Non-Phosphorylated Tau in Cerebrospinal Fluid is a Marker of Alzheimer’s Disease Continuum in Young Urbanites Exposed to Air Pollution

Abstract

Long-term exposure to fine particulate matter (PM_2.5) and ozone (O₃) above USEPA standards is associated with Alzheimer’s disease (AD) risk. Metropolitan Mexico City (MMC) children exhibit subcortical pretangles in infancy and cortical tau pre-tangles, NFTs, and amyloid phases 1-2 by the 2nd decade. Given their AD continuum, we measured in 507 normal cerebrospinal fluid (CSF) samples (MMC 354, controls 153, 12.82±6.73 y), a high affinity monoclonal non-phosphorylated tau antibody (non-P-Tau), as a potential biomarker of AD and axonal damage. In 81 samples, we also measured total tau (T-Tau), tau phosphorylated at threonine 181 (P-Tau), amyloid-β_1-42, BDNF, and vitamin D. We documented by electron microscopy myelinated axonal size and the pathology associated with combustion-derived nanoparticles (CDNPs) in anterior cingulate cortex white matter in 6 young residents (16.25±3.34 y). Non-P-Tau showed a strong increase with age significantly faster among MMC versus controls (p = 0.0055). Aβ_{1 - 42} and BDNF concentrations were lower in MMC children (p = 0.002 and 0.03, respectively). Anterior cingulate cortex showed a significant decrease (p = <0.0001) in the average axonal size and CDNPs were associated with organelle pathology. Significant age increases in non-P-Tau support tau changes early in a population with axonal pathology and evolving AD hallmarks in the first two decades of life. Non-P-Tau is an early biomarker of axonal damage and potentially valuable to monitor progressive longitudinal changes along with AD multianalyte classical CSF markers. Neuroprotection of young urbanites with PM_2.5 and CDNPs exposures ought to be a public health priority to halt the development of AD in the first two decades of life.

Keywords

air pollution Alzheimer’s disease axonal damage BDNF cerebrospinal fluid children combustion-derived nanoparticles insulin leptin Mexico City non-phosphorylated tau vitamin D Wallerian degeneration white matter

INTRODUCTION

The notion that in the scenario of high exposures to air pollution, Alzheimer’s disease (AD) pathology starts very early, progresses relentlessly in the first two decades of life, and is indicative of an active individual complex response to environmental and genetic factors is at the core of our research [1 –17]. We have identified subcortical pretangle stage b [18] and olfactory bulb hyperphosphorylated tau and Lewy neurites in <12-month-old residents in the highly polluted Metropolitan Mexico City (MMC) [15, 16]. Cortical tau pre-tangles, neurofibrillary tangles (NFT) Stages I-II, amyloid phases 1-2, tau phosphorylated at threonine 181 (P-Tau) in substantia nigra and auditory, oculomotor, and trigeminal nuclei are identified by the 2nd decade and progression to NFT stages III-V is documented in 24.8 % of 30–40-year-old subjects [18, 19]. Apolipoprotein E allele 4 (APOE4) carriers have 23.6 times higher odds of NFT V (p < 0.0001) versus APOE4 non-carriers having similar cumulative fine particulate matter (CPM_{2 ·5}) exposure, and age [15]. The issue of combustion-derived nanoparticles (CDNPs) associated with early and progressive damage to the neurovascular unit and pathology in mitochondria, endoplasmic reticulum (ER), mitochondria-ER contacts (MERCs), axons, and dendrites in MMC residents and the extensive literature of nanoparticles (NPs), their transportation to the brain and their direct association with aggregation and propagation of abnormal proteins are key for our current work [20 –28]. Ambient air NPs can be very high and even the smallest NPs, called nanocluster aerosol particles, emitted by road transportation (1.3–3.0 nm) can contribute more than 50% to the ambient air particle number concentration [22].

We have shown that MMC children cerebrospinal fluid (CSF) samples have significant reductions in amyloid-β (Aβ)₁ _{- 42} compared to clean air controls [13], and significant increases in CSF Macrophage Inhibitory Factor (MIF), IL6, IL1ra, and IL-2, versus controls, strongly suggest there is an important neuro-immune and inflammatory intrathecal component at play in MMC children [7]. Eighty-seven percent of MMC children also exhibit severe deficiency of serum 25-hydroxyvitamin D (<30 ng/mL) and given the role of vitamin D in immune regulation, cognitive impairment, dementia, and the association between maternal 25(OH) D status with childhood attention and executive function, we also pursued the measurements of vitamin D in CSF samples in this work [29 –31]. Given that tau alterations [total tau (T-Tau), P-Tau] are at the core of CSF diagnostic biomarkers in AD [32], and since CSF tau occurs as a subset of distinct tau species [33], we explored the importance of non-P-Tau epitopes, described as potential biomarkers for axonal damage and AD [34, 35] in the CSF of MMC versus lower air pollution controls, knowing beforehand that both T-Tau and P-Tau are not significantly different in MMC children versus controls [10].

Non-P-Tau CSF has been described in high concentrations in Creutzfeldt-Jakob disease and is elevated in AD [34]. Non-P-Tau has been suggested as a reliable biomarker for AD in stage of mild cognitive impairment (MCI) or dementia [35]. The work of Lewczuk and Ermann describe non-P-Tau with high sensitivity and specificity for discrimination of the targeted groups [34, 35].

Thus, our selection of the monoclonal tau antibody with strong affinity for two KTTP motifs without phosphorylation: T175 and T181 [35]. Since the epitope resides in the N-terminal region of the tau protein, the selected monoclonal is favorable for CSF detection, because C-terminal tau fragments are almost absent in CSF [33].

Since young residents in Mexico City [15] have the biomarker profiles corresponding to the AD continuum [36], to further examine the damage to the supratentorial white matter (WM) [37 –39], we did transmission electron microscopy in frontal samples from previously staged AD autopsy cases [15] (Supplementary Tables 1 and 2) and measured axonal diameters in anterior cingulate cortex (ACC) WM from 4 MMC and 2 controls (age 16.16±2.92 y).

Our results strongly suggest that non-P-Tau discriminates highly exposed young urbanites from low pollution controls and more importantly puts tau research into focus in the early AD stages, 5 to 7 decades away from the late NFT stages and terminal AD pathology. The increases in non-P-Tau support that tau changes are early, potentially related to axonal damage and the accumulation of cortical hyperphosphorylated tau [37 –39] and seen across tau species in highly exposed populations.

METHODS

This prospective pilot study was approved by the review boards and ethics committees at the hospitals involved. Normal CSF samples were obtained from two cohorts: 1) children admitted to the Mexico City hospital from a clean air city or Mexico City, with a work up diagnosis of acute lymphoblastic leukemia (ALL) entering a clinical protocol, which included a spinal tap and 2) young adults admitted to the hospital for a work-up that required a spinal tap, with varied diagnoses including potential neoplastic and infectious involvement of the CNS, leukemia, and migraine headache. The cohort of young adults also included permanent residents in clean environments and lifetime residents in highly polluted Mexico City. The normal CSF samples used for this study were destined to be destroyed after the diagnosis of normal CSF was completed.

Study cities and air quality

Children and adult cohorts included residents in MMC and small cities in Mexico (Puerto Escondido; Boca del Río; Tlaxcala). The control cities are characterized by concentrations of the six criteria air pollutants (ozone, particulate matter, sulfur dioxide, nitrogen oxides, carbon monoxide, and lead) below the current US EPA standards [40]. MMC is an example of extreme uncontrolled urban growth and environmental pollution [41 –44].

The metropolitan area of over 2,000 km² lies in an elevated basin 7,400 feet above sea level surrounded on three sides by mountain ridges. MMC has approximately 25 million inhabitants, over 50,000 industries, and >5 million vehicles consuming more than 50 million liters of petroleum fuels per day [44]. MMC motor vehicles produce abundant amounts of primary particular matter (PM_2.5) elemental carbon, particle-bound polycyclic aromatic hydrocarbons, carbon monoxide, and a wide range of air toxins, including lipopolysaccharides, formaldehyde, acetaldehyde, benzene, toluene, and xylenes [45, 46]. The high altitude and tropical climate facilitate ozone production all year and contribute to the formation of fine secondary particulate matter. Air quality is worse in the winter, when rain is scarce and thermal inversions are frequent. Mexico City vehicle transit-imposed regulations restricting Saturdays fail to improve air quality, and no evidence that the restriction was successful in getting drivers to switch to lower-emitting forms of transportation [47]. Children from MMC were residents in the northern-industrialized and southern-residential zones. Southern Mexico City children have been exposed to significant concentrations of ozone, secondary tracers (NO₃-), and particles with lipopolysaccharides, while northern children have been exposed to higher concentrations of volatile organic compounds, PM_2.5, and its constituents: organic and elemental carbon including secondary inorganic aerosols (SO₄^{2 -}, NO₃ ^-, NH₄ ⁺), and metals (Zn, Cu, Pb, Ti, Mn, Cr, V) [41 –44]. MMC residents are also exposed to polycyclic aromatic hydrocarbons (PAHs), complex mixtures containing over 100 compounds, associated with fine particles. These PAHs are abundant in indoor and outdoor air, landfill biogases, petrochemical complex emissions, coke industry and steel metallurgy soil contamination, busy roadways, frying oils, and in a wide range of occupational exposures [43]. MMC residents are involuntarily exposed on daily bases to outdoor high concentrations of total PAHs and benzo[a]pyrene [43].

Pediatric cohort for the measurement of AD, brain-derived neurotrophic factor, inflammatory, and metabolic markers

This work includes data from a pediatric cohort: 55 children from Mexico City (21F/34M, Mean age = 10.94 years, SD = 5.4) and 26 control children (12F/14M, Mean age = 12.8 years, SD = 4.0). The selected children had no previous oncologic and/or hematologic treatments, their CSF samples were read as normal, and CNS involvement was ruled out at the time of their hospitalization. Children’s clinical inclusion criteria were: negative smoking history and environmental tobacco exposure, lifelong residency in MMC or the control city, residency within 5 miles of the city monitoring stations, full term birth, and unremarkable clinical histories prior to their admission to the hospital. We specifically excluded children with a history of active participation in team sports with high incidence of head trauma, including soccer. Mothers had unremarkable, full term pregnancies with uncomplicated vaginal deliveries and took no drugs, including alcohol and tobacco. These children had a history of breast feeding for a minimum of 6 months and were introduced to solid foods after age 4 months. Participants were from middle class families, living in single-family homes with no indoor pets, used natural gas for cooking, and kitchens were separated from the living and sleeping areas. Low and high pollution exposed participants were matched by age, gender, and socioeconomic status.

Children and adult cohorts for the measurement of non-P-Tau

This work includes data from children and adults from low and high polluted cities. A total of 127 controls (57F, 67M) (Mean age 14.83 years, SD = 8.6) and 299 Mexico City subjects (104F/189M) (Mean age = 12.74 years, SD = 8.85), for the entire cohort Mean age = 12.82, SD = 6.73). The inclusion criteria for this 426 combined cohort were the same as per the pediatric cohort.

Cerebrospinal fluid samples

Spinal tap was performed in the supine position from lumbar levels using a standard 22 spinal needle. Spinal taps were performed between 8 and 10 am. CSF was collected dripping in free air in1 ml aliquot into Nalge Nunc polypropylene CryoTubes. Lumbar puncture samples were collected during non-traumatic, non-complicated procedures. CSF were stored at –80°C immediately after examination to determine hematological involvement and kept frozen until the current analysis. CSF pleocytosis was defined as CSF white blood cell counts of >7 cells per mm³. We performed the T-tau, P-tau_181P, and Amyloid-β 1-42HS from Fujirebio-US, Seguin, TX. Human Metabolic Hormone plex Discovery Assay (Insulin, Leptin) and the Human Cytokine plex assay (TNF-α, IL-1β, IFN γ) were custom made human Multiplexing Laser Bead Technology, Bio-Rad Human Diabetes (Eve Technologies Corporation, Calgary, Alberta, Canada). Brain-derived neurotrophic factor (BDNF) was done using the Boster Biology Tech ELISA kit EK0307, Pleasanton, CA, USA. Total 25-OH vitamin D was measured by ELISA, Kit # 90340, Crystal Chem, Elk Grove Village, IL, USA and pTAU rel ELISA (Analytik Jena, AJ Roboscreen GmbH, Leipzig, Germany) for measurement of non-P-Tau.

Human brain frontal anterior cingulate sections

Postmortem ACC tissue was obtained from four teens and young adults (two females) and two age-matched controls (one female), average age 16.16±2.92 y. Demographics, axonal measurements, and neuropathological diagnosis [15] are summarized in Supplementary Tables 1 and 2. We excised small blocks (∼2×2 cm) of WM fixed in a mixture of 2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, post-fixed in 1% osmium tetraoxide and embedded in Epon. Semi-thin sections (1μm) were cut and stained with toluidine blue for light microscopic examination. Ten blocks from left ACC WM were cut and examined in each case. Each toluidine blue 1μm section (30 slides in each case) was examined under a microscope Carl Zeiss Axioskop 2 Plus equipped with an AxioVision REL 4.8 imaging system. Mean value, standard deviation (SD), median, maximum and minimum value of axonal diameters (inner diameter of myelinated axons) in μm [48] and the AD staging of the autopsy selected cases are shown in Supplementary Tables 1 and 2. The measurements were taken from photomicrographs at 8x with a final amplification of 13,300X. We measured 180 axons per case. One EM researcher was in charge of the measurement of the myelinated axons. This researcher (RRR) was blind to the identification of the cases. Two researchers (AGM and LCG) reviewed the sections and the pictures, without access to the identification codes.

Data analysis

Firstly, we calculate the summary statistics such as sample mean and sample standard deviation of all continuous variables within the Mexico City and Control group. Next, we perform two-sample t-tests to check the equality of the average of all variables in these two groups. We also tested the validity of assuming normal distribution for the concerned variables and found that a number of those behave significantly differently than normal distribution. Therefore, we performed Wilcoxon rank-sum tests, the non-parametric equivalent of two-sample t-tests, as well. We then calculate Pearson’s correlation coefficients of all pairs of the concerned variables within each group after adjusting age and gender, and then calculate the p-values for testing significance of each of those. After that, we focus on the non-phosphorylated tau and its relationship with age, gender, and the subject’s residency. We performed linear regression of non-phosphorylated tau with age, gender, and residency, and found that the assumption of heteroscedasticity was not met. Therefore, we search for an acceptable transformation of the response variable and found that logarithm transformation worked well. Therefore, we performed linear regression of the logarithm of non-phosphorylated tau with all one-way, two-way, and three-way interactions of age, gender, and residency. Next, we checked the equality of four regression lines: logarithm of non-phosphorylated tau with age for all four combinations of gender and residency. We used F-test for nested model for this purpose. We also tested the effect of gender on logarithm of non-phosphorylated tau after adjusting age and residency. Finally, we tested the equality of the regression lines of the logarithm of non-phosphorylated tau on age for two groups: Mexico City and Control. The axonal measurements were tested for the significance of differences in axonal diameters in the control versus Mexico City cohort by using a Two-sample t-test. We also prepared figures to present the findings visually. All tests were two-sided and significance was assumed when a p-value was less than 0.05. The statistical analyses were performed using the statistical software ‘R’.

RESULTS

Air pollution levels

Exposures to significant concentrations of PM_2.5 and O₃ in MMC residents are chronic and have been above the current USEPA standards for the last three decades [41 –45]. Fine particulate matter data clearly show the two major sources are diesel vehicles and industry, while the population is massively exposed to very high concentrations of PAHs like benzo[a]pyrene [45]. MMC subjects in this study have significant accumulated concentrations of PM_2.5 above the USEPA standards, regardless of where they live.

Cerebrospinal fluid results

CSF samples were colorless, with a normal opening pressure and a mean white blood cell count of 4.4±1 cells per mm³. Tables 1 and 2 show the Mean±SD results and the T-test and Wilcoxon rank sum test for key CSF variables in control (n: 26) versus MMC (n: 55) children. MMC subjects show significantly lower concentrations of Aβ at p = 0.002 versus controls, while IFN γ is significantly higher in controls (p = 0.002 Wilcoxon) (Table 2). BDNF lower values in MMC samples reaches significance with Wilcoxon p = 0.03 (Table 2). Supplementary Tables 3 and 4 show the Controls and MMC Pearson’s correlations between key variables. The Control correlations are different from the MMC results; the most striking were the correlations between non-P-Tau and total tau (r² = 0.80) and P-Tau (r² = 0.81) and leptin/insulin (r² = 0.54). In contrast, in the MMC cohort, non-P-Tau and total tau (r² = 0.38) were still significant (p = 0.001) but the correlation with P-Tau (r² = 0.06) was nonexistent p = 0.66. The correlation between leptin and insulin (r² = 0.49) was significant p = 0.0002. Vitamin D correlated negatively with BDNF (p = 0.0294) (Supplementary Table 4B).

Table 1

CSF Alzheimer biomarkers, cytokines and metabolic variables results (Mean±SD) in Control versus Metropolitan Mexico City area children and young adults. Children’s cohort results in 81 CSF samples (A) and children and young adult cohort results in 426 CSF samples (B)

CSF variables pg/ml	Controls n = 26 (12F, 14M) Age 12.8±4.0 y	MMC children n = 55 (21F, 34M) Age 10.94±5.47 y
A
Non-P-Tau	10.01±6.79	12.18±15.86
Vitamin D	81.91±30	79.31±26.13
PrP^C	2.9±3.8	2.2±2.4
Amyloid-β 1-42	312±106	224±99
Total tau	9.89±6.9	10.48±8.2
PTau	13.88±9.5	13.9±8.2
IL1β	0.43±0.10	0.41±0.1
IFN γ	0.28±0.09	0.22±0.04
Leptin	57.3±40.4	49.8±25.0
Insulin	60.1±24	60.2±30.7
BDNF	49.5±65.6	24.7±22.1
CSF variables pg/ml	Controls n = 127 (57F, 67M) Age 14.83±8.6 y	MMC n = 299 n = 299 (104F, 189M) Age 12.74±8.85 y
B
Non-P-Tau	13.46±10.24	32.68±41.6

Table 2

T-test and Wilcoxon rank sum test for the key CSF variables low air pollution controls versus MMC children’s samples

Test	Non-P-Tau	Vit D	PrP^C	Aβ	Total Tau	P-Tau	BDNF	IFN γ	IL1β	Leptin	Insulin
t-test	0.4420	0.727	0.427	0.002	0.751	0.922	0.122	0.005	0.293	0.545	0.949
Wilcoxon	0.65	0.400	0.517	0.002	0.578	0.685	0.03	0.002	0.05	0.392	0.693

Supplementary Figure 1 shows the Fitted regression lines (log (non-P-Tau) versus Age) for each pair of the cohorts: Gender and residency (Mexico City versus Control). The four regression lines are significantly different (p < 0.0001). Non-P-Tau tends to increase with age significantly faster among the MMC young residents compared to controls in the large CSF cohort. To be specific, the slopes of the regression lines: logarithm of non-P-Tau on age for the two groups are significantly different (F = 7.80, d.f. = (1, 403), p = 0.0055) (Supplementary Figure 2).

Myelinated axonal diameters in ACC controls versus MMC

Axonal diameter (inner diameter) between MMC and controls showed a significant difference in the ACC WM (Two-sample t-test p = <0.0001) (Supplementary Table 1). The average diameter of axons in the MMC WM was 0.825±0.405μm versus 1.28±0.6 in controls. We also found differences in maximal (2.02 versus 3.4μm) and minimal sizes (0.36 versus 0.65μm) in MMC versus controls.

Electron microscopic axonal findings

The major finding in Mexico City residents was the extensive WM axonal changes, the widened Virchow-Robin spaces and the deposition of NPs in axonal organelles as well in glial cells (Figs. 1 and 2). NPs in the size range of 13–73 nm, average size 43.9±23 nm, exhibited the iron-rich strongly magnetic CDNP morphology: rounded particles, reflecting crystallization upon cooling from an initially heated, iron-bearing source material [49]. CDNPs were seen in mitochondria, ER, MERCs, axons, and dendrites (Fig. 2).

Fig. 1.

Representative one micron toluidine blue anterior cingulate white matter sections from Metropolitan Mexico City and low pollution control subjects in the second decade of life. A) Fourteen-year-old control male (CTL1) with unremarkable white matter and unremarkable myelinated large, medium and small axons. 1μm Toluidine blue. Scale bar 10μm. B) Fourteen-year-old female (MMC2) with areas where large and medium myelinated axons are few at the same power as the clean air child (A). 1μm Toluidine blue. Scale bar 10μm. C) Thirteen-year-old female (MMC1) in an area where myelinated axons are few (arrows) and mostly small. Several areas of the neuropil show no structures (^*). Three glial cells, presumably oligodendrocytes (OL), show round nuclei with heterochromatin beneath the nuclear envelope and patches of heterochromatin throughout the nucleoplasm. The cytoplasm in any of them is not clearly defined. 1μm Toluidine blue. Scale bar 10μm. D) Twenty-year-old male (MMC4) with widened Virchow-Robin spaces and neuropil spaces with no visible structures (^*). Blood vessels (BV) are seen along oligodendroglia-like nuclei (OL), and few small and medium size axons (arrows). 1μm Toluidine blue. Scale bar 10μm. E) Seventeen-year-old male, APOE 3/4, showing extensive enlargement of the Virchow-Robin spaces and empty neuropil spaces (^*). Few small and medium size axons (arrows) and larger axons (arrowheads) are seen. 1μm Toluidine blue. Scale bar 10μm.

Fig. 2.

A) Fourteen-year-old female (MMC2), a small number of medium (arrowheads) and small myelinated axons (arrows) in a loose neuropil. Empty neuropil spaces (^*). Scale bar 2μm. B) Twenty-year-old male (MMC4) with abundant lipofuscin (Lf) in pericytes. The red blood cell in the lumen of the vessel is marked RBC and empty neuropil spaces (^*) are numerous. Smaller myelinated axons are few (arrows). Scale bar 2μm. C) Close-up of the lipofuscin (Lf) in pericytes in a 20-year-old male. Scale bar 2μm. Nanoparticles are seen associated to Lf and throughout the Lf (close-up of the white rectangle). Mag 30, 000x. Scale bar 500 nm. D) Seventeen-year-old APOE4 male (MMC3), myelinated axons with round nanoparticles (arrowhead). Scale bar 500 nm. E) Fourteen-year-old girl (MMC2) several nanoparticles (arrows) are free in the axoplasma. Focal myelin sheet disintegration is present in several places (arrowheads). Scale bar 500 nm. F) Seventeen-year-old male (MMC3), oligodendrocyte with numerous nanoparticles inside the nucleus (arrowheads), in the cytoplasm (arrows) and inside mitochondria (M). Scale bar 500 nm. G) Same 17-year-old (MMC3) with numerous nanoparticles inside mitochondria (M) and in ill-defined structures in the axoplasm (arrowheads). The larger NP (upper left) measures 73 nm. Scale bar 100 nm.

DISCUSSION

Non-P-Tau CSF concentrations are increasing significantly with age in a cohort of 507 normal CSF samples from MMC versus clean air controls, age 12.82±6.73 y. This finding is in accordance with our previous reports of early axonal damage associated with CDNPs and the evolving AD neuropathology in the first two decades of life [15 –17]. The early development of AD pathology (the AD continuum [36]) and the ubiquitous brain presence of iron-rich CDNPs in Mexico City residents [49] associated with axonal mitochondria, ER, and MERCs damage is a plausible explanation for the increases in axonal damage biomarkers in children, teens, and young adults. In the context of CSF biomarkers and PM_2.5 research we have shown Aβ₁ _{- 42}, BDNF, MIF, Cellular prion protein (PrP^C), IL6, IL1ra, and IL-2, are key in differentiating young MMC versus clean air controls [7, 13]. These results point into at least three directions: 1) classically accepted AD biomarkers [32] including the low concentrations of Aβ₁ _{- 42} in young MMC residents: a prime example of a marker associated with the deposit of extracellular amyloid previously shown in MMC children and teens autopsy cases [15, 16]; 2) increased concentrations of inflammatory and cytokine markers such as MIF, IL6, IFN γ, and IL2 suggesting a dysregulated neural immune response in keeping with the brain early and significant inflammatory changes [3, 6]; and last but not least, 3) the strong evidence that a number of neuroprotectors are no longer accomplishing their functions in highly exposed children (cellular prion protein and BDNF fall in this category) [50 –54].

The issue of WM pathology, through a number of pathways, and its contribution to functional decline and AD pathogenesis is at the core of this work. Small blood vessel disease, amyloid angiopathy, neurovascular instability resulting in WM ischemic damage, neurofilament light alterations, and hypoperfusion could be critical in highly exposed individuals [55 –58]. Endothelial cell dysfunction is also potentially a key factor altering myelination and blocking oligodendroglial differentiation [59].

There is clear evidence that patients with MCI and AD [60] develop microstructural WM changes occurring during the AD course, APOE strongly influences regional WM axonal density loss in AD [61], and there is a correlation between CSF Aβ levels, WM lesion loads, and diffusion-weighted images suggesting the pattern characterization of WM changes might be useful for assessing AD progression [62]. The work of McAleese et al. and Mito et al. [37–39 , 63] ought to be mentioned in the context of WM damage in AD and its association with Wallerian degeneration as a consequence of P-Tau cortical burden. Mito and co-workers [63] emphasized the involvement of long association fibers, while McAlesse et al. [37] reported P-Tau cortical pathology is significantly associated or is a predictor of white matter hyperintensities (WMH) in parietal and temporal regions.

Jackson et al. [64] discussed WM tauopathy and damage of axonal myelin lamella and myelin remodeling, while Ferrer [65] discusses oligodendrocytes and their precursors, development and functions, and the importance of dysfunction of oligodendrocytes in selected neurodegenerative diseases with abnormal protein aggregation. Jackson and colleagues [64] support that WM dysfunction early in tauopathy will give rise to altered neural circuits and potentially contribute to early clinical deficits in dementia, a situation we cannot dismiss in MMC children with significant cognitive deficits as early as age 10.3±2.4 y [4], and young adult cohorts age 20.14±2.39 y with mean Montreal Cognitive Assessment scores of 22.76±2.09 (personal communication Dr. Lilian Calderón-Garcidueñas, September 25, 2018). The work of Dr. Roxana Carare [66 –68] ought to be discussed in the context of significant neurovascular unit and basement membrane damage as we have described in MMC children and young adults [3 , 69]. Specifically, the integrity of periarterial basement membrane pathways altering Intramural Peri-Arterial Drainage pathways for the elimination of interstitial fluid and solutes from the brain [66, 67]; the dilated perivascular spaces in amyloid angiopathy [68] and certainly the presence of cerebral small vessel disease [70] in our young exposed urbanites are all potential pathways to explain WM damage. Poggi et al. [71] discussed progressive hypomyelination in Mbp heterozygous mice, the unaffected parental strain of shiverer, a classical neurological mutant. In the work of Poggi et al., the key is that subtle abnormalities of CNS myelin can cause persistent cortical network dysfunction, and, as discussed by Gao et al. [72] in a rat model, WM atrophy and disruption of myelinated fibers may contribute to the pathophysiology underlying depression. Also, in the context of vascular pathology, we have described in each MMC case, extensive microvascular leakage related to severe damage to the neurovascular unit, a pathology described in diabetes mellitus patients’ brains [73]. The issue of breakdown of the blood-brain barrier is indeed relevant to highly exposed urbanites, because is one of the earliest findings in our exposed infants [15].

The Strain et al. paper [74] is also highly relevant to our work. They evaluated 59 cognitively normal subjects and 10 cognitively impaired individuals with diffusion tensor imaging, PET Aβ ([¹⁸F] AV-45 florbetapir), and PET tau ([¹⁸F] AV-1451 flortaucipir) imaging. Their results are indeed very exciting and not really a surprise: Tau, not Aβ, was associated with changes in anterior temporal WM integrity. Highly relevant to our work: WMH, a proxy for vascular damage, was strongly associated with axonal damage, and tau independently contributed to the model, suggesting an additional degenerative mechanism within tracts projecting from regions vulnerable to AD pathology. Strain et al. [74] concluded WM decline was associated with early tau accumulation, and certainly we support the same mechanism could be at play in Mexico City, where we documented WMH in 55% of children at age 10.73±2.7 y [4].

Microstructural WM changes are also significant in APOE4 carriers [61, 75] and we have shown significant interactive and additive influences of gender, body mass index, and APOE4 on cognition in MMC children, teens, and young adults [12 , 69]. Operto et al. [75] examined by diffusion-weighted imaging 532 cognitively healthy middle-aged participants including 68 APOE ɛ4 homozygotes, 207 heterozygotes, and 257 non-carriers. APOE ɛ4 homozygotes display increased WM diffusivity in regions known to be affected by AD. The effects in radial water diffusivity suggested a disruption of the myelin sheath rather than pure axonal damage and it was interpreted by the authors as resulting from a reduced capacity of the ɛ4 isoform of the APOE protein to keep cholesterol homeostasis in the brain. The work of Slattery et al. [61] is equally relevant. Diffusion tensor imaging and neurite orientation dispersion and density imaging with tract-based spatial statistics was used to investigate APOE4 modulation of WM damage in 37 patients with young onset AD (22, 59% APOE ɛ4 carriers) and 23 age-matched controls. WM disruption was more widespread in APOE4 individuals and fractional anisotropy changes were related to combinations of axonal loss and morphological change. So, indeed, APOE4 status is associated with different patterns of WM neurodegeneration [61]. What is clear so far in our cohorts: WM decline starts early, is associated with tau accumulation, and progresses with age [15 –17].

CSF alterations are critical in the early development of AD and tau biomarkers are at the core of early AD diagnosis for intervention trials and clinical practice [35]. Non-P-Tau measurements in young highly exposed air pollution cohorts puts forward a candidate biomarker that distinguishes low from highly exposed individuals along with classical AD markers (Aβ_{1 - 42}). More importantly, non-P-Tau is likely reflecting axonal damage in association with early AD development in a very young population where CDNPs are key players and hyperphosphorylated tau in infra and supratentorial locations is a key likely irreversible finding [18].

Low concentrations of Aβ_{1 - 42} are associated to diffuse cortical Aβ_{1 - 42} plaques (Aβ Phase 2 [19]) present in about 80% of children and young adults in the first two decades of life (versus 0% in clean air controls) and supported by the literature associating long-term exposure to PM_2.5 above current USEPA standards and increased AD risk [76, 77]. We know that concentrations of Aβ_{1 - 42} reach maximum abnormality level in the asymptomatic stage of AD [78], and there is a consensus in AD familial cases of a trend for the decrease in CSF Aβ_{1 - 42} at least four decades before expected onset of clinical symptoms [79]. There is also a current agreement that CSF changes in Aβ_{1 - 42}, T-tau, and P-Tau are diagnostic of AD in its prodromal stage and have proved diagnostic accuracy for MCI and AD [32 , 80–83]. On the other side of the coin, de Leon et al. [84] have shown the prediction of cognitive decline was improved by considering both high and low levels of Aβ_{1 - 42} and more importantly, their data suggest an earlier preclinical stage marked by CSF elevations in tau and accompanied by either elevations or reductions in Aβ_{1 - 42}. On the same line of discussion is the work of Merluzzi et al. [85] showing that cognitively unimpaired individuals with AD neuropathology differ from individuals with AD dementia on biomarkers of neurodegeneration, synaptic dysfunction, and glial activation. In their work, a group of individuals with p-tau/Aβ₄₂ and Aβ₄₂/Aβ₄₀ levels comparable to those of the AD-Dementia group had significantly lower levels of neurofilament light and total tau. Merluzzi et al. [85] concluded that monitoring multiple biomarkers provides a much better insight into the biological processes phenotypic of dementia. We fully agree.

Low concentrations of BDNF in young urbanites deserves comment. BDNF has key roles in cell survival, axonal and dendritic growth, and synaptic plasticity and is the most abundant neurotrophin in the brain [86 –88]. A common single-nucleotide polymorphism in the pro-region of the human BDNF gene, resulting in a valine to methionine substitution (Val66Met), has been associated with the susceptibility, incidence, and clinical features of neurodegenerative disorders [89]. The involvement of BDNF in the regulation of psychomotor speed, working memory, and executive function has been shown in healthy subjects [90]. Of key relevance for this work, CSF BDNF levels are significantly reduced in AD compared to MCI and healthy controls (p = 0.009) [88]. And equally important, logistic regression models show that lower CSF BDNF levels, lower Aβ_{1 - 42} and lower Mini-Mental State Examination scores are significantly associated with progression from MCI to AD [88]. Thus, the finding of low CSF BDNF in Mexico City young is an expected finding. Goltz et al. [91] explored the putative associations and interactions between serum BDNF and vitamin D levels with depressive symptoms and abdominal obesity in a large population-based cohort. Vitamin D was inversely associated with depression and obesity, while BDNF was associated with abdominal obesity, but not with depression. Interestingly, in the MMC cohort, vitamin D level was negatively correlated with BDNF (p = 0.0294), a finding that obligates us to run both analytes in future CSF studies in young urban residents along with APOE4 genotyping [91 –93].

Cellular prion protein (PrP^C), a conserved GPI-anchored membrane protein with neuroprotective activity, located on the surface of neurons, at both pre and post-synaptic sites showed significant differences in its correlations with T-Tau and P-Tau between MMC and control cases, raising questions about physiological and pathological interactions between PrP^C and tau under high pollution exposures. In 2009, Laurén et al. [94] identified PrP^C as a high-affinity receptor for Aβ oligomers. The role of PrP^C in protecting neurons from threatening Ca^{2 +} overloads and excitotoxicity [95], the interaction of the N-terminal domain (N-PrP) with neurotoxic ligands, such as Aβ and Scrapie prion protein [96], the modulation of glucose homeostasis and the promotion of iron uptake through divalent-metal-transporters [97], and the unexpected interaction between inflammatory mediators and reduced PrP^C on endothelium [98] certainly provides additional mechanisms by which PrP^C alterations may lead to CNS damage. The importance of PrP^C cannot be ignored in subjects exposed to air pollutants for one major reason: our previous report of significant 15-fold downregulation of PrP^C in the frontal cortex of 18±8.7 y Mexico City residents [6]. The downregulation of PrP^C in the prefrontal cortex is likely detrimental given the protective effects of PrP^C against oxidative stress and its role in cell signaling, metal interactions, memory, T cell differentiation, myelin maintenance, axonal growth and neuronal development [94 –99].

Finally, the significant differences in a T helper Th1 cytokine between MMC and controls: Interferon γ (IFN γ) deserve a comment. IFN γ is a prototypical pro-inflammatory Th1 cytokine that drives cell-mediated immunity. Ott et al. [100] described the results of altered blood-cerebrospinal fluid barrier (BCSFB) function in MCI and AD patients versus cognitively healthy controls. Paired serum and CSF samples were assayed for a panel of cytokines, chemokines, and related trophic factors and dominance analysis models were conducted to determine the relative importance of the inflammatory factors in relationship to BCSFB permeability, as measured by CSF/serum ratios for urea, creatinine, and albumin. Their results showed severe perturbations in BCSFB function in MCI and AD and IFN γ was one of the inflammatory proteins with a shifting distribution from serum to CSF [100]. Otto and coworkers’ report is key for our findings, since highly exposed children had IFN γ concentrations lower than the controls and thus dysregulation of immune responses could be an early finding in the AD continuum associated with early neurovascular unit pathology [3, 12].

Looking forward, limitations, and summary

In the natural setting of millions of people around the world involuntarily exposed to high concentrations of PM_2.5 and ultrafine PM, the development of AD hyperphosphorylated tau and amyloid plaques from infancy and the relentless progression in the first 4 decades of life needs a biological construct that takes into account air pollution. If we are going to apply the current concept that AD is a continuum [36] and we know it is in Mexico City young residents [15], we ought to do cognitive staging and look for MRI structural and volumetric changes in the first 2 decades of life. Young adults in Mexico City already have significant structural and metabolic brain changes that translate in cognitive deficits, daily life difficulties, behavioral changes, and school struggles. We know for example, overweight girls with an APOE4 allele are at the highest risk of cognition deficits [9 , 101], so we need to accelerate the research that will allow a clear understanding of the air pollutants involved, their impact in utero and the sequence of postnatal events conducing to brain structural and metabolic changes. We acknowledge our main limitation, the number of biomarkers we could run, and the missing APOE genotyping information in each CSF sample, based on budget restrictions.

We strongly recommend the concept of preclinical AD be revised and emphasize the need to define pediatric environmental, nutritional, metabolic, and genetic risk factor interactions of paramount importance to prevent AD [36]. Non-invasive neuroimaging, targeted neurocognitive testing, and APOE genotyping could be helpful in defining AD in highly exposed children, teens, and young adult populations. Non-P-Tau is potentially an early sensitive biomarker of axonal damage and AD axonal pathology and valuable to monitor longitudinal changes during disease progression along with the AD multianalyte classical CSF profile. Neuroprotection of young urbanites with high PM_2.5 and CDNPs exposures ought to be a public health priority to prevent the emergence of AD in the first two decades of life.

Footnotes

ACKNOWLEDGMENTS

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

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

References

Calderón-Garcidueñas

, Azzarelli

, Acuna

, Garcia

, Gambling

, Osnaya

Monroy

Del Tizapantzi

, Carson

, Villarreal-Calderon

, Rewcastle

(2002) Air pollution and brain damage. Toxicol Pathol 30, 373–389.

Calderón-Garcidueñas

, Maronpot

, Torres-Jardon

, Henríquez-Roldan

, Schoonhoven

, Acufla-Ayala

, Villarreal-Calderon

, Nakamura

, Fernando

, Reed

, Azzarelli

, Swenberg

(2003) DNA damage in nasal and brain tissues of canines exposed to air pollutants is associated with evidence of chronic brain inflammation and neurodegeneration. Toxicol Pathol 31, 524–538.

Calderón-Garcidueñas

, Solt

, Henríquez-Roldan

, Torres-Jardon

, Nuse

, Herritt

, Villarreal-Calderon

, Osnaya

, Stone

, Garcia

, Brooks

, González-Maciel

, Reynoso-Robles

, Delgado-Chávez

, Reed

(2008) Long-term air pollution exposure is associated with neuroinflammation, an altered innate immune response, disruption of the blood-brain-barrier, ultrafine particulate deposition, and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults. Toxicol Pathol 36, 289–310.

Calderón-Garcidueñas

, Mora-Tiscarefio

, Gomez-Garza

, Broadway

, Chapman

, Valencia-Salazar

, Jewells

, Maronpot

, Henríquez-Roldan

, Perez-Guille

, Torres-Jardon

, Herrit

, Brooks

, Osnaya-Brizuela

, Monroy

, González-Maciel

, Reynoso-Robles

, Villarreal-Calderon

, Solt

, Engle

(2008) Air pollution, cognitive deficits and brain abnormalities: A pilot study with children and dogs. Brain Cogn 68, 117–127.

Calderón-Garcidueñas

, D'Angiulli

, Kulesza

, Torres-Jardon

, Osnaya

, Romero

, Keefe

, Herritt

, Brooks

, Avila-Ramirez

, Delgado-Chávez

, Medina-Cortina

, González-González

(2011) Air pollution is associated with brainstem auditory nuclei pathology and delayed brainstem auditory evoked potentials. Int J Dev Neurosci 29, 365–375.

Calderón-Garcidueñas

, Kavanaugh

, Block

, D'Angiulli

, Delgado-Chávez

, Torres-Jardon

, González-Maciel

, Reynoso-Robles

, Osnaya

, Villarreal-Calderon

, Guo

, Hua

, Zhu

, Perry

, Diaz

(2012) Neuroinflammation, hyperphosphorylated tau, diffuse amyloid plaques, and down-regulation of the cellular prion protein in air pollution exposed children and young adults. J Alzheimers Dis 28, 93–107.

Calderón-Garcidueñas

, Cross

, Franco-Lira

, Aragon-Flores

, Kavanaugh

, Torres-Jardon

, Chao

, Thompson

, Chang

, Zhu

, D'Angiulli

(2013) Brain immune interactions and air pollution: Macrophage inhibitory factor (MIF), prion cellular protein (PrP(C)), Interleukin-6 (IL-6), interleukin 1 receptor antagonist (IL-1Ra), and interleukin-2 (IL-2) in cerebrospinal fluid and MIF in serum differentiate urban children exposed to severe vs. low air pollution. Front Neurosci 7, 183–193.

Calderón-Garcidueñas

, Franco-Lira

, Mora-Tiscarefio

, Medina-Cortina

, Torres-Jardon

, Kavanaugh

(2013) Early Alzheimer’s and Parkinson’s disease pathology in urban children: Friend versus Foe responses-it is time to face the evidence. Biomed Res Int 2013, 161–687.

Calderón-Garcidueñas

, Mora-Tiscarefio

, Franco-Lira

, Zhu

, Lu

, Solorio

, Torres-Jardon

, D'Angiulli

(2015) Decreases in short term memory, IQ and altered brain metabolic ratios in urban Apolipoprotein e4 children exposed to air pollution. APOE modulates children's brain air pollution responses. J Alzheimers Dis 45, 757–770.

10.

Calderón-Garcidueñas

, Chao

, Thompson

, Rodriguez-Diaz

, Franco-Lira

, Mukherjee

, Perry

(2015) CSF biomarkers: Low amyloid ß1-42 and BDNF and High IFNy differentiate children exposed to Mexico City high air pollution v controls. Alzheimer’s disease uncertainties. J Alzheimers Dis Parkinsonism 5, 189.

11.

Calderón-Garcidueñas

, Franco-Lira

, D'Angiulli

, Rodriguez-Diaz

, Blaurock-Busch

, Busch

, Chao

, Thompson

, Mukherjee

, Torres-Jardon

, Perry

(2015) Mexico City normal weight children exposed to high concentrations of ambient PM2.5 show high blood leptin and endothelin-1, vitamin D deficiency, and food reward hormone dysregulation versus low pollution controls. Relevance for obesity and Alzheimer disease. Environ Res 140, 579–592.

12.

Calderón-Garcidueñas

, Reynoso-Robles

, Vargas-Martinez

, Gomez-Maqueo-Chew

, Perez-Guille

, Mukherjee

, Torres-Jardon

, Perry

, González-Maciel

(2016) Prefrontal white matter pathology in air pollution exposed Mexico City Young urbanites and their potential impact on neurovascular unit dysfunction and the development of Alzheimer’s disease. Environ Res 146, 404–417.

13.

Calderón-Garcidueñas

, Avila-Ramirez

, Calderón-Garcidueñas

, González-Heredia

, Acufia-Ayala

, Chao

, Thompson

, Ruiz-Ramos

, Cortes-González

, Martinez-Martinez

, Garcia-Perez

, Reis

, Mukherjee

, Torres-Jardon

(2016) Cerebrospinal fluid biomarkers in highly exposed PM2.5 urbanites: The risk of Alzheimer’s and Parkinson’s diseases in young Mexico city residents. J Alzheimers Dis 54, 597–613.

14.

Calderón-Garcidueñas

, Reynoso-Robles

, Perez-Guilie

, Mukherjee

, González-Maciel

(2017) Combustion derived nanoparticles, the neuroenteric system, cervical vagus, hyperphosphorylated alpha synuclein and tau in young Mexico City residents. Environ Res 159, 186–201.

15.

Calderón-Garcidueñas

, González-Maciel

, Reynoso-Robles

, Delgado-Chávez

, Mukherjee

, Kulesza

, Torres-Jardon

, Avila-Ramirez

, Villarreal-Rios

(2018) Hallmarks of Alzheimer disease are evolving relentlessly in Metropolitan Mexico City infants, children and young adults. APOE4 carriers have higher suicide risk and higher odds of reaching NFT stage V at≥40 years of age. Environ Res 164, 475–487.

16.

Calderón-Garcidueñas

, González-Maciel

, Reynoso-Robles

, Kulesza

, Mukherjee

, Torres-Jardon

, Ronkko

, Doty

(2018) Alzheimer’s disease and alpha-synuclein pathology in the olfactory bulbs of infants, children, teens and adults ≥ 40 years in Metropolitan Mexico City. APOE4 carriers at higher risk of suicide accelerate their olfactory bulb pathology. Environ Res 166, 348–362.

17.

González-Maciel

Reynoso-Robles

, Torres-Jardon

, Mukherjee

, Calderón-Garcidueñas

(2017) Combustion derived nanoparticles in key brain target cells and organelles in young urbanites: Culprit hidden in plain sight in Alzheimer’s disease development. J Alzheimers Dis 59, 189–208.

18.

Braak

, Del Tredeci

(2015) The preclinical phase of the pathological process underlying sporadic Alzheimer’s disease. Brain 138, 2814–2833.

19.

Thal

, Rub

, Orantes

, Braak

(2002) Phases of A beta-deposition in the human brain and its relevance for the development of AD. Neurology 58, 1791–1800.

20.

Alvarez

, Fauerbach

, Pellegrotti

, Jovin

, Jares-Erijman

, Stefani

(2013) Influence of gold nanoparticles on the kinetics of a-synuclein aggregation. Nano Lett 13, 6156–6163.

21.

Min

, Shin

, Yu

, Yang

, David

, Yang

, Rosania

(2013) Pulsed magnetic field improves the transport of iron oxide nanoparticles through cell barriers. ACS Nano 7,2161–2171.

22.

Ronkko

, Pirjola

, Ntziachristos

, Heikkila

, Kar-jalainen

, Hillamo

, Keskinen

(2014) Vehicle engines produce exhaust nanoparticles even when not fueled. Environ Sci Technol 48, 2043–2050.

23.

Wang

, Wang

, Chen

, Zhou

, Wang

, Zhang

, Feng

(2016) Size-Dependent translocation pattern, chemical and biological transformation of nano-and submicron-sized ferric oxide particles in the central nervous system. J Nanosci Nanotechnol 16, 5553–5561.

24.

Xie

, Liu

, Cai

, Chen

, Zhou

, Wu

, Sun

, Dai

, Kong

, Liu

(2016) Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells. Int J Nanomedicine 11, 3557–3570.

25.

Sintov

, Velasco-Aguirre

, Gallardo-Toledo

, Araya

, Kogan

(2016) Metal nanoparticles as targeted carriers circumventing the blood-brain Int Rev Neurobiol 130, 199–227.

26.

Yarjanli

, Ghaedi

, Esmaeili

, Rahgozar

, Zarrabi

(2017) Iron oxide nano particles may damage to the neural tissue through iron accumulation, oxidative stress, and protein aggregation. BMC Neurosci 18, 51.

27.

Septiadi

, Crippa

, Moore

, Rothen-Rutishauser

, Petri-Fink

(2018) Nanoparticle-cell interaction: A cell mechanics perspective. Adv Mater 30, e1704463.

28.

Bourquin

, Milosevic

, Hauser

, Lehner

, Blank

, Petri-Fink

, Rothen-Rutishauser

(2018) Biodistribution, clearance, and long-term fate of clinically relevant nanomaterials. Adv Mater 30, e1704307.

29.

Schlögl

, Holick

(2014) Vitamin D and neurocog-nitive function. Clin Interv Aging 9, 559- 568.

30.

Brouwer-Brolsma

, Vrijkotte

TGM

, Feskens

EJM

(2018) Maternal vitamin D concentrations are associated with faster childhood reaction time and response speed, but not with motor fluency and flexibility, at the age of 5-6 years: The Amsterdam Born Children and their Development (ABCD) Study. BrJNutr 120, 345–342.

31.

Lerner

, Sharony

, Miodownik

(2018) Association between mental disorders, cognitive disturbances and vitamin D serum level: Current state. Clin Nutr ESPEN 23, 89–102.

32.

Lewczuk

, Riederer

, O’Bryant

, Verbeek

, Dubois

, Visser

, Jellinger

, Engelborghs

, Ramirez

, Parnetti

, Jack

Jr , Teunissen

, Ham-pel

, Lleo

, Jessen

, Glodzik

, de Leon

, Fagan

, Molinuevo

, Jansen

, Winblad

, Shaw

, Andreasson

, Otto

, Mollenhauer

, Wiltfang

, Turner

, Zerr

, Handels

, Thompson

, Johansson

, Ermann

, Trojanowski

, Karaca

, Wagner

, Oeckl

, van Waalwijk van Doorn

, Bjerke

, Kapogiannis

, Kuiperij

, Farotti

, Li

, Gordon

, Epelbaum

, Vos

SJB

, Klijn

CJM

, van Nostrand

, Minguillon

, Schmitz

, Gallo

, Lopez Mato

, Thibaut

, Lista

, Alcolea

, Zetterberg

, Blennow

, Kornhuber

; Q Members of the WFSBP Task Force Working on this Topic: Riederer

, Gallo

, Kapogiannis

, Lopez Mato

, Thibaut

(2018) Cerebrospinal fluid and blood biomark-ers for neurodegenerative dementias: An update of the Consensus ofthe Task Force on Biological Markers in Psychiatry of the World Federation of Societies of Biological Psychiatry. World J Biol Psychiatry 19, 244–328.

33.

Meredith

Jr , Sankaranarayanan

, Guss

, Lanzetti

, Berisha

, Neely

, Slemmon

, Portelius

, Zetterberg

, Blennow

, Soares

, Ahlijanian

, Albright

(2013) Characterization of novel CSF Tau and ptau biomarkers for Alzheimer’s disease. PLoS One 8, e76523.

34.

Ermann

, Lewczuk

, Schmitz

, Lange

, Knipper

, Goebel

, Kornhuber

, Zerr

, Llorens

(2018) CSF nonphosphorylated Tau as a biomarker for the discrimination of AD from CJD. Ann Clin Transl Neurol 5, 883–887.

35.

Lewczuk

, Lelental

, Lachmann

, Holzer

, Flach

, Brandner

, Engelborghs

, Teunissen

, Zetterberg

, Molinuevo

, Mroczko

, Blennow

, Popp

, Parnetti

, Chiasserini

, Perret-Liaudet

, Spitzer

, Maler

, Kornhuber

(2017) Non-phosphorylated tau as a potential biomarker of Alzheimer’s disease: Analytical and diagnostic characterization. J Alzheimers Dis 55, 159–170.

36.

Jack

Jr , Bennett

, Blennow

, Carrillo

, Dunn

, Haeberlein

, Holtzman

, Jagust

, Jessen

, Karlawish

, Liu

, Molinuevo

, Montine

, Phelps

, Rankin

, Rowe

, Scheltens

, Siemers

, Snyder

, Sperling

; Contributors (2018) NIA-AA Research Framework: Toward abiological definition of Alzheimer’s disease. Alzheimers Dement 14, 535–562.

37.

McAleese

, Firbank

, Dey

, Colloby

, Walker

, Johnson

, Beverley

, Taylor

, Thomas

, O’Brien

, Attems

(2015) Cortical tau load is associated with white matter hyperintensities. Acta Neuropathol Commun 3, 60.

38.

McAleese

, Walker

, Graham

, Moya

ELJ

, Johnson

, Erskine

, Colloby

, Dey

, Martin-Ruiz

, Taylor

, Thomas

, McKeith

, de Carli

, Attems

(2017) Parietal white matter lesions in Alzheimer’s disease are associated with cortical neurodegenerative pathology, but not with small vessel disease. Acta Neuropathol 134, 459–473.

39.

McAleese

, Walker

, Colloby

, Taylor

, Thomas

, DeCarli

, Attems

(2018) Cortical tau pathology: A major player in fiber-specific white matter reductions in Alzheimer’s disease? Brain 141, e44.

40.

U.S. Environmental Protection Agency (2014) Air and Radiation. https://www.epa.gov/learn-issues/learn-about-air, Last updated July 21,2016. Accessed August 24,2018.

41.

Rosas-Perez

, Serrano

, Alfaro-Moreno

, Baumgard-ner

, Garcia-Cuellar

, Martin del Campo

, Raga

, Castillejos

, Colin

, Osornio Vargas

(2007) Relations between PM10 composition and cell toxicity: A multivariate and graphical approach. Chemosphere 67, 1218–1228.

42.

Querol

, Pey

, Minguillon

, Perez

, Alastuey

, Viana

, T. Moreno

, Bernabe

, Blanco

, Cardenas

, Vega

, Sosa

, Escalona

, Ruiz

, and Artffiano

(2008) PM speciation and sources in Mexico during the MILAGRO-2006 Campaign. Atmos Chem Phys 8, 111–121.

43.

Valle-Hernandez

, Miigica-Alvarez

, Salinas-Talavera

, Amador-Muüoz

, Murillo-Tovar

, Villalobos-Pietrini

, De Vizcaya-Ruiz

(2010) Temporal variation of nitro-polycyclic aromatic hydrocarbons in PM10 and PM2.5 collected in Northern Mexico City. Sci Total Environ 408, 5429–5438.

44.

SMA Secretaria del Medio Ambiente del Gobierno del Distrito Federal (2014) Direccion General de Gestion de la Calidad del Aire. Sistema de monitoreo Atmosferico de la ciudad de Mexico. Direccion de Monitoreo Atmosferico. http://www.aire.cdmx.gob.mx/default.php, Accessed 24 August 2018.

45.

Dzepina

, Arey

, Marr

, Worsnop

, Salcedo

, Zhang

(2007) Detection of particle-phase polycyclic aromatic hydrocarbons in Mexico City using an aerosol mass spectrometer Int. J Mass Spectrom 263, 152–170.

46.

, Apte

, Lipsitt

, Garcia-Gonzales

, Becker-man

, de Nazalle

, Texcalac-Sangrador

, Jerrett

(2015) Populations potentially exposed to traffic-related air pollution in seven world cities. Environ Int 78, 82–89.

47.

Davis

(2017) Saturday driving restrictions fail to improve air quality in Mexico City. Sci Rep 7, 41652.

48.

Liewald

, Miller

, Logothetis

, Wagner

, Schüz

(2014) Distribution of axon diameters in cortical white matter: An electron-microscopic study on three human brains and a macaque. Biol Cybern 108, 541–557.

49.

Maher

, Ahmed

IAM

, Karloukovski

, MacLaren

, Foulds

, Allsop

, Mann

DMA

, Torres-Jardon

, Calderón-Garcidueñas

(2016) Magnetite pollution nanoparticles in the human brain. Proc Natl Acad Sci U S A 113, 10797–10801.

50.

Ulbrich

, Janning

, Seidel

, Matschke

, Gonsberg

, Jung

, Glatzel

, Engelhard

, Winklhofer

, Tatzelt

(2018) Alterations in the brain interactome of the intrinsically disordered N-terminal domain of the cellular prion protein (PrPC) in Alzheimer’s disease. PLoS One 13, e0197659.

51.

Ashok

, Singh

(2018) Prion protein modulates glucose homeostasis by altering intracellular iron. Sci Rep 8, 6556.

52.

Parrie

, Crowell

JAE

, Telling

, Bessen

(2018) The cellular prion protein promotes olfactory sensory neuron survival and axon targeting during adult neurogenesis. Dev Biol 438, 23–32.

53.

Leal

, Comprido

, Duarte

(2014) BDNF-induced local protein synthesis and synaptic plasticity. Neurophar-macology 76, 639–656.

54.

Shen

, You

, Joseph

, Mirzaei

, Klistorner

, Graham

, Gupta

(2018) BDNF polymorphism: A review of its diagnostic and clinical relevance in neurodegenerative disorders. Aging Dis 9, 523–536.

55.

Prins

, Scheltens

(2015) White matter hyperintensi-ties, cognitive impairment and dementia: An update. Nat Rev Neurol 11, 157–165.

56.

Love

, Miners

(2015) White matter hypoperfusion and damage in dementia: Post-mortem assessment. Brain Pathol 25, 99–107.

57.

Moore

, Hohman

, Badami

, Pechman

, Osborn

, Acosta

LMY

, Bell

, Babicz

, Gifford

, Anderson

, Goldstein

, Blennow

, Zetterberg

, Jefferson

(2018) Neurofilament relates to white matter microstructure in older adults. Neurobiol Aging 70, 233–241.

58.

Hase

, Chen

, Bates

, Craggs

LJL

, Yamamoto

, Gemmell

, Oakley

, Korolchuk

, Kalaria

(2018) Severe white matter astrocytopathy in CADASIL. Brain Pathol, doi: 10.1111/bpa.12621

59.

Rajani

, Quick

, Ruigrok

, Graham

, Harris

, Verhaaren

BFJ

, Fornage

, Seshadri

, Atanur

, Dominiczak

, Smith

, Wardlaw

, Williams

(2018) Reversal of endothelial dysfunction reduces white matter vulnerability in cerebral small vessel disease in rats. Sci Transl Med 10, 448.

60.

Giulietti

, Torso

, Serra

, Spano

, Marra

, Calta-girone

, Cercignani

, Bozzali

; Alzheimer’s Disease Neuroimaging Initiative (ADNI) (2018) Whole brain white matter histogram analysis of diffusion tensor imaging data detects microstructural damage in mild cognitive impairment and Alzheimer’s disease patients. J Magn Reson Imaging, doi: 10.1002/jmri.25947

61.

Slattery

, Zhang

, Paterson

, Foulkes

AJM

, Carton

, Macpherson

, Mancini

, Thomas

, Modat

, Toussaint

, Cash

, Thornton

, Henley

SMD

, Crutch

, Alexander

, Ourselin

, Fox

, Zhang

, Schott

(2017) ApoE influences regional white-matter axonal density loss in Alzheimer’s disease. Neurobiol Aging 57, 8–17.

62.

Pietroboni

, Scarioni

, Carandini

, Basilico

, Cadi-oli

, Giulietti

, Arighi

, Caprioli

, Serra

, Sina

, Fenoglio

, Ghezzi

, Fumagalli

, de Riz

, Calvi

, Triulzi

, Bozzali

, Scarpini

, Galimberti

(2018) CSF ß-amyloid and white matter damage: A new perspective on Alzheimer’s disease. J Neurol Neurosurg Psychiatry 89, 352–357.

63.

Mito

, Raffelt

, Dhollander

, Vaughan

, Tournier

, Salvado

, Brodtmann

, Rowe

, Villemagne

, Connelly

(2018) Fibre-specific white matter reductions in Alzheimer’s disease and mild cognitive impairment. Brain, doi: 10.1093/brain/awx355

64.

Jackson

, Bianco

, Rosa

, Cowan

, Bond

, Anichtchik

, Fern

(2018) White matter tauopathy: Transient functional loss and novel myelin remodeling. Glia 66, 813–827.

65.

Ferrer

(2018) Oligodendropathy in neurodegenerative diseases with abnormal protein aggregates: The forgotten partner. Prog Neurobiol 169, 24–54

66.

Dobson

, Sharp

, Cumpsty

, Criswell

, Wellman

, Finucane

, Sullivan

, Weller

, Verma

, Carare

(2017) The perivascular pathways for influx of cere-brospinal fluid are most efficient in the midbrain. Clin Sci (Lond) 131, 2745–2752.

67.

Albargothy

, Johnston

, MacGregor-Sharp

, Weller

, Verma

, Hawkes

, Carare

(2018) Con-vective influx/glymphatic system: Tracers injected into the CSF enter and leave the brain along separate periarte-rial basement membrane pathways. Acta Neuropathol 136, 139–152.

68.

MacGregor

, Bulters

, Brandner

, Holton

, Verma

, Werring

, Carare

(2018) The fine anatomy of the perivascular compartment in the human brain: Relevance to dilated perivascular spaces in cerebral amyloid angiopathy. Neuropathol Appl Neurobiol, doi: 10.1111/nan.12480

69.

Calderón-Garcidueñas

, de la Monte

(2017) Apolipoprotein E4, gender, body mass index, inflammation, insulin resistance, and air pollution interactions: Recipe for Alzheimer’s disease development in Mexico City young females. J Alzheimers Dis 58, 613–630.

70.

Horsburgh

, Wardlaw

, van Agtmael

, Allan

, Ashford

MLJ

, Bath

, Brown

, Berwick

, Cader

, Carare

, Davis

, Duncombe

, Farr

, Fowler

, Goense

, Granata

, Hall

, Hainsworth

, Harvey

, Hawkes

, Joutel

, Kalaria

, Kehoe

, Lawrence

, Lockhart

, Love

, Macleod

, Macrae

, Markus

, McCabe

, McColl

, Meakin

, Miller

, Nedergaard

, O’Sullivan

, Quinn

, Rajani

, Saksida

, Smith

, Touyz

, Trueman

, Wang

, Williams

SCR

, Work

(2018) Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology. Clin Sci (Lond) 132, 851–868.

71.

Poggi

, Boretius

, Mobius

, Moschny

, Baudewig

, Ruhwedel

, Hassouna

, Wieser

, Werner

, Goebbels

, Nave

, Ehrenreich

(2016) Cortical network dysfunction caused by a subtle defect of myelination. Glia 64, 2025–2040.

72.

Gao

, Ma

, Tang

, Liang

, Huang

, Wang

, Chen

, Wang

, Tan

, Chao

, Zhang

, Qiu

, Luo

, Xiao

, Du

, Xiao

, Tang

(2017) White matter atrophy and myelinated fiber disruption in a rat model of depression. J Comp Neurol 525, 1922–1933.

73.

Fujihara

, Chiba

, Nakagawa

, Nishi

, Murakami

, Matsumoto

, Kawauchi

, Yamamoto

, Ueno

(2016) Albumin microvascular leakage in brains with diabetes mellitus. Microsc Res Tech 79, 833–837.

74.

Strain

, Smith

, Beaumont

, Roe

, Gordon

, Mishra

, Adeyemo

, Christensen

, Su

, Morris

, Benzinger

TLS

, Ances

(2018) Loss of white matter integrity reflects tau accumulation in Alzheimer disease defined regions. Neurology 91, e313–e318

75.

Operto

, Cacciaglia

, Grau-Rivera

, Falcon

, Brugulat-Serrat

, Rodenas

, Ramos

, Moran

, Esteller

, Bargallo

, Molinuevo

, Gispert

, ALFA Study (2018) White matter microstructure is altered in cognitively normal middle-aged APOE-e4 homozygotes. Alzheimers Res Ther 10, 48.

76.

Jung

, Lin

, Hwang

(2015) Ozone, particu-late matter, and newly diagnosed Alzheimer’s disease: A population-based cohort study in Taiwan. JAlzheimersDis 44, 573–584.

77.

Chen

, Kwong

, Copes

, Tu

, Villeneuve

, van Donkelaar

, Hystad

, Martin

, Murray

, Jessiman

, Wilton

, Kopp

, Burnett

(2017) Living near major roads and the incidence of dementia, Parkinson’s disease, and multiple sclerosis: A population-based cohort study. Lancet 389, 718–726.

78.

Molinuevo

, Sanchez-Valle

, Llado

, Fortea

, Barters-Faz

, Rami

(2012) Identifying earlier Alzheimer’s disease: Insights from the preclinical and prodromal phases. Neurodegen Dis 10, 158–160.

79.

Quiroz

, Schultz

, Chen

, Protas

, Brickhouse

, Fleisher

, Langbaum

, Thiyyagura

, Fagan

, Shah

, Muniz

, Arboleda-Velasquez

, Munoz

, Garcia

, Acosta-Baena

, Giraldo

, Tirado

, Ramirez

, Tariot

, Dickerson

, Sperling

, Lopera

, Reiman

(2015) Brain imaging and blood biomarker abnormalities in children with autosomal dominant Alzheimer disease: A cross-sectional study. JAMA Neurol 72, 912–919.

80.

Blennow

, Zetterberg

(2009) Cerebrospinal fluid biomarkers for Alzheimer’s disease. J Alzheimers Dis 18, 413–417.

81.

Bertens

, Knol

, Scheltens

, Visser

, Alzheimer’s Disease Neuroimaging Initiative (2015) Temporal evolution of biomarkers and cognitive markers in the asymptomatic, MCI and dementia stage of Alzheimer’s disease. Alzheimers Dement 11, 511–522.

82.

Bocchetta

, Galluzzi

, Kehoe

, Aguera

, Bernabei

, Bullock

, Ceccaldi

, Dartigues

, de Mendonca

, Didic

, Eriksdotter

, Felician

, Frölich

, Gertz

, Hallikainen

, Hasselbalch

, Hausner

, Heuser

, Jessen

, Jones

, Kurz

, Lawlor

, Lleo

, Martinez-Lage

, Mecocci

, Mehrabian

, Monsch

, Nobili

, Nordberg

, Rikkert

, Orgogozo

, Pasquier

, Peters

, Salmon

, Sanchez-Castellano

, Santana

, Sarazin

, Traykov

, Tsolaki

, Visser

, Wallin

, Wilcock

, Wilkinson

, Wolf

, Yener

, Zekry

, Frisoni

(2015) The use of biomarkers for the etiologic diagnosis of MCI in Europe: An EADC survey. Alzheimers Dement 11, 195–206.

83.

Teunissen

, Otto

, Engelborghs

, Herukka

, Lehmann

, Lewczuk

, Lleoi

, Perret-Liaudet

, Tumani

, Turner

, Verbeek

, Wiltfang

, Zetterberg

, Parnetti

, Blennow

(2018) White paper by the Society for CSF Analysis and Clinical Neurochemistry: Overcoming barriers in biomarker development and clinical translation. Alzheimers Res Ther 10, 30.

84.

de Leon

, Pirraglia

, Osorio

, Glodzik

, Saint-Louis

, Kim

, Fortea

, Fossati

, Laska

, Siegel

, Butler

, Li

, Rusinek

, Zetterberg

, Blennow

; Alzheimer’s Disease Neuroimaging Initiative; National Alzheimer’s Coordinating Center (2018) The nonlinear relationship between cerebrospinal fluid Aß42 and tau in preclinical Alzheimer’s disease. PLoS One 13, e0191240.

85.

Merluzzi

, Carlsson

, Johnson

, Schindler

, Asthana

, Blennow

, Zetterberg, H, Bendlin

(2018) Neurodegeneration, synaptic dysfunction, and gliosis are phenotypic of Alzheimer dementia. Neurology 91, e436–e443.

86.

Thoenen

, Barde

, Davies

, Johnson

(1987) Neurotrophic factors and neuronal death. Ciba Found Symp 126, 82–95.

87.

, Peskind

, Millard

, Chi

, Sokai

, Yu

, Bekris

, Raskind

, Galasko

, Montine

(2009) Cerebrospinal fluid concentration of brain-derived neu-rotrophic factor and cognitive function in non-demented subjects. PLoS One 4, e5424.

88.

Forlenza

, Diniz

, Teixeira

, Radanovic

, Talib

, Rocha

, Gattaz

(2015) Lower cerebrospinal fluid concentrations of brain-derived neurotrophic factor predicts progression from mild cognitive impairment to Alzheimer’s disease. Neuromolecular Med 17, 326–332.

89.

Shen

, You

, Joseph

, Mirzaei

, Klistorner

, Graham

, Gupta

(2018) BDNF polymorphism: A review of its diagnostic and clinical relevance in neurodegenerative disorders. Aging Dis 9, 523–536.

90.

Wilkoic

, Szalkowska

, Skibrnska

, Zajac-Lamparska

, Maciukiewicz

, Araszkiewicz

(2016) BDNF gene polymorphisms and haplotypes in relation to cognitive performance in Polish healthy subjects. Acta Neurobiol Exp (Wars) 76, 43–52.

91.

Goltz

, Janowitz

, Hannemann

, Nauck

, Hoffmann

, Seyfart

, Volzke

, Terock

, Grabe

(2017) Association of brain-derived neurotrophic factor and vitamin D with depression and obesity: A population-based study. Neuropsychobiology 76, 171–181.

92.

Zhang

, Sokal

, Peskind

, Quinn

, Jankovic

, Kenney

, Chung

, Millard

, Nutt

, Montine

(2008) CSF multianalyte profile distinguishes Alzheimer and Parkinson diseases. Am J Clin Pathol 129, 526–529.

93.

Dursun

, Alaylioglu

, Bilgic

, Hanagasi

, Lohmann

, Atasoy

, Candaş

, Araz

, Onal

, Gurvit

, Yil-mazer

, Gezen-Ak

(2016) Vitamin D deficiency might pose a greater risk for ApoEe4 non-carrier Alzheimer’s disease patients. Neurol Sci 37, 1633–1643.

94.

Lauren

, Gimbel

, Nygaard

, Gilbert

, Strittmatter

(2009) Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature 457, 1128–1132

95.

Bertoli

, Sorgato

(2018) Neuronal pathophysiology featuring PrPC and its control over Ca2+ metabolism. Prion 12, 28–33.

96.

Ulbrich

, Janning

, Seidel

, Matschke

, Gonsberg

, Jung

, Glatzel

, Engelhard

, Winklhofer

, Tatzelt

(2018) Alterations in the brain interactome of the intrinsically disordered N-terminal domain of the cellular prion protein (PrPC) in Alzheimer’s disease. PLoS One 13, e0197659.

97.

Ashok

, Singh

(2018) Prion protein modulates glucose homeostasis by altering intracellular iron. Sci Rep 8, 6556.

98.

Megra

, Eugenin

, Berman

(2018) Inflammatory mediators reduce surface PrPc on human BMVEC resulting in decreased barrier integrity. Lab Invest 98, 1347–1359.

99.

, Kandel

(2016) The role of functional prion-like proteins in the persistence of memory. Cold Spring Harb Perspect Biol 8, a021774.

100.

Ott

, Jones

, Daiello

, de la Monte

, Stopa

, Johanson

, Denby

, Grammas

(2018) Blood-cerebrospinal fluid barrier gradients in mild cognitive impairment and Alzheimer’s disease: Relationship to inflammatory cytokines and chemokines. Front Aging Neurosci 10, 245.

101.

Calderón-Garcidueñas

, Jewells

, Galaz-Montoya

, van Zundert

, Peirez-Calatayud

, Ascencio-Ferrel

, Valencia-Salazar

, Sandoval-Cano

, Carlos

, Solorio

, Acuüa-Ayala

, Torres-Jardon

, D'Angiulli

(2016) Interactive and additive influences of gender, BMI and Apolipoprotein E on cognition in children chronically exposed to high concentrations of PM 2.5 and ozone. APOE 4 females are at highest risk in Mexico City. Environ Res 150, 411–422.