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Calderón-Garcidueñas, Franco-Lira, Henríquez-Roldán, Osnaya, González-Maciel, Reynoso-Robles, Villarreal-Calderon, Herritt, Brooks, Keefe, Palacios-Moreno, Villarreal-Calderon, Torres-Jardón, Medina-Cortina, Delgado-Chávez, Aiello-Mora, Maronpot RR, Doty RL
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Mexico City (MC) residents are exposed to severe air pollution and exhibit olfactory bulb inflammation. We compared the olfactory function of individuals living under conditions of extreme air pollution to that of controls from a relatively clean environment and explore associations between olfaction scores, apolipoprotein E (APOE) status, and pollution exposure. The olfactory bulbs (OBs) of 35 MC and 9 controls 20.8+/-8.5 years were assessed by light and electron microscopy. The University of Pennsylvania Smell Identification Test (UPSIT) was administered to 62 MC/25 controls 21.2+/-2.7 years. MC subjects had significantly lower UPSIT scores: 34.24+/-0.42 versus controls 35.76+/-0.40, p=0.03. Olfaction deficits were present in 35.5% MC and 12% of controls. MC APOE epsilon 4 carriers failed 2.4+/-0.54 items in the 10-item smell identification scale from the UPSIT related to Alzheimer’s disease, while APOE 2/3 and 3/3 subjects failed 1.36+/-0.16 items, p=0.01. MC residents exhibited OB endothelial hyperplasia, neuronal accumulation of particles (2/35), and immunoreactivity to beta amyloid betaA(42) (29/35) and/or alpha-synuclein (4/35) in neurons, glial cells and/or blood vessels. Ultrafine particles were present in OBs endothelial cytoplasm and basement membranes. Control OBs were unremarkable. Air pollution exposure is associated with olfactory dysfunction and OB pathology, APOE 4 may confer greater susceptibility to such abnormalities, and ultrafine particles could play a key role in the OB pathology. This study contributes to our understanding of the influences of air pollution on olfaction and its potential contribution to neurodegeneration.

Keywords: α synuclein, Alzheimer’s disease, amyloid β 42, air pollution, APOE, olfaction, olfactory bulb, Parkinson’s disease, ultrafine particulate matter

Introduction

Air pollution is a complex mixture of particulate matter (PM), gases, and organic compounds present in outdoor and indoor air. Children living in Mexico City (MC) exhibit evidence of chronic inflammation of the upper and lower respiratory tracts, accumulation of particulates in nasal respiratory epithelium, breakdown of the nasal respiratory epithelial barrier, systemic inflammation, brain inflammation, cognitive deficits, and MRI brain abnormalities (Calderon-Garciduenas et al., 2001a, 2003a, 2007a, b, 2008a–c). Ultrafine particulate matter (UFPMo100 nm) is found in the olfactory bulbs (OBs) of children and young adults exposed to the highly polluted atmosphere of MC (Calderon-Garciduenas et al., 2007a, 2008b). Healthy dogs living in MC also exhibit disruption of nasal and olfactory barriers, increased apurinic and apyrimidinic DNA sites in olfactory bulb and hippocampus tissues, and white matter hyperintense prefrontal lesions by MRI similar to those present in children (Calderon-Garciduenas et al., 2001b, 2003b, 2008c).

Adult residents in MC exhibit up-regulation of a powerful inflammatory gene, cyclooxygenase-2 (COX2), in the olfactory bulb and other brain regions (Calderón-Garcidueñas et al., 2004). Children and adults younger than 25 years exhibit up-regulation of COX2, interleukin 1 beta (IL1β), and the key innate immunity receptor CD14 in their olfactory bulbs (Calderón-Garcidueñas et al., 2008b). Immunoreactivity (IR) to Aβ42 is present in mitral and tufted olfactory neurons as well as olfactory ensheathing cells and astrocytes, while α synuclein IR in the form of Lewy neurites and cytoplasmic deposits is also seen in olfactory bulb neurons of young MC residents (Calderón-Garcidueñas et al., 2008b). The observation that MC teenagers with an APOE ε 4 allele accumulate Aβ42 in olfactory bulb neurons concomitantly with markers of oxidative stress, mitochondrial abnormalities, and dysfunction of the proteasomal system (Jung et al., 2007, Keller 2006) is very important. The APOE ε 4 allele is a major genetic risk factor for the development of Alzheimer’s disease and older adult ε 4 carriers perform poorly on odor identification tests (Corder et al., 1993, Graves et al., 1999, Kovacs et al., 2004,Calhoun et al., 2005, Handley et al., 2006; Olofsson et al., 2008). Also relevant to our study, Kozauer et al., has shown an association between cognitive decline and APOE ε 4 in young individuals. Specifically, ε 4 carriers first seen at an average age of 29.3 years and followed-up for 22 years scored lower on the Mini-Mental State Examination (MMSE) and three tests of verbal learning: immediate recall, delayed recall and word recognition (Kozauer et al., 2008). Kozauers’ study suggests that the association between APOE ε 4 and cognitive decline is likely an early one, emphasizing the need to explore olfactory deficits in younger individuals carrying the ε 4 allele.

The goals of this study were threefold: first, to compare the olfactory function of a cohort of healthy young adults residing in MC to that of a matched low pollution cohort; second, to assess if the carriers of an ε 4 APOE allele would have significantly more olfactory deficits compared to APOE 2/3 and 3/3 carriers in both the high and the low pollution cohorts; third, to characterize the olfactory bulb pathology in a matched cohort of low and high pollution exposed residents. We selected as our olfactory test the Spanish version of the University of Pennsylvania Smell Identification Test (UPSIT) (Doty et al., 1984; Doty, 1995). Ten items within this test have been shown to strongly predict conversion from mild cognitive impairment to Alzheimer’s disease (Tabert et al., 2005). Given that olfactory bulb pathology has been observed in MC residents exposed to high levels of pollution, this study also examined the pathology of the olfactory bulbs of 35 MC and 9 matched controls. We hypothesized that: i. odor identification scores would be lower in MC subjects compared to controls; ii. carriers of the APOE 4 allele residing in MC would perform more poorly on the 10 UPSIT items related to risk for Alzheimer’s disease; and iii. olfactory bulb pathology would be significant in the MC residents.

Methods

Study Areas

Mexico City represents an extreme of urban growth and environmental pollution (Bravo-Alvarez and Torres-Jardón, 2002; Molina et al., 2007). The Mexico City Metropolitan Area (19°25′N latitude and 99° 100′ W longitude) lies in an elevated basin at an altitude of 2240 meters above mean sea level and its urbanized area covers around 2000 km2. The basin is surrounded by high mountains ridges on the east, south, and west but with a broad opening to the north and a gap to the south-southwest. The surrounding mountains combined with the frequent morning thermal inversions contribute to trap and accumulate air pollutants inside the basin. In this geographical setting, 20 million residents, nearly 4 million vehicles, and over 40 000 industries consume more than 40 million liters of petroleum fuels per day emitting significant concentrations of primary air pollutants (Molina et al., 2007). The high altitude and tropical climate of the region is highly conductive to fast photochemistry forming secondary pollutants such as ozone (O3) and particulate matter (PM).

The northwest sector of MC -the residency area of our exposed cohort- corresponds to a mixed medium income residential and industrial area with heavy traffic. Although PM10 (particulate matter with aerodynamic diameters of less than 10 μm) and PM2.5 (particulate matter with aerodynamic diameters of less than 2.5 μm) concentrations over this sector are not the highest in the metropolitan area, their levels represent a health concern to its residents (Secretaría del Medio Ambiente del Gobierno del Distrito Federal, 2006). The typical coarse fraction (PM2.5-10) in Mexico City is ∼ 54% while the fine fraction (PM<2.5) is ∼ 46%, the latest fraction relates to traffic exhaust emissions (Vega et al., 2004;Querol et al., 2008). The control subjects lived in Polotitlán (20°13′N latitude and 99° 49′W longitude), a rural town of ∼ 3,000 inhabitants located 121 km NW of MC at 2380 m above mean sea level. Its main activity is agricultural with just a few small textile manufacturing and dairy products facilities. The prevalent wind direction at Polotitlán comes from rural not polluted areas and thus insures good air quality. The PM10 levels at Polotitlán are ∼ 5 % lower than that of the NWMC and it is estimated that the coarse PM fraction is ∼ 60% (due to the influence of local soil resuspension), while the fine fraction is ∼ 40% (Secretaría de Ecología del Gobierno del Estado de México, 2005, Querol et al., 2008). Data from the control town indicated that other criteria air pollutants (ozone, sulfur dioxide, carbon monoxide and nitrogen dioxides) were below the current EPA standards (Secretaría de Ecología del Gobierno del Estado de México, 2005). The selection of this rural control town was based on 4 key additional factors: i. access to a young adult healthy population, ii. previous clinical studies with the Polotitlán cohort that indicated that children had no evidence of air pollution-related health issues (Calderón-Garcidueñas et al., 2007b, 2008b), iii. an altitude above sea level similar to that of MC, and iv. its relative proximity to MC to facilitate clinical access of the cohorts.

Clinical study population

The MC cohort included 62 subjects, 21 women and 41 males, with a mean (SD) age of 21.1 (2.6) years. The mean number of years of education was 13.6 (0.7). Their average time spent outdoors was 4.43 (0.5) hours per day. The control subjects included 14 males and 11 females

[mean (SD) age = 21.4 (2.8) years, years of education = 13.4 (1.3)] with an average of 4.2 (1.0) hours spent daily outdoors. The ages, years of education, and exposure times did not differ significantly between the MC and control groups. All subjects reportedly slept in bedrooms with no carpeting/draperies, and had open windows for ventilation. Anthropometric values (weight and height) were within age- and gender-related normal limits. The average residency time in MC was 5.74 (0.91) years with a range of 1-28 years. The MC subjects were residents of Northwest MC and lived within 6 miles of closest air pollutant monitoring station (Tlalnepantla). The control residents had a lifelong residency in their low polluted town. The inclusion criteria applied in this study were negative personal smoking history and environmental tobacco exposure, full term birth, no known exposures to local sources of toxic substances and unremarkable clinical histories, including absence of history of hospitalizations for respiratory illnesses, ear-nose-throat (ENT) and oral symptomatology and/or surgery, head trauma, systemic or respiratory viral diseases, lower respiratory system illnesses, and personal and family histories of atopic diseases. All included subjects denied olfactory deficits and were taking no medications.

Olfactory Testing Protocol

The study was approved by the Universidad del Ejército Human Studies Committees, and written consent was obtained from all subjects. Olfactory function was quantified using the Spanish version of the University of Pennsylvania Smell Identification Test (UPSIT). This self-administered standardized test incorporates 40 microencapsulated odorants and a forced-choice multiple alternative format to establish both absolute (i.e., normosmia, anosmia, or mild, moderate or severe hyposmia) and relative (percentile ranks) indices of function (Doty, 1995). We analyzed the full 40 item score as well as the 10 Item score that strongly predicts conversion to Alzheimer’s disease (AD) on follow-up evaluation in patients with mild cognitive impairment (Tabert et al. 2005). All data were managed anonymously. Subjects were studied in December 2005 and June 2008.

Pathology Study

Autopsies and tissue preparation

The mean (SD) age of the 35 MC autopsy subjects was 19.2 (6.7) years (range 2 – 32 years), and for the 9 controls residents 21.3 (10.3) years (range 2– 40 years). The MC cohort included 16 children ranging in age from 2 to 17 years [mean (SD) = 13.5 (4.7) years] and the control cohort included a 2 y old girl and three 17y old boys [mean (SD) = 13.2 (7.5)]. Subjects were elementary, middle, high school, and college students and blue and white collar workers. Three APOE 4 carriers were identified in the MC group and none in the control group. All autopsied subjects were clinically healthy and had died suddenly.

All 44 subjects had full autopsies, including complete neuropathological examinations and were included in the immunohistochemistry (IHC) studies. The selected cases had no pathological evidence of disease processes other than the acute cause of death. Autopsies were performed 4.1 ± 1.1 h after death. The skull was opened and the olfactory bulbs and the brain removed. Brain sections were immersed in 10% neutral formaldehyde, fixed for 48 h, and transferred to 70% alcohol. Olfactory bulb and nerve sections were included for this study. Paraffin sections 5-7 μm thick were cut and routinely stained with hematoxylin and eosin, and used for immunohistochemistry. Immunohistochemistry (IHC) was performed on olfactory bulbs sections. The sections were deparaffinized, and immunostained as described previously (Calderón-Garcidueñas et al., 2004, 2008a). In this work we used 88% formic acid as an epitope retrival method for the Aβ, while for α synuclein we used a proteinase K protocol 10 (Beach et al., 2008). Negative controls included omission or substitution of primary antibodies by nonspecific, isotype-matched antibodies. Positive (Alzheimer’s patients) and negative controls were included for each antibody. Confirmation of the IR was done with different antibodies in serial sections with a minimum of 10 slides for each Ab in each case (mean 14±2.2 SD). Selected antibodies included: β amyloid 1-16 (6E10 Signet), Covance, Emeryville, CA 1:2000, β amyloid, 17-24 (4G8 Signet), Covance, Emeryville, CA, 1:1000, α-synuclein LB509 (InVitrogen, Carlsbad, CA 1:800), α-synuclein ab 2080 to aa 116-131 and ab24592 to residues Y 125 and Y 136 (Abcam Cambridge, Mass 1:1000), PHF-Tau-8 (Innogenetics, Belgium, AT-8, 1:100), and glial fibrillary acidic protein GFAP (Abcam, Cambridge, Mass 1:500). Sections were reviewed by three pathologists with no access to the codes regarding the identification data. Electron microscopy was performed in 10 olfactory bulb samples (5 controls, 5 MC) fixed in 2% paraformaldehyde and 2% glutaraldehyde in sodium phosphate buffer (0.1M, pH 7.4), post-fixed in 1% osmium tetraoxide and embedded in Epon. Semithin sections (0.5 – 1 μm) were cut, stained with toluidine blue for light microscopy examination. Ultrathin sections (60- 90 nm) were cut and collected on slot grids previously covered with formvar membrane. Sections were stained with uranyl acetate and lead citrate and examined with a Carl Zeiss EM109T (Germany) or a JEM-1011 (Japan) microscope.

Apolipoprotein E genotyping

APOE genotype information was obtained through analysis of either nasal or venous samples for the clinical participants and from brain samples in the autopsy cases. Samples were genotyped using Taqman ready to use assays from both SNP’s that constitute the APOE genotype according to TaqMan Gene Expression Assays, Applied Biosystems, 2006.

Data analysis

In the olfactory study, the primary variables of interest were the total UPSIT scores, the subset of UPSIT items known to be particularly sensitive to Alzheimer’s disease, residency time in MC, and the APOE alleles. The two-sample Wilcoxon rank sum (Mann-Whitney) test was used for comparison for variables of interest between Apo E 2/3 and 3/3 and 3/4 and 4/4 subjects. All statistical computations were performed with the use of Stata 8.3 software (Stata Corp, College Station, TX). A two-sided type I error rate of 0.05 was considered statistically significant.

Results

Olfactory Function

The mean UPSIT scores for MC residents were lower than that of their matched controls [means (SEMs) 34.24 (0.42) and 35.76 (0.40); p=0.03, with the average deficit reflecting mild microsmia. Table 1 summarizes the olfactory scores, including those for the different APOE alleles in the two cohorts. Olfaction deficits ranging from mild to severe microsmia- regardless of APOE status- were identified in 35.5% of the MC residents, while 12% of the control residents had only mild microsmia. There were no significant differences in the total UPSIT scores between the MC APOE ε 4 and the APO E 2/3 or 3/3 genotype carriers [mean (SD) = 33.3 (4.2) and 34.46 (3.0); p = 0.52]. That being said, MC residents having the APOE 4 allele failed significantly more items from the 10 UPSIT items known to be particularly sensitive to AD than their APOE 2/3 or 3/3 allele MC counterparts [respective mean (SEM) scores = 2.4 (0.54) & 1.3 (0.16), p = 0.01], a finding not observed in the control subjects (p = 0.08). This is in spite of the fact that the APOE 2 and 3 subjects lived, on average, four more years in MC than did the APOE ε 4 subjects [respective mean years (SD) = 6.54 (1.09) & 2.4 (0.6); p=0.02]. No significant UPSIT score differences were observed between Control APOE ε 4 carriers and Control APO E 3/3 carriers [respective means (SD) = 35.0 (1.1) & 35.9 (2.1); p = 0.31].

Urban Air Pollution-study-t1

Human Pathology

Gross brain examination was unremarkable in all subjects. The control olfactory bulbs were negative for Aβ42, α-synuclein and PHF-Tau-8 and were unremarkable on microscopic examination. However, this was not the case for the olfactory bulbs of the MC cohort. Two of the 16 Mexico City children-boys age 14 and 17 y- exhibited significant amounts of black particulate material in the cytoplasm of neuron specific enolase (NSE) + cells around glomeruli (Figure 1 A). Amyloid Aβ42 (with both β 6E10 and 4G8 Signet Ab), was seen in ensheathing cells, as well as in astrocytes i