Akihiko Takashima

Abstract Prior to the formation of amyloid fibrils, the pathological hallmark in tau-related neurodegenerative disease, tau monomers aggregate into a diverse range of oligomers. Granular tau oligomers, consisting of approximately 40 tau protein molecules, are present in the prefrontal cortex of patients at Braak stages I-II, preclinical stages of Alzheimer’s disease (AD). Antibodies to granular tau oligomers as antigens have not been reported. Therefore, we generated new rat monoclonal antibodies by immunization with granular tau oligomers. Three antibodies from different hybridoma clones showed stronger immunoreactivity to granular tau oligomers and tau fibrils compared with monomeric tau. Of the three antibodies, 2D6-2C6 showed 3000-fold greater immunoreactivity in P301L-tau transgenic (rTg4510) mice than in non-transgenic mice, while MC1 antibody, which detects pathological conformations of tau, showed a 5.5-fold increase. These results suggest that 2D6-2C6 recognizes aggregates more specifically than MC1. In AD subjects, 2D6-2C6 recognized neurofibrillary tangles and pretangles, and co-localized within AT8-positive cells containing phosphorylated tau aggregates. The epitope of 2D6-2C6 is the 423–430 amino acid (AA) sequence of C-terminal regions. Taken together, a novel monoclonal antibody, 2D6-2C6, generated by immunization with granular tau oligomers binds to tau aggregates at the 423–430 AA sequence.

Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

BACKGROUND: MAPT is a causative gene in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), a hereditary degenerative disease with various clinical manifestations, including progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease, and frontotemporal dementia. OBJECTIVES: To analyze genetically, biochemically, and pathologically multiple members of two families who exhibited various phenotypes of the disease. METHODS: Genetic analysis included linkage analysis, homozygosity haplotyping, and exome sequencing. We conducted tau protein microtubule polymerization assay, heparin-induced tau aggregation, and western blotting with brain lysate from an autopsy case. We also evaluated abnormal tau aggregation by using anti-tau antibody and PM-PBB3. RESULTS: We identified a variant, c.896_897insACA, p.K298_H299insQ, in the MAPT gene of affected patients. Similar to previous reports, most patients presented with atypical parkinsonism. Biochemical analysis revealed that the mutant tau protein had a reduced ability to polymerize microtubules and formed abnormal fibrous aggregates. Pathological study revealed frontotemporal lobe atrophy, midbrain atrophy, depigmentation of the substantia nigra, and four-repeat tau-positive inclusions in the hippocampus, brainstem, and spinal cord neurons. The inclusion bodies also stained positively with PM-PBB3. CONCLUSIONS: This study confirmed that the insACA mutation caused FTDP-17. The affected patients showed symptoms resembling Parkinson's disease initially and symptoms of progressive supranuclear palsy later. Despite the initial clinical diagnosis of frontotemporal dementia in the autopsy case, the spread of lesions could explain the process of progressive supranuclear palsy. The study of more cases in the future will help clarify the common pathogenesis of MAPT mutations or specific pathogeneses of each mutation.

Alzheimer's disease is a devastating disease that is accompanied by dementia, and its incidence increases with age. However, no interventions have exhibited clear therapeutic effects. We aimed to develop and characterize behavioural tasks that allow the earlier identification of signs preceding dementia that would facilitate the development of preventative and therapeutic interventions for Alzheimer's disease. To this end, we developed a 3D virtual reality task sensitive to the activity of grid cells in the entorhinal cortex, which is the region that first exhibits neurofibrillary tangles in Alzheimer's disease. We investigated path integration (assessed by error distance) in a spatial navigation task sensitive to grid cells in the entorhinal cortex in 177 volunteers, aged 20-89 years, who did not have self-reported dementia. While place memory was intact even in old age, path integration deteriorated with increasing age. To investigate the relationship between neurofibrillary tangles in the entorhinal cortex and path integration deficit, we examined a mouse model of tauopathy (P301S mutant tau-overexpressing mice; PS19 mice). At 6 months of age, PS19 mice showed a significant accumulation of phosphorylated tau only in the entorhinal cortex, associated with impaired path integration without impairments in spatial cognition. These data are consistent with the idea that path integration deficit is caused by the accumulation of phosphorylated tau in the entorhinal cortex. This method may allow the early identification of individuals likely to develop Alzheimer's disease.