Effects of Cell Cycle Inhibitors on Tau Phosphorylation in N2aTau3R Cells

Neurofibrillary tangles are one of the pathologic hallmarks of Alzheimer's disease (AD). They are composed of paired helical filaments (PHF) containing hyperphosphorylated forms of tau. Hyperphosphorylation of certain tau sites favors its dissociation from the microtubules (MT), interfering with axonal transport and compromising the function and viability of neurons. Reappearance of cell cycle proteins have been reported in neurons exhibiting tau aggregation, suggesting that an aberrant cell cycle occurs before neurons die. Cell cycle suppression in neurons is crucial to survival, thus prevention of progression through the cell cycle may offer a therapeutic approach. Using a neuroblastoma cell line overexpressing 3-repeat (3R) tau, we investigated the effects of cell cycle inhibitors on tau phosphorylation. G2/M phase inhibitors did not alter phosphorylation of tau at Ser-202 and Ser-396/404 at the lower doses, but did at higher doses. Ser-202 and Ser-396/404 are phosphorylation sites of early and late neurofibrillary tangles, respectively, in AD. Cisplatin, a G1 phase inhibitor, did not phosphorylate tau. Cyclophosphamide and phosphoramide mustard, DNA cross-linking agents, decreased tau phosphorylation at Ser-396/404 site, but increased phosphorylation at Ser-202. These studies demonstrate that the G2/M blockers have a dose-dependent effect on tau phosphorylation. This seems to be a consequence of both the disruption of MT-organization and MT-dynamics when doses are higher, but only a disruption of MT-dynamics with lower doses. These results are also in agreement with the lack of phosphorylation seen for cisplatin, another inhibitor that produces disruption of the MT-dynamics.     

Hyperosmotic stress-induced apoptosis and tau phosphorylation in human neuroblastoma cells

A characteristic hallmark of Alzheimer's disease brain is the presence of hyperphosphorylated tau; however, the mechanisms responsible for the aberrant tau phosphorylation are unknown. Recently, it has been shown that apoptotic-like processes may be involved in some of the neuronal loss in Alzheimer's disease. In consideration of these findings, the relationship between tau phosphorylation and apoptosis was examined in human neuroblastoma SH-SY5Y cells that were subjected to hyperosmotic stress. In this model caspase 3 activity, which served as an indicator of apoptosis, was increased by 30 min of osmotic stress and remained elevated through 4 hr. Hyperosmotic stress also resulted in a robust increase in tau phosphorylation at both Ser/Pro and non-Ser/Pro sites. Phosphorylation of Ser262/356 (12E8) and Ser396/404 (PHF-1) increased by 5 min and remained elevated for at least 1 hr. In contrast, phosphorylation within the Tau-1 epitope did not increase (as evidenced by decreased immunoreactivity) until 30 min after treatment but remained elevated for a much greater period of time. Treatment with insulin-like growth factor-1 delayed but did not prevent apoptotic cell death induced by osmotic stress and attenuated the increase in phosphorylation at the Tau-1 epitope. Li(+), an inhibitor of glycogen synthase kinase 3 beta, had no effect on osmotic stress-induced caspase activation, but reduced phosphorylation at the Tau-1 epitope. Complete inhibition of osmotic stress-induced caspase activation with DEVD-CHO had no effect on the increases in tau phosphorylation. The results of these studies demonstrate that tau phosphorylation is increased at the specific epitopes during apoptosis. However, the changes in tau phosphorylation likely do not significantly impact the apoptotic process but rather occur concurrently as a result of inappropriate activation of specific protein kinases. Nonetheless, there is increasing evidence of a dysregulation of protein kinases that occurs in Alzheimer's disease brain that may be part of the events of apoptosis, which could contribute to aberrant increases in tau phosphorylation.     

Delayed caffeine treatment prevents nigral dopamine neuron loss in a progressive rat model of Parkinson's disease

Parkinson's disease (PD) is characterized by a prominent degeneration of nigrostriatal dopamine (DA) neurons with an accompanying neuroinflammation. Despite clinical and preclinical studies of neuroprotective strategies for PD, there is no effective treatment for preventing or slowing the progression of neurodegeneration. The inverse correlation between caffeine consumption and risk of PD suggests that caffeine may exert neuroprotection. Whether caffeine is neuroprotective in a chronic progressive model of PD has not been evaluated nor is it known if delayed caffeine treatment can stop DA neuronal loss. We show that a chronic unilateral intra-cerebroventricular infusion of 1-methyl-4-phenylpyridinium in the rat brain for 28 days produces a progressive loss of DA and tyrosine hydroxylase in the ipsilateral striatum and a loss of DA cell bodies and microglial activation in the ipsilateral substantia nigra. Chronic caffeine consumption prevented the degeneration of DA cell bodies in the substantia nigra. Importantly, neuroprotection was still apparent when caffeine was introduced after the onset of the neurodegenerative process. These results add to the clinical relevance for adenosine receptors as a disease-modifying drug target for PD.     

Axonal disruption and aberrant localization of tau protein characterize the neuropil pathology of Alzheimer's disease

The microtubule-associated protein tau, a major antigenic component of paired helical filaments, has been demonstrated in neurofibrillary tangles and in neurites of senile plaques. With optimal fixation and histochemical methods, we show the normal axonal location of tau protein in human cerebral cortex and the striking alterations of tau distribution that affect the cortical neuropil in Alzheimer's disease. Normally, cortical tau-immunoreactive fiber bundles form a pattern resembling that seen with myelin stains. The prominence of white matter staining suggests that tau may be especially enriched in projection systems. Alzheimer's disease causes massive axonal disruption and the dislocation of tau protein from its usual axonal domain into neuronal cell bodies, dendrites, and presynaptic regions. The normal pattern of axonal staining in cortex is disrupted and white matter staining is reduced. Prominent abnormal tau-immunoreactive neuropil fibers are densely present even in cortical regions without classical neurofibrillary tangle and senile plaque formation. The striking neuropil abnormalities, revealed by the aberrant localization of tau protein, are likely to contribute to neuronal dysfunction in Alzheimer's disease.     

Hippocampal neurons predisposed to neurofibrillary tangle formation are enriched in type II calcium/calmodulin-dependent protein kinase

The microtubule-associated phosphoprotein, tau, is an integral component of paired helical filaments in Alzheimer neurofibrillary tangles (NFT). The mechanism of NFT formation is unknown but aberrant phosphorylation of tau may be contributory. Calcium/calmodulin-dependent protein kinase type II (CaM kinase II), the most abundant kinase in the brain, phosphorylates tau in vitro. We found CaM kinase II immunoreactivity concentrated in human hippocampal pyramidal neurons of CA1 and the subiculum. In Alzheimer's disease (AD) staining intensity of CA1 and subicular neurons is strikingly increased despite NFT formation and neuronal depletion. Enhanced CaM kinase II activity, possibly a result of deafferentation, may contribute to phosphorylation of tau protein leading to NFT deposition and neuronal death in AD.     

Neuroprotection by T-cells depends on their subtype and activation state

This study analyzes how the antigen specificity, the subtype, and the activation state of T cells modulate their recently discovered neuroprotective potential. We assessed the prevention from neuronal damage in organotypic entorhinal-hippocampal slice cultures after co-culture with Th1 and Th2 cells either specific for myelin basic protein (MBP) or ovalbumin (OVA). We found that MBP-specific Th2 cells were the most effective in preventing central nervous system (CNS) tissue from secondary injury. This neuroprotective T cell effect appears to be mediated by soluble factors. After stimulation with phorbol myristate acetate and ionomycin, all T cells were most effective in preventing neuronal death. Our data show that the T cell subtype and activation state are important features in determining the neuroprotective potential of these cells.     

TGF-beta 1 protects against Abeta-neurotoxicity via the phosphatidylinositol-3-kinase pathway

beta-Amyloid (A beta) injection into the rat dorsal hippocampus had a small neurotoxic effect that was amplified by i.c.v. injection of SB431542, a selective inhibitor of transforming growth factor-beta (TGF-beta) receptor. This suggested that TGF-beta acts as a factor limiting A beta toxicity. We examined the neuroprotective activity of TGF-beta1 in pure cultures of rat cortical neurons challenged with A beta. Neuronal death triggered by A beta is known to proceed along an aberrant re-activation of the cell cycle, and involves late beta-catenin degradation and tau hyperphosphorylation. TGF-beta1 was equally protective when added either in combination with, or 6 h after A beta. Co-added TGF-beta1 prevented A beta-induced cell cycle reactivation, whereas lately added TGF-beta1 had no effect on the cell cycle, but rescued the late beta-catenin degradation and tau hyperphosphorylation. The phosphatidylinositol-3-kinase (PI-3-K) inhibitor, LY294402, abrogated all effects. Thus, TGF-beta1 blocks the whole cascade of events leading to A beta neurotoxicity by activating the PI-3-K pathway.     

Amyloid-β protein precursor regulates phosphorylation and cellular compartmentalization of microtubule associated protein tau

Tau is a multifunctional protein detected in different cellular compartments in neuronal and non-neuronal cells. When hyperphosphorylated and aggregated in atrophic neurons, tau is considered the culprit for neuronal death in familial and sporadic tauopathies. With regards to Alzheimer's disease (AD) pathogenesis, it is not yet established whether entangled tau represents a cause or a consequence of neurodegeneration. In fact, it is unquestionably accepted that amyloid-β protein precursor (AβPP) plays a pivotal role in the genesis of the disease, and it is postulated that the formation of toxic amyloid-β peptides from AβPP is the primary event that subsequently induces abnormal tau phosphorylation. In this work, we show that in the brain of AD patients there is an imbalance between the nuclear and the cytoskeletal pools of phospho-tau. We observed that in non-AD subjects, there is a stable pool of phospho-tau which remains strictly confined to neuronal nuclei, while nuclear localization of phospho-tau is significantly underrepresented in neurons of AD patients bearing neurofibrillary tangles. A specific phosphorylation of tau is required during mitosis in vitro and in vivo, likely via a Grb2-ERK1/2 signaling cascade. In differentiated neuronal A1 cells, the overexpression of AβPP modulates tau phosphorylation, altering the ratio between cytoskeletal and nuclear pools, and correlates with cell death. Altogether our data provide evidence that AβPP, in addition to amyloid formation, modulates the phosphorylation of tau and its subcellular compartmentalization, an event that may lead to the formation of neurofibrillary tangles and to neurodegeneration when occurring in postmitotic neurons.     

Rosiglitazone ameliorates diffuse axonal injury by reducing loss of tau and up-regulating caveolin-1 expression

Rosiglitazone up-regulates caveolin-1 levels and has neuroprotective effects in both chronic and acute brain injury. Therefore, we postu-lated that rosiglitazone may ameliorate diffuse axonal injuryvia its ability to up-regulate caveolin-1, inhibit expression of amyloid-beta precursor protein, and reduce the loss and abnormal phosphorylation of tau. In the present study, intraperitoneal injection of rosiglitazone signiifcantly reduced the levels ofamyloid-beta precursor protein and hyperphosphorylated tau (phosphorylated at Ser404 (p-tau (S404)), and it increased the expression of total tau and caveolin-1 in the rat cortex. Our results show that rosiglitazone inhibits the expression of amyloid-beta precursor protein and lowers p-tau (S404) levels, and it reduces the loss of total tau, possibly by up-regulating caveolin-1. These actions of rosiglitazone may underlie its neuroprotective effects in the treatment of diffuse axonal injury.     

Aberrant casein kinase II in Alzheimer's disease

Abnormal protein phosphorylation has been identified in Alzheimer's disease (AD) for several proteins including a Mr 60,000 protein, a Mr 86,000 protein and a microtubule-associated protein tau. The Mr 86,000 protein is phosphorylated by protein kinase C, whereas protein kinases responsible for other aberrant phosphorylation reactions are not known. In addition to protein kinase C, another kinase, casein kinase II (CK-II), has now been shown to be aberrant in AD. The spermine-dependent CK-II activity is reduced by 84% in AD and the amount of CK-II as determined by its immunoreactivity on a Western blot is reduced by 63%. Furthermore, the distribution of CK-II in AD is altered. Although the neuronal cell body reacts well with CK-II antisera in the normal cortex, the non-tangle-bearing neurons in the AD cortex showed a 15-30% decrease in anti-CK-II immunoreactivity. The neurofibrillary tangles, on the other hand, stain very strongly with rabbit anti-CK-II and indicates that CK-II may be involved in the pathology of AD. The study of CK-II immunoreactivity for dementing diseases other than AD revealed a similar reduction, suggesting the CK-II involvement in the common process of neurodegeneration.     

Tau,prions and Aβ: the triad of neurodegeneration

This article highlights the features that connect prion diseases with other cerebral amyloidoses and how these relate to neurodegeneration, with focus on tau phosphorylation. It also discusses similarities between prion disease and Alzheimer’s disease: mechanisms of amyloid formation, neurotoxicity, pathways involved in triggering tau phosphorylation, links to cell cycle pathways and neuronal apoptosis. We review previous evidence of prion diseases triggering hyperphosphorylation of tau, and complement these findings with cases from our collection of genetic, sporadic and transmitted forms of prion diseases. This includes the novel finding that tau phosphorylation consistently occurs in sporadic CJD, in the absence of amyloid plaques.     

Molecular and cellular events of dopamine D1 receptor‐mediated tau phosphorylation in SK‐N‐MC cells

Microtubules are involved in the formation of axons and dendrites, maintenance of neuronal morphology, and cellular trafficking. Recent studies suggest that drugs affecting dopamine activity in the brain can induce cytoskeletal modifications. For instance, we have demonstrated in acute rat brain slices a molecular chain of events connecting dopamine D1 receptor to aberrant phosphorylation of the microtubule‐associated protein tau. However, the molecular and cellular effects of tau phosphorylated by means of the activation of dopamine receptors were unexplored. Here we used SK‐N‐MC cells, which express endogenously functional D1 receptors, to demonstrate that levels of phosphorylated tau at serines 199–202 or 214 are increased by a calcium‐dependent pathway subsequent to D1 receptor stimulation. Using selective pharmacological tools, we showed that enhanced intracellular calcium lead to cyclin‐dependent kinase 5 (cdk5) activation, by calpain proteolysis of p35 to p25, as well as glycogen synthase kinase 3β (GSK3β) activation, by its phosphorylation at tyrosine 216. Interestingly, while the activation of protein kinase A (PKA) led directly to the phosphorylation of tau at serine 214, tau phosphorylation at serines 199–202 was independent of PKA. In addition, inhibition of cdk5 or GSK3β prevented the decrease in cell viability induced by D1 receptor stimulation whereas PKA inhibition had no influence. Our data demonstrate that activation of cdk5 and GSK3β following D1 receptor stimulation could have profound influence on both the neuronal cytoskeletal constituent tau and cell survival in SK‐N‐MC cells. Synapse 65:69–76, 2011. © 2010 Wiley‐Liss, Inc.     

Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice

Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of beta-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimer's disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between beta-amyloid and tau alterations may take place in vivo.     

GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila

The tauopathies are a group of disorders characterised by aggregation of the microtubule-associated protein tau and include Alzheimer's disease (AD) and the fronto-temporal dementias (FTD). We have used Drosophila to analyse how tau abnormalities cause neurodegeneration. By selectively co-expressing wild-type human tau (0N3R isoform) and a GFP vesicle marker in motorneurons, we examined the consequences of tau overexpression on axonal transport in vivo. The results show that overexpression of tau disrupts axonal transport causing vesicle aggregation and this is associated with loss of locomotor function. All these effects occur without neuron death. Co-expression of constitutively active glycogen-synthase kinase-3beta (GSK-3beta) enhances and two GSK-3beta inhibitors, lithium and AR-A014418, reverse both the axon transport and locomotor phenotypes, suggesting that the pathological effects of tau are phosphorylation dependent. These data show that tau abnormalities significantly disrupt neuronal function, in a phosphorylation-dependent manner, before the classical pathological hallmarks are evident and also suggest that the inhibition of GSK-3beta might have potential therapeutic benefits in tauopathies.     

The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome

Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer’s disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00978-4.Key Words: O-GlcNAcylation, Down syndrome, OGT/OGA, APP, tau, autophagy