Alzheimer disease starts in childhood in polluted Metropolitan Mexico City. A major health crisis in progress

Journal Pre-proof Alzheimer disease starts in childhood in polluted Metropolitan Mexico City. A major health crisis in progress Lilian Calderón-Garcidueñas, Ricardo Torres-Jardón, Randy J. Kulesza, Yusra Mansour, Luis Oscar González-González, Angélica Gónzalez-Maciel, Rafael Reynoso-Robles, Partha S. Mukherjee PII:

S0013-9351(20)30029-3

DOI:

https://doi.org/10.1016/j.envres.2020.109137

Reference:

YENRS 109137

To appear in:

Environmental Research

Received Date: 18 December 2019 Revised Date:

12 January 2020

Accepted Date: 13 January 2020

Please cite this article as: Calderón-Garcidueñas, L., Torres-Jardón, R., Kulesza, R.J., Mansour, Y., González-González, L.O., Gónzalez-Maciel, Angé., Reynoso-Robles, R., Mukherjee, P.S., Alzheimer disease starts in childhood in polluted Metropolitan Mexico City. A major health crisis in progress, Environmental Research (2020), doi: https://doi.org/10.1016/j.envres.2020.109137. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Alzheimer disease starts in childhood in polluted Metropolitan Mexico City. A major health crisis in progress. Lilian Calderón-Garcidueñas MA, MD, PhD 1, 2, Ricardo Torres-Jardón PhD3 , Randy J. Kulesza PhD4, Yusra Mansour BS4, Luis Oscar González-González MD5 , Angélica Gónzalez-Maciel MS5, Rafael Reynoso-Robles BS5, Partha S. Mukherjee PhD6

1

The University of Montana, Missoula, MT 59812, USA

2

Universidad del Valle de México, 14370, México

3

Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México, 04310,

Ciudad de México, México 4

Auditory Research Center, Lake Erie College of Osteopathic Medicine, Erie, PA 16509,

USA 5

Instituto Nacional de Pediatría, 04530, Ciudad de México, México

6

Interdisciplinary Statistical Research Unit, Indian Statistical Institute, 700108, Kolkata,

India Correspondence to: Professor Lilian Calderón-Garcidueñas, MA, MD, PhD, University of Montana,32 Campus Drive, 287 Skaggs Building, Missoula, MT 59812, USA. Tel.: 406243-4785; [email protected]

Funding: This review did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

1

Abstract Exposures to fine particulate matter (PM2.5) and ozone (O3) above USEPA standards are associated with Alzheimer’s disease (AD) risk. Metropolitan Mexico City (MMC) youth have life time exposures to PM2.5 and O3 above standards. We focused on MMC residents ≤ 30 years and reviewed 134 consecutive autopsies of subjects age 20.03±6.38y (range 11months to 30y),the staging of Htau and ß amyloid, the lifetime cumulative PM2.5 (CPM 2.5)

and the impact of the Apolipoprotein E (APOE) 4 allele, the most prevalent genetic risk

for AD.We also reviewed the results of the Montreal Cognitive Assessment (MoCA) and the brainstem auditory evoked potentials (BAEPs) in clinically healthy young cohorts. Mobile sources, particularly from non-regulated diesel vehicles dominate the MMC pollutant emissions exposing the population to PM2.5 concentrations above WHO and EPA standards. Iron-rich,magnetic, highly oxidative, combustion and friction-derived nanoparticles (CFDNPs) are measured in the brain of every MMC resident. Progressive development of Alzheimer starts in childhood and in 99.25% of 134 consecutive autopsies ≤ 30 years we can stage the disease and its progression; 66% of ≤ 30 years urbanites have cognitive impairment and involvement of the brainstem is reflected by auditory central dysfunction in every subject studied.The average age for dementia using MoCA is 20.6±3.4y. APOE4 vs 3 carriers have 1.26 higher odds of committing suicide. PM2.5 and CFDNPs play a key role in the development of neuroinflammation and neurodegeneration in young urbanites. A serious health crisis is in progress with social, educational, judicial,economic and overall negative health impact for 25 million residents. Understanding the neural circuitry associated with the earliest cognitive and behavioral manifestations of AD should be defined. Air pollution control should be prioritized2

including the regulation of diesel vehicles- and the first two decades of life ought to be a targeted for neuroprotective interventions. Defining paediatric environmental, nutritional, metabolic and genetic risk factor interactions is a multidisciplinary task of paramount importance to prevent Alzheimer’s disease. Current and future generations are at risk.

Keywords: Alzheimer, APOE4, brainstem, children, combustion-derived nanoparticles, dementia, depression, hyperphosphorylated tau, metals, Mexico City, MCI, PM 2·5, suicide, violence, young adults.

Introduction Exposure to air pollutants across the world, plays a key role in the development and/or acceleration of Alzheimer's disease (AD).1-6 Children are a target of air pollutants and cognitive deficits and brain metabolic and structural changes are described in the literature.7-12 Young residents living in Metropolitan Mexico City (MMC) have life time exposures to concentrations of fine particulate matter (PM 2.5) and ozone above the USEPA standards and compared with subjects living in cleaner cities, exhibit an early brain imbalance in genes involved in oxidative stress, inflammation, and innate and adaptive immune responses.13 Supra and infratentorial neurovascular unit damage, neuroinflammation, and accumulation of misfolded proteins associated to the early stages of both Alzheimer's and Parkinson's diseases are documented in MMC youth. 1,9,14-17 We have described the presence of combustion and friction-derived nanoparticles (CFDNPs) in the brains of MMC residents and discussed potential short and long term 3

central and peripheral nervous system effects due to the unique combination of redox activity, surface charge, strongly magnetic behaviour and their pervasive distribution affecting millions of people.9,18-22 Epidemiological trends of increasing Alzheimer’s disease (AD), growing evidence environmental and lifestyle factors contribute to AD risk and pathogenesis and the early development and progression of AD in highly exposed MMC children and young adults obligates us to pay attention to air pollution, that can be controlled and thus Alzheimer disease in the scenario of air pollution is a preventable disease. 1,4-6, 9, 23,24 This review focus on the development and progression of Alzheimer histopathological hallmarks in an autopsy MMC cohort of 134 cases with an average age of 20.03±6.38 years, the relationships between cumulative lifetime concentrations of PM2.5, mode of death and APOE 4 status. We are contrasting the neuropathological findings in consecutive forensic autopsies with the cognitive deficits and abnormal brainstem auditory evoked potentials observed in a matched age and socioeconomic status cohort of lifetime residents in MMC.The potential role of brain damage associated with iron rich, magnetic nanoparticles with transitional metals and polycyclic aromatic hydrocarbons (PAH), and nanocluster aerosols emitted by road transportation in the size range of 1.3-3.0 nm are also discussed.3,9, 19,23-26 A key issue to discuss in this review is the fact we are measuring PM2.5 as our strongest proxy for PM health effects, however, most of the air pollution particles in urban areas are indeed nanoparticles (≤ 100 nm). 9,23-26 We currently do not measure or regulate nanosize PM and given the fact NPs include highly oxidative, reactive, metal-rich particles accessing every single organ in the body and having portals of entry with direct access to the brain,the 4

question is: what type of measurements do we need in order to establish NPs health effects? Is PM2.5 enough? The questions need answers in the setting of serious air pollution exposing 25 million residents in MMC and the development of Alzheimer’s neuropathological hallmarks in Metropolitan Mexico City children and young adults.1,9 The early identification of subjects at risk for developing AD in air pollution environments and understanding the mechanistic pathways involved, are at the core of our research efforts. Identifying key air pollutants impacting neural risk trajectories and more important defining the strategy to decrease culprit pollutants exposures and if measuring NPs is necessary and how to accomplish the process, would greatly facilitate multidisciplinary prevention efforts for modifying the course of Alzheimer’s disease in paediatric and young adult ages.

Lifetime exposures of air pollutants above the USEPA and WHO standards in Metropolitan Mexico City residents. Why these sustained, high exposures are critical for the brain? It is recognized that air quality has improved in MMC since the 1990’s, but the megacity still experiences episodes of high ozone and fine particulate matter concentrations (PM2.5) that exceed health limits stated by the USEPA air quality standards and the WHO guidelines (Supplemental Table 1).27-29 Despite several governmental initiatives to reduce air pollution in MMC, PM2.5 and secondary pollutants including ozone seem to have stagnated for the last 15 years.29,30 Regular measurements of PM2.5 concentrations began in

5

2004 and the levels reported on that year have remained practically the same to the present time (Figure 1).

Figure 1. Trend of maxima PM2.5 24-hour average concentrations registered in the MMC from 2004 to August 2019 and their comparison against the WHO daily mean average and the US AQI. Data correspond to measurements from the manual network of particulate matter of the SEDEMA and were obtained from: http://www.aire.cdmx.gob.mx/default.php#

A number of non-scientifically based decisions to control the air pollution origin have hindered the solution of the problem.30,31 To make matters worse, adverse meteorological conditions for the dispersion and ventilation of air pollutants in the atmospheric basin where MMC is located, have directed to a number of severe O3 episodes in March 2016 and PM2.5 in May 2019. The latter was the result of massive uncontrolled wildfires of biomass in the center and south of Mexico triggering a PM2.5 pollution crisis rarely seen in MMC last 15 years.32,33

6

The PM2.5 24-hour average concentrations reached and surpassed the range of unhealthy level of the US EPA Air Quality Index (151-200)27 during 5 continuous days with peak hourly averages up to 227 µg/m3 (Figure 2).

Figure 2. Trend of maxima PM2.5 24-hour average concentrations registered in May 2019 at MMC monitoring stations and their comparison with the US AQI. The fire event covers the period of May 4-17. Data correspond to the automatic monitoring network of the SEDEMA and were obtained from: http://www.aire.cdmx.gob.mx/default.php#

Unfortunately, the authorities delayed to face the emergency and after several days set a number of measures that did not work, showing their ineffectiveness to solve air pollution episodes in a crisis context and their failure to protect 25 million residents. Biomass burning for heating and cooking and open trash burning are typical practices in the peri- and sub-urban MMC areas although their contributions to the PM2.5 in the urban area are relatively low.34 However, during the dry-warm season, a high percent of crop lands and forest areas in Mexico are burned to prepare the soil for the next sowing season and to open 7

land for agricultural activities.33 Regional fires within a variable distance from 60-400 km from MMC center might contribute to ~10% to the PM2.5 inside the urban area.34 The May 2019 fire event resulted in PM2.5 increases ≥150% and put the entire MMC population at high risk of respiratory,cardiovascular and stroke fatal events. 35-40 There is no doubt mobile sources dominate the pollutant emissions in MMC. According to the last emissions inventory of 2016, mobile sources contribute with 89% of CO, 82% of NOx, 21% of SO2, 18% of VOC, 30% of PM10 and 35% of PM2.5. 41 NOx and VOC, and to lesser extent CO, contribute to the formation of secondary air pollutants such as O3 as well as important fractions of NO2 and PM2.5. Ozone is formed from the reactions of NOx with VOC under sunlight conditions. Secondary organic aerosols are produced through reactions of anthropogenic and biogenic volatile organic compounds (VOC) over the surface of fine particles resulting from combustion processes, while NOx and SO2 can react with NH3 to form nitrate and sulfate secondary inorganic aerosols, respectively.42 As a consequence, the chemical composition of PM2.5 is a complex mix of primary and secondary contributions, which also depend on the spatial location within the MMC. Amador-Muñoz et al.43 have presented evidence that PM2.5 in the MMC NE and SE are associated to primary sources –i.e., diesel combustion-, while fine particles in downtown area are best linked to a combination of primary (gasoline combustion) and secondary contributions. Aerosols in SW MMC are more related to secondary formation (biogenic VOC-aerosol chemistry). A recent study on the composition of PM2.5 presented evidence that its composition has not changed significantly since the early 2000’s.44 In general, over 47% of the PM2.5 is comprised of organic aerosols, 30% of secondary inorganic aerosols, 12% black carbon, 7% soil components, 1% metals and the rest mineral components. In 8

MMC average PM2.5 constitutes ~50% of PM10 mass. Strikingly important, to the health impact:~75% of the PM2.5 is smaller than 1 micron fraction (PM1) which includes the ultrafine particles (≤100nm). Organic aerosols (OA) comprise both, primary (POA) and secondary aerosols (SOA). POA chemical composition includes primary hydrocarbon-like compounds, aldehydes, carboxylic acids, substituted phenols, polycyclic aromatic hydrocarbons (PAH) and their nitrogen-derivatives among others, all of them emitted by anthropogenic and natural sources.45 It has been estimated that aproximately a third of the POA is generated by on-road mobile sources and that uncontrolled and unregulated food cooking on the streets is an important POA source.30 The diurnal profile of OA in MMC displays two peaks, the higher one during the morning is associated to primary emissions from the rush hour, while the second one in the afternoon results from secondary contributions associated to photochemical processes and dominates ≥ 70% of the total aerosols.42 Black carbon (BC) is a term used to identify the elemental carbonaceous component of particles from the incomplete combustion of organic compounds and correlates with PM2.5 in urban areas.46 One important property of BC is its porosity which provides a large surface area able to adsorb many combustion exhaust species such as PAH.47 PAH are recognized as carcinogenic, mutagenic compounds.48 As a consequence, the importance of BC is that it is a great carrier of carcinogenic substances.49 The BC concentrations in MMC follow the same spatial distribution as PM2.5, with the highest levels in the NE and the lowest towards the SW. A historical review of BC measurements in MMC shows an stability of their concentrations for the last 30 years.50 Baumgardner and colleagues51 measured BC concentrations in SW MMC during April on 2003 and 2005 and found an average 24-hour concentration of ~ 1.5 µg/m3 with a daily maximum of 5 9

µg/m3. Equivalent measurements performed in 2015 in the same monitoring site reported an average of ~ 3.2 µg/m3 and a daily maxima of 5.8 µg/m3.52 Measurements of BC performed in northern industrialized MMC in 2000 and between 2013 and 2014 reported averages of ~ 7 µg/m3, and of ~ 2.5 µg/m3.

51,53

Peralta et al.52 found that the MMC

BC/PM2.5 concentrations ratio in 2015 was around 0.14 in the SW, almost the same as inferred from the work of Edgerton et al.50 in 1997 for the downtown area. High concentrations of BC across the northern MMC sector in the last 2 decades are evident when reviewing the maximum concentrations measured in 2000 54,200655,and 2013-201453: 9.1, 9.4 and 13.1 µg/m3, respectively. The strategies to reduce BC emissions in MMC have failed. PAH are semivolatile complex organic compounds conformed by at least two benzene rings with very high molecular weight. They are associated to incomplete combustion processes of fossil fuels and biomass, so they correlate with CO, NOx and BC.48 As PAH can be absorbed onto other particles such as BC, they are known as particle –bound PAH (PPAH). Measurements of PPAH in MMC have been performed since the early 2000´s by two procedures: on-real time and off-real time. Ladino et al. 48 presented a complete summary of the historical reports of on-real time measurements and compared the levels of PPAH reported for the spring of 200356 and those found by them in the spring of 2016 in sites with similar characteristics in south MMC. The average PPAH concentration in 2003 was 11.3 ± 38.9 ng/m3 while in 2016 was 28.1±36.7 ng/m3:150% higher in 2016. The striking difference according to Ladino et al.,48 could be the result of the increase in the number of vehicles in MMC from ~ 2 millions in 2003 to ~ 4 millions in 2016. PPAH reach their maximum in the morning,coincide with the rush hour and exposes millions of 10

people in transit to work and school. PPAH correlates with CO and NOx, the correlation in MMC depends on the intensity of the emissions associated to light gasoline vehicles versus heavy diesel vehicles. Using a multiple correlation model, Ladino et al.48 found PPAH high concentrations occur in the North and Northwest MMC,reflecting the industrialized areas with heavy diesel transport traffic. Their results strongly suggest residents in the northern MMC are exposed to average PPAH levels on the order of 20 ng/m3 or higher in the morning

period.These

PPAH

exposures

ought

to

have

serious

health

consequences,including serious brain effects.PPAH off-real time measurements done in MMC since the mid-1990s by techniques such as gas chromatography-mass spectrometry (GC-MS)48 show PPAHs are complex mixtures containing >100 compounds, including benzo[a] pyrene, chrysene, benz[a]anthracene, and benzo[a]fluoranthene. Figure 3 shows a comparison of the sum of the 13 common PPAHs found in several rural and urban sites in USA, Canada and the NE and NW sectors of MMC57-59 and the ranking of benzo[a]pyrene in particles for the same sites. Villalobos-Pietrini et al.45 reported that benzo[ghi]perylene has been systematically the most abundant PPAH followed by indeno[1,2,3-cd]pyrene. Both are tracers of vehicular emissions. Amador-Muñoz et al.60 PPAHs study between 1998-1999, evaluated the particle size distribution containing the most abundant PPAHs and found the fine PM2.5 as the culprit carrier.

11

Figure 3. Averages of (a) the sum of concentrations of 13 PAHs; (b) the concentrations of benzo(a)pyrene in particulate matter in several North America cities. The sum of PAHs include: acenaphthylene, phenanthrene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(a)anthracene, fluorene, fluoranthene, pyrene, chrysene, benzo(k)fluoranthene, dibenzo(a,h)anthracene, indene(1,2,3-c,d)pyrene, and benzo(g,h,i)perylene. Data of PAHs are for the years: 2012 Canada sites; 2010 U.S.A. sites, and 2005-2007 Mexico City sites. Numbers above averages of sum of PAHs represent the population at risk.

Others contributions to PM2.5 mass include high levels of anthropogenic metals, i.e., chromium, zinc, copper, lead, vanadium, antimony, and barium. Metals exhibit strong temporal variations in concentration and are largely associated with industrial and mobile 12

sources.61 Figure 4 displays a historical MMC 2002-2013 review of the most abundant metals in PM2.5.. Elements representing urban (mostly road traffic) including Cr, Mn, Zn, and Pb, are typically associated with engine emissions, abrasion of tires and brake pads. V and Ni are interpreted as tracers of long-range transport from the use of heavy fuel oil in the northern industrial area of Tula in the State of Hidalgo.

10000

Concentration (ng/m3)

1000

100

10

1

0.1 S

Cl

K

Ca

Ti

V

Cr

Mn

Fe

Ni

Cu

Zn

Pb

1989-90 (Downtown) 1994 (NE) 1997 (NE) 2002 (NNE) 2006 (NE) 2011-2013 (Downtown)

Figure 4. Average concentration of S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn and Pb in PM2.5 in several representative areas of MCA from 1989 through 2013 based on data from short-term sampling campaigns.61-66

The impact of sustained high concentrations of complex mixtures of air pollutants including metals, PAH and endotoxins as components of fine PM2.5 and the ultrafine PM (≤100nm) and gaseous pollutants upon 25 million people residing in Metropolitan Mexico City represents a serious health problem for everyone regardless of age, gender and socioeconomic status. MMC has one of the most advanced air pollution monitoring networks in Latin America with more than 30 stations distributed across the urban and periurban area. 13

29

However, there is no yet an accessible and practical warning system to effectively inform residents of potentially unhealthy events.29 At the present, warnings are emitted in terms of an air quality index (AQI) which is based on the Mexican Air Quality Standard (MAQS). One hundred points on the Mexican AQI corresponds to the maximal limit of the air quality standard. Although the Mexican AQI is constructed following the structure of the US AQI, the exposure bands to qualify the air quality are not equal. Table 1 shows the Mexican and US air quality index bands for O3 and PM2.5. 67

AQI Index

Air quality qualification Mexico US

0 - 50 51 - 100

Good Regular

101 - 150

Bad

151 - 200

Very bad

201 - 300 301 - 400 401 - 500

Extremel y bad

O3 (ppbv 8 hr average) Mexico US

O3 (ppbv 1 hr average) Mexico US

PM2.5 (µg/m3 24 hours moving average) Mexico US

Good Moderate Unhealthy for sensitive groups

NA NA

0 - 54 55 - 70

0 - 70 71 - 95

NA NA

0 - 12 12.1 - 45

0 – 12 12.1 – 35.4

NA

71 - 85

96 - 154

125 164

45.1 – 97.4

35.5 – 55.4

Unhealthy

NA

86 - 105

Very unhealthy

NA

106 200

Hazardous

155 204 205 – 404 405 504 505 604

76 - 114 205 – 404 405 504 505 604

97.5 – 150.4 150.5 – 250.4 250.5 – 350.4 350.5 – 500.4

55.5 – 150.4 150.5 – 250.4 250.5 – 350.4 350.5 – 500.4

Table 1. Air Quality Index (AQI) Breakpoints with concentrations in Mexico and the US.

In the MMC, 150 points on the Mexican AQI is the threshold for issuing emergency pollution health alerts called “Environmental Contingencies”.67 It is based on the real time levels of O3, PM2.5, and PM10 reported by SEDEMA and the meteorological forecast.67 A contingency is activated when O3 concentration equals to 155 ppbv (hourly average), or PM2.5 reach 97.4 µg/m3 (24 hours moving average).67 A number of actions implemented by the authorities during an environmental contingency might include: banning of the 14

circulation a part of the vehicular fleet as well as reduction of the activities of highly polluting industries. However, as the Mexican AQI is based on the MAQS which in the case of PM2.5, depends on a relatively long-term average (24 hours), the issuing of the emergency is activated after several hours of exposure of the population to high levels of PM2.5 and does not involve any actions relative to heavy diesel vehicles. Therefore, the contingency program does not guarantee an effective reduction of pollution levels on the contingency day29, nor protects MMC 25 million residents.

For this review, we have focused on <2.5µm size particles and work with cumulative PM2.5 (CPM2.5) above the annual USEPA standard: 12 µg/m3, reflecting lifetime exposures above the standard. The accumulated burden of PM2.5 for each of the 134 individuals in the autopsy study-included pregnancy time-was calculated based on their urban residency (Supplementary Table 2). Historical PM2.5 levels were obtained from a combination of particulate matter data from Mexico City Government Manual Monitoring Network for five representative urban sites: Tlalnepantla (NW), Xalostoc (NE), Pedregal (SW), Iztapalapa (SE) and Merced (downtown)and an approach considering the typical PM2.5/PM10 ratio for each of the representative sites. 1 The lifetime high exposures to PM2.5 and the complex mixtures including ozone, metals, PAH, VOC, BC and endotoxins and the failure to have an effective warning system informing the residents of health-damaging events are critical to understand why we have 99.5% Alzheimer disease cases in the 134 consecutive,unselected autopsy young MMC 20.03±6.38y cohort.1,9,23, 29,41-68

15

Statistical approach of previously published papers discussed in this work For the autopsy study1 we first calculated the summary statistics in the cohorts: accident, homicide, suicide for all MMC subjects of age less than or equal to 30 years. Then, we tested the equality between the average values of each of CPM2.5, Age, APOE status, higher Htau status (>= 3), and higher Amyloid beta (>= 2) status among the three cohorts. For the cognitive studies 69 we created three groups based on MoCA scores: >= 26, between 24 and 25, and <= 23, i.e., Normal, MCI and AD in subjects ≤30y. We calculated summary statistics of various subscores of MoCA in each of the three groups. We also drew figures of cumulative probability of suicide as a function of CPM2.5, after adjusting Age for each cohort: APOE 3 and APOE 4. We performed all these analyses on Excel and R.

Neuropathological Alzheimer hallmarks in the 134 autopsy MMC cohort

Supplemental Table 2 shows the cumulative CPM2.5 values,age, gender and staging of hyperphosphorylated tau and amyloid ß phase for autopsy subjects ≤30y. 70-74 Figure 5A shows the distribution of Htau staging and Aß phase for the 134 cases by cause of death: accident, homicide and suicide and the staging for APOE4 carriers. Autopsy cases were divided by decades one to three and the Htau staging and Aß phase displayed in bars (Figure 5B). Htau is present mostly in pretangle stages 1a,1b in every single child examined in the first decade and by the second and third decades we documented neurofibrillary (NFT) stages I-V.70-72

16

17

Figure 5A: Percentages of Pretangle and NFT stages and Aβ phases within the ≤ 30y old cohort by cause of death: accident, homicide, suicide and APOE4 carriers.

APOE4 cases clearly showed advanced stages both in NFT (III-IV and V) and amyloid beta phase 3 (Figure 5A). When we compared homicide (n:45) and suicide (n:27) cohorts, APOE4 status was significantly higher (p=0.0005). The data strongly supports young APOE4 urbanites have an acceleration of the Alzheimer neuropathological hallmarks and this acceleration potentially plays a role in their higher risk of commiting suicide.1,75-77

18

Figure 5B: Percentages of Pretangle and NFT stages and Aβ phases within the cohorts of 0-10 years, 11-20 years, and 21-30 years.

Figure 5B shows the first two decades are key for the progression of Htau neurodegeneration: every single child 10y old and younger,including the 11 month old baby already have hyperphosphorylated tau in the brainstem and the Pretangle stages 1a and 1b are quickly progressing to NFT stages I-V by the second decade. Figure 6 shows the cumulative probability of suicide after adjusting for age as a function of cumulative PM2.5 exposure in each APOE3 and APOE4 cohorts. Young APOE4 carriers have 1.26 higher odds of committing suicide and require lower cumulative PM2.5 versus APOE3 subjects.

Figure 6: Age adjusted cumulative suicide probability against cumulative PM2.5 within APOE3 and APOE4 cohorts.

19

Peripheral and central auditory system dysfunction evolves across pediatric and young adult urbanites.

A critical observation in MMC toddlers is the presence of hyperphosphorylated tau (Htau) in the lower medulla.1 Thus, the accumulation of Htau and alpha synuclein in cochlear and vestibular nuclei by the second decade and the marked dysmorphology in the ventral cochlear nucleus and superior olivary complex both in humans and dogs in MMC strongly point towards the brainstem as a key neuroanatomical region for the presence of abnormal proteins.15,74, 78-80 The abnormal results of the brainstem auditory evoked potentials

(BAEPs) in MMC clinically healthy children (8.52±3.3 years) and adults (21.08±3.0 years, 42.48±8.5 years, and 71.2±6.4 years) compared to clean air controls were in keeping with the extensive neuropathology in exposed subjects.1-3,13,78-80 Figure 7 illustrates the human auditory pathway (A) and provides a schematic of the five peaks typical of the human BAEP (B). The changes in BAEPs as subjects grow-up in the polluted megacity are summarized in figure 7C-D.80 Decreased latency to wave I, delays in waves III and V, and longer latencies for interwave intervals, consistent with delayed central conduction time of brainstem neural transmission are seen in the first decade (Figure 7C), followed by significantly shortened interwave intervals I-III and I-V in teens and young adults (Figure 7D-F) and by significantly shorter wave V and interval I-V in the fifth decade (Figure 7G). Older cohort subjects (71.2±6.4years) had significant delay in all mean latencies and interwave intervals. 80

20

Figure 7. The human auditory pathway and brainstem auditory evoked potentials (BAEPs). Shown in A is a schematic of the human auditory pathway. Sound waves are transmitted through the external and middle ears to activate sensory receptors in the organ of Corti. Neurons in the spiral ganglion relay action potentials from hair cells, along the auditory nerve (AN) to the cochlear nuclei (CN). The cochlear nuclei send axons to innervate nuclei in the superior olivary complex (SOC). Both the SOC and CN project axons through the lateral lemniscus (LL) to the inferior colliculus (IC). The IC projects to the medial geniculate (MG) complex in the thalamus, which then projects to the primary auditory cortex (A1). The brainstem auditory evoked potential (BAEP, B) consists of five positive peaks (I-V) that correspond to synchronous electrical activity in each of the brainstem stations (compare I-V in A and B). Shown in figures C-H are BAEPs from an agegraded series of subjects from longlife residents in metropolitan Mexico City. In the 16-year-old male subject there are decreased latency in waves I, III and V. However, from the 19-year-old to the 64-year-old, there is progressive delay in waves I-V.

Our interpretation of the BAEPs findings in MMC residents included involvement of the brain by neuroinflammation, cochlear synaptopathy, Alzheimer Continuum with the 21

accumulation of abnormal proteins in key nuclei resulting in compensatory plasticity, and increased auditory gain. 1,2,3,7,10,13-15,18,80-88 Albers et al., 85 summarized the auditory impairments in AD subjects and emphasized hearing loss and central auditory dysfunction are associated with a high risk of conversion to dementia, while peripheral hearing loss is associated with poorer performance on both verbal and non-verbal cognitive tests. Our BAEPs findings in the older cohort are consistent with the literature 82-88 and certainly supports the use of BAEPs as a screening tool in children and young adults to identify subjects at high risk for Alzheimer’s Continuum. 79,80, 89,90 Interestingly, Jack et al.,90 studies of three imaging biomarker-based definitions of the AD spectrum: Alzheimer continuum(abnormal amyloid regardless of tau status), AD pathological change(abnormal amyloid with normal tau) and Alzheimer disease(abnormal amyloid and tau) concluded that biologically defined AD is more prevalent that clinically defined probable AD in their 60-89 year old cohorts from Olmsted County, Minnesota,USA, and critical to our MMC studies he adds “this difference is mostly driven by asymptomatic individuals with biological AD”.Thus, even in relatively low pollution

exposed Minnesota subjects, there is a significant number with biological defined AD.

The scenario in MMC is drastically different: we have 99.25% of unselected, consecutive, children and young adults with neuropathological staging of Alzheimer disease using hyperphosphorylated tau and beta amyloid 1, every resident regardless of age with abnormal BAEPs78-80, children with significant deficits in a combination of fluid and crystallized cognition tasks91 and 55% of young adults cognitively impaired 69 or 66.6% if we apply the MoCA scores as the original paper by Nasreddine et al., 92 22

The Montreal Cognitive Assessment (MoCA)

The Montreal Cognitive Assessment (MoCA)- a brief screening test that covers several cognitive domains including episodic memory, language, attention, orientation, visuospatial and executive functions 92- administered to 150 MMC urbanites (48M,102F), age 21.59± 3.56 years, with 13.65± 1.73 y of formal education showed an score of 24.27±2.62 (normal 26-30), 34% and 32.6% scored ≤ 25 and ≤23 respectively (Mild cognitive impairment MCI≤25, Dementia D≤23)(Figure 8).69

Figure 8: Three subgroups of Metropolitan Mexico City subjects age 30 years or less, based on MoCA scores.Two thirds of the population younger than 30 years is already cognitively impaired.

Table 2 shows the MoCA scores in the three categories normal cognition, MCI and dementia (D) and the differences in each variable depending on the MoCA score. 23

MoCA scores for NC, MCI and D

MoCA score

Age

Trail

Cube

Clock

Namin g

Delay memor y

DSF DSB

Letter A tappin g

Serial 7 subtract ions

Sentence repetitio n

Word fluenc y

Abst

26-30 NC n: 50 (18 M, 32 F)

27.00± 0.95

21.64±3. 26

0.94±0. 24

0.76±0. 43

2.62±0. 60

3.00±0. 00

3.62±0. 88

1.80±0. 40

0.94±0. 24

2.76±0.59

1.72±0.45

1.00±0. 00

1.82±0. 44

p-values (26-30 vs 24-25) 24-25 MCI n:51 (16 M, 35 F) p-values (24-25 vs <= 23)

<0.0001

0.24

0.64

0.0006

0.0304

0.32

0.0004

0.96

0.64

0.22

<0.0001

0.06

0.0144

24.43± 0.50

22.47±3. 78

0.96±0. 20

0.43±0. 50

2.31±0. 79

2.98±0. 14

2.82±1. 26

1.80±0. 40

0.96±0. 20

2.61±0.63

1.22±0.61

0.90±0. 36

1.53±0. 70

<0.0001

0.0126

0.13

0.44

0.0239

0.36

0.0001

0.0008

0.38

0.0001

0.30

0.0082

0.75

≤23 D n:49 (14 M, 35 F) p-values (26-30) vs <= 23)

21.33± 1.86

20.63±3. 45

0.88±0. 33

0.51±0. 51

1.90±1. 01

2.92±0. 45

1.76±1. 42

1.47±0. 54

0.92±0. 28

1.92±1.00

1.08±0.67

0.67±0. 47

1.57±0. 61

<0.0001

0.14

0.29

0.0096

<0.0001

0.21

<0.0001

0.0009

0.68

<0.0001

<0.0001

<0.0001

0.0227

Gender BMI

0.90 0.86

NA NA

0.31 0.34

0.53 0.45

0.75 0.84

0.30 0.08

0.30 0.54

0.53 0.65

0.52 0.74

0.38 0.36

0.72 0.49

0.20 0.16

0.052 0.28

Table 2. Metropolitan Mexico City MoCA raw scores in a cohort of 150 subjects age 21.59±3.56 years. The original cutoff scores for distinguishing normal cognition NC (26-30), MCI (2425) and dementia D (≤ 23) were applied here. 92 DSF and DSB digit serial forward and backward.

Progressive cognitive deficits targeted Visuospatial, Language, and Memory domains from normal cognition to MCI(copy of the cube, delay recall and sentence repetition are early targets). Strikingly,as the young subjects move towards dementia scores there is a significant impact on Language, Visuospatial/Executive and Attention. In the subjects with scores <= 23, delay recall, serial 7’s subtractions,sentence repetition and word fluency are significantly impaired and the clock copying errors are the rule. The average age for dementia MoCA scores was 20.6±3.4 y. Gender and BMI had no impact on the MoCA scoring (Table 2).

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When we divided the subjects according to the MoCA total score corresponding to NC, MCI and D and the Cognitive Domain Index Scores (Table 3) 93,94 the data strongly suggest Language Index Score (LIS) -including sentence repetition, word fluency and animal naming-, is a key early target from NC to MCI:the cutoff score of 5.5 went to 5.09±0.75. Given that the frontal lobes,the primary visual cortex bilaterally and the ventral occipital cortex to the inferior temporal area on the right side are involved in sentence repetition, word fluency and animal naming,there is a high likelihood these areas are involved in the early neurodegenerative and neuroinflammatory process associated with residency in a highly polluted megacity. 95-101,1-3,5,7-10 MMC Cohort age ≤30y and MoCA total scores for normal cognition, MCI and D

EIS Total:13 CUTOFF SCORE 10.5

LIS Total:6 CUTOFF SCORE 5.5

VIS Total:7 CUTOFF SCORE 5.5

AIS Total:18 CUTOFF SCORE 16

OIS Total:6 CUTOFF SCORE 5.5

Delay recall+EIS+V IS+LIS Total:31 CUTOFF SCORE 24

MoCA scores≥26 Normal Cognition 27.00±0.95 n:50 MoCA Scores 2524 MCI 24.43±0.50 n:51 MoCA scores≤23 D 21.33±1.86 n:49 ALL

11.880±0.940

5.720±0.4536 6.380±0.725

18.220±0.790 5.920±0.274

27.600±1.262

11.078±1.074

5.098±0.755

5.725±0.896

17.490±1.027 5.824±0.434

24.725±1.150

9.327±1.749

4.673±1.049

5.327±1.463

16.061±1.360 5.673±0.555

21.082±2.805

10.773±1.672

5.167±0.893

5.813±1.149

17.267±1.398 5.807±0.444

24.493±3.254

Table 3 Cognitive Domains Index Scores 93 The original MoCA score categories for normal cognition (2630), mild cognitive impairment (24-25) and Dementia D (≤ 23) are applied here.92Executive Index Score (EIS) is the sum of Trail making, clock drawing, digit span forward and backward, letter A tapping, serial 7’s subtraction, word fluency and abstraction.Language Index Scores (LIS): animal naming, sentence repetition and word fluency.Visuospatial Index Score (VIS): cube copy, clock drawing and animal naming. Attention Index Score (AIS):digit span forward and backward, letter A tapping, serial 7s substraction, sentence repetition and Words Recalled in Both Immediate Recall Trials. The Orientation Index Score (OIS) includes all the Orientation items (0–6 points).

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Indeed, we also recorded the sentence repetition errors in our young subjects and their characterization as ending omissions and unrelated word substitutions as described by Beales et al., 102 for Alzheimer's subjects. In the cohort with MoCA scores ≤23, EIS, LIS and VIS scores all were below the cutoff marks (Table 3).

Social, educational, judicial,economic and the overall health impact of a cognitively impaired young population

The presence of cerebral tau pathology and extracellular aggregated amyloid-β in 133/134 consecutive young brains constitute a serious health problem. All of the 134 residents in Metropolitan Mexico City had been exposed to PM2.5 values above the current USEPA standard and to significant concentrations of PAH’s, BC, NOx,VOC’s, metals and highly oxidative and ubiquitous combustion-derived nanoparticles (CDNPs) associated with early and progressive damage to the neurovascular unit, supporting they are likely suspects for cell damage,neurotoxicity and neurodegeneration.1-3, 5,14,16,23-68 There is robust evidence the speed of Alzheimer disease progression in young cohorts ≤30y of age is given at least in part by an APOE 4 allele, present in 20% of the population. 1 APOE4 increased the odds for developing NFT V1 which means in a population of 25 million, we have 5 million of APOE 4 carriers at high risk for Alzheimer’s rapid progression. The finding of Htau in 11 month old babies is relevant to Braak and coworkers comment “continual formation of abnormal tau takes place from the beginning until the end-phase of Alzheimer’s disease and is not known to be subject to remission”.72

26

So the question of: could we reverse the presence of Htau in MMC children and young adults? The answer at this time is likely not. These are the main points to remember when we read about air pollution brain effects across the world: 1.Air pollution is very complex and varies from city to city and in large cities, strikingly within the city. 2.Fine particulate matter PM2.5 is our proxy for PM pollution, keep in mind is not just a matter of concentration, is also a matter of composition,diurnal versus nocturnal,peaks,seasonal variations,etc., Thus, the PM2.5 profile, organic and inorganic composition, metals, endotoxins,PAH,VOC,etc., are to be quantified and definition of sources, unexpected events, etc., are key. Strikingly important is the relationship between PM10/PM2.5 and within PM2.5 what percentage is 1 micron PM1 which includes the ultrafine particles (≤100nm). Heavy diesel trucks are an important pollution source in MMC and excluding them from regulation is a major gap, so is uncontrolled food cooking on the streets. 3. The nanocluster aerosols emitted by road transportation in the size range of 1.33.0 nm and the magnetic nanoparticles have to be taken into the risk equation.9,23,103 Nanoparticles resulting from combustion process can be divided into three different sources according with Rönkkö and Timonen103 i.primary NPs formed in high temperature, ii. delayed primary particles formed as gaseous compounds nucleated during the cooling and dilution process and iii. secondary NPs formed from gaseous precursors via the atmospheric photochemistry. The nature of these nanoparticles is vital, these are the

27

particles capable of traversing cell membranes and all biological barriers and we should add: difficult and expensive to measure. 4. Magnetite 'nanospheres' abundant in roadside air pollution are the result of vehicle combustion and frictional brake-wear.23 These tiny airborne magnetite pollution NPs access the brain directly via the olfactory and/or trigeminal nerves, bypass and damage the neurovascular unit and certainly traverse all biological barriers.1,3,5,14,16,19-23 Since iron-rich magnetic NPs respond to external magnetic fields and are involved in cell damage by agglomeration/clustering, magnetic rotation and/or hyperthermia 9,104 it is also very important we keep track of individual lifetime occupational magnetic field exposures knowing electric power plant,electric furnace, transformer substation workers, welders,electricians,etc.,have high exposures to magnetic fields,including extremely low frequency magnetic fields (ELF MG). 105- 109 Children exposures to Extremely Low Frequency Magnetic Fields (ELF MF) is a relevant subject, in view of the fact their brains and hearts are loaded with magnetic nanoparticles. 5,9,18,104 Tognola et al., 110 applied cluster analysis to 24 h indoor personal exposures of 884 children in France, investigating how electric networks near the children’ home affected magnetic field exposure patterns residential exposure. The authors identified three indoor highest exposures patterns: children living near overhead lines of high (63-150 kV), extra-high (225 kV) and ultra-high voltage (400 kV), while children living near underground networks of low (400 V) and mid voltage (20 kV) and substations (20 kV/400 V) were characterized by mid exposures. Children living far from electric networks had the lowest level of exposure and those using electric heating appliances, or living in big buildings or in larger families had generally a higher level of personal indoor exposure.110 Mobile phones and electric vehicles should be 28

added to the list of magnetic field sources. 111-115 Thus, the potential association between Alzheimer disease and magnetic fields is biologically plausible116: the more Fe-rich magnetic nanoparticles in the brain, the higher the negative effects of magnetic effects i.e., agglomeration/clustering, magnetic rotation and/or hyperthermia.5,23,104 5.The development and progression of Alzheimer Htau and ß amyloid are evident in the autopsy cases and the reader should be aware is possible to follow the progressive deterioration of cognition in young adults. 69 In the cohort of 150 subjects, age 21.59± 3.56 years, two thirds have cognition impairment and it is possible to define the areas progressively involved94: to copy a cube you need “visual perception in the parietal-occipital lobe, planning in the frontal lobe and integration of visual and fine motor sequences in the fronto-parietal-occipital cortices”;retrival memory impairment has been associated with medial parietal and frontal white matter damage and pathology in the posterior cingulate and the hippocampal-parietalfrontal network 94,117-123;sentence repetition deficits involve preferentially the left temporalparietal-frontal circuit. 94,124,125 Indeed, we are witnessing an accelerated Alzheimer disease process with striking time lines, and disease progression pace. We have strongly suggested childhood and teen years are critical for brain damage associated to environmental pollutant exposures, and although there is no doubt considerable individual AD progression differences are likely determined by factors such as genetics, metabolism, nutrition, lifestyle, stress, lifelong cognitively stimulating behaviors, resilience, linguistic ability,occupational history, and others 2,3,8-10,12,16,126-128 the persistence and progression of Htau lesions in young children, based on classic neuropathology studies 70-74 represent a serious short and long term health problem. 29

Given all variables involved in the brain responses to air pollution it is imperative researchers and particularly physicians realize you should not compare brain effects of any sort solely on the bases of concentrations of PM2.5, for example Barcelona children are not the same as Mexico City children, nor children in Cincinnati,Boston, Norway or the Netherlands.129-134 Humans are not Balb-c mice,so taken data from one specific cohort with genetic, nutritional, lifestyle,comorbidities, etc., characteristics and applying their results to a different population is not warranted. Caruso et al., 135 has supported the concept prolonged stress predisposes susceptible individuals to a number of physiological disorders including neurodegenerative disorders. Changes in the glucocorticoid milieu are key in the impact of stress on the brain and potential neurodegeneration.135 The issue of prolongued stress is very important for residents in Metropolitan Mexico City where the epidemic violence permeates in the population.136-141 Life expectancy among males decreased from 2005 to 2010 resulting from an increase in homicides in the age range of 15-50y and to diabetes in subjects ≥40 years.138 Depression-a leading cause of morbidity in Mexico142-is defined by the Diagnostic and Statistical Manual of Mental Disorders (DSM–5) DSM 5143 as a serious mood disorder characterized by an individual experiencing five or more symptoms during the same 2week period with at least one of the symptoms being (1) depressed mood or (2) loss of interest or pleasure.Depression is associated with a number of incident traumatic events including violence and chronic pediatric adversities predictors of substance abuse disorders. 144,145

Suicide is a problem in Mexico City and the highest mortality rate is among young

males ages 20-24 and females ages 15-19y146, certainly involving individuals carrying an APOE4 and with advanced AD stages.1 Premarital violence, psychopathology and suicidal 30

thoughts and behaviours in young adults, aggression,violence,threats to security and high risk of ischemic heart disease 147-153 are part of the grim scenario in Mexico City. We ought to emphasize Mexico City children have a short silent stage and Htau is the key abnormal protein to cause early symptoms.1,7-10,17,78,80 Cognition deficits are seen early in childhood and progression to AD scores precisely at the age young adults are building skills and acquiring knowledge in the academic and work setting. 7,69 In Mexico, where we have serious educational deficiencies 154-157 the percentage of 15y olds, 2015 top performers in reading,mathematics and science (proficiency Level 5 or 6), is one of the lowest among the Program for International Student Assessment PISA156 participating countries and economies : 0.3 %, rank 60/69 ; 0.3 %, rank 63/69 , and 0.1 %, rank 62/69 respectively; where we have 22% of young adults who are neither employed nor in education or training (NEET)157;where the gross domestic product, GDP is cero and the Mexico City crime index of 69,716 /100,000 residents reflects an spiral of violence, we ought to consider the impact of pollutants -iron rich, magnetic nanoparticles with transitional metals and PAH, and nanocluster aerosols- upon brain development,cognition, brain structural changes, neurodegeneration and violence. 1,3-6, 18,69,104, 158-161 Targeted pollutant control should be prioritised along with defining clinical, laboratory, imaging, and cognitive non-invasive markers for the initial stages of the disease knowing Htau is a prime actor, the disease starts in the brainstem and the olfactory bulb, and APOE 4 carriers are at highest risk. Nanoparticles are key players in the neuroinflammatory and neurodegenerative pathology observed in early childhood with rapid progression as the child grows up in a polluted environment104, thus we urgently need to define if PM2.5 is enough to evaluate exposures or if we need to expand our measurements to ultrafine PM. 31

The fact we can measured magnetic iron-rich magnetic particles in the brain and hearts of young urbanites ought not to be ignored. Denial is not going to protect millions of people of the air pollution detrimental effects. Understanding the neural circuitry associated with the earliest cognitive and behavioral manifestations of AD should be a priority. We have commented before, early interventions should be integrated in health and educational agendas and identification of early genderspecific risk trajectories ought to be done. Paediatric environmental, nutritional, metabolic and genetic risk factor interactions ought to be defined by multidisciplinary unbiased, highly trained groups to prevent a lethal disease killing millions of people across the world.

Declaration of interests

All authors declare no competing interests.

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54

SUPPLEMENTARY TABLES Supplemental Table 1. Mexico’s and US EPA´s ambient air quality standards, and WHO’s air quality guidelines. US National Air Quality WHO Air Quality Mexico Air Quality Standards (b) Guidelines (c) Standards (a) Pollutant Max. Limit Max. Limit Max. Limit 95 ppbv (Hourly mean) O3

70 ppbv (Annual maximum moving average for 8 hours) 210 ppbv (Hourly mean)

70 ppbv (Annual 4th highest daily maximum over 3 years) 100 ppbv (98th percentile of 1-hour daily maximum concentrations, averaged over 3 years)

50 ppbv (8 hour average daily max)

-

53 ppbv (annual average)

110 ppbv (24 hour maximum average)

75 ppbv (99th percentile of 1-hour daily maximum concentrations, averaged over 3 years)

40 ppbv (Annual average) 7 ppbv (24 hour maximum average)

200 ppbv (Annual 2nd 8hour maximum average)

500 ppbv (3 hours average not to be exceeded more than once per year

190 ppbv (10 min maximum average)

25 ppbv (Annual average) -

35 ppmv (Hourly average not to be exceeded more than once per year)

-

11 ppmv (Annual maximum moving average for 8 hours)

9 ppmv (Moving average for 8 hours not to be exceeded more than once per year) 150 µg/m3 (24 hour mean not to be exceeded more

-

NO2

SO2

CO

PM10 55

75 µg/m3 (24 hour mean)

200 ppbv (1 hour average)

50 µg/m3 (24 hour mean)

than once per year) 40 µg/m3 (Annual mean)

-

45 µg/m3 (24 hour mean)

35 µg/m3 (98th percentile, of 24 hour mean averaged over 3 years)

PM2.5

20 µg/m3 (Annual mean) 50 µg/m3 (24 hour mean)

12 µg/m3 (Annual mean)

12 µg/m3 (Annual mean, 10 µg/m3 (Annual averaged over 3 years) mean) 3 3 0.15 µg/m (Rolling 3 1.5 µg/m (Rolling 3 Pb month average) month average) (a) SEDEMA (2019) Normatividad. Secretaría del Medio Ambiente. Gobierno de la Ciudad de México. http://www.aire.cdmx.gob.mx/default.php (b) US EPA NAAQS (2019) Table. https://www.epa.gov/criteria-air-pollutants/naaqs-table (c) WHO (2006) Air Quality Guidelines: Global Update 2005 Particulate matter, ozone, nitrogen dioxide and sulphur dioxide. World Health Organization, Regional Office for Europe, Copenhagen Supplementary Table 2: Autopsy data for the Metropolitan Mexico City (MMC) 134 cases ≤30 years ID #

56

Cause of death

CPM2.5

Age

Gender

APOE

A-β Phase

Htau Stage

1

20

0

1

1

0

1

2

2

24

0

1.4

0

0

2

0

3

40

0

2

1

0

1

0

4

41

0

3

1

0

2

0

5

72

0

3

1

0

2

0

6

71

1

4

1

0

2

0

7

87

0

7

1

0

2

0

8

160

0

7

0

0

2

2

9

287

0

11

1

0

2

2

10

256

0

11

1

0

2

0

11

179

1

11

0

0

1

2

12

190

0

12

1

0

4

4

13

207

2

12

0

0

2

2

14

183

2

12

1

0

2

2

15

203

2

13

0

1

2

2

16

305

1

13

0

0

2

2

17

228

1

14

0

0

2

2

18

209

0

14

0

0

2

0

19

265

2

14

1

0

2

2

20

295

1

14

1

0

2

2

21

204

0

14

1

0

2

2

22

306

2

15

1

1

2

2

23

57

330

0

15

1

0

2

2

24

234

0

15

1

0

3

2

25

226

0

15

1

0

2

2

26

246

2

15

1

0

2

2

27

226

0

15

1

0

2

2

28

220

2

15

0

0

3

2

29

274

1

16

0

0

2

2

30

380

0

16

1

0

2

2

31

236

1

16

1

0

2

2

32

1122

0

17

1

0

1

2

33

406

2

17

1

0

3

2

34

406

1

17

1

0

2

2

35

892

0

17

1

0

2

2

36

406

1

17

1

0

2

2

37

326

1

17

1

0

2

2

38

436

2

17

1

0

2

2

39

467

0

17

1

0

2

2

40

377

1

17

0

1

3

2

41

451

0

17

1

0

2

2

42

966

1

17

1

0

2

2

43

892

1

17

1

1

2

2

44

421

0

17

1

0

2

2

45

502

2

18

1

1

2

2

46

416

1

18

1

0

2

2

47

447

0

18

1

0

2

2

48

485

0

18

1

0

2

2

49

469

1

18

1

0

2

2

50

469

1

18

1

1

3

2

51

362

1

18

1

0

2

2

52

333

0

19

1

1

2

2

53

387

0

19

1

0

2

2

54

520

0

19

1

0

2

2

55

488

0

19

1

0

2

2

56

350

1

19

0

0

2

2

57

353

1

20

1

0

2

2

58

457

1

20

1

0

2

2

59

594

0

20

1

0

2

2

60

416

2

20

1

0

2

2

61

594

0

20

1

1

2

2

62

555

1

20

1

0

5

2

63

397

0

20

0

1

2

2

64

340

1

20

1

2

5

2

65

58

538

0

20

1

0

2

2

66

538

0

20

0

0

2

2

67

1086

1

20

1

0

2

2

68

1172

1

20

1

0

2

2

69

538

0

20

1

0

2

2

70

1362

0

21

1

0

2

0

71

546

0

21

1

1

2

2

72

1485

1

21

1

3

2

0

73

511

1

21

1

0

2

2

74

592

0

21

1

0

2

2

75

364

1

21

1

0

2

2

76

592

2

22

1

2

2

2

77

430

2

22

0

0

2

2

78

671

1

22

0

0

2

2

79

634

1

22

1

2

2

2

80

1684

0

22

0

0

0

0

81

522

2

22

1

0

2

2

82

399

0

23

1

0

2

2

83

442

0

23

1

0

2

2

84

442

0

23

1

0

2

2

85

428

0

23

1

0

2

2

86

428

2

23

1

0

2

2

87

605

2

23

1

0

2

2

88

615

0

24

1

0

2

2

89

438

0

24

1

0

2

2

90

686

2

24

1

0

2

2

91

484

0

24

1

0

2

2

92

753

0

24

1

0

2

2

93

546

1

24

1

0

2

2

94

656

1

24

1

0

5

2

95

707

2

24

1

1

2

2

96

686

1

24

1

0

2

2

97

438

1

24

1

0

2

2

98

686

2

24

1

2

5

2

99

438

1

24

1

0

2

2

100

1714

0

25

1

0

2

2

101

449

0

25

0

0

2

2

102

770

1

25

1

0

1

2

103

480

1

25

1

0

2

2

104

2036

2

25

1

1

5

2

105

497

1

25

0

0

2

2

106

625

2

25

1

0

2

2

107

676

1

26

1

0

2

2

108

743

0

26

0

0

2

2

109

812

2

26

1

0

2

2

110

508

2

26

1

0

2

2

111

765

1

26

1

0

2

2

112

788

0

26

1

0

2

2

113

536

1

27

1

0

2

2

114

776

0

27

1

1

2

2

115

570

1

27

1

0

2

2

116

2303

0

27

1

0

2

2

117

536

0

27

0

0

2

2

118

554

0

27

1

0

2

2

119

502

2

27

1

0

2

2

120

806

0

27

1

0

2

2

121

502

2

27

1

0

2

2

122

1846

2

27

1

0

2

2

123

785

1

28

1

0

2

2

124

873

0

28

0

1

5

3

125

583

1

29

1

0

2

2

126

881

1

29

1

0

2

2

127

881

1

29

1

0

2

2

128

692

2

29

1

1

2

2

129

502

0

29

0

0

2

2

130

583

0

30

1

0

2

2

131

881

0

30

1

0

2

2

132

703

0

30

1

0

2

2

133

935

0

30

1

0

2

2

134

961

0

30

1

0

2

2

CPM2.5 Cumulative PM2.5 calculated for age at death + pregnancy time, data are in µg/m3 Cause of death: 0=accidents, 1=homicides, 2=suicides Age: in years except #1 11months. Gender: 0=female, 1=male. APOE 0=3/3, 1=3/4, 2=4/4, 3=2/3 Htau Stage70-72: 0=absent, 1= pretangle stages a-c, 2= pretangle stages 1a,1b, 3=NFT stages I, II, 4=NFT stages III-IV, 5=NFT stages V-VI Aβ Phase73: 0=absent, 1=basal temporal neocortex, 2=all cerebral cortex, 3=subcortical portions forebrain, 4=mesencephalic components, 5=Reticular formation and cerebellum.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: