Cheng Xin Gong

Post-translational modifications of tau protein in Alzheimer’s disease

Summary.Microtubule-associated protein tau undergoes several post-translational modifications and aggregates into paired helical filaments (PHFs) in Alzheimer’s disease (AD) and other tauopathies. These modifications of tau include hyperphosphorylation, glycosylation, ubiquitination, glycation, polyamination, nitration, and proteolysis. Hyperphosphorylation and glycosylation are crucial to the molecular pathogenesis of neurofibrillary degeneration of AD. The others appear to represent failed mechanisms for neurons to remove damaged, misfolded, and aggregated proteins. This review summarizes the abnormal post-translational modifications of tau and discusses the pathophysiological relevance of hyperphosphorylation and glycosylation of tau. Total tau and phosphorylated tau levels in cerebrospinal fluid as a diagnostic biomarkers are also reviewed. Analyses of the current advances in tau modifications suggest that intervention addressing these abnormalities may offer promising therapeutic opportunities to prevent and treat neurofibrillary degeneration of AD and other tauopathies.

Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.

O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimers disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

Mechanisms of neurofibrillary degeneration and the formation of neurofibrillary tangles

Alzheimer disease (AD) has polyetiology. Independent of the etiology the disease is characterized histopathologically by the intraneuronal accumulation of paired helical filaments (PHF), forming neurofibrillary tangles, neuropil threads and dystrophic neurites surrounding the extracellular deposits of beta-amyloid in plaques, the second major lesion. The clincal expression of AD correlates with the presence of neurofibrillary degeneration; beta-amyloid alone does not produce the disease clinically. Thus arresting neurofibrillary degeneration offers a promising key target for therapeutic intervention of AD. The major protein subunit of PHF is the microtubule-associated protein tau. Tau in AD brain, especially PHF, is abnormally hyperphosphorylated and glycosylated. With maturation, the tangles are increasingly ubiquitinated. Levels of tau and conjugated ubiquitin are elevated both in AD brain and CSF. The AD abnormally phosphorylated tau (AD P-tau) does not promote microtubule assembly, but on dephosphorylation its microtubule promoting activity is restored to approximately that of the normal tau. The AD P-tau competes with tubulin in binding to normal tau, MAP1 and MAP2 and inhibits their microtubule assembly promoting activities. Furthermore, the AD P-tau sequesters normal MAPs from microtubules. The association of AD P-tau with normal tau but not with MAP1 or MAP2 results in the formation of tangles of 3.3 +/- 0.5 mm filaments. Deglycosylation of Alzheimer neurofibrillary tangles with endoglycosidase F/N-glycosidase F untwists the PHF resulting in tangles of thin filaments similar to those formed by association between the AD P-tau and normal tau. Dephosphorylation or deglycosylation plus dephosphorylation but not deglycosylation alone restores the microtubule assembly promoting activity of tau. In vitro AD P-tau can be dephosphorylated by protein phosphatases PP-2B, PP-2A and PP-1 but not PP-2C and all the three tau phosphatases are present in brain neurons. Tau phosphatase activity is decreased by approximately 30% in AD brain. Inhibition of PP-2A and PP-1 activities in SY5Y neuroblastoma by 10 nM okadaic acid causes breakdown of microtubules and the degeneration of these cells. It is suggested (I) that a defect(s) in the protein phosphorylation/dephosphorylation system(s) leads to a hyperphosphorylation of tau, (ii) that this altered tau causes disassembly of microtubules and consequently a retrograde neuronal degeneration; (iii) a pharmacological approach to AD is to enhance the tau phosphatase activity; and (iv) that CSF tau and conjugated ubiquitin levels are promising markers of AD brain pathology.

Mechanism of neurofibrillary degeneration and pharmacologic therapeutic approach

Neurofibrillary degeneration is a key histopathological brain lesion of Alzheimer disease (AD) and related neurodegenerative disorders such as frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), commonly referred to as tauopathies. Microtubule associated protein (MAP) tau, which is a major MAP of a normal mature neuron is abnormally hyperphosphorylated in tauopathies and is the major protein subunit of paired helical filaments (PHF)/straight filaments (SF) which accumulate in the soma (as neurofibrillary tangles) and dystrophic neurites (as neuropil threads and as dystrophic neurites surrounding the beta-amyloid core in neuritic plaques in AD) of the affected neurons. Unlike normal tau which stimulates assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau inhibits assembly and disrupts microtubules. The abnormally hyperphosphorylated tau competes with tubulin/microtubules in associating with normal tau, MAP1 and MAP2. This sequestration of normal MAPs by the abnormal tau results in the breakdown of the microtubules. The association of the abnormal tau with normal tau and not with MAP1 or MAP2 results in the formation of tangles of tau filaments. All these toxic properties of the abnormally hyperphosphorylated tau are eliminated by its enzymatic dephosphorylation. Activities of phosphoseryl/phosphothreonyl protein phosphatases (PP)-2A and PP-1 which can dephosphorylate the abnormal tau to a normal-like state are compromised in AD brain. Dephosphorylation by PP-2A and PP-2B and to a lesser extent by PP-1 restores the normal microtubule assembly promoting activity in AD P-tau in vitro. Neurofibrillary tangles of PHF isolated from AD brain are also dissociated on in vitro dephosphorylation with PP-2A, and the tau released by this treatment can stimulate microtubule assembly. Thus, it appears that the abnormal hyperphosphorylation of tau leads to neurodegeneration through breakdown of the microtubule network and that the abnormal tau on association with normal tau forms neurofibrillary tangles of tau filaments i.e. PHF/SF. Increase in tau phosphatase activity is a promising approach to inhibit neurofibrillary degeneration and thereby the diseases characterized by this lesion.

Pharmacological targets to inhibit alzheimer neurofibrillary degeneration

Neurofibrillary degeneration appears to be required for the clinical expression of Alzheimer disease (AD) and related tauopathies. Given the polyetiology of these diseases and the pivotal involvement of neurofibrillary degeneration in their pathogenesis, inhibition of this lesion offers a promising therapeutic target. Studies from our laboratories have shown that there is a protein phosphorylation/dephosphorylation imbalance and that the microtubule associated protein tau is abnormally hyperphosphorylated in the brain of patients with AD and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles (PHF/NFT). The abnormal tau which is polymerized into PHF/NFT neither promotes or inhibits in vitro microtubule assembly. In contrast the cytosolic abnormally hyperphosphorylated tau from AD brain, the AD P-tau neither associates with tubulin nor promotes in vitro microtubule assembly but instead it sequesters normal tau, MAP1 and MAP2 and inhibits microtubule assembly. The AD P-tau readily self-assembles in vitro into tangles of PHF/straight filaments under physiological conditions of protein concentration, pH, ionic strength and reducing conditions and this self assembly requires the abnormal hyperphosphorylation of this protein. The activity of phosphoseryl/phosphothreonyl protein phosphatase (PP)-2A which regulates the phosphorylation of tau, is compromised in AD brain. Thus, modulation of the activities of protein phosphatase-2A and tau kinases and inhibition of the sequestration of normal MAPs by AD P-tau offer promising therapeutic opportunities to inhibit neurofibrillary degeneration and the diseases characterized by this lesion.

TDP-43 suppresses tau expression via promoting its mRNA instability

Abstract In the brains of individuals with Alzheimers disease (AD) and chronic traumatic encephalopathy, tau pathology is accompanied usually by intracellular aggregation of transactive response DNA-binding protein 43 (TDP-43). However, the role of TDP-43 in tau pathogenesis is not understood. Here, we investigated the role of TDP-43 in tau expression in vitro and in vivo. We found that TDP-43 suppressed tau expression by promoting its mRNA instability through the UG repeats of its 3΄-untranslated region (3΄-UTR). The C-terminal region of TDP-43 was required for this function. Neurodegenerative diseases-causing TDP-43 mutations affected tau mRNA instability differentially, in that some promoted and others did not significantly affect tau mRNA instability. The expression levels of tau and TDP-43 were inverse in the frontal cortex and the cerebellum. Accompanied with cytoplasmic accumulation of TDP-43, tau expression was elevated in TDP-43M337V transgenic mouse brains. The level of TDP-43, which is decreased in AD brains, was found to correlate negatively with the tau level in human brain. Our findings indicate that TDP-43 suppresses tau expression by promoting the instability of its mRNA. Down-regulation of TDP-43 may be involved in the tau pathology in AD and related neurodegenerative disorders.

Transactive response DNA-binding protein 43 (TDP-43) regulates alternative splicing of tau exon 10: implications for the pathogenesis of tauopathies

Hyperphosphorylation and aggregation of the neuronal protein tau are responsible for neurodegenerative diseases called tauopathies. Dysregulation of the alternative splicing of tau exon 10 results in alterations of the ratio of two tau isoforms, 3R-tau and 4R-tau, which have been seen in several tauopathies. Transactive response DNA-binding protein of 43 kDa (TDP-43) is involved in the regulation of RNA processing, including splicing. Cytoplasmic aggregation of TDP-43 has been observed in the brains of individuals with chronic traumatic encephalopathy or Alzheimers disease, diseases in which neurofibrillary tangles of hyperphosphorylated tau are hallmarks. Here, we investigated the role of TDP-43 in tau exon 10 splicing. We found that TDP-43 promoted tau exon 10 inclusion, which increased production of the 4R-tau isoform. Moreover, TDP-43 could bind to intron 9 of tau pre-mRNA. Deletion of the TDP-43 N or C terminus promoted its cytoplasmic aggregation and abolished or diminished TDP-43-promoted tau exon 10 inclusion. Several TDP-43 mutations associated with amyotrophic lateral sclerosis or frontotemporal lobar degeneration with ubiquitin inclusions promoted tau exon 10 inclusion more effectively than wild-type TDP-43 but did not affect TDP-43 cytoplasmic aggregation in cultured cells. The ratio of 3R-tau/4R-tau was decreased in transgenic mouse brains expressing human TDP-43 and increased in the brains expressing the disease-causing mutation TDP-43M337V, in which cytoplasmic TDP-43 was increased. These findings suggest that TDP-43 promotes tau exon 10 inclusion and 4R-tau expression and that disease-related changes of TDP-43, truncations and mutations, affect its function in tau exon 10 splicing, possibly because of TDP-43 mislocalization to the cytoplasm.

P2-148: Involvement of 9G8 in alternative splicing of tau exon 10

Shao Hong Ding, Khalid Iqbal, Inge Grundke Iqbal, Cheng Xin Gong, Fei Liu, School of Public Health , Nantong University, Nantong, China; Department of Occupational and Enviromental Health, School of Public Health, Nantong University, Nantong, China; New York State Institute for Basic Research in Developmental Disability, New York, NY, USA; Neuroregeneration Key Laboratory of Jiangsu, Nantong University, Nantong, China. Contact e-mail: dingshaohong_2000@yahoo.com.cn