Received: 8 March 2021 | Accepted: 29 July 2021 DOI: 10.1111/bpa.13016 R E SEA RCH A RTICLE Association of CD2AP neuronal deposits with Braak neurofibrillary stage in Alzheimer’s disease Jessica Camacho1,2 | Alberto Rábano3 | Paula Marazuela4 | 4 2,5 1 Anna Bonaterra-­Pastra | Garazi Serna | Teresa Moliné | 1,2 1,2 Santiago Ramón y Cajal | Elena Martínez-­Sáez | Mar Hernández-­Guillamon4 1 Pathology Department, Vall d’Hebron University Hospital, Barcelona, Spain Abstract 2 Morphological Science Department, Genome-­w ide association studies have described several genes as genetic sus- Universitat Autonoma de Barcelona, Barcelona, Spain ceptibility loci for Alzheimer's disease (AD). Among them, CD2AP encodes 3 Neuropathology Department, CIEN CD2-­associated protein, a scaffold protein implicated in dynamic actin remod- Foundation, Alzheimer’s Centre Queen Sofía Foundation, Madrid, Spain eling and membrane trafficking during endocytosis and cytokinesis. Although 4 Neurovascular Research Laboratory, Vall a clear link between CD2AP defects and glomerular pathology has been de- d’Hebron Research Institute, Universitat Autonoma de Barcelona, Barcelona, Spain scribed, little is known about the function of CD2AP in the brain. The aim 5 Molecular Oncology Group, Vall of this study was to analyze the distribution of CD2AP in the AD brain and d’Hebron Institute of Oncology (VHIO), Barcelona, Spain its potential associations with tau aggregation and β-­amyloid (Aβ) deposition. Correspondence First, we performed immunohistochemical analysis of CD2AP expression in Mar Hernández-­Guillamon, brain tissue from AD patients and controls (N = 60). Our results showed granu- Neurovascular Research Laboratory, Institut de Recerca, Pg. Vall d´Hebron, 119-­ lar CD2AP immunoreactivity in the human brain endothelium in all samples. 129, Hospital Universitari Vall d’Hebron, Barcelona 08035, Spain. In AD cases, no CD2AP was found to be associated with Aβ deposits in ves- Email: mar.hernandez.guillamon@vhir.org sels or parenchymal plaques. CD2AP neuronal inclusions similar to neurofi- Elena Martínez-­Sáez, Pathology brillary tangles (NFT) and neuropil thread-­like deposits were found only in Department, Vall d’Hebron University Hospital, Barcelona, 119-­129, Pg. Vall AD samples. Moreover, immunofluorescence analysis revealed that CD2AP d’Hebron, Barcelona 08035, Spain. Email: eamartinez@vhebron.net colocalized with pTau. Regarding CD2AP neuronal distribution, a hierarchi- cal progression from the entorhinal to the temporal and occipital cortex was Funding information This work was funded by Instituto de detected. We found that CD2AP immunodetection in neurons was strongly and Salud Carlos III (ISCIII) (PI17/00275, PI20/00465), cofinanced by the European positively associated with Braak neurofibrillary stage, independent of age and Regional Development Fund (FEDER). other pathological hallmarks. To further investigate the association between The Neurovascular Research Laboratory is part of the INVICTUS+ network, pTau and CD2AP, we included samples from cases of primary tauopathies ISCIII, Spain (RD16/0019/0021). M.H.-­G. is supported by the Miguel Servet (corticobasal degeneration [CBD], progressive supranuclear palsy [PSP], and Programme, ISCIII, Spain (CPII17/00010) Pick's disease [PiD]) in our study. Among these cases, CD2AP positivity was only found in PiD samples as neurofibrillary tangle-­like and Pick body-­like deposits, whereas no neuronal CD2AP deposits were detected in PSP or CBD samples, which suggested an association of CD2AP neuronal expression with 3R-­Tau-­diseases. In conclusion, our findings open a new road to investigate This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­c ommercial and no modifications or adaptations are made. © 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology. Brain Pathology. 2022;32:e13016.  wileyonlinelibrary.com/journal/bpa | 1 of 16 https://doi.org/10.1111/bpa.13016 2 of 16 |    CAMACHO et al. the complex cellular mechanism underlying the tangle conformation and tau pathology in the brain. K EY WOR DS Alzheimer's disease, CD2AP, neurofibrillary tangles, Pick's disease, tau, tauopathies 1 | I N T RODUC T ION in AD pathogenesis, inducing or intensifying the generation of NFT, neuronal loss, vascular damage, More than 50 million people live with dementia worldwide, and neuroinf lammation [11]. The Aβ peptide is the and it is estimated that this figure will reach 152 million product of the processing of the amyloid precursor by 2050 [1]. Alzheimer's disease (AD) is the most common protein (APP) by β-­ and γ-­s ecretases, which gives cause of dementia and is characterized by impairment of rise mainly to the Aβ40 and Aβ42 peptides. Some learning and memory. Neuropathologically, the intracel- of the evidence supporting the “amyloid cascade lular accumulation of hyperphosphorylated tau protein hypothesis” is the early occurrence of pathology (pTau) as neurofibrillary tangles (NFT) and neuropil in individuals carrying autosomal-­d ominant muta- threads, together with the extracellular accumulation of tions in genes encoding APP or the γ-­s ecretase com- amyloid-­β (Aβ) in the core of the neuritic plaque, are con- plex proteins presenilin 1/2 (PSEN1/2) and a high sidered the molecular and morphologic signatures of AD AD prevalence in individuals with Down syndrome, [2,3]. Currently, the NIA-­AA criteria, which include the an event attributed to the triplication and overex- Thal phase, Braak neurofibrillary stage, and CERAD pression of the APP gene located on chromosome plaque score are used for the neuropathological diagno- 21 [11]. However, Aβ aggregation and accumulation sis of AD [3]. Although the Thal phase [4] and CERAD correlate poorly with cognitive symptoms and neu- plaque score [5] apply to the presence and distribution of rodegeneration [12-­15]. The assessment of amyloid Aβ deposits, the Braak neurofibrillary stage [] refers to the and tau by PET imaging suggests that the rate of hierarchical progression of neurofibrillary tau pathology amyloid deposition predicts the onset of tau accu- (NFP). mulation, whereas the degree of tau deposition is AD belongs to the group of tauopathies which are related to cognitive impairment [16,17]. Indeed, dif- diseases characterized by abnormal aggregation of ferent studies have shown that cognitive decline in the microtubule-­a ssociated protein tau (MAPT) in AD starts when tau spreads from the entorhinal cor- neurons and/or glial cells [7]. In neurons, tau plays tex into the neocortex [18,19], and the stage of tau an essential role in stabilizing microtubules, but it pathology strongly correlates with the progression is also implicated in other cellular functions such as of cognitive impairment [14,15,20]. Taken together, signal transduction, interaction with the actin cyto- these findings suggest that neurodegeneration and skeleton, neuronal activity, and synaptic plasticity cognitive impairment in AD might be driven by the [8]. Tauopathies are classified according to the pre- onset and spread of tau pathology. dominant tau isoform pattern. There are six known Through genome-­w ide association studies (GWAS), isoforms of tau protein expressed in the adult human different susceptibility loci have been identified for AD, brain. There are equal amounts of tau isoforms with including variants in the CD2AP gene [21-­26], which en- three or four repetitive (3R or 4R) domains in the codes the CD2-­associated protein (CD2AP). CD2AP is normal brain, while the ratio is altered in neurode- a scaffold protein that controls actin dynamics and is generation [7,9]. For example, AD belongs to Class expressed in neurons [27] and endothelial cells of blood–­ I (tau lesions contain equal amounts of 3R and 4R brain vessels [28-­30]. A link between defects in CD2AP isoforms), progressive supranuclear palsy (PSP), and and glomerular pathology has been described [31]; corticobasal degeneration (CBD) belongs to Class II however, little is known about the function of CD2AP (tau lesions mainly composed of 4R isoforms), and, in the brain. In particular, beyond the association of finally, Pick's disease (PiD) belongs to Class III (tau several CD2AP polymorphisms with AD risk in both lesions mainly composed of 3R isoforms) [7]. While early onset (rs9473117 [32] and rs9381563 [33]) and late AD only shows pTau inclusions in neurons [9,10], onset AD (rs9349407 [23,34,35], rs10948363 [25,36,37] other tauopathies, such as PiD, PSP and CBD, dis- rs9296559 [23,38] and rs9349407 [39]) the precise role of play both neuronal and extensive glial pTau inclu- the CD2AP protein in this disease is still undiscovered. sions [9]. From a clinical point of view, these diseases In this regard, the main goal of the present study was to can present as frontotemporal dementias or atypical analyze CD2AP distribution in the AD brain and the parkinsonisms [7]. potential association of CD2AP with tau aggregation, Although still under debate, different evidences Aβ deposition, and localization in AD, as well as in support the idea that Aβ deposition is a major event other tauopathies. CD2AP NEURONAL DEPOSITS IN AD    | 3 of 16 2 | M AT E R I A L A N D M ET HOD S p-­value <0.001 0.580 2.1 | Selection of patients Brain tissue samples were obtained from 81 autopsies car- 75.1 ± 9.7* 3 (42.9%) Demographic characteristics of Cohort A (Vall d’Hebron University Hospital), Cohort B (CIEN Foundation) and the compilation of the two cohorts (total cohort) ried out at the Pathology Department of Vall d’Hebron University Hospital (HUVH, Barcelona) (Cohort A) and PiD the Center for Research in Neurological Diseases (CIEN B 7 Foundation, Madrid) (Cohort B). This series of patients included 45 with AD neuropathological change (≥B1) and 21 with a tauopathy other than AD (seven cases of PiD, 72.4 ± 8.6 2 (40.0%) nine cases of PSP, and five cases of CBD); 15 cases with- CBD out pTau deposition were included as control cases. All B 5 patient characteristics are presented in Table 1. Patients included in this study, or their relatives, expressed their willingness to donate brain tissue for research purposes 74.2 ± 9.1 * 4 (44.4%) and were donors to the Neurological Tissue Bank of the HUVH or to the CIEN Foundation Brain Bank. The PSP project was approved by the Clinical Research Ethics B 9 Note: Statistical differences were analyzed among total cohorts between disease groups. Bold numbers indicate statistically significant differences. Committee of the Vall d’Hebron University Hospital, Barcelona, Spain (PR (IR) 173/2019). For specific comparative purposes, patients with 61.4 ± 10.0 7 (46.7%) other neurodegenerative diseases were analyzed, all the Total brain donors to one of the two biobanks. Two cases of 15 Parkinson disease (PD), two cases of Multisystem at- rophy (MSA), one case of Amyotrophic lateral scle- 62.9 ± 8.6 rosis (ALS) without cognitive impairment, one case 5 (55.6%) of Frontotemporal lobar degeneration-­ ALS with C9orf72 mutation and p62 positive inclusions in the B 9 cerebellar granular layer (FTLD-­ALS), and two cases of primary age-­related tauopathy (PART) with a Braak 59.2 ± 12.4 2 (33.3%) Control III neurofibrillary stage (B2). Clinic-­pathologic data of these patients are summarized in Table S1. A 6 2.2 | pTau and Aβ assessment in AD 78.9 ± 10.8*** 28 (62.2%) cases and controls Total All samples were obtained 2–­20 hours after death and 45 fixed in 10% buffered formalin from 4 to 10 weeks. Macroscopic examination of the brain was then per- ***p < 0.001 vs the control group.; *p < 0.05 vs the control group. formed, and 27 cortical and subcortical areas, including 79.6 ± 12.2 18 (64.3%) the brainstem and cerebellum, were selected for rou- tine histologic diagnosis. The paraffin blocks were cut into 3 µm sections and stained with hematoxylin and 28 B Alzheimer's disease eosin. From each case, sections from the frontal, tempo- ral, parietal, and occipital lobes and the hippocampus, amygdala, brain stem, and cerebellum were analyzed by 10 (58.8%) 77.8 ± 8.3 immunohistochemistry using a mouse anti-­A myloid-­β antibody (Clone 6F/3D 1: 500, Dako, Glostrup, Denmark) 17 A and a mouse anti-­Tau antibody (Clone AT8 1:20, Thermo Scientific, Rockford, USA). For the AD patients, AD neuropathologic changes Sex (females) were evaluated and staged according to the NIA-­A A TA BL E 1 criteria [3]. Accordingly, cases with Braak I or II neu- Cohort rofibrillary stages were reclassified as B1, Braak III or Age N IV as B2, and Braak V or VI as B3. Aβ deposits initially 4 of 16 |    CAMACHO et al. scored from 0 to 5 (Thal phases [4] were reclassified as A0 a cocktail of HRP-­labeled antibodies (mouse and rab- [Thal 0], A1 [Thal 1 or 2], A2 [Thal 3], or A3 [Thal 4 or bit antibodies to IgG and IgM). The resulting complex 5]). CERAD plaque score A to C were transformed into was then visualized using DAB Chromogen and finally C1 (CERAD A), C2 (CERAD B), or C3 (CERAD C) [5]. counterstained with Hematoxylin and Bluing Reagent. The combination of these three grading systems gives an The positivity of the staining was confirmed by the ab- “ABC score” that indicates a low, intermediate, or high sence of signal when the primary antibody was omitted probability that dementia was caused by AD changes or (negative controls). CD2AP expression was evaluated in that there were no AD-­related changes [3]. vascular walls (meningeal and cortical arteries and cap- illaries), neurons, astrocytes, oligodendrocytes, endothe- lium, and parenchymal deposits in all samples. In these 2.3 | pTau assessment in other tauopathies cases, CD2AP evaluation was performed blinded to the specific disease. Twenty-­one cases of sporadic tauopathies other than AD To study the possible association of neuronal CD2AP were included in the study: seven cases of PiD, nine cases deposition with pTau protein, the “B” score of the of PSP, and five cases of CBD. PiD, PSP, and CBD cases NIA-­ A A guidelines [3,6] was used to grade CD2AP were diagnosed when characteristic pathologic changes deposits in AD and control cases. Thus, the score were found. PiD is a 3R tauopathy histologically defined CD2AP-­0 was given to cases without any CD2AP de- by the presence of round neuronal argyrophilic inclu- posits, CD2AP-­1 was given to cases with Braak Stage sions known as Pick bodies. Thorn-­shaped and ramified I-­analog (NFP of at least low density in the transento- astrocytes and small globular oligodendroglial inclu- rhinal region) or Braak Stage II-­analog (NFP of at least sions may be found. PSP and CBD are 4R tauopathies moderate density in the outer layers of the entorhinal with different topographies and pathologic changes, al- region and at least low density in the inner layers of the though some overlap in regional distribution may exist. entorhinal region) CD2AP distribution, CD2AP-­2 was PSP pathology is predominant in the hindbrain, and its given to cases with Braak Stage III-­analog (NFP of at characteristic findings include neuronal (globose tan- least moderate density in the superficial and deep layers gles) and astrocytic pTau inclusions (tufted astrocytes). of the occipitotemporal gyrus, even in a small region of CBD pathology predominates in the forebrain and is the occipitotemporal gyrus adjoining the transentorhi- characterized mainly by astrocytic pTau positivity (as- nal region) or Braak Stage IV-­analog (NFP of at least trocytic plaques). PSP and CBD both exhibit pretangles moderate density in the superficial and/or deep layers in neurons and coiled bodies in oligodendroglial cells of the middle temporal gyrus) CD2AP distribution, and [9]. In these cases, pTau immunohistochemistry was per- finally, CD2AP-­3 was given to cases with Braak Stage formed as described in Section 2.2. V-­analog (NFP of at least moderate density in the su- perficial and deep layers of the peristriate and often also the parastriate area) or Braak Stage VI-­analog (NFP of 2.4 | CD2AP Immunohistochemistry at least moderate density in layer V of the striate area) CD2AP distribution. All cases were evaluated blinded to Four brain regions were selected to evaluate CD2AP im- Braak neurofibrillary stage. munostaining in AD and control cases: the anterior hip- pocampus, posterior hippocampus, temporal lobe, and occipital lobe. In PiD, PSP, and CBD cases, different 2.5 | Immunofluorescence brain regions were selected: the frontal and parietal lobes for all cases and areas with a strong load of pathology: The presence of CD2AP and pTau was evaluated by im- midbrain for CBD, midbrain, basal ganglia, and pons for munofluorescence in 3-­µm-­thick paraffin-­embedded PSP, and posterior hippocampus for PiD cases. We se- human brain slides. Brain sections were deparaffinized, lected substantia nigra from PD cases, cerebellum from hydrated, and incubated with citrate buffer for 30 min MSA, the spinal cord from ALS cases, and cerebellar at 95°C to improve antigen exposure. The sections were cortex from the FTLD-­ALS with C9orf72 mutation case blocked for 1 h at room temperature (RT) and incubated and posterior hippocampus from PART cases. Paraffin overnight with mouse anti-­Tau AT8 antibody (5 μg/ml, blocks of the selected areas were cut into 3 µm sec- Thermo Fisher) or mouse anti-­AT100 antibody (2 µg/ml, tions and stained with the rabbit anti-­CD2AP antibody Thermo Fisher) and rabbit anti-­CD2AP antibody (1 μg/ (Clone HPA003326, 1:200, Sigma-­ A ldrich, St. Louis, ml, Sigma-­A ldrich). After rinsing, the samples were in- USA) using the Benchmark XT staining module with cubated with Alexa Fluor-­568 anti-­mouse IgG and Alexa the ultraView Universal DAB Detection Kit (Ventana Fluor-­ 488 anti-­rabbit IgG secondary antibodies (Life Medical Systems). Deparaffinization was carried out Technologies) diluted 1:500 in blocking solution for 1 h using EZ PrepTM solution. The primary antibody was at room temperature. To reduce brain tissue autofluo- incubated for 40 min at a 1/200 dilution and localized rescence, brain sections were then incubated with 0.3% by a secondary antibody HRP Multimer containing Sudan Black B (Sigma-­ A ldrich) for 10 min. Finally, CD2AP NEURONAL DEPOSITS IN AD    | 5 of 16 samples were dehydrated and mounted on coverslips 3 | R E SU LT S using Vectashield with 4′,6-­diamidino-­2-­phenylindole (DAPI, Vector Laboratories). Double immunofluo- 3.1 | CD2AP immunopositivity in AD cases rescence images were captured with a Zeiss LSM980 confocal microscope. The percentage of CD2AP im- The distribution of CD2AP immunoreactivity and its po- munoreactivity was evaluated as the total number of tential association with pTau and Aβ pathology were ana- CD2AP positive neuronal inclusions in pTau-­AT8 posi- lyzed in cortical areas of postmortem AD brain samples tive NFT in five fields (20x magnification). from Cohort A. CD2AP neuronal deposits resembling neurofibrillary tangles and occasional tortuous fibers resembling neuropil threads were found in cases with 2.6 | Next generation immunohistochemistry AD pathology in regions showing pTau pathology, as de- picted in consecutive sections of the entorhinal region in Next generation immunohistochemistry (NGI) was used Figure 1A,B. However, no evident CD2AP-­positive dys- for coexpression analyses. Briefly, NGI consists of se- trophic neurites were found (Figure 1C,D). No vascular quential immunohistochemical stainings on the same tis- wall or parenchymal CD2AP deposits were identified sue section by destaining the alcohol-­soluble chromogen in areas with parenchymal and vascular Aβ deposition between the stainings, digitalization, and alignment of (as shown in consecutive cortical levels in Figure 1E–­H). the images to finally obtain the information of different However, in all cases, mild granular endothelial and red biomarkers in the same cells (virtually assigning colors blood cells CD2AP positivity was detected. To further for each antibody). This methodology is already vali- study the association found, immunofluorescence dou- dated and used for different panels [40,41]. Antibodies ble staining of CD2AP and pTau-­AT8 was performed and protocols used in this study are as follows: mouse in AD cortical sections (Figure 2). The results con- anti-­ 3R-­ TAU antibody (Clone 05-­ 803, 1:50, Sigma–­ firmed that CD2AP colocalized with pTau-­AT8 (Ser202/ Aldrich, St. Louis, USA), mouse anti-­4R-­TAU antibody Thr305), as shown by cytoplasmic intraneuronal depos- (Clone 05-­804, 1:50, Sigma-­A ldrich, St. Louis, USA), and its in pyramidal neurons and occasional neuropil thread-­ rabbit anti-­CD2AP antibody (Clone HPA003326, 1:200, like structures (Figure 2A,B). In AD cases, no CD2AP Sigma-­A ldrich, St. Louis, USA). immunoreactivity was found independent of pTau-­AT8 and the percentage of CD2AP positivity in pTau-­AT8 de- posits was 76 ± 6%. At a higher magnification, this colo- 2.7 | Statistical analysis calization with pTau was shown to be incomplete, and CD2AP expression displayed a discontinuous granular All statistical analyses were conducted using SPSS pattern (Figure 2B). Furthermore, double immunofluo- Statistics, version 21 (IBM Corporation). The association rescence staining of CD2AP and pTau-­AT100 indicated of categorical variables was analyzed using contingency that CD2AP also co-­localized with pTau (Thr212/Ser214) tables and a chi-­squared test with the Pearson p-­value. in AD sections (Figure 2C). The distribution of age was tested using the Kolmogorov–­ The next step was to confirm the association between Smirnov test. When the data were normally distributed, pTau and CD2AP distribution in AD cases and controls. variables were expressed as the mean ± standard devia- For this purpose, we enlarged the series of patients by tion (SD), and an ANOVA test with a Bonferroni cor- adding a second cohort of AD and non-­AD patients rection for multivariate analysis was applied. When (Cohort B, Table 1). Equal numbers of cases from dif- the data were not normally distributed, variables were ferent Braak neurofibrillary stages were included in the expressed as the median (interquartile range), and a total cohort (N = 60). No significant differences regard- Mann–­W hitney U test was applied. Univariate asso- ing age or sex were found between cohorts. Considering ciations of the CD2AP score with categorical variables the two cohorts together, AD patients were significantly were also determined using contingency tables and a older than control patients (78.9 ± 10.8 vs 61.4 ± 10, chi-­squared test. In this case, the Pearson p-­value was p < 0.001), but no differences were found regarding sex used for sex, whereas the Linear-­by-­Linear Association (Table 1). Braak neurofibrillary stage evaluation with p-­value was used for Thal phase, Braak neurofibrillary Thal phase and CERAD plaque score allowed the as- stage, CERAD plaque score, and AD risk. The asso- signment of “ABC scores” and an AD risk evaluation [3]. ciation of the CD2AP score with the continuous vari- The two cohorts were neuropathologically equivalent, as able age was tested by applying a Kruskal–­Wallis test. no significant differences were found in the frequency of Regression analysis of categorical data (CATREG) was cases classified according to Thal phase, Braak neuro- performed using only the significant variables of the uni- fibrillary stage, or CERAD plaque score, considering variate analysis. The standardized coefficients beta with AD cases and controls separately (Table 2). Based on the corresponding estimate of standard error (SE) and p-­ these evaluations, 48.4% of total cases (29/60) fulfilled value were obtained and displayed. A p-­value below 0.05 the criteria for intermediate (16/60) or high (13/60) levels was considered statistically significant. of AD neuropathologic changes. 6 of 16 |    CAMACHO et al. F I G U R E 1 Representative images of consecutive sections from AD brains (A–­B, C–­D, E–­F and G–­H). (A and C) pTau-­AT8 was detected in neurofibrillary tangles in pyramidal neurons and neuropil threads in dendrites (red and green arrows, respectively) and dystrophic neurites (red asterisks). (B and D). CD2AP-­positive inclusions were present in pyramidal neurons (red arrows) and occasional neuropil thread-­l ike deposits (green arrows), and granular CD2AP positivity was detected in endothelial cells (black arrowheads). No dystrophic neurites of neuritic plaques were stained with CD2AP. (E, G) Aβ immunostaining demonstrated parenchymal and vascular deposits. (F, H) CD2AP immunohistochemistry showed no parenchymal or vascular positivity in those areas. Only endothelial and red blood cells were positive for CD2AP (black and green arrowheads, respectively). C, D, G, and H: Scale bars = 20 µm. A, B, E, and F: Scale bars = 50 µm Next, the CD2AP score was evaluated in AD and of the categorical data, only the association of CD2AP control cases. The results obtained from both cohorts expression with Braak neurofibrillary stage remained sta- (A and B) were also comparable (Table 2). CD2AP de- tistically significant (p = 0.006) (Table 3). In this context, posits were rated similar to Braak neurofibrillary stage none of the control cases (B0) showed CD2AP deposi- (Figure 3A) and, as described before, neuronal CD2AP tion, whereas 33.33% of the B1 cases (5/15) were CD2AP-­1 positivity closely resembled the NFT identified with pTau. and 66.67% did not show any CD2AP immunoreactivity Interestingly, neuropil thread-­like CD2AP positivity was (10/15). All B2 and B3 cases presented CD2AP positivity: significantly lower than pTau expression. The intensity of 93.33% of B2 cases (14/15) were graded as CD2AP-­1, and CD2AP neuronal immunostaining was homogenous, in- 6.67% were graded as CD2AP-­2 (1/15) and 13,33% of B3 dependent of AD risk, although when it expanded, it fol- cases were graded as CD2AP-­1 (2/15), 33.33% were scored lowed the pathologic distribution of pTau. An example of CD2AP-­2 (5/15), and 53.33% of B3 cases (8/15) showed the Braak neurofibrillary stages and CD2AP score is shown highest expansion of CD2AP (CD2AP-­3). This hierarchical in Figure 3B. After assessment, 41.7% of the cases did progression of CD2AP distribution regarding Braak neu- not show CD2AP deposits (25/60), 35% of the cases were rofibrillary stage is represented in Figure 4. graded as CD2AP-­1 (21/60), 10% of the cases were rated as CD2AP-­2 (6/60), and finally, 13.3% of the cases fulfilled criteria for CD2AP-­3 score (8/60) (Table 2). 3.2 | CD2AP immunopositivity in other Statistical univariate analyses revealed that CD2AP ex- tauopathies pression was significantly associated with age, Thal phase, Braak neurofibrillary stage, CERAD plaque score, and AD To analyze whether the association between pTau risk (p < 0.001) (Table 3). However, after regression analysis and CD2AP occurs in other tauopathies beyond AD, CD2AP NEURONAL DEPOSITS IN AD    | 7 of 16 F I G U R E 2 Confocal evaluation of CD2AP/pTau-­AT8 (A and B) and CD2AP/pTau-­AT100 (C) distribution in cortical brain sections from AD patients. Representative confocal images showing the colocalization of pTau (red) and CD2AP (green) in the neuronal cytoplasm and neuropil threads in a case with B3 and CD2AP-­3 scores. Merged images show the colocalization of pTau and CD2AP (yellow). Scale bars = 10 μm CD2AP distribution was studied in nine cases of PSP, 3.3 | CD2AP immunopositivity in other five cases of CBD, and seven cases of PiD (Table 1). neurodegenerative diseases The PSP and PiD patients were also significantly older than the control patients (PSP = 74.2 years ± 9.1 and In order to evaluate whether neuronal CD2AP inclu- PiD = 75.1 years ± 9.7 vs Controls = 61.4 years ± 10, sions were present in other neurodegenerative diseases, p < 0.001). No significant differences were found re- CD2AP immunostaining was also determined in alpha-­ garding sex (Table 1). synucleinopathies (PD and MSA) and TDP43-­pathies The presence of CD2AP was evaluated in neurons (ALS, FTLD-­ALS with p62 positive inclusions in cer- and glial and oligodendroglial cells. Our results evi- ebellar granular layer). Results showed the absence of denced only spherical neuronal CD2AP inclusions neuronal or glial CD2AP positivity, preserving CD2AP resembling Pick bodies in cortex and hippocampus expression in endothelial and red blood cells in these (Figure 5E,F,K,L, respectively), and some NFT-­ cases (Figure 7A–­H). PART cases were also evaluated, like and neuropil thread-­ l ike inclusions in 6/7 PiD as it is considered a 3R/4R-­tauopathy, and results con- cases. No CD2AP neuronal or glial inclusions were firmed CD2AP neuronal positivity, similar to AD cases found in any CBD or PSP cases (Figure 5A–­D,G–­J ). (Figure 7I–­J). Remarkably, no pretangle-­l ike neuronal granular pos- itivity was detected. Given this suggestive association with 3R-­ Tau-­ a ssociated diseases, we then compared 4 | DI SC US SION CD2AP and 3R-­Tau expression in AD and PiD cases using NGI (Figure 6). The results showed CD2AP Advances in human genetics have revealed different ge- colocalization in certain but not all 3R-­Tau positive netic variants involved in different neurologic diseases. NFT in AD. Interestingly, CD2AP immunodetection In particular, GWAS have identified common genetic was evident in those NFT with a less intense positiv- variations at numerous loci associated with AD risk. ity of 3R-­Tau isoform (Figure 6A). In contrast, colo- Some of these are variants in genes involved in choles- calization between CD2AP and 4R-­Tau was negligible terol transport (ABCA7, APOE4, and INPP5D), oth- (Figure 6B). The association between CD2AP and 3R-­ ers are linked to the clearance of nonnative proteins Tau positivity was confirmed in PiD (Figure 6C). As by immune cells (TREM2 and CLU), and others are expected, no 4R-­Tau immunoreactivity was found in related to endocytic and lysosomal transport (BIN1, Pick bodies (Figure 6D). CD2AP, PICALM, RIN3, SORL1, GRN, and PLD3) 8 of 16 |    CAMACHO et al. T A B L E 2 Demographic and pathologic description of Alzheimer's diseases (AD) cases and controls from Cohort A (Vall d’Hebron University Hospital) and Cohort B (CIEN Foundation). Alzheimer's disease Controls p-­value (AD Cohort A Cohort B p-­value Total Cohort A Cohort B p-­value Total vs Controls) N 17 28 45 6 9 15 Braak 0.910 N/A <0.001 B0 —­ —­ —­ 6 (100%) 9 (100%) 15 (100%) B1 6 (35.3%) 9 (32.1%) 15 (33.3%) —­ —­ —­ B2 6 (35.3%) 9 (32.1%) 15 (33.3%) —­ —­ —­ B3 5 (29.4%) 10 (35.7%) 15 (33.3%) —­ —­ —­ Thal 0.068 0.205 <0.001 A0 4 (23.5%) 3 (10.7%) 7 (15.6%) 5 (83.3%) 9 (100%) 14 (93.3%) A1 1 (5.9%) 5 (17.9%) 6 (13.3%) 1 (16.7%) —­ 1 (6.7%) A2 7 (41.2%) 4 (14.3%) 11 (24.4%) —­ —­ —­ A3 5 (29.4%) 16 (57.1%) 21 (46.7%) —­ —­ —­ CERAD 0.123 0.205 <0.001 C0 4 (23.5%) 10 (35.7%) 14 (31.1%) 5 (83.3%) 9 (100%) 14 (93.3%) C1 6 (35.3%) 2 (7.1%) 8 (17.8%) —­ —­ —­ C2 2 (11.8%) 4 (14.3%) 6 (13.3%) 1 (16.7%) —­ 1 (6.7%) C3 5 (29.4%) 12 (42.9%) 17 (37.8%) —­ —­ —­ AD risk 0.254 0.205 <0.001 Nonrelated 5 (29.4%) 3 (10.7%) 8 (17.8%) 5 (83.3%) 9 (100%) 14 (93.3%) Low 2 (11.8%) 6 (21.4%) 8 (17.8%) 1 (16.7%) —­ —­ Moderate 7 (41.2%) 9 (32.1%) 16 (35.6%) —­ —­ —­ High 3 (17.6%) 10 (35.7%) 13 (28.9%) —­ —­ —­ CD2AP 0.422 N/A <0.001 0 4 (23.5%) 6 (21.4%) 10 (22.2%) 6 (100%) 9 (100%) 15 (100%) 1 9 (52.9%) 12 (42.9%) 21 (46.7%) —­ —­ —­ 2 3 (17.6%) 3 (10.7%) 6 (13.3%) —­ —­ —­ 3 1 (5.9%) 7 (25.0%) 8 (17.8%) —­ —­ —­ Note: The expression of CD2AP was classified according to the score described in Section 2.4 of Materials and Methods and schematically represented in Figure 3A. Statistical differences were analyzed among cohorts for each diagnosis group. Statistical differences between disease groups were also analyzed. Abbreviation: N/A, not applicable. [21,24,25,26,31]. Although the association of different podocytes, which compose the protein filtration barrier CD2AP genetic variants with AD risk has been well doc- of the kidney. Its critical role in renal function was con- umented in multiple cohorts [21,23,24,25,26,31], the role firmed, as mice lacking CD2AP developed congenital of the expression of the CD2AP protein in this patho- nephrotic syndrome [43] because of its role in a special- genesis is still unclear. To determine how CD2AP con- ized junction known as a slit diaphragm [45]. Moreover, tributes to AD, we studied its distribution in brain tissue human CD2AP mutations in which the product is a from AD patients and controls in a multicenter study truncated CD2AP protein have been linked to nephrotic with the participation of two Spanish biobank centers. syndrome and focal segmental glomerulosclerosis in CD2AP is a multidomain scaffolding protein impli- children [31,46,47]. Although one of these mutations cated in multiple physiologic and disease processes. It was identified in a young adult with focal segmental glo- was first described as a protein involved in T-­c ell acti- merulosclerosis and mild dementia [48], the long-­term vation that stabilizes the interaction between T cells and neurologic consequences of CD2AP haploinsufficiency antigen-­presenting cells [42]. CD2AP contains a coiled in adults carrying CD2AP mutations have not been de- coil domain and three Src homology 3 (SH3) domains scribed thus far. CD2AP immunostaining studies in mice that serve as attachment sites for other proteins [43] showed the expression of this protein mostly in epithe- and facilitate its role in dynamic actin remodeling and lial cells but also in the endothelium of different organs, membrane trafficking during endocytosis and cytoki- including the central nervous system [28,29]. Indeed, in nesis [44]. CD2AP is primarily expressed in glomerular the brain, a more recent study in rodents also detected CD2AP NEURONAL DEPOSITS IN AD    | 9 of 16 F I G U R E 3 (A) Schematic representation of the CD2AP score established according to the positivity of CD2AP in neurons at different areas studied for the Braak neurofibrillary stage assessment. (B) Representative images of consecutive sections stained with anti-­ pTau-­AT8 and anti-­CD2AP in different brain areas. Scale bars = 50 µm T A B L E 3 CD2AP score associations with demographic and pathologic CATREG regression Univariate analysis characteristics in AD patients and controls CD2AP ASSOCIATION p-­value Beta ± SE p-­value of both cohorts Age <0.001 −0.097 ± 0.081 0.237 Sex (female) 0.680 —­ —­ Thal phase <0.001 0.190 ± 0.374 0.773 Braak neurofibrillary stage <0.001 0.495 ± 0.230 0.006 CERAD plaque score <0.001 0.045 ± 0.167 0.790 AD risk <0.001 0.334 ± 0.436 0.561 Note: Categorical regression (CATREG) was assessed using only the significant variables of the univariate analysis. Beta ± SE: standardized coefficients beta with the corresponding estimate of standard error. Bold numbers indicate statistically significant differences. CD2AP protein in Purkinje cells of the cerebellum and production and release, CD2AP loss of function did not in cortical neurons [27]. Thus, CD2AP in the brain has affect total Aβ levels o amyloid plaque burden in an AD been linked to synapse function through its involvement mouse model in vivo [51]. In AD patients, the possible in vesicle trafficking [25,39,49], axonal sproutings [27], role of CD2AP in β-­amyloidosis is reinforced by data and maintenance of the blood–­brain barrier [50]. showing that the CD2AP variants rs9349407 [35] and In our study, we confirmed granular immunopos- rs10948363 [36] are associated with a greater load of neu- itivity for CD2AP in the human brain endothelium. ritic plaques. However, in our AD cohort, brain CD2AP However, our most interesting finding was the presence immunodetection was not associated with vascular or of CD2AP-­ i mmunoreactive neuronal inclusions sim- parenchymal Aβ deposits. In contrast, the protein was ilar to neurofibrillary tangles in the hippocampus and specifically present in regions with an evident presence temporal and occipital cortex of AD brains. In this of NFT. Indeed, we confirmed by immunofluorescence pathologic context, CD2AP has previously been linked that CD2AP colocalized at an intraneuronal level with with Aβ metabolism and clearance. Downregulation of pTau protein in the AD brain. CD2AP in neuroblastoma cells decreased extracellular Based on this result, we next analyzed the expres- Aβ levels [51]. Additionally, CD2AP was implicated in sion of CD2AP in a cohort of brains from two different APP trafficking and processing [52], and its overexpres- centers including an equal number of cases from dif- sion induced decreased levels of APP in neurons by ac- ferent Braak neurofibrillary stages [6]. First, we found celerating the lysosome-­dependent degradation of APP that none of the control samples showed intraneuro- [53]. In contrast to the in vitro effects of CD2AP on Aβ nal CD2AP deposits. In contrast, in the AD samples, 10 of 16 |    CAMACHO et al. immunodetection of CD2AP in neurons was strongly stage, independent of age, and other pathologic hall- and positively associated with Braak neurofibrillary marks. In fact, although CD2AP expression was signifi- cantly linked with AD risk in the univariate analysis, the results from the logistic regression confirmed that this association was attributable to the strong associa- tion of CD2AP expression with Braak neurofibrillary stage. Our observations suggest that the alteration in CD2AP accumulation is directly linked to tau pathol- ogy following a hierarchical progression in the studied areas (hippocampus, temporal cortex, and occipital cortex). Remarkably, CD2AP positivity was never found beyond pTau deposition, suggesting that the appearance of CD2AP neuronal deposits would follow a slower pro- gression than pTau in AD. Different studies have already proposed that CD2AP regulation might be related to tau toxicity and neuronal apoptosis. For example, the loss of the fly ortholog of CD2AP, Cindr, exacerbated tau-­mediated toxicity in a Drosophila model [54] and was required for neuronal ho- meostasis and synaptic function in this model [55]. These studies suggest that enhanced expression of CD2AP may F I G U R E 4 Bar diagram depicting the CD2AP score according be protective against AD. Nonetheless, the authors of to pTau Braak neurofibrillary stage the latter study described ubiquitous expression of Cindr F I G U R E 5 Representative images of consecutive sections from different tauopathies: CBD (A–­B, G–­H), PSP (C–­D, I–­J), and PiD (E–­F, K–­L). (A, C, E, G, I, and K) pTau-­AT8 immunostaining showed diffuse granular cytoplasmic immunoreactivity in neurons (pretangle, black arrow) and astrocytic plaques (red arrow) in the CBD (A and G), tufted astrocytes (black arrowhead), pretangles (black arrow) and globose tangle (green arrow) in PSP samples (C and I) and neurofibrillary tangles (red asterisk), and Pick bodies (red arrowheads) in PiD samples (E and K). (B, D, F, H, J, and L) CD2AP immunohistochemistry failed to show any neuronal or glial positivity in CBD or PSP samples (B, D, H, and J), whereas CD2AP-­positive Pick body-­l ike spherical inclusions (red arrowheads) were observed in PiD samples (F and L). Images corresponding to CD2AP staining showed red blood cells and endothelial positivity. BG: basal ganglia, MD: midbrain, DG: dentate gyrus. A–­L: Scale bars = 20 µm CD2AP NEURONAL DEPOSITS IN AD    | 11 of 16 F I G U R E 6 Representative images of NGI technology of CD2AP, 3R-­Tau, and 4R-­Tau in AD (A–­B) and PiD (C–­D) cases. The last column corresponds to virtually assigned colors: red for CD2AP, green for 3R-­Tau, or 4R-­Tau and blue for negative nuclei, showing a yellow signal in the colocalization of CD2AP and 3R-­Tau or 4R-­Tau. (A) Hippocampal cortex from an AD patient sequentially stained with CD2AP antibody and 3R-­Tau antibody, and merge (CD2AP red, 3R-­Tau green) showing coexistence of both positivities with colocalizing foci (white asterisks). (B) Hippocampal cortex from an AD patient sequentially stained with CD2AP antibody and 4R-­Tau antibody, and merge (CD2AP red, 4R-­Tau green) showing coexistence of both positivities with negligible colocalization. (C) Dentate gyrus from a PiD patient sequentially stained with CD2AP antibody and 3R-­Tau antibody, and merge (CD2AP red, 3R-­Tau green) showing coexistence of both positivities with colocalizing foci (white asterisks). (D) Dentate gyrus from a PiD patient sequentially stained with CD2AP antibody and 4R-­Tau antibody, and merge (CD2AP red, 4R-­Tau green, nuclei highlighted in blue) confirming the presence of CD2AP positivity and the absence of 4R-­t au expression. A–­D: Scale bars = 50 µm. in glial cells and neurons, with a particular enrichment protein expression load in patients carrying these CD2AP at presynaptic terminals [55]. As this expression pattern genetic variants has not been specifically determined. clearly differs from the distribution that we found in However, the results from available data sets from the human tissue, which was restricted to endothelial cells, Genotype-­ Tissue Expression Project, which describes isolated neurites and NFT-­like neuronal inclusions, the genetic effects on gene expression levels [57] show that function of Cindr in Drosophila may not be exactly com- CD2AP risk alleles are associated with increased gene parable with the function of the protein in the patho- expression in various tissues including the brain. These logic human brain. Indeed, in humans, previous reports observations, together with our results showing negligi- have also suggested a link between CD2AP and tau ble levels of neuronal CD2AP expression in cases with pathology, as CD2AP was detected as a susceptibility lower Braak neurofibrillary stages and higher presence locus for cerebrospinal fluid (CSF) tau biomarkers [56], of CD2AP neuronal accumulation in cases with a higher and the CD2AP rs9381563 variant was associated with Braak neurofibrillary stage and AD score risk, open the altered pTau levels in the CSF [33]. Moreover, CD2AP possibility that elevated CD2AP expression may be in- rs10948363 showed a suggestive association with NFT volved in the pTau pathology in AD, in contrast to the [37]. To our knowledge, the association between CD2AP previous hypothesis [38,54,55]. However, the total or 12 of 16 |    CAMACHO et al. F I G U R E 7 Representative images of consecutive sections from other neurodegenerative diseases (A–­B, C–­D, E–­F, G–­H, and I–­J). Alfa-­ synuclein (ASYN) immunostaining showed immunoreactivity as Lewy bodies (black arrows) and Lewy neurites in Parkinson Disease (PD, A) and glial inclusions (red asterisk) in Multisystem atrophy (MSA, C). TDP-­43 showed neuronal inclusions (red arrows) in Amyotrophic lateral sclerosis (ALS) without cognitive impairment (E) and p62 showed positive inclusions (red arrowhead) in the cerebellar granular layer of FTLD-­ ALS with C9orf72 mutation (G). None CD2AP neuronal or glial inclusions were found in PD (B), MSA (D), ALS (F) neither in FTLD-­ALS (H). pTau-­AT8 showed neurofibrillary tangles (NFT, green arrows) in primary age-­related tauopathy (PART) (I) and CD2AP showed only NFT-­l ike positivity (green arrows) (J). Images corresponding to CD2AP staining showed red blood cells and endothelial positivity. Black scale bars = 20 µm, red scale bars = 10 µm soluble levels of CD2AP expression have not been exten- AD as well as those with endocytic cellular functions sively analyzed or previously described. A prior report have also been associated with tau pathology. This is the showed that CD2AP gene expression was decreased in case, for instance, for PICALM, whose levels were de- peripheral blood lymphocytes from sporadic AD cases creased in AD brains and colocalized with NFT [58]. The [38], although expression in postmortem human tissue authors of these studies showed that abnormal PICALM samples should be determined through different analyt- processing was linked to an impairment of autophagy ical techniques to underscore the implication of CD2AP in AD and in tauopathies without Aβ pathology such as in tau pathology progression. Other proteins encoded by CBD and PiD [59]. Furthermore, in postmortem brains genes identified as presenting variants associated with of familial cases of frontotemporal lobar degeneration CD2AP NEURONAL DEPOSITS IN AD    | 13 of 16 with tau-­immunoreactive inclusions (FTLD-­tau), solu- advanced NFT structure, although further studies are ble PICALM protein levels were reduced compared with needed to specifically study this topic. nondemented control brains, while PICALM detected Our study has some limitations that should be con- in the insoluble brain fraction was significantly in- sidered. Only immunodetection studies were performed, creased [60]. Interestingly, in a murine tauopathy model, whereas biochemical analysis to determine the total gene PICALM haploinsufficiency induced exacerbated tau and protein expression of CD2AP levels in the brain, as pathology via autophagy deregulation [60]. Overall, the well as in blood and CSF, would have been extremely in- balance between soluble protein levels and insoluble/pro- sightful. Despite the effort of collecting multicenter co- tein inclusions of tau-­associated proteins seems to be a horts, obtaining a significant sample size for AD cases, it critical factor influencing the toxicity of NFT and should was not possible to do so for some infrequent diseases (PSP, also be considered in the case of CD2AP in future studies. CDB, and PiD). The small sample size of these groups was Indeed, autophagic-­ endolysosomal networks perform a limitation for statistical analysis, allowing mainly qual- important functions in tau accumulation, including the itative and descriptive results. Unfortunately, systematic clearance of neurotoxic forms of tau and pTau at specific information regarding cognitive status from the whole co- sites through the ubiquitin-­proteasome system (UPS). hort was not available, which would have been extremely Dysregulation of these autophagic-­endolysosomal pro- valuable to determine functional implications of the pres- cesses has been shown in different tauopathies [61]. In ence of intraneuronal CD2AP in AD and other tauopa- this regard, CD2AP binds ubiquitin and is also proposed thies. Finally, because the APOE genotype is linked to Aβ to regulate UPS function in synaptic transmission [62]. brain accumulation [68,69] but is also a risk factor for tau Because of the observed link between CD2AP and neurodegeneration independent of Aβ [57,70,71,72,73,74], pTau pathology, the expression of the CD2AP protein future studies analyzing the effect of APOE on CD2AP in the brain of patients with other tauopathies was also neuronal deposition in tauopathies are required. analyzed. Our results showed that only PiD presented In conclusion, our immunohistochemical study CD2AP deposits resembling Pick bodies in the cyto- showed an association of CD2AP with pTau inclusions plasm of neurons. To date, there are no reports linking in AD and PiD but not in CDB or PSP. This descrip- PiD to CD2AP or to other tauopathies. PiD is dementia tive analysis cannot establish a causative relationship in which tau lesions are mainly composed of the 3R iso- between CD2AP neuronal deposits and AD risk. More forms, whereas pTau lesions in AD are composed of equal functional studies are needed to elucidate the implication amounts of 3R and 4R isoforms [7,9]. Since CD2AP neu- of the increase in CD2AP expression or its accumulation ronal immunoreactivity was not observed in coiled bod- in neurons in AD. Nevertheless, determining its role in ies, tufted astrocytes of PSP cases, astrocytic plaques or in pathologic conditions seems relevant to elucidate the NFTs of CBD cases, we next analyzed whether a 3R-­Tau molecular events involved in the formation of NFT. We isoform might be associated with CD2AP. Interestingly, are facing an opportunity to offer future perspectives for although CD2AP was not present in all 3R-­positive NFT, the diagnosis or treatment of tauopathies. In this regard, it partially colocalized with this specific tau isoform in analyzing experimentally the outcome of treatment with certain inclusions in AD and PiD brains. This result, to- CD2AP modulators, some of them previously tested for gether with the lack of CD2AP neuronal expression in the focal segmental glomerulosclerosis [48,72], appears to be 4R-­tauopathies studied, suggests an association of this a promising strategy to determine the potential modula- protein with 3R-­tau-­related disease. Different processing tion of NFT conformation and toxicity in AD and PiD. and posttranslational modifications could explain this In addition, because CSF biomarkers based on the de- disease-­specific association between CD2AP and pTau. tection of total tau and pTau have demonstrated clinical Arakhamia et al. [63] compared posttranslationally modi- utility in AD diagnosis [73,74]; our hypothesis is that the fied tau filaments in AD and CBD and found striking dif- combination of these established markers with CD2AP ferences in the processes of misfolding and self-­assembly levels in CSF might be tested as markers for AD risk and into fibrils. In fact, different electrophoretic migration contribute to the differential diagnosis of tauopathies. patterns of tau deposits have been found in CBD or PSP [64] and PiD [65] compared with the paired helical fila- AC K NOW L E D G M E N T S ments found in AD. The ultrastructural characteristics of The authors are indebted to the donors and their families tau filaments in these tauopathies were also studied [66], for their generous contributions. This work was funded revealing differences in shape, size, and conformation. by Instituto de Salud Carlos III (ISCIII) (PI17/00275, The traits of the AD paired helical filaments may be at PI20/00465), cofinanced by the European Regional the origin of this specific association between pTau and Development Fund (FEDER). The Neurovascular CD2AP. Another explanation for this disease-­specific as- Research Laboratory is part of the INVICTUS+ net- sociation could be the age of the tangle, as in AD, there work, ISCIII, Spain (RD16/0019/0021). is a shift from 4R in the pretangle to 3R in the ghost tan- gle [67]. Our results suggest that the presence of CD2AP C ON F L IC T OF I N T E R E ST neuronal inclusions could be related to a more mature or The authors declare no conflict of interest. 14 of 16 |    CAMACHO et al. 3. Hyman BT, Phelps CH, Beach TG, Bigio EH, Cairns NJ, AU T HOR C ON T R I BU T ION S Carrillo MC, et al. National Institute on Aging-­ A lzheimer’s Mar Hernández-­ Guillamon and Elena Martínez-­ Association guidelines for the neuropathologic assessment of Sáez designed and coordinated the study. Jessica Alzheimer’s disease. Alzheimers Dement. 2012;8(1):1–­13. https:// Camacho participated in the design of the study. doi.org/10.1016/j.jalz.2011.10.007 4. Thal DR, Rüb U, Orantes M, Braak H. Phases of Aβ-­deposition Santiago Ramón y Cajal and Alberto Rábano ad- in the human brain and its relevance for the development of vised on the theoretical aspects of the study. Teresa AD. Neurology. 2002;58(12):1791–­ 800. https://doi.org/10.1212/ Moliné performed the immunohistochemistry, Paula wnl.58.12.1791 Marazuela performed the immunofluorescence anal- 5. Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, ysis, Garazi Serna performed the NGI study. Jessica Brownlee LM, et al. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD): part II. Standardization of the Camacho, Elena Martínez-­Sáez, and Alberto Rábano neuropathologic assessment of Alzheimer’s disease. Neurology. selected brain samples. Jessica Camacho revised all 1991;41(4):479. https://doi.org/10.1212/wnl.41.4.479 the immunohistochemistry. Anna Bonaterra-­ Pastra 6. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici performed the statistical analysis. Jessica Camacho, K. Staging of Alzheimer disease-­associated neurofibrillary pa- Elena Martínez-­Sáez, and Mar Hernández-­Guillamon thology using paraffin sections and immunocytochemistry. Acta Neuropathol. 2006;112(4):389–­404. https://doi.org/10.1007/s0040​ wrote the manuscript. All authors read and approved 1-­0 06-­0127-­z the final manuscript. 7. Spillantini MG, Goedert M. Tau protein pathology in neurode- generative diseases. Trends Neurosci. 1998;21(10):428–­33. https:// E T H IC S A PPROVA L doi.org/10.1016/s0166​-­2236(98)01337​-­x The study was approved by the Ethical Committee of 8. 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Written informed consent was obtained Neuropharmacol. 2017;15(6):926–­35. https://doi.org/10.2174/15701​ from all participants. 59X15​66617​01161​43743 12. Arriagada PV, Growdon JH, Hedley-­ W hyte ET, Hyman BT. Neurofibrillary tangles but not senile plaques parallel duration PE R M I S S ION TO R E PRODUC E M AT E R I A L and severity of Alzheimer’s disease. Neurology. 1992;42(3):631. F ROM OT H E R S OU RC E S https://doi.org/10.1212/wnl.42.3.631 Not applicable. 13. Josephs KA, Whitwell JL, Ahmed Z, Shiung MM, Weigand SD, Knopman DS, et al. β-­a myloid burden is not associated with DATA AVA I L A B I L I T Y STAT E M E N T rates of brain atrophy. Ann Neurol. 2008;63(2):204–­12. https:// doi.org/10.1002/ana.21223 The data that support the findings of this study are avail- 14. Long JM, Holtzman DM. Alzheimer disease: an update on able upon request. pathobiology and treatment strategies. Cell. 2019;179(2):312–­39. https://doi.org/10.1016/j.cell.2019.09.001 ORC I D 15. 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