated stage. The plaque load was characterized, revealing an age-related increase. Human brain tissue affected by cerebral amyloid angiopathy was investigated and hyperscattering structures resembling amyloid beta accumulations in the vessel walls were identified. All results were in good agreement with histology. A comparison of plaque features in both human and mouse brain tissue was performed, revealing an increase in plaque load and a decrease in reflectivity for mouse as compared with human brain tissue. Based on the promising outcome of our experiments, visible light OCM might be a powerful tool for investigating microscopic features in ex-vivo brain tissue.

Systematic analysis of GSK-3 signaling pathways in aging of cerebral tissue.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase, involved in regulation of multiple physiological processes. In brain, changes in GSK-3 signaling are related to neurodegenerative issues, including Alzheimer's disease. Due to the wide range of GSK-3 cellular targets, a therapeutic use of the enzyme inhibitors entails significant risk of side effects. Thus, altering the ratio of specific pool of GSK-3 or specific substrates instead of changing the global activity of GSK-3 in brains might be a more appropriate strategy. This paper provides a comprehensive data on abundances of proteins involved in GSK-3 signaling in three regions of young and old mouse brains. It might help to identify novel protein targets with the highest therapeutic potential for treatment of age-related neurodegenerative diseases.

Aging‐induced Mitochondrial Dysfunction in the Mouse Brain Microvessels: Effect of Neuronal Nitric Oxide Synthase Inhibition

Aging‐induced cerebrovascular changes contribute to the increased risk of stroke and neurodegenerative diseases. Notably, aging‐induced impairments of cerebral microcirculation have been implicated in cognitive dysfunction. Mitochondrial dysfunction plays an important role in the age‐related cerebrovascular changes; however, mitochondrial respiratory dysfunction has never been studied in the microvasculature. Similarly, neuronal nitric oxide synthase (nNOS) is known to play a critical role in various cells of cerebrovasculature, but its effects on mitochondrial respiration in the young and aged brain microvessels have never been examined previously. Here, we studied the mitochondrial respiration and the responses to nNOS inhibition in the in the aged‐microvessels from mouse brain.MethodsWe isolated the microvessels from the saline‐perfused brains from young (3 months of age) and aged mice (18 months of age) and collected microvessels by a combination of gradient centrifugation and filtration (40μm – 300μm). Isolated microvessels were treated with saline or nNOS inhibitor, ARL‐17477 (1μM, ARL) and plated into the wells of Seahorse XFe24 microculture plate. Oxygen consumption rate (OCR) was measured at the basal level and after the injection of oligomycin, FCCP and antimycin/Rotenone. Basal and maximal respiration were measured along with ATP production, proton leak, and maximal respiration. We also measured the reactive oxygen species (ROS) levels by electron spin resonance spectrometry (ESR).ResultsAged‐microvessels showed 40% decrease in the basal respiration, whereas maximal respiration is decreased by 49% when compared with the microvessels from the young mouse brain. ATP production, proton leak, and non‐mitochondrial respiration were decreased by 36%, 40%, and 72% respectively in the aged‐microvessels when compared to the young vessels. Aged‐microvessels also showed increased ROS levels by 38.6%. ARL treatment increased the maximal respiration by 21.2% (101.8±6.4 vs 84.0±8.9 picomoles of O2/min/μg, p&lt;0.05) and ATP production by 19% (52.3±4.4 vs 43.9±3.6 picomoles of O2/min/μg) in mouse brain microvessels. Basal respiration, proton leak, and non‐mitochondrial respiration were not altered by ARL treatment in the microvessels in young mice. In contrast, none of the respiratory parameters were affected by ARL in the aged‐microvessels. Interestingly, nNOS inhibition significantly decreased ROS levels in microvessels from both young and old mice compared to the respective untreated microvessels (p&lt;0.05). Notably, ROS levels following nNOS inhibition was significantly greater in microvessels from young than aged mice (p&lt;0.05) indicating a reduction of nNOS function with aging.ConclusionsOur study showed that mitochondrial respiratory parameters were compromised in the aged‐microvessels from the mouse brain, indicating a possible role of mitochondrial dysfunction in the aging‐associated cerebral microvascular impairments. In addition, nNOS appears to negatively regulate mitochondrial respiration in the microvasculature of the young mouse brain but the nNOS effect on mitochondrial respiration was lost in aged brain microvesselsSupport or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006), and National Institute of Health: (PVK: R01NS094834)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

THE NLRC4 INFLAMMASOME CONTRIBUTES TO BRAIN INFLAMMAGING

Inflammation is a natural part of aging in a process known as inflammaging. Inflammaging has been associated with deleterious outcomes in the aging brain. It is believed that inflammaging precedes the onset of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. The inflammasome is an arm of the innate immune response involved in the activation of the inflammatory caspase-1 and the processing of the pro-inflammatory cytokines interleukin (IL)-1b and IL-18. We have previously shown that inflammasome contributes to the inflammatory response in the central nervous system after brain and spinal cord injury as well as stroke. In addition, we have shown that inflammasome inhibition in the aging brain results in improved cognitive outcomes. In this study we analyzed the brain of young (3 months old) and aged (18 months old) mice for the expression of inflammasome proteins. Our findings indicate that the inflammasome proteins NLRC4, caspase-1, ASC, and IL-18 are elevated in the cytoplasm of cortical lysates in aged mice when compared to young. Similarly, in the cytoplasmic fraction of hippocampal lysates in aged mice, we found an increase in NLRC4, caspase-1, caspase-11, ASC and IL-1b. Moreover, ASC is involved in the cell death mechanism of pyroptosis. Interestingly, the pyroptosome is elevated in the brain of aged mice. Taken together, our data indicate that the NLCR4 inflammasome contributes to brain inflammaging and that pyroptosis contributes to cell death in the aging brain; thus highlighting the inflammasome as a novel therapeutic target for the treatment of brain inflammaging.

An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.