Clearance of intracellular tau protein from neuronal cells via VAMP8-induced secretion.

In Alzheimer's disease (AD), tau, a microtubule-associated protein (MAP), becomes hyperphosphorylated, aggregates, and accumulates in the somato-dendritic compartment of neurons. In parallel to its intracellular accumulation in AD, tau is also released in the extracellular space, as revealed by its increased presence in cerebrospinal fluid (CSF). Consistent with this, recent studies, including ours, have reported that neurons secrete tau, and several therapeutic strategies aim to prevent the intracellular tau accumulation. Previously, we reported that late endosomes were implicated in tau secretion. Here, we explore the possibility of preventing intracellular tau accumulation by increasing tau secretion. Using neuronal models, we investigated whether overexpression of the vesicle-associated membrane protein 8 (VAMP8), an R-SNARE found on late endosomes, could increase tau secretion. The overexpression of VAMP8 significantly increased tau secretion, decreasing its intracellular levels in the neuroblastoma (N2a) cell line. Increased tau secretion by VAMP8 was also observed in murine hippocampal slices. The intracellular reduction of tau by VAMP8 overexpression correlated to a decrease of acetylated tubulin induced by tau overexpression in N2a cells. VAMP8 staining was preferentially found on late endosomes in N2a cells. Using total internal reflection fluorescence (TIRF) microscopy, the fusion of VAMP8-positive vesicles with the plasma membrane was correlated to the depletion of tau in the cytoplasm. Finally, overexpression of VAMP8 reduced the intracellular accumulation of tau mutants linked to frontotemporal dementia with parkinsonism and α-synuclein by increasing their secretion. Collectively, the present data indicate that VAMP8 could be used to increase tau and α-synuclein clearance to prevent their intracellular accumulation.

Tau secretion is correlated to an increase of Golgi dynamics.

Tau protein can be released by neurons, an event linked to the propagation of Tau pathology in Alzheimer'disease (AD). Neuronal hyperexcitability was shown to significantly increase Tau release by neurons. We confirmed this in the present study. In a previous study, it was demonstrated that hyperexcitability induces Golgi apparatus dynamics resulting in its fragmentation. Our present results revealed that the increase of Tau secretion upon hyperexcitability could be significantly reduced by preventing Golgi dynamics through the inactivation of cdk5. We then verified whether a Golgi fragmentation not induced by hyperexcitability could also increase Tau secretion. The suppression of Rab1A, Rab GTPase associated with the Golgi membranes, known to induce a Golgi fragmentation increased Tau secretion by both neurons and HeLa cells. Although it remains to be demonstrated whether the Golgi is directly involved in Tau secretion, the present results demonstrate that its dynamics are correlated to a modulation of Tau secretion.

Rab7A regulates tau secretion.

The axonal microtubule-associated protein TAU, involved in Alzheimer's disease (AD), can be found in the extracellular space where it could be taken up by neurons, an event that is believed to contribute to the propagation of tau pathology in the brain. Since the small GTPase Rab7A is involved in the trafficking of endosomes, autophagosomes, and lysosomes, and RAB7A gene expression and protein levels are up-regulated in AD patients, we tested the hypothesis that Rab7A was involved in tau secretion. We previously reported that both primary cortical neurons and HeLa cells over-expressing human TAU can release tau. Using these two cellular systems, we demonstrated that Rab7A regulates tau secretion. Upon Rab7A deletion, tau secretion was decreased. Consistent with this, the over-expression of a dominant negative and a constitutively active form of Rab7A decreased and increased tau secretion, respectively. A partial co-localization of tau and Rab7-positive structures in both neurons and HeLa cells indicated that a late endosomal compartment could be involved in its secretion. Collectively, the present data indicate that Rab7A regulates tau secretion and therefore the up-regulation of RAB7A reported in AD, could contribute to the extracellular accumulation of pathological TAU species that could result in the propagation of tau pathology in the AD brain.

Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors.

Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with let-7 precursors. Previous studies have described seemingly inconsistent Lin28 binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining the binding mechanism of Lin28 to the terminal loop of pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, we investigate Lin28 binding to TL-let-7g variants and short RNA fragments and identify three independent binding sites for Lin28 on TL-let-7g. We then determine that Lin28 assembles in a stepwise manner on TL-let-7g to form a stable 1:3 complex. We show that the cold-shock domain (CSD) of Lin28 is responsible for remodelling the terminal loop of TL-let-7g, whereas the NCp7-like domain facilitates the initial binding of Lin28 to TL-let-7g. This stable binding of multiple Lin28 molecules to the terminal loop of pre-let-7g extends to other precursors of the let-7 family, but not to other pre-miRNAs tested. We propose a model for stepwise assembly of the 1:1, 1:2 and 1:3 pre-let-7g/Lin28 complexes. Stepwise multimerization of Lin28 on pre-let-7 is required for maximum inhibition of Dicer cleavage for a least one member of the let-7 family and may be important for orchestrating the activity of the several factors that regulate let-7 biogenesis.

Affinity purification of RNA using an ARiBo tag.

The increased awareness of the importance of RNA in biology, illustrated by the recent attention given to RNA interference research and applications, has spurred structural and functional investigations of RNA. For these studies, the traditional purification method for in vitro transcribed RNA is denaturing polyacrylamide gel electrophoresis. However, gel-based procedures denature the RNA and can be very tedious and time-consuming. Thus, several alternative schemes have been developed for fast non-denaturing purification of RNA transcribed in vitro. In a recent report, a quick affinity purification procedure was developed for RNAs transcribed with a 3'-ARiBo tag and shown to provide RNA with exceptionally high purity and yield. The ARiBo tag contains the λboxB RNA and the glmS ribozyme, allowing immobilization on GSH-Sepharose resin via a λN-GST fusion protein and elution by activation of the glmS ribozyme with glucosamine-6-phosphate. This Chapter outlines the experimental details for affinity batch purification of RNAs using ARiBo tags. Although the procedure was originally developed for purification of a stable purine riboswitch mutant, it is demonstrated here for purification of the terminal loop of the let-7g precursor miRNA, an important target of the pluripotency factor Lin28.

Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28.

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7 g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7 g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7 g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7 g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.

Multivalent binding oligomers inhibit HIV Tat-TAR interaction critical for viral replication.

We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the association of a protein-RNA complex, Tat-TAR, that is essential for HIV replication. In vitro binding assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat-TAR binding at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-based assays. MBOs provide a framework for the development of future RNA-targeting molecules.

NMR structure of a complex formed by the carboxyl-terminal domain of human RAP74 and a phosphorylated peptide from the central domain of the FCP1 phosphatase.

Recycling of RNA polymerase II (RNAPII) requires dephosphorylation of the C-terminal domain (CTD) of the largest subunit of the polymerase. FCP1 enables the recycling of RNAPII via its CTD-specific phosphatase activity, which is stimulated by the RAP74 subunit of the general transcription factor TFIIF. Both the central (centFCP1) and C-terminal (cterFCP1) domains of FCP1 interact independently and specifically with the C-terminal domain of RAP74 (cterRAP74), suggesting that these interactions mediate the stimulatory effect of TFIIF on the CTD phosphatase activity of FCP1. Phosphorylation of FCP1 by casein kinase 2 on residues in its central (T584) and C-terminal (S942 and S944) domains stimulates its binding to RAP74 and its CTD phosphatase activity. To improve our understanding of the FCP1-RAP74 interactions, we previously determined the NMR structure of a complex formed by human cterRAP74 and cterFCP1. We now present the high-resolution NMR structure and thermodynamic characterization by isothermal titration calorimetry of a complex formed by the same cterRAP74 domain and a phosphorylated peptide from the central domain of human FCP1 (centFCP1-PO(4)). Comparison of the cterFCP1-cterRAP74 and centFCP1-PO(4)-cterRAP74 complexes indicates that centFCP1 and cterFCP1 both utilize hydrophobic and acidic residues to recognize the same groove of RAP74, but there are significant differences in the details of their interactions. These differences point to the adaptability of RAP74 to recognize the two regions of FCP1. Our NMR and thermodynamic studies further elucidate the complex molecular mechanism by which TFIIF and FCP1 cooperate for RNAPII recycling.