Leah Behar

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.

cis-acting signals and trans-acting proteins are involved in tau mRNA targeting into neurites of differentiating neuronal cells.

Tau microtubule‐associated protein is a neuron specific protein found primarily in axons and is developmentally regulated. The function of tau is in stabilization of microtubules, which is important in establishing and maintaining neuronal morphology. Axonal localization of tau involves a multistep process which is studied in differentiating primary neuronal culture. The initial step involves sorting and subcellular localization of its encoding mRNA into the proximal portion of the axon. Using the transfection assay into neuronal cells, we have demonstrated that sequences located in the 3′‐untranslated region include a cis‐acting signal which is involved in tau mRNA targeting. In addition, using ultraviolet cross‐linking assay, two RNA‐binding proteins of 43 and 38 kDa were identified, that exhibit specific binding to a minimal sequence of 91 nucleotides located within the same functional region, which is involved in targeting. The 43 and 38‐kDa RNA‐binding proteins are present in cytoplasmic extracts, prepared from neuronal cells, and in isolated microtubule preparations. Our results support a novel model in which cis‐acting signals, together with RNA‐binding proteins, are involved in the targeting of tau mRNA, that may ultimately lead to its axonal localization.

Dynamic association with polysomes during P19 neuronal differentiation and an untranslated‐region‐dependent translation regulation of the tau mRNA by the tau mRNA‐associated proteins IMP1, HuD, and G3BP1

Regulation of mRNA translation is a key step in mediating neuronal polarity during differentiation, insofar as neuronal polarity is partially determined by local translation of specific mRNA molecules as dendrites and axons are emanating. The multiplicity of mRNA‐binding proteins in neurons plays an essential role in controlling mRNA translation. These proteins are associated with ribosomes and translation factors, thereby regulating both temporally and spatially the translation process. In a previous study, we have shown an association among the tau mRNA‐binding proteins HuD, IMP1, and G3BP1 with translating polysomes in P19 neurons. In the present study, we determined the dynamics of the association among G3BP1, IMP1, and HuD with polysomes through P19 neuronal differentiation as well as the functional effect of these proteins on tau mRNA translation. We show a novel, differentiation‐dependent association of these proteins with polysomes. In addition, we show a strong, negative effect on translation of the tau mRNA by IMP1, G3BP1, and HuD proteins in HEK‐293 cells. To our knowledge this is the first observation of a direct translational role of G3BP1 for any mRNA and the first report of a translation inhibition by IMP1 and HuD on the tau mRNA in a cell system. The translation inhibition is shown to be mediated by the tau mRNA 3′untranslated regions (UTRs), thus giving a new, translational role for these sequences, which were previously implicated in mRNA stabilization. We also define a novel mechanism for IMP1 binding to tau mRNA, which suggests a conformational binding, which is not sequence dependent.

Dcp1a phosphorylation along neuronal development and stress.

Decapping protein 1a (Dcp1a) is found in P‐bodies and functions in mRNA cap removal prior to its degradation. The function and binding partners of Dcp1a have been thoroughly studied, however its expression pattern is still unclear. In this study we have monitored Dcp1a expression along brain development, neuronal differentiation and during cellular stress. We found that Dcp1a is hyperphosphorylated under these physiological conditions. We followed our observations and identified the specific amino acid residues that are phosphorylated. These findings suggest a novel post‐translational modification that may influence the function of Dcp1a in response to various physiological cues.

Novel M1 Agonists: From Symptomatic Treatment Towards Delaying the Progression of Alzheimer’s Disease

Restoration of ACh levels or replacement with an M1 (or m1) muscarinic agonist may be effective in treating at least some of the cognitive symptoms in Alzheimer’s disease (AD), (see Court and Perry, 1991; Fisher and Barak, 1994). Treatment approaches for AD have to address abnormalities occurring also along various signal transduction pathways (Harrison et al., 1991). Novel activities associated with m1 muscarinic receptors (m1 mAChR) indicate that m1 agonists may also activate hypofunctional signalling pathways in AD (Fisher and Barak, 1994; Gurwitz et al., 1994).