Fasting-mimicking diet causes hepatic and blood markers changes indicating reduced biological age and disease risk.

In mice, periodic cycles of a fasting mimicking diet (FMD) protect normal cells while killing damaged cells including cancer and autoimmune cells, reduce inflammation, promote multi-system regeneration, and extend longevity. Here, we performed secondary and exploratory analysis of blood samples from a randomized clinical trial (NCT02158897) and show that 3 FMD cycles in adult study participants are associated with reduced insulin resistance and other pre-diabetes markers, lower hepatic fat (as determined by magnetic resonance imaging) and increased lymphoid to myeloid ratio: an indicator of immune system age. Based on a validated measure of biological age predictive of morbidity and mortality, 3 FMD cycles were associated with a decrease of 2.5 years in median biological age, independent of weight loss. Nearly identical findings resulted from  a second clinical study (NCT04150159). Together these results provide initial support for beneficial effects of the FMD on multiple cardiometabolic risk factors and biomarkers of biological age.

Air pollution nanoparticle and alpha-synuclein fibrils synergistically decrease glutamate receptor A1, depending upon nPM batch activity.

Background: Epidemiological studies have variably linked air pollution to increased risk of Parkinson's disease (PD). However, there is little experimental evidence for this association. Alpha-synuclein (α-syn) propagation plays central roles in PD and glutamate receptor A1 (GluA1) is involved in memory and olfaction function. Methods: Each mouse was exposed to one of three different batches of nano-particulate matter (nPM) (300 µg/m3, 5 h/d, 3 d/week), collected at different dates, 2017-2019, in the same urban site. After these experiments, these nPM batches were found to vary in activity. C57BL/6 female mice (3 mo) were injected with pre-formed murine α-synuclein fibrils (PFFs) (0.4 µg), which act as seeds for α-syn aggregation. Two exposure paradigms were used: in Paradigm 1, PFFs were injected into olfactory bulb (OB) prior to 4-week nPM (Batch 5b) exposure and in Paradigm 2, PFFs were injected at 4th week during 10-week nPM exposure (Batches 7 and 9). α-syn pSer129, microglia Iba1, inflammatory cytokines, and Gria1 expression were measured by immunohistochemistry or qPCR assays. Results: As expected, α-syn pSer129 was detected in ipsilateral OB, anterior olfactory nucleus, amygdala and piriform cortex. One of the three batches of nPM caused a trend for elevated α-syn pSer129 in Paradigm 1, but two other batches showed no effect in Paradigm 2. However, the combination of nPM and PFF significantly decreased Gria1 mRNA in both the ipsi- and contra-lateral OB and frontal cortex for the most active two nPM batches. Neither nPM nor PFFs alone induced responses of microglia Iba1 and expression of Gria1 in the OB and cortex. Conclusion: Exposures to ambient nPM had weak effect on α-syn propagation in the brain in current experimental paradigms; however, nPM and α-syn synergistically downregulated the expression of Gria1 in both OB and cortex.

Alzheimer's disease ferroptotic associations with oxidative damage and neuronal loss.

The role of reactive iron in Alzheimer's Disease (AD) remains unresolved. Little is known of how AD may alter iron transport, glutathione-mediated oxidative repair, and their associations with ApoE alleles. Postmortem brain intravascular blood was minimized by washing minced brain (n=24/group). HNE from iron-associated lipid peroxidation increased in AD prefrontal cortex by 50% for whole tissue and in subcellular lipid rafts, where Aß-peptides are produced. HNE correlated with iron storage ferritin light chain (FTL; r=0.35); both were higher in ApoE4. Iron chelation by DFO in EFAD mice decreased HNE consistent with ferroptosis. Neuronal and synaptic loss in AD was inversely correlated to FTL (r=-0.55). AD decreased levels of ferroptosis suppressor protein 1, glutamate cysteine ligase modulator subunit (GCLM), and lipid raft glutathione peroxidase 4 (GPx4), mitigators of ferroptosis. These findings provide a mechanistic framework for iron-associated neurodegeneration during AD by impaired lipid peroxidation repair mechanisms involving glutathione.

Particulate matter exposure and chronic cerebral hypoperfusion promote oxidative stress and induce neuronal and oligodendrocyte apoptosis in male mice.

Chronic cerebral hypoperfusion (CCH) may amplify the neurotoxicity of nanoscale particulate matter (nPM), resulting in white matter injury. This study characterized the joint effects of nPM (diameter ≤ 200 nm) and CCH secondary to bilateral carotid artery stenosis (BCAS) exposure on neuronal and white matter injury in a murine model. nPM was collected near a highway and re-aerosolized for exposure. Ten-week-old C57BL/6 male mice were randomized into four groups: filtered air (FA), nPM, FA + BCAS, and nPM + BCAS. Mice were exposed to FA or nPM for 10 weeks. BCAS surgeries were performed. Markers of inflammation, oxidative stress, and apoptosis were examined. nPM + BCAS exposure increased brain hemisphere TNFα protein compared to FA. iNOS and HNE immunofluorescence were increased in the corpus callosum and cerebral cortex of nPM + BCAS mice compared to FA. While nPM exposure alone did not decrease cortical neuronal cell count, nPM decreased corpus callosum oligodendrocyte cell count. nPM exposure decreased mature oligodendrocyte cell count and increased oligodendrocyte precursor cell count in the corpus callosum. nPM + BCAS mice exhibited a 200% increase in cortical neuronal TUNEL staining and a 700% increase in corpus callosum oligodendrocyte TUNEL staining compared to FA. There was a supra-additive interaction between nPM and BCAS on cortical neuronal TUNEL staining (2.6× the additive effects of nPM + BCAS). nPM + BCAS exposure increased apoptosis, neuroinflammation, and oxidative stress in the cerebral cortex and corpus callosum. nPM + BCAS exposure increased neuronal apoptosis above the separate responses to each exposure. However, oligodendrocytes in the corpus callosum demonstrated a greater susceptibility to the combined neurotoxic effects of nPM + BCAS exposure.

Air Pollution Particulate Matter Amplifies White Matter Vascular Pathology and Demyelination Caused by Hypoperfusion.

Cerebrovascular pathologies are commonly associated with dementia. Because air pollution increases arterial disease in humans and rodent models, we hypothesized that air pollution would also contribute to brain vascular dysfunction. We examined the effects of exposing mice to nanoparticulate matter (nPM; aerodynamic diameter ≤200 nm) from urban traffic and interactions with cerebral hypoperfusion. C57BL/6 mice were exposed to filtered air or nPM with and without bilateral carotid artery stenosis (BCAS) and analyzed by multiparametric MRI and histochemistry. Exposure to nPM alone did not alter regional cerebral blood flow (CBF) or blood brain barrier (BBB) integrity. However, nPM worsened the white matter hypoperfusion (decreased CBF on DSC-MRI) and exacerbated the BBB permeability (extravascular IgG deposits) resulting from BCAS. White matter MRI diffusion metrics were abnormal in mice subjected to cerebral hypoperfusion and worsened by combined nPM+BCAS. Axonal density was reduced equally in the BCAS cohorts regardless of nPM status, whereas nPM exposure caused demyelination in the white matter with or without cerebral hypoperfusion. In summary, air pollution nPM exacerbates cerebrovascular pathology and demyelination in the setting of cerebral hypoperfusion, suggesting that air pollution exposure can augment underlying cerebrovascular contributions to cognitive loss and dementia in susceptible elderly populations.

Fasting-mimicking diet prevents high-fat diet effect on cardiometabolic risk and lifespan.

Diet-induced obesity is a major risk factor for metabolic syndrome, diabetes and cardiovascular disease. Here, we show that a 5-d fasting-mimicking diet (FMD), administered every 4 weeks for a period of 2 years, ameliorates the detrimental changes caused by consumption of a high-fat, high-calorie diet (HFCD) in female mice. We demonstrate that monthly FMD cycles inhibit HFCD-mediated obesity by reducing the accumulation of visceral and subcutaneous fat without causing loss of lean body mass. FMD cycles increase cardiac vascularity and function and resistance to cardiotoxins, prevent HFCD-dependent hyperglycaemia, hypercholesterolaemia and hyperleptinaemia and ameliorate impaired glucose and insulin tolerance. The effect of monthly FMD cycles on gene expression associated with mitochondrial metabolism and biogenesis in adipocytes and the sustained ketogenesis in HFCD-fed mice indicate a role for fat cell reprogramming in obesity prevention. These effects of an FMD on adiposity and cardiac ageing could explain the protection from HFCD-dependent early mortality.

Air Pollution Particulate Matter Exposure and Chronic Cerebral Hypoperfusion and Measures of White Matter Injury in a Murine Model.

BACKGROUND: Exposure to ambient air pollution particulate matter (PM) is associated with increased risk of dementia and accelerated cognitive loss. Vascular contributions to cognitive impairment are well recognized. Chronic cerebral hypoperfusion (CCH) promotes neuroinflammation and blood-brain barrier weakening, which may augment neurotoxic effects of PM. OBJECTIVES: This study examined interactions of nanoscale particulate matter (nPM; fine particulate matter with aerodynamic diameter ≤200 nm) and CCH secondary to bilateral carotid artery stenosis (BCAS) in a murine model to produce white matter injury. Based on other air pollution interactions, we predicted synergies of nPM with BCAS. METHODS: nPM was collected using a particle sampler near a Los Angeles, California, freeway. Mice were exposed to 10 wk of reaerosolized nPM or filtered air (FA) for 150 h. CCH was induced by BCAS surgery. Mice (C57BL/6J males) were randomized to four exposure paradigms: a) FA, b) nPM, c) FA + BCAS, and d) nPM + BCAS. Behavioral outcomes, white matter injury, glial cell activation, inflammation, and oxidative stress were assessed. RESULTS: The joint nPM + BCAS group exhibited synergistic effects on white matter injury (2.3× the additive nPM and FA + BCAS scores) with greater loss of corpus callosum volume on T2 magnetic resonance imaging (MRI) (30% smaller than FA group). Histochemical analyses suggested potential microglial-specific inflammatory responses with synergistic effects on corpus callosum C5 immunofluorescent density and whole brain nitrate concentrations (2.1× and 3.9× the additive nPM and FA + BCAS effects, respectively) in the joint exposure group. Transcriptomic responses (RNA-Seq) showed greater impact of nPM + BCAS than individual additive effects, consistent with changes in proinflammatory pathways. Although nPM exposure alone did not alter working memory, the nPM + BCAS cohort demonstrated impaired working memory when compared to the FA + BCAS group. DISCUSSION: Our data suggest that nPM and CCH contribute to white matter injury in a synergistic manner in a mouse model. Adverse neurological effects may be aggravated in a susceptible population exposed to air pollution. https://doi.org/10.1289/EHP8792.

Cerebral cortex and blood transcriptome changes in mouse neonates prenatally exposed to air pollution particulate matter.

BACKGROUND: Prenatal exposure to air pollutants is associated with increased risk for neurodevelopmental and neurodegenerative disorders. However, few studies have identified transcriptional changes related to air pollutant exposure. METHODS: RNA sequencing was used to examine transcriptomic changes in blood and cerebral cortex of three male and three female mouse neonates prenatally exposed to traffic-related nano-sized particulate matter (nPM) compared to three male and three female mouse neonates prenatally exposed to control filter air. RESULTS: We identified 19 nPM-associated differentially expressed genes (nPM-DEGs) in blood and 124 nPM-DEGs in cerebral cortex. The cerebral cortex transcriptional responses to nPM suggested neuroinflammation involvement, including CREB1, BDNF, and IFNγ genes. Both blood and brain tissues showed nPM transcriptional changes related to DNA damage, oxidative stress, and immune responses. Three blood nPM-DEGs showed a canonical correlation of 0.98 with 14 nPM-DEGS in the cerebral cortex, suggesting a convergence of gene expression changes in blood and cerebral cortex. Exploratory sex-stratified analyses suggested a higher number of nPM-DEGs in female cerebral cortex than male cerebral cortex. The sex-stratified analyses identified 2 nPM-DEGs (Rgl2 and Gm37534) shared between blood and cerebral cortex in a sex-dependent manner. CONCLUSIONS: Our findings suggest that prenatal nPM exposure induces transcriptional changes in the cerebral cortex, some of which are also observed in blood. Further research is needed to replicate nPM-induced transcriptional changes with additional biologically relevant time points for brain development.

Nanoparticulate matter exposure results in white matter damage and an inflammatory microglial response in an experimental murine model.

Exposure to ambient air pollution has been associated with white matter damage and neurocognitive decline. However, the mechanisms of this injury are not well understood and remain largely uncharacterized in experimental models. Prior studies have shown that exposure to particulate matter (PM), a sub-fraction of air pollution, results in neuroinflammation, specifically the upregulation of inflammatory microglia. This study examines white matter and axonal injury, and characterizes microglial reactivity in the corpus callosum of mice exposed to 10 weeks (150 hours) of PM. Nanoscale particulate matter (nPM, aerodynamic diameter ≤200 nm) consisting primarily of traffic-related emissions was collected from an urban area in Los Angeles. Male C57BL/6J mice were exposed to either re-aerosolized nPM or filtered air for 5 hours/day, 3 days/week, for 10 weeks (150 hours; n = 18/group). Microglia were characterized by immunohistochemical double staining of ionized calcium-binding protein-1 (Iba-1) with inducible nitric oxide synthase (iNOS) to identify pro-inflammatory cells, and Iba-1 with arginase-1 (Arg) to identify anti-inflammatory/ homeostatic cells. Myelin injury was assessed by degraded myelin basic protein (dMBP). Oligodendrocyte cell counts were evaluated by oligodendrocyte transcription factor 2 (Olig2). Axonal injury was assessed by axonal neurofilament marker SMI-312. iNOS-expressing microglia were significantly increased in the corpus callosum of mice exposed to nPM when compared to those exposed to filtered air (2.2 fold increase; p<0.05). This was accompanied by an increase in dMBP (1.4 fold increase; p<0.05) immunofluorescent density, a decrease in oligodendrocyte cell counts (1.16 fold decrease; p<0.05), and a decrease in neurofilament SMI-312 (1.13 fold decrease; p<0.05) immunofluorescent density. Exposure to nPM results in increased inflammatory microglia, white matter injury, and axonal degradation in the corpus callosum of adult male mice. iNOS-expressing microglia release cytokines and reactive oxygen/ nitrogen species which may further contribute to the white matter damage observed in this model.

Urban Air Pollution Nanoparticles from Los Angeles: Recently Decreased Neurotoxicity.

BACKGROUND: Air pollution is widely associated with accelerated cognitive decline at later ages and risk of Alzheimer's disease (AD). Correspondingly, rodent models demonstrate the neurotoxicity of ambient air pollution and its components. Our studies with nano-sized particulate matter (nPM) from urban Los Angeles collected since 2009 have shown pro-amyloidogenic and pro-inflammatory responses. However, recent batches of nPM have diminished induction of the glutamate receptor GluA1 subunit, Iba1, TNFα, Aß42 peptide, and white matter damage. The same methods, materials, and mouse genotypes were used throughout. OBJECTIVE: Expand the nPM batch comparisons and evaluate archived brain samples to identify the earliest change in nPM potency. METHODS: Batches of nPM were analyzed by in vitro cell assays for NF-κB and Nrf2 induction for comparison with in vivo responses of mouse brain regions from mice exposed to these batches, analyzed by PCR and western blot. RESULTS: Five older nPM batches (2009-2017) and four recent nPM batches (2018, 2019) for NF-κB and Nrf2 induction showed declines in nPM potency after 2017 that paralleled declines of in vivo activity from independent exposures in different years. CONCLUSION: Transcription-based in vitro assays of nPM corresponded to the loss of in vivo potency for inflammatory and oxidative responses. These recent decreases of nPM neurotoxicity give a rationale for evaluating possible benefits to the risk of dementia and stroke in Los Angeles populations.

Age, sex, and cerebral microbleeds in EFAD Alzheimer disease mice.

Cerebral microbleeds (MBs) increase at later ages in association with increased cognitive decline and Alzheimer Disease (AD). MB prevalence is also increased by APOE4 and hypertension. In EFAD mice (5XFAD+/-/human APOE+/+), cerebral cortex MBs are most prevalent in E4 females at 6 months, paralleling plaque amyloid. We evaluated MBs at 2, 4, and 6 months in relation to amyloid in plaques and cerebral amyloid angiopathy (CAA) by age, sex, APOE allele, and blood pressure. At 2 mo, MBs were 50% more numerous than plaques, followed by decreased ratio of MBs:Aß plaques with female excess to 6 mo. The stable size of MBs suggests MBs arise as single events of extravasation, which may "seed" plaque formation. Blood pressure was normal from 2 to 6 months, minimizing a role of hypertension. Memory, assessed by fear conditioning, decreased with age in correlation with MBs and amyloid. Cortical layer analysis showed prevalent MBs and plaque in layers 4 and 5. Contrarily, CAA was prevalent in layers 1 and 2, discounting its contribution to MBs.

Age-related alteration in HNE elimination enzymes.

4-hydroxynonenal (HNE, 4-hydroxy-2-nonenal) is a primary α,ß-unsaturated aldehyde product of lipid peroxidation. The accumulation of HNE increases with aging and the mechanisms are mainly attributable to increased oxidative stress and decreased capacity of HNE elimination. In this review article, we summarize the studies on age-related change of HNE concentration and alteration of HNE metabolizing enzymes (GCL, GST, ALDHs, aldose reductase, and 20S-proteasome), and discuss potential mechanism of age-related decrease in HNE-elimination capacity by focusing on Nrf2 redox signaling.

The APOE gene cluster responds to air pollution factors in mice with coordinated expression of genes that differs by age in humans.

Little is known of gene-environment interactions for Alzheimer's disease (AD) risk factors. Apolipoprotein E (APOE) and neighbors on chromosome 19q13.3 have variants associated with risks of AD, but with unknown mechanism. This study describes novel links among the APOE network, air pollution, and age-related diseases. Mice exposed to air pollution nano-sized particulate matter (nPM) had coordinate responses of Apoe-Apoc1-Tomm40 in the cerebral cortex. In humans, the AD vulnerable hippocampus and amygdala had stronger age decline in APOE cluster expression than the AD-resistant cerebellum and hypothalamus. Using consensus weighted gene co-expression network, we showed that APOE has a conserved co-expressed network in rodent and primate brains. SOX1, which has AD-associated single nucleotide polymorphisms, was among the co-expressed genes in the human hippocampus. Humans and mice shared 87% of potential binding sites for transcription factors in APOE cluster promoter, suggesting similar inducibility and a novel link among environment, APOE cluster, and risk of AD.

Effects of ambient particulate matter on vascular tissue: a review.

Fine and ultra-fine particulate matter (PM) are major constituents of urban air pollution and recognized risk factors for cardiovascular diseases. This review examined the effects of PM exposure on vascular tissue. Specific mechanisms by which PM affects the vasculature include inflammation, oxidative stress, actions on vascular tone and vasomotor responses, as well as atherosclerotic plaque formation. Further, there appears to be a greater PM exposure effect on susceptible individuals with pre-existing cardiovascular conditions.

Adult mouse hippocampal transcriptome changes associated with long-term behavioral and metabolic effects of gestational air pollution toxicity.

Gestational exposure to air pollution increases the risk of autism spectrum disorder and cognitive impairments with unresolved molecular mechanisms. This study exposed C57BL/6J mice throughout gestation to urban-derived nanosized particulate matter (nPM). Young adult male and female offspring were studied for behavioral and metabolic changes using forced swim test, fat gain, glucose tolerance, and hippocampal transcriptome. Gestational nPM exposure caused increased depressive behaviors, decreased neurogenesis in the dentate gyrus, and increased glucose tolerance in adult male offspring. Both sexes gained fat and body weight. Gestational nPM exposure induced 29 differentially expressed genes (DEGs) in adult hippocampus related to cytokine production, IL17a signaling, and dopamine degradation in both sexes. Stratification by sex showed twofold more DEGs in males than females (69 vs 37), as well as male-specific enrichment of DEGs mediating serotonin signaling, endocytosis, Gαi, and cAMP signaling. Gene co-expression analysis (WCGNA) identified a module of 43 genes with divergent responses to nPM between the sexes. Chronic changes in 14 DEGs (e.g., microRNA9-1) were associated with depressive behaviors, adiposity and glucose intolerance. These genes enriched neuroimmune pathways such as HMGB1 and TLR4. Based on cerebral cortex transcriptome data of neonates, we traced the initial nPM responses of HMGB1 pathway. In vitro, mixed glia responded to 24 h nPM with lower HMGB1 protein and increased proinflammatory cytokines. This response was ameliorated by TLR4 knockdown. In sum, we identified transcriptional changes that could be associated with air pollution-mediated behavioral and phenotypic changes. These identified genes merit further mechanistic studies for therapeutic intervention development.

Mouse brain transcriptome responses to inhaled nanoparticulate matter differed by sex and APOE in Nrf2-Nfkb interactions.

The neurotoxicity of air pollution is undefined for sex and APOE alleles. These major risk factors of Alzheimer's disease (AD) were examined in mice given chronic exposure to nPM, a nano-sized subfraction of urban air pollution. In the cerebral cortex, female mice had two-fold more genes responding to nPM than males. Transcriptomic responses to nPM had sex-APOE interactions in AD-relevant pathways. Only APOE3 mice responded to nPM in genes related to Abeta deposition and clearance (Vav2, Vav3, S1009a). Other responding genes included axonal guidance, inflammation (AMPK, NFKB, APK/JNK signaling), and antioxidant signaling (NRF2, HIF1A). Genes downstream of NFKB and NRF2 responded in opposite directions to nPM. Nrf2 knockdown in microglia augmented NFKB responses to nPM, suggesting a critical role of NRF2 in air pollution neurotoxicity. These findings give a rationale for epidemiologic studies of air pollution to consider sex interactions with APOE alleles and other AD-risk genes.

Air Pollution Neurotoxicity in the Adult Brain: Emerging Concepts from Experimental Findings.

Epidemiological studies are associating elevated exposure to air pollution with increased risk of Alzheimer's disease and other neurodegenerative disorders. In effect, air pollution accelerates many aging conditions that promote cognitive declines of aging. The underlying mechanisms and scale of effects remain largely unknown due to its chemical and physical complexity. Moreover, individual responses to air pollution are shaped by an intricate interface of pollutant mixture with the biological features of the exposed individual such as age, sex, genetic background, underlying diseases, and nutrition, but also other environmental factors including exposure to cigarette smoke. Resolving this complex manifold requires more detailed environmental and lifestyle data on diverse populations, and a systematic experimental approach. Our review aims to summarize the modest existing literature on experimental studies on air pollution neurotoxicity for adult rodents and identify key gaps and emerging challenges as we go forward. It is timely for experimental biologists to critically understand prior findings and develop innovative approaches to this urgent global problem. We hope to increase recognition of the importance of air pollution on brain aging by our colleagues in the neurosciences and in biomedical gerontology, and to support the immediate translation of the findings into public health guidelines for the regulation of remedial environmental factors that accelerate aging processes.

Toxicity of urban air pollution particulate matter in developing and adult mouse brain: Comparison of total and filter-eluted nanoparticles.

Air pollution (AirP) is associated with many neurodevelopmental and neurological disorders in human populations. Rodent models show similar neurotoxic effects of AirP particulate matter (PM) collected by different methods or from various sources. However, controversies continue on the identity of the specific neurotoxic components and mechanisms of neurotoxicity. We collected urban PM by two modes at the same site and time: direct collection as an aqueous slurry (sPM) versus a nano-sized sub-fraction of PM0.2 that was eluted from filters (nPM). The nPM lacks water-insoluble PAHs (polycyclic aromatic hydrocarbons) and is depleted by >50% in bioactive metals (e.g., copper, iron, nickel), inorganic ions, black carbon, and other organic compounds. Three biological models were used: in vivo exposure of adult male mice to re-aerosolized nPM and sPM for 3 weeks, gestational exposure, and glial cell cultures. In contrast to larger inflammatory responses of sPM in vitro, cerebral cortex responses of mice to sPM and nPM largely overlapped for adult and gestational exposures. Adult brain responses included induction of IFNγ and NF-κB. Gestational exposure to nPM and sPM caused equivalent depressive behaviors. Responses to nPM and sPM diverged for cerebral cortex glutamate receptor mRNA, systemic fat gain and insulin resistance. The shared toxic responses of sPM with nPM may arise from shared transition metals and organics. In contrast, gestational exposure to sPM but not nPM, decreased glutamatergic mRNAs, which may be attributed to PAHs. We discuss potential mechanisms in the overlap between nPM and sPM despite major differences in bulk chemical composition.