Ultrastructural alterations of Alzheimer's disease paired helical filaments by grape seed-derived polyphenols.

Abnormal folding of the microtubule-associated protein tau leads to aggregation of tau into paired helical filaments (PHFs) and neurofibrillary tangles, the major hallmark of Alzheimer's disease (AD). We have recently shown that grape seed polyphenol extract (GSPE) reduces tau pathology in the TMHT mouse model of tauopathy (Wang et al., 2010). In the present studies we assessed the impact of GSPE exposure on the ultrastructure of PHFs isolated from Alzheimer's disease brain. Transmission electron microscopy revealed that GSPE induced profound dose- and time-dependent alterations in the morphology of PHFs with partial disintegration of filaments. Filaments showed ∼2-fold enlargement in width and displayed numerous protrusions and splayed ends consistent with unfolding of tau and diminished structural stability. In addition, GSPE induced a reduction in immunogold labeling with antibodies against the C-terminal half (12E8, PHF-1) and the middle region of tau (AT8, Tau5, pSer214 tau, and AT180) but not the C-terminal end (Tau46). In comparison, labeling of N-terminus (Alz50) was enhanced. It is unlikely that alterations in immunogold labeling were due to biochemical alterations, e.g., protein phosphatase or proteolytic activities potentially stimulated by GSPE, because western blotting studies have shown the preservation of full length polypeptides of tau and their phospho-epitopes in GSPE-treated samples. The GSPE mechanism may include a noncovalent interaction of polyphenols with proline residues in the proline-rich domain of tau, with Pin1 sites at P213 and P232 most seriously affected as judged by suppression of labeling. Collectively, our results suggest that GSPE has a significant potential for therapeutic development by neutralizing phospho-epitopes and disrupting fibrillary conformation leading to disintegration of PHFs.

GSPE interferes with tau aggregation in vivo: implication for treating tauopathy.

Tauopathies are characterized by progressive neurodegeneration caused by intracellular accumulation of hyperphosphorylated tau protein aggregates in the brain. The present study was designed to test whether a grape seed polyphenolic extract (GSPE) previously shown to inhibit tau protein aggregation in vitro could benefit tau-mediated neuropathology and behavior deficits in JNPL3 transgenic mice expressing a human tau protein containing the P301L mutation. Nine-month-old JNPL3 mice were treated with GSPE delivered through their drinking water for 6 months. We found that GSPE treatment significantly reduced the number of motor neurons immunoreactive for hyperphosphorylated and conformationally-modified tau in the ventral horns of the spinal cord identified using AT100, PHF-1, AT8, and Alz50 tau antibodies. This coincided with a drastically reduced level of hyperphosphorylated and sarcosyl-insoluble tau in spinal cord fractions. Furthermore, the reduction of tau pathology was accompanied by an improvement in the motor function assessed by a wire hang test. Collectively, our results suggest that GSPE can interfere with tau-mediated neurodegenerative mechanisms and ameliorate neurodegenerative phenotype in an animal model of tauopathy. Our studies support further evaluation of GSPE for preventing and/or treating of tauopathies in humans.

Grape derived polyphenols attenuate tau neuropathology in a mouse model of Alzheimer's disease.

Aggregation of microtubule-associated protein tau into insoluble intracellular neurofibrillary tangles is a characteristic hallmark of Alzheimer's disease (AD) and other neurodegenerative diseases, including progressive supranuclear palsy, argyrophilic grain disease, corticobasal degeneration, frontotemporal dementias with Parkinsonism linked to chromosome 17, and Pick's disease. Tau is abnormally hyperphosphorylated in AD and aberrant tau phosphorylation contributes to the neuropathology of AD and other tauopathies. Anti-aggregation and anti-phosphorylation are main approaches for tau-based therapy. In this study, we report that a select grape-seed polyphenol extract (GSPE) could potently interfere with the assembly of tau peptides into neurotoxic aggregates. Moreover, oral administration of GSPE significantly attenuated the development of AD type tau neuropathology in the brain of TMHT mouse model of AD through mechanisms associated with attenuation of extracellular signal-receptor kinase 1/2 signaling in the brain.

Development of a grape seed polyphenolic extract with anti-oligomeric activity as a novel treatment in progressive supranuclear palsy and other tauopathies.

A diverse group of neurodegenerative diseases - including progressive supranuclear palsy (PSP), corticobasal degeneration and Alzheimer's disease among others, collectively referred to as tauopathies - are characterized by progressive, age-dependent intracellular formations of misfolded protein aggregates that play key roles in the initiation and progression of neuropathogenesis. Recent studies from our laboratory reveal that grape seed-derived polyphenolic extracts (GSPE) potently prevent tau fibrillization into neurotoxic aggregates and therapeutically promote the dissociation of preformed tau aggregates [J. Alzheimer's Dis. (2009) vol. 16, pp. 433]. Based on our extensive bioavailability, bioactivity and functional preclinical studies, combined with the safety of GSPE in laboratory animals and in humans, we initiated a series of studies exploring the role of GSPE (Meganatural-Az(®) GSPE) as a potential novel botanical drug for the treatment of certain forms of tauopathies including PSP, a neurodegenerative disorder involving the accumulation and deposition of misfolded tau proteins in the brain characterized, in part, by abnormal intracellular tau inclusions in specific anatomical areas involving astrocytes, oligodendrocytes and neurons [J. Neuropathol. Exp. Neurol. (2002) vol. 61, pp. 33]. In this mini-review article, we discuss the biochemical characterization of GSPE in our laboratory and its potential preventative and therapeutic role in model systems of abnormal tau processing pertinent to PSP and related tauopathies.

Characterization of paired helical filaments by scanning transmission electron microscopy.

Paired helical filaments (PHFs) are abnormal twisted filaments composed of hyperphosphorylated tau protein. They are found in Alzheimer's disease and other neurodegenerative disorders designated as tauopathies. They are a major component of intracellular inclusions known as neurofibrillary tangles (NFTs). The objective of this review is to summarize various structural studies of PHFs in which using scanning transmission electron microscopy (STEM) has been particularly informative. STEM provides shape and mass per unit length measurements important for studying ultrastructural aspects of filaments. These include quantitative comparisons between dispersed and aggregated populations of PHFs as well as comparative studies of PHFs in Alzheimer's disease and other neurodegenerative disorders. Other approaches are also discussed if relevant or complementary to studies using STEM, e.g., application of a novel staining reagent, Nanovan. Our understanding of the PHF structure and the development of PHFs into NFTs is presented from a historical perspective. Others goals are to describe the biochemical and ultrastructural complexity of authentic PHFs, to assess similarities between authentic and synthetic PHFs, and to discuss recent advances in PHF modeling.