Jianlan Gu

Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease

It has been established for a long time that brain glucose metabolism is impaired in Alzheimers disease. Recent studies have demonstrated that impaired brain glucose metabolism precedes the appearance of clinical symptoms, implying its active role in the development of Alzheimers disease. However, the molecular mechanism by which this impairment contributes to the disease is not known. In this study, we demonstrated that protein O-GlcNAcylation, a common post-translational modification of nucleocytoplasmic proteins with beta-N-acetyl-glucosamine and a process regulated by glucose metabolism, was markedly decreased in Alzheimers disease cerebrum. More importantly, the decrease in O-GlcNAc correlated negatively with phosphorylation at most phosphorylation sites of tau protein, which is known to play a crucial role in the neurofibrillary degeneration of Alzheimers disease. We also found that hyperphosphorylated tau contained 4-fold less O-GlcNAc than non-hyperphosphorylated tau, demonstrating for the first time an inverse relationship between O-GlcNAcylation and phosphorylation of tau in the human brain. Downregulation of O-GlcNAcylation by knockdown of O-GlcNAc transferase with small hairpin RNA led to increased phosphorylation of tau in HEK-293 cells. Inhibition of the hexosamine biosynthesis pathway in rat brain resulted in decreased O-GlcNAcylation and increased phosphorylation of tau, which resembled changes of O-GlcNAcylation and phosphorylation of tau in rodent brains with decreased glucose metabolism induced by fasting, but not those in rat brains when protein phosphatase 2A was inhibited. Comparison of tau phosphorylation patterns under various conditions suggests that abnormal tau hyperphosphorylation in Alzheimers disease brain may result from downregulation of both O-GlcNAcylation and protein phosphatase 2A. These findings suggest that impaired brain glucose metabolism leads to abnormal hyperphosphorylation of tau and neurofibrillary degeneration via downregulation of tau O-GlcNAcylation in Alzheimers disease. Thus, restoration of brain tau O-GlcNAcylation and protein phosphatase 2A activity may offer promising therapeutic targets for treating Alzheimers disease.

Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

Background: Dysregulation of the alternative splicing of Tau exon 10 causes several types of neurodegenerative diseases. Results: SRp55 promotes Tau exon 10 inclusion. Dyrk1A interacts with SRp55, mainly phosphorylates its proline-rich domain and inhibits its ability to promote Tau exon 10 inclusion. Conclusion: Dyrk1A suppresses SRp55-promoted Tau exon 10 inclusion. Significance: Up-regulation of Dyrk1A disrupts the alternative splicing of Tau exon 10. Tau exon 10, which encodes the second microtubule-binding repeat, is regulated by alternative splicing. Its alternative splicing generates Tau isoforms with three- or four-microtubule-binding repeats, named 3R-tau and 4R-tau. Adult human brain expresses equal levels of 3R-tau and 4R-tau. Imbalance of 3R-tau and 4R-tau causes Tau aggregation and neurofibrillary degeneration. In the present study, we found that splicing factor SRp55 (serine/arginine-rich protein 55) promoted Tau exon 10 inclusion. Knockdown of SRp55 significantly promoted Tau exon 10 exclusion. The promotion of Tau exon 10 inclusion by SRp55 required the arginine/serine-rich region, which was responsible for the subnucleic speckle localization. Dyrk1A (dual specificity tyrosine-phosphorylated and regulated kinase 1A) interacted with SRp55 and mainly phosphorylated its proline-rich domain. Phosphorylation of SRp55 by Dyrk1A suppressed its ability to promote Tau exon 10 inclusion. Up-regulation of Dyrk1A as in Down syndrome could lead to neurofibrillary degeneration by shifting the alternative splicing of Tau exon 10 to an increase in the ratio of 3R-tau/4R-tau.

Cross talk between PI3K-AKT-GSK-3β and PP2A pathways determines tau hyperphosphorylation

Glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) are the important enzymes controlling tau hyperphosphorylation. The relationship between these two enzymes and its impact on tau hyperphosphorylation are not well understood. In the present study, we determined the cross talk between PI3K-AKT-GSK-3β and PP2A pathways and found that the former regulated the methylation of PP2Ac via GSK-3β. Upregulation of GSK-3β led to an increase in the methylation and activity of PP2Ac through suppression of protein phosphatase methylesterase-1 expression and phosphorylation of leucine carboxyl methyltransferase 1. PP2A also regulated GSK-3β phosphorylation. Downregulation of PP2A enhanced Ser9 phosphorylation of GSK-3β and inhibited its kinase activity. Thus, GSK-3β and PP2A regulate each other and control tau phosphorylation both directly and indirectly through each other. Reduction of tau phosphorylation by inhibition of GSK-3β may be more than offset by inhibition of PP2A through a shift in phosphatase methylesterase-1/leucine carboxyl methyltransferase 1 balance; PP2A regulates phosphorylation of tau at Ser262/356, a required site for tau pathology. These findings suggest targeting PP2A rather than GSK-3β to inhibit tau pathology.

O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling

Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expression of AKT. We also found a dynamic elevation of protein O-GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O-GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O-GlcNAcylation in ischemic brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O-GlcNAcylation that promotes apoptosis through down-regulation of AKT activity. These findings provide a novel mechanism through which O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I A MOLECULAR MECHANISM LINKED TO TAU PATHOLOGY IN ALZHEIMER DISEASE

Background: Dyrk1A regulates alternative splicing of exon 10 and phosphorylation of Tau. Results: Calpain I proteolyzes Dyrk1A and enhances its kinase activity, which promotes exon 10 exclusion and hyperphosphorylation of Tau. Conclusion: Truncation and activation of Dyrk1A may be responsible for Tau pathology in AD brains. Significance: These findings indicate a new mechanism linked to Tau pathology in AD. Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased Vmax but not Km. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca2+/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain.

Activation of Protein Phosphatase 2B and Hyperphosphorylation of Tau in Alzheimer's Disease

Protein phosphatase 2B (PP2B) is one of the major brain phosphatases and can dephosphorylate tau at several phosphorylation sites in vitro. Previous studies that measured PP2B activity in human brain crude extracts showed that PP2B activity was either unchanged or decreased in Alzheimers disease (AD) brain. These results led to the speculation that PP2B might regulate tau phosphorylation and that a down-regulation of PP2B might contribute to abnormal hyperphosphorylation of tau. In this study, we immunoprecipitated PP2B from brains of six AD subjects and seven postmortem- and age-matched controls and then measured the phosphatase activity. We found a three-fold increase in PP2B activity in AD brain as compared with control brains. The activation was due to the partial cleavage of PP2B by calpain I that was activated in AD brain. The truncation of PP2B appeared to alter its intracellular distribution in the brain. In human brains, PP2B activity correlated positively, rather than negatively, to the levels of tau phosphorylation at several sites that can be dephosphorylated by PP2B in vitro. Truncation of PP2B in the frontal cortex was more than in the temporal cortex, and tau phosphorylation was also more in the frontal cortex. Taken together, these results indicate that truncation of PP2B by calpain I elevates its activity but does not counteract the abnormal hyperphosphorylation tau in AD brain.

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimers disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.

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.

Cyclic AMP-dependent protein kinase regulates 9G8-mediated alternative splicing of tau exon 10

9G8 physically interacts with PKA catalytic subunit by coimmunoprecipitation (View interaction)