Tau Hyperphosphorylation In Alzheimer's Disease Research Articles

Activation of TRPV4 induces intraneuronal tau hyperphosphorylation via cholesterol accumulation

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel, whose aberrant function in neurons has been reported to participate in the progression of brain disorders, including Alzheimer's disease (AD). However, the influence of TRPV4 activation on tau hyperphosphorylation in AD has not yet been elucidated. Since disturbed brain cholesterol homeostasis is considered to be related to excessive tau phosphorylation, this study aimed to explore whether dysregulation of TRPV4 affects tau phosphorylation and whether it involves cholesterol unbalance. Our data indicated that TRPV4 activation increased tau phosphorylation in the cortex and hippocampus of P301S tauopathy mouse model and aggravated its cognitive decline. In addition, we detected that TRPV4 activation upregulated cholesterol levels in primary neurons, and the elevation of cholesterol promoted hyperphosphorylation of tau. TRPV4 knockdown improved tau hyperphosphorylation by reducing intracellular cholesterol accumulation. Our results suggest that activation of TRPV4 may take part in the pathological mechanism of AD by promoting intraneuronal tau hyperphosphorylation in a cholesterol-dependent manner.

TREM2 Inhibits Tau Hyperphosphorylation and Neuronal Apoptosis via the PI3K/Akt/GSK-3β Signaling Pathway In vivo and In vitro.

Triggering receptor expressed on myeloid cells-2 (TREM2), a cell surface receptor mainly expressed on microglia, has been shown to play a critical role in Alzheimer's disease (AD) pathogenesis and progression. Our recent results showed that overexpression of TREM2 inhibited inflammatory response in APP/PS1 mice and BV2 cells. Several studies indicated that TREM2 ameliorated tau hyperphosphorylation might be ascribed to the inhibition of neuroinflammation. However, the precise signaling pathways underlying the effect of TREM2 on tau pathology and neuronal apoptosis have not been fully elucidated. In the present study, upregulation of TREM2 significantly inhibited tau hyperphosphorylation at Ser199, Ser396, and Thr205, respectively, as well as prevented neuronal loss and apoptosis. We also found that upregulation of TREM2 alleviated behavioral deficits and improved the spatial cognitive ability of APP/PS1 mice. Further study revealed that TREM2 could activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, resulting in an inhibitory effect on glycogen synthase kinase-3β (GSK-3β), which is a major kinase responsible for tau hyperphosphorylation in AD. In line with in vivo findings, TREM2-overexpressing BV2 microglia following β-amyloid (Aβ) stimulation led to a significant increase in the phosphorylation of PI3K, Akt, and GSK-3β, accompanied by a decrease in tau hyperphosphorylation and apoptosis in co-cultured SH-SY5Y cells. Furthermore, LY294002, a specific PI3K inhibitor, was observed to abolish the beneficial effects of TREM2 on tau hyperphosphorylation, neuronal apoptosis, and spatial cognitive impairments in vivo and in vitro. Thus, our findings indicated that TREM2 inhibits tau hyperphosphorylation and neuronal apoptosis, at least in part, by the activation of the PI3K/Akt/GSK-3β signaling pathway. Taken together, the above results allow us to better understand how TREM2 protects against tau pathology and suggest that upregulation of TREM2 may provide new ideas and therapeutic targets for AD.

Vascular smooth muscle cell dysfunction contribute to neuroinflammation and Tau hyperphosphorylation in Alzheimer disease

SummaryDespite the emerging evidence implying early vascular contributions to neurodegenerative syndromes, the role of vascular smooth muscle cells (VSMCs) in the pathogenesis of Alzheimer disease (AD) is still not well understood. Herein, we show that VSMCs in brains of patients with AD and animal models of the disease are deficient in multiple VSMC contractile markers which correlated with Tau accumulation in brain arterioles. Ex vivo and in vitro experiments demonstrated that VSMCs undergo dramatic phenotypic transitions under AD-like conditions, adopting pro-inflammatory phenotypes. Notably, these changes coincided with Tau hyperphosphorylation at residues Y18, T205, and S262. We also observed that VSMC dysfunction occurred in an age-dependent manner and that expression of Sm22α protein was inversely correlated with CD68 and Tau expression in brain arterioles of the 3xTg-AD and 5xFAD mice. Together, these findings further support the contribution of dysfunctional VSMCs in AD pathogenesis and nominate VSMCs as a potential therapeutic target in AD.

CK2 Phosphorylating I2PP2A/SET Mediates Tau Pathology and Cognitive Impairment.

Casein kinase 2 (CK2) is highly activated in Alzheimer disease (AD) and is associated with neurofibrillary tangles formation. Phosphorylated SET, a potent PP2A inhibitor, mediates tau hyperphosphorylation in AD. However, whether CK2 phosphorylates SET and regulates tau pathological phosphorylation in AD remains unclear. Here, we show that CK2 phosphorylating SET at Ser9 induced tau hyperphosphorylation in AD. We found that either Aβ treatment or tau overexpression stimulated CK2 activation leading to SET Ser9 hyperphosphorylation in neurons and animal models, while inhibition of CK2 by TBB abolished this event. Overexpression of CK2 in mouse hippocampus via virus injection induced cognitive deficit associated with SET Ser9 hyperphosphorylation. Injection of SET Ser9 phosphorylation mimetic mutant induced tau pathology and behavior impairments. Conversely co-injection of non-phosphorylated SET S9A with CK2 abolished the CK2 overexpression-induced AD pathology and cognitive deficit. Together, our data demonstrate that CK2 phosphorylates SET at Ser9 leading to SET cytoplasmic translocation and inhibition of PP2A resulting in tau pathology and cognitive impairments.

Endoplasmic reticulum stress mediates JNK-dependent IRS-1 serine phosphorylation and results in Tau hyperphosphorylation in amyloid β oligomer-treated PC12 cells and primary neurons

AimsEndoplasmic reticulum stress (ERS) and insulin signaling impairment are commonly observed in Alzheimer's disease (AD), but the association between these two factors in AD has not been carefully studied. In peripheral insulin signaling impairment, ERS interferes with insulin signaling through c-Jun. N-terminal kinase (JNK)-dependent insulin receptor substance-1 (IRS-1) serine phosphorylation. We conducted this study to determine whether a similar mechanism contributes to insulin signaling impairment in AD pathogenesis. MethodsChanges in the levels of ERS markers, JNK activation, the insulin signaling status and Tau hyperphosphorylation were examined in amyloid β1–42 (Aβ1–42) oligomer-treated PC12 cells and primary neurons by western blotting and real-time fluorescence quantitative PCR. Inhibitors of ERS and JNK were utilized to confirm their association. ResultsOur results demonstrated that Aβ1–42 oligomers significantly induced ERS and JNK activation. In addition, in response to Aβ1–42 oligomers, IRS-1 phosphorylation at serines 307, 318 and 612 was increased. Further, an increase in Tau hyperphosphorylation at threonine 181 was observed following Aβ1–42 oligomer treatment. Moreover, inhibition of ERS or JNK could partially reverse the changes induced by the Aβ1–42 oligomers. ConclusionsThese findings suggest that ERS may contribute to insulin signaling impairment in AD through JNK-dependent IRS-1 serine phosphorylation. The ERS/JNK/IRS-1 pathway may be involved in Aβ1–42 oligomer-induced Tau hyperphosphorylation in AD.

Morroniside-Induced PP2A Activation Antagonizes Tau Hyperphosphorylation in a Cellular Model of Neurodegeneration.

An accumulation of hyperphosphorylated tau in the brain is a hallmark of Alzheimer's disease (AD). Deficits in protein phosphatase 2A (PP2A) are associated with tau hyperphosphorylation in AD. To investigate the effects of morroniside (MOR), isolated from Cornus officinalis, on tau hyperphosphorylation and its underlying mechanisms related to PP2A. SK-N-SH cells were pretreated with 50-200 μM MOR for 24 h followed by 20 nM okadaic acid (OA) for 6 h. PP2Ac siRNA was transfected into HEK293 cells to determine the direct interaction of MOR with PP2A. Western blotting was used to measure the expression of proteins and enzymes. PP2A activity was measured by molybdenum blue spectrophotometry. Pretreatment with MOR improved the cellular morphological damage and inhibited tau hyperphosphorylation in SK-N-SH cells induced by OA, a PP2A inhibitor. Moreover, MOR increased PP2A activity, concurrent with a decrease in the expression of demethylated PP2A at Leu309 and phosphorylated PP2A at Tyr307. MOR decreased protein phosphatase methylesterase 1 (PME-1) expression and the ratio of PME-1/leucine carboxyl methyltransferase 1 (LCMT-1). Furthermore, MOR treatment decreased the phosphorylation of Src at Tyr416, which regulates the phosphorylation of PP2A. MOR had no effect on PP2Ac expression and tau hyperphosphorylation in PP2Ac siRNA-transfected cells. MOR attenuated OA-induced tau hyperphosphorylation via PP2A activation, and its mechanism might be related to the regulation of PP2Ac post-translational modification and upstream enzymes such as Src and PME-1.

The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-β Production and Tau Hyperphosphorylation

We have previously shown that the L-type calcium channel (LCC) antagonist nilvadipine reduces brain amyloid-β (Aβ) accumulation by affecting both Aβ production and Aβ clearance across the blood-brain barrier (BBB). Nilvadipine consists of a mixture of two enantiomers, (+)-nilvadipine and (-)-nilvadipine, in equal proportion. (+)-Nilvadipine is the active enantiomer responsible for the inhibition of LCC, whereas (-)-nilvadipine is considered inactive. Both nilvadipine enantiomers inhibit Aβ production and improve the clearance of Aβ across the BBB showing that these effects are not related to LCC inhibition. In addition, treatment of P301S mutant human Tau transgenic mice (transgenic Tau P301S) with (-)-nilvadipine reduces Tau hyperphosphorylation at several Alzheimer disease (AD) pertinent epitopes. A search for the mechanism of action of (-)-nilvadipine revealed that this compound inhibits the spleen tyrosine kinase (Syk). We further validated Syk as a target-regulating Aβ by showing that pharmacological inhibition of Syk or down-regulation of Syk expression reduces Aβ production and increases the clearance of Aβ across the BBB mimicking (-)-nilvadipine effects. Moreover, treatment of transgenic mice overexpressing Aβ and transgenic Tau P301S mice with a selective Syk inhibitor respectively decreased brain Aβ accumulation and Tau hyperphosphorylation at multiple AD relevant epitopes. We show that Syk inhibition induces an increased phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase-3β, a primary Tau kinase involved in Tau phosphorylation, by activating protein kinase A, providing a mechanism explaining the reduction of Tau phosphorylation at GSK3β-dependent epitopes following Syk inhibition. Altogether our data highlight Syk as a promising target for preventing both Aβ accumulation and Tau hyperphosphorylation in AD.

Akt-activated GSK3β inhibitory peptide effectively blocks tau phosphorylation

Tau hyperphosphorylation and accumulation in neurofibrillary tangles are closely associated with cognitive deficits in Alzheimer's disease (AD). Abnormal overexpression of GSK3β has been implicated in tau hyperphosphorylation, thus many GSK3β inhibitors have been developed as drug candidates for AD. However, the potent GSK3β inhibitors are prone to show side effects because they can interfere with basal GSK3β function. We have recently reported that the specific motifs in the Wnt coreceptor LRP6 are able to directly inhibit GSK3β when phosphorylated, an thus a novel GSK3β inhibitory peptide (GIP) that can be activated by Akt, was generated by combining the LRP6 motif and an Akt target sequence. In the present study, we tested the effects of a novel GIP on modulating tau phosphorylation and tauopathy. The GSK3β-inhibitory action of GIP was tested ex vivo using hippocampal brain tissue. Human neuroblastoma SH-SY5Y cells were used to determine the protective effects of GIP against Aβ-induced death and tau phosphorylation. We used 3xTg-AD mice to verify the effect of GIP on the tau hyperphosphorylation and Aβ plaques in vivo. GIP effectively blocked GSK3 β -induced tau phosphorylation in hippocampal homogenate. In addition, the GIP fused with a cell permeable sequence attenuated Aβ-induced tau phosphorylation and cell death in human neuroblastoma cells. Interestingly, high Akt activation was observed particularly in the hippocampus assuming that GIP can be activated to inhibit GSK3 β. T he designed GIP significantly reduced tau phosphorylation in the hippocampus of 3 ' Tg-AD mice without affecting levels of Aβ plaques. In conclusion, the current findings provide a novel concept for the drug development targeting tau hyperphosphorylation in AD, and GIP may have potential as a therapeutic drug in tauopathies.

Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease

Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I2(PP2A). In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I2(PP2A) is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I2NTF) and the C-terminal fragment (I2CTF). Both I2NTF and I2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I2(PP2A) translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I2(PP2A), except when I2(PP2A) was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I2(PP2A), inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I2(PP2A)-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.

Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.

Telomere Shortening and Alzheimer’s Disease

Telomeres, at the ends of chromosomes and strands of genetic material, become shorter as cells divide in the process of aging. Telomere length has been considered as a biological marker of age. Telomere length shortening has also been evidenced as the causable role in age-related neurodegenerative diseases, including Alzheimer's disease (AD). It has been demonstrated that telomere shortening has been associated with cognitive impairment, amyloid pathology and hyper-phosphorylation of tau in AD and plays an important role in the pathogenesis of AD via the mechanism of oxidative stress and inflammation. However, it seems that there is no relationship between telomere shortening and AD. Therefore, it is essential for further clarification of telomere-related pathogenesis in AD.

PP2A and Alzheimer Disease

Phosphorylation and, therefore, binding capacity of microtubule-associated protein tau is regulated by specific kinases and phosphatases. Activation of tau kinases plays a crucial role in tau- hyper-phosphorylation in Alzheimer disease (AD) and related tauopathies. Among phosphatases, protein phosphatase 2A, PP2A, is a principal tau dephosphorylating enzyme in the brain. PP2A acts as trimer composed of a catalytic (PP2A C), a scaffolding (PP2A A) and a regulatory (PP2 AB; B55α) subunit. Several abnormalities of PP2A have been reported in AD, including decreased mRNA and protein levels of the PP2A C (not replicated by other studies); decreased protein levels of the PP2A A and B55α; reduced PP2A C methylation at Leu309 due to impaired function methyltransferase type IV; increased PP2A C phosphorylation at Tyr307; up-regulation of the PP2A inhibitors I1 and I2; and loss of enzymatic activity. These observations indicate that PP2A is a putative target of therapeutic intervention considering that enhancing PP2A activity would decrease tau hyper-phosphorylation in AD. In spite of these achievements further studies are needed to replicate the reported individual different alterations converging in PP2A in AD.

Okadaic acid induces tau phosphorylation in SH-SY5Y cells in an estrogen-preventable manner

One of the pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which are composed of abnormally hyperphosphorylated tau, but the mechanism of tau hyperphosphorylation in AD is still unclear. To investigate the effects of estrogens on tau phosphorylation, SH-SY5Y cells were treated with okadaic acid (OA), a serine/threonine phosphatase inhibitor, to induce tau phosphorylation and the effects of estrogen were observed by co-treatment with 17β-estradiol (E2). We found that OA induced in vitro tau hyperphosphorylation, which was prevented by E2 in a dose-dependent manner. This effect of E2 was partially blocked by an estrogen receptor (ER) antagonist, ICI 182,780. In addition to tau hyperphosphorylation, inhibition of serine/threonine phosphorylation induced upregulation of cdk5 levels, which was attenuated by E2 in a manner that was counteracted by ICI 182,780. Our results show that cdk5 is involved in OA-induced tau hyperphosphorylation, and estrogens ameliorate the tau hyperphosphorylation, which may be mediated in part by ER.

PP2A Regulates Tau Phosphorylation Directly and also Indirectly via Activating GSK-3β

Abnormal hyperphosphorylation of tau appears to be crucial in neurofibrillary degeneration in Alzheimer's disease (AD). Previous studies suggest that a down-regulation of protein phosphatase 2A (PP2A), the major tau phosphatase in human brain, contributes to tau hyperphosphorylation in AD. However, the effects of PP2A down-regulation on site-specific tau hyperphosphorylation is not well understood. In the present study, we showed that PP2A dephosphorylated tau at several phosphorylation sites with different efficiencies. Among the sites studied, Thr205, Thr212, Ser214, and Ser262 were the most favorable sites, and Ser199 and Ser404 were the least favorable sites for PP2A in vitro. Inhibition of PP2A with okadaic acid in metabolically active rat brain slices caused inhibition of glycogen synthase kinase-3beta (GSK-3beta) via an increase in its phosphorylation at Ser9. GSK-3beta phosphorylated tau at many sites, with Ser199, Thr205, and Ser396 being the most favorable sites in cells. The overall alterations in tau phosphorylation induced by PP2A inhibition were the result of the combined effects of both reduced tau dephosphorylation due to PP2A inhibition directly and reduced phosphorylation by GSK-3beta due to its inhibition. Because the impacts of tau phosphorylation on its biological activity and on neurofibrillary degeneration are site-specific, this study provides a new insight into the role of PP2A down-regulation in neurofibrillary degeneration in AD.

Increased CRMP2 phosphorylation is observed in Alzheimer's disease; does this tell us anything about disease development?

Collapsin response mediator protein-2 (CRMP2) was recently identified as a physiological substrate for GSK3 and Cdk5, two protein kinases suggested to exhibit greater activity in Alzheimer's disease (AD). Indeed, phosphorylation of CRMP2, at the residues targeted by GSK3 and Cdk5, is relatively high in cortex isolated from human AD brain, as well as in the brains of animal models of AD, while phospho-CRMP2 is found in neurofibrillary tangles. In mouse models of AD, increased phosphorylation occurs prior to pathology. Although CRMP2 has no known enzymatic activity, a great deal of information is appearing on its importance in neuronal development and polarity, as well as in axon growth and guidance. In this mini-review, we examine what is known about CRMP2 function, how that is controlled by phosphorylation, what alterations in molecular mechanisms could lead to the abnormally high CRMP2 phosphorylation in AD, and whether this is likely to be specific to AD or occur in other forms of neurodegeneration. This will include discussion of the evidence for increased GSK3 or Cdk5 activity, for decreased phosphatase activity, or the upregulation of other CRMP2 protein kinases in AD. Importantly, we will compare the processes that may contribute to increased CRMP2 phosphorylation with those known to increase tau hyperphosphorylation in AD, and whether these are likely to be part of disease development or a useful early marker for AD.

Cyclin-dependent kinase 5 activator p35 over-expression and amyloid beta synergism increase apoptosis in cultured neuronal cells

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuronal loss, dementia and pain. Two main protein aggregates, extracellular (senile plaques, SP) and intracellular (neurofibrillary tangles, NFT), are associated with AD. NFT are mainly composed of hyperphosphorylated microtubule-associated protein tau. Nowadays several protein kinases have been implicated in the phosphorylation of tau, including glycogen synthase kinase 3 beta (GSK3β), MAP kinase, protein kinase A and cyclin-dependent kinase 5 (Cdk5). A deregulation in the activity of Cdk5 has been postulated to participate in the abnormal tau hyperphosphorylation in AD. Activation of Cdk5 occurs after its association with p35, a neuron-specific activator, predominantly in the nervous system. Therefore, in this study we used the tetracycline transactivator system to increase p35/GFP in neuronal cells, treated with amyloid beta 1-42 (Aβ1-42) peptide. These cells showed an increase of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3 staining, indicating increased apoptosis of neuronal cells. This effect could be reversed by the addition of tetracycline in the culture medium, suggesting synergistic effects of p35 over-expression and Aβ treatment in the apoptosis of neuronal cells. These results represent a linkage between amyloidogenic and cdk5 pathways leading to apoptosis of neuronal cells.

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.

Pin1-dependent Prolyl Isomerization Modulates the Stress-induced Phosphorylation of High Molecular Weight Neurofilament Protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.

Decreased glucose transporters correlate to abnormal hyperphosphorylation of tau in Alzheimer disease

Brain glucose uptake/metabolism is impaired in Alzheimer disease (AD). Here, we report that levels of the two major brain glucose transporters (GLUT1 and GLUT3) responsible for glucose uptake into neurons were decreased in AD brain. This decrease correlated to the decrease in O-GlcNAcylation, to the hyperphosphorylation of tau, and to the density of neurofibrillary tangles in human brains. We also found down-regulation of hypoxia-inducible factor 1, a major regulator of GLUT1 and GLUT3, in AD brain. These studies provide a possible mechanism by which GLUT1 and GLUT3 deficiency could cause impaired brain glucose uptake/metabolism and contribute to neurodegeneration via down-regulation of O-GlcNAcylation and hyperphosphorylation of tau in AD.