Pin1 promotes degradation of Smad proteins and their interaction with phosphorylated tau in Alzheimer's disease.

AIMS: Neurodegeneration in Alzheimer's disease (AD) is characterized by pathological protein aggregates and inadequate activation of cell cycle regulating proteins. Recently, Smad proteins were identified to control the expression of AD relevant proteins such as APP, CDK4 and CDK inhibitors, both critical regulators of cell cycle activation. This might indicate a central role for Smads in AD pathology where they show a substantial deficiency and disturbed subcellular distribution in neurones. Still, the mechanisms driving relocation and decrease of neuronal Smad in AD are not well understood. However, Pin1, a peptidyl-prolyl-cis/trans-isomerase, which allows isomerization of tau protein, was recently identified also controlling the fate of Smads. Here we analyse a possible role of Pin1 for Smad disturbances in AD. METHODS: Multiple immunofluorescence labelling and confocal laser-scanning microscopy were performed to examine the localization of Smad and Pin1 in human control and AD hippocampi. Ectopic Pin1 expression in neuronal cell cultures combined with Western blot analysis and immunoprecipitation allowed studying Smad level and subcellular distribution. Luciferase reporter assays, electromobility shift, RNAi-technique and qRT-PCR revealed a potential transcriptional impact of Smad on Pin1 promoter. RESULTS: We report on a colocalization of phosphorylated Smad in AD with Pin1. Pin1 does not only affect Smad phosphorylation and stability but also regulates subcellular localization of Smad2 and supports its binding to phosphorylated tau protein. Smads, in turn, exert a negative feed-back regulation on Pin1. CONCLUSION: Our data suggest both Smad proteins and Pin1 to be elements of a vicious circle with potential pathogenetic significance in AD.

Altered subcellular location of phosphorylated Smads in Alzheimer's disease.

A number of growth factors and cytokines, such as transforming growth factor beta 1 (TGF-beta1), is elevated in Alzheimer's disease (AD), giving rise to activated intracellular mitogenic signaling cascades. Activated mitogenic signaling involving the mitogen-activated protein kinases (MAPKs) and other protein kinases might alter the phosphorylation states of structural proteins such as tau, resulting in hyperphosphorylated deposits. Many intracellular signaling proteins are potential targets of misregulated phosphorylation and dephosphorylation. Recently, a crosstalk between MAPKs and Smad proteins, both involved in mediating TGF-beta1 signaling, has been reported. Although TGF-beta1 has previously been shown to be involved in the pathogenesis of AD, the role of Smad proteins has not been investigated. In this study we thus analysed the subcellular distribution of phosphorylated Smad2 and Smad3 in the hippocampus of both normal and AD brains. Here we report on strong nuclear detection of phosphorylated Smad2 and Smad3 in neurons of control brains. In AD brains these phosphorylated proteins were additionally found in cytoplasmic granules in hippocampal neurons, within amyloid plaques and attached to neurofibrillary tangles. Our data suggest a critical role of Smad proteins in the pathogenesis of AD.

Inducible neuronal expression of transgenic TGF-beta1 in vivo: dissection of short-term and long-term effects.

Various chronic neurological diseases are associated with increased expression of transforming growth factor-beta1 (TGF-beta1) in the brain. TGF-beta1 has both neuroprotective and neurodegenerative functions, depending on conditions such as duration and the local and temporal pattern of its expression. Previous transgenic approaches did not enable control for these dynamic aspects. To overcome these limitations, we established a transgenic mouse model with inducible neuron-specific expression of TGF-beta1 based on the tetracycline-regulated gene expression system. TGF-beta1 expression was restricted to the brain where it was particularly pronounced in the neocortex, hippocampus and striatum. Transgene expression was highly sensitive to the presence of doxycycline and completely silenced within 6 days after doxycycline application. After long-term expression, perivascular thioflavin-positive depositions, formed by amyloid fibrils, developed. These depositions persisted even after prolonged silencing of the transgene, indicating an irreversible process. Similarly, strong perivascular apolipoprotein E (ApoE) depositions were found after TGF-beta1 expression and these remained despite TGF-beta1 removal. These in vivo observations suggests that the continuous presence of TGF-beta1 as initial trigger is not necessary for the persistence and development of chronic lesions. Neuroprotective effects were observed after short-term expression of TGF-beta1. Death of striatal neurons induced by 3-nitropropionic acid was markedly reduced after induced TGF-beta1 expression.

Inverse association of Pin1 and tau accumulation in Alzheimer's disease hippocampus.

Neurofibrillary degeneration, one of the pathological hallmarks of Alzheimer's disease, is not ubiquitous to all brain regions or neurons. While a high degree of vulnerability has been documented for entorhinal cortex, hippocampal and neocortical pyramidal neurons other brain structures are largely spared. Even within highly vulnerable regions such as hippocampus neurons are affected to a variable extent. The molecular basis for this selective susceptibility remains unknown. Neurofibrillary degeneration involves hyperphosphorylation of tau which critically impairs its binding capacity to microtubule and, therefore, is believed to disrupt the axonal cytoskeleton. Recently, Lu et al. [Nature (1999) 399:784] described the ability of the peptidyl-prolyl cis-trans isomerase Pin1 to recover microtubule-binding affinity and microtubule stabilisation of phosphorylated tau. In the present study, we analysed the potential involvement of Pin1 in selective vulnerability of hippocampal neurons to neurofibrillary degeneration in Alzheimer's disease. Pin1 immunoreactivity appeared as cytoplasmic granules affecting hippocampal subfields to a different extent (CA2>subiculum>CA1>CA3/CA4). Since the main markers of granulovacuolar degeneration do not co-label Pin1-immunoreactive granules, we propose that these granules may represent a new lesion in Alzheimer's disease. Neurons containing Pin1 granules were devoid of neurofibrillary tangles. Granular accumulation of Pin1 may correspond to an absence of neurofibrillary lesions in these cells and might be associated with other mechanisms of neuronal degeneration.