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Alzheimer’s disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short (“stubby”) spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allowed us to precisely measure spine parameters. Mathematical modeling indicated that the shortening and widening of the necks should alter the electrical compartmentalization of the spines, leading to reduced postsynaptic potentials in spine heads, but not in soma. Accordingly, there was no alteration in basal synaptic transmission, but long-term potentiation and spatial memory were impaired. These results indicate that an alteration of spine morphology could be involved in the early cognitive deficits associated with AD.

In Alzheimer’s disease (AD), Tau and Aβ aggregates involve sequentially connected regions, sometimes distantly separated. These alterations were studied in the pillar of the fornix (PoF), an axonal tract, to analyse the role of axons in their propagation. The PoF axons mainly originate from the subicular neurons and project to the mamillary body. Forty-seven post-mortem cases at various Braak stages (Tau) and Thal phases (Aβ) were analysed by immunohistochemistry. The distribution of the lesions showed that the subiculum was affected before the mamillary body, but neither Tau aggregation nor Aβ deposition was consistently first. The subiculum and the mamillary body contained Gallyas positive neurofibrillary tangles, immunolabelled by AT8, TG3, PHF1, Alz50 and C3 Tau antibodies. In the PoF, only thin and fragmented threads were observed, exclusively in the cases with neurofibrillary tangles in the subiculum. The threads were made of Gallyas negative, AT8 and TG3 positive Tau. They were intra-axonal and devoid of paired helical filaments at electron microscopy. We tested PoF homogenates containing Tau AT8 positive axons in a Tau P301S biosensor HEK cell line and found a seeding activity. There was no Aβ immunoreactivity detected in the PoF. We could follow microcryodissected AT8 positive axons entering the mamillary body; contacts between Tau positive endings and Aβ positive diffuse or focal deposits were observed in CLARITY-cleared mamillary body. In conclusion, we show that non-fibrillary, hyperphosphorylated Tau is transported by the axons of the PoF from the subiculum to the mamillary body and has a seeding activity. Either Tau aggregation or Aβ accumulation may occur first in this system: this inconstant order is incompatible with a cause-and-effects relationship. However, both pathologies were correlated and intimately associated, indicating an interaction of the two processes, once initiated.

Microduplications of the 17q21.31 chromosomal region encompassing the MAPT gene, which encodes the Tau protein, were identified in patients with a progressive disorder initially characterized by severe memory impairment with or without behavioral changes that can clinically mimic Alzheimer disease. The unique neuropathological report showed a primary tauopathy, which could not be unanimously classified in a given known subtype, showing both 4R- and 3R-tau inclusions, mainly within temporal cortical subregions and basal ganglia, without amyloid deposits. Recently, two subjects harboring the same duplication were reported with an atypical extrapyramidal syndrome and gait disorder. To decipher the phenotypic spectrum associated with MAPT duplications, we studied ten carriers from nine families, including two novel unrelated probands, gathering clinical (n = 10), cerebrospinal fluid (n = 6), MRI (n = 8), dopamine transporter scan (n = 4), functional (n = 5), amyloid (n = 3) and Tau-tracer (n = 2) PET imaging data as well as neuropathological examination (n = 4). Ages at onset ranged from 37 to 57 years, with prominent episodic memory impairment in 8/10 patients, associated with behavioral changes in four, while two patients showed atypical extrapyramidal syndrome with gait disorder at presentation, including one with associated cognitive deficits. Amyloid imaging was negative but Tau imaging showed significant deposits mainly in both mesiotemporal cortex. Dopaminergic denervation was found in 4/4 patients, including three without extrapyramidal symptoms. Neuropathological examination exclusively showed Tau-immunoreactive lesions. Distribution, aspect and 4R/3R tau aggregates composition suggested a spectrum from predominantly 3R, mainly cortical deposits well correlating with cognitive and behavioral changes, to predominantly 4R deposits, mainly in the basal ganglia and midbrain, in patients with prominent extrapyramidal syndrome. Finally, we performed in vitro seeding experiments in HEK-biosensor cells. Morphological features of aggregates induced by homogenates of three MAPT duplication carriers showed dense/granular ratios graduating between those induced by homogenates of a Pick disease and a progressive supranuclear palsy cases. These results suggest that MAPT duplication causes a primary tauopathy associated with diverse clinical and neuropathological features.

Extracellular accumulation of Aβ peptides and intracellular aggregation of hyperphosphorylated tau proteins are the two hallmark lesions of Alzheimer disease (AD). The senile plaque is made of a core of extracellular Aβ surrounded by phospho-tau positive neurites. It includes multiple components such as axons, synapses, glial fibers and microglia. To visualize the relationships of those elements, an original technique was developed, based on the dilation of interstitial water during freezing. Samples of neocortex, hippocampus and striatum were taken from formalin-fixed brains (one control case; three cases with severe Alzheimer disease). The samples were subjected to various numbers of freezing/thawing cycles (from 0 to 320) with an automated system we devised. The samples were embedded in paraffin, cut and stained with haematoxylin-eosin or immunostained against Aβ, phospho-tau, and antigens enriched in axons, synapses, macrophages or astrocytes. Microcryodissection induced the dissociation of tissue components, especially in the grey matter where the neuropil formed an oriented “mesh”. The size of the empty spaces separating the fiber bundles and cells increased with the number of cycles. The amyloid core of the senile plaque separated from its neuritic crown at around 300 freezing/thawing cycles. The dissected core remained associated with macrophages containing Aβ in their cytoplasm. Phospho-tau positive axons were distinctly seen projecting from the neuritic crown to the isolated amyloid core, where they ended in large synapses. The microcryodissection showed astrocytic processes stuck directly to the core. The original method we developed—microcryodissection—helped understanding how histological components were assembled in the tissue.

Cerebral amyloid angiopathy (CAA) is characterized by amyloid beta (Aβ) deposition in cerebrovasculature. It is prevalent with aging and Alzheimer’s disease (AD), associated with intracerebral hemorrhage, and contributes to cognitive deficits. To better understand molecular mechanisms, CAA(+) and CAA(−) vessels were microdissected from paraffin-embedded autopsy temporal cortex of age-matched Control ( n  = 10), mild cognitive impairment (MCI; n  = 4), and sporadic AD ( n  = 6) cases, followed by label-free quantitative mass spectrometry. 257 proteins were differentially abundant in CAA(+) vessels compared to neighboring CAA(−) vessels in MCI, and 289 in AD ( p  < 0.05, fold-change > 1.5). 84 proteins changed in the same direction in both groups, and many changed in the same direction among proteins significant in at least one group ( p  < 0.0001, R 2  = 0.62). In CAA(+) vessels, proteins significantly increased in both AD and MCI were particularly associated with collagen-containing extracellular matrix, while proteins associated with ribonucleoprotein complex were significantly decreased in both AD and MCI. In neighboring CAA(−) vessels, 61 proteins were differentially abundant in MCI, and 112 in AD when compared to Control cases. Increased proteins in CAA(−) vessels were associated with extracellular matrix, external encapsulating structure, and collagen-containing extracellular matrix in MCI; collagen trimer in AD. Twenty two proteins were increased in CAA(−) vessels of both AD and MCI. Comparison of the CAA proteome with published amyloid-plaque proteomic datasets identified many proteins similarly enriched in CAA and plaques, as well as a protein subset hypothesized as preferentially enriched in CAA when compared to plaques. SEMA3G emerged as a CAA specific marker, validated immunohistochemically and with correlation to pathology levels ( p  < 0.0001; R 2  = 0.90). Overall, the CAA(−) vessel proteomes indicated changes in vessel integrity in AD and MCI in the absence of Aβ, and the CAA(+) vessel proteome was similar in MCI and AD, which was associated with vascular matrix reorganization, protein translation deficits, and blood brain barrier breakdown.

APOE ε4 is the major genetic risk factor for sporadic Alzheimer’s disease (AD). Although APOE ε4 is known to promote Aβ pathology, recent data also support an effect of  APOE  polymorphism on phosphorylated Tau (pTau) pathology. To elucidate these potential effects, the pTau interactome was analyzed across  APOE  genotypes in the frontal cortex of 10 advanced AD cases ( n  = 5  APOE ε3/ε3 and n  = 5  APOE ε4/ε4 ), using a combination of anti-pTau pS396/pS404 (PHF1) immunoprecipitation (IP) and mass spectrometry (MS). This proteomic approach was complemented by an analysis of anti-pTau PHF1 and anti-Aβ 4G8 immunohistochemistry, performed in the frontal cortex of 21 advanced AD cases ( n  = 11  APOE ε3/ε3 and n  = 10  APOE ε4/ε4 ). Our dataset includes 1130 and 1330 proteins enriched in IP PHF1 samples from APOE ε3/ε3 and APOE ε4/ε4 groups (fold change ≥ 1.50, IP PHF1 vs IP IgG ctrl ). We identified 80 and 68 proteins as probable pTau interactors in APOE ε3/ε3 and APOE ε4/ε4 groups, respectively (SAINT score ≥ 0.80; false discovery rate (FDR) ≤ 5%). A total of 47/80 proteins were identified as more likely to interact with pTau in APOE ε3/ε3 vs APOE ε4/ε4 cases. Functional enrichment analyses showed that they were significantly associated with the nucleoplasm compartment and involved in RNA processing. In contrast, 35/68 proteins were identified as more likely to interact with pTau in APOE ε4/ε4 vs APOE ε3/ε3 cases. They were significantly associated with the synaptic compartment and involved in cellular transport. A characterization of Tau pathology in the frontal cortex showed a higher density of plaque-associated neuritic crowns, made of dystrophic axons and synapses, in APOE ε4 carriers. Cerebral amyloid angiopathy was more frequent and severe in APOE ε4/ε4 cases. Our study supports an influence of APOE genotype on pTau-subcellular location in AD. These results suggest a facilitation of pTau progression to Aβ-affected brain regions in APOE ε4 carriers, paving the way to the identification of new therapeutic targets.

APOE.sup.[epsilon]4 is the major genetic risk factor for sporadic Alzheimer's disease (AD). Although APOE.sup.[epsilon]4 is known to promote A pathology, recent data also support an effect of APOE polymorphism on phosphorylated Tau (pTau) pathology. To elucidate these potential effects, the pTau interactome was analyzed across APOE genotypes in the frontal cortex of 10 advanced AD cases (n = 5 APOE.sup.[epsilon]3/[epsilon]3 and n = 5 APOE.sup.[epsilon]4/[epsilon]4), using a combination of anti-pTau pS396/pS404 (PHF1) immunoprecipitation (IP) and mass spectrometry (MS). This proteomic approach was complemented by an analysis of anti-pTau PHF1 and anti-A 4G8 immunohistochemistry, performed in the frontal cortex of 21 advanced AD cases (n = 11 APOE.sup.[epsilon]3/[epsilon]3 and n = 10 APOE.sup.[epsilon]4/[epsilon]4). Our dataset includes 1130 and 1330 proteins enriched in IP.sub.PHF1 samples from APOE.sup.[epsilon]3/[epsilon]3 and APOE.sup.[epsilon]4/[epsilon]4 groups (fold change [greater than or equal to] 1.50, IP.sub.PHF1vs IP.sub.IgG ctrl). We identified 80 and 68 proteins as probable pTau interactors in APOE.sup.[epsilon]3/[epsilon]3 and APOE.sup.[epsilon]4/[epsilon]4 groups, respectively (SAINT score [greater than or equal to] 0.80; false discovery rate (FDR) 5%). A total of 47/80 proteins were identified as more likely to interact with pTau in APOE.sup.[epsilon]3/[epsilon]3 vs APOE.sup.[epsilon]4/[epsilon]4 cases. Functional enrichment analyses showed that they were significantly associated with the nucleoplasm compartment and involved in RNA processing. In contrast, 35/68 proteins were identified as more likely to interact with pTau in APOE.sup.[epsilon]4/[epsilon]4 vs APOE.sup.[epsilon]3/[epsilon]3 cases. They were significantly associated with the synaptic compartment and involved in cellular transport. A characterization of Tau pathology in the frontal cortex showed a higher density of plaque-associated neuritic crowns, made of dystrophic axons and synapses, in APOE.sup.[epsilon]4 carriers. Cerebral amyloid angiopathy was more frequent and severe in APOE.sup.[epsilon]4/[epsilon]4 cases. Our study supports an influence of APOE genotype on pTau-subcellular location in AD. These results suggest a facilitation of pTau progression to A-affected brain regions in APOE.sup.[epsilon]4 carriers, paving the way to the identification of new therapeutic targets.

Hyperphosphorylated tau (pTau) in Alzheimer's disease (AD) brain tissue is a complex mix of multiple tau species that are variably phosphorylated on up to 55 epitopes. Emerging studies suggest that phosphorylation of specific epitopes may alter the role of tau. The role of specific pTau species can be explored through protein interaction (\"interactome\") studies. These provide knowledge about the role of individual pTau species and critical context for tau-focused biomarker and drug-discovery studies. The aim of this study was to analyse the interactome of pTau217, which biomarker studies suggest is one of the earliest accumulating tau species in AD. pTau217 interactors were identified in fresh-frozen human brain tissue from 10 cases of advanced AD using affinity purification-mass spectrometry. Cases were balanced for ApoE ε3/ε3 and ε4/ε4 genotypes (n = 5 each) to explore if ApoE influences pTau protein interactions. Results were compared to our previous pTauS396/S404 interactome dataset generated using the same cases to determine if individual pTau species have different interactomes. 579 proteins were significantly enriched by pTau217 co-immunoprecipitation in comparison to control IgG (p < 0.05; fold change >1.5). Of these, 23 proteins were identified as bona fide pTau217 interactors (SAINT score > 0.65), including known pTau interactors SQSTM1 and ubiquitin. Phosphorylation analysis of tau enriched by pTau217 vs pTau396/S404 co-immunoprecipitation confirmed enrichment of a different pools of tau, with pTau217 displaying fewer phosphorylated epitopes, hinting that pTau217 may be an earlier generated species. Despite these differences, 15 bona fide pTau217 tau interactors also interacted with pTauS396/S404 suggesting close similarities in interactomes (Fisher's exact p = 2.3 × 10 ). Common interactors notably included five subunits of an E3 ubiquitin ligase not previously been linked to tau or AD. 46 and 28 pTau217 interactors were identified in ApoE ε3/ε3 and ApoE ε4/ε4 cases respectively and these significantly overlapped (16 common interactors; Fisher's exact p = 4.3 × 10 ). pTau217 interacts with many similar proteins to pTauS396/S404. Our results highlight a strong interaction between multiple pTau species and a novel E3 ubiquitin ligase, which may have an important role in AD and potentially tau degradation.

Background Hyperphosphorylated tau (pTau) in Alzheimer’s disease (AD) brain tissue is a complex mix of multiple tau species that are variably phosphorylated on up to 55 epitopes. Emerging studies suggest that phosphorylation of specific epitopes may alter the role of tau. The role of specific pTau species can be explored through protein interaction (“interactome”) studies. These provide knowledge about the role of individual pTau species and critical context for tau‐focused biomarker and drug‐discovery studies. The aim of this study was to analyse the interactome of pTau217, which biomarker studies suggest is one of the earliest accumulating tau species in AD. Methods pTau217 interactors were identified in fresh‐frozen human brain tissue from 10 cases of advanced AD using affinity purification‐mass spectrometry. Cases were balanced for ApoE ε3/ε3 and ε4/ε4 genotypes (n = 5 each) to explore if ApoE influences pTau protein interactions. Results were compared to our previous pTauS396/S404 interactome dataset generated using the same cases to determine if individual pTau species have different interactomes. Results 579 proteins were significantly enriched by pTau217 co‐immunoprecipitation in comparison to control IgG (p < 0.05; fold change >1.5). Of these, 23 proteins were identified as bona fide pTau217 interactors (SAINT score > 0.65), including known pTau interactors SQSTM1 and ubiquitin. Phosphorylation analysis of tau enriched by pTau217 vs pTau396/S404 co‐immunoprecipitation confirmed enrichment of a different pools of tau, with pTau217 displaying fewer phosphorylated epitopes, hinting that pTau217 may be an earlier generated species. Despite these differences, 15 bona fide pTau217 tau interactors also interacted with pTauS396/S404 suggesting close similarities in interactomes (Fisher’s exact p = 2.3 × 10‐14). Common interactors notably included five subunits of an E3 ubiquitin ligase not previously been linked to tau or AD. 46 and 28 pTau217 interactors were identified in ApoE ε3/ε3 and ApoE ε4/ε4 cases respectively and these significantly overlapped (16 common interactors; Fisher’s exact p = 4.3 × 10‐24). Conclusions pTau217 interacts with many similar proteins to pTauS396/S404. Our results highlight a strong interaction between multiple pTau species and a novel E3 ubiquitin ligase, which may have an important role in AD and potentially tau degradation.

Hyperphosphorylated tau (pTau) in Alzheimer’s disease (AD) brain tissue is a complex mix of multiple tau species that are variably phosphorylated. The emerging studies suggest that phosphorylation of specific residues may alter the role of tau. The role of specific pTau species can be explored through protein interactome studies. The aim of this study was to analyse the interactome of tau phosphorylated at T217 (pT217), which biomarker studies suggest is one of the earliest accumulating tau species in AD. pT217 interactors were identified in fresh-frozen human brain tissue from 10 cases of advanced AD using affinity purification-mass spectrometry. The cases included a balanced cohort of APOE ε3/ε3 and ε4/ε4 genotypes ( n  = 5 each) to explore how apolipoprotein E altered phosphorylated tau interactions. The results were compared to our previous interactome dataset that profiled the interactors of PHF1-enriched tau to determine if individual pTau species have different interactomes. 23 proteins were identified as bona fide pT217 interactors, including known pTau interactor SQSTM1. pT217 enriched tau was phosphorylated at fewer residues compared to PHF1-enriched tau, suggesting an earlier stage of pathology development. Notable pT217 interactors included five subunits of the CTLH E3 ubiquitin ligase (WDR26, ARMC8, GID8, RANBP9, MAEA), which has not previously been linked to AD. In APOE ε3/ε3 cases pT217 significantly interacted with 46 proteins compared to 28 in APOE ε4/ε4 cases, but these proteins were significantly overlapped. CTLH E3 ubiquitin ligase subunits significantly interacted with phosphorylated tau in both APOE genotypes. pT217 interactions with SQSTM1, WDR26 and RANBP9 were validated using co-immunoprecipitation and immunofluorescent microscopy of post-mortem human brain tissue, which showed colocalisation of both protein interactors with tau pathology. Our results report the interactome of pT217 in human Alzheimer’s disease brain tissue for the first time and highlight the CTLH E3 ubiquitin ligase complex as a significant novel interactor of pT217 tau.