An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer's disease (AD). The extract can also promote complete disassembly of recombinant tau filaments and cause substantial alteration of the morphology of paired-helical filaments isolated from AD brain. Cinnamon extract (CE) was not deleterious to the normal cellular function of tau, namely the assembly of free tubulin into microtubules. An A-linked proanthocyanidin trimer molecule was purified from the extract and shown to contain a significant proportion of the inhibitory activity. Treatment with polyvinylpyrolidone effectively depleted all proanthocyanidins from the extract solution and removed the majority, but not all, of the inhibitory activity. The remainder inhibitory activity could be attributed to cinnamaldehyde. This work shows that compounds endogenous to cinnamon may be beneficial to AD themselves or may guide the discovery of other potential therapeutics if their mechanisms of action can be discerned.

Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.

Alzheimer's disease (AD) is characterized by the intracellular accumulation of the neurofibrillary tangles comprised mainly of the microtubule-associated protein, tau. A critical aspect of understanding tangle formation is to understand the transition of soluble monomeric tau into mature fibrils by characterizing the structure of intermediates along the aggregation pathway. We have carried out multidimensional NMR studies on a C-terminal fragment of human tau (tau (187)) to gain structural insight into the aggregation process. To specifically monitor intermolecular interaction between tau molecules in solution, we combined (15)N- and (14)N-labeled tau, the latter of which was modified with a paramagnetic nitroxide spin label (MTSL). Paramagnetic relaxation enhancement (PRE) of (15)N-tau by interaction with MTSL- (14)N-tau allowed identification of low molecular weight oligomers of tau (187) that formed in response to heparin-induced aggregation. Two regions, VQIINK (280) and VQIVYK (311), were exclusively broadened by MTSL located at varied positions in the tau molecule. We propose that soluble oligomers of tau (187) are generated via intermolecular interactions at these motifs triggered by heparin addition. However, the associated line broadening at these motifs cannot be due to interaction between tau (187) and heparin directly. Instead, these specific interactions necessarily occur between tau molecules and are intermolecular in nature. Our data support the idea that VQIINK (280) and VQIVYK (311) are the major, if not sole, critical regions that directly mediate intermolecular contact between tau molecules during the early phases of aggregation.

We present a generally applicable approach for monitoring protein aggregation by detecting changes in surface hydration water dynamics and the changes in solvent accessibility of specific protein sites, as protein aggregation proceeds in solution state. This is made possible through the Overhauser dynamic nuclear polarization (DNP) of water interacting with stable nitroxide spin labels tethered to specific proteins sites. This effect is highly localized due to the magnetic dipolar nature of the electron–proton spin interaction, with >80 % of their interaction occurring within 5 Å between the unpaired electron of the spin label and the proton of water. We showcase our tool on the aggregation of tau proteins, whose fibrillization is linked to neurodegenerative disease pathologies known as taupathies. We demonstrate that the DNP approach to monitor local changes in hydration dynamics with residue specificity and local contrast can distinguish specific and neat protein-protein packing leading to fibers from non-specific protein agglomeration or precipitation. The ability to monitor tau assembly with local, residue-specific, resolution, under ambient condition and in solution state will help unravel the mechanism and structural characteristics of the gradual process of tau aggregation into amyloid fibers, which remains unclear to this day.

CDK5/p35 is a cyclin-dependent kinase essential for normal neuron function. Proteolysis of the p35 subunit in vivo results in CDK5/p25 that causes neurotoxicity associated with a number of neurodegenerative diseases. Whereas the mechanism by which conversion of p35 to p25 leads to toxicity is unknown, there is common belief that CDK5/p25 is catalytically hyperactive compared to CDK5/p35. Here, we have compared the steady-state kinetic parameters of CDK5/p35 and CDK5/p25 towards both histone H1, the best known substrate for both enzymes, and the microtubule-associated protein, tau, a physiological substrate whose in vivo phosphorylation is relevant to Alzheimer's disease. We show that the kinetics of both enzymes are the same towards either substrate in vitro. Furthermore, both enzymes display virtually identical kinetics towards individual phosphorylation sites in tau monitored by NMR. We conclude that conversion of p35 to p25 does not alter the catalytic efficiency of the CDK5 catalytic subunit by using histone H1 or tau as substrates, and that neurotoxicity associated with CDK5/p25 is unlikely attributable to CDK5 hyperactivation, as measured in vitro.Alzheimer's disease | neurotoxicity | NMR | protein kinase | proteolysis C DK5/p25 was first identified as a tau kinase (1, 2) that displayed a CDK1 (formerly p34 cdc2 ) -like substrate specificity in brain (3, 4). Of particular interest, phosphorylation of normal tau by CDK5/p25 could partially recapitulate several characteristics inherent to PHF-tau associated with Alzheimer's disease (1, 2). The CDK5 catalytic subunit was homologous to other CDKs and displayed ubiquitous expression (5), but p25 was originally discovered as a unique protein expressed predominantly in neurons, and generated by proteolytic cleavage of a larger protein precursor, p35 (6-9). Whereas CDK5/p35 is associated with normal neuron development and function (10), endogenous cleavage of p35 to p25 is associated with neuronal cell death, neuropathology, and is implicated in the progression of Alzheimer's disease (11-18), Parkinson's disease (19), and ALS (20). CDK5/p25, but not CDK5/p35, is toxic when overexpressed in transformed cell lines or when generated by cleavage of endogenous p35 in neurons (11,21,22). The cellular mechanism by which cleavage causes toxicity is not known, but in theory could relate to one or more known key differences between these two enzyme forms, including differences in subcellular distribution (11, 23), stability of the protein (11, 24), and/or cellular substrate specificity (11,25).There is common belief that the catalytic activity of CDK5/p25 is significantly elevated in comparison to CDK5/p35, and therefore that p25 causes "hyperactivity" of CDK5 compared to p35, contributing to its toxicity (10). To rigorously demonstrate its hyperactivity, however, it is necessary to show that the catalytic parameters associated with steady-state phosphorylation of substrates are different between the two enzymes. Hashiguchi et al. (26) previously conducted such experiments,...