Tau Phosphorylation and Aggregation in the Developing Human Brain

Abstract

Tau hyperphosphorylation, mostly at serine (Ser) or threonine (Thr) residues, plays a key role in the pathogenesis of Alzheimer disease (AD) and other tauopathies. Rodent studies show similar hyperphosphorylation in the developing brain, which may be involved in regulating axonal growth and plasticity, but detailed human studies are lacking. Here, we examine tau phosphorylation by immunohistochemistry and immunoblotting in human fetal and adult autopsy brain tissue. Of the 20 cases with sufficient tissue preservation, 18 (90%) showed positive staining for S214 (pSer214), with the majority also positive for CP13 (pSer202), and PHF-1 (pSer396/pSer404). AT8 (pSer202/pThr205) and RZ3 (pThr231) were largely negative while PG5 (pSer409) was negative in all cases. Immunoblotting showed tau monomers with a similar staining pattern. We also observed phospho-tau aggregates in the fetal molecular layer, staining positively for S214, CP13, and PHF1 and negative for thioflavin S. These corresponded to high-molecular weight (∼150 kD) bands seen on Western blots probed with S214, PHF1, and PG5. We therefore conclude that fetal phosphorylation overlaps with AD in some residues, while others (e.g. T231, S409) appear to be unique to AD, and that tau is capable of forming nontoxic aggregates in the developing brain. These findings suggest that the fetal brain is resilient to formation of toxic aggregates, the mechanism for which may yield insights into the pathogenesis of tau aggregation and toxicity in the aging brain.

INTRODUCTION

Although best known for its role in Alzheimer disease (AD) and related dementias, the tau protein is expressed throughout human development (1). In vitro studies in cultured mouse and hamster neurons suggest that tau is involved in axonogenesis and neuronal circuit formation (2, 3), although interestingly, MAPT (tau) knockout mice appear to develop neurologic deficits only late in life despite having abnormal axonal microtubule organization (4, 5). Tau is also thought to be involved in glutamatergic signaling and to play a role in excitotoxicity and epileptogenesis via a FYN and PSD-95 mediated mechanism (6–8). Despite this, the developmental role of the tau protein remains unclear due to the difficulties inherent in translating mouse data to human brain development and in genetic manipulation of animals with more complex brain development.

Tau is a predominantly axonal microtubule-associated protein that undergoes extensive post-translational modification, including phosphorylation at more than 20 distinct epitopes, most of which are threonines (pThr) or serines (pSer). Phosphorylation of tau is mediated by multiple kinases and phosphatases including GSK3β, DYRK1A, and PP2A, among others. Abnormally hyperphosphorylated tau is the principal component of neurofibrillary tangles and other toxic aggregates in neurodegenerative tauopathies including AD, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP) (reviewed in [9]). The classic antibodies used for neuropathologic diagnosis of AD include PHF1, which targets pSer396/pSer404 and AT8, which targets pSer202/pThr205 (10). Tau phosphorylation is thought to play a key role in modulating microtubule binding affinity, allowing axonal growth and plasticity in the developing brain (11–14), but in vitro and animal studies suggest that hyperphosphorylation also predisposes to liquid-liquid phase separation and aggregation (15–17).

Interestingly, however, rodents show tau hyperphosphorylation during fetal brain development, with many of the same epitopes that are seen in Alzheimer and other neurodegenerative diseases, but without significant toxicity (18–25). Limited human data suggest that human fetal tau is also hyperphosphorylated, but this is based on studies in small numbers of cerebrospinal fluid and brain homogenate samples (18, 26, 27). Detailed data on specific epitopes and on anatomic and temporal variation in tau phosphorylation during early human development are lacking. It is also unclear whether the phosphorylation pattern in the developing fetal brain differs from that seen in neurofibrillary tangles and other aggregates of neurodegenerative disease and aging (e.g. the pS202/pThr205 sites recognized by the AT8 antibody commonly used for Braak staging in neuropathology) (10). Understanding such key differences would provide important insights into mechanisms triggering toxic tau aggregation, and might help identify potential therapeutic targets for neurodegenerative diseases including AD.

We have previously shown that splicing changes in the developing human brain are similar but not identical to those seen in rodents (28). Here we present, to our knowledge, the first in depth analysis of tau phosphorylation in the human fetal brain using a combination of immunohistochemistry and Western blotting, showing phospho-epitopes that are unique to Alzheimer-type pathology. We also report, to our knowledge for the first time, the finding of apparently nontoxic tau aggregates in the developing fetal brain.

MATERIALS AND METHODS

Tissue Samples

Formalin-fixed, paraffin-embedded tissue was obtained from the autopsy archives of the Mount Sinai Medical Center and University of Iowa Hospitals and Clinics. Snap-frozen fetal brain tissue was obtained from the autopsy service at the Mount Sinai Medical Center. Frozen adult control and AD specimens were obtained from the Mount Sinai Neuropathology Brain Bank & Research Core. In all cases, brains with CNS malformation or neuropathologic evidence of hypoxic-ischemic injury were excluded. Tissue collection protocols were approved by the Mount Sinai Institutional Review Board (Protocol IRB-17-01313). Protocols at the University of Iowa were reviewed by the University of Iowa Institutional Review Board and determined to be exempt from review (Project 201706772). All methods were carried out in accordance with the relevant guidelines, laws, and regulations.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue blocks were cut at 4–5 μm in thickness, placed on charged slides and baked overnight at 70°C. Immunohistochemistry was performed on a Ventana Benchmark XT according to manufacturer’s directions. Antigen retrieval was done using CC1 (Tris/Borate/EDTA buffer, pH 8.0–8.5, Roche Diagnostics, Basel Switzerland) for 1 hour. All primary antibodies were diluted in Antibody dilution buffer (ABD24, Roche Diagnostics) and the dilutions are shown in Table 1. Primary antibodies were incubated for either 24 minutes (AT8, S214) or 32 minutes (RZ3, PHF1, CP13, PG5) at 37°C. Primary antibodies were visualized using either the Ultraview or Optiview detection kit (Roche Diagnostics) as an indirect biotin-free system to detect mouse primary antibodies according to manufacturer’s directions. All slides were counterstained with hematoxylin for 8 minutes and a coverslip was added. Slides were visualized using an Olympus BX40 brightfield microscope with an Olympus DP27 camera and CellSens software (both from Olympus, Tokyo, Japan). All slides were scored by an experienced developmental neuropathologist (M.M.H.) blinded to gestational age using a semiquantitative scoring system as follows: no staining (−), weak (+/−), strong (+), and very strong (++). Neuropathologically confirmed cases of AD, CBD, PSP, and primary age-related tauopathy were used as controls.

TABLE 1.

Primary Antibodies and Concentrations Used

Antibody	Epitope	Source	Concentration
			IHC	WB
RZ3	pThr231	Dr Peter Davies	1:500	1:200
PHF1	pSer396, pSer404	Dr Peter Davies	1:500	1:500
CP13	pSer202	Dr Peter Davies	1:500	1:500
AT8	pSer202, pThr205	Fisher Scientific	1:2000	1:1000
S214	pSer214	Invitrogen	1:1000	1:1000
PG5	pSer409	Dr Peter Davies	1:500	1:200

pThr, phosphothreonine; pSer, phosphoserine; IHC, immunohistochemistry; WB, Western blotting.

Biochemical Studies

Western blotting was done as previously described in (28). Briefly, fresh-frozen brain tissue was homogenized with a glass-Teflon homogenizer at 500 rpm in 10 volumes (wt/vol) of ice-cold tissue homogenization buffer containing 20 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). For each sample, 30 μg of proteins were run on 10% PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA), blotted to nitrocellulose membranes, and stained with the same antibodies indicated in Table 1. Horseradish peroxidase-labeled secondary anti-mouse or rabbit antibody (Vector Labs, Burlingame, CA) both at 1:20 000 dilution, was detected by SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific). To quantify and standardize protein levels without reliance on specific housekeeping proteins, total protein was detected with Amido Black (Sigma-Aldrich, St. Louis, MO) as previously described in (28). We elected to use total protein quantification because levels of housekeeping proteins such as GAPDH and α-tubulin change during development (29, 30). We ourselves found that levels of β-actin vary markedly with development despite equivalent protein loads, making this problematic as well (data not shown). Total protein quantification with amido black has been validated as an alternative control for quantitation of Western blots (31). Chemiluminescence was measured in an LAS-4000 Intelligent Dark Box imager (Fuji Film, Valhalla, NY), and relative optical densities were determined by using AlphaEaseFC software, version 4.0.1 (Alpha Innotech, San Jose, CA), normalized to total protein loaded. Human AD brain homogenate and normal aging adults were used as controls.

Thioflavin S Staining

Formalin-fixed, paraffin embedded tissue blocks were cut at 4–5 μm thickness and baked at 70°C for 1 hour. The tissue was deparaffinized with 100% xylene (2 5-minute washes) followed 50%/50% ethanol/xylene (3 minutes) and then hydrated using a series of ethanol dilutions (100%, 95%, 70%, 50%, 3 minutes each). The slides were then washed in water (2 3-minute washes). Thioflavin-S (AC213150250, Arcos Organics, Geel, Belgium) was reconstituted in water to make a 1% aqueous solution and filtered to remove particles. Slides were incubated in Thioflavin-S for 8 minutes at room temperature in the dark followed by a second series of ethanol dilutions (80%, 80%, 95%, 3 minutes each) and 3 washes with distilled water. Slides were mounted with VectaMount Mounting Medium (Cat. No H-5000, Vector Laboratories) and visualized using a Nikon Eclipse E600 microscope (Nikon, Melville, NY) with a Lumencor Solar Light Engine (Lumencor, Beaverton, OR), SPOT RT Slider camera (SPOT Imaging, Sterling Heights, MI) and associated software. A human AD brain section was used as a positive control.

Statistics and Data Analysis

Densitometry data were compared using 1-way ANOVA with Tukey’s test for pairwise comparisons of groups. Statistical analysis and graph production were done in RStudio using the R programming environment.

RESULTS

Patient Population

We initially identified 40 fetal cases with available formalin-fixed, paraffin-embedded tissue. Of these cases, 20 showed no staining with any of the antibodies used and were therefore excluded from further analysis since protein degradation due to prolonged postmortem interval could not be excluded. This left 20 fetal cases remaining, ranging from 14 to 38 postconceptional weeks (PCW). All subsequent data are reported as a fraction of these 20 cases. The causes of death included placental abruption (n = 2), cervical incompetence (n = 1), chorioamnionitis (n = 9), extreme prematurity (n = 2), termination for hand-heart syndrome (n = 1), complications of maternal diabetes (n = 1), sepsis (n = 2), and idiopathic spontaneous miscarriage (n = 2). Frozen tissue for Western blotting included 3 fetal cases ranging in age from 19 to 21 PCW. Cause of death was elective termination for unspecified reasons in all 3 cases. None had central nervous system malformations by report or at autopsy. Adult neurodegenerative cases and controls (16 formalin-fixed, paraffin-embedded, 6 frozen) were obtained from the autopsy archives of the Mount Sinai Medical Center and are summarized in Table 2.

TABLE 2.

Tau Expression in the Developing Fetal Brain by Immunohistochemistry

	Age	Diagnosis	S214	PHF1	CP13	RZ3	AT8	PG5
Fetal 1	14 pcw	Control	++	++	++	−	+	−
Fetal 2	17 pcw	Control	++	+/−	−	−	++	−
Fetal 3	18 pcw	Control	++	+	++	−	−	−
Fetal 4	18 pcw	Control	++	++	++	−	++	−
Fetal 5	19 pcw	Control	++	+/−	+	−	−	−
Fetal 6	19 pcw	Control	−	−	++	−	−	−
Fetal 7	19 pcw	Control	++	++	++	−	−	−
Fetal 8	20 pcw	Control	++	++	++	++	−	−
Fetal 9	20 pcw	Control	++	+	+	−	−	−
Fetal 10	20 pcw	Control	++	++	++	−	++	−
Fetal 11	20 pcw	Control	++	+	++	−	−	−
Fetal 12	21 pcw	Control	+	++	++	−	−	−
Fetal 13	21 pcw	Control	++	+	+	−	−	−
Fetal 14	22 pcw	Control	++	++	++	++	−	−
Fetal 15	22 pcw	Control	++	+	−	−	−	−
Fetal 16	22 pcw	Control	++	+	−	−	−	−
Fetal 17	24 pcw	Control	++	−	+/−	−	−	−
Fetal 18	26 pcw	Control	++	+/−	+	−	−	−
Fetal 19	37 pcw	Control	++	+	+	−	−	−
Fetal 20	38 pcw	Control	−	−	−	−	++	−
Adult 7	57 years	Control	−	−	−	−	−	−
Adult 8	50 years	Control	−	−	−	−	−	−
Adult 9	42 years	Control	−	−	−	−	−	−
Adult 10	89 years	Control	−	−	−	−	−	−
Adult 11	60 years	Control	−	−	−	−	−	−
Adult 12	66 years	Control	−	−	−	−	−	−
Adult 1	71 years	FTLD-tau	++	++	++	++	++	+
Adult 2	75 years	CBD	++	+	++	++	++	+
Adult 3	72 years	CBD	++	++	++	++	++	++
Adult 5	64 years	PSP	−	++	+	−	++	−
Adult 6	56 years	PSP	+	++	++	++	++	+/−
Adult 4	95 years	AD (A2B3C2)	++	++	++	++	++	+
Adult 14	82 years	AD (A2B3C2)	−	++	−	−	−	−
Adult 15	82 years	AD (A2B3C2)	++	++	++	++	++	+
Adult 16	66 years	AD (A2B3C2)	++	++	++	++	++	++
Adult 17	78 years	AD (A2B3C2)	+	−	++	+	++	+

IHC-stained slides were scored semiquantitatively by a blinded observer. Tau antibodies are shown in Table 1.

ABC scores as defined in (15).

PCW, postconceptional weeks; AD, Alzheimer disease; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; FTLD, fronto-temporal lobar degeneration.

Fetal Brain Shows Immunoreactivity for Multiple Phospho-Tau Epitopes

Of the 20 fetal cases that showed immunoreactivity for at least one of the antibodies tested, 18/20 (90%) showed positive staining for phosphorylation at serine 214 (S214, pSer214) (Table 2). Sixteen of 20 (80%) cases showed positive staining for CP13 (pSer202), all of which were also positive for S214 (Table 2; Fig. 1). Seventeen cases showed positive staining for PHF1 (pSer396, pSer404), with some cases showing positive staining for only PHF1 (n = 3) or only CP13 (n = 2). AT8 (pSer202/pThr205) and RZ3 (pThr231) were positive in 4 and 2 cases, respectively. PG5 (pSer409) was negative in all cases examined. There was robust staining for S214, CP13, and PHF1 starting at 14 PCW, with decreased staining for PHF1 and CP13 at ∼22 PCW, and persistent positive staining for S214 until ∼38 PCW (Table 2; Fig. 2). Tissue microarrays consisting of sections of neocortex from adult tauopathies (AD, FTLD-tau, PSP, and CBD) were used as positive controls for all antibodies. Because of expected variability in staining due to the small size of individual cores, multiple specimens for each diagnosis were included in the array. PHF-1, CP13, and AT8 were positive in 9/10 cases, while S214, RZ3, and PG5 were positive in 8/10 cases (Table 2, bottom).

FIGURE 1.

Phospho-tau immunohistochemistry in a 20-postconceptional week human fetal brain. There is strong positive staining for CP13 and PHF1, as well as weaker staining for AT8. Staining for S214 is limited to the molecular layer (better seen in Fig. 2). Insets show gray-white matter junction at high power for each stain with cortical plate to the left. Large bar = 2 mm, small bar = 20 μm. ml, molecular layer; cp, cortical plate; wm, white matter; stf, stratified transitional field; svz, subventricular zone; lv, lateral ventricle.

FIGURE 2.

Phospho-tau expression patterns show minimal variation with gestational age during the second trimester. Positive staining for S214 in the molecular layer remains essentially invariant from 14 PCW to 24 PCW, with gradual decrease in staining intensity by 37 PCW. Top row shows negative (normal adult brain) and positive (PSP, AD) controls. PSP, progressive supranuclear palsy; AD, Alzheimer disease; PCW, postconceptional weeks; ml, molecular layer; cp, cortical plate. Black bar = 50 μm.

Distribution of Staining Differs Between Epitopes

The distribution of staining varied between epitopes, but neurogenic regions (subventricular zone, germinal matrix) were negative in all cases. Staining for S214 was limited to the molecular layer (cortical layer I) in 16 of 18 cases (Figs. 1 and 2). Three cases showed strong positive cytoplasmic staining in a subset of cells in the subpial granule cell layer (Fig. 3, black arrowheads) in addition to granular staining in the molecular layer (Fig. 3, white arrowhead). The frequency of such staining is however difficult to assess as in many cases this later was artifactually disrupted and could not be assessed. CP13 and PHF1 were also strongly positive in the molecular layer, but in the majority of cases (12/16 and 11/17 cases respectively), also showed positive staining in the white matter (Fig. 1). Only 2 cases showed positive staining for RZ3, and in 1 case this was limited to cytoplasmic staining in the basal ganglia, of unclear significance (data not shown). There was no positive staining for PG5 in any of the examined fetal cases.

FIGURE 3.

Phospho-tau positive aggregates are seen in the molecular layer. The aggregates are strongly positive for S214, with lesser staining for PHF-1 and CP13. White and black arrowheads indicate aggregates in the molecular layer and subpial cytoplasmic staining, respectively. Representative images from a 19 PCW fetus. CP, cortical plate; ML, molecular layer; SGL, subpial granule cell layer. Black bar = 50 μm.

Fetal Brain Shows Formation of Phospho-Tau Aggregates

We observed aggregates, predominantly in the molecular layer, that stained positively for S214, with weaker staining for CP13 and PHF-1 (Fig. 4, black arrow). These aggregates were negative for thioflavin S (Fig. 5) and were seen in all cases that showed positive staining for S214. We then used Western blotting with the same antibodies used for immunohistochemistry to confirm our findings. As expected, we saw bands corresponding to tau monomers staining positive for CP13 and PHF1 that were not present in adult brain (Fig. 4). When we probed with our S214 antibody, however, we saw strong high-molecular weight bands similar to those seen in the AD cases (Fig. 4, black arrow). These bands showed weak positive staining for AT8 but were otherwise negative. Quantification of bands is shown in Figure 6. All except PG5 were positive by 1-way ANOVA, with Tukey’s HSD showing a significant difference between fetal and AD in AT8, CP13, RZ3, and PHF1. S214 showed a strong, statistically significant difference between fetal and adult brain (p = 1.3 × 10⁻³ by Tukey’s HSD). Differences between fetal and adult in CP13 and PHF1 were significant by simple Student t-test (p < 0.05 for both) but not when corrected for multiple comparisons.

FIGURE 4.

Tau aggregates in the molecular layer do not represent amyloids. S214-positive aggregates in the molecular layer do not show positive staining by thioflavin S, while an adult AD positive control shows staining in both neurofibrillary tangles (filled arrowheads) and neuritic plaques (unfilled arrowhead).

FIGURE 5.

Fetal brain shows S214-positive high-molecular weight aggregates. High-molecular aggregates show strong positive staining for S214 (black arrowhead) with weaker staining for CP13 and PG5. Aggregates from AD control brains (right 3 columns) show strong positive staining for all epitopes. Immunoblotting of fetal, adult, and AD brain homogenate with PG5, CP13, RZ3, AT8, PHF1, and S214.

FIGURE 6.

Quantification of phospho-tau Western blots. S214-positive staining is significantly higher than both adult control and AD brains. AT8, CP13, PHF1, and RZ3 are significantly lower in fetal than in AD brain. Density of all bands quantified relative to total protein (amido black staining) as previously described in (28), p values are indicated above the graphs and were calculated using a 1-way ANOVA with pairwise comparison by Tukey's test. All bars represent means ± standard error.

Conclusions

Our findings suggest that, while tau is extensively phosphorylated in human fetal brain, the phosphorylation pattern is different from that seen in AD and related tauopathies. Specifically, 4 serine residues (Ser214, Ser396, Ser404, and Ser202) appear to be phosphorylated in both conditions. Threonine 231 (Thr231) shows variable phosphorylation in fetal brain but is strongly positive in AD. Serine 409, however, appears to be unique to AD. The strong positive staining for CP13 (pSer202) and weak staining for AT8 (pSer202/pThr205) suggests that Thr205 is also likely unique to AD and related tauopathies. In addition, recent data suggest that AT8 has the highest affinity for tau phosphorylated at Ser202, Ser205, and Ser208, with the latter having the greatest effect on affinity (32). This would suggest that Ser205 and Ser208 are minimally phosphorylated in fetal brain, which is supported by recent in vitro data suggesting that these residues are critical in forming toxic aggregates (15).

Our human fetal brain samples showed aggregates strongly positive for Ser214, Ser202, Ser396, and Ser404 by immunohistochemistry. These appear to correspond to high-molecular weight tau aggregates seen on Western blotting with a similar phosphorylation profile. Although their molecular weight is similar to that seen in AD control cases, they have a distinct phosphorylation profile similar to that seen by immunohistochemistry. Of note, the positive signal for pS214 is far stronger in the fetal brain than in the adult AD controls. In addition to their distinct phosphorylation profile, these aggregates do not appear to represent an amyloid, given that they are negative for thioflavin S. While it is possible that these aggregates are triggered by hypoxic-ischemic injury or other physiologic stress, immunoblotting was performed on cases with minimal perimortem hypoxic-ischemic injury (see Patient Population section), making this unlikely. This finding has not been previously reported, to our knowledge, in fetal human or animal brains.

Our data suggest that human fetal brain shares some phospho-epitopes with rat (S202, S214, S396, S404) but lacks phosphorylation at pS409, which is seen in fetal rat brain (27). AT8 (S202/T205) was positive in studies of both fetal rat (27) and feline brains (33). Interestingly the feline brain data show a pattern of AT8 staining similar to that seen by us for S214 (33). There are, to the best of our knowledge, no previously published systematic studies of human fetal tau phosphorylation. A single human fetal brain sample was included in a larger study by Brion et al (23), which showed bands positive for PHF-1 and AT8 on Western blot. Since this study only included a single case, this is difficult to interpret.

Although the developmental role of tau remains poorly understood, the existing evidence suggests a key role in axonogenesis. Tau ablation causes abnormal neurite morphology in primary mouse hippocampal cultures and malformed axonal growth cones in hamster primary neuronal cultures (2, 3). Tau knockout (MAPT−/−) mice develop muscle weakness, poor balance, hyperactivity, and impaired contextual fear conditioning (5). The developmental role of phosphorylation has not been studied to the same degree, but phosphorylation is known to decrease tau microtubule binding affinity, leading to the hypothesis that tau phosphorylation is necessary to allow the microtubule plasticity necessary for axonogenesis during development (11–14). In animal model, decreased GSK3β (34) or DYRK1A (35) impairs neuronal maturation, and DYRK1A is overexpressed in patients with Downs syndrome, who have abnormal developmental tau phosphorylation (36) and develop early AD-type neuropathology (37–39). The mechanistic link between abnormal tau phosphorylation and these developmental phenotypes remains unclear however.

Our study is the first to systematically examine tau phosphorylation in the human fetal brain. Due to the high-volume autopsy services of our institutions, we were able to retrospectively collect a large number of fetal, neonatal, and adult autopsies. Because of the high volume, we were able to exclude cases with evidence of maceration, autolysis, hypoxic-ischemic injury, or central nervous system malformations, reducing potential confounders. We were also able to include a broad range of gestational ages, with cases as early as 14 PCW.

Due to the retrospective nature of our study, we were, however, limited by tissue sampling performed by the original pathologist and detailed data on postmortem intervals, particularly time from fetal demise to delivery in cases of stillbirth, was generally not available. We cannot therefore definitively exclude the possibility that some antibodies might have been differentially affected by autolytic changes. We also cannot exclude the possibility that more subtle hypoxic-ischemic injury may be responsible for some of the variation in tau phosphorylation, particularly that seen in AT8 and RZ3. We were forced to exclude a subset of our formalin-fixed, paraffin-embedded cases that had no detectable staining for any tau phosphoepitopes. The remaining cases showed a staining pattern similar to that seen by immunoblotting in a separate cohort of frozen tissue from cases with very short postmortem intervals, which strongly suggests that this loss of staining is due to poor tissue preservation, although we cannot definitively exclude other causes such as hypoxic-ischemic injury. Our study also relies on the specificity of the phosphoepitope-specific antibodies. The CP13, PFH1, PG5, RZ3, and S214 antibodies used in the current study have, however, been extensively validated and their target epitopes mapped in detail (40–43). Recent data suggest that AT8 (pSer202/pSer205) may also detect pSer208 tau as noted above, but this does not affect the conclusions of this study (32).

Our findings suggest that the developing fetal brain contains hyperphosphorylated tau with epitopes that overlap with, but are not identical to, those seen in AD. In addition, it appears that fetal tau is able to form aggregates. These aggregates show a phosphorylation pattern distinct from those seen in neurodegenerative disease and do not appear to form an amyloid. We cannot, however, exclude the possibility that the formation of these aggregates plays a role in physiologic neuronal loss during development. Further progress in this area will require more detailed studies of developmental tau phosphorylation by mass spectroscopy or other methods that permit broader assessment of tau phosphorylation and other forms of post-translational modification. The presence of tau aggregates will also require further exploration to further characterize their timing, phosphorylation status, and role in brain development.

In conclusion, we report that human fetal tau shows hyperphosphorylation at multiple sites overlapping with those see in AD and related dementias, and that it is able to form aggregates which do not, however, lead to AD-like tau pathology.

This work was supported by the National Institutes of Health [UL1TR002537 to M.H., R01AG054008 and R01NS095252 to J.F.C. and F32AG056098 to K.F.], the United States Department of Defense [13267017 to J.F.C.], the Tau Consortium, the Williams Cannon Foundation [to M.M.H.], and an Alexander Saint-Amand Scholarship [to J.F.C.]. The authors also wish to acknowledge the Mount Sinai Neuropathology Brain Bank & Research Core, supported by funding from the Nash Family Department of Neuroscience at the Icahn School of Medicine at Mount Sinai.

The authors have no duality or conflicts of interest to declare.