Aß∗56 is a stable oligomer that impairs memory function in mice.

Amyloid-ß (Aß) oligomers consist of fibrillar and non-fibrillar soluble assemblies of the Aß peptide. Aß∗56 is a non-fibrillar Aß assembly that is linked to memory deficits. Previous studies did not decipher specific forms of Aß present in Aß∗56. Here, we confirmed the memory-impairing characteristics of Aß∗56 and extended its biochemical characterization. We used anti-Aß(1-x), anti-Aß(x-40), anti-Aß(x-42), and A11 anti-oligomer antibodies in conjunction with western blotting, immunoaffinity purification, and size-exclusion chromatography to probe aqueous brain extracts from Tg2576, 5xFAD, and APP/TTA mice. In Tg2576, Aß∗56 is a ∼56-kDa, SDS-stable, A11-reactive, non-plaque-dependent, water-soluble, brain-derived oligomer containing canonical Aß(1-40). In 5xFAD, Aß∗56 is composed of Aß(1-42), whereas in APP/TTA, it contains both Aß(1-40) and Aß(1-42). When injected into the hippocampus of wild-type mice, Aß∗56 derived from Tg2576 mice impairs memory. The unusual stability of this oligomer renders it an attractive candidate for studying relationships between molecular structure and effects on brain function.

Aß*56 is a stable oligomer that correlates with age-related memory loss in Tg2576 mice.

Amyloid-ß (Aß) oligomers consist of fibrillar and non-fibrillar soluble assemblies of the Aß peptide. Tg2576 human amyloid precursor protein (APP)-expressing transgenic mice modeling Alzheimer's disease produce Aß*56, a non-fibrillar Aß assembly that has been shown by several groups to relate more closely to memory deficits than plaques. Previous studies did not decipher specific forms of Aß present in Aß*56. Here, we confirm and extend the biochemical characterization of Aß*56. We used anti-Aß(1-x), anti-Aß(x-40), and A11 anti-oligomer antibodies in conjunction with western blotting, immunoaffinity purification, and size-exclusion chromatography to probe aqueous brain extracts from Tg2576 mice of different ages. We found that Aß*56 is a ∼56-kDa, SDS-stable, A11-reactive, non-plaque-related, water-soluble, brain-derived oligomer containing canonical Aß(1-40) that correlates with age-related memory loss. The unusual stability of this high molecular-weight oligomer renders it an attractive candidate for studying relationships between molecular structure and effects on brain function.

Caspase-2 mRNA levels are not elevated in mild cognitive impairment, Alzheimer's disease, Huntington's disease, or Lewy Body dementia.

Caspase-2 is a member of the caspase family that exhibits both apoptotic and non-apoptotic properties, and has been shown to mediate synaptic deficits in models of several neurological conditions, including Alzheimer's disease (AD), Huntington's disease (HD), and Lewy Body dementia (LBD). Our lab previously reported that caspase-2 protein levels are elevated in these diseases, leading us to hypothesize that elevated caspase-2 protein levels are due to increased transcription of caspase-2 mRNA. There are two major isoforms of caspase-2 mRNA, caspase-2L and caspase-2S. We tested our hypothesis by measuring the levels of these mRNA isoforms normalized to levels of RPL13 mRNA, a reference gene that showed no disease-associated changes. Here, we report no increases in caspase-2L mRNA levels in any of the three diseases studied, AD (with mild cognitive impairment (MCI)), HD and LBD, disproving our hypothesis. Caspase-2S mRNA showed a non-significant downward trend in AD. We also analyzed expression levels of SNAP25 and ßIII-tubulin mRNA. SNAP25 mRNA was significantly lower in AD and there were downward trends in MCI, LBD, and HD. ßIII-tubulin mRNA expression remained unchanged between disease groups and controls. These findings indicate that factors besides transcriptional regulation cause increases in caspase-2 protein levels. The reduction of SNAP25 mRNA expression suggests that presynaptic dysfunction contributes to cognitive deficits in neurodegeneration.

Blocking Site-Specific Cleavage of Human Tau Delays Progression of Disease-Related Phenotypes in Genetically Matched Tau-Transgenic Mice Modeling Frontotemporal Dementia.

Studies have recently demonstrated that a caspase-2-mediated cleavage of human tau (htau) at asparate-314 (D314) is responsible for cognitive deficits and neurodegeneration in mice modeling frontotemporal dementia (FTD). However, these animal studies may be confounded by flaws in their model systems, such as endogenous functional gene disruption and inequivalent transgene expression. To avoid these weaknesses, we examined the pathogenic role of this site-specific htau cleavage in FTD using genetically matched htau targeted-insertion mouse lines: rT2 and rT3. Both male and female mice were included in this study. rT2 mice contain a single copy of the FTD-linked htau proline-to-leucine mutation at amino acid 301 (htau P301L), inserted into a neutral site to avoid dysregulation of host gene expression. The similarly constructed rT3 mice harbor an additional D314-to-glutamate (D314E) mutation that blocks htau cleavage. We demonstrate that htau transgene expression occurs primarily in the forebrain at similar levels in rT2 and rT3 mice. Importantly, expression of the cleavage-resistant D314E mutant delays transgene-induced tau accumulation in the postsynaptic density, brain atrophy, hippocampal neurodegeneration, and spatial memory impairment, without altering age-related progression of pathologic tau conformation and phosphorylation. Our comprehensive investigation of age-dependent disease phenotypes associated with the htau P301L variant in precisely engineered FTD-modeling mice unveils a transiently protective effect of blocking htau cleavage at D314. Findings of this study advance our understanding of the contribution of this tau cleavage to the pathogenesis of FTD, and aid the development of effective dementia-targeting therapies.SIGNIFICANCE STATEMENT A site-specific and caspase-2-mediated cleavage of human tau plays a pathologic role in dementia. In this study, we investigate the contribution of this cleavage to the pathogenesis of frontotemporal dementia (FTD) using two genetically matched, tau-transgene targeted-insertion mouse lines that differ only by a cleavage-resistant mutation. The use of these mice avoids confounding effects associated with the random integration of tau transgenes to the mouse genome and allows us to comprehensively evaluate the impact of the tau cleavage on FTD phenotypes. Our data reveal that blocking this tau cleavage delays memory impairment and neurodegeneration of FTD-modeling mice. These findings improve our understanding of the pathogenic mechanisms underlying FTD and will facilitate the development of effective therapeutics.

Phosphorylation in two discrete tau domains regulates a stepwise process leading to postsynaptic dysfunction.

KEY POINTS: Tau mislocalization to dendritic spines and associated postsynaptic deficits are mediated through different and non-overlapping phosphorylation sites. Tau mislocalization to dendritic spines depends upon the phosphorylation of either Ser396 or Ser404 in the C-terminus. Postsynaptic dysfunction instead depends upon the phosphorylation of at least one of five residues in the proline-rich region of tau. The blockade of both glycogen synthetase kinase 3ß and cyclin-dependent kinase 5 is required to prevent P301L-induced tau mislocalization to dendritic spines, supporting redundant pathways that control tau mislocalization to spines. ABSTRACT: Tau protein consists of an N-terminal projection domain, a microtubule-binding domain and a C-terminal domain. In neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia, the hyperphosphorylation of tau changes its shape, binding partners and resulting function. An early consequence of tau phosphorylation by proline-directed kinases is postsynaptic dysfunction associated with the mislocalization of tau to dendritic spines. The specific phosphorylation sites leading to these abnormalities have not been elucidated. Here, using imaging and electrophysiological techniques to study cultured rat hippocampal neurons, we show that postsynaptic dysfunction results from a sequential process involving differential phosphorylation in the N-terminal and C-terminal domains. First, tau mislocalizes to dendritic spines, in a manner that depends upon the phosphorylation of either Ser396 or Ser404 in the C-terminal domain. The blockade of both glycogen synthetase kinase 3ß and cyclin-dependent kinase 5 prevents tau mislocalization to dendritic spines. Second, a reduction of functional AMPA receptors depends upon the phosphorylation of at least one of five residues (Ser202, Thr205, Thr212, Thr217 and Thr231) in the proline-rich region of the N-terminal domain. This is the first report of differential phosphorylation in distinct tau domains governing separate, but linked, steps leading to synaptic dysfunction.

Author Correction: A soluble truncated tau species related to cognitive dysfunction is elevated in the brain of cognitively impaired human individuals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A soluble truncated tau species related to cognitive dysfunction is elevated in the brain of cognitively impaired human individuals.

Neurofibrillary tangles are a pathological hallmark of Alzheimer's disease, and their levels correlate with the severity of cognitive dysfunction in humans. However, experimental evidence suggests that soluble tau species cause cognitive deficits and memory impairment. Our recent study suggests that caspase-2 (Casp2)-catalyzed tau cleavage at aspartate 314 mediates synaptic dysfunction and memory impairment in mouse and cellular models of neurodegenerative disorders. Δtau314, the C-terminally-truncated cleavage products, are soluble and present in human brain. In addition, levels of Δtau314 proteins are elevated in the brain of the cognitively impaired individuals compared to the cognitively normal individuals, indicating a possible role for Δtau314 proteins in cognitive deterioration. Here we show that (1) Δtau314 proteins are present in the inferior temporal gyrus of human brains; (2) Δtau314 proteins are generated from all six tau splicing isoforms, (3) levels of both Casp2 and Δtau314 proteins are elevated in cognitively impaired individuals compared to cognitively normal individuals, and (4) levels of Δtau314 proteins show a modest predictive value for dementia. These findings advance our understanding of the characteristics of Δtau314 proteins and their relevance to cognitive dysfunction and shed light on the contribution of Casp2-mediated Δtau314 production to cognitive deterioration.

Developmental Pathogenicity of 4-Repeat Human Tau Is Lost with the P301L Mutation in Genetically Matched Tau-Transgenic Mice.

Tau is a microtubule-associated protein that becomes dysregulated in a group of neurodegenerative diseases called tauopathies. Differential tau isoforms, expression levels, promoters, and disruption of endogenous genes in transgenic mouse models of tauopathy make it difficult to draw definitive conclusions about the biological role of tau in these models. We addressed this shortcoming by characterizing the molecular and cognitive phenotypes associated with the pathogenic P301L tau mutation (rT2 mice) in relation to a genetically matched transgenic mouse overexpressing nonmutant (NM) 4-repeat (4R) human tau (rT1 mice). Both male and female mice were included in this study. Unexpectedly, we found that 4R NM human tau (hTau) exhibited abnormal dynamics in young mice that were lost with the P301L mutation, including elevated protein stability and hyperphosphorylation, which were associated with cognitive impairment in 5-month-old rT1 mice. Hyperphosphorylation of NM hTau was observed as early as 4 weeks of age, and transgene suppression for the first 4 or 12 weeks of life prevented abnormal molecular and cognitive phenotypes in rT1, demonstrating that NM hTau pathogenicity is specific to postnatal development. We also show that NM hTau exhibits stronger binding to microtubules than P301L hTau, and is associated with mitochondrial abnormalities. Overall, our genetically matched mice have revealed that 4R NM hTau overexpression is pathogenic in a manner distinct from classical aging-related tauopathy, underlining the importance of assaying the effects of transgenic disease-related proteins at appropriate stages in life.SIGNIFICANCE STATEMENT Due to differences in creation of transgenic lines, the pathological properties of the P301L mutation confers to the tau protein in vivo have remained elusive, perhaps contributing to the lack of disease-modifying therapies for tauopathies. In an attempt to characterize P301L-specific effects on tau biology and cognition in novel genetically matched transgenic mouse models, we surprisingly found that nonmutant human tau has development-specific pathogenic properties of its own. Our findings indicate that overexpression of 4-repeat human tau during postnatal development is associated with excessive microtubule binding, which may disrupt important cellular processes, such as mitochondrial dynamics, leading to elevated stability and hyperphosphorylation of tau, and eventual cognitive impairments.

A soluble tau fragment generated by caspase-2 is associated with dementia in Lewy body disease.

Lewy body diseases are neurodegenerative disorders characterized by Lewy bodies in the brain. Lewy body dementia (LBD) refers to two forms of Lewy body disease: Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). Tau is a cytoskeletal protein found in neurofibrillary tangles, but not Lewy bodies. The gene encoding tau, MAPT, is a well-established genetic risk factor for LBD; odds ratios of the H1:H2 MAPT haplotypes have been reported in the range of 2 to 4. Despite this genetic association, the mechanism by which tau contributes to dementia is unclear. Recently, a soluble form of tau, Δtau314, which is generated when caspase-2 (Casp2) cleaves tau at Asp314, was reported to be associated with impaired cognition in mice modeling frontotemporal dementia, and with mild cognitive impairment and Alzheimer's disease (AD) in humans. To investigate whether Δtau314 is associated with dementia in Lewy body disease, we compared Δtau314 levels in aqueous extracts from the superior temporal gyrus of pathologically confirmed LBD (n = 21) and non-dementia Parkinson's disease (PD) (n = 12). We excluded subjects with AD or microvascular pathology, which could mask potential associations of Δtau314 with LBD.Using a Δtau314-specific ELISA, we found ~ 2-fold higher levels of Δtau314 in LBD relative to PD (p = 0.009). Additionally, we found ~40% lower levels of soluble total tau and the neuronal marker ß-III-tubulin in LBD. These results suggest that in LBD, there is substantial neuron loss or axonal degeneration in the neocortex but disproportionately high levels of Δtau314 in the surviving neurons.Our results indicate an association between Δtau314 and dementia in Lewy body disease. Cleavage of tau by Casp2 promotes the mislocalization of tau to dendritic spines leading to a reduction in postsynaptic AMPA receptors and excitatory neurotransmission, which suggests a mechanism of the synaptic dysfunction underlying cognitive impairment in LBD. These findings support the potential of Casp2 as a novel drug target for treating LBD.

Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice.

The tauopathy-like phenotype observed in the rTg4510 mouse line, in which human tauP301L expression specifically within the forebrain can be temporally controlled, has largely been attributed to high overexpression of mutant human tau in the forebrain region. Unexpectedly, we found that in a different mouse line with a targeted-insertion of the same transgene driven by the same tetracycline-TransActivator (tTA) allele, but with even higher overexpression of tauP301L than rTg4510, atrophy and tau histopathology are delayed, and a different behavioral profile is observed. This suggests that it is not overexpression of mutant human tau alone that contributes to the phenotype in rTg4510 mice. Furthermore we show that the tauopathy-like phenotype seen in rTg4510 requires a ~70-copy tau-transgene insertion in a 244 kb deletion in Fgf14, a ~7-copy tTA-transgene insertion in a 508 kb deletion that disrupts another five genes, in addition to high transgene overexpression. We propose that these additional effects need to be accounted for in any studies using rTg4510.

Grape seed polyphenolic extract specifically decreases aß*56 in the brains of Tg2576 mice.

Amyloid-ß (Aß) oligomers, found in the brains of Alzheimer's disease (AD) patients and transgenic mouse models of AD, cause synaptotoxicity and memory impairment. Grape seed polyphenolic extract (GSPE) inhibits Aß oligomerization in vitro and attenuates cognitive impairment and AD-related neuropathology in the brains of transgenic mice. In the current study, GSPE was administered to Tg2576 mice for a period of five months. Treatment significantly decreased brain levels of Aß*56, a 56-kDa Aß oligomer previously shown to induce memory dysfunction in rodents, without changing the levels of transgenic amyloid-ß protein precursor, monomeric Aß, or other Aß oligomers. These results thus provide the first demonstration that a safe and affordable intervention can lower the levels of a memory-impairing Aß oligomer in vivo and strongly suggest that GSPE should be further tested as a potential prevention and/or therapy for AD.