Naruhiko Sahara

GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimers disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.

Hyperphosphorylated tau in parahippocampal cortex impairs place learning in aged mice expressing wild-type human tau

To investigate how tau affects neuronal function during neurofibrillary tangle (NFT) formation, we examined the behavior, neural activity, and neuropathology of mice expressing wild‐type human tau. Here, we demonstrate that aged (>20 months old) mice display impaired place learning and memory, even though they do not form NFTs or display neuronal loss. However, soluble hyperphosphorylated tau and synapse loss were found in the same regions. Mn‐enhanced MRI showed that the activity of the parahippocampal area is strongly correlated with the decline of memory as assessed by the Morris water maze. Taken together, the accumulation of hyperphosphorylated tau and synapse loss in aged mice, leading to inhibition of neural activity in parahippocampal areas, including the entorhinal cortex, may underlie place learning impairment. Thus, the accumulation of hyperphosphorylated tau that occurs before NFT formation in entorhinal cortex may contribute to the memory problems seen in Alzheimers disease (AD).

Molecular chaperone-mediated tau protein metabolism counteracts the formation of granular tau oligomers in human brain.

Intracellular accumulation of filamentous tau proteins is a defining feature of neurodegenerative diseases termed tauopathies. The pathogenesis of tauopathies remains largely unknown. Molecular chaperones such as heat shock proteins (HSPs), however, have been implicated in tauopathies as well as in other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates. To search for in vivo evidence of chaperone‐related tau protein metabolism, we analyzed human brains with varying degrees of neurofibrillary tangle (NFT) pathology, as defined by Braak NFT staging. Quantitative analysis of soluble protein levels revealed significant positive correlations between tau and Hsp90, Hsp40, Hsp27, α‐crystallin, and CHIP. An inverse correlation was observed between the levels of HSPs in each specimen and the levels of granular tau oligomers, the latter of which were isolated from brain as intermediates of tau filaments. We speculate that HSPs function as regulators of soluble tau protein levels, and, once the capacity of this chaperone system is saturated, granular tau oligomers form virtually unabated. This is expressed pathologically as an early sign of NFT formation. The molecular basis of chaperone‐mediated protection against neurodegeneration might lead to the development of therapeutics for tauopathies.

Identification and characterization of presenilin I-467, I-463 and I-374.

We cloned a novel isoform of presenilin I (presenilin I‐374) besides previously published presenilin I‐467 and I‐463 in human lymphocytes. Presenilin I‐463 was identical to presenilin I‐467 except a 12 bp nucleotides deletion in its amino terminal region. Another isoform, presenilin I‐374 was produced by an alternative splicing with an additional exon consisting of 92 bp nucleotides (exon 11), which resulted in the frame shift with a stop codon to generate a truncated presenilin consisting of 374 amino acids. The transcripts for presenilin I‐4671463 was ubiquitously detected while that for presenilin I‐374 was selectively detected in liver, spleen, kidney. Abnormal behavior of presenilin I on gel electrophoresis was found with affinity‐purified antibodies against presenilin I.We cloned a novel isoform of presenilin I (presenilin I-374) besides previously published presenilin I-467 and I-463 in human lymphocytes. Presenilin I-463 was identical to presenilin I-467 except a 12 bp nucleotides deletion in its amino terminal region. Another isoform, presenilin I-374 was produced by an alternative splicing with an additional exon consisting of 92 bp nucleotides (exon 11), which resulted in the frame shift with a stop codon to generate a truncated presenilin consisting of 374 amino acids. The transcripts for presenilin I-4671463 was ubiquitously detected while that for presenilin I-374 was selectively detected in liver, spleen, kidney. Abnormal behavior of presenilin I on gel electrophoresis was found with affinity-purified antibodies against presenilin I.

Active c-jun N-terminal kinase induces caspase cleavage of tau and additional phosphorylation by GSK-3β is required for tau aggregation

Neurofibrillary tangles (NFTs), comprising human intracellular microtubule‐associated protein tau, are one of the hallmarks of tauopathies, including Alzheimer’s disease. Recently, a report that caspase‐cleaved tau is present in NFTs has led to the hypothesis that the mechanisms underlying NFT formation may involve the apoptosis cascade. Here, we show that adenoviral infection of tau into COS‐7 cells induces activation of c‐jun N‐terminal kinase (JNK), followed by excessive phosphorylation of tau and its cleavage by caspase. However, JNK activation alone was insufficient to induce sodium dodecyl sulfate (SDS)‐insoluble tau aggregation and additional phosphorylation by GSK‐3β was required. In SH‐SY5Y neuroblastoma cells, overexpression of active JNK and GSK‐3β increased caspase‐3 activation and cytotoxicity more than overexpression of tau alone. Taken together, these results indicate that, although JNK activation may be a primary inducing factor, further phosphorylation of tau is required for neuronal death and NFT formation in neurodegenerative diseases, including those characterized by tauopathy.

Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies

Diverse neurodegenerative disorders are characterized by deposition of tau fibrils composed of conformers (i.e. strains) unique to each illness. The development of tau imaging agents has enabled visualization of tau lesions in tauopathy patients, but the modes of their binding to different tau strains remain elusive. Here we compared binding of tau positron emission tomography ligands, PBB3 and AV-1451, by fluorescence, autoradiography and homogenate binding assays with homologous and heterologous blockades using tauopathy brain samples. Fluorescence microscopy demonstrated intense labelling of non-ghost and ghost tangles with PBB3 and AV-1451, while dystrophic neurites were more clearly detected by PBB3 in brains of Alzheimers disease and diffuse neurofibrillary tangles with calcification, characterized by accumulation of all six tau isoforms. Correspondingly, partially distinct distributions of autoradiographic labelling of Alzheimers disease slices with 11C-PBB3 and 18F-AV-1451 were noted. Neuronal and glial tau lesions comprised of 4-repeat isoforms in brains of progressive supranuclear palsy, corticobasal degeneration and familial tauopathy due to N279K tau mutation and 3-repeat isoforms in brains of Picks disease and familial tauopathy due to G272V tau mutation were sensitively detected by PBB3 fluorescence in contrast to very weak AV-1451 signals. This was in line with moderate 11C-PBB3 versus faint 18F-AV-1451 autoradiographic labelling of these tissues. Radioligand binding to brain homogenates revealed multiple binding components with differential affinities for 11C-PBB3 and 18F-AV-1451, and higher availability of binding sites on progressive supranuclear palsy tau deposits for 11C-PBB3 than 18F-AV-1451. Our data indicate distinct selectivity of PBB3 compared to AV-1451 for diverse tau fibril strains. This highlights the more robust ability of PBB3 to capture wide-range tau pathologies.

PET Quantification of Tau Pathology in Human Brain with 11C-PBB3

Tau accumulation in the brain is a pathologic hallmark of Alzheimer disease and other tauopathies. Quantitative visualization of tau pathology in humans can be a powerful method as a diagnostic aid and for monitoring potential therapeutic interventions. We established methods of PET quantification of tau pathology with 11C-PBB3 (2-((1E,3E)-4-(6-(11C-methylamino)pyridin-3-yl)buta-1,3-dienyl) benzo[d]thiazol-6-ol), considering its radiometabolite entering the brain. Methods: Seven Alzheimer disease patients and 7 healthy subjects underwent dynamic 11C-PBB3 PET scanning. Arterial blood was sampled to obtain the parent and metabolite input functions. Quantification of 11C-PBB3 binding was performed using dual-input models that take the brain metabolite activity into consideration, traditional single-input models without such considerations, and the reference tissue model (MRTMO) and standardized uptake value ratio (SUVR). The cerebellar cortex was used as the reference tissue for all methods. Results: The dual-input graphical models estimated binding parameter (BPND*) stably (∼0.36 in high-binding regions). The MRTMO BPND* matched the corresponding BPND* by the dual-input graphical model (r2 = 1.00). SUVR minus 1 correlated well with MRTMO BPND* (r2 > 0.97). However, BPND by the single-input models did not correlate with BPND* by the dual-input graphical model (r2 = 0.04). Conclusion: The dual-input graphical model BPND* is consistent with the reference tissue BPND* and SUVR-1, suggesting that these parameters can accurately quantify binding of 11C-PBB3 despite the entry of its radiometabolites into the brain.

Tau oligomers, what we know and what we don't know

Neurofibrillary tangles, composed of intracellular aggregates of tau protein, are a key neuropathological feature of Alzheimer’s disease and other neurodegenerative diseases, collectively termed tauopathies. Tau research has become one of the central players in the investigation of neurodegenerative diseases. Tau protein has several unique characteristics such as natively unfolded conformation, thermo-stability, acid-stability, and capability of post-translational modifications. We still do not know whether tau itself is toxic. With certain triggers, tau may transit into toxic forms. Researchers are now looking for “tau oligomers” as toxic components. Because “tau oligomers” contain variable species of tau protein [e.g., dimer (disulfide bond-dependent or -independent), multimer (more than dimer), granular (defined as EM or AFM) and perhaps small filamentous aggregates] (Figure u200b(Figure1),1), it is important to have a consensus regarding the definition, terminology, and methodology for the identification of “tau oligomers” (1–6). n n n nFigure 1 n nSchematic illustration of the central role of tau oligomers in tauopathies. Figure taken from Gerson and Kayed (1). n n n nRecently, “prion-like” toxicity and propagation mechanisms underlying the progression of disease have been proposed. With this concept, tau may have the ability to translocate between neurons and amplify toxic components (7). Although we do not know the exact forms of toxic tau oligomers, accumulating evidence has shown the probability of tau oligomer propagation (6). n nTau is an intracellular microtubule-associated protein. The mechanism of tau transmission from cell to cell is still unknown. Research focusing on extracellular tau will open potential new avenues for discovering the mechanism of tau propagation (8). n nAbnormally hyperphosphorylated tau is a key feature of human tauopathies. Although we are not sure whether phosphorylation rather than oligomerization of tau is an initial molecular event in tau pathogenesis, investigating the regulatory mechanisms of tau phosphorylation will be essential (9–11). n nHere, we provide an overview of the current understandings of “tau oligomers” (1–12). Efforts toward the identification of neurotoxic tau species will ultimately lead to the translational research for developing novel therapeutic strategies for tauopathies.

Amyloid-β-protein isoforms in brain of subjects with PS1-linked, βAPP-linked and sporadic alzheimer disease

To determine whether similar abnormalities of various soluble full-length and N-terminal truncated Abeta peptides occur in postmortem cerebral cortex of affected PS1 mutation carriers, we examined the amounts of two amyloid species ending at residue 40 or at residues 42(43) using sandwich ELISA systems. Our results indicate that PS1 mutations effect a dramatic accumulation in brain of the highly insoluble potentially neurotoxic long-tailed isoforms of the Abeta peptide such as Abeta1-42(43) and Abetax-42(43). This enhancing effect of PS1 mutation on Abetax-42(43) deposition was highly similar to that of a betaAPP mutation (Val717Ile) but the effects on Abetax-40 production were significantly different between these two causal genes. In contrast to previous studies of soluble Abeta in plasma and in supernatants from cultured fibroblasts of subjects with PS1 mutations, our studies also show that there is an increase in insoluble Abetax-40 peptides in brain of subjects with PS1 mutations.

Calcium dysregulation contributes to neurodegeneration in FTLD patient iPSC-derived neurons.

Mutations in the gene MAPT encoding tau, a microtubules-associated protein, cause a subtype of familial neurodegenerative disorder, known as frontotemporal lobar degeneration tauopathy (FTLD-Tau), which presents with dementia and is characterized by atrophy in the frontal and temporal lobes of the brain. Although induced pluripotent stem cell (iPSC) technology has facilitated the investigation of phenotypes of FTLD-Tau patient neuronal cells in vitro, it remains unclear how FTLD-Tau patient neurons degenerate. Here, we established neuronal models of FTLD-Tau by Neurogenin2-induced direct neuronal differentiation from FTLD-Tau patient iPSCs. We found that FTLD-Tau neurons, either with an intronic MAPT mutation or with an exonic mutation, developed accumulation and extracellular release of misfolded tau followed by neuronal death, which we confirmed by correction of the intronic mutation with CRISPR/Cas9. FTLD-Tau neurons showed dysregulation of the augmentation of Ca2+ transients evoked by electrical stimulation. Chemogenetic or pharmacological control of neuronal activity-relevant Ca2+ influx by the introduction of designer receptors exclusively activated by designer drugs (DREADDs) or by the treatment with glutamate receptor blockers attenuated misfolded tau accumulation and neuronal death. These data suggest that neuronal activity may regulate neurodegeneration in tauopathy. This FTLD-Tau model provides mechanistic insights into tauopathy pathogenesis and potential avenues for treatments.