Functional gains in energy and cell metabolism after TSPO gene insertion.

Recent loss-of-function studies in tissue-specific as well as global Tspo (Translocator Protein 18 kDa) knockout mice have not confirmed its long assumed indispensability for the translocation of cholesterol across the mitochondrial inter-membrane space, a rate-limiting step in steroid biosynthesis. Instead, recent studies in global Tspo knockout mice indicate that TSPO may play a more fundamental role in cellular bioenergetics, which may include the indirect down-stream regulation of transport or metabolic functions. To examine whether overexpression of the TSPO protein alters the cellular bioenergetic profile, Jurkat cells with low to absent endogenous expression were transfected with a TSPO construct to create a stable cell line with de novo expression of exogenous TSPO protein. Expression of TSPO was confirmed by RT-qPCR, radioligand binding with [3H]PK11195 and immunocytochemistry with a TSPO antibody. We demonstrate that TSPO gene insertion causes increased transcription of genes involved in the mitochondrial electron transport chain. Furthermore, TSPO insertion increased mitochondrial ATP production as well as cell excitability, reflected in a decrease in patch clamp recorded rectified K channel currents. These functional changes were accompanied by an increase in cell proliferation and motility, which were inhibited by PK11195, a selective ligand for TSPO. We suggest that TSPO may serve a range of functions that can be viewed as downstream regulatory effects of its primary, evolutionary conserved role in cell metabolism and energy production.

Epigenetic Silencing of the Human 18 kDa Translocator Protein in a T Cell Leukemia Cell Line.

The mitochondrial membrane 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is constitutively expressed in most organs, most abundantly in hormonal tissue and cells of mononuclear phagocyte lineage, while in the brain, TSPO expression is induced in the wake of injury, inflammation, and neurodegeneration. Increased TSPO expression is also prominent in several cancerous tissues where it appears to correlate with the degree of malignancy. Currently, TSPO is thus actively investigated as a generic biomarker for disease activity and a therapeutic target for a wide range of diseases. In this study, we report a Jurkat human T cell leukemia cell line that has only trace expression of TSPO mRNA. Through the use of bisulphite genomic sequencing, we show that the Jurkat TSPO promoter is highly methylated except for CpG sites that are adjacent to the transcription start site. Control measurements in HEK-293, HeLa, and U87-MG cells with high TSPO mRNA expression showed low levels of TSPO promoter methylation. Demethylation with 5-aza-2'-deoxycytidine (5-aza-dC) caused a dose-dependent increase in TSPO mRNA with a corresponding demethylation of the TSPO promoter in Jurkat cells. Treating HeLa and U87-MG cells with 5-aza-dC caused no change in the level of TSPO mRNA. These observations confirm the epigenetic regulation of TSPO and suggest it to be a more common mechanism by which the differential expression of TSPO in various cell types and in health and disease may be explained.

Positron emission tomography and functional characterization of a complete PBR/TSPO knockout.

The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs.

The 18 kDa translocator protein, microglia and neuroinflammation.

The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is expressed in the injured brain. It has become known as an imaging marker of "neuroinflammation" indicating active disease, and is best interpreted as a nondiagnostic biomarker and disease staging tool that refers to histopathology rather than disease etiology. The therapeutic potential of TSPO as a drug target is mostly based on the understanding that it is an outer mitochondrial membrane protein required for the translocation of cholesterol, which thus regulates the rate of steroid synthesis. This pivotal role together with the evolutionary conservation of TSPO has underpinned the belief that any loss or mutation of TSPO should be associated with significant physiological deficits or be outright incompatible with life. However, against prediction, full Tspo knockout mice are viable and across their lifespan do not show the phenotype expected if cholesterol transport and steroid synthesis were significantly impaired. Thus, the "translocation" function of TSPO remains to be better substantiated. Here, we discuss the literature before and after the introduction of the new nomenclature for TSPO and review some of the newer findings. In light of the controversy surrounding the function of TSPO, we emphasize the continued importance of identifying compounds with confirmed selectivity and suggest that TSPO expression is analyzed within specific disease contexts rather than merely equated with the reified concept of "neuroinflammation."

Effects of ionizing radiation on mitochondria.

The current concept of radiobiology posits that damage to the DNA in the cell nucleus is the primary cause for the detrimental effects of radiation. However, emerging experimental evidence suggests that this theoretical framework is insufficient for describing extranuclear radiation effects, particularly the response of the mitochondria, an important site of extranuclear, coding DNA. Here, we discuss experimental observations of the effects of ionizing radiation on the mitochondria at (1) the DNA and (2) functional levels. The roles of mitochondria in (3) oxidative stress and (4) late radiation effects are discussed. In this review, we summarize the current understanding of targets for ionizing radiation outside the cell nucleus. Available experimental data suggest that an increase in the tumoricidal efficacy of radiation therapy might be achievable by targeting mitochondria. Likewise, more specific protection of mitochondria and its coding DNA should reduce damage to healthy cells exposed to ionizing radiation.

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

Predicted ionisation in mitochondria and observed acute changes in the mitochondrial transcriptome after gamma irradiation: a Monte Carlo simulation and quantitative PCR study.

It is a widely accepted that the cell nucleus is the primary site of radiation damage while extra-nuclear radiation effects are not yet systematically included into models of radiation damage. We performed Monte Carlo simulations assuming a spherical cell (diameter 11.5 µm) modelled after JURKAT cells with the inclusion of realistic elemental composition data based on published literature. The cell model consists of cytoplasm (density 1g/cm(3)), nucleus (diameter 8.5 µm; 40% of cell volume) as well as cylindrical mitochondria (diameter 1 µm; volume 0.5 µm(3)) of three different densities (1, 2 and 10 g/cm(3)) and total mitochondrial volume relative to the cell volume (10, 20, 30%). Our simulation predicts that if mitochondria take up more than 20% of a cell's volume, ionisation events will be the preferentially located in mitochondria rather than in the cell nucleus. Using quantitative polymerase chain reaction, we substantiate in JURKAT cells that human mitochondria respond to gamma radiation with early (within 30 min) differential changes in the expression levels of 18 mitochondrially encoded genes, whereby the number of regulated genes varies in a dose-dependent but non-linear pattern (10 Gy: 1 gene; 50 Gy: 5 genes; 100 Gy: 12 genes). The simulation data as well as the experimental observations suggest that current models of acute radiation effects, which largely focus on nuclear effects, might benefit from more systematic considerations of the early mitochondrial responses and how these may subsequently determine cell response to ionising radiation.

Green fluorescent protein alters the transcriptional regulation of human mitochondrial genes after gamma irradiation.

Green fluorescent proteins (GFP), extensively used as reporters in biological and imaging studies, are assumed to be mostly biologically inert. Here, we test the assumption in regard to the transcriptional regulation of 18 mitochondrially encoded genes in GFP expressing human T-cell line (JURKAT cells) exposed to gamma radiation. Using quantitative polymerase chain reaction, we demonstrate that wild type and GFP expressing JURKAT cells have different baseline mitochondrial transcript expression (10 out of the 18 tested genes) and after a single dose of radiation (100 Gy) show a significantly different transcriptional regulation of their mitochondrial genes. While in wild type cells, ten of the tested genes are up-regulated in response to radiation exposure, GFP expressing cells show less transcriptional regulation with a small down-regulation in five genes. Our results indicate that the presence of GFP in the cytoplasm can alter the cellular response to ionizing radiation.

A comparative assessment of PM2.5 exposures in light-rail, subway, freeway, and surface street environments in Los Angeles and estimated lung cancer risk.

According to the U.S. Census Bureau, 570000+ commuters in Los Angeles travel for over 60 minutes to work. Studies have shown that a substantial portion of particulate matter (PM) exposure can occur during this commute. This study represents the integration of the results from five commute environments in Los Angeles. Personal PM exposures are discussed for the: (1) METRO gold line, a ground-level light-rail route, (2) METRO red line, a subway line, (3) the 110, a high volume freeway with low heavy-duty vehicle (HDV) fraction, (4) the 710, a major corridor for HDVs from the Port of Los Angeles, and (5) Wilshire/Sunset Boulevards, major surface streets. Chemical analysis including total and water-soluble metals and trace elements, elemental and organic carbon (EC/OC), and polycyclic aromatic hydrocarbons (PAHs) was performed. The focus of this study is to compare the composition and estimated lung cancer risk of PM2.5 (dp < 2.5 µm) for the five differential commute environments. Metals associated with stainless steel, notably Fe, Cr, and Mn, were elevated for the red line (subway), most likely from abrasion processes between the rail and brakes; elements associated with tire and brake wear and oil additives (Ca, Ti, Sn, Sb, and Pb) were elevated on roadways. Elemental concentrations on the gold line (light-rail) were the lowest. For water-solubility, metals observed on the red line (subway) were the least soluble. PAHs are primarily derived from vehicular emissions. Overall, the 710 exhibited high levels of PAHs (3.0 ng m−3), most likely due to its high volume of HDVs, while the red and gold lines exhibited low PAH concentrations (0.6 and 0.8 ng m−3 for red and gold lines, respectively). Lastly, lung cancer risk due to inhalation of PAHs was calculated based on a commuter lifetime (45 years for 2 hours per workday). Results showed that lung cancer risk for the 710 is 3.8 and 4.5 times higher than the light-rail (gold line) and subway (red line), respectively. With low levels of both metal and PAH pollutants, our results indicate that commuting on the light-rail (gold line) may have potential health benefits when compared to driving on freeways and busy roadways.

Atmospheric ultrafine particles promote vascular calcification via the NF-κB signaling pathway.

Exposure to atmospheric fine particulate matter (PM(2.5)) is a modifiable risk factor of cardiovascular disease. Ultrafine particles (UFP, diameter <0.1 µm), a subfraction of PM(2.5), promote vascular oxidative stress and inflammatory responses. Epidemiologic studies suggest that PM exposure promotes vascular calcification. Here, we assessed whether UFP exposure promotes vascular calcification via NF-κB signaling. UFP exposure at 50 µg/ml increased alkaline phosphatase (ALP) activity by 4.4 ± 0.2-fold on day 3 (n = 3, P < 0.001) and matrix calcification by 3.5 ± 1.7-fold on day 10 (n = 4, P < 0.05) in calcifying vascular cells (CVC), a subpopulation of vascular smooth muscle cells with osteoblastic potential. Treatment of CVC with conditioned media derived from UFP-treated macrophages (UFP-CM) also led to an increase in ALP activities and matrix calcification. Furthermore, both UFP and UFP-CM significantly increased NF-κB activity, and cotreatment with an NF-κB inhibitor, JSH23, attenuated both UFP- and UFP-CM-induced ALP activity and calcification. When low-density lipoprotein receptor-null mice were exposed to UFP at 359.5 µg/m(3) for 10 wk, NF-κB activation and vascular calcification were detected in the regions of aortic roots compared with control filtered air-exposed mice. These findings suggest that UFP promotes vascular calcification via activating NF-κB signaling.

Characterization of organic, metal and trace element PM2.5 species and derivation of freeway-based emission rates in Los Angeles, CA.

On-road particulate matter (PM) was collected during a sampling campaign in March-April of 2011 on two major Los Angeles freeways, I-710 and Route 110. I-710 is a major route for heavy-duty vehicles (HDVs) traveling to and from the Ports of Long Beach and Los Angeles, while Route 110 has a much lower HDV fraction -3.9% versus 11.4%. Two sets of samples were collected for each roadway, each set representing approximately 50°h of on-road sampling. Concurrent sampling at a fixed site at the University of Southern California's (USC) downtown Los Angeles campus provided estimates of urban background levels. Chemical analysis was performed for elemental carbon (EC), organic carbon (OC), polycyclic aromatic hydrocarbons (PAHs), hopanes and steranes, and metals and trace elements. Freeway-based emission rates (ERs) - mass per kilometer of freeway per hour - were calculated using mass concentrations, fuel characteristics, and traffic flow rates. These ERs are presented such that freeways could be treated as a line source of emissions for use in predictive models of population exposure for nearby communities. This data could also be used to assess the exposure of commuters to traffic-related PM2.5 emissions. ERs are compared to data from a previous fixed-site roadside study of I-710 as well as to reconstructed values from a tunnel study. ERs were generally lower (or comparable) on the gasoline-vehicle dominated freeway (Route 110) than the freeway with more diesel trucks (I-710), with EC and pyrene being notably lower on Route 110, findings consistent with the Route 110's lower HDV fraction. We found EC emission rates decreased over time suggesting that efforts to reduce diesel emissions from HDVs at the Ports of Los Angeles and Long Beach have been successful. While ERs for most of the organic species were within the range of values reported by previous studies, the present study found much higher ERs for metals and trace elements. This suggests that the sampling methods employed in this campaign are more efficient at capturing particles from sources such as resuspended road dust and wear from tires and brakes, which are usually not included in traditional sampling methodologies for assessing vehicular emissions (e.g. dynamometer studies).

The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice.

The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3)H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1), 499±106 Bq x mg(-1) in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3)H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1)). Further, our study includes technical feasibility data on [(18)F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18)F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18)F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.

Chemical characterization and redox potential of coarse and fine particulate matter (PM) in underground and ground-level rail systems of the Los Angeles Metro.

A campaign was conducted to assess personal exposure of coarse (2.5 µm < d(p) < 10 µm) and fine (d(p) < 2.5 µm) PM for two lines of the L.A. Metro-a subway (red) and light-rail (gold) line. Concurrent measurements were taken at University of Southern California (USC) to represent ambient conditions. A comprehensive chemical analysis was performed including total and water-soluble metals, inorganic ions, elemental and organic carbon, and organic compounds. Mass balance showed that in coarse PM, iron makes up 27%, 6%, and 2% of gravimetric mass for the red line, the gold line, and USC, respectively; in fine PM, iron makes up 32%, 3%, and 1%. Ambient air is the primary source of inorganic ions and organic compounds for both lines. Noncrustal metals, particularly Cr, Mn, Co, Ni, Mo, Cd, and Eu, were elevated for the red line and, to a lesser degree, the gold line. Mo exhibited the greatest crustal enrichment factors. The enriched species were less water-soluble on the red line than corresponding species on the gold line. Bivariate analysis showed that reactive oxygen species (ROS) activity is strongly correlated with water-soluble Fe (R(2) = 0.77), Ni (R(2 )= 0.95), and OC (R(2 )= 0.92). A multiple linear regression model (R(2) = 0.94, p < 0.001) using water-soluble Fe and OC as predictor variables was developed to explain the variance in ROS. In addition, PM from the red line generates 65% and 55% more ROS activity per m(3) of air than PM from USC and the gold line, respectively; however, one unit of PM mass from the gold line may be as intrinsically toxic as one unit of PM from the red line.

Glutamatergic neurons in rodent models respond to nanoscale particulate urban air pollutants in vivo and in vitro.

BACKGROUND: Inhalation of airborne particulate matter (PM) derived from urban traffic is associated with pathology in the arteries, heart, and lung; effects on brain are also indicated but are less documented. OBJECTIVE: We evaluated rodent brain responses to urban nanoscale (< 200 nm) PM (nPM). METHODS: Ambient nPM collected near an urban freeway was transferred to aqueous suspension and reaerosolized for 10-week inhalation exposure of mice or directly applied to rat brain cell cultures. RESULTS: Free radicals were detected by electron paramagnetic resonance in the nPM 30 days after initial collection. Chronic inhalation of reaerosolized nPM altered selected neuronal and glial activities in mice. The neuronal glutamate receptor subunit (GluA1) was decreased in hippocampus, whereas glia were activated and inflammatory cytokines were induced [interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα)] in cerebral cortex. Two in vitro models showed effects of nPM suspensions within 24-48 hr of exposure that involved glutamatergic functions. In hippocampal slice cultures, nPM increased the neurotoxicity of NMDA (N-methyl-d-aspartic acid), a glutamatergic agonist, which was in turn blocked by the NMDA antagonist AP5 [(2R)-amino-5-phosphonopentanoate]. In embryonic neuron cultures, nPM impaired neurite outgrowth, also blocked by AP5. Induction of IL-1α and TNFα in mixed glia cultures required higher nPM concentrations than did neuronal effects. Because conditioned media from nPM-exposed glia also impaired outgrowth of embryonic neurites, nPM can act indirectly, as well as directly, on neurons in vitro. CONCLUSIONS: nPM can affect embryonic and adult neurons through glutamatergic mechanisms. The interactions of nPM with glutamatergic neuronal functions suggest that cerebral ischemia, which involves glutamatergic excitotoxicity, could be exacerbated by nPM.