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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Brain Pathol. Author manuscript; available in PMC 2017 May 1.
Published in final edited form as:
Published online 2015 August 24. doi:  10.1111/bpa.12289
PMCID: PMC4720581
NIHMSID: NIHMS725304

Precortical phase of Alzheimer’s disease (AD)-related tau cytoskeletal pathology

Abstract

Alzheimer’s disease (AD) represents the most frequent progressive neuropsychiatric disorder worldwide leading to dementia and accounts for 60 to 70% of demented individuals. In view of the early appearance of neuronal deposits of the hyperphosphorylated cytoskeletal protein tau in the transentorhinal and entorhinal regions of the allocortex (i.e. in Braak and Braak AD stage I in the evolution of the AD-related cortical tau cytoskeletal pathology) it has been believed for a long time that these allocortical regions represent the first brain targets of the AD-related tau cytoskeletal pathology. However, recent pathoanatomical studies suggested that the subcortical brain nuclei that send efferent projections to the transentorhinal and entorhinal regions may also comprise AD-related cytoskeletal changes already at very early Braak and Braak AD stages. In order to corroborate these initial results we systematically investigated the presence and extent of the AD-related cytoskeletal pathology in serial thick tissue sections through all the subcortical nuclei known to send efferent projections to these vulnerable allocortical regions of three individuals with Braak and Braak AD stage 0 and fourteen individuals with Braak and Braak AD stage I by means of immunostainings with the anti-tau antibody AT8. These investigations revealed consistent AT8 immunoreactive neuronal tau cytoskeletal pathology in a subset of these subcortical nuclei (i.e. medial septal nucleus, nuclei of the vertical and horizontal limbs of the diagonal band of Broca, basal nucleus of Meynert; claustrum; hypothalamic ventromedial, tuberomamillary and supramamillary nuclei, perifornical region and lateral area; thalamic central medial, laterodorsal, subparafascicular, and central lateral nuclei, medial pulvinar and limitans-suprageniculate complex; peripeduncular nucleus, dopaminergic substantia nigra and ventral tegmental area, periaqueductal gray, midbrain and pontine dorsal raphe nuclei, locus coeruleus, and parabrachial nuclei) in the Braak and Braak AD stage 0 individuals and in all of these subcortical nuclei in the Braak and Braak AD stage I individuals. The widespread affection of the subcortical nuclei in our Braak and Braak AD stage I individuals shows that the extent of the subcortical tau cytoskeletal pathology in this AD stage has been considerably underestimated during the last decades. In addition, our novel findings in the Braak and Braak AD stage 0 individuals support the concept that subcortical nuclei become already affected during an early ‘pre-cortical’ evolutional phase before the first AD-related cytoskeletal changes occur in the well-known cortical predilection sites of the mediobasal temporal lobe (i.e. transentorhinal and entorhinal regions). In addition, our new findings indicate that the AD-related tau cytoskeletal pathology by no means is confined to single subcortical nuclei of Braak and Braak AD stage 0 individuals, but may develop in a large variety of their subcortical nuclei interconnected with the allocortical entorhinal and transentorhinal regions. Accordingly, these very early involved subcortical brain regions may represent the origin of the AD-related tau cytoskeletal pathology, from where the neuronal cytoskeletal pathology takes an ascending course towards the secondarily affected allocortex and spreads transneuronally along anatomical pathways and interconnectivities in predictable and stereotypical sequences

Keywords: Allocortex, Alzheimer’s disease, cytoskeletal pathology, entorhinal region, subcortical nuclei, tau protein, prion-like diseases

Introduction

Alzheimer’s disease (AD) is a progressive neuropsychiatric disorder and represents the most frequent dementing disorder accounting for 60 to 70% of demented individuals [11, 18, 38, 46, 51, 52, 71, 74, 89]. Owing to its estimated frequency of approximately 25 million patients worldwide in 2001 and the occurrence of approximately 5 million new AD cases per year, AD is a tremendous burden not only for diseased individuals, their families and caregivers, but also for health syste ms and social economies [8, 11, 18, 38, 46, 52, 58, 71].

The neuropathology of AD is characterized by neuronal loss at specific cortical and subcortical brain sites (i.e. transentorhinal and entorhinal regions, hippocampus, amygdala, medial septal nucleus, nuclei of the diagonal band of Broca, basal nucleus of Meynert, compact part of the substantia nigra, locus coeruleus, midbrain and pontine raphe nuclei), as well as intraneuronal and extraneuronal deposits of abnormal proteins [1315, 18, 25, 28, 29, 33, 35, 43, 46, 52, 93]. The extraneuronal deposits are formed by the insoluble β-amyloid protein and the intraneuronal deposits by the abnormally phosphorylated tau protein [4, 5, 8, 11, 1315, 18, 23, 27, 29, 33, 35, 38, 4749, 52, 58, 59, 74, 92, 93, 96].

Tau, an axonal cytoskeletal and the major microtubule associated protein (MAP), becomes hyperphosphorylated in nerve cells of AD brains due to an imbalance of multiple protein kinases and phosphatases..In the disease state hyperphosphorylation of tau leads to (1) a reduced affinity for microtubules and ineffective microtubule polymerization, assembly, and stabilization, (2) loss of microtubule rails and impairments of intra-axonal transport mechanisms, and (3) to tau insolubility, its self-aggregation and deposition in affected neurons [5, 8, 11, 12, 18, 23, 26, 27, 29, 32, 37, 38, 46, 48, 49, 58, 59, 62, 71, 74, 79, 89, 91, 92, 96].

Neuronal aggregations of the tau protein acquire the form of paired helical filaments (PHF) which represent the ultrastructural basis of the AD-related cytoskeletal pathology and coalesce into neurofibrillary tangles (NFT) in neuronal perikarya and into dendritic neuropil threads (NT) originally described by Alois Alzheimer and Oskar Fischer [46, 8, 1114, 18, 22, 23, 29, 32, 35, 3739, 46, 48, 49, 52, 58, 59, 62, 65, 74, 89, 96]. The stereotypical, gradual and hierarchical recruitment of regions of the cerebral cortex by the AD-related hyperphosphorylated tau cytoskeletal pathology has allowed to establish a staging procedure that describes its increasing severity, distribution and topographical spread (i.e. Braak and Braak AD stages in the evolution of the AD-related cortical cytoskeletal pathology). The predictable temporal and spatial sequences of the expansion of the AD-related cortical tau cytoskeletal pathology start from the transentorhinal region of the mediobasal temporal lobe, reach out to the immediately adjacent entorhinal region, extend into the hippocampus and ultimately affect all portions of the cerebral neocortex. This reproducible and fairly stereotypic progession also implies a long-lasting preclinical phase of approximately three to four decades during which everyday relevant cognitive deficits are not yet evident [4, 911, 13, 14, 1618, 27, 29, 30, 32, 35, 44, 52, 58, 59, 65, 67].

Owing to the early appearance of tau cytoskeletal pathology in neurons of the transentorhinal and entorhinal regions it has been believed for a long time that the cerebral allocortex represents the first brain target of the AD-related cytoskeletal pathology [9, 10, 12, 14, 1618, 32, 33, 35, 39, 55, 71, 78, 89, 96]. However, several studies suggested that the AD-related tau cytoskeletal pathology also occurs rather early in specific nuclei of the basal forebrain, thalamus and brainstem, all of which are anatomically connected with the allocortical transentorhinal and entorhinal regions via fiber tracts and were not included in the original Braak and Braak AD staging scheme [13, 14, 29, 30, 39, 40, 68, 71, 72, 8083, 88, 89]. Although, the original staging system has been recently extended, our preliminary findings of pilot studies indicated that the very early subcortical tau pathology may be more widespread than described in the revised Braak and Braak AD staging system and in addition to the locus coeruleus may also affect a large variety of other subcortical nuclei with projections to the early affected entorhinal and transentorhinal regions [13, 14, 18, 21].

It is well-known that all of the subcortical nuclei with efferent projections to the allocortical transentorhinal and entorhinal regions consistently exhibit a marked neuronal tau pathology in demented AD patients burdened by Braak and Braak AD stage V or VI tau cytoskeletal pathologies [2, 3, 15, 18, 29, 33, 40, 56, 57, 68, 72, 73, 8183, 86, 88] (Supplementary Figs. 1–5). Considering our preliminary findings we therefore hypothesized that the high vulnerability of the subcortical sites with efferent projections to the transentorhinal and entorhinal regions for the AD-related tau cytoskeletal pathology is not only mirrored by their marked and consistent involvement in the late Braak and Braak AD stages V and VI, but may also be reflected by their very early involvement during the evolution of the AD-related tau pathology. Accordingly, we tested the hypothesis by investigating the presence and extent of the AD-related tau cytoskeletal pathology in all subcortical nuclei sending efferent projections to the transentorhinal and entorhinal regions [45, 61] in individuals with Braak and Braak AD stage I or 0 cortical tau cytoskeletal pathology (Supplementary Figs. 1–5).

Patients and methods

A total of forty-four brains from patients without neurological or psychiatric diseases in their medical histories and intact cognitive functions upon clinical examination (22 females, 22 males, age at death: 54.20 ± 17.70 years) submitted to autopsy at the Department of Pathology and Medical Biology at the University Medical Center Groningen, the Netherlands, were screened for this study. The brains of all forty-four individuals were examined macroscopically, fixed in 4% non-buffered formaldehyde, investigated for purposes of neuropathological diagnosis by an experienced neuropathologist (W den D) and eventually classified using the Braak and Braak AD staging procedure distinguishing stages I-VI in the distribution and severity of the AD-related cortical tau cytoskeletal pathology [4, 10, 13, 14, 16, 18, 27, 29, 30, 32, 35, 44, 65]. This neuropathological classification revealed the presence of three brains with Braak and Braak AD stage 0 (1 female, 2 males; age at death: 29.0 ± 11.36 years) and fourteen with Braak and Braak AD stage I (6 females, 8 males; age at death: 43.4 ± 12.00 years) cortical cytoskeletal pathology among the screened forty-four control brains (Supplementary Table 1). Autopsies were performed in these Braak and Braak AD stage 0 and I individuals within 13.94 ± 9.9 h postmortem (Supplementary Table 1).

For comparison and additional demonstration of their exceptionally high vulnerability we also documented the AD-related cytoskeletal pathology in the subcortical nuclei sending efferent projections to the transentorhinal and entorhinal regions [43, 59] of seven patients in Braak and Braak AD stages V or VI (2 females, 5 males; age at death: 74.0 ± 5.48 years) [13, 14, 18] which were clinically diagnosed as demented or suffered from incipient dementia (Supplementary Table 2).

Tissue processing

For purposes of neuropathological classification of their AD-related cortical cytoskeletal pathology according to the Braak and Braak AD staging system, at least six tissue sections through recommended brain regions of all forty-four control individuals were treated with the monoclonal antibody AT8 to visualize the hyperphosphorylated cytoskeletal protein tau (1:2000; Thermo Scientific Pierce Products, Rockford, USA) [1214, 18, 29, 39, 44, 65, 83]. The AT8 antibody recognizes phosphorylated tau epitopes at serine 202 and threonine 205 and reveals a robust and reliable immunoreactivity for hyperphosphorylated tau protein in human brain autopsy material regardless of the length of the fixation time in formaldehyde and/or the condition of the preserved tissue [12, 13, 29, 39]. The specificity of the immunostaining was analyzed by omission of the primary antibody. Incubation of free-floating sections with the AT8 antibody was carried out for 40 h at 4°C. Incubation with the second biotinylated antibody (anti-mouse IgG) was performed for 2 h. Immunoreactions were visualized by means of the AB complex (Vectastain, Vector Laboratories, Burlingame, CA, USA) and 3,3- diaminobenzidine-tetra-HCl/H2O2 (DAB D5637, Sigma, Taufkirchen, Germany).

Subsequent to the neuropathological classification of their AD-related cortical cytoskeletal pathology tissue blocks from the right cerebral hemispheres and the rostral brainstem of all Braak and Braak AD stage 0 and I individuals including the basal forebrain, mediobasal temporal lobe (with the amygdala, hippocampus, transentorhinal and entorhinal regions), claustrum, hypothalamus, thalamus, midbrain and pons were embedded in polyethylene glycol (PEG 1000, Merck, Darmstadt, Germany) [14, 15, 20, 90] and cut into five equidistant series of 100 µm thick tissue sections (cerebral blocks: frontal sections; brainstem blocks: horizontal sections). All tissue stainings were performed on free floating sections. In each individual, every fifth of the 100 µm thick serial sections were stained for lipofuscin pigment with aldehyde-fuchsin and for Nissl material with Darrow red and used for anatoical orientation [20]. Parallel sections were stained with the anti-tau antibody AT8 (1:2000; Thermo Scientific Pierce Products, Rockford, USA) [1214, 18, 29, 39, 44, 65].

For purposes of pathoanatomcial examination of their subcortical tau cytoskeletal pathology at least five tissue sections through the following subcortical brain sites of all Braak and Braak AD stage 0 and I individuals were investigated: medial septal nucleus, nuclei of the vertical and horizontal limbs of the diagonal band of Broca, basal nucleus of Meynert, amygdala, claustrum, hypothalamus, thalamus, peripeduncular nucleus, dopaminergic substantia nigra and ventral tegmental area, periaqueductal gray, dorsal raphe nuclei, locus coeruleus, and parabrachial nuclei. The presence of the AT8-immunoreactive tau pathology in the perikarya, perikarya and processes or only in the processes of nerve cells of these subcortical brain sites and their subnuclei (amygdala: cortical, accessory basal, lateral, and laterobasal nuclei, cortical transitional area; hypothalamus: dorsomedial, ventromedial, tuberomamillary and supramamillary nuclei, perifornical region and lateral area; thalamus: central medial, paraventricular, laterodorsal, subparafascicular, and central lateral nuclei, medial pulvinar and limitans-suprageniculate complex; dorsal raphe nuclei: supratrochlear and caudal compact parts) (Supplementary Figs. 1–5) of the Braak and Braak AD stage 0 and I individuals was semi-quantitatively assessed by UR and categorized into: AT8 immunoreactive perikarya and/or processes of nerve cells not detectable even after a time period of 4 min of careful investigation of a given brain nucleus and application of 100×magnification (−), only scarce numbers of AT8 immunoreactive perikarya and/or processes of nerve cells and only detectable after very careful examination and application of 100 × magnifiication (+), a number of AT8 immunoreactive perikarya and/or processes of nerve cells and quickly detectable light microscopically at 250 × magnification (++) (Tables 1 and and2).2). Inter-rater agreement between UR and KS regarding the detection of AT8 immunoreactive nerve cell perikarya or processes was assessed by means of the weighted kappa coefficient Κw [BiAS for Windows, version 9.14, Epsilon, Darmstadt, Germany].

Table 1
Distribution and extent of the AT8-immunoreactive Alzheimer’s disease AD)-related tau cytoskeletal pathology in nuclei of the basal forebrain, amygdala and hypothalamus with efferent projections to the transentorhinal and entorhinal
Table 2
Distribution and extent of the AT8-immunoreactive Alzheimer’s disease (AD)-related tau cytoskeletal pathology in nuclei of the thalamus and brainstem with efferent projections to the transentorhinal and entorhinal regions in individuals with Braak ...

Routine neuropathological investigation of the brains of the Braak and Braak stage V or VI individuals was performed by W den Dunnen and included assessment of their AD-related cortical tau cytoskeletal pathology on recommended thin tissue sections according to the Braak and Braak AD staging procedure [13, 14, 18] (Supplementary Table 2). For the pathoanatomical demonstration of the advanced subcortical tau cytoskeletal pathology selected tissue blocks from the right cerebral hemisphere and the rostral brainstem of these Braak and Braak AD stage V or VI individuals were embedded in polyethylene glycol (PEG 1000, Merck, Darmstadt, Germany) [14, 15, 20, 90] and cut into five equidistant series of 100 µm thick tissue sections (cerebral blocks: frontal sections; brainstem blocks: horizontal sections). The first, fifth, tenth etc. of the 100 µm thick free floating cerebral and brainstem serial tissue sections were immunostained with the AT8 anti-tau antibody (1:2000; Thermo Scientific Pierce Products, Rockford, USA) [1214, 18, 27, 29, 39, 44, 65, 83].

In all of our Braak and Braak AD stage 0, I, V and VI individuals select thin tissue sections through recommended brain sites were either immunostained with a monocloncal anti-Aβ antibody (1:400; DAKO, Heverlee, Belgium) to visualize β-amyloid deposits [93] or with a monoclonal antibody against alpha-synuclein (1:40; Novocastra, Newcastle upon Tyne, United Kingdom) for the detection of Parkinson’s disease (PD)-related Lewy bodies (LB) and/or Lewy neurites (LN) [19]. Furthermore, thin tissue sections through well-known brain predilection sites (i.e. amygdala and cortex of the medial temporal lobe) were also immunlabeled with a polycloncal antibody against the pathological form of the predominantly nuclear protein TDP-43 (1:6400; Bioconnect, Huissen, The Netherlands) to demonstrate the presence of abnormal aggregations of TDP-43 in nerve or glial cells [97]. Neuropathological classification of the AD-related β-amyloidosis was performed on the immunostained tissue sections according to the acknowledged Thal staging system that distinguishes five phases in the evolution of the AD-related β-amyloidosis [93]. The tissue sections immunostained for alpha-synuclein were used to classify the PD-related neuronal aggregation pathology according to the internationally accepted Braak et al. PD staging procedure [19].

The AD-related tau cytoskeletal pathology [18] and β-amyloidosis [77], as well as the PD- related brain pathology [19] commonly affect the diseased brains bilateral symmetrically. In view ot the bilateral symmetrical brain distribution of the AD and PD-related pathologies we perfomed all of our neuropathological classifications and pathanatomical studies of the subcortical l tau cytoskeletal pathology on brain tissue from one cerebral hemisphere (i.e. right cerebral hemisphere) of our Braak and Braak AD stage 0, I, V and VI individuals Genomic DNA was extracted from formaldehyde-fixed and paraffin-embedded brain tissues to determine APOE genotypes of all Braak and Braak AD stage 0, I, V, and VI individuals according to the method described previously [34]. The frequencies of the APOE alleles (ε2, ε3 and ε4) were obtained by counting the alleles from the observed genotypes (Supplementary Tables 1 and 2).

Brain tissue used for our pathoanatomical investigation of the subcortical tau cytoskeletal pathology was processed by M Bouzrou and APOE genotyping was performed by E Ghebremedhin. D Del Turco and K Seidel produced the figures of the AT8 immunostained tissue sections. H Heinsen, HW Korf, L Grinberg, J Bohl and S Wharton were involved in our initial pilot studies and in the design of our pathoanatomical investigations. In addition, they provided ongoing intellectual neuroanatomical and neuropathological support of our study and were significantly involved in the preparation of our manuscript.

The study was approved by the Ethical board of the Faculty of Medicine at the Goethe University of Frankfurt/Main.

Results

APOE genotypes

The distribution of the APOE genotypes in our Braak and Braak AD stage 0 and I individuals and in our Braak and Braak AD stage V and VI individuals is depicted in Supplementary Tables 1 and 2. Briefly, the distribution of the allele frequencies of APOE ε2, ε3 and ε4 were 0.071, 0.429 and 0.5 in the group of our Braak and Braak AD stage V and VI individuals as compared to 0.059, 0.765 and 0.177 in our group of Braak and Braak AD stage 0 and I individuals. As expected, the frequency of the APOE ε4 allele in our group of Braak and Braak AD stage V and VI individuals (50%) was higher than in our group of Braak and Braak AD stage 0 and I individuals (17.7%) indicating the ε4 allele conferred increased risk for the development of AD [24, 34].

AD-related tau cytoskeletal pathology in subcortical nuclei with efferent projections to the transentorhinal and entorhinal regions in Braak and Braak AD stages V and VI

In all Braak and Braak AD stage V or VI individuals all of the subcortical nuclei with efferent projections to the transentorhinal and entorhinal regions showed a marked to serious AT8 immunoreactive tau pathology (Fig. 1 A–C; Fig. 2 A–F; Fig. 3 A–C; Fig. 4 A, B; Fig. 5 A–C; Supplementary Fig. 6 A–C; Supplementary Fig. 7 A–D; Supplementary Fig. 8 A–C). Due to the even distribution of tau immunoreactive nerve cells throughout the affected subcortical nuclei, their localization in the brain and anatomical outlines were readily identifiable in the AT8 immunolabeled tissue sections.

Figure 1
AT8-immunoreactive Alzheimer’s disease (AD)-related tau cytoskeletal pathology in the medial septal nucleus, claustrum, and in the nuclei of the vertical and horizontal limbs of the diagonal band of Broca
Figure 2
AT8-immunoreactive Alzheimer’s disease (AD)-related tau cytoskeletal pathology in the hypothalamus and amygdala
Figure 3
AT8-immunoreactive Alzheimer’s disease (AD)-related tau cytoskeletal pathology in the thalamic laterodorsal and subparafascicular nuclei, as well as in the midbrain peripeduncular nucleus
Figure 4
AT8-immunoreactive Alzheimer’s disease (AD)-related tau cytoskeletal pathology in the dopaminergic substantia nigra and ventral tegmental area
Figure 5
AT8-immunoreactive Alzheimer’s disease (AD)-related tau cytoskeletal pathology in the supratrochlear and caudal compact parts of the dorsal raphe nucleus and in the locus coeruleus

AD-related tau cytoskeletal pathology in subcortical brain sites with efferent projections to the transentorhinal and entorhinal regions in Braak and Braak AD stage 0

Despite careful investigation, no AT8 immunoreactive AD-related tau pathology was observed in the allocortical transentorhinal and entorhinal regions of our three Braak and Braak AD stage 0 individuals. However, these individuals consistently showed AT8 immunoreactive AD-related tau pathology (Tables 1 and and2)2) in all cholinergic nuclei of the basal forebrain (Supplementary Fig. 6 E, F), the claustrum, in select nuclei of the thalamus (central medial, laterodorsal, subparafascicular, central lateral nuclei, medial pulvinar and limitans-suprageniculate complex) (Fig. 3 D, F; Supplementary Fig. 7 E, G; Supplementary Fig. 8 D) and hypothalamus (ventromedial, tuberomamillary and supramamillary nuclei, perifornical and lateral regions), as well as in the midbrain (peripeduncular nucleus, dopaminergic substantia nigra and ventral tegmental area, periaqueductal gray, supratrochlear part of the dorsal raphe nucleus) (Fig. 4 C, E; Fig. 5 G) and pons (compact caudal part of the dorsal raphe nucleus, locus coeruleus, parabrachial nuclei) (Fig. 5 D, I). Notably, the subnuclei of the amygdala with efferent projections to the transentorhinal and entorhinal regions were either spared or only inconsistently affected at Braak and Braak stage 0 (Table 1). In all Braak and Braak AD stage 0 individuals the AT8 immunoreactive cytoskeletal pathology in the cholinergic basal forebrain nuclei and claustrum was present in the perikarya and processes of affected nerve cells. In the affected nuclei of the amygdala and hypothalamus, as well as in the majority of their involved thalamic and brainstem nuclei the tau cytoskeletal pathology was confined to nerve cell processes (Tables 1 and and22),

AD-related tau cytoskeletal pathology in subcortical brain sites with efferent projections to the transentorhinal and entorhinal regions in Braak and Braak AD stage I

In all of the Braak and Braak AD stage I cases studied all of the subcortical nuclei studied exhibited AT8 immunoreactive nerve cells (Fig. 1 E–H; Fig. 2 G–L; Fig. 3 E, G, H; Fig. 4 D, F; 5 E, F, H, J; Supplementary Fig. 6 D, G, H; Supplementary Fig. 7 F, H; Supplementary Fig. 8 E–H; Tables 1 and 2) along with a few AT8 immunoreactive projection nerve cells in the pre-alpha layer of the allocortical transentorhinal and entorhinal regions. The AT8 immunoreactivity was either found in the perikarya of affected nerve cells (Fig. 3 E), or in their perikarya and proximal processes and dendritic trees (Fig. 1 E, G, H; Fig. 2 G, I–L; Fig. 3 F, G; Fig. 5 F, H–J; Supplementary Fig. 6 D, G, H; Supplementary Fig. 7 F, H; Supplementary Fig. 8 E, G, H), but also occurred as isolated short, thick, swollen or club-shaped nerve cell processes (Tables 1 and and2).2). Most of the AT8 immunoreactive nerve cell processes were thin, slender, filamentous, elongated, serpentine or sausage-like (Fig. 1 E–H; Fig. 2 G–L; Fig. 3 D, F–H; Fig. 4 C–F; Fig. 5 D–J; Supplementary Fig. 6 D–H; Supplementary Fig. 7 E–H; Supplementary Fig. 8 D–H) Frequently, the nerve cell processes containing hyperphosphorylated tau could be traced up to the brain surface and adjacent ventricular surface or subarachnoidal space (Fig. 1 G, H; Fig. 5 D, E; 6; Fig. Supplementary Fig. 6 G). In the claustrum, dopaminergic substantia nigra and ventral tegmental area, the medial pulvinar and limitans-suprageniculate complex of the thalamus, AT8 immunoreactive nerve cells were selectively present at those topographical subunits from which projections to the transentorhinal and entorhinal regions were shown to originate in non-human primates [45].

In the majority of the Braak and Braak AD stage I individuals the affected nerve cells in the cholinergic basal forebrain nuclei, claustrum and amygdala showed tau immunoreactivity in both their perikarya and processes (Table 1). In one half of the Braak and Braak AD stage I individuals the tau immunoreactive cytoskeletal changes were also simultaneously present in the perikarya and processes of affected hypothalamic nerve cells, while in the thalamus and brainstem the affected nerve cells of most individuals displayed only tau immunoreactivity in their processes (Tables 1 and and22).

In all of the Braak and Braak stage 0 and I individuals studied the nuclei immediately adjacent to the subcortical brain sites with efferent projections to the transentorhinal and entorhinal regions (e. g. striatum, pallidum, thalamic ventral anterior, ventrolateral and mediodorsal nuclei, thalamic medial and lateral geniculate bodies) (Supplementary Figs. 1–5) were free of AT8 immunreactive nerve cells.

Related tissue changes

In all fo our Braak and Braak AD stage 0, I, V and VI individuals no PD-related immunoreactive LB and/or LN or abnormal TDP-43 nerve or glial cell aggregations were detected. In addition, except of one Braak and Braak AD stage I individual who showed some neocortical β-amyloid deposits corresponding to phase 1 of the AD-related brain β-amyloidosis [93] (case 10; Supplementary Table 1), no brain β-amyloid deposits were observed in our Braak and Braak AD stage 0 and I individuals.

The brain β-amyloidosis of our Braak and Braak AD stage V individuals corresponded to phase 3 (case 5; Supplementary Table 2), phase 4 (cases 2 and 7; Supplementary Table 2) or 5 (cases 3 and 6; Supplementary Table 2) of the AD-related brain β-amyloidosis and in our Braak and Braak AD stage VI individuals to phase 3 (case 1; Supplementary Table 1) or phase 4 (case 4; Supplementary Table 2) [93].

Statistical analysis

Calculation of the weighted kappa coefficient revealed a high inter-rater reliability and a substantial agreement between UR and KS regarding the detection of AT8 immunoreactive nerve cell perikarya or processes (Κw = 0.76; p < 0.0001).

Discussion

Early AD-related cytoskeletal pathology in the subcortical nuclei with efferent projections to the allocortical transentorhinal and entorhinal regions

In the present study, we demonstrate the very early and substantial AT8 immunoreactive tau cytoskeletal pathology in the subcortical nuclei with efferent projections to the allocortical transentorhinal and entorhinal regions in individuals with Braak and Braak stage 0 or I AD-related cortical cytoskeletal pathology (Supplementary Figs. 1–5). All of these affected nuclei are important relay stations in the circuits of the limbic system of the human brain, are highly interconnected anatomically via fiber tracts [1, 7, 13, 41, 42, 54, 60, 63, 64, 66, 69, 75, 8085, 94, 98]. are classically recognized as being highly vulnerable to and bear the brunt of the immunoreactive brain tau cytoskeletal pathology in demented AD patients individuals burdened by AD-related Braak and Braak AD stage V or VI cortical tau cytoskeletal pathology [2, 3, 15, 18, 29, 33, 40, 56, 57, 68, 72, 73, 8083, 8689] (Supplementary Figs. 1–5). The present study not only confirms the exceptional high vulnerability and severe affection of these subcortical nuclei during the final stages of the evolution of the AD-related cytoskeletal pathology (i.e. Braak and Braak AD stages V and VI). It also demonstrates for the first time (1) that all of these subcortical nuclei are very early affected by the immunoreactive tau pathology during the initial stages of the evolution of the AD-related cytoskeletal pathology and (2) that the extent of the subcortical tau cytoskeletal pathology present in Braak and Braak AD stages 0 und I has been considerably underestimated in the past [13, 14, 18, 21, 29, 39, 8083, 88, 89].

Although the intraneuronal tau pathology and the extracellular deposits of the insoluble β-amyloid protein apparently belong to the same underlying disease process and represent two sides of the same coin the present study (1) was intended to provide new insights into the early evolution of the AD-related tau cytoskeletal pathology and (2) was not designed to elucidate the enigmatic association between the appearance of the AD-related neuronal and extraneuronal deposits. This association was specified many years ago in the ‘amyloid cascade theory’, which postulated that an increase in β-amyloid secretion and subsequent extraneuronal aggregation of this protein in the diseased brains induces and triggers a cascade of deleterious changes which ultimately lead to the development of the AD-related tau cytoskeletal pathology and neuronal death. In all of our Braak and Braak AD stage 0 individuals and thirteen of our Braak and Braak AD stage I individuals subcortical cytoskeletal changes occurred in the absence of any brain β-amyloid deposits. Together with the findings of a number of previous morphological investigations these findings give rise to additional doubts regarding the valididty of the ‘amyloid cascade theory’ and support the opinion that the accumulations of the β-amyloid protein do not represent the initial AD-related pathological alterations which secondarily provoke the appearance of the AD-related tau cytoskeletal pathology [18, 29, 39, 79].

The presence of the APOE ε4 allele is a well-established risk factor for the development of AD [24, 34]. Consistent with prevailing assumptions, our findings show an increased prevalence of the APOE ε4 allele in our Braak and Braak AD stage V and VI individuals implicating ε4 allele as a strong risk marker for the development of AD.

Recently it has been suggested to create a separate neurodegenerative disease entity called PART (i. e. primary age-associated tauopathy), which has been considered to occur independent from AD and includes individuals with a tau cytoskeletal pathology more or less confined to the allocortical entorhinal region and hippocampus either without or mininmal β-amyloid deposits in the brain [28, 53]. Although the subcortical tau pathology that may occur in PART currently is incompletely known and described [53], considering the new concept of PART one may be inclined to regard the early tau pathology described in the present study as a manifestation of PART. For the following reasons, however, we regard the early subcortical immunoreactive Braak and Braak stage 0 and I tau pathologies observed in our study as an intergral component of the AD continuum rather as a part of a separate disease entity or tauopathy: (1) These subcortical pathologies are confined to the subcortical nuclei with efferent projections to the transentorhinal and entorhinal region all of which are well known to undergo severe tau cytoskeletal pathology in demented AD patients burdened by Braak and Braak AD stage V or VI tau cytoskeletal pathologies [2, 3, 15, 18, 29, 33, 40, 56, 57, 69, 72, 73, 8183, 86, 88]. Since the topographical distribution of the early subcortical Braak and Braak AD stage 0 and I tau cytoskeletal pathology documented in our study represents a mirror of the subcortical distribution pattern of the full-blown subcortical AD-related cytoskeletal pathology in Braak and Braak AD stages V and VI, it is hard to image that the selective and early affection of the subcortical nuclei with efferent projections to the transentorhinal and entorhinal region is purely by chance. Instead, owing to the very close correlation between the anatomical distribution patterns of the subcortical cytoskeletal pathology in Braak and Braak and AD stages 0 and I and that in Braak and Braak AD stages V and VI it appears more plausible that the very early tau cytoskeletal pathology of the subcortical nuclei with efferent projections to the transentorhinal and entorhinal regions in contrast to the allocortical tau cytoskeletal pathology [53] can serve as a predictor of the progress of the underlying disease process to fully developed AD. In addition this forerunner role of the early subcortical cytoskeletal pathology is supported by the fact that the mean ages at death of our Braak and Braak AD stage I individuals and that of the Braak and Braak AD stage V and VI individuals differed by thirty years and that this time period of thirty years is in a good agreement with the estimated time interval, which the continuous disease process needs for its progression from Braak and Braak AD stage I to stage V [83].

For a long time the allocortical transentorhinal or entorhinal regions were considered as the first and sole brain regions that display AD-related cytoskeletal pathology already in Braak and Braak AD stage I and it has been suggested that the AD-related cortical cytoskeletal pathology spreads to other brain regions that become affected in later Braak and Braak AD stages [11, 1318, 30, 59]. According to recent epidemiological estimations we have to assume that the in vivo transition from the Braak and Braak AD stage I to stage II takes several years [29, 31, 83]. Therefore, the concomitant presence of the tau cytoskeletal pathology in subcortical nuclei sending efferent projections to the transentorhinal and entorhinal regions and in their allocortical projection targets in the brains of individuals with neuropathological Braak and Braak AD stage I does not allow to distinguish whether the vulnerability of the brain areas follow a simultaneous or a sequential temporal pattern in vivo with one of these brain sites presenting the initial and the other the secondarily induced AD-related tau pathology [39, 83].

Subcortical origin and ascending propagation of the AD-related tau cytoskeletal pathology

In addition to the consistent affection of all subcortical nuclei with efferent projections to the allocortical transentorhinal and entorhinal regions in individuals with Braak and Braak stage I AD-related cortical tau cytoskeletal pathology the present pathoanatomical study also demonstrates a considerable immunoreactive tau pathology in a subset of these limbic nuclei in three individuals devoid of AD-related cortical cytoskeletal pathology (i.e. Braak and Braak AD stage 0) (Figs. 3 D, F; 4 C, E; 5 D, G, I; Supplementary Fig. 6 E, F; Supplementary Fig. 7 E, G; Supplementary Fig. 8 D). The consistent involvement of a subset of these subcortical limbic nuclei (i.e. medial septal nucleus, nuclei of the vertical and horizontal limbs of the diagonal band of Broca, basal nucleus of Meynert; claustrum; hypothalamic ventromedial, tuberomamillary and supramamillary nuclei, perifornical region and lateral area; thalamic central medial, laterodorsal, subparafascicular, and central lateral nuclei, medial pulvinar and limitans-suprageniculate complex; peripeduncular nucleus, dopaminergic substantia nigra and ventral tegmental area, periaqueductal gray, midbrain and pontine dorsal raphe nuclei, locus coeruleus, and parabrachial nuclei) by the AD-related tau cytoskeletal pathology in the absence of any tau immunoreactive cytoskeletal pathology in the allo- or neocortex (i.e. pre-cortical Braak and Braak stage 0 in the evolution of the AD-related tau pathology) supports the concept that the subcortical nuclei under consideration play an important pathophysiological role in the initial stage of the disease process of AD and may represent the port of entry from which the AD-related cytoskeletal pathology ascends towards the cerebral allocortex. This concept is based on the following hypotheses about the development of the AD-related cytoskeletal pathology during the disease progression: (1) the AD-related tau cytoskeletal pathology originates in subcortical brain sites, in which the first nerve cells in the brain sustain accumulations of the hyperphosphorylated cytoskeletal protein tau [39, 83, 89], (2) the AD-related cytoskeletal pathology develops first and only in nerve cells of anatomically interconnected cortical and subcortical regions of select functional systems and networks of the human brain [19, 25, 29, 33, 55, 59, 7173, 78, 87, 89, 96], (3) the anatomical interconnectivities of a given cortical or subcortical brain region establish its vulnerability for or resistance to the AD-related cytoskeletal pathology [83, 89], and (4) the pathological process underlying the AD-related cytoskeletal pathology uses these anatomical pathways and interconnectivities for its transneuronal spread throughout the brain [18, 25, 29, 33, 39, 55, 59, 71, 73, 78, 83, 96].

Transneuronal spread of the AD-related tau cytoskeletal pathology

The evolution and progressive expansion of the AD-related tau cytoskeletal pathology in the human brain is not a random or arbitrary process, but (1) target only select subcortical nuclei, layers and areas of the cerebral cortex, (2) follow highly predictable chronological and spatial sequences and (3) ultimately lead to a stereotypical topographic brain progression and characteristic nuclei-specific, area-specific, layer-specific and cell-type specific distribution pattern in the diseased brain [4, 11, 1416, 18, 33, 44, 55, 65, 86, 89]. Explaining the highly stereotypical targeting and propagation of the AD-related tau cytoskeletal pathology is still one of the most important and enduring challenges in clinic neuroscience worldwide and remains the first–rate goal of contemporary and future AD research [87]. Previous explanations for the high vulnerability of select cortical and subcortical brain sites were either based on their anatomical affiliation to the limbic system, their integration into specific brain neurotransmitter systems, the specific molecular biochemical phenotypes of the affected nerve cells, or on the low degree or absence of myelinization of the particular long axons of their affected nerve cells [12, 16, 18, 27, 78, 96].

More and more evidence points to the anatomical interconnectivities of a given brain region (1) as a pivotal factor accounting for its susceptibility for or resistance to the AD-related tau cytoskeletal pathology, and (2) as the structural base for its strikingly well-ordered and stereotypical progressive brain expansion and characteristic distribution pattern [18, 25, 29, 33, 39, 67, 78, 83, 87, 89, 96]. Although the exact basic molecular mechanisms of the disease still are unclear, many renowned researchers in the field favor the pathomechanistic hypothesis that the degenerative process of the AD-related cytoskeletal pathology (1) follows anatomical pathways for its brain propagation, (2) spreads transneuronally via these pathways in predictable, inter-individually consistent chronological and topographical sequences, and (3) leads to a stereotypical and specific brain distribution pattern of the immunoreactive tau pathology which represents an exact mirror of the complex anatomical interconnectivities within the brain networks and circuits involved [27, 29, 33, 55, 58, 65, 67, 71, 89, 96].

Although these pathomechanistic hypothesis are currently widely held further empirical evidences can only strengthen the underlying theoretical concept. The subcortical nuclei consistently affected in our Braak and Braak AD stage 0 and I individuals by the immunoreactive tau pathology share common allocortical projection targets (i.e. transentorhinal and entorhinal regions) [45, 61], are intimately interconnected with each other via reciprocal brain fiber tracts [1, 7, 13, 42, 54, 63, 64, 66, 69, 75, 80, 84, 85, 94, 98], and receive substantial afferent input from or project to the midbrain raphe nuclei [41, 60, 63, 64, 69, 75, 80, 84]. Accordingly, the findings of the present post-mortem AD study are in agreement with these pathoanatomical hypothesis and meta-analytical considerations and point to the pivotal role of intact anatomical interconnectivities for the ascend of the AD-related cytoskeletal pathology towards the cerebral cortex and its highly predictable and well-ordered chronological and topographic spread [27, 29, 33, 55, 58, 65, 71, 89, 96].

To the best of our knowledge Saper and colleagues were among the first neuroscientists who recognized and pointed to the strikingly well-ordered, predictable chronological and topographical sequential orders of the spread of the AD-related tau cytoskeletal pathology and tried to explain this stereotypical propagation with a transneuronally spread of the underlying disease process [87]. This firstly very abstractly formulated and ill-defined idea of a transneuronal propagation of the AD-related cytoskeletal pathology, however, has been recently specified and supplemented by the proposal that (1) AD may be among the diseases sharing prion-like pathological mechanisms and (2) that the directed brain spread of the AD-related tau cytoskeletal pathology via neuron-to-neuron transmission and transsynaptic transport from affected neurons to anatomically interconnected healthy nerve cells may follow a prion-like corruptive protein templating and seeding-like pathomechanism which induces the templated misfolding, self-aggregation and propagation of the altered tau protein within the frame work of an injurious auto-catalytic c ascade of pathological events [36, 50, 55, 70, 71, 79, 96].

Human neurodegenerative protein misfolding diseases assigned to the so-called ‘prion-like’ diseases are characterized neuropathologically by their begin in circumscribed brain areas, the directed, transneuronal spread and stereotypical brain propagating of the underlying disease process in predictable sequences along anatomical pathways, as well as by molecular pathological mechanisms in the initiation, proliferation, intercellular transfer and spread of insoluble ‘non-prion’ aggregates of the aberrantly conformed and misfolded disease protein that are reminiscent to that of proper prion diseases [5, 36, 55, 71, 76, 79, 95]. Although the presumed begin of AD in circumscribed subcortical brain regions and the possible targeted transneuronal spread of its underlying disease process along anatomical pathways may be in support of the currently prevailing idea of AD as a chronic prion-like neurodegenerative disease, further studies are required to substantiate this new scientific approach and to prove unequivocally the suggested prion-like nature and behavior of the hyperphosphorylated tau protein [36, 50, 55, 70, 71, 79, 96].

Implications of the early subcortical AD-related tau cytoskeletal pathology for the modified Braak and Braak AD staging system

The subcortical AD-related tau cytoskeletal pathology for a long time has been regarded as a secondary phenomenon that takes place subsequent to and is triggered by the involvement of the cerebral cortex and has such attracted only minor attention in previous neuropathological AD research [12, 18, 30, 33, 72]. The worldwide practiced Braak and Braak staging procedure of the AD-related tau cytoskeletal pathology relates to the distribution, extent and progression of this pathology in the cerebral cortex and represents an acknowledged, simple and reliable procedure for the rapid post-mortem assessment of the distribution and degree of the AD-related cortical tau pathology upon diagnostic neuropathological examination [9, 10, 13, 14, 16, 18, 2931, 43, 44, 52, 58, 59, 65]. Moreover, the Braak and Braak AD staging system correlates well with the progressive cognitive impairments of AD patients [9, 10, 18, 30, 52, 59, 79]. Although the original staging system has been recently revised our preliminary findings indicated that the very early subcortical tau cytoskeletal pathology in addition to the pontine locus coeruleus may also affect a large variety of other subcortical nuclei with projections to the early affected allocortical entorhinal and transentorhinal regions [13, 14, 18, 21, 29]. The results of the present pathoanatomical study confirmed our initial impressions and showed that the extent of the very early AD-related subcortical tau cytoskeletal pathology of individuals with Braak and Braak AD stages 0 or I has been considerably underestimated in the past and is clearly more widespread than believed so far. In view of their potential pathogenetic relevance [39, 83, 89] and the widespread distribution of the very early AD-related subcortical tau cytoskeletal pathology observed in our Braak and Braak AD stage 0 individuals we suggest to re-evaluate the modified Braak and Braak AD stages and to perform further cross-sectional studies of a large cohort of individuals with Braak and Braak AD stage 0 cytoskeletal pathology with the goal to establish the real extent of the very early pre-cortical phase subcortical tau cytoskeletal pathology [18, 21]. These additional cross-sectional studies may also help (1) to further parcellate the precortical Braak and Braak AD stage 0 into sequential phases in the early evolution of the AD-related tau cytoskeletal pathology, (2) to stepwise narrow down the true portal of entry of the disease process underlying AD and (3) ultimately facilitate the identification of the initial cascades of pathological events leading to the first AD-related tau cytoskeletal changes and their exact brain location [39, 50].

Considering the presumably long pre-clinical phase of AD of approximately thirty to fourty years, unequivocal identification of the exact brain location of the first AD-related tau cytoskeletal pathology by improved in vivo imaging techniques could (1) lead to the establishment of a reliable biomarker for AD onset and (2) open a window of therapeutical opportunities that extends practically the whole lifetime of affected individuals [18, 22, 23, 46, 67]. These subcortical brain sites with the first AD-related tau cytoskeletal pathology most likely represent the induction sites of the pathological process of AD, which, once started, spreads transneuronally via anatomical pathways in highly predictable, inter-individually consistent chronological and topographical sequences throughout the brain. Therefore, these subcortical induction sites should be envisaged as the first therapeutic intervention targets for imaging-guided immunotherapies capabale to interrupt the transeuronal brain propagation of the AD-related disease process [22, 23, 36, 39, 55]. Since the extent and distribution of the AD-related cortical tau aggregation pathology correlates significantly with the evolution of the disease intrinsic dementia [911, 23, 29, 48, 49, 52, 55, 58, 59, 65] such early immunotherapeutical interventions may be suitable to effectively prevent dementia and other AD-related disease symptoms several decades before their onset.

Supplementary Material

01

Acknowledgements

This study was supported by grants from the Dr. Senckenbergische Stiftung Frankfurt/Main, Germany). The skillful assistance of D. von Meltzer (secretary), C. Mauer tissue cutting), I. Szasz (graphics) and H. Korff (APOE genotyping) is thankfully acknowledged. LTG is supported by NIH grants R01AG040311 and P50AG023501. The authors also like to thank Jean-Paul Vonsattel (The New York Brain Bank/Taub Institute, The Presbyterian Hospital and Columbia University, New York, USA) for his critical revision of our manuscript and his valuable criticism and comments.

Footnotes

Disclosure statement

All authors have no actual or potential conflicts of interest to disclose, including financial, personal, or other relationships with other people or organizations, within three years of beginning the work submitted.

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