Truncation of Tau selectively facilitates its pathological activities.

Neurofibrillary tangles of abnormally hyperphosphorylated Tau are a hallmark of Alzheimer's disease (AD) and related tauopathies. Tau is truncated at multiple sites by various proteases in AD brain. Although many studies have reported the effect of truncation on the aggregation of Tau, these studies mostly employed highly artificial conditions, using heparin sulfate or arachidonic acid to induce aggregation. Here, we report for the first time the pathological activities of various truncations of Tau, including site-specific phosphorylation, self-aggregation, binding to hyperphosphorylated and oligomeric Tau isolated from AD brain tissue (AD O-Tau), and aggregation seeded by AD O-Tau. We found that deletion of the first 150 or 230 amino acids (aa) enhanced Tau's site-specific phosphorylation, self-aggregation, and binding to AD O-Tau and aggregation seeded by AD O-Tau, but deletion of the first 50 aa did not produce a significant effect. Deletion of the last 50 aa was found to modulate Tau's site-specific phosphorylation, promote its self-aggregation, and cause it to be captured by and aggregation seeded by AD O-Tau, whereas deletion of the last 20 aa had no such effects. Among the truncated Taus, Tau151-391 showed the highest pathological activities. AD O-Tau induced aggregation of Tau151-391in vitro and in cultured cells. These findings suggest that the first 150 aa and the last 50 aa protect Tau from pathological characteristics and that their deletions facilitate pathological activities. Thus, inhibition of Tau truncation may represent a potential therapeutic approach to suppress Tau pathology in AD and related tauopathies.

Phosphorylation of trans-active response DNA-binding protein-of 43 kDa promotes its cytoplasmic aggregation and modulates its function in tau mRNA stability and exon 10 alternative splicing.

Trans-active response DNA-binding protein of 43 kDa (TDP-43) promotes tau mRNA instability and tau exon 10 inclusion. Aggregation of phosphorylated TDP-43 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Casein kinase 1ε (CK1ε) phosphorylates TDP-43 at multiple sites, enhances its cytoplasmic aggregation, and modulates its function in tau mRNA processing. To determine roles of TDP-43 site-specific phosphorylation in its localization, aggregation, and function in tau mRNA processing, TDP-43 was mutated to alanine or aspartic acid at Ser379, Ser403/404, or Ser409/410 to block or mimic phosphorylation. Site-specific phosphorylation of TDP-43 and its mutants by CK1ε was studied in vitro and in cultured cells. Cytoplasmic and nuclear TDP-43 and phospho-TDP-43 were analyzed by western blots. Aggregation of TDP-43 was assessed by immunostaining and level of radioimmunoprecipitation assay buffer-insoluble TDP-43. Green florescent protein tailed with tau 3'-untranslated region and mini-tau gene pCI/SI9-LI10 were used to study tau mRNA stability and alternative splicing of tau exon 10. We found that phospho-blocking mutations of TDP-43 at Ser379, Ser403/404, or Ser409/410 were not effectively phosphorylated by CK1ε. Compared with TDP-43, higher level of phosphorylated TDP-43 in the cytoplasm was observed. Phospho-mimicking mutations at these sites enhanced cytoplasmic aggregation of TDP-43. Green florescent protein expression was not inhibited by phospho-blocking mutants of TDP-43, but tau exon 10 inclusion was further enhanced by phospho-blocking mutations at Ser379 and Ser403/404. Phosphorylation of TDP-43 at Ser379, Ser403/404, or Ser409/410 primes its phosphorylation by CK1ε, promotes TDP-43 cytoplasmic aggregation, and modulates its function in tau mRNA processing in site-specific manner.

TDP-43 suppresses tau expression via promoting its mRNA instability.

In the brains of individuals with Alzheimer's 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 Alzheimer's 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.

O-GlcNAcylation modulates PKA-CREB signaling in a manner specific to PKA catalytic subunit isoforms.

O-GlcNAcylation is a post-translational modification of proteins. Protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling plays critical roles in multiple biological processes. Isoforms α and ß of PKA catalytic subunit (PKAc) and CREB are modified by O-GlcNAcylation. In the present study, we determined the role of O-GlcNAcylation in PKAc isoform-specific CREB signaling. We found that up-regulation of O-GlcNAcylation enhanced CREB phosphorylation, but suppressed CREB expression in exogenous PKAc isoform-unspecific manner. PKAc isoforms affected exogenous expression of OGT or OGA and protein O-GlcNAcylation differently. Up-regulation of O-GlcNAcylation did not significantly affect net PKAcα-CREB signaling, but enhanced PKAcß-CREB signaling. The role of O-GlcNAcylation in PKA-CREB signaling was desensitized by insulin treatment. This study suggests a role of O-GlcNAcylation in PKA-CREB signaling by affecting phosphorylation of CREB in a PKAc isoform-specific manner.

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

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 Ca(2+)/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain.

LINC00894 inhibited neuron cellular apoptosis and regulated activating transcription factor 3 expression.

LINC00894 may be associated with synaptic function, but its biology function in neural cells is still unknown. In this study, LINC00894 knockdown decreased the EdU incorporated into newly synthesized DNA and cell viability in MTT or CCK-8 assay in HEK-293T and BE(2)-M17 (M17) neuroblastoma cells. And LINC00894 knockdown increased cellular apoptosis in Annexin V-FITC staining, the expression of activated Caspase3 and the level of reactive oxygen species (ROS) both in HEK-293T and M17 cells. Moreover, LINC00894 also protected cells from hydrogen peroxide induced apoptosis in in vitro models. Utilizing RNA sequencing (RNA-seq) integrated with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblot, we identified that LINC00894 affected activating transcription factor 3 (ATF3) expression in HEK-293T, M17, and SH-SY5Y neuroblastoma cells. Finally, we found that ectopic expression of ATF3 restored cell proliferation and inhibited cell apoptosis in LINC00894 downregulated M17 cells. While knockdown of ATF3 also significantly increased the cell viability inhibition and apoptosis promotion induced by LINC00894 knockdown in M17 cells. Our results from in vitro models revealed that LINC00894 could promote neuronal cell proliferation and inhibit cellular apoptosis by affecting ATF3 expression.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) modulates serine/arginine-rich protein 55 (SRp55)-promoted Tau exon 10 inclusion.

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.

Casein Kinase 1δ Phosphorylates TDP-43 and Suppresses Its Function in Tau mRNA Processing.

BACKGROUND: Neurofibrillary tangle aggregated from anomalous hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD). Trans-active response DNA-binding protein of 43 kDa (TDP-43) enhances the instability and exon (E) 10 inclusion of tau mRNA. Cytoplasmic inclusion of hyperphosphorylated TDP-43 in the neurons constitutes the third most prevalent proteinopathy of AD. Casein kinase 1δ (CK1δ) is elevated in AD brain and phosphorylates TDP-43 in vitro. OBJECTIVE: To determine the roles of CK1δ in phosphorylation, aggregation, and function of TDP-43 in the processing of tau mRNA. METHODS: The interaction and colocalization of TDP-43 and CK1δ were analyzed by co-immunoprecipitation and immunofluorescence staining. TDP-43 phosphorylation by CK1δ was determined in vitro and in cultured cells. RIPA-insoluble TDP-43 aggregates obtained by ultracentrifugation were analyzed by immunoblots. The instability and E10 splicing of tau mRNA were studied by using a reporter of green fluorescence protein tailed with 3'-untranslational region of tau mRNA and a mini-tau gene and analyzed by real-time quantitative PCR and reverse transcriptional PCR. RESULTS: We found that CK1δ interacted and co-localized with TDP-43. TDP-43 was phosphorylated by CK1δ at Ser379, Ser403/404, and Ser409/410 in vitro and in cultured cells, which was mutually enhanced. CK1δ overexpression promoted the aggregation of TDP-43 and suppressed its activity in enhancing the instability and E10 inclusion of tau mRNA. CONCLUSION: CK1δ phosphorylates TDP-43, promotes its aggregation, and inhibits its activity in promoting the instability of tau mRNA and inclusion of tau E10. Elevated CK1δ in AD brain may contribute to TDP-43 and tau pathologies directly or indirectly.

Two simple assays for assessing the seeding activity of proteopathic tau.

The regional distribution of neurofibrillary tangles of hyperphosphorylated tau aggregates is associated with the progression of Alzheimer's disease (AD). Misfolded proteopathic tau recruits naïve tau and templates its misfolding and aggregation in a prion-like fashion, which is believed to be the molecular basis of propagation of tau pathology. A practical way to assess tau seeding activity is to measure its ability to recruit/bind other tau molecules and to induce tau aggregation. Based on the properties of proteopathic tau, here we report the development of two simple assays to assess tau seeding activity ----- capture assay in vitro and seeded-tau aggregation assay in cultured cells. In the capture assay, proteopathic tau was applied onto a nitrocellulose membrane and the membrane was incubated with cell lysate containing HA-tagged tau151-391 (HA-tau151-391). The captured tau on the membrane was determined by immuno-blots developed with anti-HA. For the seeded-tau aggregation assay, HEK-293FT cells transiently expressing HA-tau151-391 were treated with proteopathic tau in the presence of Lipofectamine 2000 and then lysed with RIPA buffer. RIPA-insoluble fraction containing aggregated tau was obtained by ultracentrifugation and analyzed by immuno-blot developed with anti-HA. To validate these two assays, we assessed the seeding activity of tau in the middle frontal gyrus, middle temporal gyrus and basal forebrain of AD and control brains and found that AD, but not control, brain extracts effectively captured and seeded tau151-391 aggregation. Basal forebrain contained less phospho-tau and tau seeding activity. The levels of captured tau or seeded-tau aggregates were positively correlated to the levels of phospho-tau, Braak stages and tangle sores. These two assays are specific and sensitive and can be carried out in a regular biomedical laboratory setting by using routine biochemical techniques.

Excess folic acid supplementation before and during pregnancy and lactation activates ß-catenin in the brain of male mouse offspring.

Folic acid (FA) supplementation in early pregnancy is recommended to protect against birth defects. But excess FA has exhibited neurodevelopmental toxicity. We previously reported that the mice treated with 2.5-fold the dietary requirement of FA one week before mating and throughout pregnancy and lactation displayed abnormal behaviors in the offspring. Here we found the levels of non-phosphorylated ß-catenin (active) were increased in the brains of weaning and adult FA-exposed offspring. Meanwhile, demethylation of protein phosphatase 2 A catalytic subunit (PP2Ac), which suppresses its enzyme activity in regulatory subunit dependent manner, was significantly inhibited. Among the upstream regulators of ß-catenin, PI3K/Akt/GSK-3ß but not Wnt signaling was stimulated in FA-exposed brains only at weaning. In mouse neuroblastoma N2a cells, knockdown of PP2Ac or leucine carboxyl methyltransferase-1 (LCMT-1), or overexpression of PP2Ac methylation-deficient mutant decreased ß-catenin dephosphorylation. These results suggest that excess FA may activate ß-catenin via suppressing PP2Ac demethylation, providing a novel mechanism for the influence of FA on neurodevelopment.

Tau seeding activity in various regions of down syndrome brain assessed by two novel assays.

Propagation of tau pathology via the seeding of naive tau aggregation underlies the progression of Alzheimer's disease (AD) and related tauopathies. Individuals with Down syndrome (DS) develop tau pathology at the fourth decade of life, but tau seeding activity in DS brain has not yet been determined. To measure tau seeding activity, we developed capture assay and seeded-tau aggregation assay with truncated tau151-391. By using brain extracts from AD and related tauopathies, we validated these two methods and found that the brain extracts from AD and related tauopathies, but not from controls and the diseases in which tau was not hyperphosphorylated, captured in vitro and seeded 3R-tau151-391 and 4R-tau151-391 to aggregate in cultured cells similarly. Captured tau151-391 levels were strongly correlated with the seeded-tau151-391 aggregation. Employing these two newly developed assays, we analyzed tau seeding activity in the temporal (TC), frontal (FC), and occipital cortex (OC); corpus callosum (CC); and cerebellar cortex (CBC) of DS and control brains. We found that the extracts of TC, FC, or OC, but not the CC or CBC of DS or the corresponding brain regions of control cases, captured tau151-391. Levels of the captured tau151-391 by brain extracts were positively correlated with their levels of phosphorylated tau. Extracts of cerebral cortex and CC, but not CBC of DS with a similar tau level, induced more tau151-391 aggregation than did the corresponding samples from the control cases. Thus, higher tau seeding activity associated with tau hyperphosphorylation was found in the TC, FC, and OC of DS compared with the corresponding control regions as well as with the CBC and CC of DS. Of note, these two assays are sensitive, specific, and repeatable at a low cost and provide a platform for measuring tau seeding activity and for drug screening that targets tau propagation.

Dephosphorylation Passivates the Seeding Activity of Oligomeric Tau Derived From Alzheimer's Brain.

Accumulation of intracellular neurofibrillary tangles (NFTs), which are constituted of abnormally phosphorylated tau, is one of the neuropathological hallmarks of Alzheimer's disease (AD). The oligomeric aggregates of tau in AD brain (AD O-tau) are believed to trigger NFT spreading by seeding normal tau aggregation as toxic seeds, in a prion-like fashion. Here, we revealed the features of AD O-tau by Western blots using antibodies against various epitopes and determined the effect of dephosphorylation on the seeding activity of AD O-tau by capture and seeded aggregation assays. We found that N-terminal truncated and C-terminalhyperphosphorylated tau species were enriched in AD O-tau. Dephosphorylation of AD O-tau by alkaline phosphatasediminished its activity in capturing tau in vitro and ininducing insoluble aggregates in cultured cells. Our resultssuggested that dephosphorylation passivated the seeding activity ofAD O-tau. Inhibition of phosphorylation may be a potentstrategy to prevent the spreading of tau patho3logy.

Seeding-Competent Tau in Gray Matter Versus White Matter of Alzheimer's Disease Brain.

BACKGROUND: Neurofibrillary pathology of abnormally hyperphosphorylated tau spreads along neuroanatomical connections, underlying the progression of Alzheimer's disease (AD). The propagation of tau pathology to axonally connected brain regions inevitably involves trafficking of seeding-competent tau within the axonal compartment of the neuron. OBJECTIVE: To determine the seeding activity of tau in cerebral gray and white matters of AD. METHODS: Levels of total tau, hyperphosphorylation of tau, and SDS- and ß-mercaptoethanol-resistant high molecular weight tau (HMW-tau) in crude extracts from gray and white matters of AD frontal lobes were analyzed by immuno-blots. Tau seeding activity was quantitatively assessed by measuring RIPA buffer-insoluble tau in HEK-293FT/tau151-391 cells treated with brain extracts. RESULTS: We found a comparable level of soluble tau in gray matter versus white matter of control brains, but a higher level of soluble tau in gray matter than white matter of AD brains. In AD brains, tau is hyperphosphorylated in both gray and white matters, with a higher level in the former. The extracts of both gray and white matters of AD brains seeded tau aggregation in HEK-293FT/tau151-391 cells but the white matter showed less potency. Seeding activity of tau in brain extracts was positively correlated with the levels of tau hyperphosphorylation and HMW-tau. RIPA-insoluble tau, but not RIPA-soluble tau, was hyperphosphorylated tau at multiple sites. CONCLUSION: Both gray and white matters of AD brain contain seeding-competent tau that can template aggregation of hyperphosphorylated tau, but the seeding potency is markedly higher in gray matter than in white matter.

Alzheimer's disease brain contains tau fractions with differential prion-like activities.

Neurofibrillary tangles (NFTs) made of abnormally hyperphosphorylated tau are a hallmark of Alzheimer's disease (AD) and related tauopathies. Regional distribution of NFTs is associated with the progression of the disease and has been proposed to be a result of prion-like propagation of misfolded tau. Tau in AD brain is heterogenous and presents in various forms. In the present study, we prepared different tau fractions by sedimentation combined with sarkosyl solubility from AD brains and analyzed their biochemical and pathological properties. We found that tau in oligomeric fraction (O-tau), sarkosyl-insoluble fractions 1 and 2 (SI1-tau and SI2-tau) and monomeric heat-stable fraction (HS-tau) showed differences in truncation, hyperphosphorylation, and resistance to proteinase K. O-tau, SI1-tau, and SI2-tau, but not HS-tau, were hyperphosphorylated at multiple sites and contained SDS- and ß-mercaptoethanol-resistant high molecular weight aggregates, which lacked the N-terminal portion of tau. O-tau and SI2-tau displayed more truncation and less hyperphosphorylation than SI1-tau. Resistance to proteinase K was increased from O-tau to SI1-tau to SI2-tau. O-tau and SI1-tau, but not SI2-tau or HS-tau, captured tau from cell lysates and seeded tau aggregation in cultured cells. Heat treatment could not kill the prion-like activity of O-tau to capture normal tau. Hippocampal injection of O-tau into 18-month-old FVB mice induced significant tau aggregation in both ipsilateral and contralateral hippocampi, but SI1-tau only induced tau pathology in the ipsilateral hippocampus, and SI2-tau and HS-tau failed to induce any detectable tau aggregation. These findings suggest that O-tau and SI1-tau have prion-like activities and may serve as seeds to recruit tau and template tau to aggregate, resulting in the propagation of tau pathology. Heterogeneity of tau pathology within AD brain results in different fractions with different biological and prion-like properties, which may pose a major challenge in targeting tau for development of effective therapeutic treatments.

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 Alzheimer's disease. Recent studies have demonstrated that impaired brain glucose metabolism precedes the appearance of clinical symptoms, implying its active role in the development of Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's disease. Thus, restoration of brain tau O-GlcNAcylation and protein phosphatase 2A activity may offer promising therapeutic targets for treating Alzheimer's disease.

Tau in Alzheimer's Disease: Pathological Alterations and an Attractive Therapeutic Target.

Alzheimer's disease (AD) is an age-related neurodegenerative disease with two major hallmarks: extracellular amyloid plaques made of amyloid-ß (Aß) and intracellular neurofibrillary tangles (NFTs) of abnormally hyperphosphorylated tau. The number of NFTs correlates positively with the severity of dementia in AD patients. However, there is still no efficient therapy available for AD treatment and prevention so far. A deeper understanding of AD pathogenesis has identified novel strategies for the generation of specific therapies over the past few decades. Several studies have suggested that the prion-like seeding and spreading of tau pathology in the brain may be a key driver of AD. Tau protein is considered as a promising candidate target for the development of therapeutic interventions due to its considerable pathological role in a variety of neurodegenerative disorders. Abnormal tau hyperphosphorylation plays a detrimental pathological role, eventually leading to neurodegeneration. In the present review, we describe the recent research progresses in the pathological mechanisms of tau protein in AD and briefly discuss tau-based therapeutic strategies.

High Mobility Group Box 1 Ameliorates Cognitive Impairment in the 3×Tg-AD Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia. Studies indicate that neuroinflammation plays an important role in the pathophysiology of AD. High-mobility group box 1 (HMGB1) is an important chromatin protein. It can be secreted by immune cells and passively released from damaged cells to promote inflammation. HMGB1 also can recruit stem cells and promote their proliferation and tissue repairing. However, the role of HMGB1 in the progression of AD is currently unknown. OBJECTIVE: The aims were to investigate the effect of HMGB1 on the AD-related pathologies and cognitive function using 3×Tg-AD mouse model. METHODS: Female 5-month-old 3×Tg-AD mice were intracerebroventricularly injected with 4.5 µg of HMGB1 or with saline as a control. The levels of interesting protein were assessed by western blots or immunofluorescence. The effect of HMGB1 on the cognitive function was evaluated by one-trial novel object recognition test and Morris water maze. RESULTS: Intracerebroventricular injection of recombinant HMGB1 ameliorated cognitive impairment in 5-6-month-old 3×Tg-AD mice. The levels of synapsin 1, synaptophysin, MAP2, NeuN, and phosphorylated CREB were increased in HMGB1-treated 3×Tg-AD mouse brains. HMGB1 decreased intracellular amyloid-ß level but did not affect tau phosphorylation. HMGB1 treatment also promoted neurogenesis in the dentate gyrus and increased the level of GFAP in the 3×Tg-AD mouse brains. CONCLUSION: These results reveal a novel function of HMGB1 in enhancing neuroplasticity and improving cognitive function in 3×Tg-AD mice.

Elevation of casein kinase 1ε associated with TDP-43 and tau pathologies in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence of extracellular amyloid ß plaques and intraneuronal neurofibrillary tangles of hyperphosphorylated microtubule-associated protein tau in the brain. Aggregation of transactive response DNA-binding protein of 43 kDa (TDP-43) in the neuronal cytoplasm is another feature of AD. However, how TDP-43 is associated with AD pathogenesis is unknown. Here, we found that casein kinase 1ε (CK1ε) phosphorylated TDP-43 at Ser403/404 and Ser409/410. In AD brains, the level of CK1ε was dramatically increased and positively correlated with the phosphorylation of TDP-43 at Ser403/404 and Ser409/410. Overexpression of CK1ε promoted its cytoplasmic aggregation and suppressed TDP-43-promoted tau mRNA instability and tau exon 10 inclusion, leading to an increase of tau and 3R-tau expressions. Levels of CK1ε and TDP-43 phosphorylation were positively correlated with the levels of total tau and 3R-tau in human brains. Furthermore, we observed, in pilot immunohistochemical studies, that the severe tau pathology was accompanied by robust TDP-43 pathology and a high level of CK1ε. Taken together, our findings suggest that the elevation of CK1ε in AD brain may phosphorylate TDP-43, promote its cytoplasmic aggregation and suppress its function in tau mRNA processing, leading to acceleration/exacerbation of tau pathology. Thus, the elevation of CK1ε may link TDP-43 to tau pathogenesis in AD brain.

Cyclic AMP-Dependent Protein Kinase Phosphorylates TDP-43 and Modulates Its Function in Tau mRNA Processing.

Trans-active response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved and ubiquitously expressed nuclear protein. As a member of heterogeneous ribonucleoproteins, TDP-43 plays pivotal roles in mRNA processing. We recently found that TDP-43 promoted tau mRNA instability via acting on the 3'-untranslated region of its mRNA and enhanced tau exon 10 inclusion. TDP-43 is a phospho-protein. The function and the pathological aggregation of TDP-43 are regulated by the phosphorylation. In the present study, we determined phosphorylation of TDP-43 by cyclic AMP-dependent protein kinase (PKA). We found that TDP-43 was co-immunoprecipitated by and co-localized with PKA in the nucleus. PKA phosphorylated TDP-43 at Ser379, Ser403/404, and Ser409/410 in vitro and in cultured cells. Phosphorylation of TDP-43 at these sites enhanced mutually their phosphorylation by PKA in vitro and in cultured cells. Overexpression of PKA suppressed TDP-43's activity in promoting tau mRNA instability and tau exon 10 inclusion. These findings shed light on the role of PKA in phosphorylation and function of TDP-43. Downregulation of PKA signaling in AD brain may attenuate the impact of TDP-43 pathology in tau pathogenesis.