Effects of cyclin-dependent kinase-5 activity on apoptosis and tau phosphorylation in immortalized mouse brain cortical cells

Cyclin-dependent kinase-5 (CDK5), a unique CDK family member, is active primarily in the central nervous system (CNS). Previous studies suggest that CDK5 is proapoptotic and contributes to tau hyperphosphorylation and neurodegeneration in Alzheimer's disease. The objective of this study was to examine CDK5 effects on apoptotic progression and tau phosphorylation. Immortalized embryonic mouse brain cortical cells were used to establish a stable cell line that overexpressed wild-type human tau. In these studies, thapsigargin, which induces endoplasmic reticulum stress and can cause accumulation of misfolded proteins, was used to induce apoptosis. Caspase-3 activity and poly-(ADP-ribose)-polymerase (PARP) cleavage, as measures of apoptosis, were significantly increased 24 and 48 hr after thapsigargin treatment, and these events were unaffected by tau expression. Although transient coexpression of CDK5 and its activator, p25, increased CDK5 activity greater than tenfold, increases in caspase-3 activity in response to thapsigargin treatment were unaffected by the presence of CDK5/p25. Tau phosphorylation at the PHF-1 epitope, but not the Tau-1 epitope, was increased significantly in CDK5/p25-transfected cells compared to cells transfected with dominant negative CDK5 (DNCDK5). The PHF-1 epitope remained phosphorylated until 48 hr after thapsigargin treatment in the CDK5/p25-transfected cells. Over the course of apoptosis in this model, phosphorylation of the Tau-1 epitope was unaffected in cells transfected with DNCDK5, vector, or CDK5/p25. In summary, these results demonstrate that CDK5 does not have a significant impact on tau phosphorylation and thapsigargin-induced apoptosis in this neuronal cell model.     

Pathological effects of glyoxalase I inhibition in SH-SY5Y neuroblastoma cells

In Alzheimer's disease (AD), in aging, and under conditions of oxidative stress, the levels of reactive carbonyl compounds continuously increase. Accumulating carbonyl levels might be caused by an impaired enzymatic detoxification system. The major dicarbonyl detoxifying system is the glyoxalase system, which removes methylglyoxal in order to minimize cellular impairment. Although a reduced activity of glyoxalase I was evident in aging brains, it is not known how raising the intracellular methylglyoxal level influences neuronal function and the phosphorylation pattern of tau protein, which is known to be abnormally hyperphosphorylated in AD. To simulate a reduced glyoxalase I activity, we applied an inhibitor of glyoxalase I, p-bromobenzylglutathione cyclopentyl diester (pBrBzGSCp(2)), to SH-SY5Y neuroblastoma cells to induce chronically elevated methylglyoxal concentrations. We have shown that 10 microM pBrBzGSCp(2) leads to a fourfold elevation of the methylglyoxal level after 24 hr. In addition, glyoxalase I inhibition leads to reduced cell viability, strongly retracted neuritis, increase in [Ca(2+)](i), and activation of caspase-3. However, pBrBzGSCp(2) did not lead to tau "hyper"-phosphorylation despite activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase but rather activated protein phosphatases 2 and induced tau dephosphorylation at the Ser(202)/Thr(205) and Ser(396)/Ser(404) epitopes. Preincubation with the carbonyl scavenger aminoguanidine prevented tau dephosphorylation, indicating the specific effect of methylglyoxal. Also, pretreatment with the inhibitor okadaic acid prevented tau dephosphorylation, indicating that methylglyoxal activates PP-2A. In summary, our data suggest that a reduced glyoxalase I activity mimics some changes associated with neurodegeneration, such as neurite retraction and apoptotic cell death.     

Phospholipid transfer protein reduces phosphorylation of tau in human neuronal cells

Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer's disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau and increases levels of the inactive form of glycogen synthase kinase‐3β (GSK3β) in HCN2 cells. Furthermore, inhibition of phosphatidylinositol‐3 kinase (PI3K) reversed the PLTP‐induced increase in levels of GSK3β phosphorylated at serine 9 (pGSK3β_Ser9) and partially reversed the PLTP‐induced reduction in tau phosphorylation. We provide evidence that the PLTP‐induced changes are not due to activation of Disabled‐1 (Dab1), insofar as PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin‐like growth factor 1 (IGF1) receptor (IGFR) reverses the PLTP‐induced increase in levels of phosphorylated Akt (pAkt_Thr308 and pAkt_Ser473), suggesting that PLTP‐mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons. © 2009 Wiley‐Liss, Inc.     

Tau and HMW tau phosphorylation and compartmentalization in apoptotic neuronal PC12 cells

In the Alzheimer disease brain, the microtubule-associated protein tau is hyperphosphorylated. There is also evidence that apoptotic-like processes may contribute to the neuronal loss in AD. In an apoptotic model that involves replating neuronal PC12 cells without serum and nerve growth factor (NGF), tau was hyperphosphorylated. During replating, however, neurites are removed. Here, differentiated cells were maintained in serum-free media before growth factor removal, thus maintaining neuritic processes during the apoptotic process and allowing for evaluation of neuritic changes. Tau phosphorylation, evaluated by immunoblotting and immunocytochemistry, was compared with various measures of cell death. Compared with control, NGF-deprived cells exhibited gradual and consistent increases of lactate dehydrogenase release over a 5-day period and a peak of caspase-3 activity at Day 2 after NGF removal. Nuclear staining demonstrated chromatin condensation in NGF-deprived cells. Apoptotic cells had thickened, tortuous, and shortened neuritic processes compared with control cells. Immunoblotting showed an increase in both tau and high molecular weight (HMW) tau phosphorylation during the apoptotic process. Immunoreactivity of both tau isoforms shifted from the detergent insoluble cytoskeleton to the detergent soluble compartment in the apoptotic cells. The microtubule binding of both tau isoforms from apoptotic cells also was impaired. Immunoblotting of purified plasma membrane showed preferential association of HMW tau with the plasma membrane during apoptosis. Also, plasma membrane-associated HMW tau was more phosphorylated during apoptosis. Immunocytochemistry demonstrated increased tau phosphorylation in most apoptotic cells, especially in the neurites. Tau was, however, dephosphorylated cells in the last stages of apoptosis.     

Protective effects of erythropoietin on tau phosphorylation induced by beta-amyloid

Neuropathological studies have demonstrated that the presence of neurofibrillary tangles (NFTs) is one of the most prominent pathologic characteristics of Alzheimer's disease (AD). The microtubule-associated protein tau is the major component of NFTs, and its abnormal hyperphosphorylation leads to the destabilization of microtubules, impaired axonal transport, and eventual death of the neurons. The hematopoietic cytokine erythropoietin (Epo) is now considered as a viable agent with regard to central nervous system injury in a variety of cellular systems. Here we report that Epo prevented tau hyperphosphorylation in SH-SY5Y cells exposed to the beta-amyloid peptide and that this effect may depend on the PI3K/Akt-GSK-3beta pathway. This study provides new molecular insight into the neuroprotective effect of Epo and suggests its possible therapeutic role in the management of AD.     

Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation

Tungstate treatment increases the phosphorylation of glycogen synthase kinase-3beta (GSK3beta) at serine 9, which triggers its inactivation both in cultured neural cells and in vivo. GSK3 phosphorylation is dependent on the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) induced by tungstate. As a consequence of GSK3 inactivation, the phosphorylation of several GSK3-dependent sites of the microtubule-associated protein tau decreases. Tungstate reduces tau phosphorylation only in primed sequences, namely, those prephosphorylated by other kinases before GSK3beta modification, which are serines 198, 199, or 202 and threonine 231. The phosphorylation at these sites is involved in reduction of the interaction of tau with microtubules that occurs in Alzheimer's disease.     

S/P and T/P phosphorylation is critical for tau neurotoxicity in Drosophila

The microtubule-associated protein tau is hyperphosphorylated abnormally in AD and related neurodegenerative disorders. Many phospho epitopes created by proline directed kinases (SP/TP sites) show relative specificity for disease states. To test whether phosphorylation at the disease-associated SP/TP sites affects tau toxicity in vivo, we expressed a form of tau in Drosophila in which all SP/TP sites are mutated to alanine. We find that blocking phosphorylation at SP/TP motifs markedly reduces tau toxicity in vivo. Using phosphorylation-specific antibodies, we identify a positive correlation between increased phosphorylation at disease-associated sites and neurotoxicity. We use the phosphorylation-incompetent version of tau to show that kinase and phosphatase modifiers of tau neurotoxicity, including cdk5/p35, the JNK kinase hemipterous and PP2A act via SP/TP phosphorylation sites. We provide direct evidence in an animal model system to support the role of phosphorylation at SP/TP sites in playing a critical role in tau neurotoxicity.     

Effect of cortistatin on tau phosphorylation at Ser262 site

The development of intraneuronal lesions as a result of the progressive deposition of hyperphosphorylated tau at specific brain regions (such as hippocampus and cortex) plays a key role in the pathological process of Alzheimer's disease. However, the mechanisms by which tau phosphorylation is regulated, mainly in the pathology found in the cortex, are still poorly understood. Here, we analyzed the effect of cortistatin, a cortical neuropeptide related to somatostatin, on tau phosphorylation at Ser262 in cultures of murine cortical neurons. Both somatostatin and cortistatin induce tau phosphorylation at Ser262, a site modified in Alzheimer's disease, although with different kinetics in cortex. The effect of cortistatin likely is mediated by heterodimeric receptors composed of somatostatin receptor subtypes 2 and 4 and also by protein kinase C signaling. Cortistatin-deficient mice show decreased tau phosphorylation at Ser262 in the cortex but not in other brain regions tested. Our results suggest an important role for cortistatin in the regulation of tau phosphorylation that may be associated with the pathophysiology of Alzheimer's disease in regions such as the cerebral cortex.     

ER stress is involved in Abeta-induced GSK-3beta activation and tau phosphorylation

Intracellular neurofibrillary tangles, one of the characteristic hallmarks of Alzheimer's disease (AD), are mainly composed of hyperphosphorylated tau. The abnormal tau phosphorylation seems to be related to altered activity of kinases such as glycogen synthase kinase-3beta (GSK-3beta). Tau pathology is thought to be a later event during the progression of the disease, and it seems to occur as a consequence of amyloid-beta (Abeta) peptide accumulation. The aim of this work was to investigate whether soluble Abeta1-42, particularly oligomers that correspond to the neurotoxic species involved early in the development of AD, triggers tau phosphorylation by a mechanism involving activation of tau-kinase GSK-3beta. Several studies suggest that GSK-3beta plays a central role in signaling the downstream effects of endoplasmic reticulum (ER) stress. Therefore, the involvement of ER Ca(2+) release in GSK-3beta activation and tau phosphorylation induced by Abeta1-42 oligomers was evaluated using dantrolene, an inhibitor of Ca(2+) release through channels associated with ER ryanodine receptors. We observed that Abeta1-42 oligomers increase tau phosphorylation and compromises cell survival through a mechanism mediated by GSK-3beta activation. We also demonstrated that oligomeric Abeta1-42 induces ER stress and that ER Ca(2+) release is involved in oligomer-induced GSK-3beta activation and tau phosphorylation. This work suggests that GSK-3beta can be a promising target for therapeutic intervention in AD.     

p35/Cdk5 pathway mediates soluble amyloid-beta peptide-induced tau phosphorylation in vitro

Alzheimer's disease (AD) is pathologically characterized by deposition of amyloid-beta peptides (Abeta) as senile plaques and by the occurrence of neurofibrillary tangles (NFTs) composed primarily of hyperphosphorylated tau protein. Activation of cyclin-dependent kinase 5 (Cdk5) via its potent activator p25 has recently been shown to promote phosphorylation of tau at AD-specific phosphoepitopes, and increased cleavage of p35 to p25 has been demonstrated in AD patients, suggesting that Cdk5 may represent a pathogenic tau protein kinase. We were interested in the potential effect of soluble forms of Abeta on Cdk5-mediated AD-like tau phosphorylation, insofar as previous studies of human biopsies and aged canine and primate brains have shown that dystrophic neurites appear before the formation of neuritic plaques. We transfected N2a cells with a p35 vector (N2a/p35 cells) and, after differentiation, challenged these cells with Abeta(1-42) peptide in soluble form (sAbeta(1-42)). Results show that sAbeta(1-42) at relatively low levels (1-5 microM) dose-dependently increases tau phosphorylation at AD-specific phosphoepitopes in differentiated N2a/p35 cells compared with controls, an effect that is blocked by antisense oligonucleotides against p35. sAbeta(1-42)-induced tau phosphorylation is concomitant with an increase in both p25 to p35 ratio and Cdk5 activity (but not protein levels). Additionally, blockade of L-type calcium channels or inhibition of calpain completely abolishes this effect. Taken together, these data indicate that sAbeta is a potent activator of the p25/Cdk5 pathway, resulting in promotion of AD-like tau phosphorylation in vitro.     

Akt activity in presenilin 1 wild-type and mutation transfected human SH-SY5Y neuroblastoma cells after serum deprivation and high glucose stress.

The majority of early-onset familial Alzheimer disease cases are caused by mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations have been hypothesised to cause Alzheimer disease either by altering amyloid precursor protein metabolism or by increasing the vulnerability of neurons to undergo death by apoptosis. We showed previously that PS1 exon 9 deletion (PS1 DeltaE9) and L250S mutations predispose SH-SY5Y neuroblastoma cells to high glucose stress-induced apoptosis and that the anti-apoptotic effect of insulin-like growth factor I (IGF-I) is compromised by these mutations. The present study investigates whether the susceptibility of PS1 mutation transfected SH-SY5Y cells to undergo apoptosis is likely due to a downregulation of Akt/protein kinase B (Akt), a key intermediate in the phosphatidylinositol 3 (PI3)-kinase arm of the IGF-I signaling pathway. We used two methods to determine the regulation of Akt in response to the pro-apoptotic stimuli of serum deprivation and high glucose stress, as well as treatment with IGF-I. We also looked at the phosphorylatiom state of GSK-3beta at Ser9. Using a kinase assay with immunoprecipitated Akt, we detected an increased Akt activity in PS1 L250S cells at 1 hr after the combination of 20 mM glucose plus 10 nM IGF-I, when compared to the other cell types. This effect, however, was transient in that no mutation related differences were seen at either 6- or 24-hr post-treatment. Immunoblotting for Phospho-Akt as a ratio of total Akt, as well as for GSK-3beta phosphorylated at Ser9 revealed no apparent between cell type and treatment differences. This data strongly indicates that PS1 wt and mutant cells show no major differences in the pattern of Akt regulation after exposure to the pro-apoptotic stimuli of either serum deprivation or high glucose stress, or treatment with IGF-I. It is suggested that another component of IGF-I signaling is likely disrupted in these cells to increase their vulnerability to undergo death by apoptosis.     

Caspases‐2 and ‐8 are involved in the presenilin1/γ‐secretase‐dependent cleavage of amyloid precursor protein after the induction of apoptosis

The presenilin/γ‐secretase protease cleaves many type‐I membrane proteins, including the amyloid β‐protein (Aβ) precursor (APP). Previous studies have shown that apoptosis induces alterations in Aβ production in a caspase‐dependent manner. Here, we report that staurosporine (STS)‐induced apoptosis induces caspase‐8 and/or‐2‐dependent γ‐secretase activation. Blocking of caspase activity with caspase‐8 inhibitor z‐IETD‐fmk, and caspase‐2 inhibitor z‐VDVAD‐fmk reduced Aβ production by STS in H4 cells expressing the Swedish mutant of APP (HSW) or APP‐C99 (H4‐C99). There was no inhibitory effect of other caspases (‐1, ‐3, ‐5, ‐6, ‐9) on Aβ production by STS. This finding was further supported by evidence that siRNA transfection, depleting caspase‐2 or ‐8 levels, lowered Aβ production in HSW and H4‐C99 cells without affecting expression of APP or γ‐secretase complex. In addition, Aβ production by STS was decreased by JNK inhibitors, SP600125. These results suggest that caspase‐2 and/or ‐8 is involved in presenilin/γ‐secretase activation and Aβ production in apoptosis. © 2010 Wiley‐Liss, Inc.     

Microtubule assembly competence analysis of freshly-biopsied human tau,dephosphorylated tau,and Alzheimer tau

Phosphorylation of the microtubule-associated protein tau regulates its binding to microtubules; highly phosphorylated tau is also a prime component of paired helical filaments (PHFs) of Alzheimer's disease (AD). Tau from freshly biopsied human, monkey, and rat brain share similar electrophoretic mobility patterns and overlapping phosphorylated epitopes when compared to AD tau isolated from AD brain. We compared the microtubule reassembly competence of fresh isolates of phosphorylated tau to that of maximally dephosphorylated tau and tau from AD brain. A rapid procedure was developed which permitted the enrichment of phosphorylated and dephosphorylated tau from human biopsies in the absence of protein kinase and phosphatase activity. Microtubule assembly assays, using a spectrophotometric measure and purified bovine brain tubulin, were used to correlate assembly competence with states of tau electrophoretic mobility. Maximally dephosphorylated human biopsy-derived tau and monkey tau were assembly competent; tau from AD brain was virtually unable to direct microtubule assembly. Unmodified, biopsy-derived tau from non-AD brain was intermediate in assembly competence relative to AD tau and dephosphorylated tau. Several lines of evidence were used to correlate phosphorylation states of tau with microtubule assembly. First, in vitro dephosphorylation of human biopsy-derived tau with either PP2A or PP2B alone or in combination led to increasing assembly competence as the electrophoretic mobility of tau increased. Second, addition of the protein phosphatase inhibitor okadaic acid (10 μM) to brain-slice preparations slowed electrophoretic mobility of tau and decreased binding competence. We suggest that tau derived from freshly-biopsied brain exists in a range of phosphorylated states, and that dephosphorylation by PP2A and/or PP2B increases microtubules assembly competence. © 1996 Wiley-Liss, Inc.     

Amino- and carboxyl-terminal mutants of presenilin 1 cause neuronal cell death through distinct toxic mechanisms: Study of 27 different presenilin 1 mutants

Presenilin (PS)1 and its mutants, which consist of the N-terminal and C-terminal fragments, cause certain familial forms of Alzheimer's disease (FAD). Our earlier studies found that FAD-linked M146L-PS1 causes neuronal cell death through nitrogen oxide synthase (NOS) and that FAD-linked N141I-PS2, another member of the PS family, causes neuronal cell death through NADPH oxidase. In this study, we examined 27 different FAD-linked mutants of PS1, and found that PS1 mutants with mutations in the N-terminal fragment caused NOS inhibitor (NOSI)-sensitive neuronal cell death; in contrast, the PS1 mutants with mutations in the C-terminal fragment caused NOSI-resistant neuronal cell death. The former toxicity was resistant to the specific NADPH oxidase inhibitor apocynin and was inhibited by Humanin (HN), a newly identified neuroprotective factor against Alzheimer's disease (AD)-relevant insults, but not by insulin-like growth factor-I (IGF-I). In contrast, the latter toxicity was sensitive to apocynin and inhibited by both IGF-I and HN. This study indicates for the first time that N- and C-terminal fragment PS1 mutants can generate distinct neurotoxic signals, which will provide an important clue to the understanding of the entire array of neurotoxic signals generated by FAD-causative mutations of PS1.     

Characterization of catechol-thioether-induced apoptosis in human SH-SY5Y neuroblastoma cells

Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.     

Activation of PP2A-like phosphatase and modulation of tau phosphorylation accompany stress-induced apoptosis in cultured oligodendrocytes

In a number of neurodegenerative diseases, tau-positive glial cytoplasmic inclusions (GCIs), immunochemically labeled with antibodies to the small heat shock protein (HSP) alphaB-crystallin, occur in oligodendrocytes. The microtubule-associated protein tau is functionally modulated by phosphorylation. We have shown previously that oxidative stress (OS) and heat shock (HS) induce apoptotic cell death in oligodendrocytes. The present study was undertaken to test whether stress responses in oligodendrocytes cause abnormalities in the expression and posttranslational modification of tau proteins, and whether the dynamic phosphorylation and dephosphorylation of tau are involved in the pathogenesis of glial cells. Cultured rat brain oligodendrocytes were subjected to OS, exerted by hydrogen peroxide, or HS (44 degrees C, 30 min). Immunoblot analysis with a panel of phosphorylation-dependent antibodies shows that OS and HS caused the rapid dephosphorylation of tau proteins at multiple sites, before characteristic features of apoptosis were observed. Concomitantly, ERK1,2 (extracellular signal-regulated kinase) was activated. Tau phosphorylation and rephosphorylation after stress was mediated by glycogen synthase kinase 3beta (GSK-3beta), and not by ERK1,2 and could be suppressed by lithium chloride, a specific inhibitor of GSK-3beta. Stress-induced dephosphorylation could be mimicked by alkaline phosphatase and suppressed by the protein phosphatase inhibitor okadaic acid (OA), indicating that PP2A in oligodendrocytes is activated by stress. OA at low concentrations could prevent stress-induced DNA fragmentation, but eventually exerted cytotoxic effects. Hence, stress-induced activation of PP2A in oligodendrocytes and tau dephosphorylation constitute a major feature of the response to injury in these cells, which eventually undergo apoptotic cell death.     

Small heat shock proteins Hsp27 or alphaB-crystallin and the protein components of neurofibrillary tangles: tau and neurofilaments

The heat-shock proteins (HSPs) Hsp27 and alphaB-crystallin are up-regulated in Alzheimer's disease (AD), but the extent of this and the consequences are still largely unknown. The HSPs are involved in protein degradation and protection against protein aggregation, and they interact with several cytoskeletal components such as microtubules (MT) and neurofilaments (NF). AD pathology includes aggregated proteins (tau, NF), decreased protein degradation, and cytoskeletal disruption. It is thus of interest to investigate more closely the possible roles of the HSPs in AD pathology. The expressions of Hsp27 and alphaB-crystallin in AD brain samples were significantly increased (by approximately 20% and approximately 30%, respectively) and correlated significantly with phosphorylated tau and NF proteins. To investigate the consequences of increased HSP levels on tau and NF regulation, N2a cells were transfected with Hsp27 or alphaB-crystallin constructs, and overexpression of the HSPs was confirmed in the cells. Increased tau phosphorylation at the Ser262 site in the N2a cells was regulated by Hsp27 overexpression (possibly through p70S6k), whereas the overexpression of alphaB-crystallin resulted in decreased levels of phosphorylated tau, NF, and GSK-3beta. It was also shown that overexpression of HSPs causes an increase in the percentage of cells present in the G(1) phase. The results presented suggest that a cellular defense against dysregulated proteins, in the form of Hsp27 and alphaB-crystallin, might contribute to the cell cycle reentry seen in AD cells. Furthermore, Hsp27 might also be involved in AD pathology by aggravating MT disruption by tau phosphorylation.     

Effect of acetylcholine on tau phosphorylation in human neuroblastoma cells

Alzheimer's disease (AD) is a senile dementia characterized by a progressive loss of memory, together with cognitive and behavioral impairments. In the past it was indicated that the disease was associated with a loss of acetylcholine (ACh) in the cerebral cortex (Bowen et al., 1976; Davies and Maloney, 1976); afterward, it was indicated that the severity of dementia was correlated with the extent of cholinergic loss (Perry et al., 1981). Because one of the biochemical features of AD is modification by phosphorylation of the microtubule-associated protein tau (for review, see Avila et al., 2004), in this work we indicate the effect of ACh on tau phosphorylation at specific sites recognized by 12E8 and PThr50 antibodies in human neuroblastoma SH-SY5Y cells. Two sites in which modification might regulate the binding of tau to microtubules (Novak et al., 1991; Feijoo et al., 2004).     

Phospholipids alter tau conformation,phosphorylation, proteolysis,and association with microtubules: Implication for tau function under normal and degenerative conditions

Discerning the in situ functions of the microtubule-associated protein (MAP) tau is of interest both in terms of neuronal differentiation and homeostasis as well as in terms of neurodegenerative conditions such as Alzheimer's disease. In the present study, exposure to excess phosphatidyl serine (PS) for <1 min induced antigenic alterations in multiple N-terminal, C-terminal and central epitopes of purified human brain tau. Notably, “AD-like” epitopes (PHF-1, ALZ-50, AT-8) were decreased by PS; other epitopes (e.g., 5E2, Tau-1) increased and others remained relatively unchanged. Inclusion of γ-AT[³²P] during incubations did not reveal any contaminating kinase activity. Direct addition of chloroform:methanol (CM; the initial PS solvent) demonstrated that these changes were not derived from CM-mediated tau denaturation. Phosphatidyl choline induced similar antigenic changes, while phosphatidyl inositol did not. PS inhibited MAP-kinase generation of phospho-dependent tau epitopes and incorporation of phosphates by tau. Inclusion of PS during coincubation of tau and tubulin reduced the extent of cosedimentation of tau with MTs. Finally, PS enhanced the ability of calpain-mediated tau proteolysis. These data suggest that tau antigenicity in situ may be derived from phospholipid-dependent alterations in tau conformation in addition to tau phosphorylation state. These data further suggest that disruption of the normal association of tau with phospholipids may foster accumulation of tau and, in doing so, render tau more susceptible to hyperphosphorylation. J. Neurosci. Res. 50:114–122, 1997. © 1997 Wiley-Liss, Inc.