Anyang Sun

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production

Neuropathological hallmarks of Alzheimers disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-β peptides (Aβ) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Aβ production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimers disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimers disease. However, the role of Pin1 in APP processing and Aβ production is unknown. Here we show that Pin1 has profound effects on APP processing and Aβ production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Aβ secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Aβ42 (a major toxic species) in brains in an age-dependent manner, with Aβ42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimers disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Aβ production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimers disease.

Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration

The neuropathological hallmarks of Alzheimers disease and other tauopathies include senile plaques and/or neurofibrillary tangles. Although mouse models have been created by overexpressing specific proteins including β-amyloid precursor protein, presenilin and tau, no model has been generated by gene knockout. Phosphorylation of tau and other proteins on serine or threonine residues preceding proline seems to precede tangle formation and neurodegeneration in Alzheimers disease. Notably, these phospho(Ser/Thr)-Pro motifs exist in two distinct conformations, whose conversion in some proteins is catalysed by the Pin1 prolyl isomerase. Pin1 activity can directly restore the conformation and function of phosphorylated tau or it can do so indirectly by promoting its dephosphorylation, which suggests that Pin1 is involved in neurodegeneration; however, genetic evidence is lacking. Here we show that Pin1 expression is inversely correlated with predicted neuronal vulnerability and actual neurofibrillary degeneration in Alzheimers disease. Pin1 knockout in mice causes progressive age-dependent neuropathy characterized by motor and behavioural deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Thus, Pin1 is pivotal in protecting against age-dependent neurodegeneration, providing insight into the pathogenesis and treatment of Alzheimers disease and other tauopathies.

Comparative Analysis of an Improved Thioflavin-S Stain, Gallyas Silver Stain, and Immunohistochemistry for Neurofibrillary Tangle Demonstration on the Same Sections

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimers disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.

Pin1 has opposite effects on wild-type and P301L tau stability and tauopathy.

Tau pathology is a hallmark of many neurodegenerative diseases including Alzheimer disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Genetic tau mutations can cause FTDP-17, and mice overexpressing tau mutants such as P301L tau are used as AD models. However, since no tau mutations are found in AD, it remains unclear how appropriate tau mutant mice are as an AD model. The prolyl isomerase Pin1 binds and isomerizes tau and has been implicated in protecting against neurodegeneration, but whether such Pin1 regulation is affected by tau mutations is unknown. Consistent with earlier findings that Pin1 KO induces tauopathy, here we demonstrate that Pin1 knockdown or KO increased WT tau protein stability in vitro and in mice and that Pin1 overexpression suppressed the tauopathy phenotype in WT tau transgenic mice. Unexpectedly, Pin1 knockdown or KO decreased P301L tau protein stability and abolished its robust tauopathy phenotype in mice. In contrast, Pin1 overexpression exacerbated the tauopathy phenotype in P301L tau mice. Thus, Pin1 has opposite effects on the tauopathy phenotype depending on whether the tau is WT or a P301L mutant, indicating the need for disease-specific therapies for tauopathies.

Localization of β-Secretase Memapsin 2 in the Brain of Alzheimer's Patients and Normal Aged Controls

Chronic accumulation of beta-amyloid in the brain has been shown to result in complex molecular and cellular changes that accompany neurodegeneration in Alzheimers disease (AD). In this study, we examined the expression of a newly identified beta-secretase, memapsin 2 (M2) or beta-site APP cleaving enzyme in deparaffinized sections from 10 AD patients and 10 aged matched controls and in frozen samples of parietal cortex from 11 AD and 8 controls. M2 is mainly expressed in neurons, with high levels in CA4 to CA2 regions and transentorhinal cortex and low or intermediate levels in CA1, subiculum, and granule cells of the dentate gyrus. The majority of AD brains showed an increase of M2 expression in the CA1, but a decrease in the transentorhinal cortex. A subset of controls and AD patients had high M2 expression in parietal neocortex. Double-staining revealed that senile plaques are not directly associated with the soma of M2-expressing neurons. Neurofibrillary tangles were associated with lower M2 expression in AD. These data indicate that beta-secretase M2 may not be straightforwardly involved in amyloid plaque formation in AD brain.

Expression of microsomal epoxide hydrolase is elevated in Alzheimer's hippocampus and induced by exogenous β-amyloid and trimethyl-tin

The brain is a potential target for drugs and environmental toxins. Microsomal epoxide hydrolase (mEH) is one of several critical biotransformation enzymes in xenobiotic metabolism and detoxification. In the present study, we report that the expression of mEH is significantly elevated in the hippocampus and associated cortex, but not in the cerebellum, in Alzheimers disease (AD) patients. A large proportion of the mEH‐positive cells are located around β‐amyloid plaques. The mEH‐positive‐staining cells are astrocytes and pyramidal neurons. Western blotting analysis confirmed increased expression of mEH in AD hippocampal tissues. In primary hippocampal glial culture, β‐amyloid aggregation stimulated mEH expression in the astrocytes, which displayed a patchy distribution. An environmental neurotoxic agent, trimethyl‐tin, also activated mEH expression in rat hippocampus and entorhinal cortex. The present study demonstrates a significant increase in mEH expression in the AD hippocampus, a region showing abundant neuropathology in AD. The expression of mEH could also be elevated by exposure to exogenous β‐amyloid in vitro and environmental toxins in vivo. Our studies suggest that mEH may play a role in pathogenesis of neurodegeneration in response to environmental stress.

A novel fluorescent method for direct visualization of neurofibrillary pathology in Alzheimer's disease

Methods currently available for detecting neurofibrillary pathology are indirect and depend on staining with exogenous chemicals or antibodies. In the present study, we report a novel method named intrinsic fluorescence induction (IFI), which allows direct visualization of neurofibrillary tangles (NFTs), neuropil threads (NTs), and neuritic plaques (NPs) in tissue sections of Alzheimers disease (AD) brain. The IFI method is based on both induction of a red intrinsic fluorescence and quenching red background autofluorescence. The IFI procedure includes sustained hydrophobic treatment, protein secondary structure enhancement and incubation in high concentration of phosphate buffer. Following this procedure, a unique red fluorescence is generated from the structures of NFTs, NTs, and NPs in brain sections from AD patients. Sequential application of mild permanganate oxidation and 1% sodium borohydride selectively removes the red background autofluorescence, while the latter enhances the intrinsic fluorescence of neurofibrillary pathology. Comparative studies reveal that the IFI method is as sensitive as Gallyas silver staining, and more sensitive than Bielschowsky silver staining or PHF-1 immunostaining in detecting NFTs in the pre-alpha layer of entorhinal cortex and the pri-alpha layer of the entorhinal/transentorhinal cortex. Furthermore, the IFI method is sensitive in displaying plaque neurites and threads, but not NFTs in the hippocampus. This novel finding provides a direct method for detecting neurofibrillary pathology in particular regions of AD brain and a novel tool for AD research.

Biophysical and biochemical characterization of the intrinsic fluorescence from neurofibrillary tangles

Recently, we developed a novel fluorescent method named intrinsic fluorescence induction that allows direct visualization of neurofibrillary pathology without introducing exogenous chromogens. In the present study, we further characterized the properties of this novel red fluorescence biophysically, biochemically, and neuropathologically. In vitro spectrofluorometry and in situ emission scan show that the intrinsic fluorescence of neurofibrillary tangles has a long emission wavelength peak at 620 nm and a large Stokes shift of 70 nm. Dephosphorylation of Alzheimers disease brain sections with alkaline phosphatase or denaturation with guanidine only causes a subtle reduction in the induced fluorescence of neurofibrillary tangles, while hydrofluoric acid or formic acid completely eliminates the fluorescence. Chemical modification of residue serine, but not tyrosine or tryptophan, reduced the intensity of induced fluorescence significantly. The induced fluorophore, thus, has unique properties, and its generation likely depends on the particular conformation of paired helical filaments, which may in turn depend on tau hyperphosphorylation.

Erratum: The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-Β production (Nature (2006) 440, (528-534))

This corrects the article DOI: 10.1038/nature04543