Irith Ginzburg

Subcellular localization of tau mRNA in differentiating neuronal cell culture: Implications for neuronal polarity

A primary neuronal cell culture derived from whole brains of fetal rats was used to analyze the subcellular localization of tau mRNA, employing nonisotopic detection by in situ hybridization. The culture exhibited a developmental differentiation pattern previously described for neuronal cells in vivo; i.e., a transition from immature to mature tau isoforms as well as segregation of tau into the axons. Our results demonstrate that unlike tubulin mRNA, which is confined to cell bodies, or MAP2 mRNA, which extends into dendrites, tau mRNA was observed to enter the proximal portion of the axon. This sorting of tau mRNA might explain how the tau protein could be selectively delivered to the axon and could have important implications for the development of neuronal polarity.

Embryonic Lethal Abnormal Vision-Like RNA-Binding Proteins Regulate Neurite Outgrowth and Tau Expression in PC12 Cells

The embryonic lethal abnormal vision (ELAV)-like proteins are mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family are required for neuronal differentiation. We identified the binding region of purified HuD protein to a target neuronal mRNA encoding for the tau microtubule-associated protein and demonstrated an in vivo interaction between the ELAV-like protein and its target tau mRNA. We show that treatment of neuronal cells with antisense oligodeoxynucleotides directed against HuD blocks the induction of neurite outgrowth and decreases the levels of tau mRNAs, indicating that the ELAV-like proteins are required for neuronal differentiation.

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimers disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m1 muscarinic acetylcholine receptor. Stimulation of m1 receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation‐dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule‐associated protein.

The insulin-like growth factor mRNA binding-protein IMP-1 and the Ras-regulatory protein G3BP associate with tau mRNA and HuD protein in differentiated P19 neuronal cells

Tau mRNA is axonally localized mRNA that is found in developing neurons and targeted by an axonal localization signal (ALS) that is located in the 3′UTR of the message. The tau mRNA is trafficked in an RNA–protein complex (RNP) from the neuronal cell body to the distal parts of the axon, reaching as far as the growth cone. This movement is microtubule‐dependent and is observed as granules that contain tau mRNA and additional proteins. A major protein contained in the granule is HuD, an Elav protein family member, which has an identified mRNA binding site on the tau 3′UTR and stabilizes the tau message and several axonally targeted mRNAs. Using GST‐HuD fusion protein as bait, we have identified four proteins contained within the tau RNP, in differentiated P19 neuronal cells. In this work, we studied two of the identified proteins, i.e. IGF‐II mRNA binding protein 1 (IMP‐1), the orthologue of chick β‐actin binding protein‐ZBP1, and RAS‐GAP SH3 domain binding protein (G3BP). We show that IMP‐1 associates with HuD and G3BP‐1 proteins in an RNA‐dependent manner and binds directly to tau mRNA. We also show an RNA–dependent association between G3BP‐1 and HuD proteins. These associations are investigated in relation to the neuronal differentiation of P19 cells.

BAG-1 Associates with Hsc70·Tau Complex and Regulates the Proteasomal Degradation of Tau Protein

Intraneuronal accumulation of phosphorylated Tau protein is a molecular pathology found in many forms of dementia, including Alzheimer disease. Research into possible mechanisms leading to the accumulation of modified Tau protein and the possibility of removing Tau protein from the system have revealed that the chaperone protein system can interact with Tau and mediate its degradation. Hsp70/Hsc70, a member of the chaperone protein family, interacts with Tau protein and mediates proper folding of Tau and can promote degradation of Tau protein under certain circumstances. However, because Hsp70/Hsc70 has many binding partners that can mediate its activity, there is still much to discover about how Hsp70 acts in vivo to regulate Tau protein. BAG-1, an Hsp70/Hsc70 binding partner, has been implicated as a mediator of neuronal function. In this work we show that BAG-1 associates with Tau protein in an Hsc70-dependent manner. Overexpression of BAG-1 induced an increase in Tau levels, which is shown to be due to an inhibition of protein degradation. We further show that BAG-1 can inhibit the degradation of Tau protein by the 20 S proteasome but does not affect the ubiquitination of Tau protein. RNA-mediated interference depletion of BAG-1 leads to a decrease in total Tau protein levels as well as promoting hyperphosphorylation of the remaining protein. Induction of Hsp70 by heat shock enhanced the increase of Tau levels in cells overexpressing BAG-1 but induced a decrease of Tau levels in cells that were depleted of BAG-1. Finally, BAG-1 is highly expressed in neurons bearing Tau tangles in a mouse model of Alzheimer disease. This data suggests a molecular mechanism through which Tau protein levels are regulated in the cell and possible consequences for the pathology and treatment of Alzheimer disease.

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg2+ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide. Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin. The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.

Brain‐derived neurotrophic factor induces a rapid dephosphorylation of tau protein through a PI‐3Kinase signalling mechanism

The microtubule‐associated protein tau is essential for microtubule stabilization in neuronal axons. Hyperphosphorylation and intracellular fibrillar formation of tau protein is a pathology found in Alzheimers disease (AD) brains, and in a variety of neurodegenerative disorders referred to as ‘taupathies’. In the present study, we investigated how brain‐derived neurotrophic factor (BDNF), an extracellular factor that is down‐regulated in AD brains, affects tau phosphorylation. BDNF stimulation of neuronally differentiated P19 mouse embryonic carcinoma cells resulted in a rapid decrease in tau phosphorylation, at phosphorylation sites recognized by Tau1, AT8, AT180 and p262‐Tau antibodies. K252a, a tyrosine receptor kinase (Trk) inhibitor, attenuated this dephosphorylation event, suggesting that BNDF activation of TrkB is responsible for the tau dephosphorylation. In addition, BDNF had no affect on tau phosphorylation in the presence of wortmannin, a PI‐3Kinase inhibitor, or lithium, a GSK3β inhibitor, suggesting that these two kinases are part of the signaling transduction cascade leading from TrkB receptor activation to tau dephosphorylation. These results suggest a link between a correlate of AD, decrease in BDNF levels and an AD pathology, tau hyperphosphorylation.

Microtubules are involved in the localization of tau mRNA in primary neuronal cell cultures

Subcellular localization of neuronal mRNAs contributes to the development of identifiable microdomains. In differentiated neurons, tau mRNA is localized in the cell body and the proximal portion of the axon, and MAP2 mRNA is localized in the cell body and dendrites, whereas tubulin mRNA is restricted to the cell body. To investigate the mechanism(s) leading to segregation of mictrotubule-associated protein mRNA, we examined the role of the cytoskeleton in this process. Detergent extraction of primary neuronal cells in culture followed by in situ hybridization analysis demonstrated that tau mRNA remains bound to cytoskeleton of the treated cells. In addition, biochemical fractionation showed that tau and MAP2 mRNAs are preferentially associated with the fraction of assembled microtubules. In contrast, mRNAs restricted to the neuronal cell body, such as those of tubulin, the 68 kDa neurofilament, and mouse GAPDH, are preferentially found in the supernatant. Using cytoskeletal inhibitors, we demonstrate that tau mRNA is associated with the microtubule system, and not with the actin filaments, thus supporting the hypothesis that the mechanism of mRNA localization is a multistep pathway in which the microtubules play a crucial role.

GAP‐43 regulates NCAM‐180‐mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth‐associated protein (GAP‐43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP‐43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP‐43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP‐43. We demonstrate that phosphorylation of GAP‐43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM‐induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP‐43, NCAM‐induced neurite outgrowth requires functional association of NCAM‐180/spectrin/GAP‐43, whereas in the absence of GAP‐43, the NCAM‐140/non‐receptor tyrosine kinase (Fyn)‐associated signaling pathway is pivotal. Thus, expression of GAP‐43 presumably acts as a functional switch for NCAM‐180‐induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP‐43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM‐180/spectrin‐mediated modulation of the actin cytoskeleton, NCAM‐140‐mediated activation of Fyn, or both.

Identification of 3'UTR region implicated in tau mRNA stabilization in neuronal cells.

Tau, a neuronal microtuble-associated protein (MAP) plays an important role in the formation and maintenance of neuronal polarity. Tau mRNA is a stable message and exhibits a relatively long half-life in neuronal cells. The regulation of mRNA stability is a crucial determinant in controlling mRNA steady-state levels in neuronal cells and thereby influences gene expression. The half-lives of specific mRNAs may be dependent on specific sequences located at their 3′untranslated region (UTR), which in turn, may be recognized by tissue-specific proteins.To identify the sequence elements involved in tau mRNA stabilization, selected regions of the 3′UTR were subcloned downstream to c-fos reporter mRNA or to the coding region of the tau mRNA. Using stably transfected neuronal cells, we have demonstrated that a fragment of 240 bp (H fragment) located in the 3′UTR can stabilize c-fos and tau mRNAs. Analysis of stably transfected cells indicated that the transfected tau mRNAs are associated with the microtubules of neuronal cells, suggesting that this association may play a role in tau mRNA stabilization. This step may be a prerequisite in the multistep process leading to the subcellular localization of tau mRNA in neuronal cells.